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S.L. Donovan, J. Zhang, J. Gray, M.A. Dyer; Rb Acts Upstream Of Nrl During Rod Photoreceptor Development . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4188.
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Three of the best–characterized transcription factors that regulate rod photoreceptor development are Nrl, Nr2e3, and Crx. The Rb tumor suppressor gene was recently added to this list of transcriptional regulators that are required for rod development. To determine where Rb functions in the rod development pathway, we have mated conditional Rb knockout mice (Chx10cre;Rblox/–) to Nrl knockout mice to generate double knockout mice (DKO). If Nrl acts upstream of Rb, then we expect the retinal phenotype of the DKO to resemble the single Nrl knockout and contain more S–cones. However, if Nrl is downstream of Rb, then the DKO will resemble the single Rb knockout and fail to develop rods, with no change in the composition of other cell types.
To determine where in the rod photoreceptor transcriptional network Rb is required.
P12 DKO retinal sections and dissociated cells were processed for fluorescent immunostaining with a panel of retinal cell–type specific antibodies. In some cases, multiple antibodies that recognize either rod and/or cone photoreceptors were used. TUNEL and BrdU (10mg/kg, 1 h prior to sacrificing) assays were carried out to determine whether cell death or proliferation, respectively, contribute to the observed effects. To determine the percentage of each of the 7 main classes of retinal cell types, dissociated immunostained retinae were scored in five independent samples for each genotype. RNA was isolated from contralateral retinae and analyzed by real–time RT–PCR. To assess for localization of mRNA, we performed in situ hybridization on P12 DKO retina for a variety of photoreceptor specific genes.
The staining patterns in the DKO retina resemble the single Rb KO.
Rb is upstream of Nrl or may act in a parallel pathway.
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