Wasf3 is required for photoreceptor sensory cilia (PSC) formation

Abstract

 

Purpose

 

Inherited retinal degenerations (IRDs) are important causes of blindness. These disorders are now recognized to be a sub-class of the larger group of ciliopathies caused by dysfunction of cilia. Over 190 IRD disease genes have been identified to date, yet mutations in these genes account for only 50-60% of IRD patients. To help identify additional IRD disease genes, we are evaluating the locations and functions of novel proteins in the photoreceptor sensory cilium (PSC) proteome. In these studies, we evaluated the role of Wasf3, a member of the WAS family of proteins associated with cytoskeleton and actin complexes, in PSC biology.

 

Methods

 

The human WASF3 ORF was cloned into expression vectors with V5 or Flag tag and expressed in rat retinas via sub-retinal injection and in vivo electroporation. The location of tagged WASF3 protein was evaluated 21 days after transfection. The location of mouse endogenous Wasf3 protein was verified using anti-Wasf3 antibodies. The tissue distribution of Wasf3 was studied using immunoblot analyses. Morpholino oligonucleotide (MO) - mediated knockdown of wasf3 was investigated in zebrafish and the phenotype was evaluated by histology analysis.

 

Results

 

In vivo expression of V5 or Flag tagged WASF3 revealed its location at the junction between the base of axoneme and the distal tip of the transition zone, with a clear gap to the distal end of inner segment marked by EGFP. Immunostaining of mouse endogenous Wasf3 confirmed its location at the proximal end of axoneme that co-stained with Rp1 antibody. Immunoblot demonstrated that Wasf3 protein was expressed mainly in the retina and brain. wasf3 morphants displayed smaller eyes than those of wild type zebrafish, and a curved body appearance. Histologic analysis showed that wasf3 morphants exhibited compromised photoreceptor ciliogenesis, with only a few shortened outer segments present.

 

Conclusions

 

Our results indicate that Wasf3 is a novel cilia protein in vertebrate retina. Loss of wasf3 function prevents normal morphogenesis or maintenance of PSC outer segments. This suggests that WASF3 plays an important role in the biologic function of photoreceptor cells and screening WASF3 for mutations in IRD patients is warranted.

 

 

Wasf3 (Red) and Rp1 (Green) co-staining in mouse retina

 

 

Immunostaining with anti-V5 antibody (Red) in rat retina 3 weeks after subretinal injection of V5-WASF3 plasmid and then followed electroporation.

 

Keywords: 689 retina: distal (photoreceptors, horizontal cells, bipolar cells) • 648 photoreceptors