Ibrahim H and Winklbauer R (2001), Mechanisms of mesendoderm internalization in th...

XB-ART-7826

Dev Biol 2001 Dec 01;2401:108-22. doi: 10.1006/dbio.2001.0459.

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Mechanisms of mesendoderm internalization in the Xenopus gastrula: lessons from the ventral side.

Ibrahim H , Winklbauer R .

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Two main processes are involved in driving ventral mesendoderm internalization in the Xenopus gastrula. First, vegetal rotation, an active movement of the vegetal cell mass, initiates gastrulation by rolling the peripheral blastocoel floor against the blastocoel roof. In this way, the leading edge of the internalized mesendoderm is established, that remains separated from the blastocoel roof by Brachet's cleft. Second, in a process of active involution, blastopore lip cells translocate on arc-like trails around the tip of Brachet's cleft. Hereby the lower, Xbra-negative part of the lip moves toward the interior, to contribute mainly to endoderm. In contrast, the upper, Xbra-expressing part moves toward the blastocoel roof-apposed surface of the involuted mesoderm, and eventually becomes inserted into this surface. Vegetal rotation and active mesoderm surface insertion persist over much of gastrulation ventrally. Both processes are also active dorsally. In fact, internalization processes generally spread from dorsal to ventral, though at different rates, which suggests that they are independently controlled. Ventrally and laterally, mesoderm occurs not only in the marginal zone, but also in the adjacent blastocoel roof. Such blastocoel roof mesoderm shares properties with the remaining, ectodermal roof, that are related to its function as substratum for mesendoderm migration. It repels involuted mesoderm, thus contributing to separation of cell layers, and it assembles a fibronectin matrix. These properties change as the blastocoel roof mesoderm moves into the blastopore lip during gastrulation.

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Species referenced: Xenopus laevis
Genes referenced: bcr fn1 pim1 tbxt

