Upper-layer Neurons Research Articles

Comparative single-cell multiome identifies evolutionary changes in neural progenitor cells during primate brain development.

Understanding the cellular and genetic mechanisms driving human-specific features of cortical development remains a challenge. We generated a cell-type resolved atlas of transcriptome and chromatin accessibility in the developing macaque and mouse prefrontal cortex (PFC). Comparing with published human data, our findings demonstrate that although the cortex cellular composition is overall conserved across species, progenitor cells show significant evolutionary divergence in cellular properties. Specifically, human neural progenitors exhibit extensive transcriptional rewiring in growth factor and extracellular matrix (ECM) pathways. Expression of the human-specific progenitor marker ITGA2 in the fetal mouse cortex increases the progenitor proliferation and the proportion of upper-layer neurons. These transcriptional divergences are primarily driven by altered activity in the distal regulatory elements. The chromatin regions with human-gained accessibility are enriched with human-specific sequence changes and polymorphisms linked to intelligence and neuropsychiatric disorders. Our results identify evolutionary changes in neural progenitors and putative gene regulatory mechanisms shaping primate brain evolution.

Reducing Filamin A Restores Cortical Synaptic Connectivity and Early Social Communication Following Cellular Mosaicism in Autism Spectrum Disorder Pathways.

Communication in the form of nonverbal, social vocalization, or crying is evolutionary conserved in mammals and is impaired early in human infants that are later diagnosed with autism spectrum disorder (ASD). Defects in infant vocalization have been proposed as an early sign of ASD that may exacerbate ASD development. However, the neural mechanisms associated with early communicative deficits in ASD are not known. Here, we expressed a constitutively active mutant of Rheb (RhebS16H), which is known to upregulate two ASD core pathways, mTOR complex 1 (mTORC1) and ERK1/2, in Layer (L) 2/3 pyramidal neurons of the neocortex of mice of either sex. We found that cellular mosaic expression of RhebS16H in L2/3 pyramidal neurons altered the production of isolation calls from neonatal mice. This was accompanied by an expected misplacement of neurons and dendrite overgrowth, along with an unexpected increase in spine density and length, which was associated with increased excitatory synaptic activity. This contrasted with the known decrease in spine density in RhebS16H neurons of 1-month-old mice. Reducing the levels of the actin cross-linking and adaptor protein filamin A (FLNA), known to be increased downstream of ERK1/2, attenuated dendrite overgrowth and fully restored spine properties, synaptic connectivity, and the production of pup isolation calls. These findings suggest that upper-layer cortical pyramidal neurons contribute to communicative deficits in a condition known to affect two core ASD pathways and that these mechanisms are regulated by FLNA.

Loss of Zmiz1 in mice leads to impaired cortical development and autistic-like behaviors.

De novo mutations in transcriptional regulators are emerging as key risk factors contributing to the etiology of neurodevelopmental disorders. Human genetic studies have recently identified ZMIZ1 and its de novo mutations as causal of a neurodevelopmental syndrome strongly associated with intellectual disability, autism, ADHD, microcephaly, and other developmental anomalies. However, the role of ZMIZ in brain development or how ZMIZ1 mutations cause neurological phenotypes is unknown. Here, we generated a forebrain-specific Zmiz1 mutant mouse model that develops brain abnormalities, including cortical microcephaly, corpus callosum dysgenesis, and abnormal differentiation of upper-layer cortical neurons. Behaviorally, Zmiz1 mutant mice show alterations in motor activity, anxiety, communication, and social interactions with strong sex differences, resembling phenotypes associated with autism. Molecularly, Zmiz1 deficiency leads to transcriptomic changes disrupting neurogenesis, neuron differentiation programs, and synaptic signaling. We identified Zmiz1-mediated downstream regulation of key neurodevelopmental factors, including Lhx2, Auts2, and EfnB2. Importantly, reactivation of the EfnB2 pathway by exogenous EFNB2 recombinant protein rescues the dendritic outgrowth deficits in Zmiz1 mutant cortical neurons. Overall, our in vivo findings provide insight into Zmiz1 function in cortical development and reveal mechanistic underpinnings of ZMIZ1 syndrome, thereby providing valuable information relevant to future studies on this neurodevelopmental disorder.

