Vestigial Brain Melanocyte Development During Embryogenesis of an Anural Ascidian. (anural...

Develop. Growth & Differ., 34 (1 ), 17-25 ( 1 992) Development Growth & Differentiation

Vestigial Brain Melanocyte Development During Embryogenesis of an Anural Ascidian (anural development/ brain melanocyte drfferentiation/ovoviviparrty/evolutron)

Billie J. Swalla and William R. Jeffery Department of Zoology and Bodega Marine Laboratory, University of California, Davis, P 0 Box 247, Bodega Bay, CA 94923 U S A

Anural development was examined in the ascidian Bostrichobranchus digonas using specific markers for differentiated urodele ascidian larval cells and tissues. In this ovoviviparous anural ascidian, eggs, embryos and developing juveniles were present in the gonads, brood sacs, and atrial cavity, respectively. Morphological studies indicated that 6. digonas embryos do not develop into tailed larvae with an extended notochord and differentiated muscle cells. In addition, these embryos lack detectable expression of the muscle-specific markers acetylcholinesterase, alpha actin, and myosin heavy chain. In striking contrast to other anural ascidian embryos, however, B. digonas embryos can develop tyrosinase in several melanocyte precursor cells and eventually form a brain pigment cell. The melanocyte does not become part of a definitive brain sensory organ (otolith) and subsequently disappears during metamorphosis. A period of tyrosinase expression was also observed following metamorphosis in which many tyrosinase-positive cells appear in the body of the developing juvenile. The results demonstrate that different urodele features can be uncoupled during the evolution of anural development. The development of a vestigial brain melanocyte also suggests that B. digonas evolved from a urodele ancestor rather than from another anurai ascidian lacking a brain pigment cell.

Introduction

Ascidians exhibit two distinct phases in their life cycle (see 5 for review). The adult phase is a sessile filter-feeding zooid which exists either in a solitary state or associated with other individuals in a colony. Most solitary and all compound ascidians develop from a motile, non-feeding larva. The tadpole larva consists of an anterior trunk, which contains a dorsal brain with pigmented sensory organs (the otolith and the ocellus), and a posterior tail, which is comprised primarily of a central notochord with several rows of flanking muscle cells (12). Most of the larval muscle cells develop autonomously, presumably due to the inheritance of segregated factors from the egg (see 20, 25, 29 for reviews). In contrast, brain sensory organs are specified by inductive cell interactions initiated during gastrulation (1 7-1 9). Following a free-swimming period of variable dura- tion, the tadpole larva attaches to a hard substrate and metamorphoses into an adult. During metamorphosis the tail is retracted into the trunk, the larval specific tissues are destroyed, and adult structures, including a new system of siphon and body wall muscles, differentiate in the developing

17

juvenile. Although the vast majority of ascidian species

exhibit a life cycle that includes a tailed (or urodele) larva, a few species (mostly in the family Molguli- dae) have modified the conventional mode of development (see 3, 9 for reviews). In these species, embryonic development occurs without the formation of a brain sensory organ (urodele larvae in the family Molgulidae contain an otolith but no ocellus; see 3 , 4), a notochord, or tail muscle cells, resulting in a tailless (or anural) larva, which subsequently metamorphoses directly into an adult. Most anural ascidian species are found in relatively uniform habitats, such as sand or mud flats, where there is presumably no selective advantage for a motile larval phase. A sub-set of anural ascidians is thought to have re-colonized hard substrata in regions of swift current or wave action (2, 3, 30). Because of the predominance of ascidian species with conventional urodele development, species with anural development are thought to be derived from urodele ancestors (3).

The phylogenetic distribution of anural species suggests that this mode of development has evolved independently in several different ascidian families. Multiple origins of anural development

Copyright 0 by The Japanese Society of Developmental Biologists 1992 All rights of reproduction in any form reseNed.

18 B. J. Swalla and W. R. Jeffery

would suggest that a relatively simple sequence of events mediates this change in life history. Unfor- tunately, however, anural development has been investigated in detail only in a single subfamily of molgulid ascidians, the Molgulinae (2, 6, 22, 28, 30). In the present study, we describe anural development in Bostrichobranchus digonas (1 ), which belongs to another molgulid subfamily, the Eugyrinae. The most significant result of this study is that tyrosinase-positive neural cells and a brain melanocyte were shown to develop during embryogenesis of this anural ascidian indicating that development of different urodele features can be uncoupled during the evolution of anural development. The development of a vestigial brain melanocyte in B. digonas embryos also suggests that the anural Eugyrinae evolved from a urodele ancestor rather than another anural molgulid lacking a brain pigment cell.

