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. 2016 Nov;9(2):169–175. doi: 10.15283/ijsc16020

Fig. 2.

Fig. 2

The intrinsic molecular axes and their involvement in the neurogenic potential of MG. (A) A schematic diagram shows the virus-mediated ectopic expression in enriched MG to perturb Lin28a (Lin28a-let7-Hmga2/Ascl1) and miR-124 and miR-9/9* (Rest-miR-124/miR-9-9*-Sox9/Hes). (B) Lin28a overexpression caused a significant decrease and increase in transcripts characterizing MG (Glast) and neurons (beta-tubulin and Map2), respectively. Neuronal differentiation of transduced MG could also be ascertained by their neuronal morphology and expression of immunoreactivities corresponding to beta-tubulin and Map2. Neuronal differentiation of MG was accompanied by a significant increase and decrease in the expression of positive (miR-124, Hmga2, and Ascl1) and negative (REST and Hes1) regulators of neurogenesis. (C) Similarly, miR-124-9-9* overexpression led to a decrease in levels of glia-specific (Glast) and an increase in neuron-specific (beta-tubulin and Map2) transcripts. As observed in Lin28a overexpression, transduced MG displayed neuronal morphology and expressed beta-tubulin and Map2 immunoreactivities. A significant increase and decrease in the expression of positive (Lin28a and Ascl1) and negative (REST and Hes1) regulators of neurogenesis was accompanied by miR-124-9-9* mediated neuronal differentiation of MG. Levels of Hmga2 transcripts remained unchanged. That the two axes might intersect and influence each other was demonstrated by the increase of miR-124 transcript levels in Lin28 overexpression experiment and vice versa (enclosed graph, B, C). MG were enriched as described in Das et al., (6). They were transduced with Lin28a retrovirus (24) or miR124-9-9* lentivirus (27) and the effects of perturbations on gene expression and phenotype were examined by Q-PCR and immunocytochemical analyses, respectively, as described in Parameswaran et al., (22) and Xia et al., (17). Controls included MG transduced with empty retrovirus/lentivirus. Levels of transcripts and miRNA are presented after normalization with that of GAPDH and U6, respectively. Arrowheads indicate transduced MG (GFP+/RFP+) expressing neuronal markers, beta-tubulin or Map2. Scale bar, 50 μm. Data are mean±s.e.m.