Biallelic null variants in PNPLA8 cause microcephaly by reducing the number of basal radial glia.

Patatin-like phospholipase domain-containing lipase 8 (PNPLA8), one of the calcium-independent phospholipase A2 enzymes, is involved in various physiological processes through the maintenance of membrane phospholipids. Biallelic variants in PNPLA8 have been associated with a range of paediatric neurodegenerative disorders. However, the phenotypic spectrum, genotype-phenotype correlations and the underlying mechanisms are poorly understood. Here, we newly identified 14 individuals from 12 unrelated families with biallelic ultra-rare variants in PNPLA8 presenting with a wide phenotypic spectrum of clinical features. Analysis of the clinical features of current and previously reported individuals (25 affected individuals across 20 families) showed that PNPLA8-related neurological diseases manifest as a continuum ranging from variable developmental and/or degenerative epileptic-dyskinetic encephalopathy to childhood-onset neurodegeneration. We found that complete loss of PNPLA8 was associated with the more profound end of the spectrum, with congenital microcephaly. Using cerebral organoids generated from human induced pluripotent stem cells, we found that loss of PNPLA8 led to developmental defects by reducing the number of basal radial glial cells and upper-layer neurons. Spatial transcriptomics revealed that loss of PNPLA8 altered the fate specification of apical radial glial cells, as reflected by the enrichment of gene sets related to the cell cycle, basal radial glial cells and neural differentiation. Neural progenitor cells lacking PNPLA8 showed a reduced amount of lysophosphatidic acid, lysophosphatidylethanolamine and phosphatidic acid. The reduced number of basal radial glial cells in patient-derived cerebral organoids was rescued, in part, by the addition of lysophosphatidic acid. Our data suggest that PNPLA8 is crucial to meet phospholipid synthetic needs and to produce abundant basal radial glial cells in human brain development.

Epistatic interactions between NMD and TRP53 control progenitor cell maintenance and brain size.

Mutations in human nonsense-mediated mRNA decay (NMD) factors are enriched in neurodevelopmental disorders. We show that deletion of key NMD factor Upf2 in mouse embryonic neural progenitor cells causes perinatal microcephaly but deletion in immature neurons does not, indicating NMD's critical roles in progenitors. Upf2 knockout (KO) prolongs the cell cycle of radial glia progenitor cells, promotes their transition into intermediate progenitors, and leads to reduced upper-layer neurons. CRISPRi screening identified Trp53 knockdown rescuing Upf2KO progenitors without globally reversing NMD inhibition, implying marginal contributionsof most NMD targets to the cell cycle defect. Integrated functional genomics shows that NMD degrades selective TRP53 downstream targets, including Cdkn1a, which, without NMD suppression, slow the cell cycle. Trp53KO restores the progenitor cell pool and rescues the microcephaly of Upf2KO mice. Therefore, one physiological role of NMD in the developing brain is to degrade selective TRP53 targets to control progenitor cell cycle and brain size.

Cell type specific roles of FOXP1 during early neocortical murine development.