Materials and Methods

Biological materials: The anural ascidian Bos- trichobranchus digonas (1) was provided by Gulf Specimen Co. (Panacea, FL.). Adults were maintained in aquaria containing Instant Ocean or run- ning sea water at 18°C. Eggs, embryos and developing juveniles were obtained by dissection from the gonads, brood sacs, and atrial cavity, respectively. As reported for other ovoviviparous ascidians (4), embryonic development was arrested after zygotes were removed from the body cavity. Therefore, embryos and juveniles were photographed or processed for further analysis immediately after dissection from the adult.

These experiments used several urodele species as controls, including Styela claw (collected at Woods Hole, MA), Molgula occidentalis (obtained from Gulf Specimen Co.), Molgula citrina (collected in the Cape Cod Canal, Bourne, MA), MolguIa oculata (collected at Roscoff, France) and Halocynthia roretzi (sections of fixed specimens were obtained from N. Satoh, Kyoto University, Japan). Adults were maintained and embryos were prepared and cultured as described pre- viously (8, 22, 23).

General histology: For histological observations and in situ hybridization, embryos and adults (removed from the tunic) were fixed in 3 : 1 ethanol : acetic acid on ice for 30 min. The fixed specimens were dehydrated through an ethanol series to 100% ethanol, cleared in toluene, and embedded in Paraplast. Sections were cut at 8 p m and

attached to gelatin-subbed glass microscope slides. The sections were stained with Harris hematoxylin or hybridized in situ with a radioactive RNA probe (see below).

Enzyme histochemistry: Embryos were fixed for 20min with 5% formalin-sea water (4°C) and then washed with several volumes of sea water (tyrosinase) or 0.1 M sodium phosphate buffer (pH 6.0; acetylcholinesterase) before histochemistry. Acetylcholinesterase (AChE) activity was determined by the method of Karnovsky and Roots (1 1). Tyrosinase activity was determined by the method of Laidlaw (13).

Preparation of RNA probes: RNA probes were transcribed from SpMA3C, a cDNA subcloned into pBluescribe which contains part of the coding region of a Styela plicata alpha actin gene (24), or pcM 111, a cDNA subcloned into pGEM which contains part of the coding region of a Halocynthia roretzi myosin heavy chain (MHC) gene (14, 15). SpMA3C plasmid was linearized with EcoR1 and transcribed with T3 polymerase, whereas pcM 11 1 was linearized with BamHl and transcribed with T7 polymerase to obtain labelled anti-sense RNA probes. RNA probes were transcribed from the linearized DNA using 5 pI of [35S]-ATP (3000 Ci/ mmole; New England Nuclear) in a 25 pl reaction mixture containing: 5 pI of 5 x transcription buffer (0.2 M Tris-HCI, pH 7.5, 0.03 M MgCI2, 0.01 M spermidine, 0.05 M NaCI); 1 pl each of 0.01 M CTP, 0.01 M UTP, 0.01 M GTP; 1 pI of 0.075 M dithiothreitol (DTT); and 1 p g of linearized DNA template in diethylpyrocarbonate (DEP)-treated water. After synthesis, 25 pI of DEP-treated water was added and the reaction mixture was passed over a Minispin column (Worthington Biochemical Corp., Freehold, NJ). The RNA probes were pre- cipitated overnight in 0.3 M Na acetate and 70% ethanol (-20°C). After washing and drying the precipitate, the labelled probes were re- suspended in 10 pI of 0.01 M Tris-HCI, pH 8.0, 0.001 M EDTA before adding to hybridization buffer (see below).

in situ hybridization: In situ hybridization and autoradiography were carried out as described by Jeffery (8) using [35S]-labelled RNA probes and a hybridization buffer containing 50% formamide, 0.01 M Tris-HCI, pH 8.0, 0.001 M EDTA, 0.3 M NaCI, 0.1 M DTT, 1 x Denhardt’s solution, 500 p g / ml tRNA, 500pg/ml poly A, and 10% dextran sulfate. Following hybridization, the slides were

Brain Melanocyte Development in a n Anural Ascidian 19

treated with RNase and then washed successively in 4xSSC (0.1 M DTT was added to reduce back- ground), 2xSSC and 1 xSSC (0.15 M NaCI, 0.015 M Na citrate) at 20"C, autoradiographed, developed, and stained through the emulsion with Harris hematoxylin.

