Intermediate Progenitor Cells Research Articles

Cascade diversification directs generation of neuronal diversity in the hypothalamus.

The hypothalamus contains an astounding heterogeneity of neurons that regulate endocrine, autonomic, and behavioral functions. However, its molecular developmental trajectory and origin of neuronal diversity remain unclear. Here, we profile the transcriptome of 43,261 cells derived from Rax+ hypothalamic neuroepithelium to map the developmental landscape of the mouse hypothalamus and trajectory of radial glial cells (RGCs), intermediate progenitor cells (IPCs), nascent neurons, and peptidergic neurons. We show that RGCs adopt a conserved strategy for multipotential differentiation but generate Ascl1+ and Neurog2+ IPCs. Ascl1+ IPCs differ from their telencephalic counterpart by displaying fate bifurcation, and postmitotic nascent neurons resolve into multiple peptidergic neuronal subtypes. Clonal analysis further demonstrates that single RGCs can produce multiple neuronal subtypes. Our study reveals that multiple cell types along the lineage hierarchy contribute to fate diversification of hypothalamic neurons in a stepwise fashion, suggesting a cascade diversification model that deconstructs the origin of neuronal diversity.

Molecular Profiling Reveals Involvement of ESCO2 in Intermediate Progenitor Cell Maintenance in the Developing Mouse Cortex.

SummaryIntermediate progenitor cells (IPCs) are neocortical neuronal precursors. Although IPCs play crucial roles in corticogenesis, their molecular features remain largely unknown. In this study, we aimed to characterize the molecular profile of IPCs. We isolated TBR2-positive (+) IPCs and TBR2-negative (−) cell populations in the developing mouse cortex. Comparative genome-wide gene expression analysis of TBR2+ IPCs versus TBR2− cells revealed differences in key factors involved in chromatid segregation, cell-cycle regulation, transcriptional regulation, and cell signaling. Notably, mutation of many IPC genes in human has led to intellectual disability and caused a wide range of cortical malformations, including microcephaly and agenesis of corpus callosum. Loss-of-function experiments in cortex-specific mutants of Esco2, one of the novel IPC genes, demonstrate its critical role in IPC maintenance, and substantiate the identification of a central genetic determinant of IPC biogenesis. Our data provide novel molecular characteristics of IPCs in the developing mouse cortex.

#71: Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Neuroprogenitor Cells and Cerebral Organoids

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not observed prior to the recent epidemics in French Polynesia and the Americas, despite evidence that the Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neuroprogenitor cells and cerebral organoids, which recapitulate complex three-dimensional neural architecture. We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results In neuroprogenitor cells, single-cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these are largely suppressed in cells infected with ZIKV within the same culture dish. In our organoid model, single-cell sequencing allows us to identify multiple cellular populations, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. In this model of the developing brain, we identify preferred tropisms of ZIKV infection. Our data additionally reveal differences in cell-type frequencies and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. Conclusions These findings may help explain phenotypic differences attributed to the viruses, including variable propensities to cause microcephaly. Overall, our work provides insight into normal and diseased human brain development and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

The expression of aristaless-related homeobox in neural progenitors of gyrencephalic carnivore ferrets

Aristaless-related homeobox (ARX) has important functions in the development of various organs including the brain. Mutations of the human ARX gene have been associated with malformations of the cerebral cortex such as microcephaly and lissencephaly. Although the expression patterns of ARX in the lissencephalic cerebral cortex of mice have been intensively investigated, those in expanded gyrencephalic brains remained unclear. Here, we show the expression patterns of ARX in the developing cerebral cortex of gyrencephalic carnivore ferrets. We found that ARX was expressed not only in intermediate progenitor (IP) cells but also in outer radial glial (oRG) cells, which are neural progenitors preferentially observed in the gyrencephalic cerebral cortex. We found that the majority of ARX-positive oRG cells expressed the proliferating cell marker Ki-67. These results may indicate that ARX in oRG cells mediates the expansion of the gyrencephalic cerebral cortex during development and evolution.

Progenitor cell diversity in the developing mouse neocortex

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.

Hes1 overexpression leads to expansion of embryonic neural stem cell pool and stem cell reservoir in the postnatal brain.

