A kinase-independent function of cyclin-dependent kinase 6 promotes outer radial glia expansion and neocortical folding

The neocortex, the center for higher brain function, first emerged in mammals and has become massively expanded and folded in humans, constituting almost half the volume of the human brain. Primary microcephaly, a developmental disorder in which the brain is smaller than normal at birth, mainly results from the number of neurons in the neocortex being reduced because of defects in neural progenitor cells (NPCs). Outer radial glia (oRGs), NPCs that are abundant in gyrencephalic species but rare in lisencephalic species, are thought to play key roles in the expansion and folding of the neocortex. However, how oRGs expand, whether they are necessary for neocortical folding, and whether defects in oRGs cause microcephaly remain important questions in the study of brain development, evolution, and disease. Here, we show that oRG expansion in mice, ferrets, and human cerebral organoids requires cyclin-dependent kinase 6 (CDK6), the mutation of which causes primary microcephaly via an unknown mechanism. In a mouse model in which increased Hedgehog signaling expands oRGs and intermediate progenitor cells and induces neocortical folding, CDK6 loss selectively decreased oRGs and abolished neocortical folding. Remarkably, this function of CDK6 in oRG expansion did not require its kinase activity, was not shared by the highly similar CDK4 and CDK2, and was disrupted by the mutation causing microcephaly. Therefore, our results indicate that CDK6 is conserved to promote oRG expansion; that oRGs are necessary for neocortical folding; and that defects in oRG expansion may cause primary microcephaly. Significance Statement Primary microcephaly, a disorder in which the brain is smaller than normal at birth, disproportionately affects the neocortex. Although outer radial glia (oRGs) expansion is hypothesized to be important in neocortical expansion and folding, it remains unknown whether oRGs are necessary for neocortical folding and whether defective oRGs cause microcephaly. Moreover, how oRGs expand is not well understood. A mutation in CDK6 causes microcephaly via an unknown mechanism. Here, we show that CDK6 promotes oRG expansion and neocortical folding. This function of CDK6 does not require its kinase activity but is disrupted by a mutation that causes microcephaly. Our findings show that CDK6 is conserved to expand oRGs and provide evidence that oRG defects disrupt neocortical growth and folding.