Mancilla A and Mayor R (1996), Neural crest formation in Xenopus laevis: mecha...

XB-ART-17898

Dev Biol 1996 Aug 01;1772:580-9. doi: 10.1006/dbio.1996.0187.

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Neural crest formation in Xenopus laevis: mechanisms of Xslug induction.

Mancilla A , Mayor R .

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A study of the induction of the prospective neural crest in Xenopus laevis embryos has been carried out, using the expression of Xslug as a specific marker for the neural crest. We have analyzed the competence and the specification of the neural crest. The competence to express Xslug was analyzed using two different approaches: (1) in vitro culture of conjugates of dorsal mesoderm and ectoderm taken from embryos at different ages and (2) grafts of equivalent pieces of ectoderm in the neural fold region of a gastrula or a neurula. Similar results were obtained with both methods: the ectoderm loses the competence to respond to neural fold induction at the end of gastrulation. Neural crest specification was analyzed by culturing a region of the ectoderm that contained the prospective neural crest and analyzing Xslug expression. Our results show that neural folds are specified autonomously to express Xslug by the end of gastrulation. By grafting labeled neural plate into lateral epidermis we have shown that neural crest can be induced by an interaction between neural plate and epidermis. Furthermore, neural crest cells come from both tissues. We have discarded the possibility that these neural crest cells are induced by a signal coming from the underlying lateral plate, by a homeogenetic signal coming from the host neural plate, or by regeneration of crest cells from the dissected neural plate. We propose a model to explain how the neural crest cells are induced at the border of the neural plate in X. laevis.

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Species referenced: Xenopus laevis
Genes referenced: snai2

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	FIG. 1. Analysis of the competence of the ectoderm to respond to neural fold and neural plate induction. (a, d– g) Ventral animal caps were taken at different stages as indicated in the figure and combined with a piece of dorsal mesoderm taken from stage 10.5 (yellow in a). The conjugates were cultured until stage 17, fixed, and examined by whole-mount in situ hybridization using an Xslug antisense probe. (d) Stage 10 ectoderm; notice the strong Xslug induction (arrows). (e) Stage 11 ectoderm; notice the Xslug induction (arrows). (f) Stage 12 ectoderm; notice the weak Xslug induction (arrows). (g) Stage 13 ectoderm; no induction was detected, although it should be noticed that this photograph was taken under different magnification and light conditions than in (d–f). (h–k) Similar experiment to that described in (a) but Xsox-2 expression was analyzed. (h) Control embryo showing Xsox-2 expression in the neural plate. (i) Control of ectoderm taken from a stage 10 embryo, cultured in vitro until stage 17, fixed, and analyzed for Xsox-2 expression. No expression was detected. (j) Stage 12 and (k) stage 13 ectoderm; notice the Xsox-2 induction (arrows). (b, l– s) Ventral ectoderm taken from embryos injected with RLDx (magenta in b) and transplanted into stage 13 embryos. The embryos were cultured until stage 17 and analyzed for Xslug expression by whole-mount in situ hybridization. (l–o) Xslug in situ hybridization; (p– s) fluorescent image of the corresponding in situ hybridization shown. Graft of a stage 10 (l, p) or 11 (m, q) ectoderm; note the normal Xslug expression in both neural folds (l, m) and the Xslug expression in the region of the fluorescent transplant (p, q, arrow in l and m). Graft of a stage 12 (n, r) or 13 (o, s) ectoderm; notice the gap of Xslug expression in one of the folds (n, o) and the absence of Xslug expression in the region of the fluorescent transplant (n, r, o, s). (t) Control of ventral ectoderm taken from a stage 10 embryo, cultured in vitro until stage 17, fixed, and analyzed for Xslug expression. No expression was detected. (c, u–w) Ventral ectoderm taken from an embryo previously injected with FLDx (green in c) was transplanted into the prospective folds of a stage 11.5 embryo. The embryo was cultured until stage 17, fixed, analyzed for Xslug expression, and sectioned. Only the transplant of the stage 13 embryo is shown (u); notice the interruption of the Xslug expression in one of the folds (u; arrowhead shows the normal Xslug expression) and the absence of Xslug expression in the region of the fluorescent transplant (v; in w: asterisk, transplanted ectoderm; arrowheads, host’s Xslug expression).
	FIG. 2. Specification of Xslug in the neural crest. (a) Animal cap explants containing the prospective neural crest, dissected at different stages (b, 10; c, 11; and d, 13) were cultured until the equivalent of stage 17, fixed, and analyzed for Xslug expression. Notice the absence of Xslug expression when the ectoderm is dissected before stage 12 (b, c) and the visible Xslug expression when the ectoderm is taken from a stage 13 embryo (arrows in d).
	FIG. 3. Induction of the neural crest by an interaction between neural plate and epidermis. (a–d) A piece of anterior neural plate taken from a stage 13 embryo previously injected with RLDx was transplanted to the ventral region of a normal stage 13 embryo. The host embryo was cultured until stage 23 (b) or 17 (c, d) and fixed and the Xslug expression was analyzed. (b) Upper embryo: control showing the normal Xslug expression in the neural fold. Lower embryo shows the expression in a ring around the graft (arrow). Fluorescence (c) and Xslug in situ hybridization (d) of the same embryo; notice that the induced crest cells that express Xslug at the border of the graft come from the fluorescent neural plate (arrows in d) and from the unlabeled epidermis (arrowheads in d). Some RLDx-labeled cells have left the graft but they do not express Xslug. (e–h) Pieces of different tissues were dissected from a stage 13 embryo and conjugated with another tissue or cultured in vitro until the equivalent of stage 17. Then, they were fixed and Xslug expression was analyzed. (f) Neural plate cultured alone never expressed Xslug. (g) A conjugate of neural plate and lateral mesoderm never expressed Xslug. (h) A conjugate of anterior neural plate and lateral epidermis showed a strong Xslug expression (arrows). (i –k) A piece of stage 10 animal cap was grafted into the lateral region of a stage 13 embryo. The embryo was cultured until stage 17 and Xslug (j) and Xsox-2 (k) expression was analyzed by whole-mount in situ hybridization. The dotted line surrounds the transplant; notice that Xsox-2 (k), but no Xslug (l) expression was detected in the graft. np, anterior neural plate; ep, epidermis; m, lateral plate mesoderm.
	FIG. 4. Model of neural crest induction in Xenopus. We propose that during gastrulation a hypothetical signal coming from the lateral mesoderm (dashed line from L) starts the induction of the neural crest. At the same time the neural plate is induced. Later, at the end of gastrulation, the induced neural plate interacts with the epidermis to complete the induction of the neural crest. Dotted area: mesoderm. Black area: prospective neural crest. A, animal pole; Vg, vegetal pole; D, dorsal side; V, ventral side.