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W. Mao, R.-T. Yan, S.-Z. Wang; Proneural Bhlh Gene Ash1 Inhibits Ngn2 Expression in the Developing Chick Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3075.
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© ARVO (1962-2015); The Authors (2016-present)
To gain new insight into the regulatory control over the generation of retinal neural diversity by delineating the cross-regulatory relationship between bHLH genes ash1 and ngn2 in the chick embryonic retina.
For gain-of-function experiments, replication-competent retrovirus RCAS was used to drive chick Ash1 or Ngn2 overexpression in the developing chick retina. For loss-of-function experiments, the active repressor domain of Drosophila Engrail (En) was fused with Ash1 (En-Ash1) or Ngn2 (En-Ngn2) at the N-terminus and the fusion protein was expressed in retinal cells through RCAS transduction. Chick retinas were collected at embryonic day 8 for in situ hybridization with digoxigenin labeled anti-ngn2 or anti-ash1 RNA probes. After in situ hybridization, the same retinas were immunolabeled with anti-P27 antibody, which recognizes a viral protein of RCAS.
The developing chick retina expresses ngn2 and ash1 during active neurogenesis. Although both genes are expressed in progenitor cells, their spatial patterns differ, suggesting that some cells may express one but not the other. To minimize ambiguity associated with comparing different retinal sections, E8 retinas partially infected with the virus were used. In the uninfected regions of retinas partially infected with RCAS-Ash1, ngn2 mRNA was detected in a large number of cells, most of which were localized within the "germinal zone," where retinal progenitor cells reside at this developmental stage. However, in the adjacent, infected regions, ngn2 mRNA was detected in fewer cells and those positive cells contained weaker in situ hybridization signals. No changes were observed with EN-Ash1. In retinas partially infected with RCAS-ngn2, ash1 expression remained unchanged in regions infected with the virus compared to the adjacent, uninfected region. Likewise, no alteration of ash1 expression was observed with En-Ngn2.
The results showed that ash1 inhibited ngn2 expression, but ngn2 did not affect ash1 expression. This one way inhibition between ash1 and ngn2 may be an integral part of a regulatory network governing neuron diversity in the retina.
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