A Different Type of Amphibian Mesoderm Morphogenesis in Ceratophrys Ornata

Development 117, 307-317 (1993) 307 Printed in Great Britain © The Company of Biologists Limited 1993

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 Prospective somitic tissue ingresses ﬁrst from two lat- signiﬁcantly different pattern of early morphogenesis eral zones, followed by ingression of prospective noto- than does the most studied amphibian, Xenopus laevis. chord from the medial zone and tailbud mesoderm from Time-lapse videomicroscopy, scanning electron the circumblastoporal zone. This is unlike X. laevis, in microscopy, histological sections and lineage tracers which no cells with constricted apices are present on the have shown that, in C. ornata, some prospective noto- dorsal surface of the archenteron, nor do any cells chord, somite and tailbud mesoderm cells leave the sur- ingress into the deep mesodermal layers from the surface epithelium of the archenteron by ingression. After face layer. 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. Key words: mesoderm, notochord, ingression, amphibian

INTRODUCTION from X. laevis. Since Keller published the fate map of X. laevis in 1975, some have speculated that Vogt’s and Pas- It was long thought that early and fundamental processes teels’ fate maps may be incorrect (Løvtrup, 1975; of development such as gastrulation, were conserved fea- Nieuwkoop and Sutasurya, 1976). tures of development since their alteration would have To begin resolving this issue, we have analyzed gastru- repercussions on later events. But it appears that gastrula- lation movements and mesoderm morphogenesis in the tion and mesoderm morphogenesis are in fact highly vari- Argentinean horned frog, Ceratophrys ornata, a species of able processes (Ballard, 1981). In the amphibians, an impor- frog not closely related to X. laevis (Duellman and Trueb, tant issue is the tissue fates and organization of the marginal 1986). The embryo of C. ornata differs from that of X. zone. There has been a controversy for decades over laevis in having prospective mesoderm in the surface layer whether the origin of the mesoderm is the same in all anuran at the gastrula stage. These findings are significant for amphibians (frogs and toads) (Løvtrup, 1966, 1975; understanding the cellular mechanisms of morphogenesis Nieuwkoop and Sutasurya, 1976, 1979; Hanken, 1986). and pattern formation in anurans, and for understanding the Some ectoderm of the marginal zone is induced to form evolution of morphogenetic processes that occur in early mesoderm by the subblastoporal endoderm during the late vertebrate embryogenesis. blastula stages in amphibian embryos (Nieuwkoop, 1969; Sudarwati and Nieuwkoop, 1971). The marginal zone invo- lutes through the blastopore during gastrulation, internaliz- ing the endodermal and mesodermal layers. In the anuran MATERIALS AND METHODS Xenopus laevis, only prospective endoderm and ectoderm are found on the surface of the pregastrula embryo. The Obtaining and handling of embryos mesoderm forms in the deep layers and remains there Fertilized Ceratophrys ornata eggs were obtained from Bob’s throughout gastrulation (Nieuwkoop and Florshütz, 1950; Happy Fish (Rt 4 Box 605, Woodland, CA 95695) and were kept Sudarwati and Nieuwkoop, 1971; Keller, 1975, 1976). 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 Keller’s fate map of X. laevis is different from earlier fibrous jelly coat and the vitelline envelope were removed with fate maps of other anurans. Vogt’s fate map of Bombina - sharpened forceps, and the inner jelly coat was removed by soak- tor (Bombina) (1929) and Pasteels’ map of Discoglossus ing in a solution of 3.5% cysteine hydrochloride at pH 7.9 for 2 (1942) show surface mesoderm in the marginal zones of to 5 minutes. All manipulations were done in 35 mm plastic Petri these anuran embryos, implying that these species differ dishes, some with a base of 2% agarose. 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 Fig. 1. Explant technique for videomicroscopy of the archenteron tailbud stage. Recordings were made using a Zeiss upright com- roof and for SEM is shown. For videomicroscopy, stage 14 pound microscope with a Nikon 4´ or a Zeiss 10´ plan objective, embryos were cut between the lateral edge of the archenteron roof low angle epi-illumination and a DAGE-MTI 81 high resolution and the ventral yolk mass. The dorsal piece was removed, turned video camera. Images were processed by averaging 32 frames and upside down and placed under a coverslip for filming such that the using contrast control features of an Image One video image epithelium of the archenteron could be seen. The coverslip was processor (Universal Imaging, Media, PA), and recorded on a positioned using vacuum grease. For SEM preparations, embryos Panasonic TQ-2028F optical memory disk recorder (OMDR). were fixed first at different stages and then the explants were made as shown before dehydration. bp, blastopore, a, anterior, d, dorsal. 