Antibody staining and immunofluorescence : For antibody staining, embryos were fixed in 100% methanol (-20°C) for 20 min, post-fixed in 100% ethanol (-20°C) for 20 min, and embedded in polyester was as described by Swalla et a/. (23). Sections were cut at 8pm, de-waxed in absolute ethanol, hydrated, and stained with "18 monoclonal antibody (1 :25 dilution in PBS; clone "18; ICN Immunobiologicals, Lisle, IL). After staining, the specimens were rinsed in 0.02M PBS and treated with rhodamine-conjugated goat anti- mouse IgG (Cappel Laboratories Inc., Downing- town, PA) diluted 1 :40 in PBS. After rinsing several times in 0.02 M PBS, the specimens were mounted in glycerol, and examined and photographed using a Leitz epifluorescence microscope.

Results

Anural development Selected developmental stages obtained from

the gonads, brood sacs, and the atrial cavity of B. digonas adults are shown in Figure 1. The uncleaved egg is about 190 pm in diameter and filled with opaque yolk granules (Fig. 1A). It is sur- rounded by a chorion, but appears to be lacking test and follicle cells. Fertilization occurs in the gonad, and cleavage is initiated before the zygote is deposited in the brood sac. In contrast to other anural ascidians (2, 3, 6, 22, 28), cleavage is unequal after the first two divisions (Fig. 1B and data not shown). Because of the opaque yolk granules, the movements of gastrulation and neurulation could not be evaluated, but it was clear from observations of living (Fig. 1C-E) and sectioned material (Fig. 28; Fig. 5B; Fig. 6B, C) that development is anural (1).

Developmental stages between gastrulation and metamorphosis were classified according to the shape of the embryo, the size and number of blastomeres, and the extent of ampullae (respira- tory appendages of the juvenile) formation. Stage 1 embryos are elliptical and contain relatively large cells (Fig. 1C); stage 2 embryos are also elliptical but contain smaller and more numerous cells and have elaborated several blunt epidermal thicken-

Fig. 1. Development of 6. digonas. A. An uncleaved egg. B. A cleaving embryo at the 16 cell stage showing rnicromeres and macromeres. C. A stage 1 embryo. D. A stage 2 embryo beginning to extend ampullae (arrow). E. A star-shaped, stage 3 embryo with ampullae distending the test (arrows). F. A stage 4 embryo which has expanded within the test. G. A young juvenile showing a residual ampulla (arrow) and developing siphons. H. A more advanced juvenile with elongate siphons (s). Scale bar in A is 100 pm; magnification is the same in each frame.

ings (Fig. 1D); stage 3 embryos are star-shaped with multiple elongate ampullae which distend the shape of the test (Fig. lE), and stage 4 embryos are spherical and larger than the earlier stages because the body cavity of the juvenile swells during metamorphosis.

Sections of stage 1-4 embryos were stained


Fig. 2. lmmunofluorescence of sectioned embryos stained with "18 antibody. A. A parasaggital section of a mid tailbud-stage M. citrina (urodele) embryo showing stained tail muscle (m) and neural (n) cells. B-C. Bright- (B) and dark- (C) field images of a frontal section through a stage 1 6. digonas embryo showing slightly elevated staining in the posterior region. The posterior region of the embryo faces the bottom of B and C. The edge of the archenteron is indicated by the x. Scale bar is 40 prn

Scale bar is 40 pm.

with the monoclonal antibody "18 and compared with a urodele control (Fig. 2). "18 detects an antigen localized primarily in neural and tail muscle cells of urodele embryos (23; Fig. 2A). Although a slight elevation in "18 staining was observed in the posterior region of B. digonas embryos, distinct cells could not be identified as members of the muscle lineage either by their location in the embryo (Fig. 28 and data not shown) or by antibody staining (Fig. 2C). A mass of relatively large cells was apparent in sections of stage 2 embryos (see Fig. 6B), however, which probably corres- ponds to the undifferentiated notochord placode described in other anural embryos (6, 22, 28). A elongate notochord composed of a single row of cuboidal cells, as seen in embryos of all urodele species, was not observed during B. digonas development. Despite the absence of differentiated muscle and notochord cells, careful observations sometimes revealed a melanin pigment spot in living stage 2 and 3 embryos (Fig. 3). Embryos with pigment spots were observed in 5 of 15 different clutches.

