Delaune E et al. (2005), Neural induction in Xenopus requires early FGF ...

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Development 2005 Jan 01;1322:299-310. doi: 10.1242/dev.01582.

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Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition.

Delaune E , Lemaire P , Kodjabachian L .

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Neural induction constitutes the first step in the generation of the vertebrate nervous system from embryonic ectoderm. Work with Xenopus ectodermal explants has suggested that epidermis is induced by BMP signals, whereas neural fates arise by default following BMP inhibition. In amniotes and ascidians, however, BMP inhibition does not appear to be sufficient for neural fate acquisition, which is initiated by FGF signalling. We decided to re-evaluate in the context of the whole embryo the roles of the BMP and FGF pathways during neural induction in Xenopus. We find that ectopic BMP activity converts the neural plate into epidermis, confirming that this pathway must be inhibited during neural induction in vivo. Conversely, inhibition of BMP, or of its intracellular effector SMAD1 in the non-neural ectoderm leads to epidermis suppression. In no instances, however, is BMP/SMAD1 inhibition sufficient to elicit neural induction in ventral ectoderm. By contrast, we find that neural specification occurs when weak eFGF or low ras signalling are combined with BMP inhibition. Using all available antimorphic FGF receptors (FGFR), as well as the pharmacological FGFR inhibitor SU5402, we demonstrate that pre-gastrula FGF signalling is required in the ectoderm for the emergence of neural fates. Finally, we show that although the FGF pathway contributes to BMP inhibition, as in other model systems, it is also essential for neural induction in vivo and in animal caps in a manner that cannot be accounted for by simple BMP inhibition. Taken together, our results reveal that in contrast to predictions from the default model, BMP inhibition is required but not sufficient for neural induction in vivo. This work contributes to the emergence of a model whereby FGF functions as a conserved initiator of neural specification among chordates.

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Species referenced: Xenopus
Genes referenced: admp bmp4 cdx4 cer1 chrd dkk1 fgf4 fgfr4 gsc hhex krt12.4 ncam1 nodal2 nog nr2c2 otx2 smad1 smad6 smad6.2 snai2 sox17a sox2 tbxt vegt zic1

