DEVELOPMENT AND EVOLUTIONARY ORIGINS OF VERTEBRATE SKELETOGENIC AND ODONTOGENIC TISSUES (3)...

B i d . Rev . (19901, 65. PP . 277-373 Printed in Great Britain

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DEVELOPMENT AND EVOLUTIONARY ORIGINS OF VERTEBRATE SKELETOGENIC AND ODONTOGENIC

TISSUES

BY MOYA M . SMITH AND BRIAN K . HALL*+ Unit of Anatomy in Relation to Dentistry. United Medical and Dental Schools of

Guy's and S t Thomas's Hospitals. London Bridge S E I 9 R T . U.K.

(Received 13 November 1989. revised and accepted 20 February 1990)

C O N T E N T S I . Introduction . . . . . . . . . . . . . . .

I1 . T h e evolutionary sequence in which tissues appeared amongst the lower taxa of vertebrates . . . . . . . . . . . . . . . (I) Introduction . . . . . . . . . . . . . . (2) Problematica . . . . . . . . . . . . . . (3) Primitive craniates -conodonts . . . . . . (4) Primitive craniates - early agnathans . . . . . . . . .

(a) T h e tuberculated exoskeleton of Middle and Upper Ordovician specimens .

(c) Astraspis and Pycnaspis . . . . . . . . . . . (b) Eriptychius . . . . . . . . . . . . . .

( 5 ) Heterostracans . . . . . . . . . . . . . . (a) Tubercles . . . . . . . . . . . . . . (b) Aspidin . . . . . . . . . . . . . .

(6) Anaspids . . . . . . . . . . . . . . . (7) Thelodonts . . . . . . . . . . . . . . (8) Galeaspids . . . . . . . . . . . . . .

(a) Tubercles . . . . . . . . . . . . . . (10) Gnathostomes . . . . . . . . . . . . . .

(9) Osteostracans . . . . . . . . . . . . . .

(a) Chondrichthyans . . . . . . . . . . . . (b) Acanthodians . . . . . . . . . . . . .

( I I ) Summary and discussion . . . . . . . . . . . . (a) Cellular-acellular bone . . . . . . . . . . . (b) Cartilage versus bone . . . . . . . . . . . (c) Types of dentine . . . . . . . . . . . . (d) Developmental perspectives . . . . . . . . . .

111 . T h e neural crest origin of cranial skeletogenic and odontogenic tissues in extant vertebrates . . . . . . . . . . . . . . .

(c) Osteichthyans . . . . . . . . . . . . .

(I) Introduction . . . . . . . . . . . . . . (2) Dentine . . . . . . . . . . . . . . . (3) Bone . . . . . . . . . . . . . . . (4) Cartilage . . . . . . . . . . . . . . .

IV . T h e neural-crest origin of trunk skeletogenic and odontogenic tissues in extant vertebrates . . . . . . . . . . . . . . .

278

325 325 328 329 329

332

* Permanent address : Department of Biology. Life Sciences Centre. Dalhousie University. Halifax. Nova

f No significance should be attached to the order of authorship . Scotia. Canada B3H4J I .

278 MOYA M. SMITH AND B. K. HALL 1.. Regulation of the development of skeletogenic and odontogenic tissues in extant

\ ertebrates . . . . . . . . . . . . . . . ( I ) De\ elopmental principles . . . . . . . . . . .

(3) Epithelial-mesenchymal interactions and the differentiation of dermal bone . .

(4) Epigenetic cascades . . . . . . . . . . . . . (5) Epithelial~mesench!.mal interactions and inhibition of differentiation . . .

V l . 1Iaintenance o f developmental interactions regulating skeletogenic/odontogenic differentiation across vertebrate taxa . . . . . . . . . . . ( I ) Introduction . . . . . . . . . . . . . . ( 2 ) I,oss of skeletal elements. . . . . . . . . . . . (3) Epithelial-mesenchymal interactions and heterochrony (1) Transformations of odontogenic tissues . . . . . . . . . (j) Loss/reappearance of odontogenic tissues . . . . . . . . (6) Loss/reappearance of skeletogenic tissues

V l I . The neural-crest origin of skeletogenic and odontogenetic tissues in the first vertebrates :

(z) Epithelial ~mesenchymal interactions . . . . . . . . .

i z postulates . . . . . . . . . . . . . . . ( I ) Dentine . . . . . . . . . . . . . . ( 2 ) Bone o f attachment . . . . . . . . . . . . (3) Basal bone . . . . . . . . . . . . .

(j) L'ncalcitied cartilage . . . . . . . . . . . . (6) Perichondral bone . . . . . . . . . . . . . (7) Trunk neural crest . . . . . . . . . . . . . (8) Trunk endoskeleton. . . . . . . . . . . . ( 0 ) Initiation of differentiation . . . . . . . . . . .

( 10) Timing of epigenetic cascades . . . . . . . . ( I I ) Heterochronic shifts . . . . . . . . . . .

(4) Calcified cartilage . . . . . . . . . . . . .

. .

( i 2 ) Epithelial-mesenchymal interactions . . . . . . . . . Y l l l . 1)iscussion . . . . . . . . . . . . . .

I S . Summar! . . . . . . . . . . . . . . . N. .lckno\\ Iedgemcnts . . . . . . . . .

S I . References . . . . . . . . . .

335 3 3 i 336 3 3 8 339 3-10

34' 341 342 3-12 3 4-1 346 348

I. I N T R O D U C T I O N

This review deals with the development and evolution of vertebrate skeletogenic and odontogenic tissues. Included in our treatment is the association between these tissues and the evolutionary sequence in which they appeared in the earliest vertebrates; the separate evolutionary origin of the exo- and endoskeletons ; the neural crest origin of skeletogenic and odontogenic tissues ; the processes that regulate their development and nhether these are conserved across vertebrate taxa. We derive 1 2 postulates that encompass our knowledge of the evolutionary and developmental origin of these ti s u e s .

A few examples from the range of skeletal tissues found in extant and fossil vertebrates are illustrated in Fig. I . T h e endoskeleton and exoskeleton are amazingly shown at the same time in the intact Jurassic fossil actinopterygian, Mesodon (Fig. I D), which as a normal occurrence only has exoskeleton over the anterior half of the fish, leaving the endoskeleton exposed in the posterior half, both types are well ossified. T h e t\vo components of the cranial endoskeleton, chondrocranium and visceral arches (Fig. I A), in osteichthyan fishes are invested with exoskeletal dermal bones and themselves become converted into bone. In contrast, the endoskeleton of chondrichthyan fishes is completely cartilaginous (see later for exceptions to this), and the exoskeleton is without

Skeletal development and evolution 279

dermal bones, just placoid denticles. This exoskeleton consists of separate non- articulated placoid scales/denticles (Fig. I B , C), each one is considered to be an odontode (homologous with oral odontodes/teeth) consisting of enameloid, dentine and bone of attachment (Fig. I B, see later for debate on this point, also Fig. 5 ) . These may form an ordered, overlapping array in fossil sharks (Fig. I C), comparable to that shown in the exoskeleton of the fossil actinopterygian (Fig. I D). It is possible to find in some fossil collections of isolated scales, a growth series (Fig. I E, F), in which the dentine of the scale gradually becomes thicker at the expense of the pulp chamber, and the bone of the base thickens with each increment of growth; a normal pattern in odontogenesis. Growth of the endoskeleton and its conversion from cartilage to bone (endochondral ossification) is part of skeletogenesis, as is fusion, addition, and remodelling in the exoskeleton. We classify and define skeletogenic and odontogenic tissues as given in Table I .

In reviewing the fossil evidence for the earliest vertebrate skeletogenic and odontogenic tissues we use the following :

I . Previous postulates on the evolution of skeletal tissues by Moss ( I 968 a-c), Schaeffer (1977), Brvig (1951, 1957, 1965, 1968), Hall (1975, 1978) and Reif (1982).

2. A seminal paper by Patterson (1977) on the distinction between dermal bone and membrane bone on the one hand and between the endoskeleton and exoskeleton on the other.

3. Janvier’s (1981) cladogram of the craniates and extrapolation of the occurrence of skeletal tissues to vertebrate phylogeny.

4. Maisey’s (I 988) recent analysis of the phylogeny of vertebrate skeletal tissues and of the evolution of the developmental processes that produced those tissues.

In reviewing the developmental basis for the evolution and diversification of vertebrate skeletal tissues we start with the assumption that, both from a consideration of development and from evolution, the exoskeleton and endoskeleton have separate origins. Although not the first to challenge the assumption that the two were immutably linked [Moss ( I 968 c) and Halstead (I 969) having put forth the same proposition], Patterson ( I 977) marshalled the most impressive evidence and arguments for the dermal skeleton and endoskeleton having always been distinct. In setting down the evidence we also have to consider the possibility of a less direct relationship, in which one follows the other and is influenced by the other during development. There is no evidence for such a causal association ; in fact, there is evidence for disassociation of exo- and endoskeletal elements, especially under hormonal control during amphibian metamorphosis (Trueb, 1985; Hanken & Hall, 1988a, b).

The second assumption is that all the cranial and trunk exoskeleton and the greater part of the cranial endoskeleton are derived from neural crest. All these depend for their differentiation on reciprocal interactions between neural-crest cells and ectoderm or endoderm, whereas the postcranial endoskeleton is derived entirely from mesoderm and, although initiated through tissue interactions that may involve the ectoderm, it is not tied to the ectoderm as is the neural-crest-derived skeleton.

A third assumption is that fossil skeletogenic and odontogenic tissues can be identified on the basis of tissues in extant vertebrates and that sequences in development reflect sequences in evolution (in particular, where there is a causal relationship in the sequence, see Alberch, I 985) ; in the exoskeleton, dentine-enamel-bone of attachment-

280 MOYA M. SMITH AND B. K. HALL

Cranial endoskeleton

Chondrocranium ----------

Visceral skeleton I ar bh hh ch bbrhbrCDr Exoskeleton

(') Placoid scale rows

I Endoskeleton I I-) Trunk caudal I

, -..-.y...-

I

I

Exoskeleton -..?&

Cranial trunk I 4 - 1 (D)

Developmental series placiod scales

Fig. I . For legend see opposite

Skeletal development and evolution

Table I . ClassiJcation of skeletogenic and odontogenic tissues

Exoskeleton Endoskeleton

28 I

Dermocranial and trunk Skeletogenic Odontogenic

Cartilage (secondary) Dentine

Chondro-viscerocranial Axial, appendicular

Skeletogenic

Cartilage (primary) Bone

Bone Dermal Enamel Perichondral Perichondral -

Endochondral Cementum Membrane Acellular/cellular Bone of attachment Acellular/cellular

Endochondral

dermal bone-fusion of all elements (secondary cartilage in some groups); in the endoskeleton, cartilage-perichondral bone-endochondral bone ; later membrane bones (Patterson I 977). Phylogenetic series have already been proposed by developmental studies on the dentition of teleost fishes (Huysseune, 1983), but these were on the pharyngeal dentition, a possibly specialized dentate region. An evolutionary sequence of the occurrence of skeletal tissues can be predicted by cladistic taxonomy, as proposed by Janvier ( I 981, I 984) and Maisey ( I 988), but often skeletal characters are relied upon in forming the cladogram.

The topics that we discuss are the following: I . The subdivision of the skeleton into exo- and endoskeleton and which appeared

first in the vertebrate record ; which tissues were initially and subsequently present in exo- and endoskeletons ; whether the same tissues (e.g. cartilage, bone) necessarily appeared in the same sequence in both the exo- and endoskeleton, the topographic association between skeletal and dental tissues.

Fig. I (A-F). The main categories of skeletal tissues, are illustrated, exoskeleton and endoskeleton, as well as the odontogenic/dental component of the exoskeleton (placoid scales). (A) chondrocranium and visceral arch skeleton, the two components of the cranial endoskeleton, in a teleostome fish (after Goodrich, 1930, fig. 283). (B) Vertical section of a placoid scale of an extant shark, lateral, and surface views (after Romer, 1971 , fig. 71) . (C) View from inside of symmetrically arranged scale bases in the skin of a Silurian shark, Elegestolepis grossi, in which the overlap area of the button-shaped denticles is indicated. These scale bases are shown in vertical section and horizontal views in (E) and (F) (Karatajute- Talimaa, 1973, figs j N , gA-E). (D) Lateral view, restoration of Mesodon macropus, Upper Jurassic, with cheek plates removed, dermal bones of the face and jaws in place, and a cover of articulated scales form the exoskeleton, but the caudal region is as found, ‘destitute of scales’, leaving exposed the deeper endoskeleton (after Woodward, 1898, fig. 74). (E, F) Growth series of scales of Elegestolepis grossi in vertical sections with blood vessels indicated (E), to show the increase in thickness of dentine and the development of the neck canal. Base of scale (F) from below to show the growth of basal tissue (bone/cement) and the obliteration of the basal canal (Karatajute-Talimaa, 1973, fig. jA-E). (C, E, F reproduced by permission from the author and publisher, E. Schweizerbart’sche, Verlagsbuchhandlung.) Abbreviations: ac, auditory capsule; an, antorbital process; ar, articular region; ba, base; bbr, basibranchial ; bh, basihyal ; bpr, basal process ; btp, basitrabecular process ; cbr, ceratobranchial ; ch, ceratohyal; d , dentine; dd, durodentine ; ddk,’dentine canals; e, enamel; ebr, epibranchial; fr, frontal; ha, neck ; hbr, hypobranchial ; hh, hypohyal ; hk, main canals; hm, hyomandibular ; kr, crown ; lc, lateral commissure; m, mandible; md, mesodentine; m.eth, mesethmoid; nc, nasal capsule; op, operculum; oca, occipital arch; orb, orbit; orc orbital; ot, otic process; pa, patietal; pbr pharyngobranchial; pc, pulp cavity; pfc, prefacial commissure; pl,, basal pulp canal; pl,, neck canal; pmx, premaxilla; p.op, preoperculum; pq, palatoquadrate; q, quadrate; s , symplectic region; sn, nasaal septum; s.ocs, supraoccipital ; spc, spiracular canal ; squamosal ; st, stylohyal ; tr, trabecula; v, prevomer.

I * B R E 65

282 M O Y ~ M. SMITH AND B. K. HALL T h e proposition in this section, following Reif (1982), Janvier (1981) and Patterson

(1977) will be ( a ) that exoskeletal dentine and bone of attachment appeared earliest in the fossil record and that cartilage developed only subsequently and secondarily in the exoskeleton ; ( b ) that initially cartilage alone appeared within the endoskeleton, followed by perichondral bone (prismatic calcified cartilage may be an alternative perichondral tissue) and then endochondral bone ; (c) that dental tissues were restricted to the exoskeleton and that dentine was covered by enamel and supported by bone of attachment; (4 that the basal bone on which dentine and bone of attachment rest is a dermal bone and part of the exoskeleton.

Evidence will come from fossils placed in hierarchical order and from the sequence of skeletogenesis during development. Also discussed will be to what extent it is acceptable to use order of appearance in development to predict fossil sequences, and the limitations ; modification of developmental strategies to achieve the adult phenotype in a restricted time scale. As Alberch (1985) has stated so cogently, only causal developmental sequences [i.e. sequences where one developmental event depends for its appearance on a previous developmental event(s) occurring] will be conserved phylogenetically.

This will involve discussing (i) whether cartilage or bone appeared first in the endo- exoskeletons; (ii) whether acellular bone was the first bone tissue; (iii) whether and why dentine occurred before bone (the sensory function of dentine) ; (iv) the association of dentine with bone of attachment in the first instance (the odontode); (v) time of appearance of basal bone and whether exo- or endoskeletal; (vi) presence of enamel (enameloid) in the first exoskeleton, the structure of odontodes ; (vii) whether the first vertebrate skeleton was cranial or t runk; we will argue that the first skeleton was exoskeletal, both cranial and trunk.

2. Whether the first vertebrate skeleton was of neural crest or mesodermal origin. Because [as proposed in I . (vii) above] the first vertebrate skeleton was exo-skeletal, we will argue that it was also neural-crest derived; in fact, that a neural-crest skeleton is the primitive (fundamental) vertebrate skeleton and that the neural crest is a quintessential vertebrate tissue. Evidence for this position comes from experimental studies on the origin of the skeleton in all major groups of extant vertebrates from agnathans to mammals.

3. As exoskeleton appeared in the trunk as dermal denticles, the trunk exoskeleton was also of neural crest origin. But tradition dictates that, among extant vertebrates, only cranial neural crest is skeletogenic/odontogenic. We review evidence for segregation of cranial/trunk neural crest, discuss cranial/trunk neural crest in relation to the production of ectomesenchyme, and whether the ability to produce ectomesenchyme is equivalent to the ability to form skeletal/dental tissues.

4. Whether the mechanisms that regulate skeletal development differ between the skeletogenic and odontogenic tissues on the one hand and between exo- and endoskeleton on the other, and whether these mechanisms are maintained across the vertebrates. j. I t is impossible to establish an independent phylogeny of the early vertebrates

based upon skeletal/dental developmental characters alone, because the majority of the characters available to establish any phylogeny of the early vertebrates are skeletal and dental. Every phylogeny of the early fossil vertebrates must therefore rely heavily

Skeletal development and evolution 283 on skeletal and dental characters that subsume development. However, the polarity of the skeletal characters, if established independently, could influence the hierarchy developed from a wider character base.

11. T H E EVOLUTIONARY SEQUENCE IN WHICH T H E TISSUES APPEARED AMONGST T H E LOWER TAXA OF VERTEBRATES

(I) Introduction

Initially we start with a group by group summary of the skeletal tissues in early fossil vertebrates to precede a discussion based on established phylogenies of Janvier (1981, 1984), Reif (1982), Blieck (1982, 1984), Halstead (1982), Forey (1984) and Maisey (1988).

An important tool in understanding the structure of the skeletogenic and odontogenic tissues is the ability to examine thin slices of the whole animal and sections through the fossil tissues for light microscopy and for both transmission and scanning electron microscopy, Halstead Tarlo (I 964 6, I 969) provided an excellent history of studies on the calcified tissues in the earliest agnathan vertebrates, beginning with Agassiz’s ( I 845) and Pander’s (1857) identification of dentine in Psammosteus. Huxley’s key paper of 1858 on the skeletal microstructure first allowed Lankester (I 868) to separate the Heterostraci from the Osteostraci on the basis of the character, ‘skeletal tissues lacking bone cell spaces ’, and then Traquair ( I 898) to place Psammosteus in the Heterostraci. Halstead Tarlo (19646, 1969) continues with Gross’s (1930) naming of aspidin, and then with detailed discussion of the microstructure of aspidin, dentine, cartilage and intermediates between aspidin-bone, dentine-aspidinlbone, or cartilage-bone. Clearly the identification of aspidin is central to the theme of the origin of cellular bone, whether in a sequence from the acellular variety, prior to it, or separate from it.

Detailed microscopy of fossil fish tissues originated with Agassiz and Pander, but more recently the most extensive reporting and discussion of patterns and proposed mechanisms of vertebrate skeletal evolution has been by 0rvig (1951, 1957, 1965, 1968). By comparing the histology, we can classify the tissues present and examine their topographical relationships to one another, although it is often difficult to localize them to particular regions of the body; they are often only available as isolated fragments. When preserved as whole animals they are often only as moulds in the rock matrix, although in many the skeletal tissues are also present.

As a general statement we can say that examination of such fossil tissues indicates a degree of complexity and sophistication perhaps unexpected in animals that are half a billion years old, and reveals a variety of tissue types not found in extant forms. We can be sure of this complexity, and these differences, because we can classify such tissues on the basis of the skeletal tissues of extant vertebrates ; bone, cartilage, dentine, cement and enamel, with the additional advantage that they can be more clearly identified from a knowledge of their development and growth. We do not give a description of each tissue because of a general acceptance of each type (although there is debate about many of the specific examples). Where appropriate a detailed description will be given alongside the discussion of evolutionary consequences (section VI I I). Examples of each type can be found in the illustrations throughout the paper (Figs 4-10, 12-16).

It is true that there are ambiguous tissues present in these early vertebrates, that do T I - 2


C

Fig. z(X-C). Large dorsal (A) and ventral (B) shields, together with lateral ( C ) and rostra1 plates, and a range of small bones, scales and spines form a complete armour to the head, trunk and tail in this reconstruction of a psammosteid heterostracan agnathan Drepanaspis gemuendenszs, x 0.3 (Gross, 1963, figs I O A , B, I I A). (Reproduced by permission of the publisher, E. Schweizerhart’sche, Verlangs- huchhandlung).

not fall readily into the categories established from extant forms, perhaps because of plasticity of development. This situation lead one of us to stress the importance of ‘ intermediate ’ tissues that have features of two of the readily classified skeletal or dental tissues (Hall, 1975, 1978). That the number of such tissues declined as vertebrates evolved is taken as evidence for a consolidation of skeletogenic and odontogenic lineages as individual tissues were found by natural selection to be best suited to particular tasks, cartilage for rapid growth of the endoskeleton, bone to store mineral, to respond readily to balance levels of circulating calcium and phosphate, and to house haemopoietic tissue ; dentine for sensing tactile, temperature, osmotic and/or electrical changes in the environment. We also see a separation of skeletal from dental tissues, the former becoming specialized for support, growth and mineral storage, the latter for prey capture and mastication.

Skeletal development and evolution 285 Discussion of the tissues present in particular early vertebrate groups will be on the

basis of those tissues that are found in fossils, i.e. we will only be tabulating fossilizable skeletal and dental tissues. Other skeletal tissues not capable of being mineralized (e.g. notochord, unmineralized cartilage) may also have been present in these early vertebrates, an issue that we take up in section VIII.

Inevitably, a stratigraphic order forms the easiest initial basis for presenting a sequence of skeletal types, although one is well aware that this may not show a progression from primitive to derived types. The earliest phosphatic skeleton of craniate affinity is that of Anatolepis (Bockelie & Fortey, 1976, Repetski 1978) from the Upper Cambrian and Early Ordovician. Much debate has focused on whether it is an arthropod or vertebrate, but Janvier ( I 9 8 1 ) concluded that the material described by the latter author is vertebrate and suggested heterostracan affinities, although recently he has reverted to the opinion that it should be incertae sedis (pers. comm., 1989).

The earliest certain vertebrate fossils are agnathans of the Ordovician, consisting of head shields, trunk and tail regions as typified by the classic illustrations of Drepanaspis (Fig. z), and assemblages of exoskeletal and endoskeletal fragments, from animals that were generally small (50-300 mm in body length).

The earliest chordate is represented by a single species in the Middle Cambrian, Burgess Shale, Pikaia gracilens (Conway Morris & Whittington, 1979), described as free-swimming, with a notochordal rod and segmental muscle blocks, but no calcified skeleton. This superb preservation of a soft-bodied animal represents a major discovery early in the life-history of the phylum Chordata, of which vertebrates are later representatives, and in which mineralized skeletal structures appeared.

We attempt to describe the skeletal components of individual genera within taxa to which they have been previously assigned, for example the five taxa of fossil agnathans [(c)-(g), listed below], following closely a scheme proposed by Janvier ( 1 9 8 1 ) , but separate some of the early fossils into non-taxonomic groups, because some have been difficult to assign in previous phylogenies.

(a) Problematica - Cambrian to Ordovician (b ) Primitive craniates

(i) Conodonts - Cambrian to Triassic (ii) Early agnathans - Middle and Upper Ordovician

( c ) Heterostracans - Lower Silurian to Upper Devonian ( d ) Anaspids - Lower Silurian to Upper Devonian (e) Thelodonts - Lower Silurian to Upper Devonian (f) Galeaspids - Silurian to Upper Devonian (g) Osteostracans - Lower Silurian to Upper Devonian (h) Gnathostomes - Lower Silurian to Present

(i) Chondrichthyes (ii) Acanthodii

(iii) Osteichthyes

(2) Problematica

Although phosphatic skeletons have been described from Early Cambrian deposits (Bengtson, 1977a , b) their affinities have not been agreed upon, consequently they have been assigned to a group simply referred to as Problematica. Accounts of these skeletal

286 MOYA M. SMITH AND B. K. HALL remains, not exceeding 2 mm in dimensions, sometimes referred to as sclerites, concentrate on their morphological diversity and their possible use in correlation of Cambrian deposits. Amongst the tissues it is probable that some are homologues of vertebrate tubercles, as suggested by Nitecki et al. (1975) for some of the Ordovician phosphatic fragments from the United States, who comment that histology is critical for identification. Relevant to this, the histology has been described for Late Cambrian (Marss 1988), and for Early Cambrian forms by Bendix-Almgren & Peel (1988) and is considered as part of this review.

Whilst the vertebrate affinity of sclerites has not been universally acknowledged, we are not aware of similar tissues for any accepted non-chordate fossil or extant group. Marss (1988), did compare the sclerites of Hadimopanella and Kaimenella with the heterostracans and anaspids, on the basis of their three-layered microstructure, but Bendix-Almgren & Peel (1988) concluded that those of Hadimopanella were more comparable to sclerites of Utaphospha. Marss referred to this tissue in Hadimopanella and Kaimenella as enameloid-covered dentine tubercles (Marss, 1988, figs 3, 5). T h e former has no basal bone, whereas in the latter Kaimenella, there are clearly three tissues, the most basal being spongy bone, or at least bone with vascular canals. The superficial part is relatively acid resistant, and translucent, called dense capping by Bendix-Almgren & Peel ( I 988, fig. 7), and the core and basal portion is penetrated by very fine linear spaces above a basal lamellated layer, the latter could have formed as increments of the core tissue. T h e fine linear spaces are not tubule-like but could represent spaces between groups of large parallel crystals in an organized phosphatic skeleton. Large crystals are illustrated in this tissue by Bendix-Almgren & Peel (1988, fig. 4E). However, Bendix-Almgren & Peel (1988) considered that these spaces represent fibre spaces and further suggested that the fibres anchored the sclerites to the inter-sclerite wall. They also drew an analogy with lower vertebrate dermal odontodes, and considered two possibilities ; that the sclerites of Hadimopanella represented ectodermal skeletal units either ( I ) of early vertebrates, or (2) of primitive chordates. In the latter taxon, they compared the sclerites with ascidian spicules, in particular those of the fossil and extant species of Cystodytes. Their tentative conclusion was that these sclerites belong to early Paleozoic relatives of the Urochordata, and they suggested that this represents a separation of the bioinorganic evolution of hard substances between these and the cephalochordates to which some of the Anatolepis material (Repetski, 1978) is referred. However a major problem remains to be explained because the mineral composition of tunicate spicules is different, being aragonite or vaterite, not hydroxyapatite (van den Boogaard, I 989). Another major difference between the groups would be that only the epithelium is involved in the formation of sclerites, not mesenchyme. This would mean that neural crest is not involved in the urochordate line in the production of the exoskeleton, a very important consideration in any discussion of the evolution of vertebrate skeletal tissues.

T h e skeletal fragments of Anatolepis from Upper Cambrian and Lower Ordovician rocks (Fig. 3) , show parts (trunk or cranial) where a thin sheet of tissue is covered by rows of closely spaced, lancelate, rhombic structures, and these have been assigned as craniates with heterostracan affinities (Bockelie & Fortey, 1976 ; Janvier, 1981), although their position is still considered as problematic by some (incertae sedis, Janvier 1989, pers. comm.). Whole animals have been described from the Upper Cambrian with scales in situ (Repetzki, 1978), and sections through a row of scales of Anatolepis


Fig. 3(A-C). Three SEMs of the dermal skeleton of Anatolepis heinzei showing flattened scales in oriented rows adjacent to an edge with a tooth-like row (A, B). T h e scales (25-100 ,urn) are located on an extremely thin basal sheet of mineralized tissue, arrows (C). (A) x 195; (B) x 1 5 0 ; (C) x500. Interpolated scales often occur in the rows, suggestmg constant infilling or replacement during growth by individual units (scale odontodes). [Photographs provided by Richard Fortey, British Museum of Natural History (BMNH).]

heintzi described by Bockelie & Fortey (1976, fig. 3) from the Early Ordovician of Spitzbergen. Slightly earlier (Late Cambrian) deposits from Western Newfoundland also contained fragments of the integument of Anatolepis (Fortey et al., 1982; fig. 9X, Y, AA). The fauna was fully marine and the specimens found only 1-2 mm, although considered to be parts of larger plates. The small rhomboid scales (zoo pm long) have a polarity (possibly antero-posterior) and are arranged in alternating closely spaced rows (Bockelie & Fortey, 1976, figs. I , z). From the histology they tentatively suggested that the tubercles of the scales were dentine above a spongy layer, all superimposed on a continuous lamellar layer. The suggestion was made by Bockelie & Fortey (1976) that

288 MOYA M. SMITH AND B. K. HALL skeletization occurred all at once in the integument. We interpret this to mean that tubercles (odontodes) formed throughout the epithelium and fused together via their bone of attachment with a thin sheet of basal tissue. It can only be based on speculation to suggest that the exoskeleton was non-growing and replaced all at once in phases of remodelling.

