&p.1:Abstract The special morphological features of fresh-water planarians make them an attractive and informativemodel for studying the processes of regeneration andpattern formation. In this work, we investigate patternformation and maturation of the planarian pharynx dur-ing regeneration in tail fragments. Using three monoclo-nal antibodies (TCAV-1, TF-26 and TMUS-13) specificfor epithelial, secretory and muscle cells, respectively,we followed the sequence and timing of differentiationand maturation of these three cell types within the regen-erating pharynx. Two of these monoclonal antibodies,TCAV-1 and TMUS-13, also labelled morphologicallyimmature cells that appear to be committed to the differ-entiation pathway leading to their respective adult celltypes. Our results show that the cells forming the newpharynx come from undifferentiated cells through prolif-eration and differentiation processes rather than fromdifferentiated cells of the old stump. We describe threestages of pharynx regeneration according to the immuno-reactivity shown: (1) no immunoreactivity, correspond-ing to the accumulation of undifferentiated cells thatform the pharynx primordium; (2) immunoreactivity toTCAV-1 and TMUS-13, corresponding to the re-buildingof the pharynx; and (3) immunoreactivity to TF-26, cor-responding to a fully mature and functional pharynx. Thesequence of differentiation of these three cell types sug-gests that the pharynx grows by intercalation of new un-differentiated cells coming from the parenchyma be-tween the older pharyngeal cells, in agreement with ex-isting models of pharynx regeneration. Finally, our re-sults suggest an intercalary model for pharynx epithelialcell renewal.

&kwd:Key words Planaria · Pattern formation · Pharynx re-generation · Cell renewal · Monoclonal antibodies&bdy:

Introduction

Freshwater planarians (Platyhelminthes, Turbellaria, Tri-cladida) have a remarkable regenerative capacity. Whenthey are cut into several pieces, each tiny fragment isable to regenerate and become a new, complete individu-al (for general reviews, see Brøndsted 1969 and Baguñàet al. 1990, 1994). Because of their high regenerativepower, planarians are useful organisms for studying thecellular and molecular mechanisms leading to patternrestoration.

Morphologically, freshwater planarians have a simplethree-layered flattened structure with marked antero-posterior and dorso-ventral polarities. They possess apharynx situated in the middle of the body, between thehead and tail. This cylindrical structure is placed withinthe pharynx cavity, which opens to the ventral surface,and is attached to the rest of the body by its anteriorend. This organ has been commonly used as a morpho-logical reference marker in regeneration studies. Severalinvestigators have published valuable papers on thestructure and ultrastructure of mature and regeneratingpharynx (Skaer 1961; Kido 1961; Ishii 1962, 1964;Brøndsted 1969; Bowen and Ryder 1973; Baguñà 1973;Assai 1990, 1991).

Planarian regeneration requires a population of small,undifferentiated, self-renewing cells called neoblastswhich give rise to all differentiated cell types (for a gen-eral review, see Baguñà et al. 1990). They are scatteredthroughout the body except within the pharynx: neitherneoblasts nor mitoses have been seen in the pharynx, ex-cept in its proximal third where the presence of somecells similar to neoblasts has been reported (Baguñà1976; see Fig. 1 for terminology). Thus, it is thought thatcells involved in pharynx regeneration and cell renewalcome from neoblasts of the pharynx implantation zone(the region where the pharynx is joined to the rest of thebody). Planarian fragments lacking a pharynx will regen-erate a new pharynx completely de novo. It is not known,however, whether all cells forming the new pharynxcome from undifferentiated cells through differentiation

Edited by D. Weisblat

D. Bueno (✉) · L. Espinosa · J. Baguñà · R. RomeroDepartament de Genètica, Facultat de Biologia,Universitat de Barcelona, Av. Diagonal 645, E-08071 Barcelona,Catalonia, Spain&/fn-block:

Dev Genes Evol (1997) 206:425–434 © Springer-Verlag 1997

O R I G I N A L A RT I C L E

&roles:David Bueno · Lluis Espinosa · Jaume BaguñàRafael Romero

Planarian pharynx regeneration in regenerating tail fragments monitoredwith cell-specific monoclonal antibodies

&misc:Received: 30 September 1996 / Accepted: 6 December 1996

processes or whether some of them come from recycledcells from the nearby parenchyma.