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. the surface epithelium and ingress (Figs 2,3). The bound- Embryos were cut with a microscalpel to expose the archenteron aries between zones I and II are substantive in that cells roof; ventral halves were discarded (Fig. 1). Some embryos were were not seen to cross this boundary, although cell cut transversely or obliquely to expose the deep cell layers. rearrangement was minimal in any case. Cells in the medial Embryos were then critical-point dried with liquid CO2, mounted on stubs with silver paint, coated with platinum by standard meth- and lateral zones first constrict their apices (arrows, Fig. ods, and viewed on an ISI-DS130 scanning electron microscope 2A,B), and then leave the epithelial layer (Fig. 2A,C). This with an accelerating voltage of 15 kV. happens rapidly, occurring within a few hours at room tem- perature. In the recordings, only cells with tightly con- Histology stricted apices leave the epithelium, and all constricted cells Embryos were placed in Smith’s fixative for 12 hours at 4°C, eventually disappear. The high correlation between these dehydrated using an ethanol series, embedded in Paraplast using two cell behaviors suggests that apical constriction may be Histosol and cut into 10 mm sections. Sections were stained with necessary for ingression to occur. Cells with apical con- giemsa stain, and mounted with Permount. Embryos were fixed striction appear dark due to concentration of apical pigment at gastrula (stage 10) through tailbud (stage 18). granules into a smaller area. At the completion of ingres- Vital dye marking sion, the non-ingressing cells of the lateral archenteron surface meet at the midline. These cells do not constrict their Vital dye marking of the surface cells was done using Nile blue dye in agarose chips (Keller, 1975), which were placed up against apices (arrowhead, Fig. 2A,C). This process was observed the embryo for 15 seconds. This is long enough for the surface in four recordings, each using an embryo from a different cells under the chip to take up the dye. Control embryos were dis- batch, with no significant differences between them. sected at stage 13, before any ingression occurred, to be sure only Fig. 3 shows tracings made from the video recording surface cells were taking up the Nile blue. Embryos were marked shown in Fig. 2, with selected cells outlined and numbered at stage 10 and dissected at stage 15,16,17 or 18 to locate the dye to illustrate the points made above. The two lateral zones marks. of ingression are shaded. The medial zone and the non- ingressing cells are unshaded. In the first 25 minutes of the recording (Fig. 3A,B), cells in the lateral zones, numbered RESULTS 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 Cell movements recorded by time-lapse have ingressed completely, and the other lateral zone is sig- videomicroscopy nificantly smaller. Videorecordings of the dorsal surface of the archenteron In the second 25 minutes of the recording (Fig. 3B,C), and the blastopore region in explants from stage 13 to stage cells in the other lateral zone complete their ingression, 18 show that apically constricted cells in four zones leave leaving only cells in the medial zone, which have not fin- Mesoderm morphogenesis in C. ornata 309 ished ingressing. Cells numbered 10,15 and 16 in the medial decrease their apical surface area. Non-ingressing cells do zone have left the surface layer, and the remaining medial not leave the surface in any of four recordings made. The zone cells have decreased their apical surface area. In Fig. cells numbered 1, 2, 3, 5, 6 and 18-21 in the two lateral 3A, the non-ingressing cells numbered 4 and 22 are separ- zones disappear before the cells numbered 7-17 in the ated from the midline by many rows of cells, however, they medial zone. end up quite close to it (Fig. 3C). It is significant that these preparations are explants with The recordings show clearly that cells with constricted most of the surrounding tissue removed (Fig. 1), and still apices leave the surface of the epithelium. During the period ingression occurs. In appears that the non-ingressing cells illustrated in this tracing, cells numbered 1-3, 5, 6, 10, 15, are dragged together by the ingressing cells, as they do not 16, and 18-21 disappear from the surface layer. The non- show any protrusive activity characteristic of migrating ingressing cells, numbered 4 and 22, do not significantly 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 mar- gins 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 consti- tutes 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. 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 transi- tion 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.

