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A study of the molecules noggin and fibroblast growth factor (FGF) and its receptor in the induction of the prospective neural crest in Xenopus laevis embryos has been carried out, using the expression of the gene Xslu as a marker for the neural crest. We show that when a truncated FGF receptor (XFD) was expressed ectopically in order to block FGF signaling Xslu expression was inhibited. The effect of XFD on Xslu was specific and could be reversed by the coinjection of the wild-type FGF receptor (FGFR). Inhibition of Xslu expression by XFD is not a consequence of neural plate inhibition, as was shown by analyzing Xsox-2 expression. When ectoderm expressing XFD was transplanted into the prospective neural fold region of embryos Xslu induction was inhibited. The neural crest can also be induced by an interaction between neural plate and epidermis. As this induction is suppressed by the presence of XFD in the neural plate and not in the epidermis, it suggests that the neural crest is induced by FGF from the epidermis. However, treatment of neural plate with FGF was not able to induce Xslug expression, showing that in addition to FGF other non-FGF factors are also required. Previously we have suggested that the ectopic ventral expression of Xslu produced by overexpression of noggin mRNA resulted from an interaction of noggin with a ventral signal. Overexpression of XFD inhibits this effect, suggesting that FGF could be one component involved in this ventral signaling. Overexpression of FGFR produced a remarkable increase in the expression of Xslu in the posterior neural folds and around the blastopore. Injections in different blastomeres of the embryo suggest that the target cells of this effect are the ventral cells. Finally, we proposed a model in which the induction of the neural crests at the border of the neural plate requires functional FGF signaling, which possibly interacts with a neural inducer such as noggin.
FIG. 1. Inhibition of Xslu expression by XFD and rescue by FGFR. (A) Control embryo injected with an unrelated mRNA (0 .2 ng of /3-
gal mRNA) showing an abnormal closure of the blastopore. (B) Embryo injected at the 1-cell stage with 0.4 ng of FGFR mRNA, n = 24.
(C) Embryo injected in one blastomere at the 2-cell stage with 0.2 ng of XFD mRNA. Arrow: injected side. 97% inhibition, n = 31. (D)
Embryo injected at the 1-cell stage with 0.4 ng of XFD and 0.4 ng of FGFR. 9% inhibition, n = 22.
FIG. 2. Neural plate induction in XFD-injected embryos. (A) Cont rol embryo showing Xsox-2 expression in t he neural tube. (B) Embryo
injected in one blastomere at the 2-cell stage with 0.2 ng of XFD mRNA. Not ice the abnormal closure of the blastopore but t he normal
Xsox-2 expression at both sides of the embryo, n = 56.
FIG. 3. Inhibition of Xslu by overexpression of XFD in the ectoderm. (A) Embryos were injected with RLDx at the 1-cell stage and with
0.2 ng of XFD mRNA at the 2-cell stage. The injected embryos were cultured until stage 10.5 and a piece of ectoderm was dissected and
grafted into the anterior neural fold of a normal stage 13 embryo. As controls ectoderm was dissected from normal stage 10.5 embryos
and grafted as described. The grafted embryos were cultured until stage 17 and Xslu expression was analyzed. (B) The grafts of normal
(N) or XFD-injected (XFD) ectoderm were recognized by fluorescence and the Xslu expression in the graft was scored. For N, n = 18; for
XFD, n = 12. (C, D) T he embryos were sectioned and photographed under white light (D) for comparison with fluo rescent images (C).
Arrow, normal neural fold; arrowhead, grafted neural fold. (E) Animal caps taken at stage 10.5 and cultured until the equivalent of stage
17, when Xslug expression was analyzed. (F) Conjugates of animal caps taken from a normal stage 10.5 embryo and dorsal mesoderm
taken from an embryo injected at the 2-cell stage with 0.2 ng of XFD per blastomere. The conjugates were cultured until the equivalent
of stage 17, when Xslug expression was analyzed. Arrows, Xslug expression induced in the conjugate.
