Zebrafish Rpgr is required for normal retinal development and plays a role in dynein-based retrograde transport processes

Abstract

Mutations in the human RPGR gene cause one of the most common and severe forms of inherited retinal dystrophy, but the function of its protein product remains unclear. We have identified two genes resembling human RPGR (ZFRPGR1, ZFRPGR2) in zebrafish (Danio rerio), both of which are expressed within the nascent and adult eye as well as more widely during development. ZFRPGR2 appears to be functionally orthologous to human RPGR, because it encodes similar protein isoforms (ZFRPGR2(ORF15), ZFRPGR2(ex1-17)) and causes developmental defects similar to other ciliary proteins, affecting gastrulation, tail and head development after morpholino-induced knockdown (translation suppression). These defects are consistent with a ciliary function and were rescued by human RPGR but not by RPGR mutants causing retinal dystrophy. Unlike mammals, RPGR knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. Developmental abnormalities in the eye included lamination defects, failure to develop photoreceptor outer segments and a small eye phenotype, associated with increased cell death throughout the retina. These defects could be rescued by expression of wild-type but not mutant forms of human RPGR. ZFRPGR2 knockdown also resulted in an intracellular transport defect affecting retrograde but not anterograde transport of organelles. ZFRPGR2 is therefore necessary both for the normal differentiation and lamination of the retina and to prevent apoptotic retinal cell death, which may relate to its proposed role in dynein-based retrograde transport processes.

The zebrafish eye—a paradigm for investigating human ocular genetics

“…77 The rpgr zebrafish morphant presents with a similar retinal phenotype as a consequence of cell death in the dysplastic retina. 78 These zebrafish morphant phenotypes can be rescued by injection of human RP2 or RPGR mRNA, respectively, indicative of a functional role for RP2 and RPGR in the pathogenesis of human X-linked RP. 78 Mutations in the Rhodopsin (RHO) gene are the most common cause of human autosomal dominant RP.…”

“…78 These zebrafish morphant phenotypes can be rescued by injection of human RP2 or RPGR mRNA, respectively, indicative of a functional role for RP2 and RPGR in the pathogenesis of human X-linked RP. 78 Mutations in the Rhodopsin (RHO) gene are the most common cause of human autosomal dominant RP. Rhodopsin is a member of the G-protein-coupled receptor family and has a role in phototransduction in rod photoreceptors.…”

Effects of acute exposure to polybrominated diphenyl ethers on retinoid signaling in zebrafish larvae

“…Whole 120 hpf larvae were first fixed in 4% paraformaldehyde and permeabilized in proteinase K at room temperature. After blocked in 2% goat serum, the larvae were then incubated in diluted mouse primary antibody raised to rhodopsin (1:1000), which has been verified reactive with zebrafish rhodopsin (Shu et al, 2010). This incubation was performed for 20 h at room temperature with gentle rotation.…”

The 1D4 Antibody Labels Outer Segments of Long Double Cone But Not Rod Photoreceptors in Zebrafish

“…This antibody is commercially available (ab5417; Abcam, Cambridge, UK; R5403; Sigma-Aldrich, St. Louis, MO; sc-57,432; SCBT, Santa Cruz, CA; MAB5356; Millipore, Billerica, MA) and according to the supplier is used to detect rhodopsin also in zebrafish. Accordingly, two recent zebrafish publications used the 1D4 antibody as a marker to examine rhodopsin localization and to describe rod deformations in studies characterizing two RP related genes, RPGR 17 and ZNF513. 18 In our study, we used histologic analysis of a rod-specific transgenic reporter line and surprisingly found that 1D4 does not stain rod outer segments in zebrafish.…”