Vital dye conﬁrmation of cell fates Fig. 3. Cell tracings of video frames in Fig. 2. The dotted line represents the midline. Lateral zone cells are shaded, the medial Nile blue marks were used to conﬁrm that surface cells zone and non-ingressing cells are unshaded. Selected cells are from the gastrula marginal zone had actually ingressed into numbered in each region. (A) Cells 4 and 22 are non-ingressing, the somites, notochord and tailbud mesoderm by the tail- 1-3, 5, 6 and 18-21 are in the two lateral zones, and cells 7-17 are bud stage, as suggested by SEMs and histological sections. in the medial zone. (B) The two lateral zones have almost For dye marking experiments, the embryo was divided into completely disappeared. The numbered cells that are no longer longitudinal quadrants; the results were assigned to one of present have ingressed from the epithelium into the deep layer. three sections, depending upon the quadrant in which the (C) The lateral zones have completely ingressed, and many of the cells from the medial zone have also ingressed. All the medial initial mark was made (Fig. 8). Quadrant 1 is centered on cells remaining in the surface layer have smaller apices than they the blastopore lip, which forms on the dorsal side at stage did at the beginning of the recording. The two non-ingressing 10. The left and right lateral quadrants are labelled 2, and cells, 4 and 22, have not appreciably decreased their apical surface the ventral-most quadrant, in which the blastopore lip forms area. a, anterior, p, posterior. last, is labelled 3. 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 ing marginal zone contains prospective endoderm, posterior in the posterior half of the notochord, with labelled cells notochord, somite and tailbud mesoderm. The blastopore primarily in the ventral portion of the notochord. The sur- lip forms on the dorsal side in typical amphibian fashion, face mesoderm in the rest of the marginal zone was found and involution of the marginal zone begins. The lip forms in the medial portion of the somites next to the notochord, closer to the equator of the blastula in C. ornata than in and in the tailbud mesoderm (Table 1). Rarely, dye was Xenopus laevis. The lip then extends to the lateral and ven- found in lateral plate mesoderm near the tailbud. Control tral sides. The blastopore then closes down and the yolk embryos were dissected at stage 13, before any ingression plug is internalized. After gastrulation is complete, the had occurred to conﬁrm that only the surface layer was dorsal and lateral surfaces of the archenteron are lined with labelled. the surface layer of the involuting marginal zone, and the ventral surface is lined with cells from the subblastoporal Summary of Ceratophrys gastrulation and endoderm, as in X. laevis (Keller, 1986). The involuted roof mesoderm morphogenesis of the archenteron consists of suprablastoporal endoderm In the pregastrula embryo, the surface layer of the involut- anteriorly, and prospective somitic mesoderm, prospective 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. 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. The mechanics of ingression The cells with constricted apices are likely the driving force Fig. 7. Scanning electron micrographs of ingressing cells in lateral in the process of ingression for four reasons. First, apical zones of C. ornata. (A) Parasagittal fracture through zone II, constriction has been shown to be an active, force-generat- showing cells ingressing into the somitic tissue. These cells have a ing process in a variety of systems, including neural tube bottle shape, with constricted apices and dilated bases. The formation (Burnside, 1973; Löfberg, 1974; Schoenwolf and arrowhead indicates a cell that had just left the epithelial layer. Franks, 1984), optic cup formation (Owaribe et al. 1981), The arrow indicates a cell with its apex still attached to the amphibian blastopore formation (Hardin and Keller, 1988; epithelium. The open arrows indicate the broken edge of the Baker, 1965; reviewed by Ettensohn, 1985) and ventral epithelium. Note also the visibly uneven distribution of extracellular matrix, which appears as white spheres after furrow formation in Drosophila (Leptin, 1991). Second, the dehydration. The matrix becomes more dense towards the basal non-ingressing cells, which will end up meeting along the end of the cells. (B) Transverse cut showing an ingressing cell midline, do not show activity indicative of migration in the from zone II. Notice the very constricted apex and broad basal videorecordings and so are unlikely to be crawling over the end. Filiform protrusions can be seen on the basal end of the cell ingressing cells. Third, SEMs show many ﬁliform and indicated by the arrowhead. Notice that the basal end of the lamelliform protrusions on the deep side of the ingressing ingressing cell curves away from the notochord. Open arrows cells. These types of protrusions are associated with cell indicate the broken edge of the epithelium. Scale bar, 10 µm. n, movement (Trinkaus, 1984) and are a fair indication of the notochord, s, somite. direction that the cells are moving. Finally, and most impor- tantly, the cells constrict and ingress even in an explant, later stages into several tissue types. These ingressing meso- when most of the surrounding tissue has been removed derm cells add to the mesoderm cells already in the deep (such as in the videomicroscopy results). Thus, it is unlikely layer (Fig. 9). that those surrounding tissues actively squeeze ingressing cells out of the surface layer.

