A different type of amphibian mesoderm morphogenesis in Ceratophrys ornata

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1 Development 117, (1993) Printed in Great Britain The Company of Biologists Limited A different type of amphibian mesoderm morphogenesis in Ceratophrys ornata Susan M. Purcell* and Ray Keller Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA *Present address: Department of Pharmacology, School of Medicine, University of Washington, Seattle, WA 98195, USA SUMMARY Ceratophrys ornata, the Argentinean horned frog, has a significantly different pattern of early morphogenesis than does the most studied amphibian, Xenopus laevis. Time-lapse videomicroscopy, scanning electron microscopy, histological sections and lineage tracers have shown that, in C. ornata, some prospective notochord, somite and tailbud mesoderm cells leave the surface epithelium of the archenteron by ingression. After gastrulation, SEM reveals cells with constricted apices and a bottle shape in three zones on the archenteron roof and in a fourth zone around the blastopore. Prospective somitic tissue ingresses first from two lateral zones, followed by ingression of prospective notochord from the medial zone and tailbud mesoderm from the circumblastoporal zone. This is unlike X. laevis, in which no cells with constricted apices are present on the dorsal surface of the archenteron, nor do any cells ingress into the deep mesodermal layers from the surface layer. Key words: mesoderm, notochord, ingression, amphibian INTRODUCTION It was long thought that early and fundamental processes of development such as gastrulation, were conserved features of development since their alteration would have repercussions on later events. But it appears that gastrulation and mesoderm morphogenesis are in fact highly variable processes (Ballard, 1981). In the amphibians, an important issue is the tissue fates and organization of the marginal zone. There has been a controversy for decades over whether the origin of the mesoderm is the same in all anuran amphibians (frogs and toads) (Løvtrup, 1966, 1975; Nieuwkoop and Sutasurya, 1976, 1979; Hanken, 1986). Some ectoderm of the marginal zone is induced to form mesoderm by the subblastoporal endoderm during the late blastula stages in amphibian embryos (Nieuwkoop, 1969; Sudarwati and Nieuwkoop, 1971). The marginal zone involutes through the blastopore during gastrulation, internalizing the endodermal and mesodermal layers. In the anuran Xenopus laevis, only prospective endoderm and ectoderm are found on the surface of the pregastrula embryo. The mesoderm forms in the deep layers and remains there throughout gastrulation (Nieuwkoop and Florshütz, 1950; Sudarwati and Nieuwkoop, 1971; Keller, 1975, 1976). Keller s fate map of X. laevis is different from earlier fate maps of other anurans. Vogt s fate map of Bombina - tor (Bombina) (1929) and Pasteels map of Discoglossus (1942) show surface mesoderm in the marginal zones of these anuran embryos, implying that these species differ from X. laevis. Since Keller published the fate map of X. laevis in 1975, some have speculated that Vogt s and Pasteels fate maps may be incorrect (Løvtrup, 1975; Nieuwkoop and Sutasurya, 1976). To begin resolving this issue, we have analyzed gastrulation movements and mesoderm morphogenesis in the Argentinean horned frog, Ceratophrys ornata, a species of frog not closely related to X. laevis (Duellman and Trueb, 1986). The embryo of C. ornata differs from that of X. laevis in having prospective mesoderm in the surface layer at the gastrula stage. These findings are significant for understanding the cellular mechanisms of morphogenesis and pattern formation in anurans, and for understanding the evolution of morphogenetic processes that occur in early vertebrate embryogenesis. MATERIALS AND METHODS Obtaining and handling of embryos Fertilized Ceratophrys ornata eggs were obtained from Bob s Happy Fish (Rt 4 Box 605, Woodland, CA 95695) and were kept in 15% Steinberg s solution or 15% modified Barth s solution. Eggs range in size from 1.5 to 2.0 mm in diameter. The outer fibrous jelly coat and the vitelline envelope were removed with sharpened forceps, and the inner jelly coat was removed by soaking in a solution of 3.5% cysteine hydrochloride at ph 7.9 for 2 to 5 minutes. All manipulations were done in 35 mm plastic Petri dishes, some with a base of 2% agarose.