Cortical development is a tightly controlled process and any deviation during development may increase the susceptibility to neurodevelopmental disorders, such as autism spectrum disorders (ASD). Numerous studies identified mutations in FOXP1, a transcription factor enriched in the neocortex, as causal for ASD and FOXP1 syndrome. Our group has shown that Foxp1 deletion in the mouse cortex leads to overall reduced cortex thickness, alterations in cortical lamination, and changes in the relative thickness of cortical layers. However, the developmental and cell type-specific mechanisms underlying these changes remained unclear. This work characterizes the developmental requirement of neocortical Foxp1 at key embryonic and perinatal ages using a conditional knock-out of Foxp1. We find that Foxp1 deletion results in accelerated pseudo-age during early neurogenesis, increased cell cycle exit during late neurogenesis, altered gene expression and chromatin accessibility, and selective migration deficits in a subset of upper-layer neurons. These data explain the postnatal differences observed in cortical layers and relative cortical thickness. We also highlight genes regulated by FOXP1 and their enrichment with high-confidence ASD or synaptic genes. Together, these results underscore a network of neurodevelopmental disorder-related genes that may serve as potential modulatory targets for postnatal modification relevant to ASD and FOXP1 syndrome.

Altered projection-specific synaptic remodeling and its modification by oxytocin in an idiopathic autism marmoset model.

Alterations in the experience-dependent and autonomous elaboration of neural circuits are assumed to underlie autism spectrum disorder (ASD), though it is unclear what synaptic traits are responsible. Here, utilizing a valproic acid-induced ASD marmoset model, which shares common molecular features with idiopathic ASD, we investigate changes in the structural dynamics of tuft dendrites of upper-layer pyramidal neurons and adjacent axons in the dorsomedial prefrontal cortex through two-photon microscopy. In model marmosets, dendritic spine turnover is upregulated, and spines are generated in clusters and survived more often than in control marmosets. Presynaptic boutons in local axons, but not in commissural long-range axons, demonstrate hyperdynamic turnover in model marmosets, suggesting alterations in projection-specific plasticity. Intriguingly, nasal oxytocin administration attenuates clustered spine emergence in model marmosets. Enhanced clustered spine generation, possibly unique to certain presynaptic partners, may be associated with ASD and be a potential therapeutic target.

Spatiotemporal expression of thyroid hormone transporter MCT8 and THRA mRNA in human cerebral organoids recapitulating first trimester cortex development

Thyroid hormones (TH) play critical roles during nervous system development and patients carrying coding variants of MCT8 (monocarboxylate transporter 8) or THRA (thyroid hormone receptor alpha) present a spectrum of neurological phenotypes resulting from perturbed local TH action during early brain development. Recently, human cerebral organoids (hCOs) emerged as powerful in vitro tools for disease modelling recapitulating key aspects of early human cortex development. To begin exploring prospects of this model for thyroid research, we performed a detailed characterization of the spatiotemporal expression of MCT8 and THRA in developing hCOs. Immunostaining showed MCT8 membrane expression in neuronal progenitor cell types including early neuroepithelial cells, radial glia cells (RGCs), intermediate progenitors and outer RGCs. In addition, we detected robust MCT8 protein expression in deep layer and upper layer neurons. Spatiotemporal SLC16A2 mRNA expression, detected by fluorescent in situ hybridization (FISH), was highly concordant with MCT8 protein expression across cortical cell layers. FISH detected THRA mRNA expression already in neuroepithelium before the onset of neurogenesis. THRA mRNA expression remained low in the ventricular zone, increased in the subventricular zone whereas strong THRA expression was observed in excitatory neurons. In combination with a robust up-regulation of known T3 response genes following T3 treatment, these observations show that hCOs provide a promising and experimentally tractable model to probe local TH action during human cortical neurogenesis and eventually to model the consequences of impaired TH function for early cortex development.