DISCUSSION Zones of ingression I and II are tissue specific Before ingression begins, the boundary between notochord Several aspects of the development of Ceratophrys ornata and somite is already well formed in the deep tissue of the are especially significant. The first is the mechanics of body axis. The boundaries between the notochord and ingression and its partially tissue-specific nature, the second somitic tissue in the deep layer are exactly in register with is comparisons of the developmental pattern in C. ornata the boundaries between zones of ingression I and II. These with those of other anurans, urodeles and other vertebrates, surface and deep boundaries are likely the same boundary. 314 S. M. Purcell and R. Keller

Table 1. Vital dye marking of surface cells in the surface layer at stage 10. It has been proposed that the X. marginal zone laevis pattern may be common to all anurans (Løvtrup, Location of dye at stage 18 1975; Nieuwkoop and Sutasurya, 1976, 1979), and that Quadrant of Total early fate maps of Bombina variegata (=pachypus) (Vogt, marginal embryos Other 1929) and Discoglossus (Pasteels, 1942) were inaccurate. zone marked marked Notochord Somite mesoderm However, Vogt’s fate maps of urodeles showing surface 1 (dorsal) 37 15 7 1 mesoderm were correct (Vogt, 1929; Smith and Malacin- 2 (lateral) 9 0 3 5 ski, 1983; Lundmark, 1986; Delarue et al., 1992). Of the 3 (ventral) 9 0 1 5 three anuran fate maps so far constructed, only the one for Summary of data from vital dye marking experiments in which marks X. laevis has no prospective mesoderm in the surface layer. fell entirely within one of the four quadrants (see Fig. 7). The embryo was Other anurans have also been reported to have surface divided into quadrants with quadrant 1 centered on the dorsal midline. For mesoderm at early gastrula stages using histology and trans- each quadrant, at least two different batches of embryos were used. “Other mission electron microscopy. These are Hyla regilla mesoderm” is mostly tailbud mesoderm and some posterior lateral plate mesoderm. Most marks also labelled archenteron endoderm. (Baker, 1965), Bufo americanus (=lentiginosus) and Rana palustris (King, 1903) and Ceratophrys ornata (Fig. 10). Ruffini (1925) analyzed the development of Rana esculenta If ingression were not tissue-specific, we would expect to and Bufo bufo (=vulgaris). He concluded that the meso- see cells crossing one or both of these boundaries during derm arose entirely from the deep layer in anurans. A reex- ingression. However, no cells were seen crossing the sur- amination of his figures reveals that B. bufo appears to have face boundary during videomicroscopy, nor were any cells zones of cells with constricted apices on the archenteron seen spanning the boundary in the histological sections. roof much like those in C. ornata. Ruffini may have mis- Cells do not normally cross the notochord-somite bound- interpreted his data from B. bufo because he saw deep meso- ary in X. laevis, along which cell behavior has been exten- derm in that species and may not have considered the pos- sively recorded (Wilson, 1991; Wilson et al., 1989; Keller sibility that the notochord could arise from both the surface et al., 1992; Shih and Keller, 1992b). and the deep layers. In C. ornata, the basal ends of ingressing cells are embedded in the deep tissue that they will join while the Surface mesoderm in urodeles apical ends are still firmly a part of the epithelium. Cells The pattern of cell movement in C. ornata differs from that appear to be directionally ingressing into the somitic tissue in previously studied amphibians both in the timing of the or notochord, as can be seen in views of the lateral zone ingression of mesoderm, and the quantity of mesoderm cells ingressing and clearly curving away from the midline. originating in the surface and deep layers (Table 2). X. In the circumblastoporal region, however, there are no laevis has no surface mesoderm and no ingression (Keller, boundaries yet formed, and so it is possible that cells are 1975). In the salamander Ambystoma mexicanum, most of ingressing prior to specification as a particular tissue type. the mesoderm is from the surface layer; the somitic cells The tailbud region, into which cells ingress, is a mes- ingress during gastrulation, and the notochord cells ingress enchymal mass, which will differentiate into several tissue after gastrulation is complete (Pasteels, 1942; Smith and types and has no apparent internal boundaries when first Malacinski, 1983; Brun and Garson, 1984; Lundmark, formed. 