2 308 S. M. Purcell and R. Keller Staging of embryos Staging of C. ornata is according to the table of normal development for this species (Purcell and Brothers, J. A., unpublished data). Stage 10: early gastrula, formation of the blastopore lip. Stage 13: blastopore closing, 1/5 original diameter. Stage 13.5: slit blastopore. Stage 14: neural plate formation. Stage 15-16: neural folds closing. Stage 17: neural tube closed, anterior somitic furrows formed. Stage 18: All somitic furrows of the body axis formed, tailbud formed. Videomicroscopy Explants of the dorsal side of the embryo were made at stage 13 and 13.5, by a modification of the method of Wilson et al. (1989), but unlike their dissection, here the epithelium of the roof of the archenteron was left intact (Fig. 1). The explants were made and cultured in 100% modified Danilchik s solution, which prevents healing and curling up in culture and supports normal development (Keller et al., 1985). Explants in this medium may live for 3-4 days, well past the end of recording at about 24 hours after fertilization. Images of explants were recorded for 9 hours, until tailbud stage. Recordings were made using a Zeiss upright compound microscope with a Nikon 4 or a Zeiss 10 plan objective, low angle epi-illumination and a DAGE-MTI 81 high resolution video camera. Images were processed by averaging 32 frames and using contrast control features of an Image One video image processor (Universal Imaging, Media, PA), and recorded on a Panasonic TQ-2028F optical memory disk recorder (OMDR). Scanning electron microscopy Embryos were fixed in a solution of 2% glutaraldehyde in 0.10 M sodium cacodylate buffer (ph 7.4) for 12 hours at 4 C. Embryos were cut with a microscalpel to expose the archenteron roof; ventral halves were discarded (Fig. 1). Some embryos were cut transversely or obliquely to expose the deep cell layers. Embryos were then critical-point dried with liquid CO2, mounted on stubs with silver paint, coated with platinum by standard methods, and viewed on an ISI-DS130 scanning electron microscope with an accelerating voltage of 15 kv. Histology Embryos were placed in Smith s fixative for 12 hours at 4 C, dehydrated using an ethanol series, embedded in Paraplast using Histosol and cut into 10 µm sections. Sections were stained with giemsa stain, and mounted with Permount. Embryos were fixed at gastrula (stage 10) through tailbud (stage 18). Vital dye marking Vital dye marking of the surface cells was done using Nile blue dye in agarose chips (Keller, 1975), which were placed up against the embryo for 15 seconds. This is long enough for the surface cells under the chip to take up the dye. Control embryos were dissected at stage 13, before any ingression occurred, to be sure only surface cells were taking up the Nile blue. Embryos were marked at stage 10 and dissected at stage 15,16,17 or 18 to locate the dye marks. RESULTS Cell movements recorded by time-lapse videomicroscopy Videorecordings of the dorsal surface of the archenteron and the blastopore region in explants from stage 13 to stage 18 show that apically constricted cells in four zones leave Fig. 1. Explant technique for videomicroscopy of the archenteron roof and for SEM is shown. For videomicroscopy, stage 14 embryos were cut between the lateral edge of the archenteron roof and the ventral yolk mass. The dorsal piece was removed, turned upside down and placed under a coverslip for filming such that the epithelium of the archenteron could be seen. The coverslip was positioned using vacuum grease. For SEM preparations, embryos were fixed first at different stages and then the explants were made as shown before dehydration. bp, blastopore, a, anterior, d, dorsal. the surface epithelium and ingress (Figs 2,3). The boundaries between zones I and II are substantive in that cells were not seen to cross this boundary, although cell rearrangement was minimal in any case. Cells in the medial and lateral zones first constrict their apices (arrows, Fig. 2A,B), and then leave the epithelial layer (Fig. 2A,C). This happens rapidly, occurring within a few hours at room temperature. In the recordings, only cells with tightly constricted apices leave the epithelium, and all constricted cells eventually disappear. The high correlation between these two cell behaviors suggests that apical constriction may be necessary for ingression to occur. Cells with apical constriction appear dark due to concentration of apical pigment granules into a smaller area. At the completion of ingression, the non-ingressing cells of the lateral archenteron surface meet at the midline. These cells do not constrict their apices (arrowhead, Fig. 2A,C). This process was observed in four recordings, each using an embryo from a different batch, with no significant differences between them. Fig. 3 shows tracings made from the video recording shown in Fig. 2, with selected cells outlined and numbered to illustrate the points made above. The two lateral zones of ingression are shaded. The medial zone and the noningressing cells are unshaded. In the first 25 minutes of the recording (Fig. 3A,B), cells in the lateral zones, numbered 1,2,3,5,6,18 and 21, disappear. None of the cells in the medial zone disappears. Cells in one of the lateral zones have ingressed completely, and the other lateral zone is significantly smaller. In the second 25 minutes of the recording (Fig. 3B,C), cells in the other lateral zone complete their ingression, leaving only cells in the medial zone, which have not fin-

3 Mesoderm morphogenesis in C. ornata 309 ished ingressing. Cells numbered 10,15 and 16 in the medial zone have left the surface layer, and the remaining medial zone cells have decreased their apical surface area. In Fig. 3A, the non-ingressing cells numbered 4 and 22 are separated from the midline by many rows of cells, however, they end up quite close to it (Fig. 3C). The recordings show clearly that cells with constricted apices leave the surface of the epithelium. During the period illustrated in this tracing, cells numbered 1-3, 5, 6, 10, 15, 16, and disappear from the surface layer. The noningressing cells, numbered 4 and 22, do not significantly decrease their apical surface area. Non-ingressing cells do not leave the surface in any of four recordings made. The cells numbered 1, 2, 3, 5, 6 and in the two lateral zones disappear before the cells numbered 7-17 in the medial zone. It is significant that these preparations are explants with most of the surrounding tissue removed (Fig. 1), and still ingression occurs. In appears that the non-ingressing cells are dragged together by the ingressing cells, as they do not show any protrusive activity characteristic of migrating cells in the video recordings. Cell