Juveniles at various stages of development (Figs. lG, H), along with stage 3 and 4 embryos, were recovered from the atrial cavity of adults, indicating that brooding continues through advanced stages of development (1). Although other urodele and anural ascidians are known to be ovoviviparous (3, 4), most of these spawn before or during metamorphosis, and juveniles subsequently attach to the substrate and develop into an adult outside the parent. The only other example of brooding juveniles was reported in the anural ascidian, Polycarpa tinctor (1 6).

Fig. 3. Pigment spot (arrow) in a stage 2 6. digonas embryo. Scale bar is 50 pm.

In summary, the results suggest that B. digonas embryos do not form a tail with differentiated notochord or muscle cells but can develop a melanin pigment spot which is typically present in the brain sensory organs of urodele ascidian larvae.

Muscle cell differentiation The morphological studies suggesting that

muscle cells do not differentiate in B. digonas embryos were substantiated by examining expression of AChE (26), alpha actin (24), and MHC (14, 15), specific markers for differentiated muscle cells in urodele ascidian embryos. B. digonas embryos were mixed with tadpole larvae from a urodele species and assayed for AChE activity. AChE was detected in the tail of the tadpole controls but not in stage 1-4 B. digonas embryos (Fig. 4 and data not shown). Sections of B. digonas embryos and adults and urodele controls were

Brain Melanocyte Development in an Anural Ascidian 21

Fig. 4. AChE activity is lacking in a stage 3 8. digonas embryo (left) but present in the tail of an M. occidentalis (urodele) tadpole (right). Scale bar is 50pm.

hybridized in situ with the alpha actin probe SpMA3C (24; Fig. 5) and the MHC probe pcM 11 1 (14; Fig. 6). Although actin and MHC mRNA were found to accumulate in tail muscle cells during embryogenesis of the urodele controls (Figs. 5A and 6A), autoradiographic signal representing these transcripts was not detected above back- ground levels in B. digonas embryos (Figs. 58 and 6B, C). The heterologous probes were capable of detecting RNA in this species, however, because signal was observed in muscle (Fig. 5C) and mesenchyme cells (Figs. 5C and 6D) of adults. In the same adult sections (see Fig. 6D), embryos located in the brood sacs did not show transcript accumulation, whereas the surrounding somatic tissues were heavily labelled, indicating that failure to detect mRNA with the cloned probes cannot be ascribed to transcript degradation during dissection of the embryos from the adult. The results of there experiments indicate that 5. digonas

adults with the alpha actin probe SpMA3C. A. A frontal section of an early tailbud S. claw (urodele) embryo showing transcript accumulation in the tail muscle cells. Scale bar is 20 pm. 6. cells. Scale bar is 40 pm.

A frontal section through a stage 2 B. digonas embryo lacking Fig. 5. In situ hybridization of sectioned embryos and transcript accumulation. Scale bar is 20pm. C. A section

through the siphon area of an adult B. digonas showing transcript accumulation in muscle (m) and mesenchyme (me)


embryos do not produce AChE or express genes encoding the major muscle contractile proteins.

Melanocyte differentiation The presence of pigment spots in B. digonas

embryos (Fig. 3) led to further experiments to determine whether a brain melanocyte is produced during embryonic development. Melanocyte development was assayed in two ways. First, sections of embryos were examined to determine the location and number of pigment granules. As shown in Figure 7, pigment granules were deposited in basophilic cells located in the neural region of stage 2 and 3 embryos. Although most embryos developed a single pigment granule (Fig. 7B), a few embryos showed two pigment granules (Fig. 7C). The melanocytes were not suspended in the cerebral cavity (which could be observed in other sections; see Fig. 6C), as they are in urodele embryos (compare Figs. 7A and 78, C), suggesting that the brain rnelanocyte is vestigial. Since molgulid ascidian larvae develop an otolith but not an ocellus (3, 4), we presume that the pigment cell

