Abstract
Purpose :
Retinal degenerative diseases are inherited disorders that result in photoreceptor death and eventual blindness. Defects in prominin-1 (prom1) and photoreceptor cadherin (prCAD) result in retinitis pigmentosa, cone-rod dystrophy, and Stargardt-like macular dystrophy, but how this occurs is unknown. Prom1 and prCAD proteins are localized uniquely to the basal outer segment (OS), and are proposed to form a complex that regulates OS disc synthesis and organization. We hypothesized that ablation of prom1 and prCAD genes in X. laevis will result in OS disc synthesis or organization defects, and subsequent retinal degeneration similar to human disease.
Methods :
CRISPR/Cas9 was used to knock-down prom1 and prCAD genes. Cas9 mRNA, GFP, and prom1- or prCAD-specific guide RNA was injected into fertilized eggs. Embryos were screened for GFP expression at 1-3 days post-fertilization (dpf) to confirm successful injection and then Sanger sequencing was performed to confirm target gene knock-down. Resultant phenotypes were examined in retinas of tadpoles aged ≥ 14 dpf using confocal and super-resolution fluorescence microscopy and transmission electron microscopy.
Results :
PrCAD-null rod OS are intact, but some discs are oriented vertically instead of horizontally within the outer segment; some cone OS have overgrown discs. Prom1-null rod OS are globular and discs are disorganized, overgrown, and form folds and whorls instead of neatly-packed stacks; laminations between discs remain normal. Prom1-null cone OS are severely elongated and fragmented. Dysmorphic rods and cones persist past 56 dpf. Formation of the axoneme and hairpin rims in OS discs remains intact; as does the normal localization of prom1 in prCAD-null and prCAD in prom1-null OS. Combination of prom1 + prCAD knock-down appears indistinguishable from the prom1-null phenotype. Other sensory cilia are unaffected by prom1-null mutation.
Conclusions :
The significant differences in phenotype and lack of mislocalization of prom1 and prCAD proteins in opposite null animals argue against a shared role for these proteins. Neither protein is essential for OS disc synthesis, but they are essential for OS disc organization. Identifying the functions of prom1 and prCAD will further our understanding of OS disc morphogenesis, a process that remains obscure, and aid in the development of novel therapies for some of the more common human retinal degenerative diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.