KCTD10 regulates brain development by destabilizing brain disorder–associated protein KCTD13

KCTD10 belongs to the KCTD (potassiumchannel tetramerization domain) family, many members of which are associated with neuropsychiatric disorders. However, the biological function underlying the association with brain disorders remains to be explored. Here, we reveal that Kctd10 is highly expressed in neuronal progenitors and layer V neurons throughout brain development. Kctd10 deficiency triggers abnormal proliferation and differentiation of neuronal progenitors, reduced deep-layer (especially layer V) neurons, increased upper-layer neurons, and lowered brain size. Mechanistically, we screened and identified a unique KCTD10-interacting protein, KCTD13, associated with neurodevelopmental disorders. KCTD10 mediated the ubiquitination-dependent degradation of KCTD13 and KCTD10 ablation resulted in a considerable increase of KCTD13 expression in the developing cortex. KCTD13 overexpression in neuronal progenitors led to reduced proliferation and abnormal cell distribution, mirroring KCTD10 deficiency. Notably, mice with brain-specific Kctd10 knockout exhibited obvious motor deficits. This study uncovers the physiological function of KCTD10 and provides unique insights into the pathogenesis of neurodevelopmental disorders.

Temporal transcriptomic dynamics in developing macaque neocortex

Despite intense research on mice, the transcriptional regulation of neocortical neurogenesis remains limited in humans and non-human primates. Cortical development in rhesus macaque is known to recapitulate multiple facets of cortical development in humans, including the complex composition of neural stem cells and the thicker supragranular layer. To characterize temporal shifts in transcriptomic programming responsible for differentiation from stem cells to neurons, we sampled parietal lobes of rhesus macaque at E40, E50, E70, E80, and E90, spanning the full period of prenatal neurogenesis. Single-cell RNA sequencing produced a transcriptomic atlas of developing parietal lobe in rhesus macaque neocortex. Identification of distinct cell types and neural stem cells emerging in different developmental stages revealed a terminally bifurcating trajectory from stem cells to neurons. Notably, deep-layer neurons appear in the early stages of neurogenesis, while upper-layer neurons appear later. While these different lineages show overlap in their differentiation program, cell fates are determined post-mitotically. Trajectories analysis from ventricular radial glia (vRGs) to outer radial glia (oRGs) revealed dynamic gene expression profiles and identified differential activation of BMP, FGF, and WNT signaling pathways between vRGs and oRGs. These results provide a comprehensive overview of the temporal patterns of gene expression leading to different fates of radial glial progenitors during neocortex layer formation.

Development and evolution of the primate neocortex from a progenitor cell perspective.

The generation of neurons in the developing neocortex is a major determinant of neocortex size. Crucially, the increase in cortical neuron numbers in the primate lineage, notably in the upper-layer neurons, contributes to increased cognitive abilities. Here, we review major evolutionary changes affecting the apical progenitors in the ventricular zone and focus on the key germinal zone constituting the foundation of neocortical neurogenesis in primates, the outer subventricular zone (OSVZ). We summarize characteristic features of the OSVZ and its key stem cell type, the basal (or outer) radial glia. Next, we concentrate on primate-specific and human-specific genes, expressed in OSVZ-progenitors, the ability of which to amplify these progenitors by targeting the regulation of the cell cycle ultimately underlies the evolutionary increase in upper-layer neurons. Finally, we address likely differences in neocortical development between present-day humans and Neanderthals that are based on human-specific amino acid substitutions in proteins operating in cortical progenitors.

Astrocytes of the Anterior Commissure Regulate the Axon Guidance Pathways of Newly Generated Neocortical Neurons in the Opossum Monodelphis domestica.

In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the corpus callosum, that interconnects both hemispheres. In this study, we aimed to examine neurogenesis during the formation of cortical upper layers, including their morphological maturation in a marsupial species, namely the opossum (Monodelphis domestica). Furthermore, we studied how the axons of upper layers neurons pass through the anterior commissure of the opossum, which connects neocortical areas. We showed that upper-layer II/III neurons were generated within at least seven days in the opossum neocortex. Surprisingly, these neurons expressed special AT-rich sequence binding protein 2 (Satb2) and neuropilin 1 interacting protein (Nrp1), which are proteins known to be essential for the formation of the corpus callosum in eutherians. This indicates that extrinsic, but not intrinsic, cues could be key players in guiding the axons of newly generated cortical neurons in the opossum. Although oligodendrocyte precursor cells were present in the neocortex and anterior commissure, newly generated upper-layer neurons sent unmyelinated axons to the anterior commissure. We also found numerous GFAP-expressing progenitor cells in both brain structures, the neocortex and the anterior commissure. However, at P12-P17 in the opossums, a small population of astrocytes was observed only in the midline area of the anterior commissure. We postulate that in the opossum, midline astrocytes allow neocortical axons to be guided to cross the midline, as this structure resembles the glial wedge required by fibers to cross the midline area of the corpus callosum in the rodent.

