Control of timing and directionality during neurodifferentiation of human induced Pluripotent Stem Cells (iPSC) via miRNA-mediated feedback and feedforward loops.

Development of neural cells follows a unidirectional path, progressing from pluripotent stem cells into a variety of highly differentiated neuronal and glial cell types. Although the intermediate progenitor cells along this path are well characterised, the mechanisms that determine their directional flow remain to be established. Here, we identify a gene regulatory network that directs progression of neuronal differentiation. Previously, it was shown that loss of the epigenetic repressor, EHMT1, leads to premature neuronal differentiation, and this correlates with elevated miRNA expression and inhibition of a neuronal gene suppressor, REST. Here, we use a combination of in silico-analysis and targeted suppression of multiple miRNAs to reveal an unexpected degree of complexity to this gene regulatory pathway. We identity four miRNA, miR26a, miR140, miR142, miR153, that cooperatively block REST at the mRNA level. In contrast to most miRNA suppressors of REST, such as miR9a and miR124, which are in turn transcriptionally repressed by REST in a negative feedback loop (FBL), miR26a, miR140 and miR142 are not REST-targets. This creates a feedforward loop (FFL) that drives developing cells to switch from a high REST, non-neuronal state to a low REST, neuronal state, but not in the reverse direction. We show that together miR26a, miR140, miR142 and miR153 are necessary for neuronal differentiation and introduction of synthetic miRNAs mimics is sufficient to increase downstream miR-9 and miR-124 expression. Finally, we note that the Neurodevelopmental Disorder (NDD) Kleefstra Syndrome (KS) is caused by the loss of EHMT1, suggesting that the molecular mechanism described above may play a significant role in human brain development.