Abstract
Abstract: :
Purpose: Identify and characterize the cellular mechanisms that enable retinal regeneration in the adult zebrafish (Danio rerio). Methods: Lesions were made in the dorsal retina of right eyes; left eyes were unlesioned controls. Fish were euthanized 1–7, 14 or 21 d after lesion; some fish were exposed to 5 mM BrdU for 2 hr immediately prior to euthanasia. Indirect immunohistochemical analysis was used to assess cellular proliferation (PCNA immunoreactivity; BrdU incorporation) and cell identity, using a marker for Müller glia (anti–zCAH; courtesy of P. Linser). Sections were screened for apoptosis with the TUNEL method, and rhodopsin expression patterns were determined with in situ hybridization. Results: A spatiotemporal profile of cellular ‘activation’ (cell cycle entry) was observed in the retina following lesion, characterized by an initial, relatively small number of activated cells at the outer and inner nuclear layers (ONL and INL) proximal to the lesion site by 3 d after lesion, assumed to include rod precursor cells in the ONL. This was followed by a large increase in the number of activated cells, particularly at the INL, extending >500 µm from the lesion edge. Activated cells at the INL were often clustered, and double label experiments revealed that they included Müller glia. During the first week post–lesion a dispersal of activated cells was observed, with PCNA– and BrdU–positive profiles extending through the plexiform layers. The profile of PCNA+/zCAH+ cells suggested that the nuclei of some activated Müller glia were located outside the INL, and activated zCAH–negative cells were observed closely associated with Müller glia processes, suggesting that activated cells exhibit evidence of intracellular nuclear kinesis and whole–cell migration. Evidence for apoptosis was observed proximal to lesion sites, corresponding to a region of rod photoreceptors not expressing rhodopsin. Few apoptotic cells were observed in inner retina, suggesting that Müller glia do not necessarily undergo programmed cell death as a consequence of activation. Conclusion: In adult zebrafish Müller glia may directly contribute to retinal regeneration in several ways, including functioning as a potential stem/precursor cell and providing a conduit for the spatial dispersal of activated stem/precursor cells.
Keywords: regeneration • Muller cells • proliferation