Abstract
Purpose :
Variants in the transcription factor MYRF have been described in a cardiac urogenital syndrome and familial and sporadic nanophthalmos, but the mechanism of pathogenesis is unclear. Using a conditional mouse model of Myrf loss of function (Myrf fl/fl) in the early eye cup (RxCre), we previously showed that loss of Myrf leads to retinal degeneration and loss melanin pigment in the retinal pigment epithelial (RPE). Here, we identify specific gene expression changes caused by MYRF deficiency using single cell RNA sequencing (scRNAseq).
Methods :
scRNAseq was conducted at 3 developmental timepoints (E13.5, E15.5, P0) using pools of eye cups from RxCre Myrf fl/fl or matched Myrf fl/fl littermates, and data were processed using CellRanger and Seurat. Histology, RNAscope in situ hybridization, and electron microscopy (EM) were used to validate gene expression findings.
Results :
Cell clustering revealed that Myrf deficiency altered cell type distributions with reductions in RPE cells at all timepoints. Cell cycle dynamics were stable, consistent with increased cell death in mutants. There was also a compensatory increase in retinal progenitor (RPC) population at P0, without alteration in overall cell cycle dynamics. Differential gene expression analysis and PANTHER gene ontology-term analysis revealed down regulation of key pathways in mutant RPE cells, including melanosome biogenesis, cytoskeleton, and extracellular matrix. EM analysis and immunofluorescence staining of RPE flatmounts confirmed structural defects in RPE and disorganization of photoreceptor outer segments, loss of melanosomes, and alterations in novel structural proteins in the apical RPE. Compensatory upgregulation of Prss56, another gene implicated in nanophthalmos, was found in the RPC population.
Conclusions :
These results suggest that MYRF plays a critical role in regulating RPE structure and function during development, which likely contributes to abnormal outer segment morphology and retinal degeneration in RxCre Myrf fl/fl mice. Compensatory gene expression changes in the retina may act maintain proper ocular size in mice. These studies inform molecular mechanisms of nanophthalmos and support a role for RPE in this condition.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.