Neural stem cells (NSCs) gradually alter their characteristics during mammalian neocortical development, resulting in the production of various neurons and glial cells, and remain in the postnatal brain as a source of adult neurogenesis. Notch-Hes signaling is a key regulator of stem cell properties in the developing and postnatal brain, and Hes1 is a major effector that strongly inhibits neuronal differentiation and maintains NSCs. To manipulate Hes1 expression levels in NSCs, we generated transgenic (Tg) mice using the Tet-On system. In Hes1-overexpressing Tg mice, NSCs were maintained in both embryonic and postnatal brains, and generation of later-born neurons was prolonged until later stages in the Tg neocortex. Hes1 overexpression inhibited the production of Tbr2+ intermediate progenitor cells but instead promoted the generation of basal radial glia-like cells in the subventricular zone (SVZ) at late embryonic stages. Furthermore, Hes1-overexpressing Tg mice exhibited the expansion of NSCs and enhanced neurogenesis in the SVZ of adult brain. These results indicate that Hes1 overexpression expanded the embryonic NSC pool and led to the expansion of the NSC reservoir in the postnatal and adult brain.

Neuronal Delamination and Outer Radial Glia Generation in Neocortical Development.

During neocortical development, many neuronally differentiating cells (neurons and intermediate progenitor cells) are generated at the apical/ventricular surface by the division of neural progenitor cells (apical radial glial cells, aRGs). Neurogenic cell delamination, in which these neuronally differentiating cells retract their apical processes and depart from the apical surface, is the first step of their migration. Since the microenvironment established by the apical endfeet is crucial for maintaining neuroepithelial (NE)/aRGs, proper timing of the detachment of the apical endfeet is critical for the quantitative control of neurogenesis in cerebral development. During delamination, the microtubule–actin–AJ (adherens junction) configuration at the apical endfeet shows dynamic changes, concurrent with the constriction of the AJ ring at the apical endfeet and downregulation of cadherin expression. This process is mediated by transcriptional suppression of AJ-related molecules and multiple cascades to regulate cell adhesion and cytoskeletal architecture in a posttranscriptional manner. Recent advances have added molecules to the latter category: the interphase centrosome protein AKNA affects microtubule dynamics to destabilize the microtubule–actin–AJ complex, and the microtubule-associated protein Lzts1 inhibits microtubule assembly and activates actomyosin systems at the apical endfeet of differentiating cells. Moreover, Lzts1 induces the oblique division of aRGs, and loss of Lzts1 reduces the generation of outer radial glia (oRGs, also called basal radial glia, bRGs), another type of neural progenitor cell in the subventricular zone. These findings suggest that neurogenic cell delamination, and in some cases oRG generation, could be caused by a spectrum of interlinked mechanisms.

Biphasic Roles of Hedgehog Signaling in the Production and Self-Renewal of Outer Radial Glia in the Ferret Cerebral Cortex.

The neocortex, the center for higher brain function, emerged in mammals and expanded in the course of evolution. The expansion of outer radial glia (oRGs) and intermediate progenitor cells (IPCs) plays key roles in the expansion and consequential folding of the neocortex. Therefore, understanding the mechanisms of oRG and IPC expansion is important for understanding neocortical development and evolution. By using mice and human cerebral organoids, we previously revealed that hedgehog (HH) signaling expands oRGs and IPCs. Nevertheless, it remained to be determined whether HH signaling expanded oRGs and IPCs in vivo in gyrencephalic species, in which oRGs and IPCs are naturally expanded. Here, we show that HH signaling is necessary and sufficient to expand oRGs and IPCs in ferrets, a gyrencephalic species, through conserved cellular mechanisms. HH signaling increases oRG-producing division modes of ventricular radial glia (vRGs), oRG self-renewal, and IPC proliferation. Notably, HH signaling affects vRG division modes only in an early restricted phase before superficial-layer neuron production peaks. Beyond this restricted phase, HH signaling promotes oRG self-renewal. Thus, HH signaling expands oRGs and IPCs in two distinct but continuous phases during cortical development.