morphologies seen in scanning electron micrographs In SEMs at the end of involution (stage 13, small blastopore), cells on the roof of the archenteron have apices of uniform size and the epithelium shows no zones of apical constriction (Fig. 4A). Epithelial cells form the lining of the archenteron as shown by the apposition of their margins to form an unbroken sheet (Fig. 4A). Cells in a broad zone on the dorsal surface of the archenteron begin to constrict their apices at stage 13.5 (slit blastopore) after the marginal zone has completely involuted and the blastopore has closed. This zone narrows as the cells progressively constrict their apices. By stage 15 (midneurula), four distinct zones of apical constriction are clearly visible in the archenteron epithelium (Figs 4B,5). A row of constricted apices appears down the midline of the archenteron, flanked by rows of less constricted cells. This medial zone of ingressing cells constitutes zone I (Fig. 5). Lateral to zone I are bilateral areas of apical constriction, zones II. Finally, there is a ring of apical constriction around the blastopore several cells wide, zone III (Fig. 5). By stage 16 (late neurula), the lateral zones (II), much of the medial zone (I) and much of the circumblastoporal zone (III) has disappeared from the surface layer (Fig. 4C). The cells in zones I and III, which remain in the epithelium at stage 16, ingress completely by stage 18, and the non-ingressing cells on either side meet along the midline of the archenteron. Fig. 2. Photos taken from the videomonitor, showing frames of a recording made using the OMDR of an explant from a stage 13.5 (closed blastopore) embryo of C. ornata. Time between photos is 25 minutes. Anterior is to the right. (A) Early neurula, stage 15. Many cells with constricted apices (dark cells) are visible. The medial zone, containing a central area of constricted cells and flanking regions of less constricted cells, and two lateral zones of apical constriction can easily be seen. The arrows indicate cells with small apices that are about to leave the epithelium. The arrowhead shows a non-ingressing cell. (B) Midneurula stage. The two lateral zones have almost completely disappeared. The medial zone is narrower and longer. (C) Late neurula stage. The lateral zones have completely ingressed, and the medial zone is much narrower. The arrow indicates a medial cell and the arrowhead shows a non-ingressing cell. The large non-ingressing cells fill the top half of the photo. The non-ingressing cells on each side will eventually meet at the midline. Scale bar, 50 µm. m, medial zone cell, l, lateral zone cell.

4 310 S. M. Purcell and R. Keller Histological sections and SEM views of the basolateral cell surfaces Analyses of serial transverse sections and SEMs of transversely fractured specimens at successive stages show that cells in the two lateral zones of ingression are extending into the somitic mesoderm, while cells in the medial zone are extending into the notochord. The deep boundaries between the notochord and somitic tissue have already formed by the midneurula stage when cells begin to leave the epithelium, and correspond to the surface boundary between zones I and II. No cells were ever seen in transition crossing the boundary between notochord and somite. Ingressing cells from the medial zone are seen extending into the notochord (Fig. 6A, arrow). The boundaries of the medial zone of ingressing cells are in register with the lateral boundaries of the notochord (Fig. 6B). Ingressing cells from the lateral zones extend into the somitic mesoderm (arrows, Fig. 6B). At late neurula stages when cells in the two lateral zones have completed their ingression, cells with small apices can be seen extending only into the central notochord area (arrow, Fig. 6C). SEMs of transversely and parasagittally fractured specimens show cells in all stages of ingression. Some appear just to have left the epithelium (arrowhead, Fig. 7A), and others have constricted apices and are still attached to the epithelium (arrow, Fig. 7A). In transversely fractured specimens, ingressing cells of the lateral zone appear to curve away from the notochord as they ingress (Fig. 7B). The basal and lateral surfaces of the ingressing cells show many protrusions and attachments to the deep cells (Fig. 7A,B). Matrix is associated with the deep end of the bottle cells (Fig. 7A), and appears as white clumps due to the dehydration process used in preparing specimens for SEM. The ingressing cells show no apparent change in volume. In video recordings of the archenteron roof, the ingressing cells appear to get smaller, while the non-ingressing cells appear to get larger. The serial transverse sections show that this is because the non-ingressing cells become more squamous, while the ingressing cells squeeze most of their surface out of contact with the lumen of the archenteron and into the basal part of the cells. After ingression, the circumblastoporal zone cells form a large mass of mesenchymal tissue around the blastopore. This mesenchymal tissue is attached to the posterior notochordal and somitic mesoderm in SEMs and histological sections of tailbud embryos. Fig. 3. Cell tracings of video frames in Fig. 2. The dotted line represents the midline. Lateral zone cells are shaded, the medial zone and non-ingressing cells are unshaded. Selected cells are numbered in each region. (A) Cells 4 and 22 are non-ingressing, 1-3, 5, 6 and are in the two lateral zones, and cells 7-17 are in the medial zone. (B) The two lateral zones have almost completely disappeared. The numbered cells that are no longer present have ingressed from the epithelium into the deep layer. (C) The lateral zones have completely ingressed, and many of the cells from the medial zone have also ingressed. All the medial cells remaining in the surface layer have smaller apices than they did at the beginning of the recording. The two non-ingressing cells, 4 and 22, have not appreciably decreased their apical surface area. a, anterior, p, posterior. Vital dye confirmation of cell fates Nile blue marks were used to confirm that surface cells from the gastrula marginal zone had actually ingressed into the somites, notochord and tailbud mesoderm by the tailbud stage, as suggested by SEMs and histological sections. For dye marking experiments, the embryo was divided into longitudinal quadrants; the results were assigned to one of three sections, depending upon the quadrant in which the initial mark was made (Fig. 8). Quadrant 1 is centered on the blastopore lip, which forms on the dorsal side at stage 10. The left and right lateral quadrants are labelled 2, and the ventral-most quadrant, in which the blastopore lip forms last, is labelled 3.

