NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors.. Mus musculus

Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor. Overall design: GFP positive cells from Nrlp-GFP mouse retinas at post-natal ages P2, P4, P6, P10, P12, P21, and P28 were isolated by flow sorting by FACSAria II (Becton Dickinson) into RNAlater (Invitrogen). Total RNA was extracted by RNeasy Mini Kit (Qiagen) and analyzed by 2100 Bioanalyzer (Agilent Technologies Genomics). Five nanograms of high quality of total RNA (RIN: >7.0) was used as input for exon array target preparation. Affymetrix Mouse Exon 1.0 ST arrays were performed using targets prepared from WT-Ovation RNA-Amplification System (NuGEN). Gene-level core RMA intensity values for each GeneChip® Mouse Exon 1.0 ST array were collected using Affymetrix Expression Console Software (Affymetrix) with MoEx-1_0-st-v1.r2.dt1.mm8.core.mps.