Atypical Neocortical Development in the Cited2 Conditional Knockout Leads to Behavioral Deficits Associated with Neurodevelopmental Disorders

The mammalian neocortex develops from a single layer of neuroepithelial cells to form a six-layer heterogeneous mosaic of differentiated neurons and glial cells. This process requires a complex choreography of temporally and spatially restricted transcription factors and epigenetic regulators. Even subtle disruptions in this regulation can alter the way the neocortex forms and functions, leading to a neurodevelopmental disorder. One epigenetic regulator that is essential for the precise development of the neocortex is CITED2 (CBP/p300 Interacting Transactivator with ED-rich termini). Cited2 is highly expressed by intermediate progenitor cells in the subventricular zone during the generation of the superficial layers of the neocortex. A forebrain-specific conditional knockout of Cited2 (cKO) exhibits reduced proliferation of intermediate progenitor cells embryonically, leading to reduced thickness of the superficial layers and reduced corpus callosum (CC) volume postnatally. Further, the Cited2 cKO display disruptions in balanced neocortical arealization, with a specific reduction in the somatosensory neocortical length, and dysregulation of precise, area-specific neuronal connectivity. Here, we explore the behavioral consequences resulting from this aberrant neocortical development. We demonstrate that Cited2 cKO mice display decreased maternal separation-induced ultrasonic vocalizations (USVs) as neonates, and an increase in rearing behavior and lack of habituation following repeated acoustic startle as adults. They do not display alterations in anxiety-like behavior, overall locomotor activity, or social interactions. Together with the morphological, molecular, and connectivity disruptions, these results identify the Cited2 cKO neocortex as an ideal system to study mechanisms underlying neurodevelopmental and neuroanatomical disruptions with relevance to human neurodevelopmental disorders.

Neuroinflammation Induction and Alteration of Hippocampal Neurogenesis in Mice Following Developmental Exposure to Gossypol.

BackgroundNeurogenesis in the neonatal period involves the proliferation and differentiation of neuronal stem/progenitor cells and the establishment of synaptic connections. This process plays a critical role in determining the normal development and maturation of the brain throughout life. Exposure to certain physical or chemical factors during the perinatal period can lead to many neuropathological defects that cause high cognitive dysfunction and are accompanied by abnormal hippocampal neurogenesis and plasticity. As an endocrine disruptor, gossypol is generally known to exert detrimental effects in animals exposed under experimental conditions. However, it is unclear whether gossypol affects neurogenesis in the hippocampal dentate gyrus during early developmental stages.MethodsPregnant Institute of Cancer Research mice were treated with gossypol at a daily dose of 0, 20, and 50 mg/kg body weight from embryonic day 6.5 to postnatal day (P) 21. The changes of hippocampal neurogenesis as well as potential mechanisms were investigated by 5-bromo-2-deoxyuridine labeling, behavioral tests, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western-blot analyses.ResultsAt P8, maternal gossypol exposure impaired neural stem cell proliferation in the dentate gyrus and decreased the number of newborn cells as a result of reduced proliferation of BLBP+ radial glial cells and Tbr2+ intermediate progenitor cells. At P21, the numbers of NeuN+ neurons and parvalbumin+ γ-aminobutyric acid-ergic interneurons were increased following 50 mg/kg gossypol exposure. In addition, gossypol induced hippocampal neuroinflammation, which may contribute to behavioral abnormalities and cognitive deficits and decrease synaptic plasticity.ConclusionsOur findings suggest that developmental gossypol exposure affects hippocampal neurogenesis by targeting the proliferation and differentiation of neuronal stem/progenitor cells, cognitive functions, and neuroinflammation. The present data provide novel insights into the neurotoxic effects of gossypol on offspring.

CD44v3-Positive Intermediate Progenitor Cells Contribute to Airway Goblet Cell Hyperplasia.