Neocortical neurogenesis in development and evolution-Human-specific features.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

Prenatal folic acid and vitamin B12 imbalance alter neuronal morphology and synaptic density in the mouse neocortex

Previous reports have provided evidence that insufficient or excessive maternal folic acid (FA) intake during pregnancy can alter neurodevelopment of the offspring by modulating prenatal neurogenesis. Furthermore, our earlier work in a mouse model confirmed long-term structural changes at the cellular level of either deficient or excessive FA supply by comparably reducing dendritic arborization of cortical projection neurons. Here, we report that excessive amounts of FA decrease arborization of deep layer projection neurons, but not upper layer neurons and that reduced complexity of deep layer neurons is not observed when folic acid is replaced by folinic acid, a stable reduced form of folate. In addition, deficiency of B12, a vitamin that critically regulates folate metabolism, causes even more marked decreases in neuronal arborization in both deep and upper layer neurons and particularly in combination with FA excess. Furthermore, both FA excess and B12 deficiency affect synaptic density and morphology. Our findings point to neurodevelopmental risks associated with insufficient amounts of prenatal B12, particularly in association with high levels of FA intake, suggesting that the neurodevelopmental program is sensitive to an imbalance in the status of these interacting micronutrients.

Single-cell analysis reveals specific neuronal transition during mouse corticogenesis.

Background: Currently, the mechanism(s) underlying corticogenesis is still under characterization. Methods: We curated the most comprehensive single-cell RNA-seq (scRNA-seq) datasets from mouse and human fetal cortexes for data analysis and confirmed the findings with co-immunostaining experiments. Results: By analyzing the developmental trajectories with scRNA-seq datasets in mice, we identified a specific developmental sub-path contributed by a cell-population expressing both deep- and upper-layer neurons (DLNs and ULNs) specific markers, which occurred on E13.5 but was absent in adults. In this cell-population, the percentages of cells expressing DLN and ULN markers decreased and increased, respectively, during the development suggesting direct neuronal transition (namely D-T-U). Whilst genes significantly highly/uniquely expressed in D-T-U cell population were significantly enriched in PTN/MDK signaling pathways related to cell migration. Both findings were further confirmed by co-immunostaining with DLNs, ULNs and D-T-U specific markers across different timepoints. Furthermore, six genes (co-expressed with D-T-U specific markers in mice) showing a potential opposite temporal expression between human and mouse during fetal cortical development were associated with neuronal migration and cognitive functions. In adult prefrontal cortexes (PFC), D-T-U specific genes were expressed in neurons from different layers between humans and mice. Conclusion: Our study characterizes a specific cell population D-T-U showing direct DLNs to ULNs neuronal transition and migration during fetal cortical development in mice. It is potentially associated with the difference of cortical development in humans and mice.

Human-biased TMEM25 expression promotes expansion of neural progenitor cells to alter cortical structure in the developing brain.

Cortical expansion has occurred during human brain evolution. By comparing human and mouse RNA-seq datasets, we found that transmembrane protein 25 (TMEM25) was much more highly expressed in human neural progenitors (NPCs). Overexpression of either human TMEM25 or mouse Tmem25 similarly promoted mouse NPC proliferation in vitro. Mimicking human-type expression of TMEM25 in mouse ventricular cortical progenitors accelerated proliferation of basal radial glia (bRG) and increased the number of upper-layer neurons in vivo. By contrast, RNA-seq analysis and pharmacological assays showed that knockdown of TMEM25 in cultured human NPCs compromised the effects of extracellular signals, leading to cell cycle inhibition via Akt repression. Thus, TMEM25 can receive extracellular signals to expand bRG in human cortical development.

