Purification and characterization of human neural stem and progenitor cells.

The human brain undergoes rapid development at mid-gestation from a pool of neural stem and progenitor cells (NSPCs) that give rise to the neurons, oligodendrocytes, and astrocytes of the mature brain. Functional study of these cell types has been hampered by a lack of precise purification methods. We describe a method for prospectively isolating ten distinct NSPC types from the developing human brain using cell-surface markers. CD24-THY1-/lo cells were enriched for radial glia, which robustly engrafted and differentiated into all three neural lineages in the mouse brain. THY1hi cells marked unipotent oligodendrocyte precursors committed to an oligodendroglial fate, and CD24+THY1-/lo cells marked committed excitatory and inhibitory neuronal lineages. Notably, we identify and functionally characterize a transcriptomically distinct THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell (GPC), which is lineage-restricted to astrocytes and oligodendrocytes, but not to neurons. Our study provides a framework for the functional study of distinct cell types in human neurodevelopment.

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.

Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution.

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.

Evolution of Cortical Neurogenesis in Amniotes Controlled by Robo Signaling Levels.

Cerebral cortex size differs dramatically between reptiles, birds, and mammals, owing to developmental differences in neuron production. In mammals, signaling pathways regulating neurogenesis have been identified, but genetic differences behind their evolution across amniotes remain unknown. We show that direct neurogenesis from radial glia cells, with limited neuron production, dominates the avian, reptilian, and mammalian paleocortex, whereas in the evolutionarily recent mammalian neocortex, most neurogenesis is indirect via basal progenitors. Gain- and loss-of-function experiments in mouse, chick, and snake embryos and in human cerebral organoids demonstrate that high Slit/Robo and low Dll1 signaling, via Jag1 and Jag2, are necessary and sufficient to drive direct neurogenesis. Attenuating Robo signaling and enhancing Dll1 in snakes and birds recapitulates the formation of basal progenitors and promotes indirect neurogenesis. Our study identifies modulation in activity levels of conserved signaling pathways as a primary mechanism driving the expansion and increased complexity of the mammalian neocortex during amniote evolution.

Temporal Control of Mammalian Cortical Neurogenesis by m6A Methylation.

N6-methyladenosine (m6A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m6A regulates mammalian brain development is unknown. Here, we show that m6A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m6A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m6A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m6A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m6A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m6A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m6A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.

Tissue and Organ Initiation in the Plant Embryo: A First Time for Everything.

Land plants can grow to tremendous body sizes, yet even the most complex architectures are the result of iterations of the same developmental processes: organ initiation, growth, and pattern formation. A central question in plant biology is how these processes are regulated and coordinated to allow for the formation of ordered, 3D structures. All these elementary processes first occur in early embryogenesis, during which, from a fertilized egg cell, precursors for all major tissues and stem cells are initiated, followed by tissue growth and patterning. Here we discuss recent progress in our understanding of this phase of plant life. We consider the cellular basis for multicellular development in 3D and focus on the genetic regulatory mechanisms that direct specific steps during early embryogenesis.

The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex.

Neural stem and progenitor cells have a central role in the development and evolution of the mammalian neocortex. In this review, we first provide a set of criteria to classify the various types of cortical stem and progenitor cells. We then discuss the issue of cell polarity, as well as specific subcellular features of these cells that are relevant for their modes of division and daughter cell fate. In addition, cortical stem and progenitor cell behavior is placed into a tissue context, with consideration of extracellular signals and cell-cell interactions. Finally, the differences across species regarding cortical stem and progenitor cells are dissected to gain insight into key developmental and evolutionary mechanisms underlying neocortex expansion.

The transcriptional co-activator Yap1 promotes adult hippocampal neural stem cell activation.

Most adult hippocampal neural stem cells (NSCs) remain quiescent, with only a minor portion undergoing active proliferation and neurogenesis. The molecular mechanisms that trigger the transition from quiescence to activation are still poorly understood. Here, we found the activity of the transcriptional co-activator Yap1 to be enriched in active NSCs. Genetic deletion of Yap1 led to a significant reduction in the relative proportion of active NSCs, supporting a physiological role of Yap1 in regulating the transition from quiescence to activation. Overexpression of wild-type Yap1 in adult NSCs did not induce NSC activation, suggesting tight upstream control mechanisms, but overexpression of a gain-of-function mutant (Yap1-5SA) elicited cell cycle entry in NSCs and hilar astrocytes. Consistent with a role of Yap1 in NSC activation, single cell RNA sequencing revealed a partial induction of an activated NSC gene expression program. Furthermore, Yap1-5SA expression also induced expression of Taz and other key components of the Yap/Taz regulon that were previously identified in glioblastoma stem cell-like cells. Consequently, dysregulated Yap1 activity led to repression of hippocampal neurogenesis, aberrant cell differentiation, and partial acquisition of a glioblastoma stem cell-like signature.

Evolution of New miRNAs and Cerebro-Cortical Development.

