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S.D. Spritzer, D.E. Mallory, J. Sand, D.L. Stenkamp; Preliminary Analysis of the Developmental Genetics of Ganglion Cell Regeneration in Zebrafish . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3223.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Adult teleost fish have the capacity to regenerate neurons lost due to trauma. When the retina is destroyed using the chemical agent ouabain, proliferative cells within the retina generate new retinal neurons (Raymond et al. 2000). Previous studies have revealed that cell–specific gene expression in regenerated retina is similar to that seen in embryonic retinal development (e.g. Hitchcock et al., 1992), while spatial patterning of the regenerated retina is distinct (Stenkamp et al., 2001). The purpose of this study is to determine if the temporal genetic cascade expressed during regeneration of a specific cell type, the ganglion cells (GCs), is similar to that expressed during retinal development. Methods: Zebrafish were anaesthetized and injected intraocularly with ouabain, then were sacrificed at time intervals of 6, 8, 12, 20, 40, and 80+ days post injection (dpi). Retinal damage was verified by monitoring the dorsal light reflex. Treated and injected eyes were fixed in 4% paraformaldehyde and frozen for cryosectioning. Sections were processed for in situ hybridization or immunocytochemistry for expression of the following markers: ath5 (expressed transiently in retinal progenitor cells and required for GC formation; Kay et al., 2001), pax6 (expressed in retinal progenitor cells and mature GCs in normal and regenerated retina; Hitchcock et al., 1996), and zn–12 (a marker of GCs and neuronal processes expressed immediately prior to GC terminal mitosis and persisting thereafter; Hu and Easter, 1999). Results: At 6 dpi, putative regenerative cells lined the retinal pigmented epithelium, but lacked lamination and had a neuroepithelial appearance. Ath5 and pax6 were sporadically localized to this regenerating tissue, but the zn–12 marker was predominantly associated with cellular debris elsewhere in the eye. At 8 dpi, ath5 was no longer expressed in regenerating retina, while pax6 expression persisted in a location consisting with the location of GCs. The zn–12 marker was expressed at this time in a pattern consistent with the location of GCs and neuronal processes, though this pattern revealed lamination abnormalities within the regenerating tissue. At 12 dpi and beyond, regenerating retina showed a zn–12 expression pattern indicating appropriate retinal structure. Conclusions: These preliminary data suggest that the broad temporal features of developmental genetics of GCs are recapitulated during retinal regeneration. However, the spatial patterns of zn–12 are not accurately recapitulated early in regeneration. Evaluation of additional GC–specific markers is underway. Support: The Glaucoma Foundation; NSF REU.
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