Launay C et al. (1996), A truncated FGF receptor blocks neural inductio...

XB-ART-18493

Development 1996 Mar 01;1223:869-80.

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A truncated FGF receptor blocks neural induction by endogenous Xenopus inducers.

Launay C , Fromentoux V , Shi DL , Boucaut JC .

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We have examined the role of fibroblast growth factor (FGF) signalling in neural induction. The approach takes advantage of the fact that both noggin and the dominant negative mutant activin receptor (delta1XAR1) directly induce neural tissues in the absence of dorsal mesoderm. A truncated FGF receptor (XFD) is co-expressed with noggin or delta1XAR1 in both whole embryos and isolated animal caps. We demonstrate that inhibition of FGF signalling prevents neural induction by both factors. Furthermore, neural induction by organizers (the dorsal lip of blastopore and Hensen's node) is also blocked by inhibiting FGF signalling in ectoderm. It has been proposed that the specification of anterior neuroectoderm, including the cement gland, occurs in a sequential manner as gastrulation proceeds. We show that the specification of the most anterior neuroectoderm by noggin may occur before gastrulation and does not require FGF signalling, since both the cement gland marker XCG-1 and the anterior neural marker Otx-2 are normally expressed in ectodermal explants co-injected with noggin and XFD RNAs, but the cement gland cells are poorly differentiated. In contrast, the expression of both genes induced by CSKA.noggin, which is expressed after the mid-blastula transition, is strongly inhibited by the presence of XFD. Therefore the noggin-mediated neural induction that takes place at gastrula stages is abolished in the absence of FGF signalling. Since inhibition of FGF signalling blocks the neuralizing effect of different neural inducers that function through independent mechanisms, we propose that FGF receptor-related-signalling is required for the response to inducing signals of ectodermal cells from gastrula.

