Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a

Abstract

Members of the Wnt family of signaling molecules are expressed differentially along the dorsal-ventral axis of the developing neural tube. Thus we asked whether Wnt factors are involved in patterning of the nervous system along this axis. We show that Wnt-1 and Wnt-3a, both of which are expressed in the dorsal portion of the neural tube, could synergize with the neural inducers noggin and chordin in Xenopus animal explants to generate the most dorsal neural structure, the neural crest, as determined by the expression of Krox-20, AP-2, and slug. Overexpression of Wnt-1 or Wnt-3a in the neuroectoderm of whole embryos led to a dramatic increase of slug and Krox-20-expressing cells, but the hindbrain expression of Krox-20 remained unaffected. Enlargement in the neural crest population could occur even when cell proliferation was inhibited. Wnt-5A and Wnt-8, neither of which is expressed in the dorsal neuroectoderm, failed to induce neural crest markers. Overexpression of glycogen synthase kinase 3, known to antagonize Wnt signaling, blocked the neural-crest-inducing activity of Wnt-3a in animal explants and inhibited neural crest formation in whole embryos. We suggest that Wnt-1 and Wnt-3a have a role in patterning the neural tube along its dorsoventral axis and function in the differentiation of the neural crest.

Figure 1

Expression of neural crest markers in animal cap explants injected with Wnt RNAs or DNAs, as seen by RNA blotting. RNAs were extracted from animal explants injected with Wnt RNAs or DNAs (CS2+Wnt), alone (−noggin), or with noggin RNA (+noggin), from whole embryos at the indicated stage and from uninjected explants (none). The blots were sequentially hybridized with Krox-20, AP-2, slug, nrp-1, and otx-2 probes. Ethidium bromide-stained 18S ribosomal RNA is shown as a loading control. In some experiments (two of six), coexpression of Xwnt-8 and noggin induced low levels of slug RNA but consistently failed to induce Krox-20 or AP-2 (b and d). See Materials and Methods for levels of RNA injected, except for d, where the amount of injected RNA in pg is indicated. a–e represent five experiments.

Figure 4

Both the axis inducing and neural crest inducing activities of Xwnt-3a are antagonized by GSK-3β. (a) GSK-3β partially inhibits axis rescue by Xwnt-3a. Partial rescue is observed in UV-treated embryos (DAI 0.1) injected with Xwnt-8 RNA (DAI 3.9) or Xwnt-3a RNA (DAI 2.6); rescue is inhibited when Xwnt-8 plus GSK-3β RNAs (DAI 1) or Xwnt-3a plus GSK-3β RNAs (DAI 1.2) are coinjected. (b) GSK-3β inhibits neural crest induction by Xwnt-3a. Northern blot from animal explants injected with Xwnt-3a or Xwnt-8 RNAs plus noggin RNA, with (+GSK-3β) or without GSK-3β RNA. Coinjection of GSK-3β and noggin RNAs leads to the same repertoire of gene expression as noggin RNA alone (data not shown). DAI, dorsoanterior index (30).

Figure 2

Expression of Krox-20 (a–c, g, and h) and slug (d–f) in Xenopus embryos (stage 20–25), as seen by whole-mount in situ hybridization. The embryos are viewed from the dorsal side, and anterior is to the top. Krox-20 expression in a control embryo (a) is in two stripes (rhombomeres 3 and 5) in the hindbrain and a stream of neural crest cells adjacent to rhombomere 5. Xwnt-3a-injected embryo (b); in the injected half (note the turquoise β-gal staining in the left half) the stream of Krox-20-expressing cells is dramatically expanded while still arising from a zone adjacent to rhombomere 5. In an Xwnt-8-injected embryo (c), the pattern of expression of Krox-20 is unaffected. Slug expression in a control embryo (d). Xwnt-3a-injected embryo (e) shows an enlargement of the domain of expression of slug within the injected right half (see β-gal staining). An Xwnt-8-injected embryo (f) presents a pattern of slug expression in the injected half (right side, β-gal staining) comparable to the uninjected half. (g) Higher magnification of the hindbrain region of the embryo presented in b. Rhombomere 3 (R3) and rhombomere 5 (R5) expression of Krox-20 is not affected; NC indicates stream of neural crest. CS2+Xwnt-3a-injected embryo (h) presents an expanded domain of expression of Krox-20 in the injected half (see β-gal staining). The dotted lines indicate the dorsal midline. Among the embryos injected in the animal pole region, 30% showed some level of axis duplication when injected with Wnt-1, Xwnt-3a, or Xwnt-8 RNAs.

Figure 3

Expression of Krox-20 after inhibition of cell proliferation by HUA. General appearance of a control embryo (a) and an HUA-treated embryo (b) at equivalent stage 35. Hematoxylin-eosin staining (c and e) and 4,6-diamidino-2-phenylindole (nuclear) staining (d and f) of sections of a control embryo (c and d) and an HUA-treated embryo (e and f). The neural crest expression of Krox-20 is unaffected in HUA-treated embryos although rhombomere expression is reduced (h) as compared with control untreated embryo (g). The increase in neural crest cells expressing Krox-20 generated upon Xwnt-3a RNA injection appears similar in untreated (i) and in HUA-treated (j) embryos.

Figure 5

Expression of Krox-20 in embryos injected with 1 ng of GSK-3β RNA, as seen by whole-mount in situ hybridization. The embryos are viewed from the dorsal side, and anterior is to the top. (a) Group of three embryos coinjected on the left side with GSK-3β and β-gal RNAs (turquoise staining). (b and c) GSK-3β-injected embryos at a higher magnification. The stream of Krox-20-positive neural crest cells (NC) arising from rhombomere 5 (R5) is absent from the injected half (left side), whereas the rhombomere expression is unaffected. When β-gal staining is not detected in the dorsal region of the injected embryo the neural crest expression of Krox-20 is unaffected, as shown in d, where β-gal staining is ventral-anterior.

Figure 6

Expression of dorsal and ventral neural genes in animal explants injected with noggin RNA, Xwnt-3a RNA, or a combination of both. Animal explants coexpressing noggin and Xwnt-3a activate a broad range of dorsally expressed genes (Krox-20, slug, wnt-1, and pax-3) but failed to activate ventrally expressed genes (shh and FKH-1).