MicroRNA Identity and Function in Astrocyte Differentiation
Astrocytes are a dominant cell type in the central nervous system (CNS), intimately associated with neuronal synapses and CNS function. Studies in recent years have yielded insight into the remarkable morphological and functional diversity of astrocytes. However, mechanisms underlying the specification and differentiation of glial-specified precursors (GPCs) into astrocytes during development are not fully understood. In Chapter 2&3, I present work to address the hypothesis that miRNAs target key regulatory genes and pathways to promote terminal astrocyte differentiation, using a conditional Dgcr8 knockout model in embryonic stem cell-derived GPCs. In Chapter 4, I show that the function of Dgcr8 is largely limited to its known roles in canonical miRNA biogenesis, supporting its use in studying miRNA function. In GPCs, the loss of Dgcr8 and subsequent loss of miRNAs prevents upregulation of astrocyte markers and activation of JAK-STAT signaling during differentiation. Using a screening approach, I discovered that two miRNA families expressed in GPCs and astrocytes, let-7 and miR-125, rescue the upregulation of GFAP during differentiation but not activation of the JAK-STAT pathways. However, forced activation of the JAK-STAT pathway is sufficient to rescue the differentiation phenotype, suggesting that let-7 and miR-125 may regulate the pathway at downstream steps. Microarray and bioinformatics analysis following add back of the two miRNAs revealed direct and indirect targets. While individual knockdown of targets is insufficient to recapitulate the effect of let-7 and miR-125, at least one let-7/miR-125 target, Plagl2, inhibits astrocyte differentiation when overexpressed in wild-type cells. Taken together, these observations strongly suggest that the coordination of multiple miRNAs effects on multiple targets is necessary to promote the cell fate transition that leads to generation of astrocytes.