Abstract
Purpose :
The SNARE complex facilitates fusion of vesicular cargo to a target membrane. Syntaxin 3 (Stx3) has been implicated in trafficking of proteins to the photoreceptor outer segment and exocytosis of synaptic vesicles. Previously, we found that stxbp1b and stx3 were essential for photoreceptor morphogenesis and survival in zebrafish. The purpose of this study was to further investigate the roles of Stx3 and Stxbp1b in retina development.
Methods :
An ENU-induced mutation of stxbp1b and a CRISPR-targeted allele of stx3 were used in this study. For rescue experiments, in vitro transcribed mRNA encoding wildtype stxbp1b, phosphomimetic mutant Y474D, non-phosphorylatable mutant Y474F, and wildtype stx3 were injected into 1-2 cell stage embryos. The optokinetic response (OKR) assay was used to screen for visual responses after 5 dpf. Immunolabeling was performed for photoreceptor-specific and synaptic markers. Fluorescence intensity was quantified using ImageJ.
Results :
Zebrafish contain two paralogues of STX3, stx3 and stx3a. In situ hybridization of embryos at 48 hours post-fertilization (hpf) shows stx3 expression in the outer nuclear layer of the retina, and stx3a is expressed in the inner retina. Immunolabeling with an antibody to rat Stx3 detected both paralogues with signal on photoreceptors, the outer plexiform layer (OPL) and inner plexiform layer (IPL). In stx3-/- retinas, labeling for Stx3 was largely restricted to the IPL. Quantitative immunolabeling revealed that expression of Stx3 was significantly downregulated in the photoreceptor layer and IPL of stxbp1b mutant retinas. Labeling for other synaptic makers were not altered in the IPL of either mutant retina. All Stxbp1b mRNA injections were enough to rescue photoreceptor morphology. However injected-larvae failed to display an OKR, and the Stx3 immunolabeling of the OPL and IPL remained significantly reduced.
Conclusions :
These data indicate that Stxbp1b is essential for Stx3 expression in photoreceptor and Stx3a expression in IPL, suggesting that Stxbp1b acts as a chaperon protein for both Stx3 paralogues. But phosphorylation of Y474 of stxbp1b is dispensable for formation of the outer segment.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.