Abstract
Purpose:
Previously, we identified the transcription factors Sox11 and Sox4 as candidate regulators of rod photoreceptor regeneration in zebrafish. Sox11 and Sox4 are members of the SoxC family of HMG-box containing transcriptional regulators. SoxC factors are known to regulate neurogenesis in the brain and spinal cord, and are also required for the proper development of several organ systems. However, their function during ocular development was not well understood. In this study, we investigated the role of Sox11 and Sox4 during ocular morphogenesis in zebrafish.
Methods:
We used a combination of morpholino-mediated gene knockdown, mRNA over-expression, reporter gene expression, and pharmacologic inhibition of Hh signaling, to analyze the role of SoxC genes during ocular development. Gene expression in zebrafish embryos was analyzed by quantitative RT-PCR, by whole mount in situ hybridization, and by fluorescent in situ hybridization on tissue sections. Retinal cell type differentiation was analyzed by immunohistochemistry and using fluorescent reporter transgenic lines.
Results:
Knockdown of sox4 or sox11 caused coloboma and a reduction in rod photoreceptors, but no reduction in other later-born cell types. These phenotypes were rescued by co-injection with full-length sox11 or sox4 mRNA. The coloboma phenotype of Sox4- and Sox11-deficient zebrafish was rescued by blocking Hh signaling. We found that Sox11 negatively regulates transcription of the Shh ligand. In contrast, Sox4 represses transcription of Indian Hedgehog, a Hh signaling ligand not previously implicated in ocular malformations. Overexpression of sox4 or sox11 resulted in reduced Ihh and Shh expression, respectively, and caused cyclopia. We validated the specificity of the SoxC-deficient phenotypes by using the CRISPR/Cas9 system, and observed coloboma in a significant proportion of founder embryos injected with sox4 or sox11 sgRNA/Cas9. Finally, we provide evidence that Sox4 and Sox11 are partially, but not completely, functionally redundant with respect to ocular morphogenesis.
Conclusions:
Our results show that Sox4 and Sox11 regulate ocular morphogenesis and photoreceptor development through negative control of Hh signaling. Taken together, our data suggest that mutations in SoxC genes could contribute to the development of coloboma or related ocular malformations.