5 Mesoderm morphogenesis in C. ornata 311 Fig. 4. Scanning electron micrographs of the archenteron roof of Ceratophrys ornata. (A) Stage 13 (late gastrula). Archenteron roof shows no cells with constricted apices. (B) Stage 15 (mid neurula). There are four zones of cells with constricted apices: one medial zone, two lateral zones, and a circumblastoporal zone (compare to Fig. 5). Open arrows indicate cells with small apices in each of the zones. Solid arrows show the lateral boundaries of the two lateral zones. Arrowheads show the lateral boundaries of the medial zone. (C) Stage 16 (late neurula). Ingression is almost complete, with only some medial and circumblastoporal cells remaining in the surface layer. Arrowheads show the lateral boundaries of the medial zone. Arrow shows an ingressing medial cell. a, anterior, bp, blastopore, p, posterior, m, medial cell, l, lateral cell, c, circumblastoporal cell. Scale bars, 50 µm; scale for A and B is the same. Most of the surface mesoderm in quadrant 1 was found in the posterior half of the notochord, with labelled cells primarily in the ventral portion of the notochord. The surface mesoderm in the rest of the marginal zone was found in the medial portion of the somites next to the notochord, and in the tailbud mesoderm (Table 1). Rarely, dye was found in lateral plate mesoderm near the tailbud. Control embryos were dissected at stage 13, before any ingression had occurred to confirm that only the surface layer was labelled. Summary of Ceratophrys gastrulation and mesoderm morphogenesis In the pregastrula embryo, the surface layer of the involuting marginal zone contains prospective endoderm, posterior notochord, somite and tailbud mesoderm. The blastopore lip forms on the dorsal side in typical amphibian fashion, and involution of the marginal zone begins. The lip forms closer to the equator of the blastula in C. ornata than in Xenopus laevis. The lip then extends to the lateral and ventral sides. The blastopore then closes down and the yolk plug is internalized. After gastrulation is complete, the dorsal and lateral surfaces of the archenteron are lined with the surface layer of the involuting marginal zone, and the ventral surface is lined with cells from the subblastoporal endoderm, as in X. laevis (Keller, 1986). The involuted roof of the archenteron consists of suprablastoporal endoderm anteriorly, and prospective somitic mesoderm, prospective

6 312 S. M. Purcell and R. Keller Fig. 5. Schematic diagram of Fig. 4B, showing the boundaries of the four zones of ingressing cells. Zone I is ingressing medial cells, the zones labeled II are ingressing lateral cells, and zone III is ingressing circumblastoporal cells. Dashed line at the anterior boundary of zone III indicates that this boundary is indistinct. a, anterior, p, posterior, bp, blastopore. notochordal mesoderm and prospective tailbud mesoderm posteriorly. About two thirds of the mesoderm of the notochord, somites and tailbud is located in the deep layers before gastrulation, where it remains and does not undergo ingression (Fig. 9). The mesoderm located in the surface layer before gastrulation ingresses after apical constriction in four zones, beginning at stage 13.5 (slit blastopore). Cells in the two lateral zones finish their ingression first, adding to the somitic tissue, then cells in the medial zone and the circumblastoporal zone finish their ingression, drawing together the endoderm cells originally located on either side. Medial cells contribute to the notochord, and the circumblastoporal cells form part of the mesenchymal tissue of the tailbud mesoderm. The tailbud will differentiate at Fig. 6. Transverse sections of paraplast embedded embryos of C. ornata. Dark pigment concentrations show apical constriction. (A) Stage 15 embryo, midbody. The arrow indicates a very elongated cell ingressing into the center of the notochord. (B) A more posterior section of a stage 15 embryo, close to the blastopore. A broad section of the ventral notochord is exposed to the lumen of the archenteron. The arrows indicate ingressing lateral cells with tightly constricted apices and their basal ends extended into the somitic tissue. (C) Stage 16 embryo. In this later stage embryo, the somitic tissue is no longer in contact with the lumen of the archenteron, and only a small portion of the notochord is still in contact with the lumen. The arrow indicates an ingressing medial cell. Scale bar, 20 µm. np, neural plate, s, somite, n, notochord, e, endoderm, a, archenteron lumen.

