The human neocortex underlies higher cognition and is the engine of complex thought. Yet our understanding of its neuronal diversity is limited by sparse access to tissue, inconsistent sampling across studies, and a lack of multiple modality data. Although single-cell transcriptomic taxonomies are an important framework for characterizing cell type diversity, transcriptomic information alone cannot reveal the cellular properties that define neuronal computations. To address this, we performed Patch-seq, a method for collecting Morphology, Electrophysiology, and Transcriptomic data from a single neuron. We focused on glutamatergic, neocortical, excitatory neurons, the principal long-range projecting neurons of the cortex, and systematically integrated their morphoelectric features with transcriptomic identity. In combination with spatial transcriptomic data, we interrogated 39 of 42 transcriptomically-defined neuron types with a layer-centric perspective. Morphoelectric properties, such as cortical depth, apical dendrite structure, and excitability clearly distinguish transcriptomic subclasses and support many finer transcriptomic types. Morphoelectric properties are influenced by spatial location in supragranular layers, while deeper layers exhibit greater heterogeneity. Cross-species comparisons reveal conserved subclass organization but pronounced differences in apical dendrite arborization between mouse and human, and surprising similarities between human and macaque. Together, these datasets provide a unified multimodal reference that advances our understanding of human cortical circuitry and establishes a foundation for experimental and computational studies of human brain function and disease.

Understanding how cell types are organized across the central nervous system (CNS) is key to uncovering neural function. Here, we integrate single-nucleus Multiome (RNA+ATAC) sequencing, spatial transcriptomics, and computational analyses to map conserved cell type signatures in the adult mouse brainstem and spinal cord. We identify a shared core of neuronal and non-neuronal cell types, alongside region-specific specializations reflecting distinct functions. Spatial data reveal conserved cellular niches across the brainstem-spinal cord boundary, indicating a continuous organizational logic. Cross-region comparisons uncover recurrent gene expression modules and signaling programs that may support shared circuit features. Chromatin accessibility profiling highlights cell-type-specific regulatory programs and implicates Hox transcription factors in positional identity. Notably, cell-type and positional identities are largely orthogonal, with varying regional influence across neuronal classes: motor neurons show strong positional coupling, whereas glutamatergic and GABAergic interneurons show minimal entrainment. This work provides a reference for the shared molecular architecture of these CNS regions.

The basal ganglia (BG) are a set of topographically organized, interconnected structures that are pivotal for regulating volitional movement and other aspects of cognitive, motivational, and affective behavior. Recently generated taxonomies of transcriptomically-defined cell types (T-types) have revealed both fine-grained distinctions in gene expression between neurons in these structures as well as continuous transcriptomic variation across similar T-types1-9, which are both related to location within a structure. However, it remains unclear to what extent these and other cellular properties co-vary with each other. Therefore, we performed Patch-seq experiments10,11 on over 900 neurons in mouse brain slices from BG to provide an integrated view of the co-variation between gene expression, location, physiology, and morphology measured from the same neurons. Medium spiny neurons (MSNs) from both the direct and indirect pathways across the dorsal and ventral striatum follow a gene expression gradient that varies in a dorsolateral to ventromedial direction; we find that this gradient also corresponds with systematic differences in action potential kinetics and dendritic arborization. Our analysis also characterizes additional multimodal dimensions of MSN variation, such as those between direct and indirect pathway neurons and between matrix and striosome neurons. Furthermore, through comparison with Patch-seq data from macaque, we demonstrate that the relationship between the transcriptomic/spatial gradient and electrophysiological and morphological properties is conserved across these two species. We also find that properties of striatal interneurons, such as action potential kinetics, vary across the striatum in a manner consistent with the MSN gradient. Outside the striatum, our multimodal Patch-seq dataset from the globus pallidus, subthalamic nucleus, and substantia nigra enabled us to characterize transcriptomically-defined types and link them to prior descriptions of cell types in these structures. Finally, we examined to what extent the MSN transcriptomic/spatial gradient persisted across different stages of the BG circuit by comparing Patch-seq neurons to reconstructed whole-neuron morphologies and the topography of their interareal projections, finding that the gradient is better preserved in GPe and GPi compared to SNr. Our study links transcriptomic variation across T-types in mouse BG to spatial localization and phenotypic differences at the level of individual cells, improving our understanding of cell type architecture in topographically organized circuits of the brain.