Apparently Anatolepis occurs in the same sediments as Arandaspis (Fortey, pers. comm.), also as a form with rows of separate pointed tubercles fused onto a continuous thin sheet of basal tissue, without tesserae. It is significant that Blieck and Janvier (pers. comm) think that Aranduspis is the most primitive of the early agnathans [see section II(4)].

(3) Primitive craniates - conodonts

Before considering types that are frequently placed with the heterostracans because of similarly shaped dermal ornament and the occurence of only acellular bone, it is necessary to present recent evidence on the conodonts, a group consisting of assemblages of phosphatic skeletal parts, represented as separate elements from as early as the Cambrian. Until Briggs, Clarkson & Aldridge (1983), Aldridge & Briggs (1986, 1989) and Aldridge et al., (1986) described the soft parts of the whole animal with conodont elements in situ from the Lower Carboniferous, their relationship to craniates was unknown. Although in the earlier paper they were able to recognise that the skeletal remains were part of a feeding apparatus, they assigned the conodonts to a new phylum Conodonta. However, in the 1986 paper with the discovery of three more specimens with the conodont apparatus in situ within the anterior central lumen, they were able to assign these to a separate group of primitive jawless craniates; because of the chevron-shaped, segmental muscle blocks, lateral flattening, and the head with an axial structure and possible sensory organs.

Aldridge et al. (1986, fig. 9) argue for a closer relationship between conodonts and hagfishes than between conodonts and lampreys, but they have recognized that ' the evidence for homology between conodont elements and dermal denticles of early vertebrates is inconclusive '. A second important point with relevance to the vertebrate skeleton, is the total lack of any evidence for a dermal skeleton in association with conodonts, and this has led Aldridge et al. (1986) to conclude that conodont elements xvere derived independently from the craniate dermal skeleton. Dzik (1976) had previously considered a possible homology between conodont elements and astraspid- like denticles, but Aldridge et al. ( I 986) suggested that further research on the histology of dermal tubercles of the earliest vertebrates was required before homology could be accepted. More specifically Dzik (1986) has suggested that the basal tissue of conodont elements was an analogue of aspidin, starting with tubules from mesodermal cells which disappeared in later stages. This is exactly the point ; they simulate teeth but are not homologues of teeth.

The suggestion has also been made that conodont elements may be homologous with teeth of the hagfish, and that, in turn, hagfish teeth may be homologous with the teeth of mammals, from morphological and molecular affinity studies by Krejsa & Slavkin (1987) and Slavkin et al. (1983). They reported that the keratinous structures of hagfish teeth had proteins in common with mammalian enamel. However, we do not accept that there is a reasonable basis for homology as there is no equivalent tissue to the dental

Skeletal development and evolution 289 papilla. Therefore on morphological grounds we do not support the view that hagfish teeth are homologous with odontodes nor that conodont elements represent the earliest form of craniate skeletal tissues. It should be recorded that recent publications by Krejsa, Bringas & Slavkin (1990) describe a sequence of tooth development in the hagfish Myxine glutinosa and Eptatretus stouti that they suggest is morphologically similar to that in lampreys and mammals. They conclude that the dental papilla is probably ectomesenchymal and that all these elements develop as true teeth from a dental lamina. This emphasises their previous suggestion (Krejsa & Slavkin, 1987) that oral dental tissue arose before the dermal skeleton, and that conodonts are (jawless) Paleozoic craniates ancestral to the hagfishes. Controversy has existed over the horny teeth of Myxine since Beard’s original account in 1889, discussed by Tomes (1904) and contrasted with the horny teeth of the platypus, a monotreme. Horny teeth develop for a number of different adaptations throughout the vertebrates, and in each the timing and mode of development is apparently different. We feel that the weight of evidence is congruent with teeth being homologous (neural-crest derived) with odontodes of the dermal skeleton, odontodes being already present before any skeletal structures in the oral cavity. In this we agree with the central postulate of Reif (1982) and Schaeffer (1977) (see Section VIII). Patterson’s (1988) conclusions, that there are more kinds of ‘ homologous ’ relation between molecular sequences (in this case, enamel proteins) than in morphology (odontode histology), leads to the suggestion that these findings in cyclostome teeth may be only an example of gene phylogeny and not organ homology with identical developmental sequences; only the latter can be used as a shared primitive character (plesiomorphy) in cladistic analysis. In other words the concept of homology must be very carefully considered [see section VI (6)]. In reality conodont elements and hagfish keratinous toothlets may both be convergently derived structures relating to special modes of feeding and are certainly not in either case primitive examples of oral odontodes.

(4) Primitive craniates - early agnathuns

Recently, the first Ordovician vertebrates have been described from Australia (Ritchie & Gilbert-Tomlinson, 1977) and from South America (Gagnier, Blieck & Rodrigo, I 986), and the oldest North American vertebrate has been reassessed (Elliott, 1987). All have trunk scales, a cephalic region, and lateral platelets generally interpreted as branchial plates. This allows us to compare the earliest accepted vertebrate tissues and formulate some conclusions about the primitive types. According to Blieck and Janvier (pers. comm.) one of the Australian forms is the most primitive, Arunduspis; both are casts only, therefore, there is no histology. In this form there are rows of separate, pointed tubercles on the trunk scales, with no tesserae, only a very thin continuous sheet beneath the closely fitting rows of tubercles. What is interesting is that some detail, in the form of rows of minute pores set in shallow grooves, can be seen on the surface of these tubercles. The other genus, Porophoraspis, also has a surface punctured by many pores, but larger than those of Aranduspis. Both could be cited as evidence that the tubercles were dentine with either wide or narrow tubules for a sensory function, and linked with the suggestion that the primary evolutionary function of the skeleton was sensory.

Northcutt & Gans (1983) previously suggested that an early function of dentine was

290 MOVA N l . SMITH AND B. K. HALL

sensory, for use as an enhancer of electroreception, not in relation to the tubules in the dentine but because of the presence of hydroxyapatite rather than calcite (the mineral in use prior to the predominance of a phosphatic skeleton), as a dielectric transduction enhancer. Bodznick & Northcutt (1981) had also suggested that the ability to detect weak electric signals was very widespread amongst lower vertebrates, including agnathans. It may be significant in considering early types of dentine that two sizes of pore or sensory opening, have been observed, and it may be possible to relate these to different sensory functions of dentine, one of which was electroreception.

The new specimens from the early Upper Ordovician of Bolivia, of Sacabambaspis (Gagnier, Blieck & Rodrigo I 986), are the earliest undoubted articulated craniates (Janvier, 1989; Gagnier, 1989a) and the specimens have retained skeletal tissue, but the histological details are ambiguous because of very poor preservation (Gagnier, I 989 6). It is hard to say if the bone is acellular but it is considered not to be aspidin (Gagnier, 1989~2). The SEMs of cut surfaces in the bone tissue show many small angular holes, interpreted as cell lacunae, but without canaliculi. The dermal skeleton of the cephalic region is described as small polygonal units each one with a studding of twelve, elongate tubercles. The basal layer is apparently not divided into the same small units and is, therefore, not tesselated.

The implications of these observations in possible sister group heterostracans are that bone was not present without odontodes, that the first dermal bone was continuous sheets, and that later these became divided into tesserae. An interesting scenario has been proposed by Gans (1989)) in which the origin of the vertebrate skeleton is related to electroreception : ossification of the dermal layer underlying the dentine sensory system had the important function of fixing the position of the receptors relative to the mouth. It would seem to us that initially this function would require a non-tessellate bone base, and therefore, this would be the primitive condition.

The basis of a reassessment of Astraspis desiderata (Elliott, 1987) was a reversal of the rostro-caudal polarity of the original description, the new reconstruction showing nine branchial plates with eight openings. Because other forms of heterostracan are not know-n to have a series of branchial plates, the heterostracan affinities were questioned and the suggestion made that the Ordovician vertebrates, including those from the Harding Sandstone (Eriptychius and Astraspis), may constitute a group of primitive craniates. Many of these forms of agnathan have been placed as heterostracans solely on the basis of acellular bone (aspidin, see Fig. 6A), but Janvier (1981, pp. 143, I 54 and 1988, pers. comm.) has suggested that not all acellular bone in these early forms is aspidin because of significant histological differences. Brvig ( I 989) has also concluded, on the basis of histology that astraspids may not belong to the heterostracans, but eriptychiids do. Calcified cartilage is present (it has been described in the Harding Sandstone, and associated with Eriptychius : ((arvig, I 95 I , 1967 ; Dension, I 967 ; Spjeldnaes, 1967), and this again is not found in heterostracans. All these observations raise questions about their heterostracan affinity. Although globular calcified cartilage has been described in Eriptychius (Denison, 1967; Halstead, 1973, fig. I 14, Brvig, 1989) and referred to as calcified endoskeletal cartilage by both Denison, Brvig and Janvier, no perichondral bone has been described in these early agnathans. We have observed good examples of this type of mineralized cartilage in sections from the Ordovician, Harding Sandstone which we assume to be from Eriptychius fragments,

Skeletal development and evolution 29 1

acc

Fig. 4(A, B, D). Mineralized globular cartilage in sections of the Ordovician Harding Sandstone (slide no. P-10624a, BMNH). (A), (B) taken in phase contrast; (D) in Nomarski differential interference contrast. Compared with a drawing of mineralized cartilage of the nasal capsule of a placoderm (C), gcc, globular calcified cartilage, PZourodosteus sp. ((arvig, 1951, fig. I ~ D ) (A) x 186; (B) x zoo; (C) x zoo; (D) x 320. (Reproduced by permission of the author and publishers, Almqvist and Wiksell.)

and find that it compares well with all previously illustrated examples in other fishes

Cellular bone has been reported in a fragment in the Harding Sandstone of a third vertebrate, and illustrated by Brvig (1965, 381, fig. I B), and also by Denison (1967, p. 185, fig. 26), and more explicitly by (Smith, 1990; in prep.). Denison made a clear statement that both lacunae and canaliculi were present in the most basal tissue of denticles in a specimen referred to as ‘vertebrate indet. A ’ and he considered that it ‘resembled bone’. Halstead (1987, p. 352) referred briefly to Brvig’s observation but in the context that Brvig had used it, to support his view that cellular bone preceded acellular. Brvig (1965) suggested that these fragments were from a primitive stem osteostracan, and Halstead & Turner (1973) that it was a primitive cephalaspid osteostracan. We consider that Denison’s account is the most apposite because, although he was uncertain of its osteostracan affinities, we see this reference to cellular bone as part of the ‘odontode complex’ in which all three tissues (enamel, dentine and cellular bone) developed very early in evolution. An example of this tissue arrangement is given in Fig. 5 . An odontode from the extant coelacanth fish, is compared with odontodes of fossil thelodonts and sharks, in which the basal tissue may be acellular cementum/bone ; and also one with superimposed odontodes in a fossil actinopterygian,

(Fig. 4).


e

bc’

Fig. 5 (A-D). Drawings of sections through single dermal odontodes (A-D) from three major taxa, and a composite scale of superimposed odontodes (E). (A) T h e extant coelacanth (sarcopterygian) has odontodes fused to the largely uncalcified base in the exposed portion of the scale (see also Fig. 17). Each odontode has enamel (e), dentine and cellular bone of attachment surrounding a central pulp cavity (pc). (B, C, F, G) Thelodont (agnathan) tubercles are free in the dermis but cover the whole fish, and many types consist of orthodentine (metadentine), but no cellular bone. T h e basal tissue has sparse tubules, Sharpey’s fibre spaces and incremental lines, characteristic of acellular cement (attachment tissue) in socketed teeth of higher vertebrates (see also Fig. 10). (D) Placoid denticle of edestid shark, each is situated free in the dermis but all form a complete cover to the fish (see also Fig. I C ) . This example has many thick dentine canals instead of a single large pulp chamber and the dentine is described as mesodentine-like, as it is less regular in the arrangement of tubules than metadentine. T h e basal tissue is like that of the thelodont scales, but has in addition to a basal canal (bc) a horizontal neck canal (nc)


where the odontode related bone (with cells) is fused to the basal cellular bone. This tissue arrangement has been confirmed by some new observations (Smith, 1990; in prep .) in which isolated tubercles of a distinct third vertebrate certainly have osteocytes with interconnecting canaliculi in the basal tissue of the denticle (Fig. 6C, D). Smith (1990; in prep.) suggested that it is the most primitive osteostracan, on the basis of three distinctive tissues, enameloid, mesodentine and cellular bone, occurring in separate denticles (odontodes). This is the earliest record of evidence of cellular bone, which exists in association with these early agnathans. The significance is that we intend to use this as evidence that the ‘odontode complex’ is the plesiomorphic state for vertebrates derived at the level of the craniates. For these reasons this material is amongst the most crucial to evaluate in considering the phylogeny of the vertebrate skeleton and will be presented in some detail, along with some other crucial specimens.

One unresolved issue is whether acellular bone (Fig. 6A) is plesiomorphic and therefore a retained primitive character for heterostracans, or whether cellular bone is primitive (Fig. 6B-D) and the acellular condition derived. Acellular bone is often a secondary feature of teleost fishes, and is secondarily derived. Chondrichthyan fishes are considered by many not to have any bone at all, and again it is suggested that this is a derived condition. This will be referred to in more detail later, but the reports do show that, bone is present, it is cellular, and perichondral in position (Peignoux- Deville et a/., 1982, 1985). As Janvier (1981, p. 154) concluded, ‘the polarity of some craniate characters will be defined more precisely by a detailed character analysis of - aspidin and dentine ’, also ‘they often comprise the only available data on the presumed most primitive representatives of the group ’. We would also conclude that the data on the topography of these tissue types is important in defining polarity.

Janvier (1981, p. 145) and also Halstead (1982) used orthodentine as one synapomorphy linking heterostracans with gnathostomes, but if it can be shown that orthodentine is a primitive character in early representatives of possible stem group heterostracans then it would be plesiomorphic (examples of orthodentine are shown in Figs 5 , 10, 1 3 F, and 15 B). This interpretation, that orthodentine is a derived shared character may be because Janvier placed importance on 0rvig’s (195 I ) assumption that bone preceded dentine in evolution, and that the scleroblastic cell type can be modulated from bone to dentine, with an intermediate tissue (mesodentine) as evidence for this transition, and that mesodentine is primitive relative to orthodentine. There is some evidence on the basis of tubule diameter and arrangement, that at least two different types of dentine were present in these early tissues. We have assumed from developmental evidence that i t is not true that bone cells can be modulated to make dentine, but that each came from separate sub-populations of stem cells in the dental

(see also Fig. IOF, G). (E) Primitive actinopterygian scale, many odontodes with attachment bone, superimposed on the cellular bony base. Each consists of orthodentine and a covering of ganoine (e, enamel). (A) Latimeria chalumnae, x 35 (Roux, 1942, fig. 5 ; Smith, 1972, fig. I ; 0rvig, 1977, fig. 3A). (Reproduced by permission of the author and publishers, the Linnean Society, London.) (B, C) Theiodus sp . , x 80, Devonian (Gross, 1967, fig, I K, zF). (D) Elegestolepis grossi, x 1 1 0 , Upper Silurian (Karatajute-Talimaa, 1973, fig. 4D). (E) Cheirolepis gracilis, x I 50, Middle Devonian. (F, G) Katoporus trivicus and Logania cuneata, x 56, Devonian [Gross, 1973, fig. 34D, and Gross, 1967, figs 9H, I I C]. [(B-G) reproduced by permission of the authors and publishers, E. Schweizerbart’sche, Verlags- buch handlung. 3


Fig. 6 ( X D). Drawings of bone tissue histology to show the two major types in early fossil fishes, compared mith photomicrographs of bone tissue in the vertebrate remains of a section of the Ordovician Harding sandstonc. (A) Hetcrostracan acellular hone. ’Traheculae of aspidin (c) with accretional aspidones (b) around the vascular canals (a). No cell spaces; the irregular elongate spaces may be due to uncalcified fibre bundles or stress zones. Pycnaspis splendens, x IOO (Drvig, 1965, fig. I A). (Reproduced by permission of the author and publishers, Almqvist and U’iksell.) (B) Dermal bone with enclosed cell spaces from basal bone of an ichthyodorulite spine. Osteostracan bone is similar but has a more irregular arrangement of lacunae and canaliculi. Gamphacampus politus, x zoo (0rvig, 1957, 13 B). (Reproduced by permission of the author and publishers, Norwegian University Press.) (C, D) Photomicrographs of osteocytes in basal bone of isolated tubercles of the third vertebrate associated with fragments of Evi~ t j~r l i i u s and Astraspis. Tissue grades into mesodentine of the enameloid covered tubercles. (C) x .+z j ,

(D) x j30 (slide no. H9-1, VllDS).

papilla and the subjacent ectomesenchyme (in development of the tooth, dentine forms first then bone) ; so that either bone and dentine evolved simultaneously or dentine came before bone. T h e new observations of the third vertebrate in the Harding Sandstone, proposed as a primitive osteostracan, Smith (1990; in prep.) show that both mesodentine and cellular bone occur together as part of the odontode complex, as

Skeletal development and evolution 29.5

postulated from the developmental evidence. These findings will be particularly important in considering the thelodont tissues.

( a ) The tuberculated exoskeleton of Middle and Upper Ordovician specimens The two forms found in the Harding Sandstone from the Middle Ordovician

(Caradoc) of Colorado, North America, are Astraspis and Eriptychius, first described by Walcott ( I 892). A related form Pycnaspis has been described from beds of the same age in Wyoming (0rvig 1958, 1989) and considered to be synonymous with Astraspis by Denison (1967, p. 164), but reaffirmed as a separate genus by 0rvig (1989). The third vertebrate described by Denison (1967) as an uncertain osteostracan, and by 0rvig (1989) as a completely different vertebrate, with bone tissue clearly separate from the acellular aspidin, has been verified from new material as a completely different type, and proposed as a new genus of primitive osteostracan (Smith, 1990 ; in prep.). This form is represented by separate denticles of three distinctive tissue types, cellular bone of attachment, mesodentine, and enameloid. These are not united to any spongy bone fragments, and are of the order of size of the smallest tubercles of Eriptychius, about 0.5 mm. 0rvig (1967) considered that the odontodes in Eriptychius were similar in their histology to those of early heterostracans (orthodentine lacking an enamel cover), and clearly believed that an external layer of ‘ enameloid substance ’ was acquired within the later Heterostraci (0rvig 1967, p. 82). Whereas the odontodes of Astraspis and Pycnaspis had no equivalents to any of the heterostracans (being composed of aspidin covered directly by a form of enameloid, and lacking dentine). It is interesting that of the three genera Denison (1967, p. 147) commented that only Eryptichius had coarse- tubed dentine, with the implication that this was homologous with orthodentine and the tissue in the other two (with very fine caliber tubules) was not homologous with dentine, as also suggested by Brvig (1967, p. 82; 1989, p. 444) who considered the spaces as due to collagen fibres and the tissue as acellular bone, and reinforced by Halstead (1987) calling it astraspidin.

(b ) Erip tychius

The most detailed and useful description of the histology is given by Denison ( I 967, p. 154), from which the central points may be taken. The bulk of the tubercle is composed of a tissue that, by virtue of its tubules spreading out from a central pulp cavity, can be called dentine. However, the tubules start off with a very wide calibre, branch many times, each branch tapering to a smaller diameter, but only have very few tiny lateral branches, normally many of these are found in mesodentine and orthodentine (see Fig. IS). Pronounced lamellae contour the pulp chamber (Fig. 7A); interestingly these continue into the marginal tissue which is without tubules or cell spaces, and is referred to as aspidin. It is, however, more logical to consider this as dentine without tubules. There are examples of reparative dentine (in higher vertebrates) in which tubules are not formed, or are present in low numbers. The wide calibre of the tubules and the large distance between them would not support a close developmental relationship with dentine in higher fishes.

Although these tubercles are described by Denison (1967, p. 154) as frequently without any enamel cover (Fig. 8B-D), he explained this was due to transformation into soluble calcite or loss due to abrasion (Fig. 8A), he also observed that it was absent


Fig. 7 (A-C). Photomicrographs of dentine tubercles of Eriptychius from the Ordovician Harding Sandstone, showing wide-calibre straight tubules with little branching and no lateral branches. In (A) Nomarski differential interference contrast was used and (C) phase optics. In these details of the very regular growth increments can be seen (arrows) and also a region of globular mineralization (asterisk, typical of dentine as opposed to bone) (compare with Figs 9, 12, 1 3 ) . Enamel is not present in these abraded examples. (A) x 3 2 0 ; (B) x 3 2 0 ; (C) x 640 (slide no. P10624c, BMNH).

from buried tubercles. Reif (1979) also stated that he did not observe enameloid in Eriptychius, although present in Astraspis. We have observed in sections of the Harding sandstone, that in some regions with favourable preservation, a translucent, birefringent layer is present on the margins of the tuberculate ridges that has more in common with enamel than enameloid (Fig. 8E, F). Also from the published accounts our conclusion is that it is much more likely to be enamel than enameloid, because enamel is formed later than dentine in the histogenesis of the tooth, and has a weak junction with the dentine. Its production could be delayed to the point where it did not form, or it was easily lost from the surface. In contrast enameloid forms first, before dentine, and if missing would probably prevent dentine formation as well.

The supporting tissue, continuous with the tubercles is relatively compact, with small canals for the soft tissue, each surrounded by concentric layers of a tissue without cell spaces or tubules but with a fine fibrous structure at right angles to the laminae (similar to that at the margins of the tubercles, Denison, 1967, p. 157). These have been compared with primary osteones but are termed aspidones (see b on Fig. 6A). It seems

298 MOYA M. SMITH AND B. K. HALL vascular canals. It is clear that the histology does not support a relationship with any of the better known groups of heterostracans ; a conclusion also reached by Denison (1967, P. 162).

( c ) Astraspis and Pycnaspis

Denison (1967, p. 164) refers Pycnaspis to the same genus as Astraspis however, Brvig (1989) has reaffirmed that they are different genera. There are interesting differences between the tissues in these forms and those of Eriptychius, in particular there is a sculptured cap of hard, translucent material forming an enamel-like covering, clearly distinguishable from the tissue below (Fig. 9 D, E ; Bryant, 1936, fig. 2 ; Denison, 1967, fig. 20, Brvig, 1958, fig. 3) . Brvig (1951, p. 381) considered the enamel-like tissue in Astraspis as similar to enameloid in elasmobranchs (Fig. 9 B). Denison ( I 967, p. 174) agreed with this and referred to it as durodentine (enameloid derived by modification of the dentine). Reif (1979) commented, from a SEM study, that it was single-crystallite enameloid on the tubercles of Astraspis. Of significance is the comment by Halstead (1987, p. 348) that ‘it is never found in any other heterostracan and seems to be unique to the Astraspidae ’, but he goes on to comment that ‘the outermost layer differs widely even within the heterostracans’. It seems that we could equally regard this outer tissue as a form of enamel similar to ganoine [see section VI(4)] especially because of the scalloped outer surface in the stellate tubercles which is not parallelled at the boundary between the dentine and this enamel (see Fig. 9D). It is relatively homogeneous but a faint radial arrangement of the elements can be seen and short splayed-out ‘ tubules ’ at the boundary zone (Fig. 9B), possibly interpreted as gaps between groups of crystals, as described in protoprismatic enamel (Smith 1989), rather than spaces caused by continuation of the dentine tubules. It is striking also (as Denison, 1967, p. 176 noted) that the boundary commonly does not coincide with the lamination of the inner tissue. Brvig ( I 989) also doubted that it was enameloid sensu stricto.

Another striking difference from Eriptychius is the lack of easily identified dentine. The tissue beneath the enamel is laminated, and because of the difficulty of recognising typical dentine tubules can only be assigned as distinctive for this reason. Although Denison (1967, p. 174) described minute, fine diameter tubules, of comparable size to the finest (< I pm) in the dentine of oral teeth in higher vertebrates, he also discussed the fact that they did not branch and had been considered as fibre spaces by others. Halstead (1987, p. 348) states that this tissue is not seen in any other heterostracan, nor even in any other vertebrate, and suggests a noncommittal term ‘astraspidin’. Our observations confirm the lack of typical dentine tubules, but suggest that radially arranged fine linear spaces represent the radial fibre component of this tissue. We also note that these do not continue across the junction with the enamel. This leads us to suggest that it may be a form of dentine in which in vivo the cell processes have not remained for long in the matrix, and tubules have not formed. In other words, an acellular tissue, but derived from dentine and not from one in which bone cell spaces have been occluded. In this sense we agree with Halstead (1987) that dentine and this tissue are related. I f astraspidin is defined as a tissue without cell spaces and without tubules and derived from or topographically related to dentine, then this would be the tissue type in Astraspis. However, aspidin in heterostracans is usually regarded as a type of bone, either preceding or following bone with enclosed cell spaces.


A B

Fig. 9(A-E). Tubercles of Astrapididae to show their variable morphology (A, C); sections through them as a drawing (B), and photomicrographs taken in phase contrast (D, E). All show the distinct junction between the enameloid cap and the aspidin (dentine, or astraspidin). Incremental lines and their discontinuity at the junction indicate the possible addition of layers from the outside. Pycnaspis splendens. (A, C) x 2 0 ; (B) x IOO ((drvig, 1958, figs za, b, 3). (Reproduced by permission of the author and the Smithsonian Institution Press, Washington, D.C.) (D, E) x 100, Harding Sandstone (slide no. P- 10624c, BMNH).

The supporting tissue is a coarse-fibred trabecular tissue made compact by concentric laminae (primary osteones), but without enclosed cells. Interestingly this tissue has fine calibre spaces inserting perpendicular to the laminae (Fig. 6A), a reason for Denison (1967) calling it trabecular dentine, which has become compact by the apposition of infilling laminae, more correctly termed denteones. Because of the lack of tubules in the tissue of the tubercles it would also be possible to consider this basal tissue as derived from or closer to dentine. Like many tissue arrangements the basal layer is laminated parallel to the surface, with coarse fibres at right angles to these (Denison, 1967, fig. 22), typical features of attachment tissue (i.e. acellular cement). All these comments are relevant to the discussion of aspidin in section II(7b), and to the possible homology with acellular cement in socketed teeth [section I1 ( I I a)], a tissue located immediately next to the dentine and induced to form by the presence of dentine.

300 MOYA M. SMITH AND €3. K. HALL

(5) Heterostracans

Heterostracans are uniquely characterized by three features of their dermal armour ; the acellular nature of the skeletal tissues underlying the dentine, the tubules within the dentine in an arrangement closely resembling orthodentine of higher vertebrates (Fig. IZ), and the honeycomb arrangement of the middle layer, sandwiched between the dentine tubercles and the basal laminated layer. Another significant feature of the skeleton is the absence of any calcified elements of the endoskeleton. As discussed in section I1 (4) (primitive craniates - early agnathans), calcified cartilage fragments have been found in Eriptychius, but none shows any perichondral bone as found in osteostracans, galeaspids and gnathostomes. Most of the debate and controversy has centred on the acellular tissue beneath the dentine, termed ‘aspidin’. It may be significant that in the first detailed study of its histology (Agassiz, 1845, cited by Halstead, 1973) it was considered closer in its histology to dentine than bone. It is this aspect of the tissues that we feel is important to reassess, as discussed in section I1 ( 5 b ) . It will be important to draw upon developmental information to choose between competing ideas of the evolution of the exoskeleton, and the primitive and derived characters.