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	FIG. 1. Xbra expression domains subdivide the lip region. (A, B) Drawings of midsagittal sections of early (A) or midgastrula stage (B) embryos,with Xbra expression (blue) indicated; animal is to the top, ventral to the left. B’s C, Brachet’s cleft; BCR-M, blastocoel roof mesoderm; UL, upper lip; LL, lower lip. (C, D) Explants of stage-10 dorsal lip and adjacent BCR were fixed after 5 (C) and 8 (D) h, respectively, and hybridized with Xbra probe. The boundary between lip and BCR was labeled with nile blue dots, explants were recorded before fixation, and positions of dots (red circles) were transfered to the photographs shown. (E) Ventral BCR from above the BCR mesoderm (left row), or with BCR mesoderm included (right row), was explanted at stage 10.5, and cultured for 3 days.
	FIG. 2. Movement and fate of ventral lower blastopore lip. DiI-labeled cells (A–D, F, G; fluorescence and epi-illumination pictures merged) or cells in SEM (E) were visualized in midsagittally fractured gastrulae. Stages are indicated, animal is to the top, ventral to the left. White arrowheads indicate the blastopore; black arrowheads the meso-endodermal cleft; (x) marks the tip of Brachet’s cleft. Small cells within the vegetal endoderm are shown yellow in (E). After 3 days, labeled cells from the former lower lip are still detected in whole larvae (H; anterior is to the top, dorsal to the right) or in cross-sections of the gut (I, J; dorsal is to the top). g, gut; p, proctodeum; sm, somitic muscle.
	FIG. 3. Movement and fate of ventral upper lip and adjacent BCR. DiI-labeled cells in midsagittally fractured gastrulae (A–C, F, G), or in whole (D) larvae are shown. Stages are indicated, animal is to the top, ventral to the left. (E) Cross-sectioned larvae, dorsal is to the top. White arrowheads indicate the blastopore; black arrowheads the meso-endodermal cleft; (x) marks the tip of Brachet’s cleft. White arrows indicate labeled cells in the superficial layer, that lag far behind the deep cells during involution. m, mesoderm around proctodeum; g, gut; p, proctodeum; sm, somitic muscle.
	FIG. 4. Summary of ventral cell movements during involution. Initial positions of labeled cells at stage 10.5 (A–C), and final positions at stage 12.5 (A9–C9) are indicated. Each outline represents a single patch of cells, from an individual experiment. Yellow, lower lip; red, upper lip (A, A9). Orange, between lower and upper lip; violet, between upper lip and BCR (B, B9). Blue, BCR mesoderm; gray, ventral ectoderm (C, C9). Arrowheads indicate the blastopore. e, endoderm; m, mesoderm.
	FIG. 5. Fibronectin matrix on BCR mesoderm, as shown by immunofluorescence staining. (A) Sagittal section of stage-10.5 embryo, ventral side. BCR-M denotes BCR mesoderm; IM, involuted mesoderm; (x), tip of Brachet’s cleft. (B) Whole-mount of BCR mesoderm, showing the FN matrix on its blastocoelic surface. (C, D) Blastocoelic surface of ectodermal BCR (AC) at animal pole, without (C) or with previous injection of 1 ng Xbra mRNA into animal blastomeres (D). Bars, 50 mm.
	FIG. 6. Loss of repulsion behavior from BCR mesoderm. (A) Experimental scheme of BCR assay. Small test explants from defined regions of donor embryo (top) are placed on the BCR of recipient embryo (bottom). After 45 min, it is determined at epi-illumination (inset at top) whether test explants have remained on the surface of the BCR (red arrowheads), or have sunken into it (blue arrowheads). (B) Small test explants from anterior involuted mesoderm (AIM) or animal cap (AC) were placed on BCR from the animal pole (AC), or on ventral BCR mesoderm (BCR-M; always two BCR-M explants were fused to obtain a larger test area). Test explants were also placed on rafts of three to four stage-12.5 ventral lips (L) that were fused edgewise to increase the test area. Percentage of explants remaining on surface of substratum are indicated. At least 30 test explants from at least 3 different batches of embryos were tested for each experiment. (C–F) A piece of involuted mesendoderm was placed on ventral BCR that was explanted together with the adjacent, outer part of the lip at stage 10.5 (C, D) or at stage 12.5 (E, F). When cultured under a coverslip for 45 min, the lip region, but not the BCR part, fuses with the involuted mesoderm (D, F). (C, E) Experimental design: involuted mesendoderm (yellow) was excised along the dotted line; BCR including the lip region was also excised along dotted lines. The BCR mesoderm at stage 10.5 (blue, region 2) has moved into the lip at stage 12.5, the lip at stage 10.5 (region 1) has involuted by stage 12.5, the ectodermal region 3 remains on the outside. Arrowheads, early blastopore; asterisks, late blastopore; (x) indicates the tip of Brachet’s cleft that developed in vitro.
	FIG. 7. Changes in separation behavior at the ventral lip. Test explants from different regions of early (A) and late (B) gastrulae were placed on stage-10.25 to -10.5 BCR, and the percentage of explants remaining on the surface was determined after 45 min. At least 30 aggregates from at least 3 different batches of embryos were tested for each experiment. AIM, anterior involuted mesoderm; PIM, posterior involuted mesoderm; BCR-E, ventral ectodermal BCR; BCR-M, BCR mesoderm; L, blastopore lip; LL, lower blastopore lip; UL, upper lip; arrowhead points at the blastopore. Large arrows indicate relationships between regions, as deduced from our fate mapping data.
