Abstract
Purpose :
Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. In this study, we characterize the expression pattern of chd7 in the developing zebrafish retina and begin to study its function using two chd7 mutant lines.
Methods :
All animal procedures were performed in accordance with IACUC and guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wild-type and transgenic zebrafish embryos or larvae were collected at 24, 48, 72 hours post fertilization (hpf) and 4, 5 days post fertilization (dpf). Retinas were sectioned, followed by immunohistochemistry with a Chd7 antibody or RNAscope with a chd7 mRNA probe, and imaged with fluorescent and confocal microscopy. A CRISPR chd7 mutant line was obtained and genotyped by Sanger sequencing to identify a 2 base pair deletion. A second mutant line generated by ENU mutagenesis was outcrossed four times, and genotyped by Sanger sequencing to identify a single point mutation.
Results :
Chd7 was expressed throughout the developing zebrafish retina at 24 and 48 hpf, when retinal progenitors are actively proliferating and early cells of the retina are beginning to differentiate. At 72 hpf, when most retinal cell types have terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Strong expression of Chd7 was also observed in the photoreceptor cells of the outer nuclear layer (ONL). By 4 and 5 dpf, when the zebrafish larvae display active swimming and visual behaviors, Chd7 expression remained strong in the ONL, where it co-localized with markers of cone and rod photoreceptors. Incrosses of chd7 heterozygous adults yielded homozygous mutant progeny suggesting that the mutations are not early embryonic lethal in zebrafish.
Conclusions :
Our results demonstrate that Chd7 is expressed throughout development of the zebrafish retina and remains expressed in some newly differentiated retinal cell types, including the photoreceptors. Further work is ongoing to investigate this specific patterning of Chd7 and chd7 mutant fish will be used to understand the function that this factor plays in retinal development.
This is a 2021 ARVO Annual Meeting abstract.