The evolutionary trends in the heterostracan dermal armour have been compre- hensively discussed by Halstead Tarlo (19676, 1982, pp. 164-I~I), starting from isolated tubercles to one with small, closely abutting tesserae, through to fusion into larger plates. These may secondarily break up into platelets, as in the psammosteids, a sister group of the pteraspids (without tesserae, Blieck pers. comm.). Although Janvier (1984, p. 353) concluded that the heterostracan exoskeleton derived from the micromeric form (isolated denticles), he was unsure whether the cancellous layer was derived from these denticles, comparable with thelodont-like scale units. He further suggested that the macromeric forms were derived from mesomeric forms such as Lepiduspis (with fused basal plates) rather than from those with small tesserae such as astraspidids and eriptychiids. We can cite information from the excellent description of Dinely & Loeffler (1976, p. 180) of Lepidaspis serrata from the Siluro-Devonian, where there is a sequence of progressive fusion of the basal plates. Both primary and secondary tubercles form on these plates and tubercles also form across the sutures between the basal plates, suggesting an independent origin of the tubercles and the basal bone.

(a) Tubercles

Most of the tubercles, or superficial layer, do not have an enamel cover (Halstead, 1969, figs 10-12) and the dentine seems to be of three types; one like orthodentine (Psammolepis, Fig. 13 D, F, sometimes also referred to as metadentines; Brvig, 1967, pp. 77-79) with sub-parallel tapering tubules, with small lateral branches, all issuing from a central pulp cavity and continuing to the surface of the dentine, e.g. Tartuosteus (Halstead Tarlo, 19646, pl. 10, figs 4, 6; 1969, fig. 12). Another dentine type has a system of anastomosing pulp canals with short, very irregular branching tubules emanating from them, e.g. Traquairaspis, Pteraspis (Fig. 13 C , E), also like that shown in Fig. I z C of Cephalaspis. The third has many short pulp canals from which sprays of tubules pass to the surface e.g. Tesseraspis (Tarlo, 19646, pl. IX, figs I, 3 , 4; Halstead 1969, fig. 1 1 ; Archegonaspis Fig. 12B). Also like the dentine shown in the shark scale


(Fig. IoG), and in the dentine of thelodontids, Katoporus (Fig. 5 F) and Logania (Fig. 5 G; Gross, 1968, fig. 5L, M). The significance of the variation in types of dentine is not at present known, but it should be possible to suggest a primitive type in each group from the histology and proposed development, and perhaps resolve the proposition that thelodonts are a paraphyletic group (Fig. I I ; Janvier, 1981).

(b) Aspidin This is traditionally known as acellular bone, from the assumption that cell spaces in

the developing tissue have become occluded, and in this sense the tissue is considered to be a secondary derivative from bone ((arvig, 1951). Denison (1963)~ however, considered it to be derived from dentine and as such never to have gone through a stage with enclosed cells. Neither of these views would allow for acellular bone to be the primitive tissue. This debate continues today, with Halstead ( I 987) recanting his earlier belief that aspidinocytes existed leaving scattered irregular lacunae ; the solution, he believed was to consider these spaces as arising from intrinsic coarse fibre bundles (Halstead, 1987, p. 353), and implicit in this, that the tissue is a primary form of acellular bone.

The arrangement of the aspidin is both as an extensive spongy network, the spaces later filled in by circular lamellae forming aspidones (Fig. 6A), and below this basal sheets of lamellar aspidin. The tissue itself is described with three types of structure present within the matrix; (i) coarse fibre bundles at the scale margins and the basal edge (extrinsic Sharpey’s fibres) serving as an attachment mechanism ; (ii) intrinsic coarse fibres associated with spindle-shaped structures (aspidinocyte lacunae, Halstead, 1973 , p. 307) ; (iii) tubules of very fine calibre leading from the soft tissue space into the hard tissue and assumed to contain cell processes from cell bodies at the forming front. Because Denison (1967, p. 180) rightly believed that aspidin as defined could not contain single-cell process spaces he declared that it should be regarded as trabecular dentine. Following this the aspidones, where they had transformed the trabecular dentine into compact dentine, could be compared with denteones, again for the reason that single cell processes could be identified. Halstead (1973, p. 305) recognized that in some forms (the earliest) there were no spaces that could be attributed to aspidinocyte lacunae. Spaces observed within the trabeculae of the spongy part were quite irregular, although in the aspidones spaces were parallel to the concentric lamellae. Halstead (1973, p. 307) emphasized several times that aspidin is like dentine, ‘it is evident that the aspidin of the earliest heterosracans was fundamentally like dentine ’, and ‘the evolution of the organization of the organic matrix in aspidin can thus be traced from a dentine-like condition to a bone-like’. Further in his detailed account of the histology of Ganosteus stellatus Halstead Tarlo (1964b, p. 53) considered that the fine canaliculi radiating from the central canal of the aspidone were left from retreating cells lining the canal surface, again a property of dentine, and he suggested an analogy with the tubercles of Astraspis. We concur with Denison (1967) and believe that the primitive tissue was trabecular dentine and that this progressed into compact dentine by infilling with denteones; as in Ganosteus (Halstead, 1969, fig. 5 ) , the example that he quoted as ‘having evolved from one with typical aspidin to one with typical trabecular dentine ’. The view that ‘difficulties in interpretation could be overcome by recognising that, at the very beginning of evolutionary history of the vertebrates, the tissues aspidin and dentine were hardly to be distinguished ’ (Halstead Tarlo, 1964b), is easily reconciled

302 MOYA M. SMITH AND B. K. HALL with the view that dentine is primitive and dentine-related tissues (aspidin/cementum) formed in close sequence, both in development, topographically and in time, and in evolution.

Essentially we believe that most of these component tissues of the dermal skeleton were developmentally closely related to dentine, either orthodentine (circumpulpal), or the varieties referred to by Brvig (1967, p. 65) as semi-dentine and mesodentine, and that further away from the tuberculate component they became trabecular, and then compact (trabeculae plus denteones, denteonal). Parts of the ‘aspidin ’ arranged as lamellae with extrinsic fibre bundles could as easily be regarded as cement or bone of attachment, the tissues that develop topographically closer to the dentine of the tooth root in higher vertebrates. The possible relationship of these tissues to the dental tissue cement is discussed in section I1 ( I I a) , along with definitions of bone and dentine tissues.

(6) Anaspids

The position of this group of jawless vertebrates within the agnathans has been much debated, as shown by the variety of cladograms produced (Forey, 1984, fig. 3). Several genera once included have now been assigned to other groups (Janvier, 1981, p. 139; Forey, 1984, p. 336); some are forms with a reduced dermal skeleton (Endeiolepis), or, as proposed by Forey & Gardiner (1981) one is lacking a dermal skeleton altogether uumoytius). Another problem with regard to commenting on their skeleton in any postulated evolutionary series is the scarcity of histological information. Two interesting points can be stressed, firstly the dermal skeleton of both head and trunk, is either regarded as consisting entirely of laminated acellular bone (Forey, 1984, p. 336), or superficially a single layer of odontodes assumed to be made of dentine (Reif, 1982, p. 301, cited by Denison, 1963); although Gross (1958, cited by Reif, 1982) considered that they were made of acellular bone.

The debate about the skeleton of Jamoytius kerwoodi White has been sharply focused on the carbonized segmental structures, either muscle blocks (Forey & Gardiner, 1981) or dermal ossifications (Ritchie, 1960, 1984). The original description by White (1946) was that of a naked fish, also without an endoskeleton, a primitive acraniate chordate. Ritchie (1960) discussed the idea that there are scale rows resembling those of anaspids, and presented new evidence that the structures interpreted by White ( I 946) as muscle fibres are cross-striations of ornament on scales. Again in 1984 Ritchie strongly refuted the suggestion by Forey & Gardiner (1981) that there is no evidence of calcified scales, although he does suggest a strong link with petromyzontids, the naked extant form. Halstead (1982) in a general discussion on the skeleton of the most primitive vertebrates, while accepting that cyclostomes are naked, and that Jamoytius is closely related to lampreys, also supports the view that Jurnoytius is covered by well developed ornamented scales. These two views necessitate the explanation that there was a secondary loss of armour in this group, but as Halstead (1982, p. 171) admits ‘the evidence of nakedness and armouredness is not easy to assess’. Discovery of another enigmatic chordate Conopiscius (Briggs & Clarkson, I 987) has also involved acceptance of evidence for an exoskeleton in Jamoytius with which they suggest affinities. The character they site is a covering of V-shaped scales in the trunk, reflecting the morphology of the underlying myomeres. The craniate affinity is preferred because the presence of these mineralized scales excludes it from the cephalochordates.


The second controversial topic concerns the endoskeleton. There is a calcified endoskeleton in the trunk, endoskeletal radials of the anal and caudal fins (Jarvik, 1959, p. s), said by Maisey (1988, p. 19) to be equivalent to neural-crest-derived structures in recent gnathostomes, but there is no evidence for the post-cranial endoskeleton being neural-crest derived in any extant vertebrate, and there is also doubt about their endoskeletal origin. The alternative explanation is that they are not endoskeleton but exoskeleton sunk deep below the site of its formation from the epidermis, but this involves acceptance of the theory of delamination (Holmgren, 1940) and we accept Patterson’s (1977) conclusion that this theory lacks support. The status of the fin skeleton is, therefore, in need of revision before it can be useful in cladistic analysis.

Janvier ( I 98 I ) places anaspids as a sister group of the Petromyzonidae, assuming that the latter have a reduced dermal skeleton, and both are the sister group of osteostracans. The exclusion from the Anaspida of the two species ramoytius kerwoodi and Endeiolepis aneri on the basis of skeletal characters is a good example of the use of the assumed evolutionary sequence of skeletal transformations to arrange groups in a cladogram.

(7 ) Thelodonts

The majority within this group are represented by isolated scales although a few are whole bodies with separate scales (Ritchie, 1986). Because none are fused together the skeleton of thelodonts is considered to be a micromeric exoskeleton. The record of their histology (Gross, 1967 ; KaratajutC-Talimaa, 1978), shows only dentine with a basal acellular tissue, but two types can be broadly distinguished as illustrated in Fig. 5B, C cf. F. G; those with a single undivided pulp chamber (theolodonts), and others with several small pulp chambers, katoporids, both elegantly illustrated by Karatajute-Talimaa (1978, pp. 21 6-219). The thelodontids have many branching tubules in a subparallel arrangement (see Fig. 5 B, C), resembling orthodentine, whereas the katoporids have sprays of tubules emanating from each small pulp canal (Fig. 5 F) ; Janvier ( I 98 I , p. I 5 I ) refers to the latter type of tissue as mesodentine, but this does not seem valid as there is no interconnecting meshwork of tubules, and multitubate dentine might be more appropriate. A third type is represented by Logania, also with a small pulp canal but with many irregular, branching small canals, and tubules (Fig. 5G). Orvig (1951, fig. IOA-C) refers to this in separate denticles of the Ludlow bone beds as mesodentine, and compares it with osteostracan tissue in cephalaspids. This is an area of tissue classification in need of clarification because certain types of dentine are considered exclusive for major taxons, such as semidentine, as exclusive for placoderms (Gouget, I 984), but mesodentine occurs in osteostracans and acanthodians.

Janvier illustrates the problem of the relationships of thelodonts in his cladogram (Fig. 1 1 ; Janvier, 1981, fig. 16), commenting that they are a paraphyletic group, the micromeric form of the skeleton representing a primitive character found at the roots of several monophyletic groups. This would account for the three varieties of dentine in thelodonts. It would be expected that histology would clarify relationships but no clear solution has emerged. It seems impossible to find criteria to decide which of the tissues is primitive, although Janvier ( I 98 I ) has assumed that the theolodontids are more primitive, because the dentine resembles that of heterostracans and the tissue beneath is similar to aspidin, but this assumes that the condition in heterostracans is

304 MOYA M. SMITH

C

AND B. K. HALL

Fig. io(A-I). Drawings to show the whole scale in thelodonts and sharks and the two types of arrangement of dentine, a single pulp chamber in thelodont scale, and a single basal canal opening (A-E). Multiple pulp canals in the shark scale, from a single pulp cavity, also with many horizontal neck canals (F~-G), opening at the surface between dentine and basal tissue. Photomicrographs (H, I) of the type in (A)- (E) with a single undivided pulp chamber. All show the dentine and basal attachment tissue. No enamel is present hut dentine tubules become very much finer in the outer layer (double arrow). T h e pump cavity has become smaller with growth of the dentine (shown in C, D), evidence of centripetal gro\vth is seen by the incremental lines in ( I ) (arrows). T h e basal tissue grades into the dentine and has

Skeletal development and evolution

Craniata

Vertebrata I 1

305

I I

Myx. Heter. Gal. Petr. Anas. Ost. Gnath.

V 1

Fig. I I , A theory of the phylogenetic interrelationships of the Craniata as proposed by Janvier (1981, fig. 16). (Reproduced by permission of the author and the Journal of Vertebrate Paleontology.) Solid lines indicate single relationships ; dashed lines indicate equally possible alternative relationships. The position of the thelodonts is problematic, considered to be a paraphyletic group, as shown by the shaded area, hatched for thelodontids, stippled for katoporids. The two alternative positions for galeaspids can be resolved as only B, because a cancellous middle layer is not present. The 1 3 major derived characters on which this phylogeny is based are listed in Janvier (1981, fig. 16). We only show those that relate to skeletal characters. I , neural crest; 3, mineralized dermal skeleton; 4. cancellous middle layer of exoskeleton; 5, perichondral bone; 12, cellular bone. Anas., anaspids; Gal., galeaspids; Gnath., gnathostomes; Heter., heterostracans ; Myx., myxinoids ; Ost., osteostracans ; Petr., petromyzontids.

primitive, a circular argument. It appears to us that little has been resolved by comparison of the histology and that further study might be of benefit. The dentine and basal tissue of Thelodus is illustrated in Fig. IOH, I, where the straight tubules of orthodentine terminate in very fine branches in the outer layer. The basal tissue is sparse in tubules but has regions where Sharpey’s fibres leave spaces and incremental lines contour the outer surface, features of acellular attachment tissue (cement) (Fig. gB, C, IoH). The undulating incremented lines of the dentine are clearly seen, and indicate regular deposition from the pulp cavity. These are all features of an odontode, assumed to be plesiomorphic for the exoskeleton of vertebrates.

Whole bodies have been recorded by Traquair ( I 899), Ritchie ( I 967) and by Dineley & Loeffler (1976, p. 152) with the squamation in place, and there is no evidence of any dermal bone. One of the heterostracans Lepidaspis serrata (Fig. I 3 B) is a type with a micromeric skeleton but in this form basal plates of bone are also present beneath the dentine ridges, each separated by sutures (see next section; Dineley & Loeffler, 1976,

sparse tubules and Sharpey’s fibre spaces, but no bone cell spaces (H, D, E, G). (A-E) Theloduspurwidens, (F, G) Pristis sp., x 5 0 (Gross, 1966, fig. I) . (Reproduced by permission of the publishers, E. Schweizerbart’sche, Verlagsbuchhandlung.) (H, I) Turiniu pugei, x 40, x IOO respectively (slide no. P- 31489, BMNH).

MOYA M. SMITH AND B. K. HALL

A I

D

Fig. IZ(X-II). Xgnathan dermal ornament to show the variation in agreement of the dentine tubules. (A) Pteraspis, x 170; ( B ) Arrhegonaspis, x 100; ( C ) Cephalaspis, ~ 7 0 ; (D) Psammosteus, x7o. (0rvig, 1 9 j 1 , fig. j ( A .D), (Reproduced by permission of the author and the publishers, Almqvist and Wiksell.)

pl. 28; described by them as agnathan, incertae sedis). They suggested that it is an ancestral morphotype, and as such could be related to one of the thelodonts. T h e histology has only been recently reported by Halstead (1987, p. 353) who regarded it as a key piece of evidence in interpreting aspidin, concluding that none of the spindle- shaped spaces could be interpreted as due to cells, rather to coarse fibre bundles. His only comment on relationships was 'that the genus was a primitive tessellated heterostracan that could represent a structural stage linking thelodonts with heterostracans '. Our own observations suggest that the dentine could as easily be compared with that of a cephalaspid osteostracan, except that bone cells have not been observed, but neither does it look like typical aspidin. Janvier (1981) comments that the micromeric dermal skeleton with dentine in the scales and acellular basal bone is what one would expect in the hypothetical heterostracan ancestor.


Fig. 13 (A-F). Heterostracan dermal ornament, tubercles of varying shape superimposed on bone (A, B) and on older generations of tubercles (arrows, D). The histology in the photomicrographs shows the variation in tubule arrangement of the dentine (cf. Fig. 12). A single central pulp cavity and orthodentine are seen in (F), but with few lateral branches [the same genus as in (D)]. In (C) and (E) there are many small pulp canals, from which arise many irregular branches and tappering tubules. (A) Traquairaspis plana, x 30 ((arvig, 1961, fig. 3). (B) Lepiduspzs serrata, Upper Silurian, Arctic Canada, x 50 . (C) Traquairaspis sp., x 125 (slide no. P-199601, BMNH). (D) Psammolepis paradoxa, 3D diagram (0rvig, 1968, fig. 5A). (E) Pteraspis sp., x 125 (slide no. P-25098, BMNH). (F) Psammolepzs sp., x 125 (slide no. P-17662, BMNH). [(A, D) Published by permission of the author and publishers, Almqvist and Wiksell.]


(8) Galeaspids

These represent a little known group of Silurian to Upper Devonian agnathans from North and South China that possesses massive endoskeletal and exoskeletal shields similar to those of osteostracans. They have been known for certain since the early 1960s and the view expressed that they represented a major, isolated evolutionary radiation, independently from the rest of the agnathans (Halstead et aE., 1979). The debate has oscillated between affinities with heterostracans or osteostracans (see Fig. 11). Halstead (1979) decided, from very well preserved soft tissue replicas that they were closest to the cephalaspids, unfortunately no skeletal tissue remains so that histological characters are not known. The endoskeletal inner shield, as in osteostracans is made of perichondral bone, interestingly assumed by Janvier (1984) to be cellular, with the implicit assumption that perichondral bone is always cellular, in this case a type of bone primitively with cells. The histology of the exoskeletal tissue is not at present known, because of poor preservation, for instance what type of bone it is, although the dentine of the tubercles has been claimed to be mesodentine (Halstead, 1982). The best information we have on the structure of the exoskeleton is that depicted by Janvier (1984, fig. 3B, C), as tubercles, each with a central pulp cavity, but with one variety (more primitive) open at the base, the other closed. We have no reliable information at present on the histology of these tubercles and cannot say whether or not they are made of normal dentine (the variety termed orthodentine in higher vertebrates but metadentine by Brvig in his 1967 review). In a paper in press Wang (1990) reports cellular bone in Polybranchiaspis and uses this character as one uniquely shared by osteostracans, galeaspids, and gnathostomes in his cladogram. It is clearly important to establish if this type of hard tissue has cells included or not, and whether or not it occurs in both exo- and endoskeletons.

(9) Osteostracans

This group of fossil agnathans is by far the best known because ossification of the head is much more extensive. Endoskeletal perichondral bone lines all the spaces of the inner shield, and is retained in position because parts of this region of bone are closely apposed to the relatively thick dermal skeleton. Between the outermost and the innermost layer of this perichondral bone it is assumed that there was uncalcified cartilage occupying the space between the blood vessels and nerves. The position of this endoskeletal tissue (cranial-visceral skeleton) is best appreciated in the account by Janvier ( I 98 I , fig. I 2 E I ) in which the exoskeleton and endoskeleton of osteostracans is compared with the exoskeleton of heterostracans (Fig. 14). Janvier (1989, p. 27, fig. 14, C2) also considered the problem of the homology of the endoskeleton and opted for the view that the inner shield (not including visceral arches) was uniquely of neurocranial origin. The significance of this in terms of neural-crest skeletogenic ability will be discussed in section 111.

The other significant feature of the skeletal tissues is that beneath the dentine tubercles of the dermal armour there is a calcified tissue in which there are numerous spaces (lacunae) for enclosed cells, each with many canaliculi leading from them (Fig. 6B). For these reasons it is easily recognized as cellular bone. This bone forms part of the spongy tissue of the middle layer and the basal lamellated layer of the cranial


Fig. 14. Comparison between cross-sections of whole-body skeleton of an osteostracan (El) with a heterostracan (EJ, to show the presence of cartilage and perichondral bone in El and only dermal skeleton in E, (from Janvier, 1981, fig. IzE). bp, Branchial plates; ds, dorsal shield; 0, orbit; obc, orobranchial chamber; spf, socket for paired fin; vs, ventral shield (hatching, dermal skeleton; stipple, cartilage; solid line, perichondral bone; dashed line, body cavity, gill unit). (Reproduced by permission of the author and the publishers, Journal of Vertebrate Paleontology.)

and trunk exoskeleton. Because the osteostracans are considered to be a more derived group of agnathans than the heterostracans and occur later in the stratigraphic record, the cellular variety of bone (at least in the exoskeleton) is generally taken to be secondary, but there is still a problem with accepting this (see discussion for the developmental evidence for acellular bone being secondary to cellular). Janvier ( I 98 I ,

p. 143) cites 0rvig (1965) as indicating that acellular dermal bone is found in highly derived Osteostraci ( A h p i s ) and in some teleosts. Reif (1982, p. 299) also noted that acellular bone is present in these Upper Devonian genera, and concluded that in both osteostracans and teleosts acellular bone must be derived, and cellular tissue primitive. In our opinion the heterostracan condition (acellular ‘bone’) would also have to be considered as derived relative to the primitive craniate condition. This is particularly true in the light of confirmation of a vertebrate in the Harding Sandstone that has osteocytes in the bone of the tuberculate exoskeleton (Smith, 1990; in prep.).

It may be of some importance to these considerations that the perichondral bone, occurring here at the earliest time in the vertebrate record, also has enclosed cells. Although, perichondral bone (Fig. 1 1 , character 5 ) is considered to be a derived character shared by osteostracans, anaspids, petromyzontids and gnathostomes (Fig. 1 1 , character 12; Janvier, 1981, p. 134). However, perichondral bone was also considered by Janvier ( I 981, p. I 34) to be present in the Galeaspida, a group considered to be alternately the sister group of Osteostraci plus Gnathostomata, or of heterostracans (Fig. I I , character 5 ) . It is clear from these examples that once again correctly observing the skeletal tissues, perichondral bone and cancellous middle layer of the exoskeleton, is important in cladistic analysis.

There is one record of calcified cartilage in this group, by 0rvig (1957, p. 295-299), in a cephalaspid Benneviaspis from the Lower Devonian, in what 0rvig calls

MOYA M. SMITH AND B. K. HALL endoskeleton of the shoulder girdle, continuous with the head shield. It can only be speculation, with so little evidence, but it could represent the part of the posterior cranial endoskeleton that forms from neural-crest cells in gnathostomes. If this is accepted then it is not evidence of the first mesodermal calcified skeleton. Similarly the rostra1 calcified cartilage elements in Eriptychius, described by Denison (1967, p. 142) as the internal skeleton, would be considered to have derived from cranial neural crest.

There is general agreement, although not unanimous, that there is no endochondral bone in this group. Cancellous endochondral bone was reported by Brvig (195 I , p. 430) in a footnote to confirm that it was true bone in this Lower Devonian cephalaspid Boreaspis, and he concluded that it was an example early in the fossil record of ossification in the cartilage (i.e. endochondral bone). He further stated ((arvig, 1957, p. 295) that there is cancellated endochondral bone in the endoskeleton and cites Wangsjo (1952, p. 5 2 ) and Stensio (1927, p. 296). Both Janvier (1984, p. 3 5 1 ) and Gardiner (1984, p. 185) considered this to be a mistaken interpretation. We felt this was sufficiently important, because endochondral bone is considered to be a later derived character, found within gnathostomes (see next section), to examine the sections for ourselves. Our own observations suggest that it is not endochondral bone but heavily remodelled bone deriving from the dermal skeleton because the thin trabeculae show remains of both Sharpey fibre bone and dentine tubercles. Although the cancelli are of mixed sizes and the trabeculae extremely thin, it does not show any evidence of having replaced cartilage.

T h e affinities of the Osteostraci and Gnathostomata as sister groups are favoured by Janvier (1984, p. 355) based on five synapomorphies, two of which are skeletal, cellular bone and perichondral bone, but these are also shared by galeaspids, as the sister group of the other two together.

( a ) Tubercles

Tubercles, composed of mesodentine, a tissue intermediate in appearance between bone and orthodentine, ornament both the cranial and trunk regions of the exoskeleton (Fig. I ~ C ) . This type of dentine also appears in the katoporid thelodonts and some gnathostomes, its position as a genuine intermediate type (prior to the appearance of orthodentine) depends upon accepting that bone preceded dentine in evolution, a view \ve do not hold, either from the phylogenetic or developmental evidence. We think it is a primitive type of dentine, occurring, either together with, or before bone with cells. There does, however need to be an explanation of the different types of dentine, from a developmental or functional point of view. This is dealt with in section I1 ( I I c).

( I 0) Gnathostomes

As discussed in the previous sections most of the vertebrate skeletal tissues evolved within the agnathans, except endochondral bone, a derived character within the gnathostomes. Apart from the two major groups based on the predominance of cartilage (Chondrichthyes) or bone (Osteichthyes), two other groups represented entirely be fossil forms are acanthodians and placoderms, both of which have been difficult to place in a cladistic analysis (Maisey, 1986). T h e systematic position debated by Young (1986, fig. 19) is that placoderms are either the sister group of all gnathostomes (chondrichthyans and osteichthyans), and are the most primitive (acanthodians are

Skeletal development and evolution 3 1 1

grouped with osteichthyans as primitive teleostomes) ; or they are the sister group of chondrichthyans alone, in which case osteichthyans are the most primitive gnathostome group. However, Maisey (1988, fig. 3), proposed a phylogeny in which chondrichthyans are the most primitive group, followed by acanthodians, leaving placoderms as the sister group of the osteichthyans, a scheme previously rejected by Young (1986). It is clear from these papers that there is little agreement on the interrelationships of these groups, and any one of the three has been suggested as the primitive one. For the purposes of this discussion of the origins of skeletal tissues, Maisey’s scheme will be followed, with chondrichthyans as the primitive group, because the plesiomorphic characters for gnathostomes are considered to be, separate denticles, (the micromeric condition and not a macromeric dermal ossification as proposed by Maisey, 1988; see Schaeffer, 1977), composed of three tissues, and calcified cartilage (endochondral calcification), with perichondral bone evolving later independently from that in osteostracans. One fascinating question, largely unresolved, is whether the first osseous mesodermal skeletal tissues occur in gnathostomes, in addition to those of the endoskeleton derived from neural crest.

With the evolution of jaws the major changes in the skeleton concerned an adaptation of the dermal denticles (odontodes) to become modified for feeding, prey retention, cutting and crushing, and with this the developmental innovation of a dental lamina to provide a continuous supply of teeth in advance of their requirement, and prior to there being any space at the functional surface. Reif (1982) has elegantly discussed this in a review of the evolution of the dermal skeleton and dentition in vertebrates, and proposed that a dental lamina was present in all groups except placoderms, placed on his cladogram as the sister group to all other gnathostomes (Reif, 1982, fig. I ) .

The other major innovation was the development of endochondral bone, a character that Maisey (1988) identifies as plesiomorphic for gnathostomes (Maisey, 1988, fig. 3, character I39), further stating that ‘ in Recent elasmobranchs the endoskeletal calcification is endochondral ’. This is a cladistically contentious point, because endochondral bone is considered by others as either an osteichthyan synapomorphy (Rosen et al., 1981; Gardiner, 1984), or a synapomorphy uniting placoderms and osteichthyans (Forey, 1980), and for this reason it is important to establish the salient features. Identification of endochondral bone in fossil tissues requires a knowledge of topography (internal to a sheath of perichondral bone), macrostructure (spongy bone), and development or histology (bone deposited on a framework of calcified cartilage). Using developmental data from the talpid mutant chick, Patterson (1977, p. 82) drew attention to the epigenetic dependence between perichondral bone and endochondral bone, without the former the latter will not form, and prior to this, perichondral bone formation is dependent on inductive signals from hypertrophic cartilage (i.e. calcified cartilage). This would indicate that we might find that a similar sequence had occurred in evolution, although the steps might be so tightly linked that all may have occurred at the same time. The evidence for this in fossils arranged in systematic order of the acquisition of derived characters, would be calcified cartilage alone, or together with perichondral bone, in lower clades, then endochondral bone plus perichondral bone. The absence of endochondral bone in chondrichthyans would be because it was absent in the stem group and only derived at the level of osteichthyans as a synapomorphy with placoderms.


