DOT1L activity affects neural stem cell division mode and reduces differentiation and ASNS expression.

Cortical neurogenesis depends on the balance between self-renewal and differentiation of apical progenitors (APs). Here, we study the epigenetic control of AP's division mode by focusing on the enzymatic activity of the histone methyltransferase DOT1L. Combining lineage tracing with single-cell RNA sequencing of clonally related cells, we show at the cellular level that DOT1L inhibition increases neurogenesis driven by a shift of APs from asymmetric self-renewing to symmetric neurogenic consumptive divisions. At the molecular level, DOT1L activity prevents AP differentiation by promoting transcription of metabolic genes. Mechanistically, DOT1L inhibition reduces activity of an EZH2/PRC2 pathway, converging on increased expression of asparagine synthetase (ASNS), a microcephaly associated gene. Overexpression of ASNS in APs phenocopies DOT1L inhibition, and also increases neuronal differentiation of APs. Our data suggest that DOT1L activity/PRC2 crosstalk controls AP lineage progression by regulating asparagine metabolism.

Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals.

Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Forebrain Organoids to Model the Cell Biology of Basal Radial Glia in Neurodevelopmental Disorders and Brain Evolution.

The acquisition of higher intellectual abilities that distinguish humans from their closest relatives correlates greatly with the expansion of the cerebral cortex. This expansion is a consequence of an increase in neuronal cell production driven by the higher proliferative capacity of neural progenitor cells, in particular basal radial glia (bRG). Furthermore, when the proliferation of neural progenitor cells is impaired and the final neuronal output is altered, severe neurodevelopmental disorders can arise. To effectively study the cell biology of human bRG, genetically accessible human experimental models are needed. With the pioneering success to isolate and culture pluripotent stem cells in vitro, we can now routinely investigate the developing human cerebral cortex in a dish using three-dimensional multicellular structures called organoids. Here, we will review the molecular and cell biological features of bRG that have recently been elucidated using brain organoids. We will further focus on the application of this simple model system to study in a mechanistically actionable way the molecular and cellular events in bRG that can lead to the onset of various neurodevelopmental diseases.

Inheritance and flexibility of cell polarity: a clue for understanding human brain development and evolution.

Cell polarity is fundamentally important for understanding brain development. Here, we hypothesize that the inheritance and flexibility of cell polarity during neocortex development could be implicated in neocortical evolutionary expansion. Molecular and morphological features of cell polarity may be inherited from one type of progenitor cell to the other and finally transmitted to neurons. Furthermore, key cell types, such as basal progenitors and neurons, exhibit a highly flexible polarity. We suggest that both inheritance and flexibility of cell polarity are implicated in the amplification of basal progenitors and tangential dispersion of neurons, which are key features of the evolutionary expansion of the neocortex.

The Ferret as a Model System for Neocortex Development and Evolution.

The neocortex is the largest part of the cerebral cortex and a key structure involved in human behavior and cognition. Comparison of neocortex development across mammals reveals that the proliferative capacity of neural stem and progenitor cells and the length of the neurogenic period are essential for regulating neocortex size and complexity, which in turn are thought to be instrumental for the increased cognitive abilities in humans. The domesticated ferret, Mustela putorius furo, is an important animal model in neurodevelopment for its complex postnatal cortical folding, its long period of forebrain development and its accessibility to genetic manipulation in vivo. Here, we discuss the molecular, cellular, and histological features that make this small gyrencephalic carnivore a suitable animal model to study the physiological and pathological mechanisms for the development of an expanded neocortex. We particularly focus on the mechanisms of neural stem cell proliferation, neuronal differentiation, cortical folding, visual system development, and neurodevelopmental pathologies. We further discuss the technological advances that have enabled the genetic manipulation of the ferret in vivo. Finally, we compare the features of neocortex development in the ferret with those of other model organisms.

Roots of the Malformations of Cortical Development in the Cell Biology of Neural Progenitor Cells.

The cerebral cortex is a structure that underlies various brain functions, including cognition and language. Mammalian cerebral cortex starts developing during the embryonic period with the neural progenitor cells generating neurons. Newborn neurons migrate along progenitors' radial processes from the site of their origin in the germinal zones to the cortical plate, where they mature and integrate in the forming circuitry. Cell biological features of neural progenitors, such as the location and timing of their mitoses, together with their characteristic morphologies, can directly or indirectly regulate the abundance and the identity of their neuronal progeny. Alterations in the complex and delicate process of cerebral cortex development can lead to malformations of cortical development (MCDs). They include various structural abnormalities that affect the size, thickness and/or folding pattern of the developing cortex. Their clinical manifestations can entail a neurodevelopmental disorder, such as epilepsy, developmental delay, intellectual disability, or autism spectrum disorder. The recent advancements of molecular and neuroimaging techniques, along with the development of appropriate in vitro and in vivo model systems, have enabled the assessment of the genetic and environmental causes of MCDs. Here we broadly review the cell biological characteristics of neural progenitor cells and focus on those features whose perturbations have been linked to MCDs.

