Abstract
Purpose :
HESX1 is a transcription factor important for retinal and pituitary development in both mouse and human, and mutations in this gene are associated with septo-optic dysplasia (SOD), a form of congenital blindness. Although associated with human disease, there is a gap in knowledge surrounding its role in early retinal development. We tested the hypothesis that knocking out Hesx1 in mouse embryonic stem cells would limit their ability to differentiate towards retinal neuroectoderm using a mouse embryonic stem cell (mESC)-to-3D retinal organoid platform.
Methods :
A mESC line carrying a knock-in Rx:GFP allele, which highlights early retinal progenitor cell differentiation, underwent CRISPR-Cas9 targeting to knockout Hesx1. Single cell-derived Hesx1 KO clones were isolated and induced to retinal progenitor differentiation. For rescue complementation, molecular cloning of wild-type HA-Hesx1 into a piggybac constitutive plasmid was performed and used to transfect clones of the Hesx1 KO mESCs. Flow cytometry was then performed on replicates of the corresponding day 6 retinal neuroectoderm organoids for quantification of GFP+ cells.
Results :
Hesx1 KO mESC clones D2 and E5, both harboring exon 1 frameshifts leading to nonsense mutations, display impaired Rx:GFP+ retinal neuroectoderm differentiation. Complementation with expression of full-length HA-Hesx1 partially rescued Rx:GFP+ retinal organoid differentiation, as measured by flow cytometry.
Conclusions :
Our data indicates that HESX1 is essential for proper retinal development in our mouse retinal neuroectoderm organoid model. Future studies will focus on determining how SOD-associated mutations alter retinal differentiation, as well as the functional transcriptional targets of HESX1 that enable proper retinal development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.