Apart from the development of teeth and their location on the marginal bones of the jaws, the reduction of the dermal skeleton, in those groups that evolved from a macromeric condition, is a trend amongst many of the groups (a functional explanation would be that it facilitated greater body flexibility in active predation). Also amongst the plac-oderms a variety of types of tissue (dentine/bone intermediates) are found in the tubercles of the dermal skeleton, i.e. semidentine, considered by Denison (1978, p. IS), and Goujet ( I 984) as a characteristic (synapomorphy) of placoderms. A further type of dentine has been described in gnathostomes by Brvig ( I 975, I 980) as mesodentine, one occurring in other agnathan groups, so clearly these varieties of dentine are important concepts to discuss in the evolution of vertebrate hard tissues, in particular because of major publications on the histology of placoderm and elasmobranch hard tissues by @rvig (1951, 1957, 1975, 1980).

( a ) Chondrichthyans

Two central issues are contentious in this group: ( I ) Does the skeleton contain any bone, and if so what type and what is its relation to the exo- or endoskeleton ? (2) Is the skeleton primitive or derived, and if so which components are primitive and which derived? Maisey (1988, p. 24) considered that both the dermal and endoskeletons are derived because they are micromeric. This assumes a macromeric primitive condition for gnathostomes, but, this condition (few large dermal bones) is thought to have arisen independently four times (Schaeffer, 1977, p. 39), and the view has been expressed (Schaeffer, 1977, p. 39; citing Nelson, 1970) that the micromeric condition is primitive for vertebrates. Also, Maisey ( I 986, p. 240) concludes that ‘we do not know whether the micromeric (chondrichthyan) or macromeric (osteichthyan) condition is derived among gnathostomes - since both micromerism and macromerism also prevail in agnathans ’. The condition in chondrichthyans therefore, could be the primitive one for vertebrates.

Concerning the tissues of the dermal skeleton, bone has been described as the basal plate of the teeth and dermal denticles, and although contiguous with the dentine of the odontode, Reif (1980, p. 283) concluded that it was acellular bone formed by osteoblasts from the fibrous layer basal to the developing odontodes. The function of this tissue is to attach collagen fibres of the supporting soft connective tissue to the odontodes. We would suggest that this is the same as the tissue we describe in the agnathan dermal skeleton, as homologous with the acellular, ‘ Sharpey fibre cement ’ (aspidin) derived from a sub-population of the dental papilla, and developed in a causal interdependent sequence with the dentine. Clements (1986, p. 243) also commented that ‘it is valid to describe the outermost (basal) tissue as cement rather than acellular bundle bone ’.

It is relevant to refer to Reif‘s (I 979) discussion where he cites Hertwig ( I 874) for the observation that the basal plate of shark’s placoid scales consists of cementum and not dentine. However he comments that ‘the interpretation was wrong because it is formed by osteoblasts and is an acellular bone and not cementum’. Without the relevant critical developmental information it is not possible to decide on the homology of this tissue, but using developmental information from vertebrate odontogenesis in general we conclude that it is certainly odontode related attachment tissue, probably homologous with extrinsic fibre (Sharpey’s fibre) acellular cement. The drawings in Figs. 5 B-D and 10 G illustrate this tissue with Sharpey’s fibres perpendicular to the basal surface. The

Skeletal development and evolution 3’3

observation that the base is thickened by many depositional layers (Reif, 1979, p. 27) could equally apply to cementum as to bone. Relevant to this is the information from some of the earliest recorded placoid scales of sharks (Lower Silurian) that the basal tissue contains cell spaces (Karatajutd-Talimaa, I 989).

In chondrichthyans, if we accept the above proposed homology, the primitive odontode related part of the dermal skeleton is represented, but the basal supporting bone is not. We believe that basal supporting bone is derived from a sub-population of neural crest cells, and arises later in evolution. Our observations may seem to go some way towards supporting a suggestion made by Turner (1985) that thelodonts are possibly the sister group of chondrichthyans, non-growing, non-osseous-supported scales, would be one shared character; but our contention is that it is a plesiomorphic condition derived at the craniate level and not a synapomorphy of thelodonts and chondrichthyans. One interesting observation by Reif (1980, p. 286) of the pattern of formation in both the dentition and squamation of sharks, is that unlike osteichthyans, there is no ‘initiator tooth’, all the first generation odontodes form simultaneously in a given region. It is interesting to speculate that this may be the primitive patterning mechanism (earlier we commented that the skeleton of Anatolepis heintzi had been assumed to have arisen all at once, Bockelie & Fortey, 1976), but its link with the sequence of epigenetic events in the migration of neural crest cells is not known.

New observations by Karatajutd-Talimaa (unpublished communication, I 989, 2nd Colloqium on Paleozoic Fishes, Tallinn, Estonia) that edestid shark scales, of a type illustrated in Fig. 5 D, are found in Lower Silurian, Upper Llandovery, deposits, has implications both for the earliest types of dentine and the earliest occurrence and type of gnathostome skeletal tissues. It is important to record that she has found at least three types of placoid scales of primitive sharks in the same deposits. Two of these have acellular basal tissue but one has cell spaces in the basal tissue, and all three have a different type of dentine. Other scales belonging to the oldest recorded shark, also from the Lower Silurian, consist of single, double and triple odontodes and resemble those of Eugenodus richardsoni (Zangerl, 1981, fig. I). The single scales are found on the belly skin and more compound scales on the dorsal surface, according to Zangerl(1981) each odontode (equated with a lepidomorium) is a simple cone of orthodentine, without an enameloid cover and lacking a bony base. The compound scales have dentine above either cellular or fibrous basal tissue, and as shown in Fig. 5 D of Elegestolepis grossi this tissue forms after the dentine. We would interpret this as indicating that both forms of attachment tissue, acellular and cellular cement, were present and quote from Zangerl ( I 968) on the histology of the basal tissue of scales of Holmesella and Orodus from the Upper Carboniferous - ‘ It is at least possible that these cells were not true osteocytes, but cells of intermediate character between odontoblasts and osteoblasts and that the tissue that they formed is not bone but an intermediate between dentine and bone’ - i.e. cementum. The implications of these findings are that already early in the stratigraphic record of gnathostomes the simplest form of dermal odontode formed part of the dermal skeleton and there was a variety of types of dentine and basal tissue, some with cell spaces included.

The second region where the presence of bone is contentious is in association with the endoskeleton. Two key sets of observations claim to show the presence of perichondral bone. ( I ) Peignoux-Deville et al. (1981, 1982, 1985) and Peignoux-

I 2 B R E 65

3 I4 MOYA M. SMITH AND B. K. HALL

Deville & Janvier (1984) and Bordat (1987) describe the apposition of lamellar bone in the perichondrium on the inner surface of the neural arches, which they claim is non- tesserate, (2) Kemp & Westrin (1979) described calcified tesserae on the outer border of the endoskeleton as ‘calcified cartilage which in their later stages are surmounted by a thin veneer of bone’. The latter is much more tenuous evidence of the presence of bone than the former, and Clements (1986, p. 334) strongly denies the presence of bone in this position. However, the observation (Peignoux-Deville, I 982) that hypertrophic chondrocytes appear in close proximity to the perichondral bone fits with the developmental evidence that the two are causally related ; osteogenesis is elicited from the perichondrium by cell hypertrophy or matrix calcification. The interpretation by hlaisey (1988, p. 21) that endochondral calcification is somehow (not explained) equivalent to endochondral bone, and therefore, ‘ osteichthyan endochondral bone represents a cladistically primitive calcification pattern (ossification) - rather than a novelty of bony fishes ’ cannot be supported. The evidence is entirely missing. One step in this process may be represented, i.e. perichondral bone, but no replacement of calcified cartilage by bone has ever been demonstrated. In fact such replacement would appear to be impossible because osteoclastic resorption is apparently missing from chondrichthyan calcified tissues (Clements, 1986, p. 335).

( b ) Acanthodians

Of the four major reviews of vertebrate skeletal tissues two deal exclusively with the dermal skeleton (Schaeffer, 1977; Reif, 1982), and two with all aspects of the skeleton (Patterson, 1977; Maisey, 1988). Of these only Maisey comments on acanthodians, hence appraisal of the status of the skeleton in this group is sparse. In a general account of acanthodians, Denison (1979), considers them as the earliest gnathostomes, and makes two statements which are of importance to this review, namely, that there is no endochondral bone, only perichondral bone, in the paired fins of the axial skeleton, and no calcified cartilage ; and that in the endoskeletal vertebral column only the neural and haemal arches are ossified (perichondral), with dorsal spines and posterior haemal spines. Although the visceral skeleton was calcified (perichondral bone over calcified cartilage, examples of these tissues in placoderms are shown in Fig. 16C, D), this is neural crest derived, and it is interesting that the teeth are fused to the bone of jaws. Denison (1979) believed that the endocranium was primitively unmineralized, and that perichondral bone was acquired later. It seems clear that no endochondral bone was present in this group, and that the major innovation was ankylosed teeth. This conclusion clearly supports Forey’s (I 980) proposal that endochondral bone is a synapomorphy uniting placoderms and osteichthyans (excluding acanthodians).

Although Denison (I 979) stated that there was apparently no loss from shedding and no replacement, Reif (1982; p. 342) concluded that ‘it is very likely that both types of single teeth were regularly replaced ’ although he admitted that ‘ direct evidence is missing’. The two types of tooth he referred to were single ankylosed teeth and single teeth anchored by fibres. If the former is a derived character then perhaps it was not yet universal in the dentition. The presence of tooth spirals was used by Reif (1982, p. 342) as evidence of a dental lamina; he proposed that the members of one tooth family (teeth formed at successive times) had become fused together by the bone of attachment. The main evidence being that the size of the teeth gradually increases from the inner to


C

B

Fig. I 5 (A-D). Examples of different types of dentine in gnathostome

D

ermal skeletons to s ow (A) semi- dentine in a placoderm; (B) metadentine (orthodentine) in a cladodont shark; (C, D) mesodentine in acanthodian tissues. (A) Ohioaspis tumulosa, x 230 (Gross, 1973, fig. 3C). (B) Maplemillia costata, X 230 (Gross, 1973, fig. 18F). (C) Nostolepis striata, x 230 (Gross, 1971, fig. 1 3 D). (D) Acanthodes bronni, x 2 1 5 (Gross, 1947, fig. 18 C). (All reproduced by permission of the publishers E. Schweizerbarts’sche, Verlagsbuchhandlung.)

the outer end of the spiral, but it also testifies to the lack of shedding in this series of teeth.

The fin spines, bones covering the head, and body scales are all part of the dermal skeleton, and are ornamented with odontodes, the dentine of which is described as mesodentine (Fig. I 5 C, D). The fin spines are not based on cartilage, the central tissue may be a type of dentine called osteodentine where vascular spaces fill in with denteones. The very small body scales grow throughout their life by the addition of layers of odontodes and bone of attachment, concentric with the embryo scale; resorption does not seem to occur. The growth of the individual scales is a feature not present in chondrichthyans, and may represent a derived character shared with all other gnathostomes. The presence of mesodentine is hard to explain given that later acanthodians are said to have metadentine (Fig. I ~ B , and Denison, 1979). Generally

12-2


lam lac / I I

lac can

Fig. I ~ ( A - D ) . Diagrams to illustrate the types of bone in the endoskeleton of gnathostomes (placoderms, Plourdosteus canadensis). (A, B) vertical and horizontal views of cellular perichondral bone. (C, D) Perichondral bone with vascular canals, surrounding calcified, globular cartilage of the endoskeleton. can, canaliculi; cg, calcified globules; cms, cell matrix space; lac, lacunae; lam, laminae; os, osteone; spc, subperichondral bone; pcb, perichondral bone; vc, vascular canal. (A, B) x 280 (@r\-ig, 1951, fig. 16.4, B). (C) x 100; (D) x280 (0rvig, 1951, fig. I S A , B). (Reproduced by permission of the author and publishers, Almqvist and U’iksell.)

much more needs to be known about the skeletal tissues in this fossil group before it can be evaluated.

( c ) Osteichthyans

It is beyond the scope of this review to deal with many aspects of the remaining three groups of the Osteichthyes, namely placoderms, actinopterygians and sarcopterygians. Most evolution has occurred through specializations within existing tissue types, through accretion, reduction or remodelling of the tissues. Reif (I 982) has dealt very adequately with the dermal skeleton, and proposed developmental mechanisms to explain the variation in the scales; he makes the point that both dermal bones and dermal scales are growing and both have a different pattern of evolution. Non-growing tesserae or scales are unknown, Cellular bone is accepted as plesiomorphic in both endo- and exoskeletons (Fig. 16). Resorption of the scale tissue prior to growth episodes is a derived character; this phenomenon is found only in advanced clades (Reif, 1982, p. 31 I). An example of a growing but non-resorbing scale is seen in Fig. 5 E, of a primitive actinopterygian. The innovation in this group of fishes is endochondral bone, perhaps linked to the resorptive ability found in the dermal skeleton (osteoclasts are a multicellular derivative of blood-borne monocytes). Brvig (195 I, pp. 409, 41 I, 1957, p. 3 18) provides the most direct evidence for endochondral bone in some groups of


arthrodires (placoderms), and as it is clearly present in most osteichthyans we regard it as a synapomorphy of osteichthyes.

As outlined in the general account of gnathostome tissues, the difficulty of identifying endochondral bone may account for lack of certainty on its presence. Young (1986) has discussed previous work reporting the presence of endochondral bone in placoderms and concluded that it is a plesiomorphic character for placoderms. Those groups where it is absent together with absence of perichondral bone would be derived. However he reached this conclusion also by accepting that endochondral bone is present in acanthodians, the evidence for which we would dispute. Gardiner (1984, p. 185) also discusses the occurrence or absence of endochondral bone in placoderms and concludes from macroscopic evidence alone that in placoderms it is probably primitively absent, and that ‘endochondral bone must be considered a specialization of osteicthyans (not including placoderms) ’. These views highlight the importance of accurate observations on placoderm hard tissues to provide realistic data for cladistic analyses of interrelationships amongst vertebrate taxa.

Clearly it is important to know whether or not this particular type of bone (defined on its topography and development) is present or absent in both of the entirely fossil groups, placoderms and acanthodians. The presence of endochondral bone in extant forms is also linked with the presence of red bone marrow, a site for the genesis of osteoclast precursor cells. We would suggest that this was an important innovation of mineralized tissues, occurring in osteichthyan fishes.

( I I ) Summary and discussion

The above group by group analysis indicates the following : The exoskeleton of thelodonts, heterostracans, osteostracans, galeaspids, and

chondrichthyan gnathostomes, consists of dentine and bone ; but, only bone of attachment closely associated with dentine in thelodonts, some early agnathans, and chondrichthyans ; and, only acellular aspidin in heterostracans (of various types, some serving as part of the tubercle and bone of attachment) and cellular bone, as basal bone, bone of attachment, and perichondral bone in osteostracans and galeaspids.

An enamel covering to the dentine is only seen in one of the very early agnathans (Eriptychius) of uncertain affinities, and the status of the outer layer in all groups of agnathans is uncertain, although clearly there is a ‘glassy cap’ to the tubercles of Astraspis (Fig. 9B, D, E) called durodentine by Denison (1967, p. 174) and enameloid by Brvig (1967, p. 80). Dzik (1986) suggests that the distinction between dentine and enamel was primitive in vertebrates, and that even more primitive sclerites have a homologue of enamel as the dominant tissue. Most developmental information suggests that dentinogenesis commences and then induces enamel formation, but new molecular studies may show that enamel proteins are encoded at the same time, so that at least both tissues could arise simultaneously. Cartilage (uncalcified) is inferred in heterostracans from impressions in the skeleton, and present as a calcified tissue as fragments with globular mineralization (Fig. 4A-D) in one very early agnathan (Eriptychius) not assigned to a group, and assumed to be present (uncalcified) in between the perichondral bone of the cephalic skeleton in osteostracans and galeaspids.

Clearly, the taxa of Ordovician agnathans demonstrate almost the full range of vertebrate skeletal and dental tissues ; cement is the only tissue classically considered

MOYA M. SMITH AND B. K. HALL

C D E Fig. I ~ ( X , B). SEMs of foetal scales in Latimeria chalumnae. (A) Two early odontodes each attached via hone to the scale base, and in (B) only the ring of bone of attachment is shown after the loosely attached odontode has been removed. (A) Bar, 60 p m ; (B) bar, zg y m (Smith, 1979, fig. 2). (Reproduced by permission of the author and publishers, Pergamon Press.) (C-E) Drawings of odontodes on the scales of catfishes to show the bone of attachment below each odontode. ( C ) x 2 5 0 ; (D) x 55 ; (E) x 1 2 0 (Bhatti, 1938; figs. 28, ,+y, 100; p. 78, for all abbreviations). (Reproduced by permission of the publishers, Zoological Society of London.)

not to be present, but we discuss this below. We agree with Brvig ( 1 9 5 1 , p. 381) that all five tissue types are represented : enamel/enameloid, dentine (two kinds), bone (no enclosed cells), bone with enclosed cells, and globular calcified cartilage.

Evidence from the fossils supports the idea that, in a phylogeny of vertebrates, odontodes with their bone of attachment arise earlier than, and separately from the basal plate of bone (0rvig, 1977, p. 71), and that the first skeletogenic ability was that of forming these odontodes denticles (Westoll, 1967, p. 94). There is limited information available from the development of dermal denticles in extant forms but Smith ( I 979, fig. 2) demonstrated rings of bone developing in relation to each odontode in scales of the foetal coelacanth (Fig. 17A, B). Bhatti (1938) also described odontodes of the scales of the armoured catfishes developing independently from the bone of the


scale, each with a ring of bone of attachment between the odontode and the dermal bone (Fig. 17C-E, p).

The arrangement of these tissue types, their relative proportions and the fate of the cells that produced them may vary throughout evolution. Moss (1964) tabulated four trends in skeletal evolution, all of which relate to skeletal elements and to alterations in skeletal growth, but none of which relates to the appearance of new skeletal tissues. Moss’s four trends are :

(i) a decrease in the range of skeletal tissues; (ii) a decrease in the amount of bone;

(iii) a decrease in the absolute number of bones; (iv) a more restricted localization of bone within the body. We consider that these may have been related to a more restricted range of cell

differentiation, and to functions requiring greater flexibility of the body, and then more rigidity with movement limited to joints.

However, concerning new skeletal types, or innovations from that in existence at the time, we need to resolve the problems concerning the identification of aspidin. Central to this is the origin of cellular bone, whether it evolved before, after, or at the same time as acellular bone.

( a ) Cellular-acellular bone The contemporaneous appearance of cellular bone, although not abundantly

preserved, and acellular bone in heterostracans and assumed primitive osteostracans (Tarlo, 1967 a; Smith, 1990 in prep.) during the Ordovician can only be used to support one hypothesis or another, as to the primitive state, based on ontogenetic data or on robust phylogenetic hypotheses with many congruent characters. Three possible positions have been argued, namely

( a ) that cellular bone is primitive (i.e. that acellular bone evolved from cellular bone), 0rvig (1957, 1968), Maisey (1988);

(b) that acellular bone is primitive (i.e. that cellular bone evolved from acellular), Denison ( I 963), Janvier ( I 981, p. I 37 and ref. therein), Maisey (I 988) ;

(c) that the two bone types arose independently (0rvig, 1965; Moss, 1 9 6 8 ~ ) . For reviews of this issue see Tarlo (1964a), 0rvig (1965), Halstead (1969), Hancox

(197z), Maisey (1986, 1988), and Figs 6 and 16 for examples of the tissues. Halstead (I 987) in his most recent paper has recanted his earlier suggestion that the

elongated spindle-shaped spaces in aspidin represented the in vivo vacated sites of bone-forming cells. This would lend support to the view we hold that this tissue is a tooth attachment tissue (cementum). More significantly, Halstead (1987) has stated that in two of the key Ordovician genera, each of the three tissues of the tubercle is widely different, or potentially a special derived character. Two of the tissues in Astraspis, previously described by Denison (I 967) as dentine (compact) and trabecular dentine, and used by Halstead (I 969) to argue the close evolutionary relationship between dentine and aspidin, have now been designated by Halstead ( I 987) as a unique tissue with a new name, astraspidin. To make such statements as ‘acellular bone preceded cellular bone in evolution’ on the basis of these specialized and varied tissues must surely be questioned and re-examined.

We consider that qualifications need to be made to these statements, on the basis that

MOYA M. SMITH AND B. K. HALL the topographic site within the body needs to be specified; for instance, in the dermal skeleton, the bone of attachment, and basal bone, and in the endoskeleton the perichondral bone, may each be subjected to different functional requirements. Also we agree with Brvig (1977, p. 70) that the basal plate of odontodes is not sufficient alone to account for the massive dermal ossifications, so that the three sites of bone (attached to dentine, basal to odontodes, and perichondral) could change differently in evolution. Some of these tissues with different topography could be used as characters in cladistic analyses.

What light does development throw on this longstanding controversy ? Experimental studies on tooth germ development in the mouse provide some very interesting results that are pertinent to this problem. Explants of recombinations of mouse dental papilla, or dental follicle, with denuded enamel organ in an embryonic time series (Palmer & Lumsden, 1987), reveal the developmental potential of the papilla cells and follicle cells (both of ectomesenchymal origin). At the earliest stage of the tooth germ when a papilla can be separated from the epithelial cap and the surrounding follicular mesenchyme removed, the papilla cells will make all the collagen-based tissues of the tooth root; that is, dentine, and the attachment tissues - cement, and periodontal ligament attached to the cement - but not alveolar bone. This only develops if follicular mesenchyme cells are also included (Fig. 18) .

These results of Palmer & Lumsden ( I 987) differed from the initial study by Yosikawa & Kollar (1981) . Palmer and Lumsden believed it was because follicle cells were not included in their grafts (these are assumed to contain the osteoblast progenitor cells as well as progenitor cementoblasts and fibroblasts). Mesenchymal cells of the papilla appear to have a choice in differentiation ; those in contact with the inner epithelium become odontoblasts, and those with the outer epithelium cementoblasts. Assuming stability of the histogenetic pathways through evolution it is not difficult to project this back to the earliest tubercles of the exoskeleton. Namely, the papilla cells produce dentine on the inner surface of the dental cap, and at the basal margins, migrating cells when on the outside of the enamel cap produce acellular cement with attachment fibres (i.e. the basal tissue of thelodont scales, placoid scales of chondrichthyans, and some aspidin; primary cement, in mammalian teeth, is always acellular, first to form, and topographically closest to the dentine, and secondary cement mostly cellular). Only if a second population of cells is able to separate from the papilla cells (i.e. follicle) early in development, does the bone of attachment form and fuse the separate odontodes together, or fuse them to a separate ossification centre (basal bone). In Palmer and Lumsden’s study, recombination of follicle alone with enamel organ at the earliest age, also produced all the tissues; the follicle cells had apparently not lost their early potential to make dentine, and were also able to make cement, periodontal ligament, and bone.

The interpretation arising from the developmental studies is that the early vertebrate tissues were dentine, close to the odontogenic epithelium, and a tissue without cells and with attachment fibres, homologous with cement (i.e. aspidin, or basal tissue of thelodont tubercles; Fig. 5B, C), or further from epithelial influence, bone of attachment with enclosed cells (i.e. some chondrichthyan placoid scales, eriptychiid tuberculate tesserae). This scenario would allow aspidin to be interpreted as a form of attachment tissue, or at least closer to dentine than to dermal bone in its development


and evolution, as also would the basal tissue of thelodont scales and placoid scales be closer to attachment tissues (cement). Lumsden (1987, p. 291) has also suggested that ‘ aspidin evolved first as a supporting substrate to maintain the spacing of the sensory receptors’; we would say that it evolved also as a basis for attachment of the sensory receptor complex to the underlying connective tissue.

The argument to be made on the basis of development of bone in extant vertebrates (gnathostomes), is that developmentally, acellular bone cannot be primitive, because cellular bone is always the first type to be formed, i.e. in the development of membrane bone, perichondral bone, and in the primary spongiosa of endochondral bone, cells are trapped initially in the first formed bone trabeculae.

As summarized in an extensive series of studies by Moss (1961 a, 1963, 1965) acellular bone is commonly found in teleosts inhabiting both fresh water and marine environments, Moss ( I 964) described three modes of development of acellular teleost bone; from periosteal cells, within tendons, or by metaplasia from cartilage, i.e. acellular bone always develops from a cellular tissue. Only one species (Albula vulpes, Moss, 1961) has been reported as having both cellular and acellular bone, the operculum and branchial arch skeletons being acellular.

Parenti ( I 986) has provided a valuable analysis of the role of teleost acellular bone in calcium metabolism, of the intimate relationship, both topographically and developmentally, between dermal bone and teeth (Moss’s integumental skeleton) and argued for greater account to be taken of acellular bone in teleost phylogeny. Most recently, Ekanayake & Hall ( I 987, I 988) have used fluorescence microscopy after tetracycline- labelling and transmission electron microscopy to document the pattern of activity of osteoblasts that remain on the surface of acellular bone (synthesis of extracellular matrix is polarized, matrix only being secreted at the surface of the periosteal osteoblast facing the bone). Although vertebral bone of the Japanese medaka, Oryzias latipes, has a lining of cells on both the inner (toward the notochord) and the outer (toward muscle) surfaces, matrix is only deposited by the cells on the outer surface, i.e. only the outer layer is a periosteum with preosteoblasts and osteoblasts. The same is true for the example of bone in chondrichthyans, where bone is formed from one surface only by apposition onto calcified cartilage, but this is on the inner surface of the neural arch (Peignoux-Deville, Lallier 8z Vidal, 1982). This could be related to the fact that there has been no invasion of blood vessels into the notochordal side, in the medaka, and, therefore, no osteogenic cells have arrived in that region. One could argue that osteogenic cells are closely associated with vessels in evolution and development of bone, i.e. the paravascular cells are always necessary to achieve endochondral ossification, and only do so with vascular invasion of the cartilage model. The first bone in the evolutionary record of skeletal tissues is that associated with vessels at the base of the odontode (neck canals, etc., Fig. 5A).

Maisey (1988, pp. 6, 16, 19, 24) takes a stand with regard to this question that we find difficult to support. He dismisses the developmental evidence for acellular bone being secondary with the statement that ‘ Nevertheless, it must be remembered that cellular and acellular bone are both secondary ontogenetic by-products of skeletogenesis ’ (ibid. p. 6). It is difficult to understand what this statement means because clearly it cannot apply to dermal or membrane bones which develop as primary structures directly in the connective tissue. Even in perichondral ossification bone develops initially and directly

3 22 MOYA M. SMITH AND B. K. HALL

Fig. 18. For legend see upposlte


in the connective tissue ‘ membrane ’ of the perichondral sheath around the cartilage ; ossification is not a ‘ secondary by-product ’. Only in endochondral ossification is the primary spongiosa a site where bone develops secondarily to calcified cartilage, and incidentally, secondary to perichondral bone. Maisey goes on to argue (p. 16) that, based on observed tissue distribution among early craniate taxa, acellular bone is ‘cladistically primitive ’. But the cladogram has already been erected leaving out the example of cellular bone found at the same stratigraphic level. Maisey (ibid. p. 10)

discarded the evidence of cellular bone with the statement ‘ ... the earliest scraps of cellular bone are of uncertain affinity and consequently have no bearing on the present phylogenetic analysis! ’. Given that the earliest cellular bone consists of more than scraps (Denison, 1967, who relates it to the base of the denticles), and can be assigned to an individual agnathan group (Smith, 1990 in prep.), we fail to see how cellular bone can be discarded, how acellular bone can be ‘cladistically primitive’ (ibid. p. 16) or how ‘ . . . parsimony suggests that acellular bone is primitive ’ (ibid. p. 19). The developmental data show it to be no more primitive than cellular bone, and the assignment as primitive involves a priori (and circular) arguments that heterostracans are primitive (ibid. pp. 16, A ) , forcing the conclusion of ‘the secondary acquisition of cellular bone by osteostracans (including galeaspids ?) and gnathostomes . . . ’ (ibid. p. 23). It could be proposed that heterostracans had not developed the tissue ‘bone’ per se, certainly not perichondral bone or endochondral bone, and that the dermal bone was always ‘tooth associated ’, a product related to tooth attachment and support, a role taken by cement and alveolar bone in mammals. Sometimes this bone had cells included and sometimes not (as cellular and acellular cement).