In allergic airway diseases, intermediate progenitor cells (IPCs) increase in number in the surface epithelium. IPCs arise from basal cells, the origin of hallmark pathological changes, including goblet cell hyperplasia and mucus hypersecretion. Thus, targeting IPCs will benefit future treatment of allergic airway diseases. However, the lack of adequate cell surface markers for IPCs limits their identification and characterization. We now show that CD44 containing exon v3 (CD44v3) is a surface marker for IPCs that are capable of both proliferating and generating differentiated goblet cells in allergic human nasal epithelium. In primary human nasal epithelial cells that had differentiated at an air-liquid interface, IL-4 upregulated mRNA expression of three CD44v variants that include exon v3 (CD44v3-v6, CD44v3,v8-v10, and CD44v3-v10), and it induced expression of CD44v3 protein in the basal and suprabasal layers of the culture. FACS analysis revealed two subpopulations differing in CD44v3 concentrations, as follows: CD44v3low cells expressed high amounts of proliferative and basal cell markers (Ki-67 and TP63), whereas CD44v3high cells strongly expressed progenitor and immature and mature goblet cell markers (SOX2, CA2, and SPDEF). Importantly, a blocking anti-CD44 antibody suppressed IL-4-induced mucin production by human nasal epithelial cells. Furthermore, CD44v3 was coexpressed with TP63, KRT5, or SOX2 and was upregulated in the basal and suprabasal layers of the nasal surface epithelium of subjects with allergic rhinitis. Taken together, these data demonstrate that high CD44v3 expression contributes to goblet cell hyperplasia in inflammation of the allergic airway.

Developmental exposure to diesel exhaust upregulates transcription factor expression, decreases hippocampal neurogenesis, and alters cortical lamina organization: relevance to neurodevelopmental disorders

BackgroundExposure to traffic-related air pollution (TRAP) during development and/or in adulthood has been associated in many human studies with both neurodevelopmental and neurodegenerative diseases, such as autism spectrum disorder (ASD) and Alzheimer’s disease (AD) or Parkinson’s disease (PD).MethodsIn the present study, C57BL/6 J mice were exposed to environmentally relevant levels (250+/−50 μg/m3) of diesel exhaust (DE) or filtered air (FA) during development (E0 to PND21). The expression of several transcription factors relevant for CNS development was assessed on PND3. To address possible mechanistic underpinnings of previously observed behavioral effects of DE exposure, adult neurogenesis in the hippocampus and laminar organization of neurons in the somatosensory cortex were analyzed on PND60. Results were analyzed separately for male and female mice.ResultsDevelopmental DE exposure caused a male-specific upregulation of Pax6, Tbr1, Tbr2, Sp1, and Creb1 on PND3. In contrast, in both males and females, Tbr2+ intermediate progenitor cells in the PND60 hippocampal dentate gyrus were decreased, as an indication of reduced adult neurogenesis. In the somatosensory region of the cerebral cortex, laminar distribution of Trb1, calbindin, and parvalbumin (but not of Ctip2 or Cux1) was altered by developmental DE exposure.ConclusionsThese results provide additional evidence to previous findings indicating the ability of developmental DE exposure to cause biochemical/molecular and behavioral alterations that may be involved in neurodevelopmental disorders such as ASD.

Cell-type-specific 3D epigenomes in the developing human cortex.

Lineage-specific epigenomic changes during human corticogenesis have remained elusive due to challenges with sample availability and tissue heterogeneity. For example, previous studies used single-cell RNA sequencing to identify at least nine major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here, we characterize cell type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational human cortex samples. We show that chromatin interactions underlie multiple aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell type-specific manner. In addition, promoters with significantly increased levels of chromatin interactivity, termed super interactive promoters, are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a novel technique integrating immunostaining, CRISPRi, RNAscope, and image analysis for validating cell type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our study presents the first cell type-specific characterization of 3D epigenomes in the developing human cortex, advancing our understanding of gene regulation and lineage specification during this critical developmental window.

Mitochondria Control Cortical Cell Fate after Mitosis.

During brain development, the daughter cells of neural stem cells have to make a choice - either to become new stem cells or to differentiate into neurons. In a recent issue of Science, Iwata etal. (2020) show that these fate decisions can be determined after mitosis by mitochondrial remodeling.

Early Chronic Intermittent Maternal Hyperoxygenation Impairs Cortical Development by Inhibition of Pax6-Positive Apical Progenitor Cell Proliferation.

