Abstract:
In the developing retina, cell cycle exit must be carefully coordinated with commitment to a particular cell fate to ensure that each cell type is made in the correct proportion. Previously, we have shown that Rb cell autonmously regulates retinal progenitor cell cycle exit and mediates rod development. Other studies have identified candidate genes (Nrl, Nr2e3 and Crx) required for rod development. In Rb–deficient retinae, rod photoreceptors not only fail to differentiate, but also show decreased expression of Nrl, Nr2e3 and Crx. Moreover, inactivation of Rb using the Chx10–Cre transgenic mouse line, creates a mosaic pattern of inactivation, such that rods fail to form in the stripes where Chx10 is expressed (see figure – white dashes). We sought to determine whether Rb is required for the expression of known candidate genes that control rod development and identify novel Rb–regulated genes. To determine whether Rb function is upstream of Nrl, Nr2e3 or Crx, we generated and analyzed compound knockout mice (Chx10–Cre;RbLox/–;Nrl–/–; Chx10–Cre;RbLox/–;Nr2e3–/–; Chx10–Cre;RbLox/–;Crx–/–). Analysis includes immunofluorescence with 36 different antibodies to cell type specific proteins, proliferation analysis with BrdU, and apoptosis analysis with TUNEL at various stages of retinal development. To identify putative targets of Rb by conditional inactivation, we electroporated YFP–tagged Cre–expressing DNA plasmids into RbLox/– or RbLox/Lox retinae at P0. Retinae were cultured for several days, dissociated and FACS sorted. RNA was isolated from FACS sorted cells and analyzed with microarray hybridization and real–time RT–PCR. Rb inactivation leads to a reduction in the expression of Nrl and Nr2e3, suggesting that Rb is one of the earliest genes important for rod development identified to date. Based on our data, we propose that Rb acts very early in the rod development program and is required for the expression of candidate genes that are crucial for proper rod development.
Keywords: retinal development • photoreceptors • genetics