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 sur- face 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 devel- opment 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 develop- ment (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 sur- face 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.