Because of the complex histological organization ofthe pharynx; it is difficult to examine the spatial andtemporal sequence of pattern restitution during pharynxregeneration using standard histological procedures. Toimprove our understanding of the process of pharynx re-generation and how its pattern unfolds, molecular mark-ers are clearly needed. To obtain such markers, we gen-erated a planarian-specific hybridoma library (Bueno1994, 1997) and screened it for monoclonal antibodies(MAbs) that bind to different pharyngeal cell types touse them as tools for monitoring pharynx regeneration.This approach has been used successfully in other sys-tems, including regenerating hydra (Javois et al. 1986;Bode et al. 1988).The present paper utilizes cell-type-specific MAbs to study the sequence and timing of ex-pression of molecular markers associated with cellulardifferentiation during planarian pharynx regeneration intail regenerates. Two of these MAbs (TCAV-1 andTMUS-13, specific for epithelial cells of the pharynxand pharynx cavity, and muscle cells, respectively) alsoreact with the precursors of these cell types. The otherMAb used (TF-26, specific for pharynx secretory cells)only recognizes morphologically differentiated cells.Based upon our data we also discuss an alternative hy-pothesis about normal pharynx epithelial cell renewal inadult organisms.

Materials and methods

Species and culture conditions

The planarians used belong to an asexual race (class A; Ribas etal. 1989) of the species Dugesia (Girardia) tigrinacollected nearBarcelona. They were maintained in spring water in the dark at4–6°C and fed once a month with beef liver. Specimens chosen forimmunization and immunocytochemical procedures were shiftedto 17 ± 1°C and starved for 15 days before use. Organisms,9–10 mm long, were cut at caudal level (level E: Bueno et al. 1996,Fig. 6). Regenerants were maintained at 17 ± 1°C in spring water.A minimum of five organisms were analysed for each MAb andeach studied stage.

Terminology

We use the term blastema to refer to the small unpigmentedmound of tissue made of small undifferentiated cells (neoblasts;Baguñà 1981) that forms and grows above the wound. Stump re-fers to the pigmented old body below the wound and, operational-ly, it is composed of a small region near the wound (the postblas-tema) and a larger region that covers the rest of the regenerate.Planarians have a simple body plan with a main antero-posterioraxis between two terminal structures (the head and tail) clearly ho-mologous to the antero-posterior axis of the rest of the bilaterians.As the new pharynx forms, its anterior end is attached to the sur-rounding old stump at what we call the pharynx implantationzone. Thus, we will consider the pharynx implantation zone as theproximal area of the pharynx and the opposite zone (the apicalzone) as the distal area. For further details, see Saló and Baguñà(1989).

Monoclonal antibody production

We followed a protocol modified from that described by Harlow andLane (1988). For immunization, we used total and pharyngeal ex-tracts of planarians and total macerated cells. The immunization andfusion procedures are described in Romero et al. (1991) and Buenoet al. (1997). Hybridomas producing IgG MAbs were screened us-ing an enzyme-linked immunosorbent assay (ELISA) method(Miles and Hales 1968a,b) and their specificity was tested by immu-nocytochemical procedures on paraffin sections and on maceratedcells (Romero et al. 1991; Bueno 1997). Hybridomas producing themonoclonal antibody of the desired specificity were cloned by limit-ing dilution, amplified and stored in liquid nitrogen.

Immunocytochemical procedures

Paraffin sections

Immunostained paraffin sections were obtained as described byRomero et al. (1991). The presence of linked antibody was detect-ed with the Avidin-Biotin Complex method (ABC, peroxidaseconjugated; Vector) and revealed with diaminobenzidine tetrahy-drochloride (DAB; Sigma).

Immunogold semithin sections

Immunostained semithin sections were obtained as described byBueno et al. (1996). The presence of linked antibody was detect-ed with 5 nm gold-conjugated goat anti-mouse IgG (British Bio-technology) and the gold signal was amplified with silver en-hancement (Silver Enhancing Kit; BioCell). The sections werecounterstained with 0.1% toluidine blue.

Planarian maceration

Immunostained macerated cells were obtained as previously de-scribed (Espinosa 1993; Bueno et al. 1996, 1997). The presence oflinked antibody was detected with the Avidin-Biotin Complexmethod and revealed with diaminobenzidine tetrahydrochloride.

