Neural progenitor cells (NPCs, also known as radial glial progenitors) produce neurons and then glial cells such as astrocytes during development of the mouse neocortex. Given that this sequential generation of neural cells is critical for proper brain formation, the neurogenic potential of NPCs must be precisely controlled. Here, we show that the transcription factor Plag1 plays an important role in the regulation of neurogenic potential in mouse neocortical NPCs. We found that Hmga2, a key neurogenic factor in neocortical NPCs, induces expression of the Plag1 gene. Analysis of the effects of over‐expression or knockdown of Plag1 indicated that Plag1 promotes the production of neurons at the expense of astrocyte production in embryonic neocortical cultures. Furthermore, over‐expression of Plag1 promoted and knockdown of Plag1 suppressed neuronal differentiation of neocortical NPCs in vivo. Transcriptomic analysis showed that Plag1 increases the expression of a set of neuronal genes in NPCs. Our results thus identify Plag1 as a regulator of neuronal gene expression and neuronal differentiation in NPCs of the developing mouse neocortex.

22During the early stage of mammalian neural development, neuroepithelial cells (NECs) 23 proliferate and increase their pool size before switching their fate to become neurogenic 24 radial glial progenitors (RGPs). The timing of this expansion-to-neurogenic phase 25 transition is strictly regulated so as to determine the proper number of progenitors and 26 differentiated cell types that constitute the brain. The molecular mechanism underlying 27 this switch has remained poorly understood, however, in part because of the difficulty 28 associated with manipulation of gene expression in NECs before their transition to 29 RGPs, which occurs before neural tube closure. We have now developed a simple and 30 efficient method to manipulate gene expression in mouse neocortical NECs in the 31 expansion phase by in utero injection of viral vectors at embryonic day 7.0 to 8.0. With 32 the use of this method, we found that knockdown of the chromatin-associated protein 33Hmga2 in NECs inhibited the onset of the neurogenic phase in the neocortex. This 34 effect of Hmga2 knockdown was accompanied by inhibition of the expression of a set 35 of genes targeted by Polycomb group proteins, which repress neurogenic genes in 36 cortical progenitors. Our study thus establishes a novel method for manipulation of gene 37 expression in the early stage of mouse brain development as well as uncovers a key 38 molecular player in the generation of neurogenic progenitors in the developing mouse 39 neocortex. 40 41 E8.0. This method is based on in utero injection of viral vectors into amniotic fluid and 130 is associated with a high survival rate without inducing gross developmental 131 abnormalities. Moreover, as a proof of concept, we have identified a key regulator of 132 the E-to-N transition with the use of this new method. We found that Hmga2 expression 133 in neocortical NPCs peaks at around the E-to-N transition (at E10), and that knockdown 134 of Hmga2 from E8.0 delayed the onset of neurogenesis and shifted the transcriptional 135 profile of NPCs at this transition closer to that in the expansion phase. Importantly, 136 knockdown of Hmga2 from E8.0 attenuated the expression of a subset of PcG target 137 genes in NPCs at E11.0. Our results suggest that an increase in Hmga2 expression 138 triggers the onset of the neurogenic phase in part through activation of neurogenesis-139