produced during 5. digonas embryogenesis is part of a degenerate otolith. Second, B. digonas embryos were stained for tyrosinase, the key enzyme in the pathway for melanin synthesis which is restricted to melanocyte precursors in urodele ascidian embryos (27). In this experiment, clutches of embryos that lacked pigment spots were selected for examination to avoid confusion between differentiated melanocytes and tyrosinase products produced as a result of the assay proce- dure. Although stage 1 embryos did not show tyrosinase activity (data not shown), the majority of stage 2 and 3 embryos isolated from 5 different adults contained tyrosinase positive cells (Fig. 8A). These results suggest that most, if not all, 5. digonas embryos develop tyrosinase activity. Thus, terminally-differentiated melanocytes may not have been detected in every clutch of developing embryos either because pigment cell differentiation is incomplete in some embryos or because the differentiated melanocyte is present only during a relatively brief interval preceeding metamorphosis, and some clutches do not contain

Fig. 6. In sifu hybridization of sectioned embryos and adults with the MHC probe pcM 11 1. A. A parasagittal section through a mid-tailbud H. roretzi (urodele) embryo showing transcript accumulation in the tail muscle cells. Scale bar is 50 pm, B-C. Stage 2 (B) and 3 (C) 6. digonas embryos showing no accumulation of transcripts. The dark spots seen in B and C are stained nuclei, n: notochord placode. c: cerebral cavity. Scale bar in B is 20prn; magnification is the same in B and C. D. A B. digonas adult showing transcript accumulation in mesenchyme (me) cells surrounding the brood sac. The unlabeled arrows show embryos in the brood sac that lack transcript accumulation. Scale bar is 100 pm.


Fig 8 Tyrosrnase activity in 6 digonas embryos and luveniles A A stage 2 embryo showing tyrosinase positive cells (arrow) B The neural region of a stage 2 embryo at higher magnification showing 4 tyrosinase positive cells (arrow- heads) C-D Young (C) and more advanced (D) juveniles showing tyrosinase positive cells (arrows) in the body cavity In C and D, the siphons are located on the top of each photograph Scale bar in A is 100 pm, magnification is the same in A, C, and D

Scale bar in B is 20 pm Fig. 7. Pigment granules in sectioned embryos. A.

The brain region of a tailbud stage M. oculafa (urodele) embryo showing a melanocyte with pigment granule suspended in the cerebral cavity. B-C. The brain region of stage 3 6. digonas embryos showing melanocytes with one (A) or two (6) pigment granules. Scale bar in A is 20 pm; magnification is the same in each frame.

the stage at which the melanocyte is expressed. Tyrosinase activity was observed in up to 4 pigment cell precursors (Fig. 8B), supporting previous studies with urodele species in which more neural cells were shown to produce tyrosinase than eventually differentiated into melanocytes (1 7, 22, 27). Although absent in stage 4 embryos (data not shown), numerous tyrosinase positive cells reap- peared in the body cavity of developing juveniles (Fig. 8C, D). These results suggest that distinct periods of tyrosinase expression occur in B. digonas before and after metamorphosis.

Discussion

We have examined development in B. digonas, a ovoviviparous ascidian which broods fertilized zygotes through advanced stages of post- metamorphic development (1). €3. digonas does

not develop a tailed larva with differentiated notochord and muscle cells. Our conclusions concerning notochord development are based on morphological observations in which notochord extension was not observed during embryogenesis. Strong evidence for lack of muscle cell differentiation was obtained by histochemical studies showing that B. digonas embryos do not develop AChE activity and in situ hybridization experiments showing that they do not accumulate detectable levels of muscle actin or MHC mRNA. Similar results have been obtained for muscle actin and MHC expression in embryos of the anural ascidian Molgula occulta (22).