Whole mounts

Immunostained whole mounts were obtained following a protocolmodified from that described by Wikgren and Reuter (1985; Espi-nosa 1993; Bueno 1994). Organisms were treated with 1% cys-teine chloride in phosphate-buffered saline (PBS; pH 7.0) for1 min to remove their mucus, washed in PBS and permeabilized in0.5µg/ml Pronase E in PBS for 15 min at room temperature. Afterwashing in PBS for 15 min, they were fixed in 4% paraformalde-hyde in PBS for 3 h, rinsed in PBS (3×8 h) and incubated with cellculture supernatant diluted in PBST buffer (PBS pH 7.2, 0.2%NaNO3, 0.1% bovine serum albumin, 0.3% Triton X-100) for 3–4days. They were then rinsed in PBST for 24 h, incubated 2–3 daysin fluorescein isothiocyanate-(FITC) conjugated goat anti-mouseIgG (Nordic) diluted 1:50 in PBST, rinsed in PBST for 24 h,mounted in 50% glycerol in PBS and examined for fluorescence-labelled cells using a photomicroscope equipped with epifluores-cence.

Results

Adult pharynx

To monitor pharynx and pharynx cavity regeneration, weselected three MAbs from our planarian-specific hybri-

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doma library: TCAV-1, TMUS-13 and TF-26. TheseMAbs are specific for epithelial cells of the pharynx andpharynx cavity, muscle cells and pharynx secretory cells,respectively (see Fig. 1 for a morphological scheme ofan adult pharynx). Given that the pharynx is essentiallycomposed of a protective epithelium, a muscle networkthat supports the structure, a nerve net and a secretorynetwork that gives it functionality, knowledge of the dif-ferentiation pathways and of the reestablishment of theadult pattern of these cell types during regenerationshould give us a comprehensive view of how the pharynxis built.

The MAb TCAV-1 binds to endoderm-derived cells(most of the epithelial cells of the pharynx, pharynx cav-ity, pharynx lumen and gastrodermis; Fig. 2A). Withinpharynx epithelial cells, the antigen is detected through-out the three cytoplasmic regions of these cells: the api-cal cytoplasmatic sheet spread over the pharynx surface,the basal region containing the nucleus, and the elongat-ed stalk cytoplasm connecting the other two parts(Fig. 2A,B). However, not all pharynx epithelial cells areimmunoreactive to TCAV-1 (Figs. 2C,D, 3A). Non-im-munoreactive cells are scattered throughout the pharynxepithelium and represent 10% of the total pharynx andpharynx cavity epithelial cells. The epitope recognizedby TCAV-1 is glycosidic as deduced by immunodetec-tion on dot-blots after treatment of the antigen with pro-teinase K and glycosilases (endoglycosilase F and H; notshown).

The MAb TMUS-13 binds to all muscle cells of theorganism, including those of the pharynx (Fig. 4A). Themuscle pharynx network consists of several successivelayers of longitudinal, circular and transverse fibres lo-cated immediately below the basal lamina (Fig. 4A). Im-munodetections on dot-blots after treatment of the anti-gen with proteinase K or glycosdases show that TMUS-13 epitope is a protein (not shown).

Finally, the MAb TF-26 binds to most pharynx secre-tory cells (Fig. 4E). These cells, located within the pa-renchyma of the pharynx, have large processes that ex-tend from the implantation zone of the pharynx (in theplanarian body) to the secretory ring around the pharynxlumen opening (Fig. 4E). The epitope detected by TF-26is a glycoprotein based upon immunodetections on dot-blots after treatment of the antigen with proteinase K andglycosidases (not shown).

Regenerating pharynx

Regeneration experiments were performed by transectionof adult animals at a postpharyngeal level (Fig. 5). Atthis level, neither pharynx nor pharynx cavity structuresremain in the tail stump. Thus, the regenerating tail willregenerate a new pharynx without the contribution of anytissue from the old pharynx. It is necessary to point outthat the MAb TCAV-1 immunoreacts with the gastroder-mis of the tail stump as it does in adults, in the same waythat MAb TMUS-13 reacts with muscle fibres in the restof the animal. However, because the morphology ofgastrodermal cells is different from that of pharyngealepithelial cells, these two different cell types are clearlydistinguishable. MAb TF-26 immunoreacts exclusivelywith cells of the pharynx, so the stump is absolutely neg-ative for TF-26 immunostaining.

