Abstract
Purpose: :
During embryogenesis, proper eye development is critical for vision function. Expression of the gene PAX2, is critical for normal ocular morphogenesis in animal models and dominant mutations in PAX2 result in renal coloboma syndrome. Previous studies in Drosophilia identified sparkling (spa) as a pax2 ortholog, spa mutants have abnormal eye development and reduced expression of the DNA binding protein cut. This observation led us to hypothesize that cux1b acts downstream of pax2 during early eye development, making it a good candidate to investigate for involvement in vertebrate retinal development.
Methods: :
The genetic and functional similarities to the mammalian eye make zebrafish (Danio rerio) an ideal model to study early vertebrate eye development. To evaluate the functional role of cux1b in retinal development, a Morpholino antisense oligonucleotide was utilized to knockdown expression in the zebrafish. Immunohistochemistry was used to assess cux1b morphant and rescued phenotypes.
Results: :
Microinjection of a morpholino against cux1b into zebrafish embryos results in a reduction of the overall eye size at 48hpf, with an absence of growth between 48hpf and 72hpf. Additionally, an increase in proliferation is observed in the 72hpf retina of cux1b morphant embryos. Although the overall eye size is smaller, the retina is fully laminated and the optic chiasm forms. While photoreceptors do form, the outer segments fail to fully develop and have reduced rhodopsin expression. Overexpression of the ciliary protein RPGRIPl1 in cux1b knockdown embryos normalizes eye size and partially restores rhodopsin expression throughout the developing retina. Moreover, in cux1b morphants, green opsin expression was not restricted to the photoreceptor outer segment, but was also found in the cell body.
Conclusions: :
These data provide strong evidence that cux1b is required for proper retinal development. Given the rescue of rhodopsin localization following RPGRIPL1 overexpression, it seems likely that cux1b functions in cilia development and/or maintenance in the retina.
Keywords: retina • retinal development