Evolutionary expansion of the human neocortex reflects increased amplification of basal progenitors in the subventricular zone, producing more neurons during fetal corticogenesis. In this work, we analyze the transcriptomes of distinct progenitor subpopulations isolated by a cell polarity-based approach from developing mouse and human neocortex. We identify 56 genes preferentially expressed in human apical and basal radial glia that lack mouse orthologs. Among these, ARHGAP11B has the highest degree of radial glia-specific expression. ARHGAP11B arose from partial duplication of ARHGAP11A (which encodes a Rho guanosine triphosphatase-activating protein) on the human lineage after separation from the chimpanzee lineage. Expression of ARHGAP11B in embryonic mouse neocortex promotes basal progenitor generation and self-renewal and can increase cortical plate area and induce gyrification. Hence, ARHGAP11B may have contributed to evolutionary expansion of human neocortex.

Complex neuronal circuitries such as those present in the mammalian cerebral cortex have evolved as balanced networks of excitatory and inhibitory neurons. Although the establishment of appropriate numbers for these cells is essential for brain function and behaviour, our understanding of this fundamental process is very fragmentary. Here we show that interneuron cell survival in mice depends on the activity of pyramidal cells during a critical window of postnatal development, in which excitatory synaptic input to individual interneurons predicts their outcome. Pyramidal cells regulate interneuron survival through the negative modulation of PTEN signalling, which effectively drives interneuron cell death during this period. Taken together, our findings indicate that activity-dependent mechanisms dynamically adjust the number of inhibitory cells in nascent local cortical circuits, ultimately establishing the appropriate proportions of excitatory and inhibitory neurons in the cerebral cortex.

The cerebral cortex contains multiple areas with distinctive cytoarchitectonic patterns, but the cellular mechanisms underlying the emergence of this diversity remain unclear. Here, we have investigated the neuronal output of individual progenitor cells in the developing mouse neocortex using a combination of methods that together circumvent the biases and limitations of individual approaches. Our experimental results indicate that progenitor cells generate pyramidal cell lineages with a wide range of sizes and laminar configurations. Mathematical modeling indicates that these outcomes are compatible with a stochastic model of cortical neurogenesis in which progenitor cells undergo a series of probabilistic decisions that lead to the specification of very heterogeneous progenies. Our findings support a mechanism for cortical neurogenesis whose flexibility would make it capable to generate the diverse cytoarchitectures that characterize distinct neocortical areas.

In spite of the high metabolic cost of cellular production, the brain contains only a fraction of the neurons generated during embryonic development. In the rodent cerebral cortex, a first wave of programmed cell death surges at embryonic stages and affects primarily progenitor cells. A second, larger wave unfolds during early postnatal development and ultimately determines the final number of cortical neurons. Programmed cell death in the developing cortex is particularly dependent on neuronal activity and unfolds in a cell-specific manner with precise temporal control. Pyramidal cells and interneurons adjust their numbers in sync, which is likely crucial for the establishment of balanced networks of excitatory and inhibitory neurons. In contrast, several other neuronal populations are almost completely eliminated through apoptosis during the first two weeks of postnatal development, highlighting the importance of programmed cell death in sculpting the mature cerebral cortex.

The evolutionary expansion of the neocortex in mammals has been linked to enlargement of the subventricular zone (SVZ) and increased proliferative capacity of basal progenitors (BPs), notably basal radial glia (bRG). The transcription factor Pax6 is known to be highly expressed in primate, but not mouse, BPs. Here, we demonstrate that sustaining Pax6 expression selectively in BP-genic apical radial glia (aRG) and their BP progeny of embryonic mouse neocortex suffices to induce primate-like progenitor behaviour. Specifically, we conditionally expressed Pax6 by in utero electroporation using a novel, Tis21–CreERT2 mouse line. This expression altered aRG cleavage plane orientation to promote bRG generation, increased cell-cycle re-entry of BPs, and ultimately increased upper-layer neuron production. Upper-layer neuron production was also increased in double-transgenic mouse embryos with sustained Pax6 expression in the neurogenic lineage. Strikingly, increased BPs existed not only in the SVZ but also in the intermediate zone of the neocortex of these double-transgenic mouse embryos. In mutant mouse embryos lacking functional Pax6, the proportion of bRG among BPs was reduced. Our data identify specific Pax6 effects in BPs and imply that sustaining this Pax6 function in BPs could be a key aspect of SVZ enlargement and, consequently, the evolutionary expansion of the neocortex.

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (%90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/ Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/ Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

Neocortex expansion during evolution is associated with the enlargement of the embryonic subventricular zone, which reflects an increased self-renewal and proliferation of basal progenitors. In contrast to human, the vast majority of mouse basal progenitors lack self-renewal capacity, possibly due to lack of a basal process contacting the basal lamina and downregulation of cell-autonomous production of extracellular matrix (ECM) constituents. Here we show that targeted activation of the ECM receptor integrin α v β 3 on basal progenitors in embryonic mouse neocortex promotes their expansion. Specifically, integrin α v β 3 activation causes an increased cell cycle reentry of Pax6-negative, Tbr2-positive intermediate progenitors, rather than basal radial glia, and a decrease in the proportion of intermediate progenitors committed to neurogenic division. Interestingly, integrin α v β 3 is the only known cell surface receptor for thyroid hormones. Remarkably, tetrac, a thyroid hormone analog that inhibits the binding of thyroid hormones to integrin α v β 3 , completely abolishes the intermediate progenitor expansion observed upon targeted integrin α v β 3 activation, indicating that this expansion requires the binding of thyroid hormones to integrin α v β 3 . Convergence of ECM and thyroid hormones on integrin α v β 3 thus appears to be crucial for cortical progenitor proliferation and self-renewal, and hence for normal brain development and the evolutionary expansion of the neocortex.

These results demonstrate that the small-molecule ghrelin receptor agonists profiled here readily cross the blood/brain barrier and elicit pro-cognitive effects in recognition and spatial learning and memory tests. Based on these observations, the central ghrelin receptor would appear to be a chemically tractable receptor and perhaps should be considered as a new drug target for therapeutic approaches to treat diseases affecting cognition.