Abstract
BackgroundTo investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs.Principal FindingsDicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia.ConclusionsThe block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation.
Highlights
Neural stem (NS) cells in the developing vertebrate embryo are multipotent, self-renewing cells that give rise to specific types of neurons in a fixed temporal order
We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation
In a developing neural stem cell-specific Dicer mutant, microRNAs persisted for several days after Dicer ablation [16], and we have found that following Dicer deletion in many cases microRNAs can persist at over 40% of wild-type levels during the period of neurogenesis
Summary
Neural stem (NS) cells in the developing vertebrate embryo are multipotent, self-renewing cells that give rise to specific types of neurons in a fixed temporal order. MicroRNAs have been proposed as candidates for regulating many aspects of neural stem cell biology, based on the functions of microRNAs in the control of developmental timing and cell fate specification in a number of biological systems, including stem cells (for reviews, see [1,2]). MicroRNAs have been proposed to regulate stem cell self-renewal, neurogenesis, cell fate determination and neuronal differentiation [3,4]. To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs
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