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	Fig. 1. BMP inhibition in vivo is not sufficient for neural induction. In all panels, Sox2 expression marks prospective neural tissue, K81 expression marks prospective epidermis, XBra expression marks mesoderm, at the late gastrula stage 13, and Slug marks neural crest progenitors at early neurula stage 15. Marker gene expression is revealed in purple, while β-galactosidase activity resulting from injection of lacZ RNA as a lineage tracer is in red. (A) Sixteen-cell embryos injected with 100 pg CABR RNA in one dorsal-most animal blastomere. BMPR activation represses the neural marker Sox2 and activates the epidermal marker K81. (B) Eight-cell embryos injected animally in the two left blastomeres with lacZ (250 pg/blastomere) and tBR (400 pg/blastomere) RNAs showing neural plate expansion and epidermis suppression (u, uninjected side). (C-G) Sixteen-cell embryos injected in one ventral-most animal blastomere with lacZ (C), lacZ and tBR (400 pg, D), or lacZ and Smad6 (1 ng in E,G; 4 ng in F) RNAs. In whole embryos, BMP inhibition by Smad6 represses epidermis, but does not induce neural tissue. Ectopic neural crest could form in embryos that received 1 ng (arrow), but not 4 ng, Smad6 RNA. (G) Animal caps explanted at blastula stage 9 express Sox2 in response to Smad6 injection in ventral ectoderm cells. The inset in the lower panel illustrates that Sox2 expression (purple) is restricted to the injected region (red). (A,B) Dorsal views, anterior towards the top. (C-F) Ventral views, anterior towards the top, except right panels in E,F, which are lateral views, anterior towards the top. Scale bars: 500 μm.
	Fig. 3. FGFR signalling is required for neural induction. (A) Four-cell embryos were treated until fixation with various concentrations of SU5402, as indicated. Phenotypic classes were defined according to the severity of axial deficiencies. Class V embryos lacked muscle, NCAM-positive neural tissue and neural crests [as revealed by 12.101 (B) and 4d (C) immunostaining at tailbud stage, or by Slug RNA hybridisation at neurula stage (D)]. Class V embryos at gastrula stages lacked prospective posterior and axial mesoderm (XBra, Xcad3, ADMP; E-G), prospective haematopoietic mesoderm (Xnr2, H), but contained prospective endodermal tissue (sox17α, I). (J-M) Sox2 (J,K) and opl (L,M) expression are shown at early (J,L) or late (K,M) gastrula stages in control and class V embryos. No neural precursors are present in class V embryos. (N,O) Thirty-two-cell embryos were injected with 250 pg lacZ RNA in one A1 blastomere (animal-most, dorsal-most). In untreated controls, injected cells populate mostly the eye and the brain and some head epidermis (N). In the presence of 120 μM SU5402, embryos were class V, and the injected cells were now found entirely in the epidermis below the cement gland (O). These cells expressed the marker K81, indicating that prospective neural cells were converted into epidermal progenitors in absence of FGF activity. SU5402 is not toxic to ectoderm cells as they remain alive, and expressβ -galactosidase and K81. (A-C) Lateral view, anterior towards the left. (N) Lateral view, anterior towards the right. (D,G,J-M) Dorsal view, anterior towards the top. In M, the embryo is slightly tilted upwards. (E,F,H) Vegetal view, dorsal towards the top. (I) Hemisectioned embryo, dorsal towards the right. O, frontal view.
	Fig. 4. Both pharmacological and antimorphic FGF inhibitors block neural development in a specific manner. (B,C) Four-cell embryos were injected with lacZ RNA (B), or lacZ and 5 pg v-ras (C) RNAs, and embryos were treated with 120 μM SU5402. Neural plate tissue is rescued by v-ras injection in FGFR-deficient embryos. (D-I) Four-cell embryos were injected with lacZ and 1 ng δR4 (dominant-negative FGFR4) (D,G), lacZ, 1 ng δR4 and 1 ng FGFR4 (E,H), or lacZ, 1 ng δR4 and 4 ng tR4 (activated FGFR4) RNAs (F,I). δR4 is not toxic to the cells as they retain β-galactosidase activity but no longer express Sox2. (G-I) β-Galactosidase reaction was omitted to optimise NCAM immunostaining at tailbud stage. Both FGFR4 and tR4 can rescue the loss of neural tissue due to δR4 misexpression. (J,K) Eight-cell embryos were injected in both animal blastomeres with lacZ and δR4 (200 pg/blastomere) RNAs in order to target presumptive neural tissue.δ R4-injected cells do not express Sox2 at early gastrula stage 10.5. u, uninjected side. Broken lines in G-I indicate the midline. (A-I) Dorsal view, anterior towards the left. (J,K) Dorsal view, anterior towards the top.
