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Development
2002 Nov 01;12922:5103-15. doi: 10.1242/dev.129.22.5103.
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Initiating Hox gene expression: in the early chick neural tube differential sensitivity to FGF and RA signaling subdivides the HoxB genes in two distinct groups.
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Initiation of Hox genes requires interactions between numerous factors and signaling pathways in order to establish their precise domain boundaries in the developing nervous system. There are distinct differences in the expression and regulation of members of Hox genes within a complex suggesting that multiple competing mechanisms are used to initiate their expression domains in early embryogenesis. In this study, by analyzing the response of HoxB genes to both RA and FGF signaling in neural tissue during early chick embryogenesis (HH stages 7-15), we have defined two distinct groups of Hox genes based on their reciprocal sensitivity to RA or FGF during this developmental period. We found that the expression domain of 5' members from the HoxB complex (Hoxb6-Hoxb9) can be expanded anteriorly in the chick neural tube up to the level of the otic vesicle following FGF treatment and that these same genes are refractory to RA treatment at these stages. Furthermore, we showed that the chick caudal-related genes, cdxA and cdxB, are also responsive to FGF signaling in neural tissue and that their anterior expansion is also limited to the level of the otic vesicle. Using a dominant negative form of a Xenopus Cdx gene (XcadEnR) we found that the effect of FGF treatment on 5' HoxB genes is mediated in part through the activation and function of CDX activity. Conversely, the 3' HoxB genes (Hoxb1 and Hoxb3-Hoxb5) are sensitive to RA but not FGF treatments at these stages. We demonstrated by in ovo electroporation of a dominant negative retinoid receptor construct (dnRAR) that retinoid signaling is required to initiate expression. Elevating CDX activity by ectopic expression of an activated form of a Xenopus Cdx gene (XcadVP16) in the hindbrain ectopically activates and anteriorly expands Hoxb4 expression. In a similar manner, when ectopic expression of XcadVP16 is combined with FGF treatment, we found that Hoxb9 expression expands anteriorly into the hindbrain region. Our findings suggest a model whereby, over the window of early development we examined, all HoxB genes are actually competent to interpret an FGF signal via a CDX-dependent pathway. However, mechanisms that axially restrict the Cdx domains of expression, serve to prevent 3' genes from responding to FGF signaling in the hindbrain. FGF may have a dual role in both modulating the accessibility of the HoxB complex along the axis and in activating the expression of Cdx genes. The position of the shift in RA or FGF responsiveness of Hox genes may be time dependent. Hence, the specific Hox genes in each of these complementary groups may vary in later stages of development or other tissues. These results highlight the key role of Cdx genes in integrating the input of multiple signaling pathways, such as FGFs and RA, in controlling initiation of Hox expression during development and the importance of understanding regulatory events/mechanisms that modulate Cdx expression.
Comparison of Hoxb4 and Hoxb9 expression in the chick neural tube. Dorsal views of different stages of embryos hybridized with Hoxb4 (A-E) or Hoxb9 (F-J). Embryos are at stage 4 (A), stage 8 (B,F), stage 9 (C,G), stage 10- (H), stage 10 (D), stage 11 (I), stage 14 (E), or stage 17 (J). Expression of Hoxb4 remains at a fixed AP boundary in the neural tube once activated, whereas that of Hoxb9 regresses posteriorly in the later stages. White arrows indicate the initial boundary of expression. HIN, Hensen's node. Horizontal black bars mark the boundary of expression in the CNS relative to the adjacent somite (s) number or rhombomere (r).
Effect of RA treatment and somite grafting on Hoxb4 and Hoxb9 expression in the neural tube. Dorsal views of stage 15 (A-C,G) stage 19 (D,H) and stage 14 (E,F) embryos hybridized with Hoxb4 (A-D) or Hoxb9 (E-H). (A,E) Untreated embryos. (B,F) Retinoic acid treated embryos. Exogenous application of retinoic acid causes anterior shift of the expression domain and creates a new anterior limit of Hoxb4 expression (black arrow in B) while Hoxb9 does not show any anterior shift (F). (C,G) Embryos electroporated with a dnRAR expressing construct unilaterally on the left side of the neural tube. dnRAR causes down-regulation of endogenous Hoxb4 expression (white arrowheads in C) while Hoxb9 expression is not affected (G). (D,H) Hoxb4 and Hoxb9 expression in grafted embryos, whereby posteriorsomites 23-25 of a stage 15 donor embryo were transposed into an anterior region of a stage 15 host embryo at the level of somite 7-9 and cultured for 36hrs. The grafted somites induce upregulation of Hoxb4 (*, D) while there is no change in the pattern of Hoxb9 expression (*, H). Black arrowheads in H show position of graft. OV, otic vesicle; SM, somite grafts.
Effect of FGF treatment on Hoxb4 and Hoxb9 expression in the neural tube. Dorsal views of stage 14-15 embryos hybridized with Hoxb4 (A-C) or Hoxb9 (D-F). Untreated embryos (A,D), embryos treated overnight with FGF2 in culture (B,E), embryos electroporated with an e-FGF-expressing construct unilaterally in the left side of the neural tube (C,F). In both cases, Hoxb9 is upregulated by exogenous FGF (E,F) while Hoxb4 shows no change (B,C). In E, note that the anterior limit of the Hoxb9 expression reaches the level just posterior to the otic vesicle. Black arrowheads in F show the extended domain of Hoxb9 on the left and the control limit on the right in the neural tube.
Effect of FGF treatment on cdxA and cdxB expression in the neural tube. Dorsal views of stage 9 embryos hybridized with cdxA (A,B) and stage 15 embryos hybridized with cdxB (C,D). Untreated control embryos (A,C) and embryos treated for 6 hours (B) or overnight (D) with recombinant FGF2 protein in culture. In B and D white arrowheads show the normal boundary of expression and black arrowheads show the anteriorized limit of expression in the neural tube upon FGF2 treatment. In controls black arrowheads show the normal boundary.
Effect of FGF2 treatment and electroporation of activated and dominant negative cdx variants on Hoxb9 expression. Dorsal views of stage 15 embryos hybridized with Hoxb9 following treatment with FGF2 and/or electroporation of Xcad constructs. D,E,F shows higher magnification of A,B,C, respectively. (A,D) An embryo treated overnight with FGF2 as a control of the effect of FGF. (B,E) Electroporation of an activated form of Xcad (XcadVP16) unilaterally in the left side of the neural tube induces an anterior expansion of Hoxb9 expression (bracket in B and arrowheads in E). (C,F) An embryo electroporated with a dominant negative form of Xcad (XcadEnR) unilaterally on the left side of the neural tube and cultured for overnight in the presence of FGF2 shows that the FGF mediated induction of Hoxb9 is reduced. Note that the anterior boundary of Hoxb9 expression on the right side (non-electroporated side) is just posterior to the otic vesicle (OV) because of the FGF treatment. The bracket in C and white arrowheads in F mark the zone where the ectopic expression of Hoxb9 caused by FGF treatment is down-regulated by electroporation of the XcadEnR construct.
