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J. Trimarchi, C. Cepko; Single Cell Gene Expression Profiling of the Developing Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5508.
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During the course of retinal development, cycling progenitor cells give rise to a variety of cell types (six neuronal and one glial). Once these distinct classes of cells are born, they progress from newly specified cells into fully differentiated neurons and glia. Although environmental cues may play a role, these cell fate decisions and cellular maturation processes are undoubtedly controlled by changes in intrinsic gene expression. Using a single cell gene expression approach, we sought to characterize the transcriptional programs of individual cells from the developing retina and through this examination hoped to gain insight into both retinal progenitor cell biology and the maturation processes of retinal ganglion and amacrine cells.
Single retinal cells were isolated from dissociated retinas at multiple early developmental stages (E12.5 through P0). These individual cells were lysed, their mRNA was reverse transcribed, the resulting cDNA was PCR amplified, and these cDNAs were hybridized to high density mouse Affymetrix arrays (430 2.0 arrays). A total of 80 single retinal cells were profiled in this manner.
Using single adult rod photoreceptor cells as controls, this single cell approach has been shown to be robust and reproducible. We have been able to characterize the 80 single cells we have isolated as either cycling progenitor cells or postmitotic neurons. Among the postmitotic neurons, we have been able to further subdivide the cells into developing ganglion, amacrine or photoreceptor cells. Using a combination of section in situ hybridization (ISH) and two color fluorescent ISH on dissociated retinas, we have been able to validate and extend our observations on the temporal gene expression patterns of developing ganglion and amacrine cells.
Single cell gene expression profiling has enabled us to examine heterogeneity of cycling retinal progenitor cells and developing ganglion and amacrine cells. Many genes have been found in subsets of retinal cells including several transcription factors. These microarray experiments have yielded both important new markers of developing ganglion and amacrine cells and new insights into retinal progenitor cell biology.
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