8. digonas embryos show several features distinct from other anural developers. First, cleavage is unequal after the 4-cell stage, resulting in the formation of micromeres and macromeres. Other anural ascidians have early cleavage pat-


terns that resemble those of equal-cleaving urodele species (2, 3, 6, 22, 28, 30). Telolecithal eggs of Arnarouciurn constellaturn (a urodele de- veloper) also cleave unequally (21 ); therefore, unequal cleavage in B. digonas is attributed to the abundance of yolk, rather than to anural development. Second, B. digonas embryos do not contain cells that resemble larval muscle cell precursors when assayed either by morphology or by immunofluorescence with N N I 8 antibody, which stains muscle lineage cells in urodele ascidians (23). Embryos of other anural species do not contain terminally-differentiated muscle cells, although they retain a vestigial muscle cell lineage (6, 10, 22, 28). Perhaps, muscle lineage cells have changed fate in B. digonas embryos, as has been shown to occur for part of this lineage in embryos of the anural ascidian M. occulta (10). Third, in contrast to the anural ascidians Molgula arenata (28) and M. occulta (10, 22), B. digonas embryos do not produce AChE. A similar lack of AChE production has been reported in embryos of the anural ascidians Bostrichobranchus pilularis (28) and M. pacifica (2). Finally, B. digonas is the only anural ascidian known to express tyrosinase and develop a brain melanocyte during embryogenesis.

The 5. digonas melanocyte is not part of a definitive brain sensory organ (otolith) and is likely to be vestigial. Indeed, there would be little use for a sensory organ involved in gravity perception in an ascidian that lacks a swimming larval stage and broods juveniles through advanced stages in development. Either the melanocyte is no longer functional and destined to be lost, as in other anural ascidians, or selective pressure acting on the adult, rather than the larva, may be responsible for maintaining tyrosinase activity and a melanocyte during embryonic development. This possi- bility is consistent with the observation that many cells in the developing juvenile express tyrosinase activity. The embryonic origin of these tyrosinase positive cells is unknown, but they do not appear to be descendants of the brain melanocyte because this cell disappears during metamorphosis. Melanocyte destruction during metamorphosis also distinguishes B. digonas from urodele molgu- lids, in which the brain sensory cell persists as a part of the juvenile's neural ganglion (3, 22).

The absence of a brain pigment cell, the notochord, and differentiated muscle cells in anural embryos suggested to Berrill (3) that these urodele features are controlled by a single developmental program that is suppressed during

anural development. Experiments showing that brain pigment cells (17-19) and most tail muscle cells (20, 25, 29) are specified by different embryonic processes, do not support this hypoth- esis. Additional evidence against this idea comes from an interspecific hybridization experiment in which a melanocyte, but no differentiated muscle cells, developed in eggs of the anural ascidian M. occulta which were fertilized with sperm of the urodele ascidian M. oculata (22). Therefore, un- coupling of different urodele features during B. digonas development suggests that multiple developmental programs are changed during the evolution of anural development.

Anural development has been described in two families of ascidians, the Styelidae and Molgu- lidae (see 9 for review). Assuming ascidians with anural development are derived from urodele ancestors, the anural species must have arisen independently in these two families because they each contain many urodele developers. It has also been suggested that anural development evolved multiple times within the family Molgulidae (3). Based on differences in adult morphology, the family Molgulidae is divided into two subfami- lies (7): the Molgulinae, which contain both urodele and anural species, and the Eugyrinae, in which only anural species have been described. Most previous studies on anural development have been carried out with Molgulinae (e.g. Molgula species), and have invariably concluded that anural embryogenesis proceeds without the formation of a brain sensory organ, a notochord, or tail muscle cells (2, 3, 6, 16, 22, 28, 30). The development of a brain melanocyte in B. digonas is significant because it suggests that the anural Eugyrinae evolved from a urodele ancestor rather than from another anural molgulid lacking a brain pigment cell. The developmental program leading to melanocyte differentiation must have eventually disappeared in the Eugyrinae because this urodele feature in absent is the anural eugyrids B. pilularis (3, 28) and Eugyra arenosa (Swalla and Jeffery, unpublished results). Independent origins of anural development in different lines of ascidians im- plies that a relatively simple sequence of events may mediate a radical change in life history.

Acknowledgments: We thank N. Satoh for sectioned H. roretzi embryos and the pcM 11 1 plasmid, C. Young for help in identifying 6. digonas, and K. Hadfield for photographic assistance. This research was supported by American Association of University Women and NIH (HD-07493) Post-doctoral Fellowships (BJS) and grants


from the NIH (HD-13970) and NSF (DCB 9196041) (WRJ).

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(Received July 8, 1991; accepted September 13, 1991)

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