June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Whirlin proteins localize at the outer limiting membrane and subapical region of zebrafish retina
Author Affiliations & Notes
  • Sabrina Toro
    Institute of Neuroscience, University of Oregon, Eugene, OR
  • Jennifer Phillips
    Institute of Neuroscience, University of Oregon, Eugene, OR
  • Monte Westerfield
    Institute of Neuroscience, University of Oregon, Eugene, OR
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 287. doi:
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      Sabrina Toro, Jennifer Phillips, Monte Westerfield; Whirlin proteins localize at the outer limiting membrane and subapical region of zebrafish retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):287.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Mutations in DFNB31 cause Usher syndrome type 2D. DFNB31 encodes Whirlin (Whrn), a scaffold protein thought to mediate interactions with other Usher proteins. We previously reported the discovery of two dfnb31 genes in zebrafish, each with multiple splice variants. Here, we investigate the subcellular localizations and functions of WhrnA and WhrnB in the zebrafish retina.

Methods: Immunohistochemistry on cryosectioned tissues was visualized by confocal microscopy. Antibodies were generated to unique regions of WhrnA and WhrnB, and the third PDZ domain (Whrn_PDZ3) common to both proteins. Antibodies against Harmonin (ush1c), Usherin (ush2a), Gpr98 (ush2c), Glutamine Synthetase, Acetylated Tubulin and Calretinin were also used. Whole embryos, larvae and adult eyes were analyzed from wild type (wt) and dfnb31a mutants. Splice blocking morpholinos for dfnb31a and dfnb31b were used to disrupt gene function, and optokinetic response assays (OKR) were performed to test visual function.

Results: WhrnA and WhrnB localize in a ring pattern at the base of the connecting cilium (CC), suggesting localization in the periciliary region. WhrnA is also observed both pre- and post-synaptically in the outer plexiform layer. Localization of Whrn_PDZ3 is restricted to the outer limiting membrane (OLM) and subapical region (SAR) where it largely overlaps with Müller cell labeling. Morpholino knockdown of dfnb31a or dfnb31b results in a reduced OKR. dfnb31a mutants are morphologically normal, viable and fertile. No differences in abundance or localization of Whrn or other Usher proteins were detected in mutant tissues.

Conclusions: Whrn localization at the base of the CC and at synapses is consistent with other species. However, localization at the OLM/SAR is a novel finding. Zebrafish Crumbs (Crb) proteins also localize at the OLM/SAR, and Whrn was previously shown to interact with the Crb pathway. Our data indicate conserved roles for zebrafish Whrn proteins at the CC and potential interaction between Whrn and Crb at the lateral cell contacts of photoreceptors. The lack of phenotype in dfnb31a mutants may indicate functional overlap with dfnb31b. However, the mutation is not predicted to affect all dfnb31a isoforms, so persistence of functional splice variants is another possibility under investigation.

Keywords: 689 retina: distal (photoreceptors, horizontal cells, bipolar cells)  
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