	FIG. 8. Vegetal rotation at the ventral side. (A–F) A midsagittal slice of the vegetal half was explanted between stages 10 and 10.25. Frames from the start of time-lapse recording (A), after 30 (B), 60 (C), 75 (D), 90 (E), and 120 min (F) are shown. Ventral is to the left, the blastocoel floor to the top. The red dotted line demarcates the border between the superficial layer (the original outer surface of the embryo) and the cut surface. The initial position (white arrows), and the downward rotating positions (red arrows) of the former site of attachment of the ventral BCR to the marginal zone are indicated. The curved black arrow in (F) that connects the white and red arrow visualizes the rotation movement of the vegetal periphery and expansion of the blastocoel floor. Red and white arrowheads point at ventral and dorsal blastopore, respectively. The red bracket delimits the field of bottle cells. Bar, 200 mm. (G–I) A similar slice explanted at stage 11.5. Ventral is to the left. Black arrows indicate movement of selected cells during consecutive 10-min intervals, as determined from video recordings. Red arrowhead and arrows are used as above. le, leading edge; the formerly BCR-apposed surface of the involuted mesoderm extends between red arrow and (le). The area below the leading edge that shows no downward movement of cells is shaded. The superficial layer is shown in brown. Bar, 200 mm.
	FIG. 9. Mesoderm surface insertion. (A–C) The blastocoel floor of BCR-less embryos (n 5 6–8 per experiment) was stained by short submersion in nile blue sulfate solution at stage 10.25. Then, BCR was added to the ventral side, and removed again after 1 (A), 2 (B), or 3 h (C). (D) Same as above, but staining was at stage 11.5, and the BCR was removed 2 h later. (A–D) Ventral view, animal is to the top. Asterisk, insertion zone; arrowheads, blastopore. (E, F) Midsagittally fractured embryos, the ventral BCR was removed at stage 11.5, and embryos were fixed immediately (E) or after 2 h, at stage 13 (F). Animal is to the top, ventral to the left. Arrowheads point at the ventral blastopore (or at bottle cells, i.e., the former blastopore, in F). Arrows (E, F) point at the site of former BCR attachment, the region between arrow and arrowhead in (E) is rotated by about 180° in (F), visualizing involution. The eye symbol above the white arrow in (E), at the position of the insertion zone, indicates the point of view taken in (G) and (H). The explant shown in (H) was excised along the dashed line in (E). a, archenteron; m, ventral mesoderm layer. (G, H) Stage-11.5 ventral involuted mesoderm above lip (eye symbol in E) was time-lapse recorded for 1 h in BCR-less embryos (G; n 5 7) or for 0.5 h in explants (H; n 5 6) excised as indicated in (E) and secured under a strip of coverslip. Movement of selected cells (arrows), and all cells intercalated during recording (gray) are indicated in the last frame, respectively. Animal is to the top. Violet, surface of involuted mesoderm; brown, superficial layer over lip region. Bars, 50 mm. (I, J) SEM of insertion zone. For numbers on cells, see text. BCR-M, BCR mesoderm; dashed line, Brachet’s cleft; x, tip of Brachet’s cleft. Bars, 40 mm.
	FIG. 10. Timing of gastrulation processes. Time-lapse recordings of slices, or of involuted mesoderm in BCR-less embryos, were evaluated to determine the duration of vegetal rotation and surface insertion, respectively. Two to four explants were recorded, for 1 h each, per time point. At critical stages, explantation was at consecutive 1-h intervals. For surface insertion, Nile blue staining data from this and our previous article (Winklbauer and Schu¨ rfeld, 1999) were included. Our unpublished data on the timing of bottle cell formation and archenteron deepening were used, data are also abundant in the literature (e.g., Nieuwkoop and Faber, 1967).
	FIG. 11. Mesendoderm internalization at the ventral blastopore lip. (A) Stage 10.5; (B) stage 11.5; (C) stage 12.5. The prospective germ layers ectoderm (blue), mesoderm (pink), and endoderm (yellow) are indicated. Yellow arrows symbolize vegetal rotation, red arrows active involution movement, and black arrows the vegetal rotation-driven movement of the blastocoel floor. The BCR mesoderm moves into the lip (blue arrows), and the archenteron deepens (brown arrow) by unknown mechanisms, respectively. The segment of the BCR-apposed mesoderm surface drawn in red consists of inserted surface; the red point indicates the position of leading edge at stage 10.5. For simplicity, the superficial layer is omitted. (D) An open-faced Keller explant, excised along the dashed lines in (B), consists of the outer part of the lip region, and adjacent BCR. Active involution movement of lip cells is interrupted at the cut surface (pale red arrows). (E) Insertion of cells into this surface—radial intercalation—deflects active involution movement (red arrows) and leads to extension of the explant.