Abstract
Purpose :
CRX is a transcription factor essential for the differentiation and functional development of photoreceptors. Two dominant missense mutations in CRX HD, p.E80A and p.K88N, are linked to early-onset blinding retinopathies. Molecular studies on knockin mouse models of these two mutations have shown profiles of altered photoreceptor gene expression via distinct gain-of-function mechanisms. This study aims to characterize the cellular phenotypes of these mouse models during postnatal retinal development.
Methods :
Hematoxylin and eosin staining of retinal cross-sections revealed the retinal morphology in mutant and wildtype samples from postnatal day (P) 0 to P21. Immunohistochemistry with specific markers reported changes in cell proliferation (Ki67), abundance and distribution of rods (Rho) and cones (RXRG and Arr3), pre- and post-synapses (Ctbp2 and VGluT1) at outer plexiform layer, retinal lamination (β-Catenin) and vasculature (CD31 and conjugated lectins).
Results :
Proliferating neurons and fated cones are present in mutant retinas of all genotypes at P0. However, cone identity is lost in K88N/N, K88N/+ and E80A/A mutants after P2. Cones are seen in E80A/+ mutants at P14 but not detected at P21. Rod identity is absent in K88N/N mutants but present in other genotypes at all tested ages. Interestingly, Rho expression is upregulated in E80A mutants at P3, supporting the gain-of-function nature of the mutation during early development. Irregular whorls and rosettes appear within ONL of K88N mutants before P10, accompanied by vascular malformations and Müller glia mislocalization. In particular, vessels infiltrate through ONL in K88N/N mutants during photoreceptor differentiation, making the lamination defect worse than that in K88N/+ mutants. Irregular whorls and rosettes also appear in E80A mutants, later and less severe in E80A/+ than in E80A/A. E80A/+ mutants elaborate shorter but functional outer segments. Mislocalized cells are not found within ONL of E80A mutants. Pre-synaptic ribbons are weakly labeled in E80A/+ mutants but not detected in other genotypes.
Conclusions :
Photoreceptor phenotypes of E80A and K88N mutants match the clinical features of cone-rod dystrophy and LCA respectively. ONL morphologies are further complicated by lamination and vascular defects. A deeper understanding of these cellular underpinnings will inform pathogenesis of CRX HD mutations.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.