RESUMO
During corticogenesis, ventricular zone progenitors sequentially generate distinct subtypes of neurons, accounting for the diversity of neocortical cells and the circuits they form. While activity-dependent processes are critical for the differentiation and circuit assembly of postmitotic neurons, how bioelectrical processes affect nonexcitable cells, such as progenitors, remains largely unknown. Here, we reveal that, in the developing mouse neocortex, ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons. Experimental in vivo hyperpolarization shifted the transcriptional programs and division modes of these progenitors to a later developmental status, with precocious generation of intermediate progenitors and a forward shift in the laminar, molecular, morphological, and circuit features of their neuronal progeny. These effects occurred through inhibition of the Wnt-beta-catenin signaling pathway by hyperpolarization. Thus, during corticogenesis, bioelectric membrane properties are permissive for specific molecular pathways to coordinate the temporal progression of progenitor developmental programs and thus neocortical neuron diversity.
Assuntos
Potenciais da Membrana , Neocórtex/embriologia , Neurônios/metabolismo , Células-Tronco/citologia , Animais , Encéfalo/citologia , Encéfalo/embriologia , Diferenciação Celular , Progressão da Doença , Eletroporação , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Fatores de Tempo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
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.
Assuntos
Neocórtex/crescimento & desenvolvimento , Neurogênese/fisiologia , Animais , Divisão Celular Assimétrica , Compartimento Celular , Linhagem da Célula , Membrana Celular/fisiologia , Núcleo Celular/fisiologia , Polaridade Celular , Líquido Cefalorraquidiano/fisiologia , Humanos , Junções Intercelulares/fisiologia , Ventrículos Laterais/embriologia , Lipídeos de Membrana/metabolismo , Microglia/fisiologia , Mitose , Neocórtex/citologia , Neocórtex/embriologia , Células-Tronco Neurais/classificação , Células-Tronco Neurais/fisiologia , Células Neuroepiteliais/citologia , Células Neuroepiteliais/fisiologia , Neurônios/fisiologia , Organelas/fisiologia , Especificidade da EspécieRESUMO
Foxg1 masters telencephalic development via a pleiotropic control over its progression. Expressed within the central nervous system (CNS), L1 retrotransposons are implicated in progression of its histogenesis and tuning of its genomic plasticity. Foxg1 represses gene transcription, and L1 elements share putative Foxg1-binding motifs, suggesting the former might limit telencephalic expression (and activity) of the latter. We tested such a prediction, in vivo as well as in engineered primary neural cultures, using loss- and gain-of-function approaches. We found that Foxg1-dependent, transcriptional L1 repression specifically occurs in neopallial neuronogenic progenitors and post-mitotic neurons, where it is supported by specific changes in the L1 epigenetic landscape. Unexpectedly, we discovered that Foxg1 physically interacts with L1-mRNA and positively regulates neonatal neopallium L1-DNA content, antagonizing the retrotranscription-suppressing activity exerted by Mov10 and Ddx39a helicases. To the best of our knowledge, Foxg1 represents the first CNS patterning gene acting as a bimodal retrotransposon modulator, limiting transcription of L1 elements and promoting their amplification, within a specific domain of the developing mouse brain.
Assuntos
Fatores de Transcrição Forkhead , Regulação da Expressão Gênica no Desenvolvimento , Neocórtex , Proteínas do Tecido Nervoso , RNA Mensageiro , Animais , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Neocórtex/metabolismo , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Retroelementos/genética , DNA/metabolismo , DNA/genética , Neurônios/metabolismoRESUMO
The mammalian cerebral cortex has an unparalleled diversity of cell types, which are generated during development through a series of temporally orchestrated events that are under tight evolutionary constraint and are critical for proper cortical assembly and function1,2. However, the molecular logic that governs the establishment and organization of cortical cell types remains unknown, largely due to the large number of cell classes that undergo dynamic cell-state transitions over extended developmental timelines. Here we generate a comprehensive atlas of the developing mouse neocortex, using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing. We sampled the neocortex every day throughout embryonic corticogenesis and at early postnatal ages, and complemented the sequencing data with a spatial transcriptomics time course. We computationally reconstruct developmental trajectories across the diversity of cortical cell classes, and infer their spatial organization and the gene regulatory programs that accompany their lineage bifurcation decisions and differentiation trajectories. Finally, we demonstrate how this developmental map pinpoints the origin of lineage-specific developmental abnormalities that are linked to aberrant corticogenesis in mutant mice. The data provide a global picture of the regulatory mechanisms that govern cellular diversification in the neocortex.