Maternal hyperoxygenation is a feasible, noninvasive method to treat fetal diseases, such as heart hypoplasia, but effects of maternal hyperoxygenation on the developing brain remain poorly understood. Previous studies showed that short-term maternal hyperoxygenation during midneurogenic phase (E14-E16) but not in earlier development (E10-E12) increases oxygen tension and enhances neurogenesis in the developing mouse cortex. We investigated effects of early chronic maternal hyperoxygenation (CMH) as a potential clinical treatment. Pregnant C57BL/6J mice were housed in a chamber at 75% atmospheric oxygen and the brains of E16 fetuses were analyzed using immunohistochemistry. The mitosis marker phH3 showed a significant reduction of proliferation in the dorsolateral cortices of CMH-treated E16 fetuses. Numbers of Tbr2-positive intermediate progenitor cells were unaffected whereas numbers of Pax6-positive apical progenitor cells were significantly reduced in CMH-treated mice. This resulted in altered cortical plate development with fewer Satb2-positive upper layer neurons but more Tbr1-positive neurons corresponding to the deeper layer 6. Thus, maternal hyperoxygenation affects the developing cortex depending on timing and length of applied oxygen. Early CMH causes a severe reduction of neuroprogenitor proliferation likely affecting cortical development. Further studies are needed to investigate the mechanisms underlying these findings and to assess the clinical and neurodevelopmental outcomes of the pups.

A Mathematical Model of Average Dynamics in a Stem Cell Hierarchy Suggests the Combinatorial Targeting of Cancer Stem Cells and Progenitor Cells as a Potential Strategy against Tumor Growth.

Simple SummaryCancer stem cell (CSC) directed therapies have been increasingly developed during the last years. However, some reported experiments using this strategy showed that although delayed tumor growth was observed, the tumor was not completely eliminated. Here, we hypothesize that the simultaneous targeting of CSCs and progenitor cells of intermediate phenotype may represent a better strategy against tumor growth. We aimed to fit a mathematical model, consistent with the CSC hypothesis, to reported experimental data resulting from CSC direct targeting. This is a minimal model of average tumor dynamics that could aid in the visualization of the overall tumor growth when different subpopulations of tumor cells are targeted. We show that combination therapy during a time lapse that ensures eradication of CSCs and progenitor cells in a stem cell hierarchy controlled tumor relapse. Testing this hypothesis in vivo may help to discriminate among other possibilities of tumor burden.The cancer stem cell hypothesis states that tumors are maintained by a small subpopulation of stem-like cells, often called cancer stem cells (CSCs) or tumor initiating cells. CSCs can self-renew and give rise to more differentiated cells, which comprise the bulk of the tumor. In addition, CSCs are resistant to conventional therapy, which suggests that they are responsible for tumor relapse. This has led researchers to increase efforts to develop directed therapies against CSCs. However, some experiments in mice have shown that the elimination of CSCs might not ensure tumor eradication. This may be due to different events, such as residual CSCs after treatment, the plasticity of cells within the tumor, the presence of different CSCs having their own hierarchy within the same tumor, and the ability of more differentiated cells to maintain the disease, among others. Trying to decipher this complexity may benefit from dissecting the whole in its parts. Here, we hypothesize that tumor relapse after the selective targeting of CSCs may be due to intermediate progenitor (P) cells that can maintain the tumor volume. In order to support the hypothesis, we implemented a mathematical model derived using pseudo-reactions representing the events of each cell subpopulation within the tumor. We aimed to test if a minimal unidirectional hierarchical model consisting of CSCs, P, and terminally differentiated (D) cells could be adjusted to experimental data for selective CSC targeting. We further evaluated therapies ranging from nonselective to specifically directed and combination therapy. We found that selective killing of the CSC compartment has a delaying effect on the overall exponential tumor growth, but was not able to eliminate the disease. We show that therapy that targets both CSCs and intermediate progenitor (P) cells with a sufficient capacity to proliferate and differentiate could represent a more efficient treatment option for tumor depletion. Testing this hypothesis in vivo may allow us to discriminate within the array of possibilities of tumor relapse, and further open the idea of combination therapy against different subpopulations of tumor cells instead of segregating CSCs and bulk tumor cells.