7 Mesoderm morphogenesis in C. ornata 313 Fig. 8. Illustration of longitudinal quadrants used in analyzing the results of the vital dye experiments. Quadrant 1 is centered on the dorsal midline, the two lateral quadrants are both identified as 2, and the ventral quadrant is 3. The involuting marginal zone, where the dye marks were made, is shaded. See Table 1 for results. a, animal pole, v, vegetal pole, b, blastopore lip on dorsal side. and third is the implications for mesoderm pattern formation during blastula and gastrula stages. The study of C. ornata gives us the opportunity to study an important cell behavior, ingression, which is absent in the development of the most commonly studied amphibian, Xenopus laevis. The absence of this cell behavior in X. laevis has implications for using that species as a model for amphibian mesoderm induction and morphogenesis. Fig. 7. Scanning electron micrographs of ingressing cells in lateral zones of C. ornata. (A) Parasagittal fracture through zone II, showing cells ingressing into the somitic tissue. These cells have a bottle shape, with constricted apices and dilated bases. The arrowhead indicates a cell that had just left the epithelial layer. The arrow indicates a cell with its apex still attached to the epithelium. The open arrows indicate the broken edge of the epithelium. Note also the visibly uneven distribution of extracellular matrix, which appears as white spheres after dehydration. The matrix becomes more dense towards the basal end of the cells. (B) Transverse cut showing an ingressing cell from zone II. Notice the very constricted apex and broad basal end. Filiform protrusions can be seen on the basal end of the cell indicated by the arrowhead. Notice that the basal end of the ingressing cell curves away from the notochord. Open arrows indicate the broken edge of the epithelium. Scale bar, 10 µm. n, notochord, s, somite. later stages into several tissue types. These ingressing mesoderm cells add to the mesoderm cells already in the deep layer (Fig. 9). DISCUSSION Several aspects of the development of Ceratophrys ornata are especially significant. The first is the mechanics of ingression and its partially tissue-specific nature, the second is comparisons of the developmental pattern in C. ornata with those of other anurans, urodeles and other vertebrates, The mechanics of ingression The cells with constricted apices are likely the driving force in the process of ingression for four reasons. First, apical constriction has been shown to be an active, force-generating process in a variety of systems, including neural tube formation (Burnside, 1973; Löfberg, 1974; Schoenwolf and Franks, 1984), optic cup formation (Owaribe et al. 1981), amphibian blastopore formation (Hardin and Keller, 1988; Baker, 1965; reviewed by Ettensohn, 1985) and ventral furrow formation in Drosophila (Leptin, 1991). Second, the non-ingressing cells, which will end up meeting along the midline, do not show activity indicative of migration in the videorecordings and so are unlikely to be crawling over the ingressing cells. Third, SEMs show many filiform and lamelliform protrusions on the deep side of the ingressing cells. These types of protrusions are associated with cell movement (Trinkaus, 1984) and are a fair indication of the direction that the cells are moving. Finally, and most importantly, the cells constrict and ingress even in an explant, when most of the surrounding tissue has been removed (such as in the videomicroscopy results). Thus, it is unlikely that those surrounding tissues actively squeeze ingressing cells out of the surface layer. Zones of ingression I and II are tissue specific Before ingression begins, the boundary between notochord and somite is already well formed in the deep tissue of the body axis. The boundaries between the notochord and somitic tissue in the deep layer are exactly in register with the boundaries between zones of ingression I and II. These surface and deep boundaries are likely the same boundary.

8 314 S. M. Purcell and R. Keller Table 1. Vital dye marking of surface cells in the marginal zone Location of dye at stage 18 Quadrant of Total marginal embryos Other zone marked marked Notochord Somite mesoderm 1 (dorsal) (lateral) (ventral) Summary of data from vital dye marking experiments in which marks fell entirely within one of the four quadrants (see Fig. 7). The embryo was divided into quadrants with quadrant 1 centered on the dorsal midline. For each quadrant, at least two different batches of embryos were used. Other mesoderm is mostly tailbud mesoderm and some posterior lateral plate mesoderm. Most marks also labelled archenteron endoderm. If ingression were not tissue-specific, we would expect to see cells crossing one or both of these boundaries during ingression. However, no cells were seen crossing the surface boundary during videomicroscopy, nor were any cells seen spanning the boundary in the histological sections. Cells do not normally cross the notochord-somite boundary in X. laevis, along which cell behavior has been extensively recorded (Wilson, 1991; Wilson et al., 1989; Keller et al., 1992; Shih and Keller, 1992b). In C. ornata, the basal ends of ingressing cells are embedded in the deep tissue that they will join while the apical ends are still firmly a part of the epithelium. Cells appear to be directionally ingressing into the somitic tissue or notochord, as can be seen in views of the lateral zone cells