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Species referenced: Xenopus laevis
Genes referenced: acta4 actl6a acvr2b fgfr1 gsc hoxa9 muc2 ncam1 nog prph tbxt
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	Fig. 1. Overexpression of RNAs encoding noggin and XFD in whole embryos. (A) Anteriorized embryos at stage 35 resulted from injection of noggin RNA. (B) Control embryo injected with RNAs encoding noggin and HAVø. The phenotype is essentially similar to that of the noggin-injected embryo. (C) Dorsal-deficient embryo resulting from co-injection of noggin and XFD RNAs. Note that a large area of cement gland pigmentation is visible. (D) An uninjected embryo at stage 35. Scale bar, 500 mm.
	Fig. 2. Whole-mount immunocytochemistry of N-CAM expression in stage-30 embryos. The embryos were labelled by antibodies directed against Xenopus N-CAM. (A) Control embryo. N-CAM is detected in the eyes and the entire central nervous system. (B) Noggin-injected embryo. Ectopic neural tubes are revealed by anti-N-CAM antibodies. (C) Embryo injected with RNAs encoding noggin and XFD. Small amounts of N-CAM-positive cells are present. (D) Embryos injected with XFD RNA in the dorso-animal blastomeres at the 8-cell stage. Small amounts of neural tissues and fused eyes (arrowheads) are present. (E) Embryos injected with XFD RNA in the dorsovegetal blastomeres at the 8-cell stage. Notice the trunk deficiency and unaffected head region with normal eyes (arrowheads). Scale bar, 500 mm.
	Fig. 3. Expression of neuralizing factors and XFD in animal caps cultured to stage 35. (A) Uninjected animal caps. (B) XFD-injected animal caps. (C) CSKA.noggin-injected animal caps. Cement gland forms in the middle of the elongated explants. (D) Animal caps coinjected with CSKA.noggin and XFD have a spherical shape and no cement gland pigmentations are visible. (E) Histological section of an explant resulting from injection of CSKA.noggin alone. Elongated cement gland cells (cg) and a mass of neural tissue (nt) are present. (F) A section from an explant injected with CSKA.noggin and XFD. This explant differentiates as atypical epidermis (ep). (G) Animal caps from embryos injected with noggin RNA. (H) Animal caps from embryos co-injected with noggin and XFD RNAs are rounded but cement gland pigmentations are visible. (I) Animal caps from embryos injected with D1XAR1 RNA. (J) Animal caps from embryos co-injected with D1XAR1 and XFD RNAs. Scale bars, 500 mm.
	Fig. 4. RNase protection analyses of gene expression in animal caps derived from embryos co-injected with neuralizing factors and XFD. Animal caps were dissected at stage 8 and cultured to stage 35. This is a representative RNase protection assay using 60 explants for each condition. An equivalent of one-half or two explants was hybridized with XCG-1 and epidermal keratin probe, respectively. N-CAM and XIF-3, and TSP-1 and Otx-2 probes, were respectively included in the same hybridization reaction. The type of injection is indicated on the top and the gene analyzed is shown on the right. EF-1a probe is used as a loading control.
	Fig. 5. Whole-mount immunostaining of N-CAM expression in explants cultured to stage 30. (A) Explants derived from embryos injected with noggin RNA. A mass of neural tissue is present on one side of the elongated explants. (B) Explants derived from embryos injected with RNAs encoding noggin and XFD are devoid of NCAM staining. Scale bar, 500 mm.
	Fig. 6. RNase protection analyses of the expression of mesodermal and neural genes to control for the absence of mesoderm in the explants. (A) At stage 11, the early mesodermal genes goosecoid (gsc) and XBra are not detected in explants derived from all kinds of injection. (B) Expression of neural and late mesodermal genes in explants cultured to stage 25. N-CAM is induced by noggin, CSKA.noggin and DX1AR1; XFD inhibited its expression. XlHbox-6 and muscle-specific (Mus.) actin are not detectable in these explants. Both EF-1a and cytoskeletal (Cyto.) actin are loading controls. (C) Rescue experiment. Co-injection of the wild-type XFR RNA reversed the inhibitory effects of XFD on N-CAM expression.
	Fig. 7. Effect of XFD on neural induction by Hensen’s node (HN) and Spemann organizer. (A) Schematic representation of these induction experiments. (B) Explants dissected from stage-9 embryos injected with HAVø RNA. HN induces the appearence of neural plates (arrows). (C) Recombinates between XFD-injected ectoderm and HN remain rounded. (D,E) Whole-mount immunostaining of the expression of N-CAM. Recombinates between HAVø caps dissected from stage-10.5 gastrula and DMZ (organizer) are intensely labelled by anti-N-CAM antibodies (D). Low levels of N-CAM are present in recombinates between XFD caps and DMZ (E). Scale bars, 500 mm. (F) RNase protection analysis. Both DMZ and HN induce the expression of N-CAM in HAVø-injected explants from stage 10.5 gastrula, while low levels of N-CAM transcripts are present in recombinates using XFD caps.
	Fig. 8. Whole-mount in situ hybridization of the expression of XCG- 1 transcripts in animal cap explants derived from embryos injected with neuralizing factors and XFD. (A,B) Explants from uninjectedand XFD-injected embryos. No XCG-1 labelling is detected. (C) Explants from CSKA.noggin-injected embryos. (D) Explants from embryos co-injected with CSKA.noggin and XFD. Small patches of XCG-1-positive cells are present. (E) Explants from embryos injected with noggin RNA. (F) Explants from embryos coinjected with noggin and XFD RNA. Notice that nearly the whole surface of these explants is XCG-1-positive. (G) Histological section cut after the chromogenic reaction from a noggin-injected explant. Notice the elongated cement gland cells. (H) Section cut from an explant corresponding to (F). The XCG-1-positive cells are not elongated. (I) Explants from embryos injected with D1XAR1 RNA. (J) Explants from embryos co-injected with D1XAR1 and XFD RNAs. The XCG-1-positive areas are reduced to small patches. Scale bars: (G,H), 100 mm; (A-F,I,J), 500 mm.