Epistasis experiments by mRNA misexpression. (A-B') Misexpression of ca-alk8 leads to a spherical embryonic shape in wild-type and MZspg embryos at the end of gastrulation, indicating ventralization. (C,C') A similar ventralization is seen after bmp2b misexpression in wild-type and MZspg embryos. (D-G) In situ hybridization analysis for pax2.1, krox20, ntl and myod expression. (D) Dorsal view of a non injected wild-type embryo. (D') Non-injected MZspg embryo, dorsal view shows ntl expression; inset shows ventral view with somitic myod expression in MZspg. (E,E') Expression of neuroectodermal genes such as krox20 and pax2.1, and of axial or paraxial markers such as ntl and myod is repressed or lost in MZspg embryos at the end of gastrulation. (F,F') A similar repression of neuroectodermal and paraxial markers is observed in bmp2b misexpressing wild-type and MZspg embryos. (G) bmp4 misexpression leads to a similar ventralization of MZspg as obtained by bmp2b misexpression. (H) Injection of bmp2b mRNA is not able to rescue endodermal sox17 expression in MZspg at tb stage. (I) Wild-type bmp2b expression. (J,K,M,N) Embryos injected with c.a.alk8-mRNA. (J) ca-alk8 misexpression slightly enhances bmp2b expression in the wild type and restores bmp2b initiation in MZspg to the wild-type expression level (K). (L) bmp4 is not expressed at sphere stage in the wild type. (M) ca-alk8 misexpression causes slight ectopic bmp4 expression at oblong-sphere stage in wild-type and MZspg embryos (N). (O-R) Embryos injected with XFD-mRNA. (O) XFD misexpression in the wild type leads to upregulation of bmp2b when compared with bmp2b expression of non-injected wild-type embryos (inset; upper embryo, 50% epiboly; lower embryo, shield stage). (P) bmp2b expression fully restores in XFD-injected MZspg embryos. (Q) bmp4 expression is not sensitive to XFD misexpression in wild-type embryos (inset shows non-injected wild-type embryos; upper embryo, 50% epiboly; lower embryo, shield stage). (R) bmp4 is not restored in XFD-injected MZspg embryos.
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