Abstract
Purpose:
Sox4 is a member of the group C SRY-box containing transcription factors, which also includes Sox11 and Sox12. Sox4 promotes differentiation of multiple cell lineages during development. Zebrafish have two co-orthologs of the mammalian sox4 gene: sox4a and sox4b. Their functions during zebrafish ocular development are not clear. The purpose of this project was to study the role of sox4 during eye development in zebrafish.
Methods:
Whole mount in-situ hybridization (WISH) and reverse transcript PCR(RT-PCR) were performed on wild type (WT) zebrafish embryos. Sox4 and control morpholinos were injected into zebrafish embryos at the 1-2 cell stage. Morphants were imaged by light microscopy and were cryosectioned for immunohistochemical analysis (IHC) with antibodies for various retinal cell types. TUNEL assay was used to examine apoptosis. Sox4 and sox11 mRNAs were co-injected along with morpholinos into 1-2 cell stage embryos. Morphant embryos were treated with 2µM cyclopamine from 5.5 hours post fertilization (hpf) to 13 hpf.
Results:
Sox4a and sox4b transcripts were expressed in the developing zebrafish embryo from 24 hpf through 2 months of age. Sox4a was expressed in the developing zebrafish retina from 24 hpf to 5 days post fertilization (dpf). As retinal development progressed, expression of sox4a was gradually restricted to the ciliary marginal zone. Sox4 knockdown caused microphthalmia , coloboma, increased cell death, and reduced numbers of rod photoreceptors. Treatment with cyclopamine or co-injection of sox11 mRNA rescued the coloboma phenotype of sox4 morphants. However, higher doses of sox11 caused cyclopia in sox4 morphants.
Conclusions:
The expression pattern of sox4 in the zebrafish suggests it plays a role during retinal development and differentiation. The rescue of the coloboma phenotype in sox4 morphants by cyclopamine indicates that sox4 knockdown results in increased Sonic hedgehog (Shh) signaling. Sox11 mRNA rescued the sox4 morphant phenotype, demonstrating that functional redundancy exists between Sox4 and Sox11. Moreover, since higher doses of sox11 mRNA caused cyclopia in sox4 morphants, this suggests that SoxC proteins negatively regulate Shh signaling during ocular morphogenesis.
Keywords: 698 retinal development •
739 transcription factors •
648 photoreceptors