Perinatal IL-1β-induced inflammation suppresses Tbr2+ intermediate progenitor cell proliferation in the developing hippocampus accompanied by long-term behavioral deficits

Meta-analyses have revealed associations between the incidence of maternal infections during pregnancy, premature birth, smaller brain volumes, and subsequent cognitive, motor and behavioral deficits as these children mature. Inflammation during pregnancy in rodents produces cognitive and behavioral deficits in the offspring that are similar to those reported in human studies. These deficits are accompanied by decreased neurogenesis and proliferation in the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. As systemically administering interleukin-1 β (IL-1β) to neonatal mice recapitulates many of the brain abnormalities seen in premature babies including developmental delays, the goal of this study was to determine whether IL-1-mediated neuroinflammation would affect hippocampal growth during development to produce cognitive and behavioral abnormalities. For these studies, 10 ​ng/g IL-1β was administered twice daily to Swiss Webster mice during the first 5 days of life, which increased hippocampal levels of IL-1α and acutely reduced the proliferation of Tbr2+ neural progenitors in the DG. In vitro, both IL-1α and IL-1β produced G1/S cell cycle arrest that resulted in reduced progenitor cell proliferation within the transit amplifying progenitor cell cohort. By contrast, IL-1β treatment increased neural stem cell frequency. Upon terminating IL-1β treatment, the progenitor cell pool regained its proliferative capacity. An earlier study that used this in vivo model of perinatal inflammation showed that mice that received IL-1β as neonates displayed memory deficits which suggested abnormal hippocampal function. To evaluate whether other cognitive and behavioral traits associated with hippocampal function would also be altered, mice were tested in tasks designed to assess exploratory and anxiety behavior as well as working and spatial memory. Interestingly, mice that received IL-1β as neonates showed signs of anxiety in several behavioral assays during adolescence that were also evident in adulthood. Additionally, these mice did not display working memory deficits in adulthood, but they did display deficits in long-term spatial memory. Altogether, these data support the view that perinatal inflammation negatively affects the developing hippocampus by producing behavioral deficits that persist into adulthood. These data provide a new perspective into the origin of the cognitive and behavioral impairments observed in prematurely-born sick infants.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Centrosome anchoring regulates progenitor properties and cortical formation.

SUMMARYRadial glial progenitors (RGPs) represent the major neural progenitors for generating neurons and glia in the developing mammalian cerebral cortex 1-3. They position their centrosomes away from the nucleus at the ventricular zone surface 4-7. However, the molecular basis and precise function of this highly unique and characteristic subcellular organization of the centrosome remain largely unknown. Here we show that apical membrane anchoring of the centrosome controls the mechanical properties of mouse cortical RGPs and consequently their mitotic behaviour and cortical size and formation. Mother centriole in RGPs specifically develops distal appendages to anchor to the apical membrane. Selective removal of Centrosomal protein 83 (CEP83) eliminates mother centriole distal appendages and disrupts centrosome apical membrane anchorage, resulting in microtubule disorganization and apical membrane stretching and stiffening. It activates mechanically-sensitive Yes-associated protein (YAP) and promotes excessive RGP proliferation and subsequent intermediate progenitor overproduction, leading to the formation of an enlarged cortex with abnormal folding. Simultaneous elimination of YAP suppresses cortical enlargement and folding caused by CEP83 removal. Together, these results uncover a previously unknown role of the centrosome in regulating the mechanical features of neural progenitors, and the size and configuration of the mammalian cerebral cortex.

Cortical Neural Stem Cell Lineage Progression Is Regulated by Extrinsic Signaling Molecule Sonic Hedgehog

SUMMARYNeural stem cells (NSCs) in the prenatal neocortex progressively generate different subtypes of glutamatergic projection neurons. Following that, NSCs have a major switch in their progenitor properties and produce γ-aminobutyric acid (GABAergic) interneurons for the olfactory bulb (OB), cortical oligodendrocytes, and astrocytes. Herein, we provide evidence for the molecular mechanism that underlies this switch in the state of neocortical NSCs. We show that, at around E16.5, mouse neocortical NSCs start to generate GSX2-expressing (GSX2+) intermediate progenitor cells (IPCs). In vivo lineage-tracing study revealed that GSX2+ IPC population gives rise not only to OB interneurons but also to cortical oligodendrocytes and astrocytes, suggesting that they are a tri-potential population. We demonstrated that Sonic hedgehog signaling is both necessary and sufficient for the generation of GSX2+ IPCs by reducing GLI3R protein levels. Using single-cell RNA sequencing, we identify the transcriptional profile of GSX2+ IPCs and the process of the lineage switch of cortical NSCs.