The basal ganglia are evolutionary ancient subcortical nuclei that form interconnected loops with the neocortex and limbic system to regulate movement, learning, habit formation, emotion, and motivation. Their dysfunction contributes to major neurological and psychiatric disorders, yet most cellular-level insights derive from rodent studies, leaving knowledge gaps in humans and translationally relevant primate species. To address this, we generated multi-modal Patch-seq data linking transcriptomic identity with morphological and electrophysiological properties in macaque striatum, the input nucleus of the basal ganglia. We found underappreciated diversity among medium spiny neurons, including non-canonical types, and variation aligned with functional gradients. Interneurons also exhibited spatial variation and even greater morphoelectric diversity, highlighting their functional modularity. Despite broad evolutionary conservation, we identified primate-specific features and key differences from rodent striatal neurons. By integrating molecular classification with cellular properties that shape network function, our findings provide insights into the functional organization of the primate striatum.

Norepinephrine (NE) is released throughout most of the central nervous system by neurons in the locus coeruleus (LC). We found a relationship between the morphologies, gene expression, and activity of LC-NE neurons in mice. Axonal projections of individual neurons were extensive but largely confined to subsets of brain regions. Axonal projections and graded gene expression correlated with locations of cell bodies in LC. In a behavioral task requiring ongoing learning from actions, neurons in dorsal LC projecting to the cerebral cortex were excited when mice made a different choice from the previous one and by reward prediction errors, a signal driving learning. Background activity of neurons in ventral LC was higher when mice ignored stimuli indicating potential reward availability. These observations reveal a topographically organized structure and function of a neurotransmitter system and show that it contains learning signals for flexible behavior.

The mammalian basal ganglia (BG) orchestrate motor, cognitive, and affective functions, yet cell type-specific genetic access remains limited, especially beyond rodents. Key structures implicated in movement and psychiatric disorders, including pallidum, subthalamic nucleus, and dopaminergic midbrain, lack scalable tools for cross-species targeting. Here, we present a comprehensive enhancer-AAV library enabling selective labeling and manipulation of major BG neuronal populations: striatal projection neuron subtypes, pallidal and subthalamic neurons, and midbrain dopaminergic and GABAergic populations. Using an evolutionarily informed discovery pipeline, we identified enhancers targeting canonical, non-canonical, and disease-relevant cell types, with validation demonstrating robust cross-species conservation of specificity between mouse and macaque. Computational modeling revealed sequence features predictive of in vivo performance, including motif grammar, chromatin accessibility, and evolutionary conservation, and identified distinct regulatory architectures across glial, projection, and interneuron lineages. This work establishes a comprehensive cross-species viral toolkit for the BG, unlocking previously inaccessible cell types for circuit dissection.

The mammalian brain consists of diverse neuron types with various functions. Recent single-cell RNA sequencing approaches have led to a whole-brain taxonomy of transcriptomically defined cell types1. Patch-seq experiments augment these cell-type descriptions by linking transcriptomic profiles with local morphological and electrophysiological properties2-7. However, linking transcriptomic identities to long-range axonal projections remains a major unresolved challenge. Here, to address this, we collected two datasets from the mouse visual cortex consisting of: (1) 1,528 excitatory Patch-seq neurons, with local morphological, electrophysiological and transcriptomic data collected from each cell, and (2) 341 excitatory, whole-neuron morphologies. From the Patch-seq data, we defined 17 morphoelectric-transcriptomic types and built a multistep classifier to integrate cell-type assignments with whole-neuron morphology and interrogate cross-modality relationships. We find that transcriptomic variation within and across morphoelectric-transcriptomic types corresponds with morphological and electrophysiological phenotypes. In addition, these gene expression patterns, along with the anatomical location of the cell, can be used to predict projection targets of individual neurons. We observed novel multimodal cell-type signatures for layer 5 intratelencephalic and extratelencephalic neurons and shed new light on their axonal circuitry, including interhemispheric intratelencephalic projections. With this approach, we establish a comprehensive, integrated taxonomy of cortical, excitatory neuron types, and create a system for high-dimensional cell-type classification that can be extended to the whole brain and potentially across species.

The basal ganglia (BG) are conserved brain regions essential for motor control, learning, emotion, and cognition, and are implicated in neurological and psychiatric disease. Yet a unified cross-species taxonomy of BG cell types is lacking, limiting translation of BG circuit mechanisms, interpretation of human genetic risk, and development of cell type-targeted tools. We present a multiomic consensus atlas of 1.8 million nuclei from human, macaque, and marmoset spanning eight BG structures. Integrating cross-species gene expression, open chromatin, and spatial profiling enables definition of conserved and divergent cell types. Alignment to existing mouse and human atlases identifies 61 homologous cell types conserved over 80 million years. We identify a STRd D2 StrioMat Hybrid medium spiny neuron (MSN) type with molecular, electrophysiological, and morphological features that clarify hybrid MSN identities. Comparative cis-regulatory analysis reveals conserved sequence grammars that encode cell identity and inform viral targeting strategies, providing a foundational resource for BG evolution, function, and disease.