1986). Although ingression occurs in the development of other organisms such as the sea urchin (primary mesenchyme Surface mesoderm in ancestral amphibians cells) and the chick (endoderm and mesoderm cells of the We can hypothesize as to what sort of pattern the ancestral primitive streak), C. ornata offers the first example of spa- anuran and ancestral amphibian had by using phylogeny. tially organized, simultaneous ingression of several tissue Traits that were present in the earliest member of a group types. are referred to as ancestral characters and traits arising later in the evolution of that group are referred to as derived Surface mesoderm in other anurans characters. X. laevis is not considered to be a primitive Differences in early development have been found among anuran, even though it has some ancestral characters (e.g., the few species of anurans examined. Keller (1975) clearly presence of dorsal ribs), because it has many derived char- showed that X. laevis has no prospective mesoderm in the acters 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 Table 2. Summary of the positions of prospective primitive. To determine which type of mesoderm formation mesoderm and the timing of ingression in three is the likely ancestral condition for anurans, a group out- amphibians side the anurans is needed. It is not possible to make comparisons solely within a group and determine the direction Anuran Urodele of evolution of a trait (Wiley, 1981). The best “out groups” tissues Ceratophrys Xenopus Ambystoma are sister taxa. Urodeles (newts and salamanders) and cae- prospective somite surface and deep deep surface cilians (legless amphibians) are sister groups to anurans prospective notochord surface and deep deep surface (Fig. 10). In newts and salamanders, most of the mesoderm ingressing somite after involution none during involution originates in the surface layer (Ruffini, 1925; Vogt, 1929; ingressing notochord after involution none after involution Pasteels, 1942; Smith and Malacinski, 1983; Lundmark, 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, 1986.

chord 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 Fig. 9. Diagram of stages of mesoderm ingression in C. ornata. lineage. The most parsimonious explanation of all the above Step 1. Early neurula stage. Shaded cells are prospective notochord and somite cells in the archenteron epithelium. Step 2. data is that the ancestral anuran and the ancestral amphib- Prospective somite cells are ingressing from the two lateral zones. ian had surface mesoderm in the pregastrula stage, and had Step 3. Late neurula. Prospective notochord cells are ingressing ingression of mesoderm from the archenteron roof: this from the medial zone. Step 4. Tailbud stage. Ingression is would require only one change during evolution, loss of complete. Shaded areas represent the location of cells that surface mesoderm in the pipid lineage. ingressed. n, notochord, s, somite. 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 1986; Delarue, 1992). Caecilians are reported to derive the involuting marginal zone, which is prospective archenteron notochord entirely from the surface layer in the urodele roof endoderm in X. laevis, has properties of the organizer. fashion (Brauer, 1897). The organizer in amphibians has previously been thought There is some controversy over whether the amphibians to be mesodermal, namely chordamesoderm. The surface are monophyletic (i.e., they have a common amphibian epithelial layer is necessary for normal pattern formation in ancestor) (Hanken, 1986), so other out groups should be the dorsal mesoderm of X. laevis (Shih and Keller, considered. A primitive fish, like the sturgeon, would be an 1992a,b). The surface epithelium in X. laevis can also form excellent out group for all the amphibians (Fig. 10). This notochord and induce a second