Biology subjects

Walsh, C. A.

Publications and source records attributed to Walsh, C. A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Cell-type-specific effects of autism-associated chromosome 15q11.2-13.1 duplications in human brain

Recurrent copy number variation represents one of the most well-established genetic drivers in neurodevelopmental disorders, including autism spectrum disorder (ASD). Duplication of 15q11.2-13.1 (dup15q) is a well-described neurodevelopmental syndrome that increases the risk of ASD by over 40-fold. However, the effects of this duplication on gene expression and chromatin accessibility in specific cell types in the human brain remain unknown. To identify the cell-type-specific transcriptional and epigenetic effects of dup15q in the human frontal cortex we conducted single-nucleus RNA-sequencing and multi-omic sequencing on dup15q cases (n=6) as well as non-dup15q ASD (n=7) and neurotypical controls (n=7). Cell-type-specific differential expression analysis identified significantly regulated genes, critical biological pathways, and differentially accessible genomic regions. Although there was overall increased gene expression across the duplicated genomic region, cellular identity represented an important factor mediating gene expression changes. Neuronal subtypes, showed greater upregulation of gene expression across a critical region within the duplication as compared to other cell types. Genes within the duplicated region that had high baseline expression in control individuals showed only modest changes in dup15q, regardless of cell type. Of note, dup15q and ASD had largely distinct signatures of chromatin accessibility, but shared the majority of transcriptional regulatory motifs, suggesting convergent biological pathways. However, the transcriptional binding factor motifs implicated in each condition implicated distinct biological mechanisms; neuronal JUN/FOS networks in ASD vs. an inflammatory transcriptional network in dup15q microglia. This work provides a cell-type-specific analysis of how dup15q changes gene expression and chromatin accessibility in the human brain and finds evidence of marked cell-type-specific effects of this genetic driver. These findings have implications for guiding therapeutic development in dup15q syndrome, as well as understanding the functional effects CNVs more broadly in neurodevelopmental disorders.

genetics↗

bioRxiv2024

High-resolution detection of copy number alterations in single cells with HiScanner

Improvements in single-cell whole-genome sequencing (scWGS) assays have enabled detailed characterization of somatic copy number alterations (CNAs) at the single-cell level. Yet, current computational methods are mostly designed for detecting chromosome-scale changes in cancer samples with low sequencing coverage. Here, we introduce HiScanner (High-resolution Single-Cell Allelic copy Number callER), which combines read depth, B-allele frequency, and haplotype phasing to identify CNAs with high resolution. In simulated data, HiScanner consistently outperforms state-of-the-art methods across various CNA types and sizes. When applied to high-coverage scWGS data from 65 cells across 11 neurotypical human brains, HiScanner shows a superior ability to detect smaller CNAs, uncovering distinct CNA patterns between neurons and oligodendrocytes. We also generated low-coverage scWGS data from 179 cells sampled from the same meningioma patient at two time points. For this serial dataset, integration of CNAs with point mutations revealed evolutionary trajectories of tumor cells. These findings show that HiScanner enables accurate characterization of frequency, clonality, and distribution of CNAs at the single-cell level in both non-neoplastic and neoplastic cells.

genomics↗

bioRxiv2024

ARX regulates interneuron subtype differentiation and migration

Mutations in aristaless-related homeobox (ARX) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARXs role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3, Cspr3), fate specification (e.g., Nkx2.1, Maf, Mef2c), and migration (e.g., Nkx2.1, Lmo1, Cxcr4, Nrg1, ErbB4). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

neuroscience↗

bioRxiv2024

Somatic cancer driver mutations are enriched and associated with inflammatory states in Alzheimer's disease microglia