The first morphological sign of regeneration of a newpharynx is the rudiment of the pharyngeal cavity, seenin saggital sections as a slit on the postblastema at the3rd day of regeneration (not shown, but see Fig. 5 for asummarizing scheme; Assai 1990). At 3.5 days of re-generation, a group of small round cells is seen at thepostblastema contacting the anterior end of the slitforming the pharynx primordium. No immunoreactionwith any of these MAbs is detected until the 4th day of

Fig. 1 Diagram summarizingthe structure and cell organiza-tion of an adult pharynx ofDugesia (G) tigrina. Note thelocation of the basal region con-taining the nucleus of the phar-ynx cavity epithelial cells (phe)underlying the adjacent longitu-dinal and circular muscle fibres.The gastrodermis (g) extendsthe length of the animal, but on-ly the part that connects to thepharynx is shown (cepharynxcavity epithelium, cmcircularmuscle fibres, cscyanofilic se-cretory cells, g gastrodermis, lelumen epithelium, lm longitudi-nal muscle fibres, p parenchy-ma cells, phpharynx, phcpha-ryngeal cavity, phepharynx epi-thelium, phl pharyngeal lumen,pphprepharynx zone, sepsub-epidermal nerve plexus, smpsubmuscular nerve plexus, tmtransversal muscle fibres)&/fig.c:

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regeneration. At this stage, a few round small cells (fiveto ten cells) immunoreactive to TMUS-13 are observedin a central location within the pharynx primordium(Fig. 4B). These cells do not have a mature muscle cellmorphology, but instead resemble neoblasts. TheseTMUS-13 immunoreactive cells are detected in increas-ing numbers during the following days, expanding radi-ally from their initial central location (Fig. 4B,C). At4.5 days of regeneration, when the pharyngeal primordi-um protrudes into the pharynx cavity, the first cells im-munoreactive to TCAV-1 are detected (Fig. 2E). Inter-estingly, these first immunoreactive cells are not gener-ated from endoderm of the old stump, but de novo fromsurface cells of the pharynx cavity and pharynx primor-dium. They are morphologically immature: their nucleusis attached to the apical cytoplasmic sheet instead of be-ing at the end of an elongated cytoplasmic stalk, and themorphology of the apical cytoplasmic sheet is irregular

Fig. 2A–F Monodonal antibody TCAV-1 labelling in intact (A, B,C, D) and regenerating organisms (E, F). A, C, D, E, F are sagit-tal sections. Anterior is to the leftand dorsal to the top. A Paraffinsection of an intact organism. Note the basal region containing thenucleus of the pharynx cavity epithelial cells and the distal epithe-lial cells of the pharynx lumen (arrows). B Macerated pharynxepithelial cell showing the three cytoplasmic regions. C Semithinsection of the proximal region of the pharynx showing immunore-active (arrows) and non-immunoreactive epithelial cells. Arrow-head indicates the labelled nuclear region. D Semithin section ofthe distal region of the pharynx showing immunoreactive (arrows)and non-immunoreactive epithelial cells. Arrowheadindicates thelabelled nuclear region. E Paraffin section of a regenerating organ-ism at 4.5 days of regeneration. F Paraffin section of a regenerat-ing organism at 5 days of regeneration. Abbreviations are as inFig. 1 (Scale bars: A, E–F, 0.5 mm; B–D, 0.05 mm)&/fig.c:

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(Figs. 2E,F, 3B). The immunoreactive cells are locatedin the most anterior surface of the pharynx cavity andpharynx primordium, where these tissues are in contact.The rest of the pharynx cavity and apical end of thepharynx primordium are not reactive. TCAV-1 immuno-reactive cells increase in number following an antero-posterior sequence so that at 5 days of regeneration theycover almost all the pharynx cavity and pharynx primor-dium (Fig. 2F). At 5.5 days of regeneration, immunore-active cells are homogeneously distributed along thepharynx and pharynx cavity, although only 50% of theepithelial cells are immunoreactive (Fig. 6). This spatialdistribution does not change as regeneration proceeds,although the percentage of these non-immunoreactive

cells exponentially decreases until the 15th day of re-generation, when the percentage is approximately thatseen in adults (10%; Figs. 3 and 6).