We present an enhancer-AAV toolbox for accessing and perturbing striatal cell types and circuits. Best-in-class vectors were curated for accessing major striatal neuron populations including medium spiny neurons (MSNs), direct- and indirect-pathway MSNs, Sst-Chodl, Pvalb-Pthlh, and cholinergic interneurons. Specificity was evaluated by multiple modes of molecular validation, by three different routes of virus delivery, and with diverse transgene cargos. Importantly, we provide detailed information necessary to achieve reliable cell-type-specific labeling under different experimental contexts. We demonstrate direct pathway circuit-selective optogenetic perturbation of behavior and multiplex labeling of striatal interneuron types for targeted analysis of cellular features. Lastly, we show conserved in vivo activity for exemplary MSN enhancers in rats and macaques. This collection of striatal enhancer AAVs offers greater versatility compared to available transgenic lines and can readily be applied for cell type and circuit studies in diverse mammalian species beyond the mouse model.

Proper brain function requires the assembly and function of diverse populations of neurons and glia. Single cell gene expression studies have mostly focused on characterization of neuronal cell diversity; however, recent studies have revealed substantial diversity of glial cells, particularly astrocytes. To better understand glial cell types and their roles in neurobiology, we built a new suite of adeno-associated viral (AAV)-based genetic tools to enable genetic access to astrocytes and oligodendrocytes. These oligodendrocyte and astrocyte enhancer-AAVs are highly specific (usually > 95% cell type specificity) with variable expression levels, and our astrocyte enhancer-AAVs show multiple distinct expression patterns reflecting the spatial distribution of astrocyte cell types. To provide the best glial-specific functional tools, several enhancer-AAVs were: optimized for higher expression levels, shown to be functional and specific in rat and macaque, shown to maintain specific activity in epilepsy where traditional promoters changed activity, and used to drive functional transgenes in astrocytes including Cre recombinase and acetylcholine-responsive sensor iAChSnFR. The astrocyte-specific iAChSnFR revealed a clear reward-dependent acetylcholine response in astrocytes of the nucleus accumbens during reinforcement learning. Together, this collection of glial enhancer-AAVs will enable characterization of astrocyte and oligodendrocyte populations and their roles across species, disease states, and behavioral epochs.

The mammalian cortex is composed of a highly diverse set of cell types and develops through a series of temporally regulated events1-3. Single-cell transcriptomics enables a systematic study of cell types across the entire timeline of cortical development. Here we present a comprehensive and high-resolution transcriptomic and epigenomic cell-type atlas of the developing mouse visual cortex. The atlas is built from a single-cell RNA sequencing dataset of 568,654 high-quality single-cell transcriptomes and a single-nucleus Multiome dataset of 200,061 high-quality nuclei, which were densely sampled across the embryonic and postnatal developmental stages (from embryonic day 11.5 to postnatal day 56). We computationally reconstructed a transcriptomic developmental trajectory map of all excitatory, inhibitory and non-neuronal cell types in the visual cortex. Branching points that mark the emergence of new cell types at specific developmental ages and molecular signatures of cellular diversification are identified. The trajectory map shows that neurogenesis, gliogenesis and early postmitotic maturation in the embryonic stage give rise to all cell classes and nearly all subclasses in a staggered parallel manner. Increasingly refined cell types emerge throughout the postnatal differentiation process, including the late emergence of many cell types during the eye-opening stage and the onset of critical period, suggesting that there is continuous cell-type diversification at different stages of cortical development. Throughout development, there are cooperative dynamic changes in gene expression and chromatin accessibility in specific cell types. We identify cell-type-specific and temporally resolved gene regulatory networks that link transcription factors and downstream target genes through accessible chromatin motifs. Collectively, our study provides a detailed dynamic molecular map directly associated with individual cell types and specific temporal events that can reveal the molecular logic underlying the complex and multifaceted cortical cell type and circuit development.

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to spinal motor neurons (SMNs) and SMN subtypes, we generated single-cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors driving a reporter fluorophore and functionally screened them in the mouse. We extensively characterized the most promising candidate enhancers in rat and macaque and developed an optimized pan-SMN enhancer virus. Additionally, we generated derivative viruses expressing iCre297T recombinase or ChR2-EYFP for labeling and functional studies, and we created a single vector with combined enhancer elements to achieve simultaneous labeling of layer 5 extratelencephalic projecting neurons and SMNs. This unprecedented SMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.