Alzheimers disease (AD) is an age-associated neurodegenerative disorder characterized by progressive neuronal loss and pathological accumulation of the misfolded proteins amyloid-{beta} and tau1,2. Neuroinflammation mediated by microglia and brain-resident macrophages plays a crucial role in AD pathogenesis1-5, though the mechanisms by which age, genes, and other risk factors interact remain largely unknown. Somatic mutations accumulate with age and lead to clonal expansion of many cell types, contributing to cancer and many non-cancer diseases6,7. Here we studied somatic mutation in normal aged and AD brains by three orthogonal methods and in three independent AD cohorts. Analysis of bulk RNA sequencing data from 866 samples from different brain regions revealed significantly higher ([~]two-fold) overall burdens of somatic single-nucleotide variants (sSNVs) in AD brains compared to age-matched controls. Molecular-barcoded deep (>1000X) gene panel sequencing of 311 prefrontal cortex samples showed enrichment of sSNVs and somatic insertions and deletions (sIndels) in cancer driver genes in AD brain compared to control, with recurrent, and often multiple, mutations in genes implicated in clonal hematopoiesis (CH)8,9. Pathogenic sSNVs were enriched in CSF1R+ microglia of AD brains, and the high proportion of microglia (up to 40%) carrying some sSNVs in cancer driver genes suggests mutation-driven microglial clonal expansion (MiCE). Analysis of single-nucleus RNA sequencing (snRNAseq) from temporal neocortex of 62 additional AD cases and controls exhibited nominally increased mosaic chromosomal alterations (mCAs) associated with CH10,11. Microglia carrying mCA showed upregulated pro-inflammatory genes, resembling the transcriptomic features of disease-associated microglia (DAM) in AD. Our results suggest that somatic driver mutations in microglia are common with normal aging but further enriched in AD brain, driving MiCE with inflammatory and DAM signatures. Our findings provide the first insights into microglial clonal dynamics in AD and identify potential new approaches to AD diagnosis and therapy.

genomics↗

bioRxiv2023

Somatic Mosaicism in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Reveals Widespread Degeneration from Focal Mutations

Although mutations in many genes cause familial amyotrophic lateral sclerosis and frontotemporal dementia, most cases are sporadic (sALS and sFTD) with unclear etiology. We tested whether somatic mutations contribute to sALS and sFTD by deep targeted sequencing of 88 neurodegeneration-related genes in postmortem brain and spinal cord samples from 399 sporadic cases and 144 controls. Predicted deleterious somatic variants in ALS/FTD genes were observed in 2.1% of sporadic cases lacking deleterious germline variants. These variants occurred at very low allele fractions (typically <2%) and were often focal and enriched in disease-affected regions. Analysis of bulk RNA-seq data from an additional cohort identified deleterious somatic variants in DYNC1H1 and LMNA, genes associated with pediatric motor neuron degeneration. Targeted long-read sequencing further identified one sFTD case with de novo somatic C9orf72 repeat expansions. Together, these findings suggest that rare, focal somatic variants can contribute to sALS and sFTD and drive widespread neurodegeneration.

genetics↗

bioRxiv2023

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

neuroscience↗

bioRxiv2023

Transcriptional Control of Neocortical Size and Microcephaly

The mammalian neocortex differs vastly in size and complexity between mammalian species, yet the mechanisms that lead to an increase in brain size during evolution are not known. We show here that two transcription factors coordinate gene expression programs in progenitor cells of the neocortex to regulate their proliferative capacity and neuronal output in order to determine brain size. Comparative studies in mice, ferrets and macaques demonstrate an evolutionary conserved function for these transcription factors to regulate progenitor behaviors across the mammalian clade. Strikingly, the two transcriptional regulators control the expression of large numbers of genes linked to microcephaly suggesting that transcriptional deregulation as an important determinant of the molecular pathogenesis of microcephaly, which is consistent with the finding that genetic manipulation of the two transcription factors leads to severe microcephaly.