( 6 ) Cartilage versus bone

The question of whether cartilage or bone arose first is confused for several reasons, notably that only calcified cartilage can be seen in fossil tissue so that presence of uncalcified cartilage has to be inferred for other reasons (as done by Gans & Northcutt, 1983 ; and discussed more fully in section VIII), and that time of appearance of cartilage may have been quite different (we would contend was quite different) in the exoskeleton and in the endoskeleton. Because in the past, prior to Patterson (1977), except for Stensio (1927), Jarvik (1959) and Denison (1963) insufficient attention was given to the separation of ( a ) the exo- from the endoskeleton, and ( 6 ) the possible neural-crest-

Fig. 18(A-E). Histology of normal (A, B) and experimental (C-E) tooth germs of Mus musculus to demonstrate that the attachment tissues, cementum, periodontal ligament and bone of attachment develop after the enamel and dentine of the crown and root. This occurs only if very early ectomesenchyme cells of the dental papilla [stage of development in (A)] or later dental follicle ectomesenchyme are included in the explant. (A) x 100, early cap stage; (B) x 25, appositional stage before root development; (C) xqo, new-born enamel organ combined with 16-day foetal papilla after 4 weeks explant in the anterior chamber of the eye. Dentine of the root has formed but no attachment tissues. (D) x 40; (E) x 250, 16-day foetal tooth germ recombined, including enamel organ, dental papilla and dental follicle. The root surface has fibres attached (acellular, extrinsic fibre cement) and bone has also formed adjacent to the root, with some fibre bundles aligned between the root and the bone (periodontal ligament). Haematoxylin and eosin. Abbreviations : bo, bone; ce, cementum; co, cornea; df, dental follicle; dp, dental papilla; en, enamel space; eo, enamel organ; pl, periodontal ligament; r, root surface (Palmer & Lumsden, 1987, fig. 5 ) . (Reproduced by permission of the authors and publishers, Pergamon Press.)


derived cephalic exoskeleton from mesodermal derived trunk endoskeleton, the vexing question of the primacy of cartilage has persisted. It seems most likely to us that in the exoskeleton of early agnathans bone preceded cartilage, but in the endoskeleton it is probable that cartilage preceded bone.

( c ) Types of dentine

The question of the relative primitiveness of dentine and bone is one that has aroused the most debate. With the assumption that bone arose earlier than dentine the variety of tissues in the superficial layers of the exoskeleton that display different degrees of enclosure of cell body and cell process, have been interpreted as intermediates between bone (cell bodies enclosed, many multidirectional cell processes) and dentine (cell bodies not enclosed in mineralized matrix, single polarized cell processes run from origin of tissue to last position of cell body housed in soft tissue space). In his review of this topic, Brvig (1967) described three types of dentine, starting with mesodentine (an interconnecting web of cell processes, see Fig. 1 5 C, D), through semi-dentine (cell body enclosed but a polarized cell process ; see Fig. I 5 A) to metadentine equivalent to orthodentine (many sub-parallel single cell processes with small lateral branches, Fig. 1 5 D). Both Baume (1980, fig. 41) and Reif (1982, fig. 2) illustrated these three types of dentine, and Reif commented that Brig’s assumed evolutionary sequence ‘was not readily testable with the data available’. Brvig (1967) assumed an evolutionary progression from mesodentine through semi-dentine to metadentine, as progressive changes in the histogenesis of this tissue (akin to bone), from enclosed cells to partly enclosed, to only a cytoplasmic process enclosed. This is inconsistent however, with a subsequent statement in which he states that typical dentine (assumed metadentine) was already present in the agnathans Eriptychius and Astraspis, derived from yet earlier forms. Logically the varieties of gnathostome dental tissues are also secondarily derived. We have argued in this paper that the earliest skeletal tissues were dental, and that dentine developed in association with a sensory function and was always the most superficial dermal tissue, only later covered by a hypermineralized layer (enamel or enameloid, formed from an ectodermal contribution). Less attention has been paid to the observation that there are three types, coarse-tubed dentine (Eriptychius), fine- tubed dentinelastraspidin (Astraspis), and mesodentine in the third vertebrate Smith (1990, in prep.), a feature listed by Brvig ( 1 9 5 1 , p. 381), and one we have suggested is related to different sensory functions, or none at all if the fine tubes or spaces housed radial fibres.

Another question that arises in discussion of the primitive types of dentine is the observation that in many of the thelodont and shark scales the types of dentine are also based on whether the pulp chamber remains as a single undivided pulp containing all the odontoblast bodies and blood vessels, or it becomes subdivided by trabeculae of dentine and smaller pulp canals form in the inner walls of the dentine housing odontoblast bodies and capillaries. This arrangement is shown in Figs I E, 5 D and IOG in shark placoid denticles, but is also typical of two major types of thelodont scale, katoporids and loganiids (Fig. 5 F, G), in contrast to the thelodontids (Fig. 5 B). It has to be acknowledged that in the Lower Silurian there were various types of dentine in the dermal armour of each taxon, heterostracans, thelodonts, sharks and acanthodians, and at present there is no solution as to which represents the primitive condition for

Skeletal development and evolution 325 each higher taxon. It will be difficult to obtain developmental information that pertains to this aspect of the dermal skeleton but it is an area for future research to find examples in extant tissues that have these varieties of dentine.

(6) Developmental perspectives T h e following summaries from the literature reflect changing views as more

developmental data accumulate and as phylogenetic methodology (cladistics) provides an explicit framework within which to pose and test hypotheses of evolutionary morphology.

The conclusions to be drawn from an examination of the histology of the bones of fossil vertebrates would appear to be as follows: The skeletal tissues of the early Ordovician vertebrates were essentially the same as are those of present-day vertebrates, although more intermediate tissues were present then than are present now. The first mineralized vertebrate tissues known from the fossil record were complex and specialized (implying earlier unfossilized and perhaps simpler ( ?)skeletal tissues). Skeletal evolution in the vertebrates, at least since the Ordovician, has not involved major changes in cell or tissue organization but rather has involved adaptive responses of already specialized and plastic tissues to new local environmental changes (Hall, 1975, p. 334).

As to the fossils, one should try to define more precisely the polarity of hard tissues characters in primitive craniates, since they often comprise the only available data on the presumed most primitive representatives of the group (Janvier, 1981, p. 1 5 5 ) .

The significance of aspidin is obscure - we need to use other than stratigraphic criteria for claiming primitiveness - and the polarity of the hard tissues is unresolved (Janvier, I 981, p. 143).

Of course, we endorse Maisey’s view (1986, p. 242, citing Hennig, 1965) that vertebrate skeletal history can best be generated by ‘ reciprocal illumination ’ of paleontology and developmental biology. T h e understanding that we might achieve from experimental data on ontogenetic pathways in extant vertebrates forms the basis of the following sections of the paper. T h e information so obtained is tested against the paleontological data as presented in this section, that is, a record persisting for at least 350-400 million years.

111. T H E NEURAL CREST ORIGIN OF CRANIAL SKELETOGENIC A N D ODONTOGENIC TISSUES IN EXTANT VERTEBRATES

( I ) Introduction

On the basis that the first vertebrate hard tissues were exoskeletal dentine and bone, and that we wish to understand the developmental origin of these tissues, because we lack developmental or ontogenetic series for Ordovician agnathans, we need to extrapolate from our knowledge of cranial skeletal development in extant vertebrates.

There are four possible tissue (cell, germ layer) sources for cranial skeletogenic and odontogenic tissues ; cranial mesoderm, cranial ectoderm, endoderm and cranial neural crest. Until relatively recently, cranial mesoderm was considered as the source of the skeletogenic cells (osteoblasts, chondroblasts) that deposit bone and cartilage, and of the odontogenic cells (odontoblasts) that deposit dentine. Enamel has long


Table 2. A summary of taxa and species f o r which knowledge of a skeletogenic andlor odontogenic neural crest is known. Numbers within parentheses refer to references a t the End qf the Table

(;roup

Agiiatha

Fish Xnuran amphibian

amphibian L-rodele

Reptile Birds

l lammals

Species

Petmmyzon marinus ( I )

Lnmpetrn fluc~iutilis ( 2 )

L . planeri ( 2 )

Oryzias latipes ( 7 ) Senopzrs laecis (3) Discoglossus pictus (4) Pleurodeles waltl ( 5 ) Triturus alpestris ( 6 ) Ambystoma mexicanum ( 6 ) Chelydra serpentina ( I I )

Gallus domestirus ( 8 ) Coturnix coturni.t

A l l t t s musculus ( 8 , 9 ) japonicn ( 8 )

Cartilage Bone

+ 1,acks + Lacks + Lacks + ~

+ + + " + + " + + ( I

+ + + + +

~

+ ? + ( t o )

Dentine

Lacks Lacks Lacks

+ , Tissue shoxvn t~ he of neurai-crest origin I,acks, Tissue not present in the group.

, No data available for this tissue. ' I Evidence available for some bones only. I , 1,angille & Hall, (1988a) , 2, Newth (1951, 1956); 3 , Sadagiani & Thiebaud ( 1 9 8 7 ) ; 4, Cusimano-Carollo

(1963, 1969, r g p ) , Cusimano et al. ( 1 9 6 2 ) ; 5 , Platt ( 1 8 9 3 , 1 8 9 7 ) ; Chibon ( 1 9 6 6 , 1967, 1 9 7 0 ) ; Cassin & Capuron [ 1 9 7 ( ) ) ; 6 , Hall & Horstadius ( 1 9 8 8 , for ref.); 7, Langille & Hall ( 1 9 8 8 6 ) ; 8 , Hall ( 1 9 8 3 a , 6 ; 1987a, for refs); 9, T a n 8: 3lorriss-Kay (1986) ; Xlorriss-Kay & Tan ( 1 9 8 7 ) ; 10, Lumsden ( 1 9 8 7 , 1988 , for refs); 11, Toerien ( 1 9 6 5 a , 6); 12; de Beer ( 1 9 4 7 ) .

been regarded as, and is, a derivative of ectodermal and sometimes endodermal ameloblasts.

The notion that neural crest cells, i.e. cells that begin their developmental lives in the neural ectoderm, might be the source of the cranial skeletal tissues was raised almost a century ago by Platt (1893, 1897) in studies on urodele amphibians, the neural crest itself having been discovered in 1868 by Wilhelm His. As elegantly summarized by Horstadius (1959) and more recently by Hall & Horstadius (1988), it took a long time for the novel notion of an ectodermal origin of skeletal tissues to sink into the collective consciousness of vertebrate biologists. Although now well accepted (see below) it must be acknowledged that even today, the experimental evidence for the neural-crest origin of cranial skeletal and dental tissues is limited to studies on a small number of species ; for man?; taxa the evidence is restricted to studies of a single species. We summarize the extent of our current knowledge in Table 2.

As will be apparent from Table 2, the database is not diverse and our knowledge of some tissues and taxa far exceeds that of others. T o highlight just several areas, we have no experimental evidence for neural-crest origin of the bony skeleton of the adult (frog) of any anuran amphibian ; our knowledge for urodeles is limited to data on the palatine and splenial bones, albeit in several species (Chibon, 1970; de Beer, 1947), and our direct experimental evidence for mammals is confined to the mouse and is based on transplantation and tissue recombination studies rather than on direct cell mapping (Lumsden, 1987) although the latter is under investigation (Morris-Kay & Tan, 1986;


Fig. 19(A-D). These diagrams show the constancy of regionalization of the skeletogenic neural crest in (A) Petromyzon marinus (a lamprey); (B) Oryzias latipes (the Japanese medaka, a teleost); ( C ) Gallus domesticus (a bird); and (D) Ambystoma mexicanum and Pleurodeles waltl (two urodele amphibians). T h e chondrocranial neural crest is both shown in black and demarcated by the arrowed bars, the viscerocranial neural crest is stippled and demarcated by arrowed bars. T h e skeletogenic cranial neural crest extends from the anterior mesencephalon (mid-prosencephalon in P. marinus) cauded to the level of somites 4 or 5 (S4, 545). Roman numerals, I -VII in (A), I-V in (B) refer to boundaries of regions excised by Langille & Hall (1988a, b ) to generate the fate maps. The angles from the mid-line in (D) represent sectors of neural crest excised from P. waltl by Chibon (1966, 1967) projected onto the fate map for A. mexicanum. Abbreviations : AR, anterior rhombencephalon ; M, mesencephalon ; M R , mid rhombencephalon ; P, prosencephalon ; PR, posterior rhombencephalon ; T, trunk neural-crest cells (Hall and Horstadius, 1988, fig. I ) . (Reproduced by permission of the author and publishers, Oxford University Press).

Smits van Prooije et a l . , 1987, 1988). On the other hand, the extensive use of cell markers, notably the quail-chick chimaera, has enabled every cartilage and bone in the neuro- and viscerocranial skeleton of the embryonic chick to be traced to a neural crest or mesodermal origin (Le Lievre, 1971 a, 6 , 1974, 1978; Noden, 1978).The Xenopus borealis - Xenopus laevis chimaeric system allowed Sadaghiani & Thiebaud ( I 987) to map the neuro- and viscerocranial skeletons of X . Zaevis. [3H]thymidine labelling has enabled the cartilaginous skeleton of the salamander, Pleurodeles waltl , to be mapped in similar detail (Chibon, 1966). For other groups where such markers are

328 MOYA M. SMITH AND €3. K. HALL

unavailable, notably fish and cyclostomes, the maps that have been generated are based on extirpation studies alone (Langille & Hall, 1988a, b). That the pattern of regionalization of the neural crest revealed by the latter technique produces maps of the neural crest that are coordinated with those produced by labelling studies in other groups gives us confidence in these methods (at least until labelling methods become available) and demonstrates the constancy of the regionalized skeletogenic neural crest across the vertebrates (Fig. 19). Very recently, Sadaghiani & Vielkind (1989) have shown that the H N K - I antibody (which is positive for mammalian and avian neural crest cells) can be used to visualize and map fish neural crest. They have used H N K - I staining to visualize the early stages of neural crest cell accumulation above the neural tube in two teleosts, Xiphophorus maculatus (the platyfish) and X . helleri (the swordtail). Unfortunately, we have too few data to produce an equivalent neural-crest map for the odontogenic neural crest.

It is not our intention to review in detail the evidence for the neural-crest origin of the cranial skeletogenic and odontogenic tissues - the papers in Maderson ( I 987) and the group-by-group discussion in Hall & Horstadius (1988) amply review that literature. Fig. I 9 summarizes the rostro-caudal extent of the skeletogenic cranial neural crest in cyclostomes, teleosts, amphibians and birds, and dramatically demonstrates how conserved the skeletogenic cranial neural crest is (has been) throughout the vertebrates. We comment in section IV on the question of a skeletogenic or odontogenic trunk neural crest. Here we merely highlight and summarize the evidence for each of the tissues.

(2) Dentine Dentine has been demonstrated to be derived from neural crest in urodele amphibians

on the basis of extirpation and/or transplantation of labelled neural crests (Platt, 1893, 1897; Chibon, 1966, 1967, 1970; Cassin & Capuron, 1979).

Dentine has been demonstrated to be derived from neural crest in the mouse embryo on the basis of tissue recombinations between cranial neural crest and mandibular epithelium (Lumsden, I 985, I 987). Such grafted tissue recombinations develop teeth with odontoblasts that produce the dentine differentiating from neural crest and the ameloblasts that produce the enamel differentiating from ectoderm. They also develop dental pulp, periodontium and alveolar bone of attachment. This association of dentine and bone-forming cells both differentiating not only from the neural crest, but from the same population of cells (although not necessarily the same cells) will be important when we consider the developmental origin of the bone of attachment associated with the dentinous exoskeleton in Ordovician agnatha.

We have no direct information on the embryological origin of the dentine and odontoblasts of the other toothed vertebrates, fish and reptiles. There is only indirect evidence for fishes, from the development of teeth (and dermal bone, scales and pigment cells) from clonal melanophore tumour cell lines (Matsumoto et al . , 1983; pigment cells are known to be of neural crest origin in all vertebrates, Hall & Horstadius, 1988). The observation of Ferguson (1981) that teeth do not form when migration of neural crest cells is blocked with the drug FuDR, in Alligator mississippienses, cannot be used as evidence of neural crest involvement in reptiles,


because it completely prevents formation of the whole lower jaw, as well as teeth. A discussion of the origin of dentine in trunk scales and denticles may be found in section IV.

(3) Bone As summarized in Table 2, evidence for neural crest origin of cranial bone in

amphibians is limited to observations on just several bones following deletion and/or transplantation studies (Hall, 1 9 8 7 ~ ; Hall & Horstadius, 1988; Lumsden, 1987 for refs). For fishes, evidence is restricted to the formation of dermal bone in the melanophore tumour cell lines described above. For birds, we have the detailed analyses of Le Litvre and Noden (Table 2) based on grafting cranial neural crest of Japanese quail embryos into embryonic chicks (quail-chick chimaeras), in which every bone in the head has been mapped. All of the viscerocranial and facial bones are neural crest-derived, but some cranial bones (caudal portion of the frontal, parietal, supraoccipital, exoccipital, postorbital, orbitosphenoid, basibranchial and columella) are mesodermal. The boundary between these two is very precise (Noden, 1987, fig. 9), and has been extrapolated to other vertebrates, including humans (Noden, 1987, fig. 15).

For mammals (i.e. mouse) evidence comes from the tissue recombination studies of Lumsden for a neural-crest origin of murine odontoblasts and dentine. When cranial neural crest was isolated from murine embryos and recombined with mandibular ectoderm, bone (and cartilage) developed in association with the dentine that formed. Here it is not specific bones that have been identified, but rather the bone of attachment of the teeth and the adjacent bone, which we can take to be dentary or maxilla. These experimental studies follow the studies of Ten Cate & Mills ( I 972) and Ten Cate ( I 975) which also supported a neural crest origin for mammalian bone of attachment, based on its development from murine tooth germs (dental organs, dental papillae, dental follicles) grafted either subcutaneously or into holes in the parietal bone. Osborn (1984) and Osborn & Price (1988) have also provided evidence using tritiated thymidine for the origin of alveolar bone from cells of the dental papilla.

A number of vertebrate taxa, including gymnophionans and anurans (amphibians), lizards and teleost fishes, possess osteoderms or scutelets in the dermis (Moss, 1969; Zylberberg et al., 1980; Ruibal & Shoemaker, 1984; Zylberberg & Castanet, 1985; Levrat-Calviac & Zylberberg, 1986; Whitear & Mittal, 1986). Osteoderms may consist of several layers of hypomineralized material (bone) in a collagenous matrix. They are regarded as ‘metaplastic ossifications ’ (Zylberberg & Castanet, 1985), i.e. as new structures, arising independently from the dermal exoskeleton in both development and evolution. Moss ( I 964, I 969) compared the uppermost glycosaminoglycan-rich layer to enamel and ganoin, although there is no association of osteoderms with a dermal papilla. Schaeffer (1977) also discusses this issue. The source of the cells which produce the osteoderms, whether neural-crest or mesodermally derived, is unknown. Reif (1982, p. 318) comments that, among mammals, only the Loricata [Cingulata; S. American edentates (armadillos)] had a well-developed postcranial dermal skeleton ; that bony plates are also found in the dermis of whales, and, citing Peyer (1931) that in both cases, the dermal elements evolved de novo and are not remnants of a dermal skeleton.


(4) Cartilage

On the other hand, except for reptilian and mammalian skeletons, our knowledge of the contribution of the neural crest to cranial and visceral cartilages is very extensive, representatives of most vertebrate taxa having been mapped ; Oryzias latipes (teleost, Langille & Hall, 1988 6) ; Petromyzon marinus (cyclostome, Langille & Hall, 1988 a) , Xenopus laevis (anuran amphibian, Sadaghiani & Thiebaud, I 987), several urodele amphibians (Table 2), and Gallus domesticus (bird, see Table 2). T h e boundary between neural crest and mesoderm shown by Noden for craniofacial bones also applies to craniofacial cartilages. A rostro-caudal regionalization of the neural crest is evident in all vertebrates studies (Fig. IS), with more rostral cartilages coming from rostral (mesencephalic) neural crest and more caudal cartilages of branchial arches from caudal (rhombencephalic) neural crest. Within the branchial arches, a subregional- ization along the rostro-caudal axis has been identified in urodeles (Horstadius, I 95o), the medaka and the lamprey (Langille & Hall, 1988a, 6). T h e entire branchial arch skeletons of both the medaka and the lamprey are of neural-crest origin (op . c i t . ) , an important point when we come to consider the earliest vertebrates, for origin from a similarly regionalized neural crest in a jawed and a jawless fish, argues for this being a primitive vertebrate condition. Furthermore, that trabecular and branchial arch cartilages are derived from a similarly regionalized neural crest in lamprey and teleost, supports a homology between the agnathan and gnathostome visceral skeletons, and increases our confidence in extrapolating such studies back to the earliest agnathan vertebrates, a topic which we take up in section \'I.

In addition to the neuro- and viscerocranial cartilages of extant vertebrates, fishes, birds and mammals possess cartilage within the cranium of the dermal skeleton, and on the clavicle. This is the so-called secondary or adventitious cartilage (Murray, 1963 ; Murray & Smiles, 1965; Hall, 1970, 1978, 1 9 8 6 ~ ; Tran & Hall, 1989; Vinkka, 1982; Benjamin, 1989~2, 6). Although one of us (B. K. H.) had previously argued that secondary cartilage was limited to birds and mammals and that the evidence for secondary cartilage in fish was equivocal, the recent developmental study by Benjamin ( I 989 a) clearly documents the development of secondary cartilage on the dentary, maxillae and cleithrum of the black molly Poecilia sphenops (Fig. 20) . Benjamin (1989b) also cites Norman (1926) and Nigrelli & Gordon (1946) as providing evidence for secondary cartilage in the eel, Anguilla aolgaris and in the jewelfish Hemichromis bimaculatus and believes that much of the hyaline cell cartilage on cranial dermal bones in many teleosts (see Benjamin, 1988) will turn out to be secondary cartilage once developmental series have been studied.

Huysseune and her colleagues have also reported the presence of calcified chondroid bone at the articulation between the upper pharyngeal jaws and the parasphenoid and basioccipital at the base of the neurocranium in an African mouth-breeding cichlid, Astatotilapia (Haplochromis) elegans (Huysseune et al., I 98 I , 1986 ; Huysseune, 1986 ; IIuysseune & Verraes, 1986). Although she considers, especially in the adult, that this tissue resembles hypertrophied secondary cartilage, Huysseune regards it as chondroid bone and not as secondary cartilage because ( a ) it forms in what would otherwise be acellular bone ( A . elegans has a skeleton of acellular bone) by the retention of osteoblasts and not from chondroblasts ; ( b ) the histochemistry of the extracellular matrix is typical

Skeletal development and evolution 33 1

Fig. zo(A-B). Histology of secondary cartilage development in the periosteum of membrane bone of the dentary (d) in (A) 3-day and (B) 3g-week-old specimens of Poecilin sphenops (the black molly, teleost). The meniscus (m) associated with the maxillo-dentary articulation has begun to separate in (A) and has hyaline cartilage in (B). A periosteal pad of secondary cartilage (+) is also present on the lateral margin of the dentary in (B). m.k, Meckel’s cartilage (A, B) x 300, Masson’s trichrome (Benjamin, 1989, figs 8, 9). (Reproduced by permission of the author and publishers, Cambridge University Press).

of bone rather than of cartilage; ( c ) it does not contain type I1 (cartilage-type) collagen, and (d) it forms on both membrane (parasphenoid) and endochondral (basioccipital, infrapharyngobranchials I I I-IV) bones. Such a tissue illustrates the detailed developmental knowledge required to distinguish secondary cartilage from cartilage or chondroid bone, emphasizes the existence in teleosts of what in ‘higher’ vertebrates would be regarded as intermediate or metaplastic tissues (Hall, 1975), the re- sponsiveness of teleost skeletogenesis to functional demand (chondroid bone allows rapid growth of the parasphenoid; Huysseune et al., 1986) and highlights the need for more detailed developmental studies on teleost skeletogenesis.

Secondary cartilage is always associated with intramembranous ossification (hence the term adventitious) and only appears after the initiation of osteogenesis (hence the term secondary) and only at sites such as sutures, joints, muscle insertions, ligament attachment, roots of teeth, or after intramembranous bone fracture, where movement evokes its formation. There is considerable evidence for secondary chondroblasts arising from the same cell population as produces the osteoblasts and being switched into chondroblastic differentiation in response to a stimulus associated with movement.

3 3 2 MOYA M. SMITH AND B. K. HALL Evidence for the latter comes from the locations of the cartilage at mobile sites, its failure to form in paralysed embryos (Murray & Smiles, 1965)~ evocation from isolated bone exposed to movement in vitro (Hall, 1968), and in mammalian mandibular condylar cartilage (Hall, 1978 ; Silbermann et al., 1987). The presence of cartilage in the alveolar bone of attachment underlying the teeth in murine embryos (Hall, 1971) is presumed to result from stimuli associated with tooth movement. Given the evidence from Lumsden ( I 987) of the ability of mouse cranial neural crest to form dentine, and alveolar bone of attachment, the cartilage found in alveolar bone of the mouse is most parsimoniously interpreted as neural-crest derived.

Secondary cartilages often have features interpretable as tissues intermediate between cartilage and bone, much like chondroid tissue or chondroid bone [although chondroid bone can be distinguished from secondary cartilage ; see above and also Hall (1978, 199ob), Goret-Nicaise (1984), Goret-Nicaise & Dhem (1982, 1985, 1987)l. For example, osteonectin is found in the mandibular secondary cartilages of the rat (Copray et al., 1986, 1989) and secondary cartilages, unlike other cartilages, contain high levels of type I collagen (Silbermann et al., 1987).

All the secondary cartilages on embryonic birds and mammals, with the exception of the cartilage on the clavicle, are on cranial bones The use of quail-chick chimaeras has shown these cranial bones to be of neural crest origin in birds and Le Litvre (pers. comm.) has identified quail cells in the avian secondary cartilages. The origin of clavicular secondary cartilage, which is present in both birds and mammals (Hall, 1986; Tran & Hall, 1989) is not yet known. The clavicle, regarded as a derivative of somatopleural mesoderm (Chevallier, 1977 ; Chevallier et al., 1977) is the only postcranial dermal bone in the avian and mammalian skeleton. It is not inconceivable that cranial neural crest could contribute to the clavicle for caudal cranial neural crest (somite level 1-4) extends as far caudad as to contribute to the heart in avian embryos (Kirby et al., 1985). Noden (1983) has shown extensive contribution to the cervical region from cranial crest, i.e. trunk-level ectomesenchyme can arise from cranial neural crest. Studies with quail-chick chimaeras could resolve this question for chick embryos. In fact such a study has just been published. Sumida e t al. (1989) replaced chick neural crest at the level of the mylencephalon to somite 4 (i.e, the most caudal cranial neural crest) with an equivalent region of quail neural crest, and demonstrated that cartilage and adjacent connective tissue of the heart (between the aorta and pulmonary trunk) was of quail i.e. cranial neural-crest origin. Therefore, cranial neural-crest cells extend at least as far caudad as the level of the heart. Alternatively, if the traditional mesodermal origin of clavicular bone (and their secondary cartilage) obtains, then secondary cartilages in avian and mammalian embryos have two embryological origins - cranial neural crest and mesoderm. In either case, secondary cartilage is restricted to the dermal skeleton in extant vertebrates.