FIG. 4. Interaction between neural plate and epidermis requires
FGF signaling. Anterior neural plate and ventral epidermis were
dissected from a stage 12.5 embryo, the tissues were conjugated
and cultured until stage 17, and then Xslu was analyzed. Some
tissues were dissected from embryos injected at the 1-cell stage
with 0.4 ng of XFD mRNA. (A) Control neural plate conjugated
with control epidermis showing Xslu induction (arrows), n = 15.
(B) Conjugates of normal epidermis and XFD-injected neural plate,
n = 12. (C) Conjugates of normal neural plate and XFD-injected
epidermis, n = 10. Photograph inC was taken with higher magnification
than A, B, and D. (D) Conjugates of XFD-injected neural
plate and XFD-injected epidermis, n = 14.
FIG. 5. An interaction of FGF and Noggin induces Xslu. (A) Embryo
injected with noggin. (rop) Lateral view; (bottom) posterior view: D,
dorsal; V, ventral; A, anterior; P, posterior; red band represents Xslu
expression. (B- D) Posterior view of emb1yos injected with Noggin as
in bottom in A (B) Noggin mRNA was injected at the 1-cell stage, the
embryos were cultured until stage 17, and Xslu expression was analyzed.
Notice a ring of expression around the embryo. d, dorsal side of
the embryo; n = 35. (C, D) Noggin mRNA was injected at the 1-cell
stage and at the 2-cell stage emb1yos were injected with 0.2 ng of
XFD per blastomere. Notice the partial (C, 47% of inhibition) or total
inhibition (50% of inhibition, D) of Xslu expression in the injected side
of the embryo (arrowhead). The remaining expression in the partially
inhibited embryos was always in the dorsal side of the embryo (arrow).
d, dorsal side of the embryo: n = 38.
FIG. 6. Effect of the overexpression of FGFR on Xslu pattern. (A)
Lateral and (C) posterior views of a stage 1 7 normal embryo showing
Xslu expression ve1y strongly in the anterior neural folds, weaker in
the posterior neural folds, and absent around the blastopore. (B) Lateral
and (D) posterior views of a stage 17 normal embryo, injected at the
1-cell stage with 1.2 ng of FGFR mRNA and hybridized for Xslu expression.
Ectopic expression was observed in the posterior neural folds and
around the blastopore (arrow); n = 22. (E, F) Embryos were injected at
the 4-cell stage with 0.4 ng of FGFR mRNA in the dorsal (E, n = 17)
or ventral (F, n = 23) blastomeres. Notice the ectopic expression of
Xslu in F. b, blastopore: arrowhead, normal Xslu expression.
FIG. 7. Competence of the neural crest induction. Ventral ectoderm
was dissected from embryo at different stages and conjugated
with dorsal mesoderm taken from a stage 10.5 embryo. The conjugates
were cultured in vitro until the equivalent of stage 17 and
Xslu expression was analyzed. The percentage of conjugated expressing
Xslu was scored. The ectoderm was taken from normal
embryos (white circles) or from embryos injected with 0.4 ng of
XFD at the 1-cell stage (black circles): n = 10 - 15 for each point.
FIG. 8. Effect of the overexpression of Noggin and FGFR on Xslu
pattern. Embryos were injected at the 1-cell stage with a combination
of 0. 1 ng of noggin mRNA and 1.2 ng of FGFR, cultured until stage
17 and Xslu expression was analyzed. (A) Lateral view and (B) ventral
view of the most extreme alteration of Xslu pattern. Notice the wide
band of extension to the ventral side of the embryo, different from
the effect induced by noggin (see Fig. SB) or FGFR (see Fig. 6B). d,
dorsal side of the embryo: v, ventral side of the embryo. 19% of ectopic
Xslu expression exhibited a similar pattern; n = 32.