Assuntos
Neocórtex/citologia , Neurogênese , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/embriologia , Proteínas do Tecido Nervoso/genética , Análise de Sequência de RNA , Análise de Célula Única , TranscriptomaRESUMO
The human brain is subdivided into distinct anatomical structures, including the neocortex, which in turn encompasses dozens of distinct specialized cortical areas. Early morphogenetic gradients are known to establish early brain regions and cortical areas, but how early patterns result in finer and more discrete spatial differences remains poorly understood1. Here we use single-cell RNA sequencing to profile ten major brain structures and six neocortical areas during peak neurogenesis and early gliogenesis. Within the neocortex, we find that early in the second trimester, a large number of genes are differentially expressed across distinct cortical areas in all cell types, including radial glia, the neural progenitors of the cortex. However, the abundance of areal transcriptomic signatures increases as radial glia differentiate into intermediate progenitor cells and ultimately give rise to excitatory neurons. Using an automated, multiplexed single-molecule fluorescent in situ hybridization approach, we find that laminar gene-expression patterns are highly dynamic across cortical regions. Together, our data suggest that early cortical areal patterning is defined by strong, mutually exclusive frontal and occipital gene-expression signatures, with resulting gradients giving rise to the specification of areas between these two poles throughout successive developmental timepoints.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/citologia , Neocórtex/embriologia , Atlas como Assunto , Sequência de Bases , Biomarcadores/metabolismo , Humanos , Neocórtex/metabolismo , Neurogênese , Neuroglia/classificação , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/classificação , Neurônios/citologia , Neurônios/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única , Fatores de TempoRESUMO
Dendritic and axonal morphology reflects the input and output of neurons and is a defining feature of neuronal types1,2, yet our knowledge of its diversity remains limited. Here, to systematically examine complete single-neuron morphologies on a brain-wide scale, we established a pipeline encompassing sparse labelling, whole-brain imaging, reconstruction, registration and analysis. We fully reconstructed 1,741 neurons from cortex, claustrum, thalamus, striatum and other brain regions in mice. We identified 11 major projection neuron types with distinct morphological features and corresponding transcriptomic identities. Extensive projectional diversity was found within each of these major types, on the basis of which some types were clustered into more refined subtypes. This diversity follows a set of generalizable principles that govern long-range axonal projections at different levels, including molecular correspondence, divergent or convergent projection, axon termination pattern, regional specificity, topography, and individual cell variability. Although clear concordance with transcriptomic profiles is evident at the level of major projection type, fine-grained morphological diversity often does not readily correlate with transcriptomic subtypes derived from unsupervised clustering, highlighting the need for single-cell cross-modality studies. Overall, our study demonstrates the crucial need for quantitative description of complete single-cell anatomy in cell-type classification, as single-cell morphological diversity reveals a plethora of ways in which different cell types and their individual members may contribute to the configuration and function of their respective circuits.
Assuntos
Encéfalo/citologia , Forma Celular , Neurônios/classificação , Neurônios/metabolismo , Análise de Célula Única , Atlas como Assunto , Biomarcadores/metabolismo , Encéfalo/anatomia & histologia , Encéfalo/embriologia , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Neocórtex/anatomia & histologia , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/metabolismo , Neurogênese , Neuroglia/citologia , Neurônios/citologia , RNA-Seq , Reprodutibilidade dos TestesRESUMO
The size and surface area of the mammalian brain are thought to be critical determinants of intellectual ability. Recent studies show that development of the gyrated human neocortex involves a lineage of neural stem and transit-amplifying cells that forms the outer subventricular zone (OSVZ), a proliferative region outside the ventricular epithelium. We discuss how proliferation of cells within the OSVZ expands the neocortex by increasing neuron number and modifying the trajectory of migrating neurons. Relating these features to other mammalian species and known molecular regulators of the mouse neocortex suggests how this developmental process could have emerged in evolution.