IV T H E NEURAL-CREST ORIGIN OF TRUNK SKELETOGENIC AND ODONTOGENIC TISSUES IN EXTANT VERTEBRATES

As outlined in section 111, cranial skeletogenic and odontogenic tissues have a neural crest origin. Because teeth are confined to the cranial region of extant amphibians, reptiles and mammals (although odontogenic tissues are found in trunk scales in fish) and because postcranial skeletal tissues develop from mesoderm and not from neural crest, developmental biologists have drawn an absolute dichotomy between a cranial


skeletogenic/odontogenic and a trunk non-skeletogenic/non-odontogenic neural crest. This affirmation rests upon the neural-crest mapping studies reported in section 111 which only demonstrate that the cranial neural crest is skeletogenic, and not that the trunk neural crest is not ; also on experiments in which trunk neural crest cells grafted into the head fail to form cartilage, bone, or teeth (Chibon, 1966; Horstadius & Sellman, 1946; Sellman, 1946). A criticism of the latter studies is that the trunk neural crest might not have been associated with the appropriate environment (epithelium, see section V) in the head and therefore a skeletogenic fate is not expressed, although the neural crest when confronted at the appropriate time would be quite capable of expression of skeletal tissues. This is illustrated by the grafting studies of Nakamura & Le Litvre (1982) in which avian trunk neural crest formed connective tissue and muscle, although not cartilage or bone, when grafted in place of cranial neural crest and allowed to mix with cranial neural-crest-derived ectomesenchyme. The two unusual findings in this study are the formation of ectomesenchymal derivatives from trunk neural crest when trunk does not form ectomesenchyme (or ectomesenchymal derivatives) in situ, and the apparent requirement both for interaction with cranial neural-crest cells and exposure to some aspect of the cranial environment for the ectomesenchymal fate to be expressed.

Although a trunk neural-crest contribution to the skeleton in avian embryos is not established (the report of Nakamura & Le Likvre, 1982 being the one exception), there are several reports of trunk neural-crest contributions to ectomesenchyme in several species of amphibians, namely dermal connective tissue of the trunk of Ambystoma (Raven, 1931, 1936; Du Shane, 1935; Holtfreter, 1935; Detwiler, 1937) and mesenchyme, including mesenchyme of the median fin-fold in Pleurodeles waltl (Chibon, 1966).

Recent convincing evidence for an odontogenic and osteogenic capability of trunk neural crest of the mouse and the need for a particular (cranial) ectodermal environment to elicit that differentiation, comes from the tissue recombination and transplantation studies, summarized in Lumsden ( I 987). His studies ( I 987, I 989) documenting the formation of cartilage, bone and dentine from mouse cranial neural crest have already been introduced.

Lumsden recombined trunk neural crest from the level of the open posterior neuropore at the level of future cervical somites (Sg-10) of mouse embryos at 8 days of gestation with mandibular ectoderm (a normal site of tooth formation) from 10-day-old embryos in a tissue recombination and grafted these tissues to the anterior chamber of the eye. Five of forty grafts formed teeth with alveolar bone (Fig. 21). None of five grafts of trunk neural crest with forelimb bud ectoderm formed bone or dental tissues, allowing Lumsden to conclude that trunk neural crest (at least caudad to Sg and possibly to SIO) could form dentine and alveolar bone in response to mandibular ectoderm, elicit ameloblast differentiation and enamel synthesis from the mandibular ectoderm and collaborate with mandibular ectoderm in forming a perfectly organized tooth (Fig. 21). Lumsden (1987, p. 287) does caution on the occasional association of mesenchyme with the control isolated mandibular ectoderm (but it is clear from his fig. 7a, 1987 that this is connected to tissues of the eye, i.e. is host derived) and that unsegmented paraxial mesoderm could have been included with the trunk neural crest, but thinks the latter unlikely.

We do have to reconcile the formation of dentine in denticles and scales along the

334 MOYA M. SMITH ,4ND B. K. HALL

Fig. ZI (A , B). Histology of explants in the anterior chamber of the eye in Mus musculus. Cervical trunk neural crest recombined with odontogenic mandibular epithelium form complete tooth germs (enamel, dentine and cusp morphology) together with alveolar bone (arrow in B), but no cartilage. (A) x 75 ; (B) x55. Lisson’s alcian blue and chlorantine fast red (Lumsden, 1987, fig. 8D-F, and 1988, fig. 7B).

(Reproduced by permission of the author and publishers, Company of Biologists, Cambridge.)

body of a number of extant vertebrates with a cranial neural-crest origin of dentine (see section 111). The dermal denticles in sharks, body scales of Polypterus and Latimeria and dentine in the fin rays of Polypterus fall into this category (Bhatti, 1938; Smith et al., 1972; Schaeffer, 1977; Smith, 1979; Meinke, 1982a, 6). As dentine of toothed vertebrates is always neural-crest derived, we fell justified in assuming that the dentine of trunk scales is also neural-crest derived, as Lumsden (1987, p. 291) also concluded and therefore that trunk neural crest in these fishes has the capacity to produce odontogenic and skeletogenic tissues. The association of bone with such trunk scales, as for example, in the trunk scales of foetal and adult coelacanths (Smith, 1979) is

Skeletal development and evolution 335 consistent with this notion of a neural-crest origin, given that dentine and bone of attachment are derived from the same population of neural crest cells in the cranial region. However, we cannot rule out the possibility that some rostra1 trunk denticles arise from the most caudal cranial neural crest migrating into the trunk (see the section on the origin of cartilage, above, for a discussion of such migrations).

V. REGULATION OF THE DEVELOPMENT OF SKELETOGENIC AND ODONTOGENIC TISSUES IN EXTANT VERTEBRATES

In this section, we discuss the fundamental mechanisms that regulate the initiation and histogenesis of the skeletogenic and odontogenic tissues. For recent reviews see Kollar (1983), Lumsden (1987), Hall (1983a, 6 , 1984a, 1986b, 1987~2, b, 1988a), Ruch ( 1984).

( I ) Developmental principles

The following are eleven general principles that apply to the four skeletal and odontogenic tissues under consideration :

(i) Those tissues that arise from the neural crest develop from ectomesenchymal cells that do not arise in situ but have migrated to their final positions where they then differentiate, although migration is not a prerequisite for differentiation.

(ii) There must therefore be one or several mechanisms, enabling neural-crest- derived cells to know when to stop migrating and where to settle down in the embryo.

(iii) Once at their final site and before cytodifferentiation can commence, ectomesenchymal cells that will form a particular skeletal or dental element (e.g. an individual cartilage, bone or tooth) aggregate together into a condensation (papilla, primordium, blastema). This is the phase of skeletogenesis termed the ‘membranous skeleton’ by Gruneberg (1963; see also Johnson, 1986).

(iv) The skeletogenic and odontogenic tissues do not self differentiate, but only differentiate following interactions with other developing tissues.

(v) The tissue interactions that regulate the initiation of these skeletogenic and odontogenic tissues are epithelial-mesenchymal (ectomesenchymal) interactions (Deuchar, 1975; Sawyer & Fallon, 1983).

(vi) Epithelial-mesenchymal interactions may regulate cessation of migration [point (ii)], proliferation of the ectomesenchyme [point (iii)] as well as the initiation of differentiation of all four tissues [point (v)] (Hall, 1988a; 199oa,c-e; Hall & Horstadius, 1988; Coffin-Collins & Hall, 1989; Hall & Coffin-Collins, 1990).

(vii) There may also be more than one epithelial-mesenchymal interaction involved in the differentiation of a particular skeletogenic or odontogenic tissue in which case we speak of an epigenetic cascade, a term used by Thorogood ( I 983) to refer to interactions involved in morphogenesis of the skeleton (see Hall & Horstadius, 1988).

(viii) For the two dental tissues (dentine and enamel) the interactions take place at the interface of apposed epithelia (future enamel organ) and ectomesenchyme (future dental papilla) such that the epithelium differentiates into ameloblasts and the ectomesenchyme into odontoblasts.

(ix) For the two skeletogenic tissues (cartilage and bone), both in the cranial and in the trunk skeleton, the responding ectomesenchyme (mesenchyme) can, in some cases, be at some distance from the initiating epithelium (ectoderm, notochord, neural tube) that permits differentiation of chondroblasts or osteoblasts. In turn, in some cases, the

3 36 MOYA M. SMITH AND B. K. HALL ectomesenchyme/mesenchyme permits continued squamous differentiation of the epithelium and prevents keratinization.

(x) Epithelial-mesenchymal interactions involve transfer of information across the epithelial basal lamina by one of three mechanisms :

( a ) ectomesenchymal or mesenchymal cell processes contact the basal lamina where they receive the necessary signal ;

(b) the epithelium releases a diffusible signal to which ectomesenchymal or mesenchymal cells some distance away (300 pm) from the epithelium respond ;

( c ) ectomesenchymal or mesenchymal cell processes and/or epithelial cell processes cross the basal lamina to establish direct contacts with the epithelial and/or ectomesenchymal/mesenchymal cells.

(xi) The details of the molecular basis of epithelial-mesenchymal interactions are not known, although extracellular matrix products and/or growth factors are involved at least in some interactions (Hall, 19863, 1988a, 199ob, d ; Thorogood, 1990).

We have insufficient knowledge of principles (i)-(iii) to comment on whether the mechanisms are similar when comparing cranial skeletogenic and odontogenic tissues, within and among taxa, except to note that the broad processes are identical : migration of ectomesenchymal cells, accumulation of ectomesenchyme at specific sites, aggregation of ectomesenchyme into condensations (cartilage, bone) or dental papillae (dentine). Nothing in these early stages contradicts the precept that the early development of cranial skeletogenic and odontogenic tissues is based on equivalent regulatory processes. In fact, we can make the same statement when comparing cranial with trunk skeletogenic tissues, despite the fact that the trunk tissues (with the probable exception of the dermal exoskeleton tissues) are derived from mesodermal mesenchyme. We see the same accumulation of cells at specific sites and the same phenomenon of aggregation into condensations in axial and appendicular skeletal development as we see in cranial (op. c i t . ) . The migration phase is very abbreviated in limb development (Hinchliffe & Johnson, 1980 ; Hall, 1978), although in vertebral development, sclerotomal cells migrate extensively from somites to surround the spinal cord and notochord (Hall, 1977).

We do have a much stronger basis of comparative knowledge for principles (iv)-(x). In fact, Krejsa (1979) has provided a summary of the similarity in basic epigenetic regulatory mechanism between a whole class of dermal tissues and structures, including scales, dermal bone, teeth, denticles, hair, feathers and finger nails. Maderson (1975, 1983) also discussed this issue.

( 2 ) Epithelial-mesenchymal interactions

As when discussing evidence for the neural crest origin of the skeletogenic and odontogenic tissues (section 111), we also provide a table summarizing the epithelial- mesenchymal interactions known to regulate skeletogenic and odontogenic differentiation, whether in the cranial or the trunk skeleton, whether ecto- or endoskeletal and whether mesodermally or ectomesenchymally derived.

Table 3 details an extensive list of interactions, even though for many species/tissues the evidence is restricted to a single study on a single element/tissue in a single species within a vertebrate group; dentine in the mouse (Lumsden, 1987), frontal bone in the salmon (Devillers, 1947) and visceral arch cartilage in the lamprey (Damas, 1951 ;

Skeletal development and evolution 337 Table 3 . A summary of the epithelial-mesenchymal interactions involved in initiation of

differentiation of skeletogenic and odontogenic tissuea Tissue Embryonic source

Dentine Cranial ectomesC

Epithelium

Branchial ecto & endoderm

Mandibular Mandibular

Branchial ecto & endoderm

Mandibular Mandibular

Group

Urodele

Mouse Mouse

Urodele

Mouse Mouse

amphibians

amphibian

Referenceb

I , 2 za 3 3

1. 2

Cranial ectomes Trunk ectomesd

Cranial ectomes Enamel

Cranial ectomes Trunk ectomesen

Dermal bones Frontal ?e

3 3

Lateral line & neuromasts

Oral Neural, mandibular, maxillary, notochord scleral

Neural, notochord

Salmon 4f

Various ?e

Various cranial ectomes Frog Chick

5 6

Parietal frontal squamosal

Dentary antlers

Cartilages Visceral arch

Visceral arch

Sensory capsule

Otic capsule

Scleral Limb Vertebral

Ectopic

Cranial meso Chick 6, 7

Cranial ectomes ?

Mandibular Antler velvet

Mouse Deer

3 , 8 9

Branchial

Pharyngeal endo ecto & endoderm

Cranial ectomes Lamprey

Cranial ectornes Amphibians

? Lateral line Shark

Meckel’s cranial

Meckel’s cranial

Cranial meso

ectomes

ectomes

Cranial Chick 6

Mandibular Mouse 3

Otic vesicle Chick, turtle

Chick All All

amphibian, mouse Cranial ectomes Lateral plate meso Sclerotomal meso

Pigmented retinal Limb bud Notochord & ventral spinal cord

Transitional of mammal urinary bladder

Epithelial cell lines

I 5 16 I7

Mesenchyme” 16, 18

a Those skeletogenic tissues that arise after epigenetic interactions that are not epithelial-mesenchymal interactions have been excluded, notably endochondral and perichondral bone (probably initiated by interaction with hypertrophic chondrocytes and requiring vascular invasion ; Scott-Savage & Hall, 1980) and secondary cartilage (initiated in response to biomechanical factors such as embryonic movement, mobility of fractures, muscle or ligament insertions; Hall, 1978).

References are keyed by number to those listed below. Only key reviews are cited unless the data is based on only one or two papers. I, Sellman (1947); 2, Chibon (1966, 1970); 3, Lumsden (1987); 4, De Villers (1947, 1965); 5, Cusimano-Carollo (1963, 1969, 1972); Cusimano et al. (1962); Hall & Horstadius (1988); 6, Hall (1987a); 7, Benoit & Schowing (1970); 8, Hall (1988); 9, Goss (1983); 10, Damas (1951); Newth (1956); 11, Horstadius (1950); 12, Patterson(1977); 13, Hall(1983a, b); 14,Toerien(1965b); Mila’re(1974); 15, Newsome(197z), Smith

Footnotes to Table 3 continued overleaf

3 3 8 MOYA M. SMITH AND B. K. HALL

Newth, 1956). Some groups such as hagfishes, reptiles (except for the snapping turtle, Chelydra serpentina), mammals (except for the mouse), teleosts (except for the salmon) and sharks (although even here the evidence is minimal; see footnote g on Table 3 ) are missing from the table altogether. In fact, for the epithelial-mesenchymal interactions involved in the initiation of differentiation of cartilage and bone we only have detailed information for the chick and to some extent for amphibians, and for equivalent interactions for dentine and enamel, only for the mouse. Although these interactions in the tooth germ are specific for an organ, the tooth, and involve at least two tissue types, dentine and enamel, the skeletogenic interactions involve differentiation of tissues (cartilage and bone) which only subsequently become organized into organs. For trunk skeletal tissues (cartilage of the appendicular and axial skeletons) we have rather more knowledge from rather more species (see Table 3) . Endochondral/perichondrial bone and secondary cartilage are excluded from this discussion, not because they are not epigenetically induced, but because the epigenetic factors are not epithelial- mesenchymal interactions (see footnote a in Table 3).

Because we do not yet fully (or even partially) understand the mechanism of a single interaction, it is very difficult to compare interactions, except, on what, once mechanisms are fully known, may turn out to be a rather superficial level. Several systems will illustrate this point.

( 3 ) Epithelial-mesenchymal interactions and the differentiation of dermal bone

The epithelial-mesenchymal interaction that initiates differentiation of dermal bones of the mandible in the embryonic chick is matrix mediated, i.e. the ectomesenchymal cells of the primordium of the lower jaw have to contact and interact with a component(s) in the basal lamina of the mandibular epithelium for osteoblasts to differentiate and for bone to be deposited (Hall, 1988 a) . T h e epithelial-mesenchymal interaction that initiates differentiation of the dermal bones of the sclera (the scleral ossicles) in the embryonic chick is diffusion-mediated ; ectomesenchymal cells some I jo p m away from the scleral epithelial basal membrane respond to an epithelial product [epidermal growth factor (EGF)] by differentiating as osteoblasts and by depositing bone (Pinto, 1989).

\$‘hat is the significance of these two different means of transmitting the epithelial signal to the ectomesenchyme ? Are the same epithelial factors involved in both

Footnotes tc) Table 3 continued from p 337.

& Thorogood ( 1 9 8 3 ) ; 16, Hinchliffe &Johnson (1980); Hall (1978); 17, Hall (1977, 1986); 18, Beresford (1981); Anderson (1976); LVlodarski (1989); Hall (1970). ‘ Ectomes, ectomesenchyme ; meso, mesoderm ; ect, ectoderm. ‘Experimental production of dentine or enamel in heterotypic tissue recomblnations. * Presumably a cranial ectomesenchymal derivative but no direct experimental evidence available (see section

111). ‘See .\Ioy-Thomas (1938, 1941) and Westoll (1941) for a contrary view, Pinganaud-Perrin (1973) for e~idence

o f dermal hone regeneration in the absence of lateral line involvement, and Hall & Hanken (1985) for a review of tlie unsettled problem of lateral line/neuromast- dermal bone interactions.

‘Patterson (1977) discusses the views of Holmgren, Jarvik, Allis and Orvig on whether there IS an inductive relationship between sensor); canals and mesenchyme ; hoivrver insufficient experimental evidence i s available to settle this question.

” Source unknunn.

Skeletal development and evolution 339 interactions, in the one case being trapped in the basal lamina, in the other diffusing through it ? If so, then epithelial specificity would reside in differing properties of the two basal laminae that allow them either to trap or to permit transfer of the same molecule(s). Alternatively, is the diffusible molecule produced by scleral epithelium quite different from the molecule(s) located in the mandibular basal lamina? (We know that it is a molecule(s) and not an ion(s) that passes from scleral epithelium to scleral mesenchyme because it fails to cross dialysis membranes of a pore size that allow ions to pass; Pinto and Hall, unpublished observations). We do know that mandibular mesenchyme can respond to scleral epithelium and vice versa when the two are recombined in direct contact with one another (Hall, 1981), that mandibular ectomesenchyme can respond to scleral epithelium in transfilter recombinations (presumably because the scleral epithelial signal diffuses through) but that scleral mesenchyme cannot respond to mandibular epithelium in similar transfilter recombinations (Pinto and Hall, unpublished observations).

The above results are consistent with scleral and mandibular epithelia producing equivalent bone-initiating factors that are differentially trapped or sieved by the basement membrane of the two epithelia. There are, in fact, numerous examples of an epithelium normally involved in one skeletogenic epithelial-mesenchymal interaction being able to elicit differentiation of tissue from another area of ectomesenchyme/ mesenchyme in the same embryo, or even from a different embryo, species, genus or even class of vertebrate (Hall, 19886, 1989). This topic is discussed further in the next section.

(4) Epigenetic cascades

We also have to consider the concept that development of the majority of skeletogenic and odontogenic tissues depends, not on a single epithelial-mesenchymal interaction, but on a sequence of interactions, in what Thorogood (1983) and Hall & Horstadius (1988) have referred to as a epigenetic cascades.

The most well-studied and understood epigenetic cascade involving odontogenic tissues is the series of epithelial-mesenchymal interactions leading to the differentiation of odontoblasts and ameloblasts and the deposition of dentine and enamel of the mammalian tooth (see Kollar, 1983 and Lumsden, 1987, for recent reviews). Initially, the oral epithelium stimulates adjacent ectomesenchyme to form a dental papilla, following which cells of the dental papilla stimulate adjacent epithelial cells to form an enamel organ, i.e. the initial signal is from epithelium to neural-crest-derived mesenchyme to trigger initiation of odontogenesis and dentine formation. Cells of the enamel organ in turn interact with the dental papilla and vice versa to initiate differentiation of preodontoblasts and preameloblasts respectively, which interact in turn to promote the transformation of preodontoblasts and preameloblasts to odontoblasts and ameloblasts. These differentiated cells then interact to initiate the deposition of predentine and dentine followed by enamel. Clearly, this is an incredibly integrated and coordinated cascade of interactions, initially between epithelium and ectomesenchyme and then progressively between their differentiated products.

Fagone (1959,1960) and Cusimano-Carollo (1962,1963,1967,1969,1972; Cusimano et al., 1962), on the basis of tissue recombinations between neural crest, epithelia and adjacent tissues, have demonstrated that differentiation of cartilage is a necessary

340 MOYA M. SMITH AND B. K. HALL prerequisite to the differentiation of the horny teeth in the anuran amphibian Discoglossus pictus. Pharyngeal endoderm initiates chondrogenesis from neural crest cells. The differentiating cartilages then interact with ectoderm to initiate differentiation of the horny structures of the anuran teeth and beak and the oral papillae.

An essentially similar cascade has been described for the salamander Pleurodeles waltl in which true teeth containing dentine and enamel rather than horny (keratinous) teeth develop (Cassin, 1975; Cassin & Capuron, 1972, 1977, 1979). Neural crest, prechordal and lateral plate mesoderms, stomodeal endoderm and ectoderm, must all interact in the appropriate sequence (and presumably for the appropriate duration) for teeth, cartilage and bone to differentiate. Thus, teeth only form after bone has differentiated, bone only differentiates after cartilage has formed, and cartilage only forms after initial interaction with pharyngeal and stomodeal endoderm (see fig. 3 in Hall & Horstadius ( I 988) for a summary of this epigenetic cascade]. Our presumption is that many more such cascades will be discovered once sufficient is known about other skeletogenic and odontogenic interactions. Timing and sequence are important in any mechanisms that involve a series of steps or sequential processes. In section VI we consider epigenetic cascades and the effect of heterochrony (shifts in timing in developmental steps/ processes that produce equivalent tissues/organs in descendent and ancestral taxa) as important mechanisms responsible for generating variability of skeletogenic and odontogenic tissues and organs.

( 5 ) Epithelial-mesenchymal interactions and inhibition of diflerentiation

A further caution, is that although we have discussed epithelial-mesenchymal interactions as if they always initiate differentiation, there are epithelial-mesenchymal interactions involving skeletogenic tissues that inhibit differentiation, notably, the inhibition of chondrogenesis by limb bud epithelium in the embryonic chick (Solursh, I 984) and the inhibition of Meckelian chondrogenesis by mandibular epithelium, also in the embryonic chick (Coffin-Collins & Hall, 1989; Hall & Coffin-Collins, 1990). It may seem anomalous that both these epithelia are listed in Table 3 as being responsible for initiation of chondrogenesis in limb and mandible, and indeed these epithelia play both an initiating and an inhibitory role at differing times during cartilage development. Thus, limb bud mesenchyme prior to H H stage 17 in the embryonic chick (the stage when the apical ectodermal ridge appears and the limb bud starts to grow) must interact with limb bud epithelium for cartilage to subsequently differentiate (Gumpel-Pinot, 1980, 1981) while from H H stage 23 onwards, limb bud epithelium inhibits chondrogenesis in its immediate vicinity, thereby ensuring localization of chondrogenesis to the centre of the developing limb (Solursh, 1984). In the developing mandible of the embryonic chick, differentiation of Meckel’s cartilage requires that an interaction occur between ectomesenchyme and cranial ectoderm early in neural-crest cell migration, differentiation of mandibular bone requires an interaction with mandibular epithelium that occurs after completion of neural-crest cell migration, but mandibular epithelium also inhibits cartilage differentiation (Coffin-Collins & Hall, 1989; Hall & Coffin-Collins, 1990).

One further mechanism for such inhibition lies in epithelial control over the proliferation of the mesenchymal condensations in which bones or cartilages subsequently develop. Reduction of a condensation below a critical minimal size can


lead to failure of initiation of chondrogenesis or osteogenesis with consequent lack of the individual cartilage or bone (Gruneberg, 1963). Such is the mechanism for loss of individual scleral ossicles from the avian eye when the corresponding epithelial scleral papilla fails to form (Hall, 1978, 1982; and see Gruneberg, 1963 and Johnson, 1986 for further examples).

This fragmentary knowledge, coupled with the equally fragmentary knowledge that neural-crest-derived ectomesenchyme itself can inhibit the differentiation of adjacent tissues (Jacobson, 1987; Jacobson & Sater, 1988)~ highlights just how much more we have to learn about regulation of the development of skeletogenic and odontogenic tissues. Nevertheless, given the current state of knowledge we can state that the regulation of skeletogenic and odontogenic tissues is based on equivalent epigenetic processes throughout the vertebrates, and that these processes apply equally to cranial and to trunk tissues, irrespective of whether the tissues are of mesodermal or of ectomesenchymal origin.

VI. MAINTENANCE OF DEVELOPMENTAL INTERACTIONS REGULATING SKELETOGENIC/ODONTOGENIC DIFFERENTIATION ACROSS VERTEBRATE TAXA

( I ) Introduction

Given the data presented in the previous section on the comparability of the control of skeletogenesis and odontogenesis within vertebrate groups - that epithelial- mesenchymal interactions regulate the initiation of skeletogenic and odontogenic differentiation in all vertebrate classes - how can we approach the question of how conserved these processes are across vertebrate groups? Some would argue that we cannot even discuss this problem until we fully understand the molecular and genetic control of epithelial-mesenchymal interactions. While we look forward to having that understanding, we do not believe that we should leave the problem entirely unconsidered until that information has been compiled. It is inevitable that we are constrained by our current understanding, but realistic to start from that basis.

That basis is the theoretical framework provided by Maderson (1975, 1983), Campbell & Richie (1983), Hall (1984)~ Alberch ( I 985) and Langille & Hall (1988 a, b, I 989) on the pivotal role of epithelial-mesenchymal interactions and of the necessity to examine, in a phylogenetic context, only those interactions or sequences that are causal, i.e. those where steps are not just sequential to, but dependent upon, a former step(s). Alberch (1985) cites the induction of the lens by the optic cup in Rana fusca as one example. This interaction is assumed to be used for lens induction in all vertebrate taxa, but in R. esculenta, a congeneric species, lens formation occurs without involvement of the optic cup. Jacobson & Sater (1988) re-examined this interaction and the requirement of an interaction between retina and ectoderm for lens formation. They found evidence for such an interaction in the mouse, 3 species of birds, 5 urodeles and 1 5 anuran species, but that no interaction occurred in 4 urodele and 8 anuran species. These examples clearly show how homologous structures can develop by non-homologous developmental sequences and indicate the depth of comparative knowledge required to unravel the developmental basis of phylogenetic morphological change.

We now consider whether we can find examples of interactions in which alterations in causal developmental sequences (epigenetic cascades) have produced skeletal or

3 42 MOYA M. SMITH AND B. K. HALL dental variation across taxa. I t will be clear from the outline in the previous section that there are potentially many steps in epigenetic cascades from mesenchyme of neural crest or mesoderm to the differentiation of a dental or skeletal tissue. Given so many steps, there must have been ample opportunity for modification of a cascade during vertebrate evolution. We do have a database from which to comment on whether altered cell migration [step (i)], cessation of migration [step (ii)] or condensation formation [step (iii), p. 591 are constant across vertebrate taxa, but the database comes, not from the developing cranial skeleton, but from the developing appendicular (limb skeleton). (Alberch, 1985; Alberch & Gale, 1983, 1985; Oster et a l . , 1983, 1988).

(2) Loss of skeletal elements

T h e model proposed by Oster et a l . (1983, 1988) applies to morphogenesis of the litnb and not explicitly to differentiation of limb skeletogenic tissues, although morphogenesis and differentiation are clearly related (Maclean & Hall, I 987).

Xlberch (1985) and Alberch & Gale (1983, 1985) have demonstrated that experimental alteration in the size of a limb bud by the in vivo application of colchicine, a mitotic inhibitor, not only produces smaller limb buds, but also produces a bud in ivhich the number of condensations for future skeletal elements is reduced. The resulting limbs have one less digit than do control limb buds, that is, smaller limb buds make fewer digits. This teratology would seem to have little relevance for evolutionary scenarios, except that the limb pattern produced after colchicine induced mitotic arrest involves the loss of the last digit specified during development (digit 1 in anurans, digit 4 in urodeles) and that the same patterns are found when amphibian digits are lost during evolution (Hanken, 1986; Alberch & Gale, 1985).