Divergent phenotypes in constitutive versus conditional mutant mouse models of Sifrim-Hitz-Weiss syndrome.

Chromatin remodellers are among the most important risk genes associated with neurodevelopmental disorders (NDDs), however, their functions during brain development are not fully understood. Here, we focused on Sifrim-Hitz-Weiss Syndrome (SIHIWES)-an intellectual disability disorder caused by mutations in the CHD4 chromodomain helicase gene. We utilized mouse genetics to excise the Chd4 ATPase/helicase domain-either constitutively, or conditionally in the developing telencephalon. Conditional heterozygotes exhibited no change in cortical size and cellular composition, and had only subtle behavioral phenotypes. Telencephalon-specific conditional knockouts had marked reductions in cortical growth, reduced numbers of upper-layer neurons, and exhibited alterations in anxiety and repetitive behaviors. Despite the fact that whole-body heterozygotes exhibited comparable growth defects, they were unaffected in these behaviors, but instead exhibited female-specific alterations in learning and memory. These data reveal unexpected phenotypic divergence arising from differences in the spatiotemporal deployment of loss-of-function manipulations, underscoring the importance of context in chromatin remodeller function during neurodevelopment.

Single-cell brain organoid screening identifies developmental defects in autism

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1–4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR–human organoids–single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR–Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

Lmo4 synergizes with Fezf2 to promote direct in vivo reprogramming of upper layer cortical neurons and cortical glia towards deep-layer neuron identities.

In vivo direct neuronal reprogramming relies on the implementation of an exogenous transcriptional program allowing to achieve conversion of a particular neuronal or glial cell type towards a new identity. The transcription factor (TF) Fezf2 is known for its role in neuronal subtype specification of deep-layer (DL) subcortical projection neurons. High ectopic Fezf2 expression in mice can convert both upper-layer (UL) and striatal projection neurons into a corticofugal fate, even if at low efficiency. In this study, we show that Fezf2 synergizes with the nuclear co-adaptor Lmo4 to further enhance reprogramming of UL cortical pyramidal neurons into DL corticofugal neurons, at both embryonic and early postnatal stages. Reprogrammed neurons express DL molecular markers and project toward subcerebral targets, including thalamus, cerebral peduncle (CP), and spinal cord (SC). We also show that co-expression of Fezf2 with the reprogramming factors Neurog2 and Bcl2 in early postnatal mouse glia promotes glia-to-neuron conversion with partial hallmarks of DL neurons and with Lmo4 promoting further morphological complexity. These data support a novel role for Lmo4 in synergizing with Fezf2 during direct lineage conversion in vivo.

BCAT1 controls embryonic neural stem cells proliferation and differentiation in the upper layer neurons

The regulation of neural stem cell (NSC) proliferation and differentiation during brain development is a precisely controlled process, with the production of different neuronal subtypes governed by strict timelines. Glutamate is predominantly used as a neurotransmitter by the subtypes of neurons in the various layers of the cerebral cortex. The expression pattern of BCAT1, a gene involved in glutamate metabolism, in the different layers of neurons has yet to be fully understood. Using single-cell data, we have identified seven different states of NSCs and found that state 4 is closely associated with the development of projection neurons. By inferring the developmental trajectory of different neuronal subtypes from NSC subsets of this state, we discovered that BCAT1 is involved in the regulation of NSC proliferation and differentiation and is specifically highly expressed in layer II/III and IV neurons. Suppression of BCAT1 through shRNA resulted in a reduction in NSC proliferation and an abnormal development of layer II/III and IV neurons. These findings provide new insights into the role of BCAT1 in the regulation of NSC behavior and neuronal development.