Assuntos
Evolução Biológica , Neocórtex/fisiologia , Animais , Movimento Celular , Humanos , Neocórtex/citologia , Neocórtex/embriologia , Células-Tronco Neurais/citologia , Neurogênese , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
The role of WNT/ß-catenin signalling in mouse neocortex development remains ambiguous. Most studies demonstrate that WNT/ß-catenin regulates progenitor self-renewal but others suggest it can also promote differentiation. Here we explore the role of WNT/STOP signalling, which stabilizes proteins during G2/M by inhibiting glycogen synthase kinase (GSK3)-mediated protein degradation. We show that mice mutant for cyclin Y and cyclin Y-like 1 (Ccny/l1), key regulators of WNT/STOP signalling, display reduced neurogenesis in the developing neocortex. Specifically, basal progenitors, which exhibit delayed cell cycle progression, were drastically decreased. Ccny/l1-deficient apical progenitors show reduced asymmetric division due to an increase in apical-basal astral microtubules. We identify the neurogenic transcription factors Sox4 and Sox11 as direct GSK3 targets that are stabilized by WNT/STOP signalling in basal progenitors during mitosis and that promote neuron generation. Our work reveals that WNT/STOP signalling drives cortical neurogenesis and identifies mitosis as a critical phase for neural progenitor fate.
Assuntos
Mitose , Neocórtex/embriologia , Neocórtex/metabolismo , Neurogênese , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Biomarcadores , Ciclo Celular , Diferenciação Celular/genética , Ciclinas/genética , Ciclinas/metabolismo , Embrião de Mamíferos , Imunofluorescência , Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mitose/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Fosforilação , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismoRESUMO
In the mammalian brain, the specification of a single axon and multiple dendrites occurs early in the differentiation of most neuron types. Numerous intracellular signaling events for axon specification have been described in detail. However, the identity of the extracellular factor(s) that initiate neuronal polarity in vivo is unknown. Here, we report that transforming growth factor beta (TGF-beta) initiates signaling pathways both in vivo and in vitro to fate naive neurites into axons. Neocortical neurons lacking the type II TGF-beta receptor (TbetaR2) fail to initiate axons during development. Exogenous TGF-beta is sufficient to direct the rapid growth and differentiation of an axon, and genetic enhancement of receptor activity promotes the formation of multiple axons. Finally, we show that the bulk of these TGF-beta-dependent events are mediated by site-specific phosphorylation of Par6. These results define an extrinsic cue for neuronal polarity in vivo that patterns neural circuits in the developing brain.
Assuntos
Axônios/metabolismo , Neocórtex/citologia , Neocórtex/embriologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Embrião de Mamíferos/metabolismo , Camundongos , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
The diverse subtypes of excitatory neurons that populate the neocortex are born from apical progenitors located in the ventricular zone. During corticogenesis, apical progenitors sequentially generate deep-layer neurons followed by superficial-layer neurons directly or via the generation of intermediate progenitors. Whether neurogenic fate progression necessarily implies fate restriction in single progenitor types is unknown. Here we specifically isolated apical progenitors and intermediate progenitors, and fate-mapped their respective neuronal progeny following heterochronic transplantation into younger embryos. We find that apical progenitors are temporally plastic and can re-enter past molecular, electrophysiological and neurogenic states when exposed to an earlier-stage environment by sensing dynamic changes in extracellular Wnt. By contrast, intermediate progenitors are committed progenitors that lack such retrograde fate plasticity. These findings identify a diversity in the temporal plasticity of neocortical progenitors, revealing that some subtypes of cells can be untethered from their normal temporal progression to re-enter past developmental states.
Assuntos
Plasticidade Celular/fisiologia , Neocórtex/embriologia , Neurogênese/fisiologia , Células-Tronco/citologia , Animais , Células Cultivadas , Embrião de Mamíferos , Camundongos , Neocórtex/citologia , Neurônios/citologia , Fatores de TempoRESUMO
The relationships between impaired cortical development and consequent malformations in neurodevelopmental disorders, as well as the genes implicated in these processes, are not fully elucidated to date. In this study, we report six novel cases of patients affected by BBSOAS (Boonstra-Bosch-Schaff optic atrophy syndrome), a newly emerging rare neurodevelopmental disorder, caused by loss-of-function mutations of the transcriptional regulator NR2F1. Young patients with NR2F1 haploinsufficiency display mild to moderate intellectual disability and show reproducible polymicrogyria-like brain malformations in the parietal and occipital cortex. Using a recently established BBSOAS mouse model, we found that Nr2f1 regionally controls long-term self-renewal of neural progenitor cells via modulation of cell cycle genes and key cortical development master genes, such as Pax6. In the human fetal cortex, distinct NR2F1 expression levels encompass gyri and sulci and correlate with local degrees of neurogenic activity. In addition, reduced NR2F1 levels in cerebral organoids affect neurogenesis and PAX6 expression. We propose NR2F1 as an area-specific regulator of mouse and human brain morphology and a novel causative gene of abnormal gyrification.