ingressing and clearly curving away from the midline. In the circumblastoporal region, however, there are no boundaries yet formed, and so it is possible that cells are ingressing prior to specification as a particular tissue type. The tailbud region, into which cells ingress, is a mesenchymal mass, which will differentiate into several tissue types and has no apparent internal boundaries when first formed. Although ingression occurs in the development of other organisms such as the sea urchin (primary mesenchyme cells) and the chick (endoderm and mesoderm cells of the primitive streak), C. ornata offers the first example of spatially organized, simultaneous ingression of several tissue types. Surface mesoderm in other anurans Differences in early development have been found among the few species of anurans examined. Keller (1975) clearly showed that X. laevis has no prospective mesoderm in the Table 2. Summary of the positions of prospective mesoderm and the timing of ingression in three amphibians Anuran Urodele tissues Ceratophrys Xenopus Ambystoma prospective somite surface and deep deep surface prospective notochord surface and deep deep surface ingressing somite after involution none during involution ingressing notochord after involution none after involution surface layer at stage 10. It has been proposed that the X. laevis pattern may be common to all anurans (Løvtrup, 1975; Nieuwkoop and Sutasurya, 1976, 1979), and that early fate maps of Bombina variegata (=pachypus) (Vogt, 1929) and Discoglossus (Pasteels, 1942) were inaccurate. However, Vogt s fate maps of urodeles showing surface mesoderm were correct (Vogt, 1929; Smith and Malacinski, 1983; Lundmark, 1986; Delarue et al., 1992). Of the three anuran fate maps so far constructed, only the one for X. laevis has no prospective mesoderm in the surface layer. Other anurans have also been reported to have surface mesoderm at early gastrula stages using histology and transmission electron microscopy. These are Hyla regilla (Baker, 1965), Bufo americanus (=lentiginosus) and Rana palustris (King, 1903) and Ceratophrys ornata (Fig. 10). Ruffini (1925) analyzed the development of Rana esculenta and Bufo bufo (=vulgaris). He concluded that the mesoderm arose entirely from the deep layer in anurans. A reexamination of his figures reveals that B. bufo appears to have zones of cells with constricted apices on the archenteron roof much like those in C. ornata. Ruffini may have misinterpreted his data from B. bufo because he saw deep mesoderm in that species and may not have considered the possibility that the notochord could arise from both the surface and the deep layers. Surface mesoderm in urodeles The pattern of cell movement in C. ornata differs from that in previously studied amphibians both in the timing of the ingression of mesoderm, and the quantity of mesoderm originating in the surface and deep layers (Table 2). X. laevis has no surface mesoderm and no ingression (Keller, 1975). In the salamander Ambystoma mexicanum, most of the mesoderm is from the surface layer; the somitic cells ingress during gastrulation, and the notochord cells ingress after gastrulation is complete (Pasteels, 1942; Smith and Malacinski, 1983; Brun and Garson, 1984; Lundmark, 1986). Surface mesoderm in ancestral amphibians We can hypothesize as to what sort of pattern the ancestral anuran and ancestral amphibian had by using phylogeny. Traits that were present in the earliest member of a group are referred to as ancestral characters and traits arising later in the evolution of that group are referred to as derived characters. X. laevis is not considered to be a primitive anuran, even though it has some ancestral characters (e.g., presence of dorsal ribs), because it has many derived characters unique to pipid frogs (Cannatella and Trueb, 1988). We cannot assume that a trait found in one species will be the ancestral anuran type, even if that species is considered primitive. To determine which type of mesoderm formation is the likely ancestral condition for anurans, a group outside the anurans is needed. It is not possible to make comparisons solely within a group and determine the direction of evolution of a trait (Wiley, 1981). The best out groups are sister taxa. Urodeles (newts and salamanders) and caecilians (legless amphibians) are sister groups to anurans (Fig. 10). In newts and salamanders, most of the mesoderm originates in the surface layer (Ruffini, 1925; Vogt, 1929; Pasteels, 1942; Smith and Malacinski, 1983; Lundmark,

9 Mesoderm morphogenesis in C. ornata 315 Fig. 10. Cladogram of selected vertebrates about which something of their surface mesoderm is known. Groups which have been reported to have surface mesoderm in the gastrula are indicated by the (*). No mark indicates no surface mesoderm. The chondrostei are sturgeons and paddlefish, the urodela are salamanders, the gymnophiona are caecilians (legless amphibians), and the anura are frogs and toads. Based on Duellman and Trueb, Fig. 9. Diagram of stages of mesoderm ingression in C. ornata. Step 1. Early neurula stage. Shaded cells are prospective notochord and somite cells in the archenteron epithelium. Step 2. Prospective somite cells are ingressing from the two lateral zones. Step 3. Late neurula. Prospective notochord cells are ingressing from the medial zone. Step 4. Tailbud stage. Ingression is complete. Shaded areas represent the location of cells that ingressed. n, notochord, s, somite. 