Foxcla Is Essential For Zebrafish Anterior-posterior Axis Patterning And Somitogenesis

The formations of anterior-posterior (AP), left-right (LR) and dorsal-ventral (DV) axes are important developmental events that are finely regulated by multiple signaling pathways such as Bmp, Notch, Nodal, Wnt, Fgf and Retinoic acid (RA) signaling pathways. RA is an important morphogen. Through binding to the heterodimers of RA receptors (RARs) and retinoid-X receptors (RXRs), it regulates expressions of target genes, leading to affect the patterning of AP, LR and dorso-ventral axes of embryos, somitogenesis, neurogenesis of central nervous system and so on. Insufficiency of RA signaling results in abnormal development of embryonic axes whereas excess RA, on the other hand, also causes teratogenesis. Although the roles of RA in vertebrate early development are well studied, the identities of its target genes and the molecular mechanisms underlying their regulation of embryonic axes formation remains largely unknown.To reveal the unknown mechanisms of how RA signaling determines embryonic axes formation through its direct target genes, we performed microarray analysis on the expression profiles of embryos treated by exogenous RA. The result showed that foxcla, encoding a member of forkhead transcription factors, is a direct target gene controlled by RA signaling. Employing TALEN (transcription activator-like effector nucleases) technology we knocked out foxc1a in zebrafish. Analyzing the phenotypes of foxcla null embryos revealed that the homozygous mutants exhibited similar abnormal anterior-posterior patterning of hindbrain to the embryos deprived of endogenous RA signaling. By examining the expressions of genes that are involved in determining RA levels in foxc1a depleted embryos, we found the expression of cyp26a1, the major gene that is responsible for metabolizing RA in embryos at early development, was significantly up-regulated whereas expressions of the others genes such as aldhla2(the major gene responsible for RA synthesis), cyp26bl (RA metabolizing gene) and cyp26cl (RA metabolizing gene) remains unchanged. Employing transcriptional assays in cultured cells and chromatin immunoprecipitation analysis on embryos, we demonstrated that cyp26al promoter owned two Foxc1a binding sites. The results suggest that Foxc1a controls the anterior-posterior patterning of hindbrain by directly binding cyp26al promoter to inhibit its transcription.Interestingly, the synchronous expressions of her1, her7or deltaC were not altered in foxc1a null embryos with reduced RA level because of abnormal up-regulation of cyp26al expression. The result suggests that foxc1a is dispensable to left-right axis formation of zebarfish embryos. However, the expressions of myoD, fg18and deltaC were all significantly reduced in the somitic mesoderm of foxc1a knock out embryos whereas the expression of aldh1a2in the somitic mesoderm was significantly increased in foxc1a depleted embryos. Furthermore, knocking down aldh1a2could partially rescue the decreased expressions of myoD,fgf8and deltaC in foxc1a knock out embryos. On the other hand, knocking down fgf8reduced expressions of myoD and deltaC whereas inhibiting Notch signaling decreased expressions of fgf8and myoD in somitic mesoderm. In contrast, neither knocking down fgf8or inhibiting Notch signaling affected the expression of aldhla2in somitic mesoderm. Taken together, the results reveal that both Fgf and Notch signaling work downstream of RA signaling to mediate the role of Foxcla in controlling zebrafish somitogenesis.In summary, the study identified for the first time that foxc1a, a forkhead transcription factor, is a new direct target gene of RA signaling. The gene controls anterior-posterior patterning of hindbrain and somitogenesis by acting upstream of cyp26a1and aldh1a2respectively to maintain RA homeostasis in zebrafish embryos. The results will help us to understand the roles of Foxcla and maintenance of RA homeostasis in vertebrate embryonic pattern formation.