embryonic axis if it is placed chondrostean fish has an embryo that looks very much like in the ventral deep layer at the gastrula stage (Shih and an amphibian embryo, with holoblastic cleavage and an Keller, 1992a). The surface layer in X. laevis also responds involuting marginal zone. Their early development resem- to a mesoderm inducer as well as or better than the deep bles that of amphibians more than it resembles that of the layer, forming notochord and somite tissue (Asashima and meroblastic teleost fishes. The fate map of the sturgeons Grunz, 1983). All these experimental results support the Acipenser stellatus and A. güldenstädti clearly shows noto- idea that the lack of ingression in X. laevis is a secondar- 316 S. M. Purcell and R. Keller ily derived character in the anurans, with the epithelium Gimlich, R. L. (1985). Cytoplasmic localization and chordamesoderm still retaining organizer properties of the ancestral chor- induction in the frog embryo. J. Embryol. Exp. Morph. 89 Supplement, damesoderm. 89-111. Gimlich, R. L. and Gerhart, J. C. (1984). Early cellular interactions The absence of prospective mesoderm in the surface of promote embryonic axis formation in Xenopus laevis. Dev. Biol. 104, X. laevis at the gastrula stage and its presence in urodeles 117-130. has been used to support a polyphyletic origin for amphib- Hanken, J. (1986). Developmental evidence for amphibian origins. ians (Hanken, 1986; Nieuwkoop and Sutasurya, 1976, Evolutionary Biology 20, 389-417. Hardin, J. and Keller, R. (1988). The behaviour and function of bottle cells 1979). That is, the similarities between anurans and urode- during gastrulation of Xenopus laevis. Development 103, 211-230. les that lead to their being classified into a single group, Keller, R. E. (1975). Vital dye mapping of the gastrula and neurula of Lissamphibia, might result from convergent evolution, with Xenopus laevis. I. Prospective areas and morphogenetic movements in the each group having evolved independently from fish ances- superficial layer. Dev. Biol. 42, 222-241. tors. Since it now seems likely that the common ancestor Keller, R. E. (1976). Vital dye mapping of the gastrula and neurula of Xenopus laevis. II. Prospective areas and morphogenetic movements in of the urodeles and anurans had surface mesoderm, the lack the deep region. Dev. Biol. 51, 118-137. of surface mesoderm in X. laevis can no longer be used as Keller, R. E. (1986). The Cellular Basis of Amphibian Gastrulation. In evidence for a polyphyletic origin of the amphibians. Developmental Biology: A Comprehensive Synthesis, Vol. 2: The Cellular Basis of Morphogenesis. (ed. L. Browder), pp. 241-327. New Implications for mesoderm pattern formation York: Plenum Press. Keller, R. E., Danilchik, M., Gimlich, R. and Shih, J. (1985). The In amphibian development, the dorsal vegetal cells induce function of convergent extension during gastrulation of Xenopus laevis. J. the marginal zone to form dorsal mesoderm and endoderm Embryol. Exp. Morph.89Supplement, 185-209. (Nieuwkoop, 1969; Sudarwati and Nieuwkoop, 1971; Gim- Keller, R., Shih, J. and Domingo, C. (1992). The patterning and lich and Gerhart, 1984; Gimlich, 1985). Any model of functioning of protrusive activity during convergence and extension of the Xenopus organiser. Development 1992 Supplement, 81-91. amphibian mesoderm induction and pattern formation King, H. D. (1903). The formation of the notochord in the amphibia. Biol. cannot ignore the different patterns of mesoderm in amphib- Bull.mar. Biol. Lab. Woods Hole 4, 287-300. ians. In urodeles, the mesoderm is derived mostly from the Leptin, M. (1991). Mechanics and genetics of cell shape changes during surface layer. In X. laevis, the mesoderm is derived from Drosophila ventral furrow formation. In Gastrulation. (ed. R. Keller et al.) pp. 199-212. New York: Plenum Press. the deep layer, but the surface layer has properties of the Löfberg, J. (1974). Apical surface topography of invaginating and organizer. In C. ornata and some other frogs, part of the noninvaginating Cells. A scanning-transmission study of amphibian mesoderm is derived from the surface layer and the rest is neurulae. Dev. 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