XB-ART-3593Development June 1, 2004; 131 (11): 2653-67.
Multiple points of interaction between retinoic acid and FGF signaling during embryonic axis formation.
Anteroposterior (AP) patterning of the developing CNS is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. We have examined the interactions between FGF and retinoic signaling pathways. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, we found that posterior expression of FGFR1 and FGFR4 was dependent on the expression of RARalpha2. Anterior expression was also altered with FGFR1 expression being lost, whereas FGFR4 expression was expanded beyond its normal expression domain. RARalpha2 is required for the expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. We conclude that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP neural patterning depends on a series of mutually interactive feedback loops among FGFs, RARs and HOX genes.
PubMed ID: 15128657
Article link: Development
Genes referenced: aldh1a2 cdx1 cdx2 cdx4 cyp26a1 fgf8 fgfr1 fgfr4 hoxb9 hoxc9-like rab40b rara rarb tbxt wnt8a
Morpholinos: rara MO2 rara MO4
Article Images: [+] show captions
|Fig. 1. Developmental expression of XRARα. Whole-mount in situ hybridization was performed on embryos from stage 9 to stage 25 using a probe that recognizes all isoforms of XRARα. (A) Dorsal (left) and ventral (right) view of a stage 9 embryo. (B,D,F,H,J) Frontal views. (C) A dorsal view of the stage 10 embryo. (E) A vegetal view of the stage 10 embryo. Note the sharp anterior border of strong staining.|
|Fig. 2. XRARα2.2 loss-of-function leads to axial truncations and reduction of HOXB9 expression. (A-D) Microinjection of RAR-MO causes anterior and posterior truncations at highest frequency when expressed in the head region (A) or dorsally (B). (C) Phenotypes are mild to undetectable when the lineage tracer is distributed laterally or ventrally. (D) Phenotypes are rescued by co-injection of XRARα2 mRNA, irrespective of where the lineage tracer is located. (E,F) Neither XBRA (E) nor XWNT8 (F) expression is affected by RAR-MO injection. (G-K) Effects of RAR-MO on the expression of HOXB9. (H-J) The types of phenotypes obtained. (K) HOXB9 expression was restored by co-injecting XRAR mRNA and RAR-MO.|
|Fig. 3. Modulating retinoid signaling affects the expression of XCAD3 [cdx4] but not XCAD1 [cdx2] or XCAD2 [cdx1] . (A-C,F-H,K-M) Embryos were treated with the indicated compound or ethanol solvent controls from the early blastula stage (stage 7) until harvesting when control embryos reached stage 18. Embryos were fixed and processed for whole-mount in situ hybridization with the indicated probes. (A,F,K) 10–6 M AGN193109 (RAR-selective antagonist), (B,G,L) ethanol solvent control, (C,H,M) 10–6 M TTNPB (RAR-selective agonist). RAR-MO was injected unilaterally at the two-cell stage with β-galactosidase lineage tracer alone (D,I,N) or together with 1 ng XRARα2 mRNA (E,J,O). Embryos were fixed when controls reached stage 18, stained for β-galactosidase activity and processed for whole-mount in situ hybridization with the indicated probes. Some embryos were used for RNA extraction and QRT-PCR analysis as described in the text.|
|Fig. 4. FGF8 cannot rescue the effects of XRARα2.2 loss-of-function on posterior marker genes. Embryos were microinjected at the two-cell stage with the indicated reagents, allowed to develop until controls reached stage 18 and processed for whole-mount in situ hybridization with either HOXB9 (A-E) or XCAD3 (F-J) probes.|
|Fig. 6. FGF gene loss of function alters the expression of RAR signaling pathway components in microinjected embryos. Embryos were microinjected unilaterally into one blastomere at the two- or four-cell stage with 1 ng of XFD mRNA and β-galactosidase mRNA as lineage tracer. Embryos were allowed to develop until controls reached stage 11 then fixed and processed for whole-mount in situ hybridization with the probes (A,B) XCAD3 [cdx4], (C,D) XRARα [rara], (E,F) RALDH2 [aldh1a2] or (G,H) CYP26 [cyp26a1].|
|Fig. 8. XRARα2.2 loss-of-function alters the expression of FGF8, FGFR4 and FGFR1. Embryos were microinjected unilaterally with β-galactosidase mRNA plus 10 ng RAR-MO or 10 ng RAR-MO plus 1 ng XRARα2 mRNA. Embryos were fixed when control uninjected embryos reached stage 18, stained for β-galactosidase activity and then processed for in situ hybridization with FGF8 (A-F), FGFR4 (G-L) or FGR1 (M-R) probes.|
|Fig. 10. XCAD3 expression requires RAR at early but not late stages. Embryos were treated with either TTNPB (E-H) or AGN193109 (I-L) at the blastula stage and cultured until controls (A-D) reached the indicated stages, fixed and processed for in situ hybridization with XCAD3. (A,C,E,G,I,K) Lateral views; (B,D,F,H,J,L) dorsal views.|