Purchase this article with an account.
James M Fadool, Dylan M Allen, Mailin Sotolongo-Lopez; The SNARE syntaxin3 is essential for photoreceptor morphogenesis and survival in zebrafish. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4452.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
SNARE complexes facilitate fusion of intracellular vesicles with target membranes. In the neural retina, SNARE function is essential for synaptic release and is implicated in vesicular trafficking in the inner segment. Previously, we reported that syntaxin binding protein 1b (stxbp1b) was essential for photoreceptor morphogenesis and survival. Based upon those data, we hypothesize that syntaxin3 (stx3), a member of the SNARE complex that binds to stxbp1, would also be essential for photoreceptor development.
gRNA targeting the second exon of stx3 and in vitro transcribed Cas9 mRNA were co-injected into 1-2 cell stage zebrafish embryos. Injected-embryos were pooled for DNA isolation or grown to adults. Adult fish were mated to generate an F1 population. Exon 2 of stx3 was amplified by PCR from genomic DNA isolated from embryos, or tail clips of F1 fish, and the product sequenced. F2 offspring from mating heterozygous carriers of novel stx3 alleles were screened for visual responses using the optokinetic response (OKR), and for histological alterations by immunolabelled with photoreceptor-specific markers.
Two stx3 orthologues previously were identified in the zebrafish genome, stx3 on Chr1 and stx3a on Chr14. In situ hybridization for stx3 revealed expression restricted to photoreceptor cell layer. Labeling for stx3a showed broad expression in the inner retina. To investigate gene function, CRISPR /Cas9 gene editing was used to target stx3 in vivo. Novel insertions and deletions were recovered that were predicted to induce frame shift mutations, in-frame deletions and non-sense mutations. The stx3fl11 allele resulted in a frameshift mutation (p.D17fs) and predicted to encode a truncated protein lacking the N-terminal regulatory domain, the SNARE motif, and TMD. Twenty-five percent of the larvae from mating heterozygous stx3fl11 carriers lacked an OKR at 6 days post-fertilization. Genotyping for stx3 showed that the non-responders were homozygous for the p.D17fs mutation. Histological sections of the mutants presented with extensive photoreceptor degeneration. In contrast, siblings that displayed an OKR were either homozygous or heterozygous for the wild-type allele and displayed no photoreceptor degeneration.
Together our findings demonstrate that SNARE-mediated vesicular events are essential for photoreceptor morphogenesis and survival in the developing zebrafish retina.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only