One of the fundamental questions of corticogenesis is how neuronal diversity is achieved. It was previously held that neuronal fate is determined by birthdate and through the “progressive restriction of competence” of a multipotent apical progenitor. Recent findings, albeit disputed, demonstrate that it might instead be specified through “predetermined fate-restriction” of apical progenitors. Here, we conducted a comprehensive single-cell expression profile of apical progenitors at embryonic day (E)13.5 to decipher the transcription factors (TFs), or TF codes, that confer progenitor identity and neuropotency. In the process of clustering single cells into known progenitor subtypes, we identified a cluster of atypical progenitors (19 out of 231 cells clustered by marker gene expression). These Nestin+/Tubb3+ progenitors did not express neuroepithelial cell (NEC) nor neurogenic differentiation markers, nullifying our initial hypothesis that they might represent a subpopulation of differentiating NECs. They did, however, express typical ventricular zone (VZ) markers, indicating that they might instead represent a novel subtype of apical progenitor. The expression of Mycl, a member of the MYC family of proto-oncogenes, was particularly high in subset of these atypical progenitors (5 out of 9 cells clustered by TF expression). We, therefore, conducted gain- and loss-of-function assays to determine its function during corticogenesis. Unexpectedly, we found that overexpression and knockdown of Mycl by in utero electroporation of E13.5 mouse cortices induced similar phenotypes. This was later attributed to the dominant-negative effects of Mycl overexpression. Both manipulations induced significant cortical thinning (23.77% and 29.01%, respectively) 24 h post-electroporation, which occurred in a cell-extrinsic manner. Through immunohistochemical analysis of Mycl knockdown cortices, we identified Mycl as a positive regulator of indirect neurogenesis. Knockdown reduced the fraction of Tbr2-positive basal intermediate progenitors (bIPs) by 72.32% and increased that of Ctip2-positive neurons by 231.02% 24 h post-electroporation, indicating a shift from indirect to direct neurogenesis. The fractions of Pax6-positive apical radial glia (aRG) and apical intermediate progenitors (aIPs), counted as pTa1-mCherry+/pBLBP-EGFP- cells in the VZ, were unaffected. We, moreover, identified Mycl as a putative regulator of projection neuron migration. Despite inducing precocious neurogenesis at E13.5, Mycl knockdown reduced the fraction of L5 neurons by 62.16% and increased that of L4 and L2/3 neurons by 38.30% and 38.95, respectively, in the postnatal cortex, a potential indication of delayed cortical plate (CP) entry. This superficial shift was accompanied by a 77.66% reduction in the fraction of Ctip2+/Satb2+ neurons and a 102.41% increase in that of RORb-positive neurons, demonstrating that L5-fated neurons redirected to higher laminae adopted superficial-layer identity. Further evidence of a migration defect came from the accumulation of electroporated neurons in the subplate (SP) and the formation of subcortical band heterotopias in 40% of brains analysed at P7. Lastly, we found that the cytoplasmic isoform of Mycl in enriched in the E14.5 mouse cortex, while the nuclear isoforms are undetectable by immunoblotting. These findings indicate that Mycl might exert its effects in a transcription-independent manner at mid-corticogenesis, a novel finding that opens numerous avenues of research.
|Date of Award||1 Apr 2020|
|Supervisor||Setsuko Sahara (Supervisor) & Evgeniy Makeyev (Supervisor)|
Investigating the role of Mycl in apical progenitors of the developing mouse neocortex
Teixeira E Rodrigues Ramos, S. (Author). 1 Apr 2020
Student thesis: Doctoral Thesis › Doctor of Philosophy