Abstract
Purpose: :
Progressive vision loss in Usher syndrome is due to progressive retinal degeneration. Previously, we showed that the Usher type 1C protein, Harmonin, is expressed in zebrafish and murine Müller glia, and that loss of Harmonin from Müller cells in zebrafish results in diminished visual function, impaired synaptic maturation, and progressive photoreceptor cell death. Here, we explore the potential role of Müller glia in maintaining photoreceptor cell integrity, and the extent to which Harmonin and other Usher proteins may contribute to this function.
Methods: :
We used antibodies to six zebrafish Usher proteins to perform immunohistochemistry on cryosectioned retinas from wild-type and ush1c mutant zebrafish of various ages. Retinal tissues were co-labeled for Glutamine synthetase or ZO-1 to visualize Müller cells or tight junctions of the outer limiting membrane (OLM), respectively. Images were obtained with a confocal microscope.
Results: :
Zebrafish Harmonin is not produced in photoreceptors before 2 weeks post-fertilization, but is present in Müller cells from 3 days post-fertilization (dpf) through adulthood. Thus, visual defects seen in the first two weeks of life in ush1c mutants result from depletion of Harmonin from Müller glia. Harmonin localizes within Müller glia from the inner limiting membrane to the OLM and in glial processes adjacent to the subapical region of photoreceptors. Harmonin also colocalizes with ZO-1 in the OLM of 5dpf retinas. The zebrafish Usher proteins, Usherin, Gpr98, Cip98a, Cip98b, and Clrn1, are all expressed in Müller glia from larval to adult stages, with localization patterns similar to Harmonin. Müller glial localization of Usherin and Gpr98 is unaffected in the ush1c mutant background.
Conclusions: :
All zebrafish Usher proteins examined colocalize in Müller cells throughout life, suggesting a sustained functional role for these proteins. Localization of Usherin and Gpr98 in Müller cells does not depend on the scaffold protein Harmonin, but may instead be mediated by Cip98a or Cip98b. In addition to the previously demonstrated requirement of glial expression of Harmonin in synaptic maturation, the presence of this and other Usher proteins at the OLM and in subapical glial processes suggests additional non-cell autonomous roles for these proteins in maintaining photoreceptor function and survival.
Keywords: retinal degenerations: cell biology • Muller cells • cell-cell communication