T h e order of digit loss during evolution is the reverse of the order in which digits form during development, and the evolutionary loss can be mimicked by reduction in limb bud size. How the reduced number of digits is specified is unclear, but what is clear is that we have in this system a model for how alterations in early prechondrogenic phases of skeletogenesis can affect skeletal pattern. That no similar examples of loss or modification of skeletogenic or odontogenic tissues are known may indicate that skeletogenesis and odontogenesis at the level of cyto- and histodifferentiation are processes so fundamental to vertebrate development that they had to be conserved with amazing fidelity across vertebrate taxa and throughout vertebrate history. In comparison with altering a skeletal or dental tissue, altering digit number may be trivial. T h e only comparison would be to find that reduction in mitosis resulted in a reduced number of teeth in the row or family ; or similarly in the number of ossification centres in membrane bones. T h e loss of third molars in mice has been investigated by Berry & Germain (1972) from the postulated basis that there was a threshold of size of the third molars, below, which they were absent, possibly due to the size of the tooth germ at a critical stage. They explored the idea that this was due to a reduction in mitosis or differentiation at the cap stage by a methotrexate-induced thymidine deficiency and prevention of D N A synthesis for a restricted period over days 3 , 4 and 5 after birth. They found that the frequency of missing third molars could be increased by this ‘environmental agent’ that inhibited DNA synthesis but concluded that the postulated threshold - small size of precursor (cap tooth germ) leading to tooth absence -\vas not an adequate explanation of this polygenic absence of a tooth. Many of the


animals showed a highly significant reduction in tooth size, with a change in shape and ‘peg’ teeth.

(3) Epithelial-mesenchymal interactions and heterochrony

What can we expect to find if we examine the developmental processes after the stage of mesenchymal or ectomesenchymal condensation ? We know from the previous section that all three ectomesenchymal tissues (cartilage, bone and dentine) arise because of epithelial-mesenchymal interactions that specify where, when and how much of the tissue will form (see previous section and Hall, 1983a, 1987). Given that these three components (where, when and how much) are readily modified by changes in the timing of development (within a species, within a lineage and/or between lineages) epithelial-mesenchymal interactions should be prime candidates as developmental regulators for changes brought about during evolution by heterochrony (Hall, 1975, 1984b, 199oe; Maderson, 1975, 1983).

An example from non-skeletal or dental tissues is the transformation of an internal to an external cheek pouch in the pocket gophers and kangaroo rats. The internal pouch of chipmunks opens into the mouth cavity and is lined with buccal epithelium, whereas the external pouch opens outside the mouth and is lined with fur. Brylski & Hall ( I 988 a, b) have proposed a scenario whereby altered timing of pouch primordium interaction and growth, allows a potentially internal pouch to remain attached to the corner of the mouth, be brought into contact with a dermal rather than an oral mesenchymal environment, switch for mucous secretion to the production of hairs and from an internal to an external pouch. Altered cytogenesis, histogenesis, and production of a new organ all follow from a simple alteration in timing of the developmental interactions between epithelium and mesenchyme.

Evidence for heterochronic shifts in the timing of epithelial-mesenchymal interactions leading to the differentiation of homologous skeletogenic (and odontogenic ?) elements in different vertebrate taxa is available. A prime example is initiation of differentiation of Meckel’s cartilage (homologous throughout the vertebrates) by different epithelia in representative amphibians, birds and mammals (see Table 3 ; Hall, 1 9 8 3 ~ ) . Thus, as shown in numerous species of anuran and urodele amphibians, Meckel’s cartilage is initiated following an interaction between migrating ectomesenchymal cells and pharyngeal endoderm (see Graveson & Armstrong, 1987, for the most recent experiments). In the embryonic chick, the equivalent epithelial- mesenchymal interaction (equivalent in that it is the prerequisite for initiation of chondrogenesis in Meckel’s cartilage) is with cranial ectoderm encountered by ectomesenchymal cells, either while in, or soon after they leave, the mesencephalic level of the neural tube (Tyler & Hall, 1977), i.e. the interaction occurs earlier than in the amphibians and involves a different epithelium and ectomesenchymal cells at a different stage in their migratory history, although from the same rostro-caudal level of the neural crest. In the embryonic mouse, the equivalent interaction involves neural-crest- derived ectomesenchymal cells from the same mesencephalic level as in amphibians and the embryonic chick, but the interaction is with mandibular epithelium encountered after the ectomesenchymal cells have reached the future lower jaw (Hall, 1980a), i.e. the interaction occurs later than in the amphibians.

One of us (Hall, 1983 c, 199oe) has previously argued that the amphibian interaction

344 MOYA M. SMITH AND B. K. HALL represents the more primitive condition for this particular epithelial-mesenchymal interaction. The two reasons for this assertion are:

( a ) T h e timing of the interaction is the same in both urodele and anuran amphibians, which despite being united as amphibians, may not share a common amphibian ancestor (Hanken, 1986; Duellman & Trueb, 1986).

(6) In cyclostomes, the visceral arch cartilages, known to be neural-crest derived (Langille & Hall, 1988 a ) , and developmentally homologous to Meckel’s cartilage, also differentiate after an interaction involving pharyngeal endoderm (Table 3).

T h e question of the homology between agnathan and gnathostome skeletal elements and/or branchial arches (jaws) has been much debated (Schaeffer & Thomson, 1980; Rieppel, 1988; Langille & Hall, 1989; Janvier, 1989). As articulated by Rieppel (1988):

The claim that the mandibular branchial arch transformed into jaws during the phylogeny of the Vertebrata, presupposes the inclusion of the Agnatha and Gnathostomata within a common taxon of higher rank, and it implies the homology of the embryonic rudiments of gnathostome jaws with the mandibular arch of adult agnathans (p. 141).

Langille & Hall (1988a, 1989) have shown that the latter both develop from a similarly regionalized cranial neural crest, establishing the homology of the skeletogenic primordia.

Parsimony leads us to conclude that because an interaction involving pharyngeal endoderm and migrating neural-crest cells occurs in these three disparate groups, especially given the early divergence of the cyclostomes from the gnathostomes (Schaeffer & Thomson, 1980; Maisey, 1986, 1988), it is a shared primitive character and is most likely to be the primitive condition for craniates. On this basis, the interaction has moved earlier in development in birds (or at least the one bird studied, the chick) and later in development in the mammals (or at least in the one mammal studied, the mouse).

(4) Transformations of odontogenic tissues

Now we turn to examples from odontogenic tissues that are amenable to interpretation, or that have been interpreted as, being based on shifts in timing during development.

One is the autoradiographic study by Shellis & Miles (1974) of the formation of dentine and enameloid in the teeth of two teleost fishes, Anguilla (the eel) and Labrus (the wrasse). They focused on enameloid because of the long-standing controversy over whether enameloid is ectodermally derived from cells of the inner dental epithelium, ectomesenchymally derived from activity of odontoblasts, or whether both epithelial and ectomesenchymal cells contribute to its synthesis and deposition. Shellis and Miles demonstrated with [3H]proline autoradiography that enameloid matrix was deposited by ectomesenchymal odontoblasts which leave cell processes within the enameloid, as they, the odontoblasts, withdraw from the surface of the enameloid. They further demonstrated that the epithelial cells of the inner dental epithelium deposit a protein into the enameloid matrix, i.e. that enameloid consists of an ectomesenchymally derived matrix into which an epithelially derived protein is deposited (see also Shellis, 1975, and Shellis & Miles, 1976). Previously Smith & Miles (1969) had shown that epithelial proteins were secreted into the cap region of larval urodele teeth, at the same time as

Skeletal development and evolution 345 dentine proteins, resulting in a composite tissue, but they offered no explanation for the different sequence in the adult urodele teeth, resulting in enamel on the surface of dentine.

If enameloid is a composite epithelial-ectomesenchymal tissue then variation in the timing of secretory activity of either cell type could disrupt enameloid deposition. Such a disruption could lead to failure of enameloid to form or, as suggested by Poole (1971) and Shellis & Miles ( I 9-74), if differentiation of the epithelial cells was delayed, dentine would begin to form before the epithelial cells could begin to synthesize the protein that they normally deposit into the enameloid matrix. Shellis & Miles (1974; see also Schaeffer, 1977) noted that the consequences of such dissociation of timing of synthetic activity of the epithelial and ectomesenchymal cells would be that the late-appearing epithelial protein would be deposited into enamel, a new exclusively epithelial-derived dental tissue, instead of enameloid. Shellis and Miles interpreted the developmental switch from enameloid to enamel between larval and adult urodele amphibians documented by Smith & Miles (1969, 1971) (see Gaunt & Miles, 1967, for an excellent review of tooth development in amphibians) as having its basis in a similar heterochronic shift. Smith (1967) had concluded that it involved a change in function of the dental epithelium from protein synthesis to absorption of matrix in the production of enameloid, more than a shift in timing of secretion. Because this involved a significant specialization of the cell membrane to produce a ruffled border (Smith & Miles, 1971 ; Fig. 6), Smith (1969) had concluded that it was a larval adaptation rather than an example of a change from a more primitive to a more advanced state. The observation was also made that the dental epithelium had a different function in the shaft region (collar) from the cap region (Smith & Miles, 1969), resulting in enamel in the collar and enameloid in the cap. This is exactly the situation in some actinopterygian teeth (Smith, 1989, 1990), but it is still a puzzle as to why this tissue combination should be found in one group of larval amphibians, a group not closely related to actinopterygians. Meinke & Thomson (1983, p. 145) have argued that a similar shift to those described above occurs between young and adult teeth in lungfish, although the data base for this is not experimental. We are of the opinion that the possibility should not be ruled out that enamel evolved first and that the shift in timing of enamel protein secretion was from a late time to an early time, relative to dentine secretion (Smith, 1990), resulting in enameloid as a derived tissue at least in osteichthyan fishes, and in some larval teeth. The situation in agnathan fishes is far from clear, although we have observed both enamel and enameloid-like tissues in the Ordovician tubercles of the Harding Sandstone fossils, suggesting that enamel is at least as primitive as enameloid and probably came first. The presence of enameloid in chondrichthyan fishes may be an example of convergence.

Meinke (1982a, 6 ) utilized a similar interpretation for development of the dental tissues in Polypterus, on the basis of histology, histochemistry, scanning electron microscopy and information on regeneration of fin spines and scales. Based on her analyses, the matrix of the tooth cap is formed by the combined action of epithelial and ectomesenchymal cells ; it contains proteins derived from both. Also, enameloid (an epithelially, ectomesenchymally derived tissue), ganoine (an epithelial derivative ; see Sire et al., 1987) and dentine (an ectomesenchymal derivative) are all found in the same specimen. Meinke (1982~2, p. 197) therefore concluded ‘that the enamel/

‘3 B R E 65

346 l\/lOYA M. SMITH AND B. K. HALL enameloid/dentine system forms a continuum of tissues that have diverged from one another by changes in the relative timing of developmental events and matrix production ’, and that although these are discrete tissues, ‘ clear developmental connections can be envisaged between these endpoints ’. She further cites (19826, pp. 201-2) three steps in the developmental cascade when altered timing could produce one odontogenic tissue versus another :

( a ) delay/prolongation in protein secretion by inner dental epithelial cells of the epithelium with the consequence of lack of ectomesenchymal collagen in the epithelially derived enamel and the production of enamel,

(6) impaired interaction between odontoblasts and epithelial cells with the consequence that epithelial proteins may or may not be deposited into the matrix produced by odontoblasts and the production of enameloid or dentine, and/or

( c ) altered timing of hypermineralization of enameloid with the consequent hypomineralization of the enameloid which with ( a ) would result in production of enamel.

Thomson ( I 975) and Meinke ( I 984, I 986) have interpreted the formation of cosmine in fossil osteichthyans using a model of shifts in timing of development between the skeletogenic tissues and the neuromasts/pore canals. Cosmine is a composite tissue consisting of several dermal skeletogenic and odontogenic tissues (bone, dentine, enamel) and several soft tissues (surface epithelium, blood vessels, ‘ sensory ’ pore canals). The epigenetic cascade proposed involves as we see it:

( a ) association of epithelium and ectomesenchyme ; (6) migration of neuromast precursors to epithelial-ectomesenchymal interfaces ; (c) association of ectomesenchyme with epithelium and with neuromast precursors

and initiation of interactions leading to differentiation of inner dental epithelium and odontoblasts ;

( d ) differentiation of neuromasts which, along with odontoblasts, sink below the epithelial surface ;

( e ) development of connected pore canals containing neuromasts ; completion of differentiation of dentine and enamel to form cosmine ; (f) association of cosmine with subjacent bone.

This scenario as proposed by Thomson (1975) requires, among other things, that neuromasts and skeletogenic/odontogenic tissues interact, an as yet unsettled issue (see Table 3, especially footnote g and p. 69) although Meinke’s (1986) proposal does not require a necessary inductive relationship between the two, only a topographic association that can be disassociated through heterochrony.

Smith ( I 989) on the basis of an ultrastructural analysis of the distribution of enamel in sarcopterygian teeth and correlation with the recent ultrastructural studies by Meunier et a / . ( I 988) on ganoine in primitive actinopterygians and the studies by Sire et al. ( I 987) on the regeneration of scales in a polypterid actinopterygian (Calamoichthys calabricus) concludes that ganoine and enamel are ectodermal and synapomorphic for both actinopterygians and sarcopterygians. Previous workers, assuming that ganoine was mesodermal (see Smith, 1989 for references) used enamel as a synapomorphy for sarcopterygians alone. This study provides a clear instance of where recent developmental evidence and a close study of the fossil tissues can provide vital systematic and phylogenetic information.


( 5 ) Losslreappearance of odontogenic tissues

Evidence for loss of a component of the epithelial-mesenchymal interaction leading to phylogenetic loss of an odontogenic tissue and its reappearance after experimental manipulation of the interactions will now be discussed. There are two examples that may be advanced in this category.

The first involves the phylogenetic loss of teeth in birds and the evidence of ameloblast differentiation and enamel synthesis by avian mandibular epithelia when recombined with mouse dental papilla ectomesenchyme (Kollar & Fisher, I 980). Mandibular arch epithelia from chick embryos of 5 days’ incubation were associated in tissue recombinations with mandibular molar mesenchyme from foetal mice of I 6-1 8 days’ gestation and the recombined tissues maintained in vitro overnight before being grafted for 1-4 weeks into the anterior eye chambers of adult, athymic mice. Complete teeth developed in 4 of the 5 5 grafts. Provided that the possibility of murine epithelium adhering to the mesenchyme can be eliminated, as Kollar and Fisher were careful to ensure, then the enamel and ameloblasts that developed in the chimaeras must have been derived from the chick mandibular epithelia in response to the mouse dental mesenchyme. This experiment indicates that the embryonic chick epithelium will respond to ectomesenchyme of the mouse dental papilla at the preodontoblast stage to make teeth. It may have retained one of the three or four epithelial components required to produce teeth, but tells us nothing about what abilities the chick ectomesenchyme may have, should it be given the correct signals from competent epithelium. The above experiment has not been repeated, and for this reason has not been completely accepted.

There are numerous other experiments of a similar type in so far as epithelium and/or ectomesenchyme from one species can be used to evoke dentinogenesis from ectomesenchyme or amelogenesis from epithelium in a species from a different vertebrate class. The experiments of Lumsden’s (1987), although the eliciting epithelium comes from the same species, show that dentine and bone form from trunk neural crest cells in the mouse (discussed in section IV) indicating that odontogenesis can be evoked from trunk neural crest, an ability assumed either to have been lost, or at least to have been retained only in those fishes with a dermal exoskelton, and in others not expressed because of lack of association with the requisite epithelium.

Examples from amphibians are discussed below. There is also the recent series of experimental studies based on tooth development in the Chilean, ovoviviparous lizards, Liolaemus tenuis and L. gravenhorsti and on heterospecific recombinations of their odontogenic tissues with those of the Japanese quail Coturnix coturnix japonica (Lemus et al . , 1980, 1983, 1986, 1987) in which developing lizard teeth can evoke dentinogenesis from quail cranial neural crest cells and lizard dental papillae can evoke ameloblast differentiation from quail fEank epithelium.

The second example is the evocation of balancers from a urodele embryo (Triton) and of adhesive organs from an anuran embryo (Bombinator) (normally urodeles have balancers and frogs adhesive organs) after transplanting ectoderm from a species within one taxon in place of ectoderm of a species from the other taxon (Spemann, 1938). The resulting heterotypic epithelial-mesenchymal interactions evoke the differentiation of a cranial structure not present in the host, in this sense a parallel to the ‘hen’s tooth’ experiment.

13-2

348 MOYA M. SMITH AND B. K. HALL In the initial experiment, in which Spemann & Schotte (1932) grafted anuran oral

ectoderm in place of urodele oral ectoderm and vice versa, urodele hosts developed adhesive organs but lacked teeth, while anuran hosts developed balancers and teeth. When neural-crest cells from embryos at the neural-fold stage were transplanted from the urodele to the anuran, the teeth that formed had donor (urodele) dentine from the neural-crest ectomesenchyme and anuran enamel derived from the host ectoderm. Therefore, a species from a group (anurans) that has horny (keratinous) teeth lacking enamel and dentine in the larvae, but true enamel-covered teeth in the adult upper jaw, can have ameloblast differentiation and enamel synthesis evoked from the oral ectoderm (in the larval stage) by neural-crest-derived ectomesenchyme from a species from a group (urodeles) that has ‘true’ teeth containing dentine and enamel. As with the avian example, only one component of the epithelial-mesenchymal interaction, namely epithelial ability to signal to the ectomesenchyme, has been lost, or temporarily delayed, in each species. As summarized by Spemann ( I 938) :

. . . the stimulus releasing the development of either organ (balancer and sucker) would still be the same as in that organ of the ancestors from which the two recent organs have been derived; whereas the reaction of the ectoderm would have changed [p. 359, and]. . . it is not the head of the Axolotl which lacks the faculty of sending out a stimulus for the formation of a balancer, but rather it is the epidermis which lacks the faculty of reacting upon it. (pp. 360-1).

Why should part (half ?) of an epithelial-mesenchymal interactive system be retained when the tissuelorgan is not formed in a species or even by members of a class of vertebrates ? T h e straightforward explanation is that the retained component may have another function which ensures its phylogenetic survival. Or, more interestingly, it may be that the odontogenic interaction is rooted in such a fundamental aspect of early vertebrate development, i.e. presence of a cranial skeletogenic/odontogenic neural crest with the ability to interact with a prepatterned instructive ectoderm; that it cannot be lost without totally disrupting early embryonic development. T h e argument would then be that given that dentine and bone of attachment arise from the same population of cells, it may not be possible to lose the odontoblast-forming population without losing osteoblast-forming capabilities as well. Given that evolution both of the neural crest and of epithelial-mesenchymal interactions have arguably been instrumental in the origin of vertebrates (Hall, 1975, 1978; Schaeffer, 1977; Schaeffer & Thomson, 1980; Reif, 1982; Gans & Northcutt, 1983; Northcutt & Gans, 1983; Langille & Hall, 1989, and see section VII) this seems a plausible possibility to us.

( 6 ) Losslreappearance of skeletogenic tissues

We cannot come up with any equivalent example in which a bone or cartilage lost in phylogeny has been made to reappear using heterotypic tissue recombinations between species from different vertebrate classes, although there is a considerable body of knowledge indicating that epithelia from one taxon can substitute for epithelia from another taxon in allowing skeletogenesis to be initiated, although such reciprocity is by no means universal (Kratochwil, 1983; Hall, 1988b, 1989; Nieuwkoop et al., 1985; section V). Within species the notochord, which elicits vertebral chondrogenesis from sclerotomal mesoderm in v i v o , can also elicit chondrogenesis from cranial mesoderm but not from limb mesenchyme; the epithelium of the otic vesicle, which elicits otic capsular chondrogenesis from otic mesenchyme, also elicits chondrogenesis from other

Skeletal development and evolution 349 head-mesodermally-derived mesenchyme, but not from sclerotomal mesoderm (Benoit, 1960; Petricioni, 1964; Schowing, 1974). Between taxa, otic vesicle epithelium from the chick evokes chondrogenesis from sclerotomal mesenchyme of the vertebral region in both amphibians and mice (Grobstein & Holtzer, 1955 ; Holtfreter, 1968); evokes chondrogenesis from amphibian and chick cranial mesenchyme and from amphibian olfactory mesenchyme (see Hall, 1989 for a detailed discussion of these and other interactions).

There are those situations which do not involve loss of skeletogenic tissues, but rather apparent phylogenetic replacement of a homologue with a skeletal element that forms by a different mode of ossification, i.e. non-homologous developmental processes producing homologous skeletal structures. Examples that are known to us are the development of vertebrae by intramembranous ossification in some fishes and amphibians and by endochondral ossification in other vertebrates (Duellman & Trueb, 1986) and the development of the orbitosphenoid as a membrane bone in the South American amphisbaenian, Leposternon microcephalum, but as an endochondral bone in all other vertebrates (Bellairs & Gans, 1983).

That homologous bones can develop by non-homologous developmental processes raises fundamental questions both for continuity of structures in phylogeny because of ‘correspondence of developmental processes ’ (Presley, 1983, p. 210) and for how developmental programmes change to allow suppression of the cartilaginous phase, direct ossification in mesenchyme and transformation of an endochondral bone (‘ cartilaginous ossification ’) to a membrane bone (intramembranous ossification). Bellairs & Gans (1983) comment on the presence of a pair of cartilaginous nodules on the embryonic orbitosphenoid undergoing perichondral ossification. Do these represent remnants of the cartilaginous model of the orbitosphenoid of the ancestors of Leposternon, secondary cartilages which have developed on the phylogenetically new membrane bone (in which case they would represent the only secondary cartilages known for reptiles), or, as Bellairs and Gans argue, vestiges of the orbital cartilages (presumably secondarily fused to the orbitosphenoid) ? A detailed developmental study could resolve these questions.

Rieppel (1988, pp. 48-9) provides a further discussion of homologous skeletal elements, especially the amphisbaenian epipterygoid, in the context of homology of developmental processes and Hall ( I 975) discusses the evolution of the gnathostome jaw and the mammalian middle ear ossicles in the context of evolving developmental processes.

VII. T H E NEURAL-CREST ORIGIN OF SKELETOGENIC AND ODONTOGENIC TISSUES IN T H E FIRST VERTEBRATES: IZ POSTULATES

Given that dentine can be positively identified in early (stratigraphically oldest) vertebrates that are also cladistically primitive ; and also that the production of dentine has never been shown to be, or thought to be from cells of mesodermal origin in extant vertebrates (dentine is formed by cells derived from the neural crest). We arrive at

POSTULATE I . That the dentine of the earliest agnathan vertebrates was of neural crest origin.

Given that Lumsden (1988) has demonstrated that bone of attachment of the mammalian tooth is derived from the sub-population of neural-crest cells from those

3 5 0 MOYA M. SMITH AND B. K. HALL which produce the dentine and the dental pulp, and that Cassin & Capuron (1979) and Cusimano-Carollo ( I 969) have shown that ectoderm, endoderm and skeletogenic neural crest interact in an epigenetic cascade to allow tooth formation (whether horny teeth of anurans or ' true ' teeth of urodeles ; see Hall & Horstadius, I 988 for a discussion of such epigenetic cascades), i.e. that the odontogenic and skeletogenic tissues are coupled in amphibians and in mammals, we arrive at

POSTVLATE 2. Tha t the bone of attachment associated with dentine in the earliest agnathans was both neural-crest derived, primary, and developmentally derived f r o m cells of a dental organ (odontode) equivalent to a dental papilla.

Given that the bone in which the teeth and bone of attachment are set is also of neural crest origin in amphibians and mammals, but, at least in mammals, is derived from a separate neural-crest population than that producing the dentine and bone of attachment, we arrive at

POSTCLATE 3 . Tha t the basal bone of fossil agnathans to which the dermal denticles are fused was of neural-crest origin and primary, but developmentally derived f rom a separate population of cells f r o m those producing the dentine and bone of attachment of the denticles.

Given that cartilage is found associated with bone in the cranial exoskeleton (but not in the trunk) of Ordovician agnathans, that such cartilage is never found unless associated with bone, that secondary cartilage in extant vertebrates always develops secondarily to dermal ossification, and with the probable exception of the clavicle, is of cranial neural-crest origin we can state

POSTULATE 4. Tha t calcified cartilage of the Ordovician agnathan dermal exoskeleton was present as a neural-crest-derived component of the exoskeleton in at least one early vertebrate ; that it like secondary cartilage, followed osteogenesis, indicates that cartilage was secondarily present in the original dermal exoskeleton, bone being the primary, skeletal, dermal, exoskeletal tissue.

T o summarize; dentine, bone of attachment, basal bone and cartilage of the Ordovician agnathan dermal skeleton were all neural crest derivatives. The neural crest is therefore the source of the vertebrate dermal exoskeleton.

Given that the cartilaginous endoskeleton (neuro-viscerocranium, branchial arches) of all modern-day vertebrates is derived from a rostro-caudally regionalized ske!etogenic cranial neural crest, we can state

POSTULATE 5. Tha t cranial endoskeletal cartilage in the earliest agnathans was neural- crest derived and uncalcified.

Given that the development of cartilage precedes the development of bone in the

POSTULATE 6. Tha t the appearance of perichondral bone in the endoskeleton of fossil neuro-viscero and branchial arch skeletons of all extant vertebrates, we can state

agnathans was secondary to the appearance of cartilage.

Skeletal development and evolution 3-51

Therefore, while bone is the primary skeletal tissue in the dermal exoskeleton (Postulate 4), cartilage is the primary skeletal tissue in the endoskeleton (Postulate 6), whether cranial (neurocranial or viscerocranial) or trunk.

It follows from these postulates that irrespective of whether cartilage or bone was the first vertebrate skeletal tissue, i.e. whether bone appeared first in a primitive exoskeleton or whether unmineralized cartilage appeared first in a primitive endoskeleton, because cranial endoskeletal cartilage and cranial exoskeletal bone are of neural-crest origin, the initial vertebrate skeleton was of neural-crest origin and not mesodermal.

Given that dentine (and bone) is found in denticles and scales of elasmobranchs, also scales of Polypterus and Latimeria, and given the ability of murine embryos to form odontogenic tissues (dentine and alveolar bone of attachment) from trunk neural crest when it is recombined with mandibular ectoderm, but that neither cartilage nor basal bone form from mammalian trunk neural crest, and that a small yo of grafts of amphibian trunk neural crest also form dentine but not cartilage (Chibon, 1966), we can state

POSTULATE 7. Tha t the extension of the dermal exoskeleton into the trunk of Ordovician agnathans was because the trunk neural crest was odontogenic, being capable of forming both dentine and bone of attachment.

Given that there is no evidence for a neural-crest contribution to the trunk endoskeleton in any extant vertebrate and that the first vertebrate skeleton was a neural crest, cranial and dermal bony exoskeleton, or a neural crest, cranial cartilagidous endoskeleton, we can state

POSTULATE 8. That a trunk endoskeleton appeared later than a dermal exoskeleton in Ordovician vertebrates and that the trunk endoskeleton of the agnathans was secondary and mesodermal and not neural crest ; therefore the primitive vertebrate skeletogenic tissue is not mesodermal but neural crest in origin.

Our discussion of the developmental processes that regulate skeletogenesis and odontogenesis provide us with the last four postulates. These relate to the cascade of epigenetic interactions that govern where neural crest cells will migrate, when and where migration will cease, formation of condensations of ectomesenchyme and initiation of differentiation.

POSTULATE 9. Initiation of differentiation of skeletogenic and odontogenic tissues, whether neural crest or mesodermally derived, cranial or trunk, exo- or endoskeletal, is controlled epigenetically by one or more epithelial-mesenchymal interaction(s) in an epigenetic cascade.

POSTULATE 10. The potential exists for alteration in the timing of one or more steps in an epigenetic cascade during development.

POSTULATE I I . Heterochronic shifts in timing of epithelial-mesenchymal interactions between taxa provides an evolutionary mechanism for altering either the type of skeletogenic-odontogenic tissue or the skeletal-dental structure to which that tissue contributes .

352 MOYA M. SMITH AND B. K. HALL POSTULATE I 2. The appearance of epithelial-mesenchymal interactions, along with the

origin of an ectomesenchymal neural crest a t the outset of vertebrate evolution provided the decelopmental mechanism for the origin of the vertebrate skeletogenic and odontogenic tissues.

