RESUMO
Cell-type determination is a complex process driven by the combinatorial effect of extrinsic signals and the expression of transcription factors and regulatory genes. MicroRNAs (miRNAs) are non-coding RNAs that, generally, inhibit the expression of target genes and have been involved, among other processes, in cell identity acquisition. To search for candidate miRNAs putatively involved in mice rod photoreceptor and Müller glia (MG) identity, we compared miRNA expression profiles between late-stage retinal progenitor cells (RPCs), CD73-immunopositive (CD73+) rods and postnatal MG. We found a close similarity between RPCs and CD73+ miRNA expression profiles but a divergence between CD73+ and MG miRNA signatures. We validated preferentially expressed miRNAs in the CD73+ subpopulation (miR-182, 183, 124a, 9(∗), 181c and 301b(∗)) or MG (miR-143, 145, 214, 199a-5p, 199b(∗), and 29a). Taking advantage of the unique capacity of MG to dedifferentiate into progenitor-like cells that can be differentiated to a rod phenotype in response to external cues, we evaluated changes of selected miRNAs in MG-derived progenitors (MGDP) during neuronal differentiation. We found decreased levels of miR-143 and 145, but increased levels of miR-29a in MGDP. In MGDPs committed to early neuronal lineages we found increased levels of miR-124a and upregulation of miR-124a, 9(∗) and 181c during MGDP acquisition of rod phenotypes. Furthermore, we demonstrated that ectopic miR-124 expression is sufficient to enhance early neuronal commitment of MGDP. Our data reveal a dynamic regulation of miRNAs in MGDP through early and late neuronal commitment and miRNAs that could be potential targets to exploit the silent neuronal differentiation capacity of MG in mammals.
Assuntos
Diferenciação Celular/fisiologia , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , 5'-Nucleotidase/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Células Ependimogliais/efeitos dos fármacos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Análise em Microsséries , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/farmacologia , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células-TroncoRESUMO
Müller cells are not only the main glial cell type in the retina but also latent progenitor/stem cells, which in pathological conditions can transdifferentiate to a neuronal phenotype and regenerate the neurons lost in a mature retina. Several signal transduction pathways can induce the dedifferentiation of mature Müller cells to a progenitor-like state, including that stimulated by glutamate. However, the precise molecular mechanisms by which terminally differentiated cells are initially primed to acquire multipotency remain unclear. In the present study, we have characterized early genetic and epigenetic events that occur immediately after glutamate-induced dedifferentiation of fully differentiated Müller cells is initiated. Using Müller cell-enriched cultures from postnatal rats, we demonstrate that glutamate triggers a rapid dedifferentiation response characterized by changes in cell morphology coupled to the induction of progenitor cell marker gene expression (e.g., nestin, lin28 and sox2) within 1h. Dedifferentiation involved the activation of N-methyl-d-aspartate and type II metabotropic glutamate receptors, as well as global DNA demethylation (evident through the decrease in methyl-CpG-binding protein 2 immunoreactivity) and an increase in gadd45-ß gene expression; although, early progenitor gene expression was only partially inhibited by pharmacological impairment of DNA methylation. Importantly, the expression of Müller glia identity genes (i.e., glutamine synthetase; cellular retinaldehyde binding protein, CRALBP) is retained through the process. Dedifferentiated Müller cells held an early neuronal differentiation potential similar to that observed in retinal progenitor-enriched cultures but, contrary to the latter, dedifferentiated Müller cells failed to further differentiate into mature photoreceptor lineages. We speculate that, in spite of the initial triggering of the dedifferentiation pathways, these cells may exhibit a certain degree of epigenetic memory that precludes them from further differentiation.