The telencephalon of the mammalian brain contains multiple regions and circuits that have adaptive and integrative roles in a variety of brain functions. GABAergic neurons in the telencephalon are diverse; they have many circuit functions, and dysfunction of these neurons has been implicated in various brain disorders1-3. Here we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished using 611,423 young adult single-cell transcriptomes and 614,569 single-cell transcriptomes collected from multiple prenatal and postnatal developmental timepoints. We present a hierarchically organized adult telencephalic GABAergic neuronal cell-type taxonomy of 7 classes, 52 subclasses, 284 supertypes and 1,051 clusters, as well as a corresponding developmental taxonomy of 1,688 clusters across ages from embryonic day 7 to postnatal day 14. Detailed charting efforts reveal extraordinary complexity whereby relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types are often found in distant and diverse brain regions, indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Moreover, we find various spatial dimensions of both discrete and continuous variation among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal, preoptic and most pallidal GABAergic neuronal types emerge in a burst during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell-type taxonomy will serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We use transcriptomic and epigenomic cortical cell-type taxonomies from mouse and human to define marker genes and putative enhancers and create a large toolkit of transgenic lines and enhancer adeno-associated viruses (AAVs) for selective targeting of cortical cell populations. We report creation and evaluation of fifteen transgenic driver lines, two reporter lines, and >1,000 different enhancer AAV vectors covering most subclasses of cortical cells. The tools reported here have been made publicly available, and along with the scaled process of tool creation, evaluation, and modification, they will enable diverse experimental strategies toward understanding mammalian cortex and brain function.

Alzheimer’s disease (AD) is clinically characterized by a progressive cognitive decline associated with stereotyped accumulation of amyloid-beta (Aβ) plaques and hyperphosphorylated Tau (pTau) tangles across brain regions. While histopathology has revealed patterns of regional involvement, the molecular and cellular events that accompany and potentially drive this progression remain incompletely understood. We applied single nucleus RNA sequencing (RNAseq), ATAC-seq, and Multiome profiling to over 7 million high-quality nuclei from 10 brain regions—spanning medial and lateral entorhinal cortices, hippocampus, multiple temporal and frontal cortical areas, and primary visual cortex—sampled from the same cohort of 84 aged human donors across the AD spectrum (3 regions from all donors, 7 from those without severe co-morbidities). We predicted the cell-type for each nucleus by mapping to an expanded BRAIN initiative cell-type taxonomy, which included AD-associated non-neuronal states and ∼70 brain region-specific neuronal types. These datasets were paired with regional quantitative measurements of Aβ (6e10), pTau (AT8), and other protein pathologies, as well as cellular stains for neurons, microglia, and astrocytes. We inferred two distinct protein pathology accumulation patterns across brain regions: neocortical areas accumulated Aβ prior to pTau, whereas hippocampus and entorhinal cortex had early and, in some cases, substantial pTau burden independent of Aβ. Among neocortical regions, accumulation of AT8 beyond the temporal medial lobe strongly associated with dementia. In analyzing cell-type abundance differences associated with higher levels of pTau pathology, we identified shared motifs of selective neuronal loss consistent with our previous observations from the middle temporal gyrus. These included loss of L2/3 and L5 intratelencephalic excitatory neurons and several types of inhibitory interneurons (e.g., Vip, Sst, Pvalb). These same vulnerable inhibitory types were also reduced in hippocampal and entorhinal regions, when present, and we observed parallel increases in astrocyte, microglial, and oligodendrocyte precursor cells. Additionally, we observed a decrease in specific, regionally specialized neuron subtypes in the hippocampus, entorhinal cortex, and visual cortex. Our multimodal, multi-region single-cell atlas reveals common and region-specific patterns of cellular vulnerability in AD. These cell-types, particularly those commonly affected in distinct neural circuits, could serve as candidate therapeutic targets and biomarkers.