At 5.5 days of regeneration, when the pharynx pri-mordium is completely protruded into the pharynx cavityand the pharynx is newly covered by TCAV-1 immunore-active cells, TMUS-13 immunoreactive cells still do not

Fig. 3A–C TCAV-1 whole mount immunostaining. Dorsal view.Anterior is to the left. A Adult organism. B Regenerating pharynxat 5 days of regeneration. C Regenerating pharynx at 8 days of re-generation. Arrows indicate TCAV-1 non-immunoreactive cells.Abbreviations are as in Fig. 1 (Scale bars0.5 mm)&/fig.c:

Fig. 4A–G Monoclonal antibodies TMUS-13 (A, B, C, D) andTF-26 (E, F, G) labelling on sagittal paraffin sections in intact andregenerating organisms. Anterior is to the leftand dorsal to thetop. A Intact pharynx. B Regenerating pharynx at 4 days of regen-eration. Arrows indicate the first TMUS-13 immunoreactive cells.C Regenerating pharynx at 5.5 days of regeneration. Arrows indi-cate TMUS-13 immunoreactive myoblasts. D Regenerating phar-ynx at 7.5 days of regeneration. Note that the pattern of adult mus-cle fibres is being restablished. E Intact organism. F Regeneratingpharynx at 7.5 days of regeneration. Arrows indicate the first TF-26 immunoreaction. G Regenerating pharynx at 9 days of regener-ation. Abbreviations are as in Fig. 1 (Scale bars: A–D, F–G,0.5 mm; E, 1 mm)&/fig.c:

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Fig. 4A–G for legend see page 429

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resemble mature muscle cells but continue to resembleneoblasts (Fig. 4C). We have not seen mature muscle fi-bres from the old stump penetrating into the developingpharynx. The first TMUS-13 immunoreactive cells witha clear muscle cell morphology are detected at 6.5 daysof regeneration. This coincides with the opening of thepharynx lumen, initially detected as a slit within the

Fig. 6 Changes in percentage of the total number of TCAV-1 non-immunoreactive cells of the pharynx and pharynx cavity during re-generation, calculated from whole mount TCAV-1 immunostainedorganisms (for each stage, n = 5 organisms). Error bars representsthe standard deviation (± S. D.)&/fig.c:

pharynx primordium. The epithelial cells that cover thisslit are TCAV-1 immunoreactive a few hours after theopening of the slit (not shown). The incipient pharynxlumen enlarges to connect the gut diverticulum with thepharynx cavity. The connection is completed by 7.5 daysof regeneration. From 6.5 to 7.5 days of regeneration,TMUS-13 immunoreactive cells progress to mature mus-cle cells. At 7.5 days of regeneration, almost all TMUS-13 immunoreactive cells have the typical muscle cellmorphology. They are seen re-establishing the adultmuscle fibres pattern (Fig. 4D). The muscle fibre net-work is completely restored by the 9th day of regenera-tion.

The first TF-26 immunoreactive cells are detected by7.5 days of regeneration. This immunoreaction is seenunderlying the pharynx epithelium at its apical endwhere the new secretory ring is being formed (Fig. 4F)and, based upon their shape and size, probably corre-sponds to the apical end of secretory cell processes at thesecretory ring zone. TF-26 labelling progressively ex-pands along the processes in a disto-proximal sequenceand, at the 9th day of regeneration, TF-26 immunoreac-tive secretory cell processes with a clearly identifiablemorphology are detected in the apical half of the phar-ynx (Fig. 4G). This progression continues until the11th–12th day of regeneration, when TF-26 immunore-active secretory cells restore their typical adult patternfrom the implantation zone of the pharynx (in the pre-pharynx) to its apical end in the secretory ring.

