Run-Tao Yan1, Li He, Shu-Zhen Wang. 1. Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0009, USA.
Abstract
PURPOSE: Better understanding of photoreceptor fate specification may lead to efficient production of photoreceptors for cell replacement studies. The authors investigated the role of proneural bHLH gene neurogenin1 (ngn1) in photoreceptor genesis using the chick retina. METHODS: In situ hybridization was used to delineate the spatial and temporal pattern of ngn1 expression. RCAS retrovirus was used to drive overexpression of ngn1 in retinal cells, and siRNA was used to reduce ngn1 expression in loss-of-function experiments. RESULTS: Chick ngn1 was transiently expressed during early phases of retinal neurogenesis, from embryonic day (E)3 to E6, with cells expressing ngn1 confined to the apical side of the retinal neuroepithelium. The time window and the anatomic location of ngn1 expression coincided with photoreceptor genesis and differed from those of other transiently expressed proneural bHLH genes, such as ash1, ath3, ath5, and ngn2. Most ngn1-expressing cells lacked BrdU incorporation and lacked phosphorylated histone H3. In low-density cell culture, ngn1 overexpression increased neuroD expression and expanded the photoreceptor population but reduced the ganglion population. Treatment of dissociated retinal cells with siRNA against ngn1 mRNA specifically reduced the photoreceptor population. Overexpression of ngn1 in the retina reduced the expression of ash1, ath5, chx10, and ngn2. CONCLUSIONS: The data suggest that ngn1 participates in a complex transcriptional network and may play a role in guiding a progenitor cell to the photoreceptor pathway.
PURPOSE: Better understanding of photoreceptor fate specification may lead to efficient production of photoreceptors for cell replacement studies. The authors investigated the role of proneural bHLH gene neurogenin1 (ngn1) in photoreceptor genesis using the chick retina. METHODS: In situ hybridization was used to delineate the spatial and temporal pattern of ngn1 expression. RCAS retrovirus was used to drive overexpression of ngn1 in retinal cells, and siRNA was used to reduce ngn1 expression in loss-of-function experiments. RESULTS:Chickngn1 was transiently expressed during early phases of retinal neurogenesis, from embryonic day (E)3 to E6, with cells expressing ngn1 confined to the apical side of the retinal neuroepithelium. The time window and the anatomic location of ngn1 expression coincided with photoreceptor genesis and differed from those of other transiently expressed proneural bHLH genes, such as ash1, ath3, ath5, and ngn2. Most ngn1-expressing cells lacked BrdU incorporation and lacked phosphorylated histone H3. In low-density cell culture, ngn1 overexpression increased neuroD expression and expanded the photoreceptor population but reduced the ganglion population. Treatment of dissociated retinal cells with siRNA against ngn1 mRNA specifically reduced the photoreceptor population. Overexpression of ngn1 in the retina reduced the expression of ash1, ath5, chx10, and ngn2. CONCLUSIONS: The data suggest that ngn1 participates in a complex transcriptional network and may play a role in guiding a progenitor cell to the photoreceptor pathway.