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F.C. Chiodini, N. Taverney, Y. Arsenijevic, F.L. Munier, J. Matter; Search for Extrinsic Cues Involved in the Differenciation of Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):509.
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Purpose: Several genes implicated in retinal ganglion cell (RGC) development have been identified in the vertebrate retina. The protein ATH5 of the bHLH transcription factor family is required for RGC differentiation. We have identified cis regulatory sequences of the ATH5 gene and their contribution to the expression pattern of the gene during the period of specification and differentiation of RGCs. A cascade of interacting bHLH factors appears to regulate ATH5 transcription during retinogenesis (Matter-Sadzinski et al. 2001; Development, 128:217-231). Little is known about extrinsic signals that might be implicated in the genetic regulation behind the differentiation and maintenance of RGCs. Here, we address the issue via retinal transplantation of identified RGC precursors. Methods: The ATH5-promoter/lacZ and the CMV-GFP plasmids were lipofected into retinal cells of stage 15-30 chick embryos. Transfected cells were injected in ovo into the eye of stage 15-30 embryos. Retinal incorporation of transplanted GFP-positive cells was monitored in vivo and LacZ positive cells were revealed at different times ranging from 1 to 6 days post-transplantation. The identity of the transplanted LacZ positive cells was determined by in situ hybridization with RGC specific markers (e.g., ATH5, ß3 nAChR). Synaptic connectivity of the lacZ positive cells was monitored by retrograde backfilling of the RGC cell bodies with fluorescent beads injected into the optic tectum of stage 32-34 chick embryos. In addition, the electrophysiological properties of the transplanted cells were recorded by patch-clamp on ATH5-GFP positive cells labeled with fluorescent beads. Results: Preliminary results showed that the capacity of transplanted ATH5-positive retinal cells to differentiate into RGCs depends on the developmental stage of both the donor and the host retina. Conclusion: These experiments will help us to determine the influence of extrinsic cues on transplanted RGCs, i.e., if they adopt a ganglion or another retinal cell phenotype.
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