1986; Delarue, 1992). Caecilians are reported to derive the notochord entirely from the surface layer in the urodele fashion (Brauer, 1897). There is some controversy over whether the amphibians are monophyletic (i.e., they have a common amphibian ancestor) (Hanken, 1986), so other out groups should be considered. A primitive fish, like the sturgeon, would be an excellent out group for all the amphibians (Fig. 10). This chondrostean fish has an embryo that looks very much like an amphibian embryo, with holoblastic cleavage and an involuting marginal zone. Their early development resembles that of amphibians more than it resembles that of the meroblastic teleost fishes. The fate map of the sturgeons Acipenser stellatus and A. güldenstädti clearly shows notochord and somitic mesoderm on the surface at the pregastrula stage (Ballard and Ginsburg, 1980). Recent evidence also indicates that this surface mesoderm ingresses from the archenteron roof after the completion of involution in A. transmontanus (Bolker, 1989). Since all three out groups have surface mesoderm, the ancestral amphibian likely had surface mesoderm. If the ancestral anuran had no surface mesoderm, like X. laevis, and the ancestral amphibian had surface mesoderm, two changes would be required in the lineage leading to C. ornata: a loss of surface mesoderm in the anuran lineage and then a reversion to surface mesoderm in a later anuran lineage. The most parsimonious explanation of all the above data is that the ancestral anuran and the ancestral amphibian had surface mesoderm in the pregastrula stage, and had ingression of mesoderm from the archenteron roof: this would require only one change during evolution, loss of surface mesoderm in the pipid lineage. Additional evidence that the X. laevis type of mesoderm formation may be a derived trait within the anurans has come from the discovery that the dorsal surface layer of the involuting marginal zone, which is prospective archenteron roof endoderm in X. laevis, has properties of the organizer. The organizer in amphibians has previously been thought to be mesodermal, namely chordamesoderm. The surface epithelial layer is necessary for normal pattern formation in the dorsal mesoderm of X. laevis (Shih and Keller, 1992a,b). The surface epithelium in X. laevis can also form notochord and induce a second embryonic axis if it is placed in the ventral deep layer at the gastrula stage (Shih and Keller, 1992a). The surface layer in X. laevis also responds to a mesoderm inducer as well as or better than the deep layer, forming notochord and somite tissue (Asashima and Grunz, 1983). All these experimental results support the idea that the lack of ingression in X. laevis is a secondar-

10 316 S. M. Purcell and R. Keller ily derived character in the anurans, with the epithelium still retaining organizer properties of the ancestral chordamesoderm. The absence of prospective mesoderm in the surface of X. laevis at the gastrula stage and its presence in urodeles has been used to support a polyphyletic origin for amphibians (Hanken, 1986; Nieuwkoop and Sutasurya, 1976, 1979). That is, the similarities between anurans and urodeles that lead to their being classified into a single group, Lissamphibia, might result from convergent evolution, with each group having evolved independently from fish ancestors. Since it now seems likely that the common ancestor of the urodeles and anurans had surface mesoderm, the lack of surface mesoderm in X. laevis can no longer be used as evidence for a polyphyletic origin of the amphibians. Implications for mesoderm pattern formation In amphibian development, the dorsal vegetal cells induce the marginal zone to form dorsal mesoderm and endoderm (Nieuwkoop, 1969; Sudarwati and Nieuwkoop, 1971; Gimlich and Gerhart, 1984; Gimlich, 1985). Any model of amphibian mesoderm induction and pattern formation cannot ignore the different patterns of mesoderm in amphibians. In urodeles, the mesoderm is derived mostly from the surface layer. In X. laevis, the mesoderm is derived from the deep layer, but the surface layer has properties of the organizer. In C. ornata and some other frogs, part of the mesoderm is derived from the surface layer and the rest is derived from the deep layer. We thank Jessica Bolker, Deborah Purcell and others in the Keller laboratory for their comments and advise on the text and figures, and Bob Barnes for providing C. ornata embryos. This work supported by NIH grant HD25594A and NSF grant to Ray Keller and NIH training grant HD07375 to Susan Purcell. REFERENCES Asashima, M. and Grunz, H. (1983). Effects of inducers on inner and outer gastrula ectoderm layers of Xenopus laevis. Differ. 23, Baker, P. C. (1965). Fine structure and morphogenetic movements in the gastrula of the tree frog, Hyla regilla. J. Cell Biol.24, Ballard, W. W. (1981). Morphogenetic movements and fate maps of vertebrates. Amer. Zool. 21, Ballard, W. W. and Ginsburg, A. S. (1980). Morphogenetic movements in acipenserid embryos. J. Exp. Zool. 213, Bolker, J. A. (1989). (abstract) Gastrulation in the white sturgeon, Acipenser transmontanus. Amer. Zool. 29, 86A. Brauer, A. (1897). I. Beiträge zur kenntniss der entwicklungsgeschichte und der anatomie der Gymnophionen. Zool. Jahrbuch. Anat.10, Brun, R. B., Garson, J. A. (1984). Notochord formation in the Mexican Salamander (Ambystoma mexicanum) is different from notochord formation in Xenopus laevis. J. Exp. Zool.229, Burnside, B. (1973). Microtubules and microfilaments in amphibian neurulation. Amer. Zool. 13, Cannatella, D. C. and Trueb, L. (1988). Evolution of pipoid frogs: intergeneric relationships of the aquatic frog family Pipidae (Anura) Zool. J. Linn. Soc.94, Delarue, M., Sanchez, S., Johnson, K. E., Darribère, T. and Boucaut, J. C. (1992). A fate map of superficial and deep circumblastoporal cells in the early gastrula of Pleurodeles waltl. Development 114, Duellman, W.E. and Trueb, L. (1986). Biology of Amphibians.New York: McGraw-Hill. Ettensohn, C. A. (1985). Mechanisms of epithelial invagination. Q. Rev. Biol. 60, Gimlich, R. L. (1985). Cytoplasmic localization and chordamesoderm induction in the frog embryo. J. Embryol. Exp. Morph. 