VIII . DISCUSSION

We have derived our postulates on the development of the first vertebrate skeletogenic and odontogenic tissues on the three-fold bases of (a ) the separation of the exo- from the endoskeletons ; (b) the neural-crest origin of the vertebrate cranial skeletogenic and odontogenic tissues, and ( c ) the epigenetic evocation of differentiation of these tissues by epithelial-mesenchymal interactions. We now discuss the views of previous workers on these three issues.

Moss ( I 968 c) and Halstead ( I 969) presented the position which was so elegantly developed by Patterson ( I 977) and which we also advance, that ‘ by differentiating between dermal and endoskeletal sites the problem (of whether cartilage or bone arose first) is capable of resolution.. . Cartilage did not precede bone in the dermal skeleton . . . Cartilage did precede bone in the endoskeleton ’ (Moss, 1968, p. 366), and that ‘Bone and cartilage arose independently of one another to subserve different functions’ (Halstead, 1969, p. 121).

Maisey’s (1988) approach to the cartilage problem is illustrative, for he clearly states : ‘ Vertebrate cartilage is endoskeletal, apart from adventitious cartilage in some birds and mammals’ (ibid. p. 5 ) . When viewed in this way, adventitious (secondary) cartilage in the exoskeleton (it is found in the dermal bones of birds and mammals (Hall, 1978 and see section 111) is seen as an exception, in late evolving vertebrate groups, to the primacy of cartilage as an endoskeletal tissue. Yet, as we discussed when considering the heterostracans and as Maisey himself implies, cartilage is present in the heterostracan exoskeleton. T o state that the early vertebrate cartilage is only endoskeletal is to take the appropriate position with regard to the separation of endo- and exoskeletons, but to fail to go on to indicate that both exo- and endoskeletons contain cartilage, and that cartilage arose before bone in the endoskeleton and after it in the exoskeleton. The existence of cartilage in the heterostracan exoskeleton should then be placed alongside the localization of (secondary) cartilage in fish, birds and mammals.

Because our focus is on the dermal skeleton and on the skeletogenic-odontogenic tissues found in the earliest agnathans, we do not deal with the question of whether cartilage in the endoskeleton arose before bone in the dermal exoskeleton. In fact, no one can say which came first, for if cartilage did arise in the endoskeleton at the same time as the first mineralized dentine - bone in the exoskeleton, then it was unmineralized, is not present in any fossils and therefore its presence can only be based on arguments other than the evidence of the fossil record. [In fact, unmineralized bone (osteoid) could also have been present in the dermal skeleton of early forms and also not appear in the fossil record, but it is clearly not profitable to speculate on this either.] Once a rim of perichondral bone is present it is possible to comment on the required presence of cartilage, even if the cartilage is unmineralized.

Janvier ( I 989, pers. comm.) considers that a cartilaginous, unmineralized endoskeleton rather than a mineralized dermal skeleton is the primitive condition for vertebrates. This is based on the assumption that hagfishes are the most primitive craniates, originating somewhere in the Lower Paleozoic (early Middle Ordovician)

Skeletal development and evolution 3 5 3 (see Janvier, 1981; Fig. I) . If one were to accept this view then a cartilaginous endoskeleton would have preceded the exoskeleton of bone and dentine. Unfortunately only one fossil exists that has been related to the hagfishes (Carboniferous, Gilpichthys, Janvier 1981), and it is difficult if not impossible to relate pre-Ordovician skeletal material (sclerites) to any form of whole-bodied remains, unless dermal odontodes are found in situ, or their presence is recorded in a cast. As Janvier has pointed out (1989, pers. comm.) ‘the fact that cartilage of an endoskeleton is not preserved in any fossil does not prove that it did not exist’, but also, the fact that a tuberculated exoskeleton has been described, and assigned to very early vertebrates (Anatolepis, Upper Cambrian; Bockelie & Fortey, 1976; Repetskie, 1978) suggests to us that at least the two skeletal systems may have co-existed, with perhaps the absence of a dermal exoskeleton being a derived character in hagfishes. Reif (1982) allows for this possibility in his cladogram (ibid., fig. I) , although he still puts the cartilaginous endoskeleton as the most primitive state.

Lacking fossils, the only basis for speculating on the time of appearance of cartilage is to use arguments from functional morphology as Gans and Northcutt have done so successfully (Gans & Northcutt, 1983, 1985; Northcutt & Gans, 1983; Gans, 1989). They make a very plausible case for the necessary appearance of pharyngeal cartilage (which would have been neural crest-derived) with the transformation of the early vertebrate pharynx from an organ of filter-feeding to an organ utilized in gas exchange, a function that could not occur without a supporting pharyngeal ‘skeleton’ during active pumping movements, using cartilage as an energy conserving system (Gans, 1989). However, one could conceive of a collagenous skeleton, analagous to the second skeleton of sharks (Wainwright et al., 1978) that could have served a supporting function, so that even pharyngeal gas exchange might not have required a cartilaginous skeleton at the outset. Gans (1989) envisages collagen as the basis of the first vertebrate supporting tissue and indeed, the pharyngeal supporting tissue of cephalochordates, urochordates and enteropneusts is collagenous (Welsch, I 975 ; Northcutt & Gans, I 983). A sequence of collagenous supporting skeleton-chondroid-unmineralized cartilage-mineralized cartilagebone is not unreasonable and would fit with the developmental data. In any event, the question of the timing of the origin of unmineralized cartilage is not part of our rubric and so will not be discussed further.

Moss ( 1 9 6 8 ~ ) stressed that ectoderm, mesoderm and ectomesenchyme were all capable of forming ‘scleroblasts ’ (although not all layers form all scleroblasts; ameloblasts are only produced from ectoderm and endoderm, and osteo- chondro-, odonto- and cementoblasts from mesoderm/ectomesenchyme) and stressed the potentially important role of ectomesenchyme and of the topographical and inductive associations between ectoderm and ectomesenchyme. Moss ends his paper with the disclaimer that he had previously centred his thoughts on the origin of vertebrate skeletal tissues primarily on mesodermal tissue and wished to redress the balance by pointing out the importance of the epidermis. We further redress the imbalance by placing emphasis, in fact, primacy, on the neural-crest-derived ectomesenchyme and its interaction with epidermis (ectoderm), the latter point having been previously stressed by one of us (Hall, 1975). We can now with the evidence from developmental biology of the past fifteen years remove ‘probably ’ from the statement and say : ‘ Much of the skeleton of the early vertebrates was (probably) of neural crest origin.’ (p. 340).

Schaeffer ( I 977) in an analysis that was both thoughtful and ahead of its time, argued

354 MOYA M. SMITH AND B. K. HALL for ectomesenchyme from the neural crest as ‘mostly, or entirely, involved in the development of unique vertebrate structures’ (p. 28). This was also the view of Gans & Northcutt (1983), Northcutt & Gans (1983), and one echoed by Maisey (1986; 1988), Schaeffer & Thomson (1980) and others. Schaeffer also argued, following Moss, that the dermal skeleton in fishes developed ‘from a single, integrated, modifiable morphogenetic system, that is initiated by an interaction between the epithelium and adjacent mesenchyme’ (1977, p. 25), a view first applied to fossil vertebrates by Moss (1968b, 19-72). Schaeffer extended his model to the dermal body scales of Polypterus, arguing that the association of enameloid, dentine and dermal bone allowed that ‘it may be postulated that ectomesenchyme is present in the dermis throughout the body, and, when the proper genetic signals are forthcoming, can be skeletogenic and form dermal bone as well as dental organs ’ (p. 37).

Lumsden (1987, p. 291) suggested that in those few gnathostomes which have retained dermal odontodes ‘the trunk neural crest presumably still expresses its odontogenic potential during normal development’. Also, Lumsden (1988) in an elegant set of experiments using recombinations of mouse neural crest with mandibular and limb bud epithelium, showed that whereas premigratory cranial neural-crest cells produced all tissues (cartilage, bone and neural) when in combination with limb bud epithelium, only teeth were produced with mandibular epithelium ; but more significantly, teeth also formed from trunk level neural crest in combination with mandibular epithelium. Lumsden (1988, p. 165) discussed the implications of his results for vertebrate trunk neural crest in general, and suggested that odontogenic potential may not be confined to the cranial region (whereas chondrogenic crest potential probably is), and that the developmental repertoire of murine trunk crest may have been expanded by the experimental association with oral epithelium. One significant result can be inferred from this work, namely that the epidermal ectoderm (oral in this case) specifies where the teeth will develop and instructs crest cells (either pre- or postmigratory) to become odontogenic. Lumsden (1988) concluded that ‘the oral epithelium is the earliest known site of tooth pattern’; and (1987; p. 289) ‘a prepattern for dental development must reside in the ectoderm as a mosaic of tooth- initiating foci ’. One can assume from this that all epithelium in lower vertebrates had the ability to specify where odontodes develop, but can only do so when confronted with potential odontogenic neural crest. With any change in the timing of migration of neural crest this initial inductive mechanism may be jeopardized, and teeth fail to be expressed. Currently we still do not know whether ectomesenchyme is present throughout the body, and await experimental studies on the origin of trunk dermal odontogenic and skeletogenic tissues in fishes.

Meinke & Thomson (1983) in discussing the utility of developmental data for phylogeny stressed the importance of proposing the phylogenetic implications of odontogenic changes so that experiments could be designed to test aspects of the phylogenetic hypotheses. They cite Graham-Smith ( I 978 a, b), his hypotheses concerning lateral line-dermal bone associations and the evolution of dermal bone in jawed fishes and how these hypotheses could be tested with experiments on lateral line- dermal bone development and interactions in living fishes.

Other developmentally testable hypotheses on the phylogeny of skeletogenesis that follow from this discussion of recent work are:

( a ) That bone of attachment and dentine of fish odontodes (teeth and denticles)

Skeletal development and evolution 3 5 5 develop from the same population of neural-crest-derived ectomesenchymal cells in the dental papilla.

(b) That basal bone underlying odontodes (teeth and scales) develops from a neural- crest cell population separate from (a).

(c) That trunk scales/denticles in extant fishes develop from trunk ectomesenchyme. ( d ) That the trunk ectomesenchymal potential of extant vertebrates is more

widespread than previously thought, but not expressed because of lack of association with a permissive ectoderm.

We now discuss the use that evolutionary biologists have made of the data indicating a fundamental similarity between the developmental processes that initiate differentiation of skeletogenic and odontogenic tissues. Moss (1964, 1968 a-c, 1969) developed the thesis in some detail that a variety of skeletogenic tissues arose from equivalent embryonic cells in response to specific epigenetic (epithelial-mesenchymal) interactions. Krejsa (1979) provided a very comprehensive evaluation of these similarities, and Hall (1975), and Maderson (1975, 1983) reviewed the evolutionary implications of the data known then. As indicated in section VI and in Postulates 9-12, variation in the timing of these interactions leads to (a) elaboration of a new odontogenic tissue (enameloid) and (b) alteration (loss) of odontogenic elements (teeth in birds).

Numerous authors have postulated that these epigenetic interactions arose at the outset of vertebrate evolution (Moss, op. c i t ; Hall, 1975, 1978; Maderson op c i t . ; Schaeffer, 1977; Schaeffer & Thomson, 1980; Reif, 1982; Meinke, 1984; Maisey, 1988 and Langille & Hall, 1989). Authors such as Schaeffer and Maderson have emphasized, as have we, the relatively small number of developmental processes involved in skeletogenesis and odontogenesis. In the thirteen years since Schaeffer proposed the hypothesis ‘ that the dermal skeleton develops from a single, modifiable morphogenetic system’ (1977, p. 26), there has accumulated a considerable experimental database from which to project development back to phylogeny.

In this review we have emphasized that the neural crest is a vertebrate synapomorphy and as Schaeffer & Thomson ( I 980, p. 20) stressed : ‘ . . . the vertebrate synapomorphies require for their development a highly specific sequence of tissue interactions observed first with the induction of the neural tube by the archenteron roof and continuing regionally to the final appearance of definitive adult structures ’. As related to the fossil record by Reif (1982, p. 331) in his developmentally based ‘Odontode Regulation Theory’, ‘the spectrum of dermal elements in vertebrates can be derived by a small number of morphogenetic and regulatory processes (the developmental data) from a simple microsquamate skeleton consisting of isolated odontodes ’ (the fossil evidence).

The data that we have presented for complexity of early agnathan skeletogenic and odontogenic tissues, for equivalent tissue interactions across the vertebrates, especially the data indicating that epithelia from representatives of one vertebrate class can evoke skeletogenesis or odontogenesis from ectomesenchyme of representatives from another vertebrate class, agree with Reif‘s theory but do not allow us to agree with either of Maisey’s ( I 988) proposals that ‘ throughout vertebrate history there has been a trend toward increased skeletal complexity, in turn brought about by increasingly complex patterns of tissue interactions’ (1988, p. 24). We see no evidence for increase in complexity of the histogenetic mechanisms or of the epigenetic interactions that produce them, although we would accept that morphogenesis may have become more

3 56 MOYA M. SMITH AND B. K. HALL complex. Whereas Maisey indicated that his conclusions depart from those of some developmental biologists and systematists ‘ in areas that were poorly defined by them phylogenetically’ (1988, p. zq), our conclusion departs from his in areas that were poorly defined by him developmentally, perhaps resulting from the collaboration of a paleohistologist and a developmental biologist in this review.

Four of the chapters in the volume that emanated from the 1985 American Society of Zoologists’ Symposium on Developmental and Evolutionary Aspects of the Neural Crest (edited by Maderson, 1987) dealt with the topics of early vertebrate skeletogenic/odontogenic tissues and/or of the role of the neural crest in their evolution, namely those by Lumsden, Thomson, Halstead and Gans.

Lumsden ( I 987) in dealing with odontogenic tissues, emphasized that the initial appearance of dentine in the agnathans was not as a dental tissue but as exoskeleton, and speculated that dentine functioned as a sensory tissue, enabling the Ordovician agnathans to detect tactile, temperature and osmotic changes (see also Tarlo, 1965; and see Northcutt & Gans, 1983 and Gans, 1987 for the view that dentine enabled the earliest vertebrates to engage in electroreception), that the neural crest was initially a sensory tissue and that it became skeletogenic by forming sensory dentine as the first vertebrate hard tissue. See also Reif (1982, p. 3 3 1 ) , for a similar conclusion.

Lumsden argues, from the similarity of agnathan and mammalian dentines, and on the basis of the neural-crest origin of amphibian and mammalian dentine, that ‘ it is reasonable to infer a common ontogenetic mechanism; dentinogenesis in the trunk skin of ostracoderms would have involved odontogenic tissue interactions between epidermal ectoderm and trunk neural crest’ (Lumsden, 1987, p. 291), clearly a view that we share.

Thomson ( I 987) also constructing his arguments from developmental data, largely the production of ectomesenchyme by trunk neural-crest cells in amphibians and lampreys, concluded that ‘the distinction between head and trunk crest may not be as great as it first appeared’ (Thomson, 1987, p. 307). He does allow (p. 308) for the possibility, commented on by us in section IV, that dentine in the trunk could arise from cranial neural-crest cells that have undergone extensive caudad migration, further highlighting the need for experimental studies on the odontogenic capabilities of the trunk neural crest in extant fishes.

Although utilizing developmental data when analysing formation of trunk scales in Ordovician agnathans, Thomson does not use the developmental data on secondary cartilage in the dermal skeleton of extant vertebrates when interpreting the presence of cartilage in the dermal skeleton of Eriptychius. Following 0rvig (1968) and Halstead (1987) Thomson regards cartilage as not arising in the dermal skeleton (‘ . . . the chondroblast, is never present in the dermal skeleton in lower vertebrates’, p. ~ I O ) , but as becoming secondarily incorporated into the dermal skeleton, having been previously produced in the endoskeleton. Murray (1963) dealt very effectively with this issue when discussing the possible origin of secondary cartilages from primary cartilages in birds. Our interpretation of the developmental data, especially Benjamin’s ( I 989 b ) recent demonstration of the development of secondary cartilage in fishes, clearly leads US to a conclusion that differs from Thomson’s, namely that cartilage is present in the dermal skeleton of some extant vertebrates where it arises from the same cell population that differentiates as osteoblasts and deposits bone, and that we therefore can regard

Skeletal development and evolution 357 cartilage in the dermal skeleton of early agnathans as having been produced in the dermal skeleton, although a secondary differentiation.

Thomson’s approach to the cartilage of the agnathan exoskeleton reflects his view, as articulated in detail in the 1987 paper, that because a common morphogenetic pattern accounts for all types of dermal skeleton in early vertebrates, ‘it is not possible to say that one phenotype is more primitive than the other’ (Thomson, 1987, p. 319) or ‘it is also impossible to know in what form the dermal skeleton first arose’ (Thomson, 1987, p. 330). For him ‘osteoblasts and odontoblasts represent the same basic cell type’ (Thomson, 1987, p. 3 3 0 ) , a position we cannot accept. Dentine and bone frequently develop in separate tissue combinations in extant and fossil vertebrates, whether formed from cranial or from trunk neurzl-crest-derived ectomesenchyme, and constitute different cell populations. We argue that although odontoblasts, and cementoblasts/osteoblasts forming the attachment tissue may arise from the same cell population in the dental papilla, as distinct phenotypes of one cell lineage; the basal dermal bone may be a separate population. While acknowledging that this may originally be from the same crest lineage, we do not believe that this dermal bone population once committed can also give rise to odontoblasts. In this sense, agreeing with Moss ( 1 9 6 8 ~ ) we regard them as two separate lineages. The multipotentiality of neural crest cells has recently been discussed by Lumsden ( I 989), against the opposing view that the crest is a mosaic of predetermined cells, and recent experiments, aimed at understanding the range of potentials of individual neural crest cells, are reviewed. Lumsden concluded that, ‘ in the emergent crest some cells are multipotent ’, but ‘the process (and presumed timing) of fate restriction is unknown’. However, some of the data were interpreted as ‘leaving open the possibility that the crest does contain some committed precursors ’. Whether the odontogenic and skeletogenic precursors are separately committed in premigratory crest cells is an open question.

We also find ourselves disagreeing with Thomson in one final area, namely that the mesoderm is the first skeletogenic tissue and that mesoderm ‘was replaced by ectomesenchyme’ (p. 328). We do agree that, given that all mesenchyme in the cephalochordates arises from mesoderm (cephalochordates lack a neural crest), much of the mesenchyme (but not the skeletogenic tissues) in the earliest vertebrates would therefore have arisen from mesoderm (as would the notochord), and we clearly agree with Thomson ‘that there was a very early dichotomy between developmental sequences leading to cartilaginous versus osseous tissues’ (p. 325); not that the processes vary, but that there is a fundamental separation of exo- and endoskeleton. But, we see no reason to postulate that ‘a mesodermal skeleton was replaced by ectomesenchyme’ (p. 328). Nor does Gans (1987, p. 369) who emphasizes, as in his earlier papers (Gans & Northcutt, 1983; Northcutt & Gans, 1983) the body of comparative anatomical knowledge in support of the vertebrate head being a new structure (‘a New Head’ as Gans and Northcutt subtitled their 1983 review) elaborated onto the front of the vertebrate ancestor, a condition in which there would be no mesoderm for the ectomesenchyme to replace. This view follows from our knowledge that all the shared-derived characters of vertebrates form from, are induced by, or are associated with, tissues derived from the neural crest. Maisey (1986, 1988) using cladistic analysis, comes to similar conclusions with regard to the central role of the neural crest in generating so many craniate features, and to a neural-crest-derived

358 MOYA M. SMITH AND B. K. HALL skeleton preceding the mesodermal skeleton (1986, pp. 209, 21 I , 242; 1988, pp. 1 5 , 23), although we know of no evidence for his statements that neural-crest-derived ectomesenchyme is involved with lateral plate mesoderm in the development of paired

Given that the initial vertebrate skeleton was cranial and trunk, that both cranial and trunk exoskeletal bone and dentine, and some cranial endoskeletal cartilages arise from the neural crest, it follows that, irrespective of whether cartilage of the cranial endoskeleton or bone/dentine of the cranial and/or trunk exoskeleton arose first, the first vertebrate skeleton would have been an ectomesenchymal derivative of the cranial neural crest and not mesodermal.

Reif (1982) developed the above view of the central role of the neural crest, but in his evolutionary scheme (fig. I , p. 289) has a cartilaginous endoskeleton (branchial basket, brain capsule) ‘most probably preceeding all other skeletons in the vertebrate ancestors ’ (p. 3 I 9). Whether this is so, or whether dermal dentine and/or bone arose first in evolution is impossible to judge. We have argued that at least some of the early phosphatic skeleton appears to be in the form of isolated odontodes and that these provide evidence of a very early dermal skeleton. The remnants of an uncalcified cartilaginous skeleton would be extremely difficult to detect. Maisey (1988, p. 15) commented that Reif had proposed that simple odontodes were the most primitive calcified tissue and that Janvier’s ( I 98 I) interpretation of thelodonts as stem vertebrates supported this, although a somewhat tautological proposal based on the premise that their micromeric dermal skeleton represents a primitive condition. We presume it could equally represent a secondarily simplified condition, although it is difficult to relate the thelodonts to any higher taxon (Fig. I I), Turner (1985) suggesting that they are stem chondrichthyans. That both odontodes and cartilage of the branchial basket are of neural-crest origin can be asserted. (As indicated above we do believe that whether dentine or bone came first in the dermal skeleton can be decided on the basis of developmental information.) We share with Reif ( I 982) the view that : ‘There may be phylogenetic information hidden in the fact that the neural crest cells not only contribute to the nervous system, but also develop into pigment cells and contribute to visceral cartilages, other endoskeletal cartilages, and the dermal skeleton. The meaning of this information is, however, completely obscure ’ (p. 3 I 9). We would confirm more recent opinions as discussed here that the meaning is clear: the invention of the neural crest and the interaction with pre-patterned ectoderm was how vertebrates developed their first skeletogenic and odontogenic tissues and the first skeleton.

This emphasis on the interpretation of the fossil record through developmental information leads onto the possibility of reassessing the primitive and derived characters of the skeleton in vertebrates, and to an independent test of the many proposed cladograms. We suggest that the primitive state of the exoskeleton is one with separate dermal denticles, with either acellular Sharpey fibre bone, or cellular bone of attachment ; i.e. thelodonts, indeterminate third vertebrate - Harding Sandstone, chondrichthyans. A derived state is either an expansion of the bone of attachment into a spongy bone, i.e. Eriptychius, or fusion onto a sheet of basal bone, i.e. Arandaspis, Lepidaspis. This allows for the probable coexistence of an uncalcified endoskeleton of neural-crest derived, cranial and visceral components, followed by calcified cartilage as in Eriptychius, and in chondrichthyans. This is a return to the classical theory of dental primacy (Moss, 1968b) or what he termed the ‘isolation theory’, agreeing with

appendages (1988, PP. 4, 19, 24, 25).

Skeletal development and evolution 359 Goodrich ( I 907) that dermal bone and odontodes arose independently. This analysis supports in broad outline the cladograms of Janvier & Blieck (1979), except that thelodonts are not shown, Janvier (1981) with thelodonts as stem vertebrates, and Reif ( I 982), except that elasmobranchs would be more primitive than all other gnathostomes, and is in contradiction to the views of Halstead (1982). Detailed cladograms could only arise with more rigorous tests for congruence with all other characters, and should be attempted from this developmentally based skeletal character polarity.

XI. SUMMARY

This review deals with the following seven aspects of vertebrate skeletogenic and

I . The evolutionary sequence in which the tissues appeared amongst the lower

2. The topographic association between skeletal (cartilage, bone) and dental (dentine,

3. The separate developmental origin of the exo- and endoskeletons. 4. The neural-crest origin of cranial skeletogenic and odontogenic tissues in extant

vertebrates. 5 . The neural-crest origin of trunk dermal skeletogenic and odontogenic tissues in

extant vertebrates. 6 . The developmental processes that control differentiation of skeletogenic and

odontogenic tissues in extant vertebrates. 7. Maintenance of developmental interactions regulating skeletogenic/odontogenic

differentiation across vertebrate taxa. We derive twelve postulates, eight relating to the earliest vertebrate skeletogenic and

odontogenic tissues and four relating to the development of these tissues in extant vertebrates and extrapolate the developmental data back to the evolutionary origin of vertebrate skeletogenic and odontogenic tissues. The conclusions that we draw from this analysis are as follows.

8. The dermal exoskeleton of thelodonts, heterostracans and osteostracans consisted of dentine, attachment tissue (cement or bone), and bone.

9. Cartilage (unmineralized) can be inferred to have been present in heterostracans and osteostracans, and globular mineralized cartilage was present in Eriptychius, an early Middle Ordovician vertebrate unassigned to any established group, but assumed to be a stem agnathan.

10. Enamel and possibly also enameloid was present in some early agnathans of uncertain affinities. The majority of dentine tubercles were bare.

I I . The contemporaneous appearance of cellular and acellular bone in heterostracans and osteostracans during the Ordovician provides no clue as to whether one is more primitive than the other.

I 2. We interpret aspidin as being developmentally related to the odontogenic attachment tissues, either closer to dentine or a form of cement, rather than as derived from bone.

I 3. Dentine is present in the stratigraphically oldest (Cambrian) assumed vertebrate fossils, at present some only included as Problematica, and is cladistically primitive, relative to bone.

odontogenic tissues.

craniate taxa.

cement, enamel) tissues in the oldest vertebrates of each major taxon.


I 4. The first vertebrate exoskeletal skeletogenic ability was expressed as denticles of dentine.

I 5 . Dentine, the bone of attachment associated with dentine, the basal bone to which dermal denticles are fused and cartilage of the Ordovician agnathan dermal exoskeleton were all derived from the neural crest and not from mesoderm. Therefore the earliest vertebrate skeletogenic/odontogenic tissues were of neural-crest origin.

16. Given the separate developmental and evolutionary origin of the cranial exo- and endoskeletons (both derivatives of the cranial neural crest) we conclude that bone (of attachment) was the primary skeletogenic tissue in the exoskeleton (cartilage being secondary), but that uncalcified cartilage was the primary skeletogenic tissue in the endoskeleton (bone - perichondral - being secondary).

17 . Using evidence from developmental biology we conclude that the trunk neural crest of Ordovician agnathans was odontogenic, forming both dentine and bone of attachment of the trunk dermal exoskeleton.

I 8. Initiation of differentiation of skeletogenic and odontogenic tissues is controlled epigenetically by one or more epithelial-mesenchymal interactions in epigenetic cascades.

19. Changes in timing of steps in these epigenetic cascades provides an evolutionary mechanism for altering the types of skeletogenic/odontogenic tissues and/or structures formed.

20. The appearance of epithelial-mesenchymal interactions and the origin of the skeletogenic/odontogenic neural crest at the outset of vertebrate evolution provided the developmental basis for the evolutionary origin of vertebrate skeletogenic and odontogenic tissues and for the appearance and evolution of the vertebrate skeleton.

X. ACKNOWLEDGEMENTS

M. M. S. gratefully acknowledges discussions on the fossil tissues with Philippe Janvier, Alain Blieck, Richard Fortey, and Derek Briggs, B.K.H. discussions on the development and evolution of skeletal tissues with Carl Gans, Jim Hanken, Phil Brylski, Rob Langille, Gerd Muller, Tom Miyake, Keith Thomson and we are both grateful to Philippe Janvier, Andrew Lumsden, Paula Mabee, and Gerd Muller for critically reading the initial manuscript. The critical reading of the final manuscript by Alain Blieck is greatly appreciated. Our thanks are due to Peter Forey and Sally Young for organizing the loan of material from the British Museum (Natural History), and Liz Loeffler for the loan of agnathan material from Arctic Canada. We would also like to thank Richard Fortey, Mike Benjamin, Richard Palmer and Andrew Lumsden for both access to, and permission to use illustrations from, their materials. The work was carried out while B. K. H. was on sabbatical leave as The Warwick James Fellow in the Unit of Anatomy in Relation to Dentistry of The United Medical and Dental Schools of Guy’s and St Thomas’s Hospitals, London.

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DEVELOPMENT AND EVOLUTIONARY ORIGINS OF VERTEBRATE SKELETOGENIC AND ODONTOGENIC TISSUES (3)...

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