neuroscience↗

bioRxiv2023

An econometric lens resolves cell-state parallax

Genome-wide association studies (GWAS) identify numerous disease-linked genetic variants at noncoding genomic loci, yet therapeutic progress is hampered by the challenge of deciphering the regulatory roles of these loci in tissue-specific contexts. Single-cell multimodal assays that simultaneously profile chromatin accessibility and gene expression could predict tissue-specific causal links between noncoding loci and the genes they affect. However, current computational strategies either neglect the causal relationship between chromatin accessibility and transcription or lack variant-level precision, aggregating data across genomic ranges due to data sparsity. To address this, we introduce GrID-Net, a graph neural network approach that generalizes Granger causal inference to detect new causal locus-gene associations in graph-structured systems such as single-cell trajectories. Inspired by the principles of optical parallax, which reveals object depth from static snapshots, we hypothesized that causal mechanisms could be inferred from static single-cell snapshots by exploiting the time lag between epigenetic and transcriptional cell states, a concept we term "cell-state parallax." Applying GrID-Net to schizophrenia (SCZ) genetic variants, we increased variant coverage by 36% and uncovered noncoding mechanisms that dysregulate 132 genes, including key potassium transporters such as KCNG2 and SLC12A6. Furthermore, we discovered evidence for the prominent role of neural transcription-factor binding disruptions in SCZ etiology. Our work not only provides a strategy for elucidating the tissue-specific impact of noncoding variants but also underscores the breakthrough potential of cell-state parallax in single-cell multiomics for discovering tissue-specific gene regulatory mechanisms.

bioinformatics↗

bioRxiv2023

Contrasting patterns of somatic mutations in neurons and glia reveal differential predisposition to disease in the aging human brain

Characterizing the mechanisms of somatic mutations in the brain is important for understanding aging and disease, but little is known about the mutational patterns of different cell types. We performed whole-genome sequencing of 71 oligodendrocytes and 51 neurons from neurotypical individuals (0.4 to 104 years old) and identified >67,000 somatic single nucleotide variants (sSNVs) and small insertions and deletions (indels). While both cell types accumulate mutations with age, oligodendrocytes accumulate sSNVs 69% faster than neurons (27/year versus 16/year) whereas indels accumulate 42% slower (1.8/year versus 3.1/year). Correlation with single-cell RNA and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These patterns highlight differences in the mutagenic processes in glia and neurons and suggest cell type-specific, age-related contributions to neurodegeneration and oncogenesis.

neuroscience↗

bioRxiv2022

Comparative transcriptomics reveals human-specific cortical features

Humans have unique cognitive abilities among primates, including language, but their molecular, cellular, and circuit substrates are poorly understood. We used comparative single nucleus transcriptomics in adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets from the middle temporal gyrus (MTG) to understand human-specific features of cellular and molecular organization. Human, chimpanzee, and gorilla MTG showed highly similar cell type composition and laminar organization, and a large shift in proportions of deep layer intratelencephalic-projecting neurons compared to macaque and marmoset. Species differences in gene expression generally mirrored evolutionary distance and were seen in all cell types, although chimpanzees were more similar to gorillas than humans, consistent with faster divergence along the human lineage. Microglia, astrocytes, and oligodendrocytes showed accelerated gene expression changes compared to neurons or oligodendrocyte precursor cells, indicating either relaxed evolutionary constraints or positive selection in these cell types. Only a few hundred genes showed human-specific patterning in all or specific cell types, and were significantly enriched near human accelerated regions (HARs) and conserved deletions (hCONDELS) and in cell adhesion and intercellular signaling pathways. These results suggest that relatively few cellular and molecular changes uniquely define adult human cortical structure, particularly by affecting circuit connectivity and glial cell function.

neuroscience↗

bioRxiv2022

Glial dysregulation in human brain in Fragile X-related disorders

AbstractWhile large trinucleotide repeat expansions at the FMR1 locus cause Fragile X Syndrome (FXS), smaller "premutations" are associated with the late-onset condition Fragile X-associated tremor/ataxia syndrome (FXTAS), which shows very different clinical and pathological features, with no clear molecular explanation for these marked differences. One prevailing theory posits that the premutation uniquely causes neurotoxic increases in FMR1 mRNA (i.e., 4-8-fold increases), but evidence to support this hypothesis is largely derived from analysis of peripheral blood. We applied single- nucleus RNA-sequencing to post-mortem frontal cortex and cerebellum from 9 individuals with Fragile X mutations as well as age and sex matched controls (n=6) to assess cell-type specific molecular neuropathology. We found robust reduction of FMR1 mRNA in FXS as expected, with modest but significant upregulation ([~]1.3 fold) of FMR1 in glial clusters associated with premutation expansions. In premutation cases we identified alterations in glia number in cortex and cerebellum. Differential expression analysis demonstrated altered cortical oligodendrocyte development, while gene ontology analysis revealed alterations in neuroregulatory roles of glia, such as glial modulation of neurotransmission and synaptic structure. We identified significant enrichment of known FMR1 protein target genes in differentially expressed gene lists in FXS as well as the premutation, suggesting FMR1 protein target pathways may represent a shared source of dysfunction in both conditions despite opposite FMR1 mRNA changes. These findings challenge existing dogma regarding FXTAS and implicate glial dysregulation as a critical facet of premutation pathophysiology, representing novel therapeutic targets directly derived from the human condition.