89 Supplement, Gimlich, R. L. and Gerhart, J. C. (1984). Early cellular interactions promote embryonic axis formation in Xenopus laevis. Dev. Biol. 104, Hanken, J. (1986). Developmental evidence for amphibian origins. Evolutionary Biology 20, Hardin, J. and Keller, R. (1988). The behaviour and function of bottle cells during gastrulation of Xenopus laevis. Development 103, Keller, R. E. (1975). Vital dye mapping of the gastrula and neurula of Xenopus laevis. I. Prospective areas and morphogenetic movements in the superficial layer. Dev. Biol. 42, Keller, R. E. (1976). Vital dye mapping of the gastrula and neurula of Xenopus laevis. II. Prospective areas and morphogenetic movements in the deep region. Dev. Biol. 51, Keller, R. E. (1986). The Cellular Basis of Amphibian Gastrulation. In Developmental Biology: A Comprehensive Synthesis, Vol. 2: The Cellular Basis of Morphogenesis. (ed. L. Browder), pp New York: Plenum Press. Keller, R. E., Danilchik, M., Gimlich, R. and Shih, J. (1985). The function of convergent extension during gastrulation of Xenopus laevis. J. Embryol. Exp. Morph.89Supplement, Keller, R., Shih, J. and Domingo, C. (1992). The patterning and functioning of protrusive activity during convergence and extension of the Xenopus organiser. Development 1992 Supplement, King, H. D. (1903). The formation of the notochord in the amphibia. Biol. Bull.mar. Biol. Lab. Woods Hole 4, Leptin, M. (1991). Mechanics and genetics of cell shape changes during Drosophila ventral furrow formation. In Gastrulation. (ed. R. Keller et al.) pp New York: Plenum Press. Löfberg, J. (1974). Apical surface topography of invaginating and noninvaginating Cells. A scanning-transmission study of amphibian neurulae. Dev. Biol. 36, Løvtrup, S. (1966). Morphogenesis in the amphibian embryo, cell type distribution, germ layers and fate maps. Acta Zool. 47, Løvtrup, S. (1975). Fate maps and gastrulation in Amphibia-a critique of current views. Can. J. Zool. 53, Lundmark, C. (1986). Role of bilateral zones of ingressing superficial cells during gastrulation of Ambystoma mexicanum. J. Embryol. Exp. Morph. 97, Nieuwkoop, P. D. (1969). The formation of the mesoderm in urodelean amphibians. I. Induction by the endoderm. Wilhelm Roux s Arch. EntwMech. Org. 162, Nieuwkoop, P. D. and Florshütz, P. A. (1950). Quelques caractères spéciaux de la gastrulation et de la neurulation de l oeuf de Xenopus laevis, Daud. et de quelques autres anoures. 1ère partie. Etude descriptive. Arch. Biol. (Liege) 61, Nieuwkoop, P. D. and Sutasurya, L. A. (1976). Embryological evidence for a possible polyphyletic origin of the recent amphibians. J. Embryol. Exp. Morph. 35, Nieuwkoop, P. D. and Sutasurya, L. A. (1979). Primordial Germ Cells in the Chordates. Cambridge University Press. Owaribe, K., Kodama, R. and Eguchi, G. (1981). Demonstration of contractility of circumferential actin bundles and its morphogenetic significance in pigmented epithelium in vitro and in vivo. J. Cell Biol.90, Pasteels, J. (1942). New observations concerning the maps of presumptive areas of the young amphibian gastrula (Amblystoma and Discoglossus). J. Exp. Zool. 89, Purcell, S. M. (1989). (abstract) A different type of anuran gastrulation and morphogenesis as seen in Ceratophrys ornata. Amer. Zool. 29, 85A. Ruffini, A. (1925). Fisiogenia. Dottor Francesco Vallardi, Milano. Schoenwolf, G. C. and Franks, M. V. (1984). Quantitative analyses of changes in cell shapes during bending of the avian neural plate. Dev. Biol. 105, Shih, J. and Keller, R. (1992a). The epithelium of the dorsal marginal zone of Xenopus has organiser properties. Development 116, Shih, J. and Keller, R. (1992b) Cell motility diving mediolateral intercalation in explants of Xenopus laevis. Development 116, Smith, J. C. and Malacinski, G. M. (1983). The origin of the mesoderm in an anuran, Xenopus laevis, and a urodele, Ambystoma mexicanum. Dev. Biol. 98, Sudarwati, S. and Nieuwkoop, P. D. (1971). Mesoderm formation in the

11 Mesoderm morphogenesis in C. ornata 317 anuran Xenopus laevis (Daudin) Wilhelm Roux Archiv. EntwMech. Org. 166, Trinkaus, J. P. (1984). Cells into Organs: The Forces that Shape the Embryo. Second edition. Englewood Cliffs NJ: Prentice-Hall Inc. Vogt, W. (1929). Gestaltungsanalyse am amphibienkeim mit ortlicher vitalfarbung. II. Teil. Gastrulation und mesodermbildung bei Urodelen und Anuren. Wilhelm Roux Arch. EntwMech.Org.120, Wilson, P. (1991). Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants. Development 112, Wilson, P. A., Oster, G. and Keller, R. E. (1989). Cell rearrangement and segmentation in Xenopus: direct observation of cultured explants. Development 105, Wiley, E. O. (1981). Phylogenetics: the Theory and Practice of Phylogenetic Systematics. New York. (Accepted 30 September 1992)