| Precise control of neurogenesis is critical for growth of the developing mammalian neocortex. Neurogenesis is regulated by the expression of both protein coding and non-coding genes. microRNAs (miRNAs) are a class of endogenous, non-coding genes that produce ~22nt mature RNA products. These small RNAs are used as guides by the RNA Induced Silencing Complex (RISC) to target numerous mRNAs for regulation. While it has been shown that miRNAs as a class are critical for cortical formation, determining which are critical, and what their precise roles may be, is an area of active study. In this thesis, I present my work describing the function of one such microRNA: microRNA-7 (miR-7). In mice, miR-7 is expressed in neural progenitors of the developing dorsal cortex, transcribed from three loci on separate chromosomes. In order to block the function of the miRNA from all loci at once, I designed and optimized a miRNA sponge - a synthetic gene containing multiple binding sites for miR-7. This transcript is able to block the function of miR-7 without affecting the function of other miRNAs in the cell. I went on to generate a transgenic mouse in which the activity of microRNA miR-7 is specifically blocked by the miR-7 ponge in the embryonic neocortex. Blocking the function of miR-7 in cortical neural progenitors resulted in impaired transition of radial glial cells (RGCs) into intermediate progenitors (IPs), and apoptosis. This loss of progenitors leads to reduced neurogenesis and reductions in all layers of cortical neurons. IIlumina RNA sequencing has revealed loss of regulation of 162 predicted miR-7 target genes in miR-7 sponge cortices, including many implicated in the p53 pathway. Ectopic expression of Akl or Cdknla (p21) - verified miR-7 targets and known p53 pathway genes - is able to recreate the reduced IP specification phenotype seen in the miR-7 sponge neocortex. My findings from this loss-of-function mouse model add to the growing body of research implicating the p53 pathway in neurogenesis, and furthermore reveal miR-7 as an important regulator of this pathway during cortical neurogenesis. |
