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Mailin Sotolongo-Lopez, Kathleen E. Kyle, Daniel L. Vera, James M Fadool; syntaxin binding protein 1b is essential for photoreceptor morphogenesis and survival. Invest. Ophthalmol. Vis. Sci. 2017;58(8):344.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal photoreceptor cells are vulnerable to mutations in genes essential for cilia formation, outer segment (OS) morphogenesis, and phototransduction. In a forward genetic screen of zebrafish larvae, we previously identified a mutation at the doughnut (dou) locus characterized by rapid degeneration of rod and cone photoreceptors. This study aims to characterize the dou locus and identify a cellular mechanism responsible for the photoreceptor cell phenotype.
Homozygous dou mutant larvae were identified in crosses of heterozygous adults by immunolabeling for photoreceptor markers or lack of an OKR. To identify the molecular lesion, DNA was pooled from 89 dou mutants and subjected to shotgun Illumina sequencing. Genetic mosaic animals were generated by blastula-stage cell transplantation. The photoreceptor structure was analyzed by immunolabeling and electron microscopy.
Whole genome sequence analysis of DNA isolated from dou mutants revealed a single base change of A>T that introduced a premature stop codon (K213*) of stxbp1b, truncating ~600 amino acid protein in domain 2. STXBP1/MUNC 18-1 facilitates synaptic vesicle docking and fusion, and haploinsufficiency causes epilepsy and encephalopathy disorders. Stxbp1/Munc-18 is expressed by rods and cones, but its specific function in photoreceptors remains unknown. In genetic chimeras photoreceptor degeneration was cell autonomous and survival of inner retinal neurons was unaffected. Ultrastructural analysis showed that photoreceptor terminals in mutant larvae lack invaginating synapses and contain floating ribbons consistent with a role for stxbp1b in synaptic function. The photoreceptor OSs formed in dou mutants, but were shorter in length compared to those of wildtype siblings. The inner segments were characterized by swollen Golgi and endoplasmic reticulum. Confocal images of embryos and larvae immunolabeled for acetylated-tubulin and intraflagellar transport proteins revealed shortened photoreceptor connecting cilium in developing photoreceptors, however no defects were observed in cilia of the nasal epithelium or sensory hair cells of the lateral line neuromasts.
These data indicate that in addition to synaptic function, stxbp1b is essential for photoreceptor survival and likely plays a role in vesicular trafficking in the photoreceptor inner segment.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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