miRNAs and their mRNA targets in neural stem cells

Project Leader: Gregory Wulczyn e-mail

 

miRNAs are now established as important actors in CNS development, in higher order neuronal functions, and in proliferative and degenerative brain disease. As the first mammalian miRNA to be discovered let-7 has proven to be a paradigm for understanding the contributions of miRNAs to the developmental control of gene expression. let-7 is perfectly conserved in the bilateral animal phyla, and let-7 family members are the most highly expressed miRNAs in the mammalian CNS. We have therefore focused on let-7 and its target mRNAs and defined an autoregulatory loop in neural stem cells involving let-7, the pluripotency factor Lin28 and a novel mouse gene Lin41 (also referred to as Trim71). These studies support a model in which Lin28 and Lin41 act as regulators of let-7 biogenesis (Lin28) and activity (Lin41) in undifferentiated stem cells. During embryonic neurogenesis, we found that let-7 directly suppresses Lin28 and Lin41 as part of its role as inhibitor of “stemness”. We hope to better understand the role of let-7 and Lin41 in cortical neuroepithelia and in the adult stem cell niche, and the developmental signals that turn let-7 expression and its targets off.

We were the first to describe E3 ligase activity and miRNA pathway associations for Lin41. Using a gene trap mouse line deficient in Lin41 we have produced embryonic stem cell lines heterozygous and homozygous for the gene trap allele. With this tool we will investigate a network of interaction partners and downstream targets we and others have identified for Lin41 during neural differentiation. We will take advantage of recently developed techniques for global ubiquitylation analysis to identify Lin41 substrates in stem cells as a function of differentiation state.

Deletion of either Lin41 or its interaction partner in the miRNA pathway, Ago2, leads to neural tube closure defects in the mouse. We will use the closure phenotype as a test for genetic interactions between Lin41 and Ago2, but also additional candidate genes, in heterozygote crosses or in rescue experiments. The results of these experiments can then be translated into a screen of spina bifida and hydrocephalus patients for mutations in Lin41 and miRNA pathway genes. In parallel, we are close to realization of a conditional knockout model for Lin41. This model will allow us to circumvent embryonic lethality of the gene trap allele to define the role of the Lin41/let-7 regulatory interaction at specific points of developmental time and in adult neurogenesis.