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N.L. Brown, T.T. Le, E. Wroblewski; Mouse embryonic retinal progenitor cells require Ath5/ATOH7 for cell cycle progression . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3423.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The bHLH transcription factor Ath5 is only expressed by retinal progenitors, not RGC neurons. Targeted deletion of Ath5 causes the complete loss of postnatal RGCs and an increase in cone and amacrine neurons, implying that retinal progenitors switch their primary fate. The goal of these experiments is to determine the underlying mechanism for changes in retinal neuron fate. Methods: A dose of BrdU was administered to pregnant mice carrying litters of wild type, Ath5+/– and Ath5–/– embryos, ages E11–E15. In birthdating experiments, adult offspring were genotyped, sacrificed and their eyes fixed, sectioned and labeled with anti–BrdU and a counterstain. In pulse–labeling experiments, embryonic litters at each age were fixed 1.5 hours after BrdU injection followed by genotyping, histological sectioning and antibody labeling with anti–BrdU and a counterstain. Birthdated neurons or S phase cells were tabulated and normalized from three independent litters at each age. Results: To determine when Ath5–/– progenitors adopt alternate fates, we compared retinal neuron birthdates between wild type and Ath5 mutants. At E11.5 there were significantly fewer Ath5–/– birthdated neurons in all retinal cell layers. At E13.5 and E15.5 mutant ONL birthdates were increased, and a smaller increase in mutant INL neurons occurred at E13.5. The E11.5 Ath5–/– progenitors that do not differentiate may keep dividing or become stalled in the cell cycle. We observed a significant loss of Ath5–/– S–phase cells at E11.5, followed by an equivalent increase in proliferation at E14.5. Between these ages, mutant progenitors appear stalled in the cell cycle. The temporal shift in proliferation was correlated with changes in the expression of retinal bHLH factors that promote amacrine (NeuroD) or photoreceptor (Ngn2 or NeuroD) fates. Ngn2 and NeuroD were upregulated in Ath5 mutants between E13–E15 in specific ways. NeuroD exhibited age–specific changes in Ath5–/– embryos since there are more NeuroD–expressing cells in the inner optic cup at E13.5, but only the outer cup has expanded NeuroD expression at E15.5. Ngn2 expression in Ath5 increased randomly between E13.5 and E15.5. Conclusions: We conclude that most Ath5 mutant progenitors fail to differentiate at E11.5 and become amacrine and cone photoreceptor neurons at E13.5 or E15.5. We propose that Ath5 is required to both trigger cell cycle exit at E11.5 and repress Ngn2 and NeuroD transcription for proper specification of RGC fate.
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