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Fig. 1. Wnt8 and Fgf8a are necessary for NC formation. (A) Embryos injected with Fgf8a (Fgf8aMO; 50 ng) or Wnt8 (Wnt8MO; 40 ng) morpholino antisense oligonucleotides exhibit a strong reduction of Pax3, Snail2, Sox8 and Sox10 expression at the neurula stage, while the expression domain of the pan-neural marker Sox2 is expanded. Embryos are viewed from the dorsal side anterior to the top. Injected side is on the right. (B) At the gastrula stage, Fgf8aMO- and Wnt8MO-injected embryos show normal expression of the mesoderm marker Xbra. Embryos are viewed from the vegetal pole. (C) At the tailbud stage the migration pattern of cranial NC cells is severely perturbed in both Fgf8aMO- and Wnt8MO-injected embryos, as revealed by expression of the cranial NC marker Ap2. Lateral views, dorsal to top. Left panels, anterior to the right (injected side); right panels, anterior to the left (control side). (D) TUNEL staining shows a similar increase in apoptotic cells in the cranial region of Fgf8aMO- and Wnt8MO-injected embryos at the tailbud stage (arrows). Embryos are viewed from the dorsal side, anterior to the right. The dotted lines indicate the position of the midline. (E) In animal explants, Wnt8 (W, 25 pg) or Fgf8a (C, 5 pg) share the same ability to induce NC markers (Pax3, Snail2 and Sox8) when co-expressed with the Bmp antagonist Chordin (C, 10 pg; C+W and C+F, respectively). In these explants the induction of NC fate occurs in the absence of mesoderm formation (mActin and Col2a1). Fgf8a also synergizes with Chordin to induce neural tissue (Sox2). Values (n=3) are presented as mean±s.e.m.; *P<0.05, versus uninjected animal explant (U). (F) The dual requirement of Fgf8a and Wnt8 suggests that these factors are acting either in parallel (1), or in the same pathway, one upstream of the other (2,3), to generate the NC. |
