Pownall ME et al. (1996), eFGF, Xcad3 and Hox genes form a molecular path...

XB-ART-17326

Development 1996 Dec 01;12212:3881-92. doi: 10.1242/dev.122.12.3881.

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eFGF, Xcad3 and Hox genes form a molecular pathway that establishes the anteroposterior axis in Xenopus.

Pownall ME , Tucker AS , Slack JM , Isaacs HV .

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Classical embryological experiments suggest that a posterior signal is required for patterning the developing anteroposterior axis. In this paper, we investigate a potential role for FGF signalling in this process. During normal development, embryonic fibroblast growth factor (eFGF) is expressed in the posterior of the Xenopus embryo. We have previously shown that overexpression of eFGF from the start of gastrulation results in a posteriorised phenotype of reduced head and enlarged proctodaeum. We have now determined the molecular basis of this phenotype and we propose a role for eFGF in normal anteroposterior patterning. In this study, we show that the overexpression of eFGF causes the up-regulation of a number of posteriorly expressed genes, and prominent among these are Xcad3, a caudal homologue, and the Hox genes, in particular HoxA7. There is both an increase of expression within the normal domains and an extension of expression towards the anterior. Application of eFGF-loaded beads to specific regions of gastrulae reveals that anterior truncations arise from an effect on the developing dorsal axis. Similar anterior truncations are caused by the dorsal overexpression of Xcad3 or HoxA7. This suggests that this aspect of the eFGF overexpression phenotype is caused by the ectopic activation of posterior genes in anterior regions. Further results using the dominant negative FGF receptor show that the normal expression of posterior Hox genes is dependent on FGF signalling and that this regulation is likely mediated by the activation of Xcad3. The biological activity of eFGF, together with its expression in the posterior of the embryo, make it a good candidate to fulfil the role of the 'transforming' activity proposed by Nieuwkoop in his 'activation and transformation' model for neural patterning.

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Species referenced: Xenopus
Genes referenced: bmp4 cdx4 fgf4 hoxa7 hoxb1 hoxb9 hoxc6 ncam1 ncoa6 odc1 otx2 post tbxt