Fig. 5 Diagram summarizingthe regeneration of the pharynxin E cut regenerates showingthe detection of TCAV-1,TMUS-13 and TF-26 immuno-reactivity and its evolution dur-ing pharynx regeneration (ddays of regeneration, ph & phc+ cells pharynx and pharynxcavity immunoreactive cells, ph& phc - cellspharynx and phar-ynx cavity non-immunoreactivecells)&/fig.c:

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Discussion

Several authors (Brøndsted 1969; Spiegelman and Dud-ley 1973; Assai 1990, 1991; Scheilman and Kreshenko1995) have analysed planarian pharynx regeneration us-ing conventional histological procedures. The analysiswe have performed uses cell-specific MAbs as molecularmarkers to monitor the origin and fate of the most rele-vant pharyngeal cell types during pharynx regeneration.This approach allows us to overcome the difficulties pro-duced by the complex histological organization of thepharynx. Two of the MAbs we have used (TCAV-1 andTMUS-13) recognize cells with immature morphology.Assuming that these MAbs stain the same cell lineage ateach time point, they allow us to clearly detect the originof these cells, as well as to follow their maturationamong the rest of the cells of the pharynx.

Assai (1990) has shown that the pharynx primordiumoriginates from an accumulation of round/polygonal un-differentiated cells. However, it was unclear whether allcells of the new pharynx come from these cells by prolif-eration and differentiation processes or whether somedifferentiated cells of the old stump also participate inthe building of the pharynx. Our results clearly showthat, during regeneration, pharynx muscle and pharynxepithelial cells are generated de novo from morphologi-cally undifferentiated cells of the pharynx primordium(muscle cells) or its surface (epithelial cells). We havenot detected mature muscle fibres from the old stumppenetrating into the developing pharynx or cells of theexisting endoderm (that is also TCAV-1 immunoreactive)forming the new pharynx cavity or its epithelium. Thus,these results reveal that, at least for these cell types, theyare generated de novo from morphologically undifferen-tiated cells of the pharynx primordium and not from dif-ferentiated cells of the old stump. Saló and Baguñà(1984) have proposed a model for planarian regenerationin which blastema formation is an epimorphic processrequiring cell proliferation, as well as morphallactic pro-cesses to restore proper proportions. Our results clearlydemonstrate the epimorphic origin of the new pharynxand are consistent with this epimorphic-morphallactic re-generation model because the new pharynx arises withinpre-existing tissues.

The timing of differentiation of these three cell typesmonitored with the MAbs TCAV-1, TMUS-13 and TF-26 allows us to distinguish some main steps during phar-ynx regeneration. In a first step, an unknown and unde-tected signal indicates the place where the new pharynxcavity should form (initially as a slit; Assai 1990) andwhere neoblasts should accumulate to form the pharynxprimordium. The presence of a molecule restricted in thecentral region of the organism has been described (Bue-no et al. 1995, 1996). At the 2nd day of regeneration,this molecule is expressed in a few cells (five to tencells) located in the place where the new pharynx pri-mordium will form. This molecule may be related to thefirst positional signal for the regenerating pharynx. In asecond stage, cells that are involved in the building of

the pharynx (epithelial and muscle cells) differentiate, asdetected with the MAbs TCAV-1 and TMUS-13. In athird step, initiated after the rebuilding of the pharynx ar-chitecture, cells involved in pharynx functionality (secre-tory cells) differentiate (as detected with the MAb TF-26). The starting point of this stage, by 8–10 days of re-generation, coincides with the recovery of food respons-es. TF-26-labelled cells have been described as cells se-creting glycoproteineic lubricant mucus (Baguñà 1973)as well as digestive enzymes (Pascolini 1967; Jennings1975). Thus, TF-26 labelling would indicate the restora-tion of secretory ability, that is, the recovery of pharynxfunctionality.

By the time that the pharynx primordium has protrud-ed into the pharynx cavity, cellular divisions are not de-tected in the pharynx primordium (Baguñà 1976). Thus,the pharynx likely grows by incorporation of new undif-ferentiated cells coming from the implantation zone.When new cells are recruited to the growing pharynx,they could follow two hypothetical paths: (1) remainclose to the base of the pharynx, so the pharynx wouldgrow by distal displacement of older cells; or (2) interca-late between the older cells. If newly incorporated cellsremain close to the base and differentiate following thesuccession of their recruitment and accumulation in thepharynx primordium, we would expect to find the oldercells (that is, the first immunoreactive cells) in the distalareas of the pharynx, following a disto-proximal se-quence of differentiation. Instead, we have seen that nei-ther pharynx epithelial cells nor pharynx muscle cellsfollow this sequence of differentiation. Immunoreactivepharynx and pharynx cavity epithelial cells differentiatefollowing a proximo-distal sequence, from areas wherethe pharynx epithelium contacts the pharynx cavity epi-thelium (that is, from the implantation zone of the phar-ynx) to the distal areas of both. Immunoreactive pharynxmuscle cells, however, are first detected in a central posi-tion within the pharynx primordium and they differenti-ate from this location to more external areas of the phar-ynx following a radial sequence. Both sequences couldbe explained by considering that (1) the newly incorpo-rated cells do not remain at the base of the pharynx and,instead, intercalate within the former pharynx primordi-um, or (2) they do not differentiate following the succes-sion of their recruitment and accumulation in the phar-ynx primordium and, instead, the sequence of their dif-ferentiation may be due to temporal and/or positionalmechanisms. Thus, in both tentative explanations, cellslocated distally in the pharynx are not necessarily older(in a later stage of differentiation) than the ones locatedproximally.