neuroscience↗

bioRxiv2022

Enrichment of somatic mutations in schizophrenia brain targets prenatally active transcription factor bindings sites

Schizophrenia (SCZ) is a complex neuropsychiatric disorder in which both germline genetic mutations and maternal factors, such as infection and immune activation, have been implicated, but how these two strikingly different mechanisms might converge on the same phenotype is unknown. During development, cells accumulate somatic, mosaic mutations in ways that can be shaped by the cellular environment or endogenous processes, but these early developmental mutational patterns have not been studied in SCZ. Here we analyzed deep (267x) whole-genome sequencing (WGS) of DNA from cerebral cortical neurons isolated from 61 SCZ and 25 control postmortem brains to capture mutations occurring before or during fetal neurogenesis. SCZ cases showed a >15% increase in genome-wide sSNV compared to controls (p < 2e-10). Remarkably, mosaic T>G mutations and CpG transversions (CpG>GpG or CpG>ApG) were 79- and 62-fold enriched, respectively, at transcription factor binding sites (TFBS) in SCZ, but not in controls. The pattern of T>G mutations resembles mutational processes in cancer attributed to oxidative damage that is sterically blocked from DNA repair by transcription factors (TFs) bound to damaged DNA. The CpG transversions similarly suggest unfinished DNA demethylation resulting in abasic sites that can also be blocked from repair by bound TFs. Allele frequency analysis suggests that both localized mutational spikes occur in the first trimester. We call this prenatal mutational process "skiagenesis" (from the Greek skia, meaning shadow), as these mutations occur in the shadow of bound TFs. Skiagenesis reflects as-yet unidentified prenatal factors and is associated with SCZ risk in a subset ([~]13%) of cases. In turn, mutational disruption of key TFBS active in fetal brain is well positioned to create SCZ-specific gene dysregulation in concert with germline risk genes. Skiagenesis provides a fingerprint for exploring how epigenomic regulation and prenatal factors such as maternal infection or immune activation may shape the developmental mutational landscape of human brain.

genomics↗

bioRxiv2021

Ultraspecific somatic SNV and indel detection in single neurons using primary template-directed amplification

Primary template-directed amplification (PTA) is an improved amplification technique for single-cell DNA sequencing. We generated whole-genome analysis of 76 single neurons and developed SCAN2, a computational method to accurately identify both clonal and non-clonal somatic (i.e., limited to a single neuron) single nucleotide variants (SNVs) and small insertions and deletions (indels) using PTA data. Our analysis confirms an increase in non-clonal somatic mutation in single neurons with age, but revises estimates for the rate of this accumulation to be 15 SNVs per year. We also identify artifacts in other amplification methods. Most importantly, we show that somatic indels also increase by at least 2 indels per year per neuron and that indels may have a larger impact on gene function than somatic SNVs in human neurons.

genomics↗

bioRxiv2021

Whole-genome analysis of de novo and polymorphic retrotransposon insertions in Autism Spectrum Disorder

Retrotransposons are dynamic forces in evolutionary genomics and have been implicated as causes of Mendelian disease and hereditary cancer, but their role in Autism Spectrum Disorder (ASD) has never been systematically defined. Here, we report 86,154 polymorphic retrotransposon insertions including >60% not previously reported and 158 de novo retrotransposition events identified in whole genome sequencing (WGS) data of 2,288 families with ASD from the Simons Simplex Collection (SSC). As expected, the overall burden of de novo events was similar between ASD individuals and unaffected siblings, with 1 de novo insertion per 29, 104, and 192 births for Alu, L1, and SVA respectively, and 1 de novo insertion per 20 births total, while the location of transposon insertions differed between ASD and unaffected individuals. ASD cases showed more de novo L1 insertions than expected in ASD genes, and we also found de novo intronic retrotransposition events in known syndromic ASD genes in affected individuals but not in controls. Additionally, we observed exonic insertions in genes with a high probability of being loss-of-function intolerant, including a likely causative exonic insertion in CSDE1, only in ASD individuals. Although de novo retrotransposition occurs less frequently than single nucleotide and copy number variants, these findings suggest a modest, but important, impact of intronic and exonic retrotransposition mutations in ASD and highlight the utility of developing specific bioinformatic tools for high-throughput detection of transposable element insertions.

