Requirement of neuroD for photoreceptor formation in the chick retina.
OBJECTIVE
The genetic control of photoreceptor cell fate in the vertebrate retina is poorly understood. Published studies suggest that the genetic program underlying photoreceptor production involves neuroD, a proneural basic helix-loop-helix (bHLH) gene. The present study investigates whether neuroD is necessary for photoreceptor cell development, by using loss-of-function analyses.


METHODS
Engrailed-mediated active repression, antisense oligonucleotides, and small interfering RNA (siRNA) were used to attenuate neuroD expression and function in embryonic chick retina. The development of the retina was subsequently analyzed to determine whether these experimental manipulations would yield photoreceptor deficits in otherwise normal retina.


RESULTS
Chick embryos infected with retroviruses expressing an active repression construct, En-NeuroDDeltaC, exhibited severe photoreceptor deficits. The outer nuclear layer (ONL) of the retina was no longer a contiguous structure, but became fragmented with regions that contained fewer or no photoreceptor cells. Photoreceptor deficiency was evident even before the retina became laminated, suggesting that active repression of NeuroD may have affected photoreceptor genesis. No deficiency was observed in other types of retinal cells. Culturing retinal cells in the presence of siRNA against neuroD resulted in a more than 50% reduction in the number of photoreceptor cells and an increase in the number of chx10+ cells. Subjecting the developing retina to antisense oligonucleotides against neuroD yielded fewer photoreceptor cells both in vivo and in vitro. Consistent with these observations, anti-NeuroD antibody specifically labeled the nuclei of the ONL.


CONCLUSIONS
The data suggest a specific and an essential role of neuroD in photoreceptor formation in the chick retina.
Yan
Run-Tao
RT
Department of Ophthalmology, University of Alabama at Birmingham, School of Medicine, Birmingham, Alabama, USA.
Wang
Shu-Zhen
SZ
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002642:Chick Embryo; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D018257:Helix-Loop-Helix Motifs / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D020419:Photoreceptor Cells, Vertebrate / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D012160:Retina / Q000196:embryology* / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C093564:Neurogenic differentiation factor 1