GSE128052
Transcriptional profile of pDyn-lineage spinal interneurons in the developing mouse
Purpose: Mounting evidence suggests that the spinal dorsal horn (SDH) contains multiple subpopulations of inhibitory interneurons that play distinct roles in somatosensory processing, as exemplified by the importance of spinal dynorphin-expressing neurons for the suppression of mechanical pain and chemical itch.  While it is clear that GABAergic transmission in the SDH undergoes significant alterations during early postnatal development, little is known about the maturation of discrete inhibitory “microcircuits” within the region.  As a result, the goal of the present study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life. Methods: We isolated nuclear RNA specifically from pDyn-lineage SDH interneurons at postnatal days 7, 21, and 80 using the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique in conjunction with Fluorescence-activated Nuclei Sorting (FANS), followed by  RNAseq analysis. Results: Over 650 genes were =2-fold enriched in adult pDyn nuclei compared to non-pDyn spinal cord nuclei, including targets with known relevance to pain such as galanin (Gal), prepronociceptin (Pnoc), and nitric oxide synthase 1 (Nos1).  In addition, the gene encoding a membrane-bound guanylate cyclase, Gucy2d, was identified as a novel and highly selective marker of the pDyn population within the SDH.  Differential gene expression analysis comparing pDyn nuclei across the three ages revealed sets of genes that were significantly upregulated (such as Cartpt encoding cocaine- and amphetamine-regulated transcript peptide) or downregulated (including Npbwr1 encoding the receptor for neuropeptides B/W) during postnatal development. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Transcriptional profiles of pDyn-lineage spinal cord neurons were generated from nuclear RNA and compared to non-pDyn lineage spinal cord nuclei at postnatal ages P7, P21, and P80.  Additionally, the transcriptional profiles of pDyn-lineage nuclei were compared between the three postnatal ages to elucidate age-dependent changes in gene expression.