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	Fig. 1. Gene expression of embryos injected with CSKA-eFGF. RNAase protection analysis of embryos injected at the 2-cell stage with 10 pg of CSKA-eFGF or uninjected control embryos were cultured until early gastrula (stage 10), late gastrula (stage 12), early neurula (stage 14), or late neurula (stage18). 5 mg of total RNA was assayed by RNAase protection analysis for the expression of a panel of regional markers. All assays shown were carried out on RNA from the same experiment. The ODC loading control is a representative example.
	Fig. 2. Analysis of eFGF expression in embryos overexpressing eFGF from the CSKA-eFGF plasmid. (A) Control stage 11.5 embryo showing normal expression of eFGF; vegetal view. (B) CSKA-eFGF embryo at stage 11.5 showing extent of ectopic eFGF expression around the blastopore; vegetal view. (C) CSKA-eFGF embryo at stage 11.5 showing the extent of ectopic eFGF expression; dorsal view. Embryos were injected at the 2-cell stage with 10 pg of CSKAeFGF. Arrowheads indicate the dorsal lip of the blastopore.
	Fig. 3. In situ hybridisation analysis of albino embryos injected at the 2-cell stage with 10 pg of CSKA-eFGF or of uninjected controls. Embryos were cultured until early neurula, late neurula or tailbud stages and were then hybridised to antisense DIG labelled probes for HoxA7 (AH); HoxB9 (I-N); Xcad3 (O-P, W-Y); and Xbra (S-V). Controls (A-D, I-K, O-P, S-T) are shown above the corresponding CSKA-eFGF embryos (E-H, L-N, Q-R, UV) except for the control for Xcad3 expression in tailbudstage embryos (W) which is shown beside the corresponding expression of Xcad3 in CSKA-eFGF tailbudstage embryos(X-Y). (Z) Ventral view of another CSKAeFGF embryo with a duplicated posterior axis hybridised to Xpo which also marks the tailbud and proctodaea. Arrowheads indicate the anterior limit of the normal expression of HoxA7 and HoxB9, respectively, at tail bud stages. Anterior is to the left is all cases.
	Fig. 4. In situ hybridisation analysis of regional markers in albino embryos injected with 2ng of mRNA encoding the dominant negative form of the FGF receptor. Embryos were cultured to early neurula (stage13) (A-C; E-G; I-K; M-O) and late neurula (stage 20) (B-D; F-H; J-L; N-P). Control embryos (A-B; E-F; I-J; M-N) are shown above embryos overexpressing the dominant negative form of the FGF receptor (C-D; G-H; K-L; O-P). The markers are HoxA7 (AD), Xcad3 (E-H), and HoxB1 (I-L) and otx2 (M-P). In all cases, anterior is to the left. Embryos are viewed dorsally in all but N which is a side view, O and P which are ventral views, and E which is a dorsal-posterior view. C,D,G,H,K,L are viewed down onto the open blastopore.
	Fig. 5. eFGF beads activate anterior expression of posterior Hox genes in mesoderm and neurectoderm. (A) In situ hybridisation analysis of HoxA7 expression in control tailbud embryo (top) or tailbud-stage embryo where an eFGF bead was implanted into the anterior neural plate at stage12.5 (bottom). Dorsal view; anterior to the left. (B) In situ hybridisation analysis of Xcad3 expression in control tailbud embryo (top) or tailbud-stage embryo where an eFGF bead was implanted into the anterior neural plate at stage12.5 (bottom). Dorsal view; anterior to the left. (C) The dorsal marginal zone region, not including the dorsal lip, was taken from stage 10 embryos. Two explants were used to make a sandwich around an eFGF bead and were cultured until stage14 (to assay expression of Xbra) or stage 20 (to assay expression of HoxC6, HoxA7, HoxB9 and NCAM) and processed for RNAase protection analyisis. (D) The anterior and middle neural plate was taken from stage 13 embryos. Two explants were used to make a sandwich around an eFGF bead, and were cultured until stage 20 at which time they were processed for RNAase protection analysis to assay the expression of HoxC6 and HoxA7.
	Fig. 6. Phenotypes and gene expression in embryos implanted with a heparin acrylamide bead loaded with eFGF protein. (A) Embryos were implanted with a bead into the dorsal blastopore lip at stage 11.5. The top embryo was implanted with a PBS bead, while each of the bottom three embryos were implanted with an eFGF bead and show head suppression. (B) Embryos were implanted with a bead into the ventral blastopore lip at stage 11.5. The top embryo was implanted with a PBS bead, while each of the bottom three embryos were implanted with an eFGF bead and show enlarged proctodaea. (C, D) In situ hybridisation of embryos. Both embryos were cultured to late neurula stages and hybridised to HoxA7 probe (C) or Xcad3 probe (D). The top embryo is a control, the bottom embryo had an eFGF bead implanted into the dorsal blastopore at stage 11.5.
	Fig. 7. Injection of HoxA7 mRNA dorsally, but not ventrally, results in head suppression in embryos. (A) The top embryo is an uninjected control while the bottom embryos have been injected with 1ng of HoxA7 into the dorsal two blastomeres at the four-cell stage. (B) All the embryos shown in this panel have been injected with 1 ng of HoxA7 into the ventral two blastomeres at the four-cell stage. (C) Stage 20 embryo co-injected with 1 ng HoxA7 and 500 pg of b- gal mRNA in to the dorsal two blastomeres at the 4-cell stage. (D) Tailbud embryo that had been co-injected with 1 ng HoxA7 and 500 pg of b-gal mRNA in to the dorsal two blastomeres at the 4-cell stage. (E) Stage 20 embryo co-injected with 1 ng HoxA7 and 500 pg of b-gal mRNA in to the ventral two blastomeres at the 4-cell stage. (F) Tailbud embryo that had been co-injected with 1 ng HoxA7 and 500 pg of b-gal mRNA into the ventral two blastomeres at the 4-cell stage. (G) Top embryo is a control, while the bottom three embryos have been injected with 200 pg of Xcad3 mRNA into the dorsal two blastomeres at the 4-cell stage. (H) Top embryo is a control, while the bottom three embryos have been injected with 200 pg of Xcad3 mRNA into the ventral two blastomeres at the 4-cell stage.
	Fig. 8. Xcad3 regulates Hox gene expression downstream of FGF signalling. (A) Injection of Xcad3 mRNA causes the early activation of HoxA7 expression, but does not effect the expression of eFGF or Xbra; 400pg of Xcad3 was injected at the 4-cell stage, RNA from embryos was collected for analysis at stage10. (B) Injection of Xcad3 mRNA can rescue the expression of HoxA7 in embryos where the FGF signalling pathway has been blocked (XFD-injected). 400 pg of Xcad3 was injected at the 4-cell stage ± 2 ng of XFD mRNA. RNA from embryos was collected for analysis at stage13. (C) Injection of HoxA7 mRNA does not affect the expression of eFGF, Xbra or Xcad3. 2 ng of HoxA7 mRNA was injected at the 4-cell stage and RNA from embryos was collected for analysis at stage12.5 (D) Injection of Xbra mRNA does not rescue the expression of HoxA7 in embryos where the FGF signalling pathway has been blocked (XFD-injected). 2 ng of Xbra mRNA was injected at the 4-cell stage ± 2 ng of XFD mRNA. RNA from embryos was collected for analysis at stage13. All hybridisations were done with 10 mg of total RNA.
	Fig. 9. A molecular pathway depicting the role of FGF signalling in patterning the anteroposterior axis during gastrula stages. FGF signalling is important in regulating the dorsal expression of members of the cdx family, which directly regulate Hox gene expression required for patterning the anterposterior axis. Brachyury expression is known to be dependent on FGF signalling and also feeds back to activate the expression of FGF during gastrula stages, however, Brachyury does not appear to be a direct regulator of Hox gene expression. FGF and other signals, such as BMP4, activate ventral expression of cdx and other posterior regulators which pattern ventroposterior mesoderm and induce the proctodaeum.