Serotonin Receptor 2A Activation Promotes Evolutionarily Relevant Basal Progenitor Proliferation in the Developing Neocortex.

Evolutionary expansion of the mammalian neocortex (Ncx) has been linked to increased abundance and proliferative capacity of basal progenitors (BPs) in the subventricular zone during development. BP proliferation is governed by both intrinsic and extrinsic signals, several of which have been identified. However, a role of neurotransmitters, a canonical class of extrinsic signaling molecules, in BP proliferation remains to be established. Here, we show that serotonin (5-HT), via its receptor HTR2A, promotes BP proliferation in an evolutionarily relevant manner. HTR2A is not expressed in embryonic mouse Ncx; accordingly, 5-HT does not increase mouse BP proliferation. However, ectopic HTR2A expression can increase mouse BP proliferation. Conversely, CRISPR/Cas9-mediated knockout of endogenous HTR2A in embryonic ferret Ncx reduces BP proliferation. Pharmacological activation of endogenous HTR2A in fetal human Ncx ex vivo increases BP proliferation via HER2/ERK signaling. Hence, 5-HT emerges as an important extrinsic pro-proliferative signal for BPs, which may have contributed to evolutionary Ncx expansion.

Basal Progenitor Morphology and Neocortex Evolution.

The evolutionary expansion of the mammalian neocortex is widely considered to be a basis of increased cognitive abilities. This expansion is a consequence of the enhanced production of neurons during the fetal/embryonic development of the neocortex, which in turn reflects an increased proliferative capacity of neural progenitor cells; in particular basal progenitors (BPs). The remarkable heterogeneity of BP subtypes across mammals, notably their various morphotypes and molecular fingerprints, which has recently been revealed, corroborates the importance of BPs for neocortical expansion. Here, we argue that the morphology of BPs is a key cell biological basis for maintaining their high proliferative capacity and therefore plays crucial roles in the evolutionary expansion of the neocortex.

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation.

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Extracellular matrix-inducing Sox9 promotes both basal progenitor proliferation and gliogenesis in developing neocortex.

Neocortex expansion is largely based on the proliferative capacity of basal progenitors (BPs), which is increased by extracellular matrix (ECM) components via integrin signaling. Here we show that the transcription factor Sox9 drives expression of ECM components and that laminin 211 increases BP proliferation in embryonic mouse neocortex. We show that Sox9 is expressed in human and ferret BPs and is required for BP proliferation in embryonic ferret neocortex. Conditional Sox9 expression in the mouse BP lineage, where it normally is not expressed, increases BP proliferation, reduces Tbr2 levels and induces Olig2 expression, indicative of premature gliogenesis. Conditional Sox9 expression also results in cell-non-autonomous stimulation of BP proliferation followed by increased upper-layer neuron production. Our findings demonstrate that Sox9 exerts concerted effects on transcription, BP proliferation, neuron production, and neurogenic vs. gliogenic BP cell fate, suggesting that Sox9 may have contributed to promote neocortical expansion.

Human-Specific ARHGAP11B Acts in Mitochondria to Expand Neocortical Progenitors by Glutaminolysis.

The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.

YAP Activity Is Necessary and Sufficient for Basal Progenitor Abundance and Proliferation in the Developing Neocortex.

Neocortex expansion during mammalian evolution has been linked to an increase in proliferation of basal progenitors in the subventricular zone. Here, we explored a potential role of YAP, the major downstream effector of the Hippo pathway, in proliferation of basal progenitors. YAP expression and activity are high in ferret and human basal progenitors, which exhibit high proliferative capacity, but low in mouse basal progenitors, which lack such capacity. Conditional expression of a constitutively active YAP in mouse basal progenitors resulted in increased proliferation of basal progenitor and promoted production of upper-layer neurons. Pharmacological and genetic interference with YAP function in ferret and human developing neocortex resulted in decreased abundance of cycling basal progenitors. Together, our data indicate that YAP is necessary and sufficient to promote the proliferation of basal progenitors and suggest that increases in YAP levels and presumably activity contributed to the evolutionary expansion of the neocortex.