Biological ageing can be defined as a gradual loss of homeostasis across various aspects of molecular and cellular function(1,2). Mammalian brains consist of thousands of cell types(3), which may be differentially susceptible or resilient to ageing. Here we present a comprehensive single-cell RNA sequencing dataset containing roughly 1.2million high-quality single-cell transcriptomes of brain cells from young adult and aged mice of both sexes, from regions spanning the forebrain, midbrain and hindbrain. High-resolution clustering of all cells results in 847 cell clusters and reveals at least 14 age-biased clusters that are mostly glial types. At the broader cell subclass and supertype levels, we find age-associated gene expression signatures and provide a list of 2,449 unique differentially expressed genes (age-DE genes) for many neuronal and non-neuronal cell types. Whereas most age-DE genes are unique to specific cell types, we observe common signatures with ageing across cell types, including a decrease in expression of genes related to neuronal structure and function in many neuron types, major astrocyte types and mature oligodendrocytes, and an increase in expression of genes related to immune function, antigen presentation, inflammation, and cell motility in immune cell types and some vascular cell types. Finally, we observe that some of the cell types that demonstrate the greatest sensitivity to ageing are concentrated around the third ventricle in the hypothalamus, including tanycytes, ependymal cells, and certain neuron types in the arcuate nucleus, dorsomedial nucleus and paraventricular nucleus that express genes canonically related to energy homeostasis. Many of these types demonstrate both a decrease in neuronal function and an increase in immune response. These findings suggest that the third ventricle in the hypothalamus may be a hub for ageing in the mouse brain. Overall, this study systematically delineates a dynamic landscape of cell-type-specific transcriptomic changes in the brain associated with normal ageing that will serve as a foundation for the investigation of functional changes in ageing and the interaction of ageing and disease.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

Allele-specific methylation (ASM) is an epigenetic modification whereby one parental allele becomes methylated and the other unmethylated at a specific locus. ASM is most often driven by the presence of nearby heterozygous variants that influence methylation, but also occurs somatically in the context of genomic imprinting. In this study, we investigate ASM using publicly available single-cell reduced representation bisulfite sequencing (scRRBS) data on 608 B cells sampled from six healthy B cell samples and 1,230 cells from 11 chronic lymphocytic leukemia (CLL) samples. We developed a likelihood-based criterion to test whether a CpG exhibited ASM, based on the distributions of methylated and unmethylated reads both within and across cells. Applying our likelihood ratio test, 65,998 CpG sites exhibited ASM in healthy B cell samples according to a Bonferroni criterion (p < 8.4 x 10(-9)), and 32,862 CpG sites exhibited ASM in CLL samples (p < 8.5 x 10(-9)). We also called ASM at the sample level. To evaluate the accuracy of our method, we called heterozygous variants from the scRRBS data, which enabled variant-based calls of ASM within each cell. Comparing sample-level ASM calls to the variant-based measures of ASM, we observed a positive predictive value of 76%-100% across samples. We observed high concordance of ASM across samples and an overrepresentation of ASM in previously reported imprinted genes and genes with imprinting binding motifs. Our study demonstrates that single-cell bisulfite sequencing is a potentially powerful tool to investigate ASM, especially as studies expand to increase the number of samples and cells sequenced.

The mammalian cortex is composed of a highly diverse set of cell types and develops through a series of temporally regulated events that build out the cell type and circuit foundation for cortical function. The mechanisms underlying the development of different cell types remain elusive. Single-cell transcriptomics provides the capacity to systematically study cell types across the entire temporal range of cortical development. Here, we present a comprehensive and high-resolution transcriptomic and epigenomic cell type atlas of the developing mouse visual cortex. The atlas was built from a single-cell RNA-sequencing dataset of 568,674 high-quality single-cell transcriptomes and a single-nucleus Multiome dataset of 194,545 high-quality nuclei providing both transcriptomic and chromatin accessibility profiles, densely sampled throughout the embryonic and postnatal developmental stages from E11.5 to P56. We computationally reconstructed a transcriptomic developmental trajectory map of all excitatory, inhibitory, and non-neuronal cell types in the visual cortex, identifying branching points marking the emergence of new cell types at specific developmental ages and defining molecular signatures of cellular diversification. In addition to neurogenesis, gliogenesis and early postmitotic maturation in the embryonic stage which gives rise to all the cell classes and nearly all subclasses, we find that increasingly refined cell types emerge throughout the postnatal differentiation process, including the late emergence of many cell types during the eye-opening stage (P11-P14) and the onset of critical period (P21), suggesting continuous cell type diversification at different stages of cortical development. Throughout development, we find cooperative dynamic changes in gene expression and chromatin accessibility in specific cell types, identifying both chromatin peaks potentially regulating the expression of specific genes and transcription factors potentially regulating specific peaks. Furthermore, a single gene can be regulated by multiple peaks associated with different cell types and/or different developmental stages. Collectively, our study provides the most detailed dynamic molecular map directly associated with individual cell types and specific developmental events that reveals the molecular logic underlying the continuous refinement of cell type identities in the developing visual cortex.