Assuntos
Fator I de Transcrição COUP/metabolismo , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Lobo Occipital/embriologia , Atrofias Ópticas Hereditárias/embriologia , Lobo Parietal/embriologia , Animais , Fator I de Transcrição COUP/genética , Modelos Animais de Doenças , Humanos , Camundongos , Neocórtex/patologia , Células-Tronco Neurais/patologia , Lobo Occipital/patologia , Atrofias Ópticas Hereditárias/genética , Atrofias Ópticas Hereditárias/patologia , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Lobo Parietal/patologiaRESUMO
Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5-10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells-generated via a CRISPR-Cas9 approach-produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.
Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Organogênese , Prosencéfalo/citologia , Prosencéfalo/embriologia , Animais , Quimera/embriologia , Quimera/genética , Proteínas de Ligação a DNA/deficiência , Proteínas do Domínio Duplacortina , Feminino , Teste de Complementação Genética , Células Germinativas/metabolismo , Hipocampo/anatomia & histologia , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/deficiência , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Neocórtex/anatomia & histologia , Neocórtex/citologia , Neocórtex/embriologia , Neocórtex/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/deficiência , Fenótipo , Prosencéfalo/anatomia & histologia , Prosencéfalo/fisiologiaRESUMO
In the mammalian neocortex, projection neuron types are sequentially generated by the same pool of neural progenitors. How neuron type specification is related to developmental timing remains unclear. To determine whether temporal gene expression in neural progenitors correlates with neuron type specification, we performed single-cell RNA sequencing (scRNA-Seq) analysis of the developing mouse neocortex. We uncovered neuroepithelial cell enriched genes such as Hmga2 and Ccnd1 when compared to radial glial cells (RGCs). RGCs display dynamic gene expression over time; for instance, early RGCs express higher levels of Hes5, and late RGCs show higher expression of Pou3f2 Interestingly, intermediate progenitor cell marker gene Eomes coexpresses temporally with known neuronal identity genes at different developmental stages, though mostly in postmitotic cells. Our results delineate neural progenitor cell diversity in the developing mouse neocortex and support that neuronal identity genes are transcriptionally evident in Eomes-positive cells.
Assuntos
Diferenciação Celular , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína HMGA2/metabolismo , Camundongos , Neocórtex/citologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Neuroglia/citologia , Neuroglia/metabolismo , Fatores do Domínio POU/metabolismo , Proteínas Repressoras/metabolismo , Proteínas com Domínio T/metabolismoRESUMO
Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of â¼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled â¼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-ß isoform combinations, including α3-ß1 in excitatory neurons and α3-ß2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.
Assuntos
Encéfalo/embriologia , Encéfalo/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Adulto , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Criança , Feminino , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Recém-Nascido , Interneurônios/metabolismo , Imageamento por Ressonância Magnética , Masculino , Mutação/genética , Neocórtex/embriologia , Neocórtex/enzimologia , Neurônios/metabolismo , Parvalbuminas/metabolismo , Fenótipo , Polimicrogiria/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
One paramount challenge for neuroscientists over the past century has been to identify the embryonic origins of the enormous diversity of cortical neurons found in the adult human neocortex and to unravel the developmental processes governing their emergence. In all mammals, including humans, the radial glia lining the ventricles of the embryonic telencephalon, more recently reclassified as apical radial glia (aRGs), have been identified as the neural progenitors giving rise to all excitatory neurons and inhibitory interneurons of the six-layered cortex. In this review, we explore the fundamental molecular and cellular mechanisms that regulate aRG function and the generation of neuronal diversity in the dorsal telencephalon. We survey the key structural features essential for the retention of the highly polarized aRG morphology and therefore impose aRG identity after cytokinesis. We discuss how these structures and associated molecular signaling complexes influence aRG proliferative capacity and the decision to undergo proliferative self-renewing symmetric or neurogenic asymmetric divisions. We also explore the intriguing and complex question of how the extensive neuronal diversity within the adult neocortex arises from the small aRG population located within the cortical proliferative zone. We further highlight the recent clonal lineage tracing and single-cell transcriptomic profiling studies providing compelling evidence that individual neuronal identity emerges as a consequence of exposure to temporally regulated extrinsic cues which coordinate waves of transcriptional activity that evolve over time to drive neuronal commitment and maturation.
Assuntos
Neocórtex/embriologia , Neurogênese/fisiologia , Neurônios/fisiologia , Animais , HumanosRESUMO
Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.