Neocortical Expansion Due to Increased Proliferation of Basal Progenitors Is Linked to Changes in Their Morphology.

The evolutionary expansion of the mammalian neocortex (Ncx) is thought to be linked to increased proliferative capacity of basal progenitors (BPs) and their neurogenic capacity. Here, by quantifying BP morphology in the developing Ncx of mouse, ferret, and human, we show that increased BP proliferative capacity is linked to an increase in BP process number. We identify human membrane-bound PALMDELPHIN (PALMD-Caax) as an underlying factor, and we show that it drives BP process growth and proliferation when expressed in developing mouse and ferret Ncx. Conversely, CRISPR/Cas9-mediated disruption of PALMD or its binding partner ADDUCIN-γ in fetal human Ncx reduces BP process numbers and proliferation. We further show that PALMD-induced processes enable BPs to receive pro-proliferative integrin-dependent signals. These findings provide a link between BP morphology and proliferation, suggesting that changes in BP morphology may have contributed to the evolutionary expansion of the Ncx.

Human-specific ARHGAP11B induces hallmarks of neocortical expansion in developing ferret neocortex.

The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex.

Extracellular Matrix Components HAPLN1, Lumican, and Collagen I Cause Hyaluronic Acid-Dependent Folding of the Developing Human Neocortex.

Neocortical expansion, thought to underlie the cognitive traits unique to humans, is accompanied by cortical folding. This folding starts around gestational week (GW) 20, but what causes it remains largely unknown. Extracellular matrix (ECM) has been previously implicated in neocortical expansion and here we investigate the potential role of ECM in the formation of neocortical folds. We focus on three specific ECM components localized in the human fetal cortical plate (CP): hyaluronan and proteoglycan link protein 1 (HAPLN1), lumican and collagen I (collectively, HLC). Addition of HLC to cultures of human fetal neocortex (11-22 GW) caused local changes in tissue stiffness, induced CP folding, increased CP hyaluronic acid (HA), and required the HA-receptor CD168 and downstream ERK signaling. Importantly, loss of HA reduced HLC-induced and 22 GW physiological nascent folds. This was altered in samples with neurodevelopmental disorders, indicating it may be a useful system to study such disorders.

Insm1 Induces Neural Progenitor Delamination in Developing Neocortex via Downregulation of the Adherens Junction Belt-Specific Protein Plekha7.

Delamination of neural progenitor cells (NPCs) from the ventricular surface is a crucial prerequisite to form the subventricular zone, the germinal layer linked to the expansion of the mammalian neocortex in development and evolution. Here, we dissect the molecular mechanism by which the transcription factor Insm1 promotes the generation of basal progenitors (BPs). Insm1 protein is most highly expressed in newborn BPs in mouse and human developing neocortex. Forced Insm1 expression in embryonic mouse neocortex causes NPC delamination, converting apical to basal radial glia. Insm1 represses the expression of the apical adherens junction belt-specific protein Plekha7. CRISPR/Cas9-mediated disruption of Plekha7 expression suffices to cause NPC delamination. Plekha7 overexpression impedes the intrinsic and counteracts the Insm1-induced, NPC delamination. Our findings uncover a novel molecular mechanism underlying NPC delamination in which a BP-genic transcription factor specifically targets the integrity of the apical adherens junction belt, rather than adherens junction components as such.

Epigenome profiling and editing of neocortical progenitor cells during development.

The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here, we present genomewide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator Eomes We use catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the Eomes locus in vivo, which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation data and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development.

Acetylated tubulin is essential for touch sensation in mice.

At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch.

CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

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.

αTAT1 is the major α-tubulin acetyltransferase in mice.

Post-translational modification of tubulin serves as a powerful means for rapidly adjusting the functional diversity of microtubules. Acetylation of the ε-amino group of K40 in α-tubulin is one such modification that is highly conserved in ciliated organisms. Recently, αTAT1, a Gcn5-related N-acetyltransferase, was identified as an α-tubulin acetyltransferase in Tetrahymena and C. elegans. Here we generate mice with a targeted deletion of Atat1 to determine its function in mammals. Remarkably, we observe a loss of detectable K40 α-tubulin acetylation in these mice across multiple tissues and in cellular structures such as cilia and axons where acetylation is normally enriched. Mice are viable and develop normally, however, the absence of Atat1 impacts upon sperm motility and male mouse fertility, and increases microtubule stability. Thus, αTAT1 has a conserved function as the major α-tubulin acetyltransferase in ciliated organisms and has an important role in regulating subcellular specialization of subsets of microtubules.