Abstract
Purpose :
In humans, a deficiency in the retinoschisin protein (RS1) leads to an inherited retinal degenerative disease called X-linked juvenile retinoschisis (XLRS). When RS1 is lacking, retinal lamination is compromised and cystic cavities appear in the retina. Due to its X-linked character, XLRS mainly affects males, often already at a young age. Beyond its structural role, RS1 is hypothesized to be involved in the development of retinal architecture. In order to study its role in retinal development, we developed a knockdown model for RS1 deficiency in zebrafish.
Methods :
Wildtype Tüpfel Longfin zebrafish larvae were raised according to ZFIN standards. RS1-deficient zebrafish larvae were created via morpholino oligo (MO)-mediated knockdown. The zebrafish genome contains two homologues for RS1; Rs1a and Rs1b. Two MOs were designed targeting rs1a and rs1b mRNA. As controls, two scrambled oligos (SCs) were developed containing identical bases without a target region in the transcriptome. MO or SC were injected at the 1-4 cell stage, or within one hour post-fertilization (hpf). At 48, 72, 96 and 120 hpf, MO- and SC-injected larvae were terminated on ice and collected along with uninjected siblings. Protein expression and localization were assessed on immunohistochemistry (IHC), and RNA was collected to determine differences in gene expression.
Results :
Successful knockdown of zebrafish Rs1 was confirmed on IHC. At 72 hpf, MO-mediated knockdown did not influence eye size, eye shape or overall gross embryo development, but did eliminate Rs1 protein production. No deformations associated with oligo overdose were observed at the chosen concentration. The effect of Rs1 knockdown on lamination and retinal development was consistent on IHC and RNA analyses. By 120 hpf, Rs1 was detectable again, but at much lower levels than SC-injected larvae or uninjected siblings.
Conclusions :
Delivery of translation-blocking MOs resulted in a successful knockdown of Rs1 protein in zebrafish larvae. Rs1 protein was eliminated in all essayed larvae up to at least 72 hpf, without deformations associated with excess oligo. This model allows a unique insight into the role of Rs1 in retinal development.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.