Assuntos
Orientação de Axônios/fisiologia , Efrina-A5/metabolismo , Neocórtex/embriologia , Vias Neurais/embriologia , Ponte/embriologia , Receptor EphA7/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/metabolismo , Vias Neurais/metabolismo , Ponte/patologiaRESUMO
Radial glial progenitor cells (RGCs) in the dorsal telencephalon directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single-cell RNA sequencing, we have studied gene expression patterns of RGCs with different neurogenic behavior at early stages of cortical development. At this early age, some RGCs rapidly produce postmitotic neurons, whereas others self-renew and undergo neurogenic divisions at a later age. We have identified candidate genes that are differentially expressed among these early RGC subpopulations, including the transcription factor Sox9. Using in utero electroporation in embryonic mice of either sex, we demonstrate that elevated Sox9 expression in progenitors affects RGC cell cycle duration and leads to the generation of upper layer cortical neurons. Our data thus reveal molecular differences between progenitor cells with different neurogenic behavior at early stages of corticogenesis and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons.SIGNIFICANCE STATEMENT The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, which has been found to be a specific regulator of this subpopulation of progenitor cells. Functional experiments perturbing expression levels of Sox9 reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages.
Assuntos
Autorrenovação Celular/genética , Células Ependimogliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Neocórtex/citologia , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/genética , Fatores de Transcrição SOX9/fisiologia , Animais , Ciclo Celular/genética , Eletroporação , Células Ependimogliais/metabolismo , Feminino , Genes Reporter , Vetores Genéticos/administração & dosagem , Injeções Intraventriculares , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neuroglia/citologia , Neurônios/citologia , Gravidez , Regiões Promotoras Genéticas/genética , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genética , Análise de Célula Única , Transcrição GênicaRESUMO
WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Ciliopatias/genética , Microcefalia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Anoftalmia/embriologia , Anoftalmia/genética , Anoftalmia/metabolismo , Apoptose/genética , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cílios/genética , Cílios/patologia , Ciliopatias/embriologia , Ciliopatias/metabolismo , Ciliopatias/patologia , Nanismo/embriologia , Nanismo/genética , Nanismo/metabolismo , Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Fibroblastos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcefalia/embriologia , Microcefalia/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Neocórtex/embriologia , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Lysophosphatidic acid (LPA) is a bioactive lipid that activates the G protein-coupled receptors, LPA1-6, which are associated with a wide number of cellular responses including proliferation, migration, differentiation, and survival. Although LPA1-6 are expressed in the developing brain, their functions in brain development are not fully understood. In the present study, we analyzed the temporal expression pattern of LPA receptors (LPARs) during neocortical development and found that LPA2 is highly expressed in neural stem/progenitor cells (NS/PCs) in the embryonic neocortex. LPA2 activation on cultured NS/PCs using GRI977143, a selective LPA2 agonist, promoted neuronal differentiation. LPA2-induced neuronal expansion was inhibited by FR180204, an extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, suggesting that LPA2 promotes neuronal differentiation via Erk1/2 signaling. In addition, LPA2 activation promotes neurite elongation and branch formation. These results suggest that LPA2 is a critical regulator of neuronal differentiation and development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neocórtex/citologia , Neuritos/fisiologia , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Diferenciação Celular , Feminino , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos Endogâmicos C57BL , Neocórtex/embriologia , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
During development, the p75 neurotrophin receptor (p75NTR) is widely expressed in the nervous system where it regulates neuronal differentiation, migration and axonal outgrowth. p75NTR also mediates the survival and death of newly born neurons, with functional outcomes being dependent on both timing and cellular context. Here, we show that knockout of p75NTR from embryonic day 10 (E10) in neural progenitors using a conditional Nestin-Cre p75NTR floxed mouse causes increased apoptosis of progenitor cells. By E14.5, the number of Tbr2-positive progenitor cells was significantly reduced and the rate of neurogenesis was halved. Furthermore, in adult knockout mice, there were fewer cortical pyramidal neurons, interneurons, cholinergic basal forebrain neurons and striatal neurons, corresponding to a relative reduction in volume of these structures. Thalamic midline fusion during early postnatal development was also impaired in Nestin-Cre p75NTR floxed mice, indicating a novel role for p75NTR in the formation of this structure. The phenotype of this strain demonstrates that p75NTR regulates multiple aspects of brain development, including cortical progenitor cell survival, and that expression during early neurogenesis is required for appropriate formation of telencephalic structures.