Brn3b/Brn-3.2/POU4f2 is a POU domain transcription factor that is essential for retinal ganglion cell (RGC) differentiation, axonal outgrowth and survival. Our goal was to establish a link between Brn3b and the downstream events leading to RGC differentiation. We sought to determine both the number and types of genes that depend on Brn3b for their expression. RNA probes from wild-type and Brn3b-/- E14.5, E16.5 and E18.5 mouse retinas were hybridized to a microarray containing 18,816 retina-expressed cDNAs. At E14.5, we identified 87 genes whose expression was significantly altered in the absence of Brn3b and verified the results by real-time PCR and in situ hybridization. These genes fell into discrete sets that encoded transcription factors, proteins associated with neuron integrity and function,and secreted signaling molecules. We found that Brn3b influenced gene expression in non RGCs of the retina by controlling the expression of secreted signaling molecules such as sonic hedgehog and myostatin/Gdf8. At later developmental stages, additional alterations in gene expression were secondary consequences of aberrant RGC differentiation caused by the absence of Brn3b. Our results demonstrate that a small but crucial fraction of the RGC transcriptome is dependent on Brn3b. The Brn3b-dependent gene sets therefore provide a unique molecular signature for the developing retina.
Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.
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