Abstract
Purpose:
Usher syndrome (USH), the leading cause of hereditary deafblindness, results from mutations in any one of a dozen genes. USH1G is a severe form caused by defects in a gene encoding the protein SANS. Animal models of USH are required to understand the complex molecular pathology of the disease as well as for testing potential therapies.
Methods:
Two zebrafish orthologs of USH1G were identified by tBLASTn search using the human protein sequence. cRNA probes assayed expression patterns. An antibody (Novus) against a human SANS epitope was used to detect Sans protein in immunohistochemistry (IHC) and Western blot (WB) experiments. Morpholinos (MO) were used to deplete ush1ga and ush1gb function. TALENs (Transcription Activator-Like Effector Nucleases) were used to induce truncating mutations in each gene. Single mutant lines were crossed to produce double mutant carriers. <br />
Results:
The zebrafish USH1G orthologs are highly homologous to each other and, based on synteny, are retained duplicates from the teleost genome duplication. In situ hybridization showed overlapping expression in sensory cells, suggesting functional redundancy. The SANS antibody localized to Müller glia, horizontal cells and photoreceptors (PRCs) in larval retinas, and to mechanosensory hair cells of the ear and lateral line. In PRCs, signal was detected at synapses, inner segments, and the subapical region. Localization at rod connecting cilia was observed in adults. All ush1ga MO-injected larvae exhibited a mild balance defect. The same phenotype was observed in some ush1ga mutants, but others were normal and homozygous viable.100% of progeny from ush1ga mutant parents exhibited this balance defect, suggesting a maternal contribution of ush1ga to early development. IHC and WB showed depletion of SANS protein in ush1ga MO and offspring from ush1ga homozygous parents, whereas mutants from heterozygous parents had normal protein levels. This indicates that the SANS antibody preferentially detects Sansa. ush1gb mutant larvae swam normally, but ush1ga;ush1gb double mutants exhibited severe hearing and balance defects.
Conclusions:
Zebrafish ush1g genes have partially overlapping function, but also some independent roles in hearing and balance. The redundancy of these genes in vestibular function enables us to study mutants through all life stages to investigate the progressive retinal degeneration characteristic of USH1G. <br />