TF-26 immunolabelling is detected when the newpharynx has almost finished its growth. Its labelling pro-gressively expands along the processes in a disto-proxi-mal sequence, from the pharynx secretory ring to thepharynx implantation zone. This sequence of immunola-belling is different from that shown by the MAbs TCAV-1 and TMUS-13. Considering that in adult pharyngesTF-26 immunoreactive secretory cells have large pro-

cesses originating in the pharynx implantation zone go-ing to the pharynx secretory ring, two tentative sugges-tions may be given to explain the sequence of TF-26 im-munoreactivity: (1) the secretory cells could first be lo-cated underlaying the secretory ring and then may dis-place its basal region from the secretory ring to the im-plantation zone as pharynx maturation proceeds; or (2)the secretory cells may attain their adult morphologywhen the first immunolabelling is detected, but the MAbmay label only the apical area of the cell processes. If so,the immunolabelling sequence could reflect the accumu-lation sequence of the recognized antigen, from the se-cretory ring to the implantation zone through the secreto-ry processes, being present (accumulated) in the entirelength of the secretory cells when adults. Our observa-tions fit better with the second alternative, as the first im-munoreactive cells detected do not have the shape andsize of mature secretory cells or undifferentiated cellsbut instead resemble the apical end of secretory cell pro-cesses. Moreover, histological observations at 8–9 daysof regeneration (not shown) show the presence of secre-tory cells within the pharynx from the implantation zoneto the apical secretory ring.

Some TCAV-1 non-immunoreactive cells interspersedbetween the immunoreactive ones are observed through-out the pharynx epithelium, decreasing in number as re-generation proceeds. To explain the presence and dy-namics of these non-immunoreactive cells, several expla-nations could be suggested. 1. These non-immunoreac-tive cells could be a subpopulation of pharynx epithelialcells that does not express this molecule, disclosing anunexpected heterogeneity within this tissue. If this is so,the percentage of these two subpopulations of cellsshould vary during regeneration, perhaps indicating adifferent functionality between them. 2. These cellscould be senile cells that have ended the expression ofthe recognized molecule. This hypothesis would not bein agreement with the decrease of non-immunoreactivecells during pharynx regeneration. 3. Finally, these cellscould be juvenile cells that do not yet express the mole-cule, corresponding to those described by Ishii (1995) bymorphological criteria. This hypothesis would be inagreement with the decrease of non-immunoreactivecells during pharynx maturation, implying that epithelialcell renewal in the pharynx occurs by incorporation ofjuvenile cells within the pharynx. The main objection tothis hypothesis is that neither neoblasts nor mitoses havebeen detected in the pharynx (Baguñà 1976), perhapsdue to a low proliferation rate within the pharynx and/orthe low number of these hypothetical juvenile cells in theadult pharynx.

&p.2:Acknowledgements We would like to thank: Eduard Batlle andMarc Aureli Soriano for their invaluable help and technical exper-tise in monoclonal antibody screening and immunocytochemicalwork; Almudena García and Nuria Cortadellas for advice aboutsemithin immunochemical techniques; Phillip Newmark for help-ful discussions and critical reading of the manuscript. The com-ments and suggestions of the referees that made the manuscripttechnically sounder and more readable are warmly acknowledged.This work was supported by grants to J.B. from the Comisión

Asesora de Investigación Científica y Técnica (Ministerio de Edu-cación y Ciéncia, España; PB89-0249 and PB 92–0532).

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