Secretoneurin A regulates neurogenic and inflammatory transcriptional networks in radial glia of the goldfish brain

Radial glial cells (RGCs) are the most abundant macroglia in the teleost brain and have established roles in neurogenesis and neurosteroidogenesis; however, their transcriptome remains unstudied, limiting functional understanding of this crucial cell type. Using cultured goldfish RGCs, RNA sequencing and de novo transcriptome assembly were performed, generating the first reference transcriptome for fish RGCs with 17,620 unique genes identified. These data revealed that RGCs express a diverse repertoire of receptors and signaling molecules, suggesting that RGCs may respond to and synthesize an array of hormones, peptides, cytokines, and growth factors. Expanding on recent neuroanatomical data and possible direct neuronal regulation of RGC physiology, differential gene expression analysis identified transcriptional networks that are responsive to the conserved secretogranin II-derived neuropeptide secretoneurin A (SNa). Pathway analysis indicated that cellular processes related to the central nervous system (e.g., neurogenesis, synaptic plasticity, glial cell development) and immune functions (e.g., immune system activation, leukocyte function, macrophage response) were preferentially modulated by SNa. These data reveal an array of new functions that are proposed to be critical to neuronal-glial interactions through the mediator SNa. Four control, three SNa (1000nM) treated and three SKF (10uM). SNa is sectretoneurin A peptide and SKF is a selective dopamine D1 receptor agonist SKF 38393. Passage three cells were exposed for 24hrs. Total RNA was extracted using the RNeasy Micro Kit (Qiagen) including an on-column DNase treatment to remove genomic DNA. The concentration of total RNA was determined using the Qubit RNA Assay Kit (Life Technologies). In order to evaluate the quality of the total RNA, RNA integrity number (RIN) was assessed using Agilent RNA 600 Nano Reagents and RNA Nano Chips in Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing of 10 RGC cultures was performed by MR DNA (www.mrdnalab.com) following the manufacturer’s guidelines (Illumina HiSeq) for paired end sequencing (151 bp). RNA with poly(A) tails were purified and fragmented into shorter sequences that were used as templates for cDNA synthesis. A total of 10 cDNA libraries were constructed using random-hexamer primers from 1 μg of total RNA from each sample using TruSeq RNA LT Sample Preparation Kits (Illumina).