Understanding stem cell maintenance and differentiation is a fundamental step for basic research that moreover paves the way to challenging medical objectives such as regenerative medicine. The central nervous system is a particularly relevant tissue both for its limited regenerative potential and for the severe diseases associated to its altered development and maintenance. Here we aim to understand cell cycle exit and differentiation in deep molecular terms, as a way to reveal novel pathways and regulations. Specifically, we focused on differentiation of GABAergic neurons, adopting the model of the adherent Neural Stem (NS) cells. We generated RNA-seq and miRNA profile data at four time points, from proliferating NS to the mature neuron stage. We examined differential expression when these cells exit the cycle, after removal of FGF. While their expression of differentiation markers and of synaptic genes progressively increases over time, cell-cycle regulators are rapidly shut off. We identified three down-(15b, 25 and 27a) and five up-(149, 652, 30c, 125b-5p and 342-3p) regulated miRs; integrating these data with DE analyses we identified some cognate target:miRNA pairs engaging in negative regulations. Next we examined alternative polyadenylation usage, a phenomenon recently implicated in proliferation and differentiation. Several (45) mRNA are shorter in differentiating NS cells and only few (5) are longer, following a known trend in other contexts. We have identified miR seed enriched in the lost 3’UTR portions, suggesting shortening as a possible way to escape specific miRNA based regulations. Finally, comparing WT with Dlx5;Dlx6 KO NS cells, where GABAergic differentiation is retarded, we will be able to focus only on the functionally relevant regulations. Overall, we hope to de-convolute RNA-based networks essential for GABA differentiation, leading to a systems biology comprehension of the molecular regulations underlying interneuron genesis and maturation.
A comprehensive transcriptome analysis of GABA-differentiating neural stem cells aimed at dissecting molecular mechanisms underlying cell cycle exit, neuronal commitment and differentiation
GRASSI, ELENA;ALA, UGO;DI CUNTO, Ferdinando;PROVERO, Paolo;OLIVIERO, Salvatore;MERLO, Giorgio Roberto
2014-01-01
Abstract
Understanding stem cell maintenance and differentiation is a fundamental step for basic research that moreover paves the way to challenging medical objectives such as regenerative medicine. The central nervous system is a particularly relevant tissue both for its limited regenerative potential and for the severe diseases associated to its altered development and maintenance. Here we aim to understand cell cycle exit and differentiation in deep molecular terms, as a way to reveal novel pathways and regulations. Specifically, we focused on differentiation of GABAergic neurons, adopting the model of the adherent Neural Stem (NS) cells. We generated RNA-seq and miRNA profile data at four time points, from proliferating NS to the mature neuron stage. We examined differential expression when these cells exit the cycle, after removal of FGF. While their expression of differentiation markers and of synaptic genes progressively increases over time, cell-cycle regulators are rapidly shut off. We identified three down-(15b, 25 and 27a) and five up-(149, 652, 30c, 125b-5p and 342-3p) regulated miRs; integrating these data with DE analyses we identified some cognate target:miRNA pairs engaging in negative regulations. Next we examined alternative polyadenylation usage, a phenomenon recently implicated in proliferation and differentiation. Several (45) mRNA are shorter in differentiating NS cells and only few (5) are longer, following a known trend in other contexts. We have identified miR seed enriched in the lost 3’UTR portions, suggesting shortening as a possible way to escape specific miRNA based regulations. Finally, comparing WT with Dlx5;Dlx6 KO NS cells, where GABAergic differentiation is retarded, we will be able to focus only on the functionally relevant regulations. Overall, we hope to de-convolute RNA-based networks essential for GABA differentiation, leading to a systems biology comprehension of the molecular regulations underlying interneuron genesis and maturation.
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