genetics↗

bioRxiv2020

The polymicrogyria-associated GPR56 promoter preferentially drives gene expression in developing GABAergic neurons in common marmosets

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

neuroscience↗

bioRxiv2020

Comprehensive identification of somatic nucleotide variants in human brain tissue

Post-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells. Here, the Brain Somatic Mosaicism Network conducted a coordinated, multi-institutional study to: (i) examine the ability of existing methods to detect simulated somatic single nucleotide variants (SNVs) in DNA mixing experiments; (ii) generate multiple replicates of whole genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual; (iii) devise strategies to discover somatic SNVs; and (iv) apply various approaches to validate somatic SNVs. These efforts led to the identification of 43 bona fide somatic SNVs that ranged in variant allele fractions from ~0.005 to ~0.28. Guided by these results, we devised best practices for calling mosaic SNVs from 250X whole genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrated that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees. Thus, this study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

genomics↗

bioRxiv2020

Role for the Na+/K+ ATPase α 3 subunit in folding and lamination of the human neocortex

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function that is catalyzed by the Na,K-ATPase alpha subunit. In vertebrates, four paralogous genes, ATP1A1-4, encode distinct alpha subunit isoforms (a1-a4), three of which (a1, a2, a3) are expressed in the brain, and two (a1, a3) predominantly in neurons. The a3 isoform, encoded by ATP1A3, is critical to neuronal physiology, and a growing spectrum of neurological diseases are associated with ATP1A3 pathogenic variants, with ages of onset ranging from early childhood to adulthood. Here, we describe ATP1A3 variants encoding dysfunctional a3 subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we established a transcriptional atlas of ATP1A3 expression during cortical development using mRNA in situ hybridization and transcriptomic profiling of ~125,000 individual cells with single-cell RNA sequencing (Drop-Seq) from various areas of the midgestational human neocortex. We find that fetal expression of ATP1A3 is restricted to a subset of excitatory neurons carrying transcriptional signatures of neuronal activity and maturation characteristic of the developing subplate. Furthermore, by performing Drop-Seq on ~52,000 nuclei from four different areas of an infant human neocortex, we show that ATP1A3 expression persists throughout early postnatal development, not only within excitatory neurons across all cortical layers, but also and more predominantly in inhibitory neurons, with specific enrichment in fast-spiking basket cells. In addition, we show that ATP1A3 expression, both in fetal and postnatal neurons, tends to be higher in frontal cortical areas than in occipital areas, in a pattern consistent with the rostro-caudal maturation gradient of the human neocortex. Finally, we discover distinct co-expression patterns linking catalytic subunit isoforms (ATP1A1,2,3) and auxiliary isoforms (ATP1B1,2,3), suggesting the ATPase pump may form non-redundant, cell-type specific -{beta} combinations. Together, the importance of the developmental phenotypes and dynamic expression patterns of ATP1A3 point to a key role for a3 in the development and function of human cortex.

neuroscience↗

bioRxiv2020

Parallel RNA and DNA analysis after Deep-sequencing (PRDD-seq) reveals cell type-specific lineage patterns in human brain

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed Parallel RNA and DNA analysis after Deep-sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables the first-ever simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows the first estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition. Significance StatementStem cells and progenitors undergo a series of cell divisions to generate the neurons of the brain, and understanding this sequence is critical to studying the mechanisms that control cell division and migration in developing brain. Mutations that occur as cells divide are known as the basis of cancer, but have more recently been shown to occur with normal cell divisions, creating a permanent, forensic map of the clonal patterns that define the brain. Here we develop new technology to analyze both DNA mutations and RNA gene expression patterns in single cells from human postmortem brain, allowing us to define clonal patterns among different types of human brain neurons, gaining the first direct insight into how they form.

neuroscience↗