	Fig. 7. Organiser gene expression in FGFR-deficient embryos. (A-H) Four-cell embryos were treated with 80 μM SU5402 until early gastrula stage 10.5 and fixed for in situ hybridisation. This treatment yielded class V embryos. In A-C, embryos were cut in two halves along the dorsoventral midline prior to hybridisation in order to improve probe penetration. The organiser genes cerberus (cerb; A), dickkopf1 (dkk1, B), hex (C), otx2 (D), goosecoid (gsc; E) and chordin (chd; F) are normally expressed in class V embryos, whereas noggin (nog; G) is not. Conversely, the epidermis inducer bmp4 is ectopically expressed in the dorsal ectoderm of class V embryos (H). Thus, BMP signalling is probably upregulated in prospective neural tissue in absence of FGF activity. (A-C) Dorsal towards the right. (D-G) Vegetal view, dorsal towards the top. (H) Dorsal view, anterior towards the top, broken lines indicate the blastopore.
	Fig. 8. Neural tissue development involves BMP/SMAD1-independent FGFR activity. (A-D) Stage 26 tailbud embryos immunostained for NCAM. (A) Untreated embryo; (B) class V embryo treated from stage 7 to 26 with 120 μM SU5402; (C) embryo treated similarly with SU5402 and which had received injection at the four-cell stage of lacZ, tBR (400 pg), noggin (250 pg) and Smad6 (4 ng) RNAs; (D) embryo treated similarly with SU5402 and injected at blastula stage 9 with 1 ng Noggin protein. BMP inhibitors converted epidermis into cement gland (cement glands are outlined in B-D), but did not rescue neural tissue in class V embryos. (E-G) These embryos received the same treatment and injection as in A-C, but were harvested at the early gastrula stage 10.5, and analysed for Sox2 expression. No rescue of Sox2 expression was obtained upon injection of the triple inhibitor combination in class V embryos. (H) Eight-cell embryos were injected in all four animal blastomeres with Smad6 RNA (1 ng/blastomere) and cultured in absence or in presence of 120 μM SU5402 from stage 7, as indicated. Animal caps were excised at blastula stage 9, further cultured in the inhibitor until late gastrula stage 13, and harvested at tailbud stage. Siblings were class V in this experiment. Neural induction by Smad6 is suppressed in absence of FGFR activity.
	Fig. 2. Neural induction in vivo by combined FGF signalling and BMP inhibition. (A-E) Sixteen-cell embryos were injected in one ventral-most animal blastomere with lacZ and 0.16 pg eFGF (A); lacZ, 0.16 pg eFGF and 1 ng Smad6 (B,C); lacZ, 0.16 pg eFGF and 200 pg tBR (D); or lacZ, 1 ng Smad6 and 1 pg v-ras RNAs (E). Low eFGF or activated ras signalling induces neural tissue, without Xbra- positive mesoderm, when combined with BMP inhibition in late gastrula stage 13 embryos. NCAM immunostaining at tailbud stage confirms the stable neural character of the induced tissue (arrow in C). (A,B,E) Ventral view, anterior towards the top; (C) lateral view, anterior towards the right; (D) lateral view, anterior towards the top. Scale bars: 500 μm.
	Fig. 6. FGF activity is required in the ectoderm for neural induction by Spemann’s organiser. (A) Ectoderm explants were taken at stage 10.25 or 11 from embryos that were treated (or not) with 80 μM SU5402. Animal caps were then washed and recombined with control organiser mesoderm explants prepared at the same stage. Conjugates were further cultured for 24 hours and analysed by immunostaining for NCAM expression. (B) Typical results obtained in such assays. NCAM expression is in blue pointed by asterisks, arrows indicate cement glands. Conjugates made with stage 11 SU5402-treated animal caps show either no NCAM signal (counted as negative) or dramatically reduced NCAM signal (counted as positive). Furthermore, these conjugates retain cement gland tissue despite the lack of neural tissue. (C) Bar graph presenting the results as the percentage of NCAM-positive conjugates over the total number of conjugates in each condition. Pre- treatment with the FGFR inhibitor considerably reduces the number of conjugates showing neuralisation.
	Fig. 9. A model for neural induction in Xenopus. See text for explanations. Coloured domains indicate prospective tissues. Orange and purple dots indicate β-catenin and VegT protein distribution in blastula nuclei, respectively. Arrows are not intended to represent direct regulation. The goal of this figure is to try and represent how the major molecular pathways known to impact on early embryonic patterning are integrated. It is not meant to give a complete view of this process. Regulatory arrows shown in black are inferred in part or totally from our work, while those shown in grey originate from other studies. Neural specification requires concomitant BMP inhibition, and low FGF signalling acting in a BMP-independent manner.
	ncam1 (neural cell adhesion molecule 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up
	dkk1 (dickkopf WNT signaling pathway inhibitor 1) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10.5, dorsal right, animal hemisphere up.