The noncoding portion of the genome, including microRNAs, has been fertile evolutionary soil for cortical development in primates. A major contribution to cortical expansion in primates is the generation of novel precursor cell populations. Because miRNA expression profiles track closely with cell identity, it is likely that numerous novel microRNAs have contributed to cellular diversity in the brain. The tools to determine the genomic context within which novel microRNAs emerge and how they become integrated into molecular circuitry are now in hand.

ADAMTS2 promotes radial migration by activating TGF-ß signaling in the developing neocortex.

The mammalian neocortex is formed by sequential radial migration of newborn excitatory neurons. Migrating neurons undergo a multipolar-to-bipolar transition at the subplate (SP) layer, where extracellular matrix (ECM) components are abundantly expressed. Here, we investigate the role of the ECM at the SP layer. We show that TGF-ß signaling-related ECM proteins, and their downstream effector, p-smad2/3, are selectively expressed in the SP layer. We also find that migrating neurons express a disintegrin and metalloproteinase with thrombospondin motif 2 (ADAMTS2), an ECM metalloproteinase, just below the SP layer. Knockdown and knockout of Adamts2 suppresses the multipolar-to-bipolar transition of migrating neurons and disturbs radial migration. Time-lapse luminescence imaging of TGF-ß signaling indicates that ADAMTS2 activates this signaling pathway in migrating neurons during the multipolar-to-bipolar transition at the SP layer. Overexpression of TGF-ß2 in migrating neurons partially rescues migration defects in ADAMTS2 knockout mice. Our data suggest that ADAMTS2 secreted by the migrating multipolar neurons activates TGF-ß signaling by ECM remodeling of the SP layer, which might drive the multipolar to bipolar transition.

Setting the clock of neural progenitor cells during mammalian corticogenesis.

Radial glial cells (RGCs) as primary neural stem cells in the developing mammalian cortex give rise to diverse types of neurons and glial cells according to sophisticated developmental programs with remarkable spatiotemporal precision. Recent studies suggest that regulation of the temporal competence of RGCs is a key mechanism for the highly conserved and predictable development of the cerebral cortex. Various types of epigenetic regulations, such as DNA methylation, histone modifications, and 3D chromatin architecture, play a key role in shaping the gene expression pattern of RGCs. In addition, epitranscriptomic modifications regulate temporal pre-patterning of RGCs by affecting the turnover rate and function of cell-type-specific transcripts. In this review, we summarize epigenetic and epitranscriptomic regulatory mechanisms that control the temporal competence of RGCs during mammalian corticogenesis. Furthermore, we discuss various developmental elements that also dynamically regulate the temporal competence of RGCs, including biochemical reaction speed, local environmental changes, and subcellular organelle remodeling. Finally, we discuss the underlying mechanisms that regulate the interspecies developmental tempo contributing to human-specific features of brain development.

From neural tube to spinal cord: The dynamic journey of the dorsal neuroepithelium.

In a developing embryo, formation of tissues and organs is remarkably precise in both time and space. Through cell-cell interactions, neighboring progenitors coordinate their activities, sequentially generating distinct types of cells. At present, we only have limited knowledge, rather than a systematic understanding, of the underlying logic and mechanisms responsible for cell fate transitions. The formation of the dorsal aspect of the spinal cord is an outstanding model to tackle these dynamics, as it first generates the peripheral nervous system and is later responsible for transmitting sensory information from the periphery to the brain and for coordinating local reflexes. This is reflected first by the ontogeny of neural crest cells, progenitors of the peripheral nervous system, followed by formation of the definitive roof plate of the central nervous system and specification of adjacent interneurons, then a transformation of roof plate into dorsal radial glia and ependyma lining the forming central canal. How do these peripheral and central neural branches segregate from common progenitors? How are dorsal radial glia established concomitant with transformation of the neural tube lumen into a central canal? How do the dorsal radial glia influence neighboring cells? This is only a partial list of questions whose clarification requires the implementation of experimental paradigms in which precise control of timing is crucial. Here, we outline some available answers and still open issues, while highlighting the contributions of avian models and their potential to address mechanisms of neural patterning and function.

Her4.3+ radial glial cells maintain the brain vascular network through activation of Wnt signaling.

During vascular development, radial glial cells (RGCs) regulate vascular patterning in the trunk and contribute to the early differentiation of the blood-brain barrier. Ablation of RGCs results in excessive sprouting vessels or the absence of bilateral vertebral arteries. However, interactions of RGCs with later brain vascular networks after pattern formation remain unknown. Here, we generated a her4.3 transgenic line to label RGCs and applied the metronidazole/nitroreductase system to ablate her4.3+ RGCs. The ablation of her4.3+ RGCs led to the collapse of the cerebral vascular network, disruption of the blood-brain barrier, and downregulation of Wnt signaling. The inhibition of Wnt signaling resulted in the collapse of cerebral vasculature, similar to that caused by her4.3+ RGC ablation. The defects in the maintenance of brain vasculature resulting from the absence of her4.3+ RGCs were partially rescued by the activation of Wnt signaling or overexpression of Wnt7aa or Wnt7bb. Together, our study suggests that her4.3+ RGCs maintain the cerebral vascular network through Wnt signaling.

An incoherent feed-forward loop involving bHLH transcription factors, Auxin and CYCLIN-Ds regulates style radial symmetry establishment in Arabidopsis.

The bilateral-to-radial symmetry transition occurring during the development of the Arabidopsis thaliana female reproductive organ (gynoecium) is a crucial biological process linked to plant fertilization and seed production. Despite its significance, the cellular mechanisms governing the establishment and breaking of radial symmetry at the gynoecium apex (style) remain unknown. To fill this gap, we employed quantitative confocal imaging coupled with MorphoGraphX analysis, in vivo and in vitro transcriptional experiments, and genetic analysis encompassing mutants in two bHLH transcription factors necessary and sufficient to promote transition to radial symmetry, SPATULA (SPT) and INDEHISCENT (IND). Here, we show that defects in style morphogenesis correlate with defects in cell-division orientation and rate. We showed that the SPT-mediated accumulation of auxin in the medial-apical cells undergoing symmetry transition is required to maintain cell-division-oriented perpendicular to the direction of organ growth (anticlinal, transversal cell division). In addition, SPT and IND promote the expression of specific core cell-cycle regulators, CYCLIN-D1;1 (CYC-D1;1) and CYC-D3;3, to support progression through the G1 phase of the cell cycle. This transcriptional regulation is repressed by auxin, thus forming an incoherent feed-forward loop mechanism. We propose that this mechanism fine-tunes cell division rate and orientation with the morphogenic signal provided by auxin, during patterning of radial symmetry at the style.

The Human Developing Cerebral Cortex Is Characterized by an Elevated De Novo Expression of Long Noncoding RNAs in Excitatory Neurons.

The outstanding human cognitive capacities are computed in the cerebral cortex, a mammalian-specific brain region and the place of massive biological innovation. Long noncoding RNAs have emerged as gene regulatory elements with higher evolutionary turnover than mRNAs. The many long noncoding RNAs identified in neural tissues make them candidates for molecular sources of cerebral cortex evolution and disease. Here, we characterized the genomic and cellular shifts that occurred during the evolution of the long noncoding RNA repertoire expressed in the developing cerebral cortex and explored putative roles for these long noncoding RNAs in the evolution of the human brain. Using transcriptomics and comparative genomics, we comprehensively annotated the cortical transcriptomes of humans, rhesus macaques, mice, and chickens and classified human cortical long noncoding RNAs into evolutionary groups as a function of their predicted minimal ages. Long noncoding RNA evolutionary groups showed differences in expression levels, splicing efficiencies, transposable element contents, genomic distributions, and transcription factor binding to their promoters. Furthermore, older long noncoding RNAs showed preferential expression in germinative zones, outer radial glial cells, and cortical inhibitory (GABAergic) neurons. In comparison, younger long noncoding RNAs showed preferential expression in cortical excitatory (glutamatergic) neurons, were enriched in primate and human-specific gene co-expression modules, and were dysregulated in neurodevelopmental disorders. These results suggest different evolutionary routes for older and younger cortical long noncoding RNAs, highlighting old long noncoding RNAs as a possible source of molecular evolution of conserved developmental programs; conversely, we propose that the de novo expression of primate- and human-specific young long noncoding RNAs is a putative source of molecular evolution and dysfunction of cortical excitatory neurons, warranting further investigation.

Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The '9+2' axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked whether there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana have an extensive RSP complement, including two divergent RSP9 orthologues, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither orthologue is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 orthologue, deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked to the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Regenerative neurogenesis: the integration of developmental, physiological and immune signals.

In fishes and salamanders, but not mammals, neural stem cells switch back to neurogenesis after injury. The signalling environment of neural stem cells is strongly altered by the presence of damaged cells and an influx of immune, as well as other, cells. Here, we summarise our recently expanded knowledge of developmental, physiological and immune signals that act on neural stem cells in the zebrafish central nervous system to directly, or indirectly, influence their neurogenic state. These signals act on several intracellular pathways, which leads to changes in chromatin accessibility and gene expression, ultimately resulting in regenerative neurogenesis. Translational approaches in non-regenerating mammals indicate that central nervous system stem cells can be reprogrammed for neurogenesis. Understanding signalling mechanisms in naturally regenerating species show the path to experimentally promoting neurogenesis in mammals.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Estrogens regulate early embryonic development of the olfactory sensory system via estrogen-responsive glia.

Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Dissecting the spatiotemporal diversity of adult neural stem cells.

Adult stem cells are important for tissue turnover and regeneration. However, in most adult systems it remains elusive how stem cells assume different functional states and support spatially patterned tissue architecture. Here, we dissected the diversity of neural stem cells in the adult zebrafish brain, an organ that is characterized by pronounced zonation and high regenerative capacity. We combined single-cell transcriptomics of dissected brain regions with massively parallel lineage tracing and in vivo RNA metabolic labeling to analyze the regulation of neural stem cells in space and time. We detected a large diversity of neural stem cells, with some subtypes being restricted to a single brain region, while others were found globally across the brain. Global stem cell states are linked to neurogenic differentiation, with different states being involved in proliferative and non-proliferative differentiation. Our work reveals principles of adult stem cell organization and establishes a resource for the functional manipulation of neural stem cell subtypes.