Alternative platelet differentiation pathways initiated by nonhierarchically related hematopoietic stem cells.

Rare multipotent stem cells replenish millions of blood cells per second through a time-consuming process, passing through multiple stages of increasingly lineage-restricted progenitors. Although insults to the blood-forming system highlight the need for more rapid blood replenishment from stem cells, established models of hematopoiesis implicate only one mandatory differentiation pathway for each blood cell lineage. Here, we establish a nonhierarchical relationship between distinct stem cells that replenish all blood cell lineages and stem cells that replenish almost exclusively platelets, a lineage essential for hemostasis and with important roles in both the innate and adaptive immune systems. These distinct stem cells use cellularly, molecularly and functionally separate pathways for the replenishment of molecularly distinct megakaryocyte-restricted progenitors: a slower steady-state multipotent pathway and a fast-track emergency-activated platelet-restricted pathway. These findings provide a framework for enhancing platelet replenishment in settings in which slow recovery of platelets remains a major clinical challenge.

Molecular architecture of the developing mouse brain.

The mammalian brain develops through a complex interplay of spatial cues generated by diffusible morphogens, cell-cell interactions and intrinsic genetic programs that result in probably more than a thousand distinct cell types. A complete understanding of this process requires a systematic characterization of cell states over the entire spatiotemporal range of brain development. The ability of single-cell RNA sequencing and spatial transcriptomics to reveal the molecular heterogeneity of complex tissues has therefore been particularly powerful in the nervous system. Previous studies have explored development in specific brain regions1-8, the whole adult brain9 and even entire embryos10. Here we report a comprehensive single-cell transcriptomic atlas of the embryonic mouse brain between gastrulation and birth. We identified almost eight hundred cellular states that describe a developmental program for the functional elements of the brain and its enclosing membranes, including the early neuroepithelium, region-specific secondary organizers, and both neurogenic and gliogenic progenitors. We also used in situ mRNA sequencing to map the spatial expression patterns of key developmental genes. Integrating the in situ data with our single-cell clusters revealed the precise spatial organization of neural progenitors during the patterning of the nervous system.

Cellular projections from sensory hair cells form polarity-specific scaffolds during synaptogenesis.

The assembly of a nervous system requires the extension of axons and dendrites to specific regions where they are matched with appropriate synaptic targets. Although the cues that guide long-range outgrowth have been characterized extensively, additional mechanisms are required to explain short-range guidance in neural development. Using a complementary combination of time-lapse imaging by fluorescence confocal microscopy and serial block-face electron microscopy, we identified a novel type of presynaptic projection that participates in the assembly of the vertebrate nervous system. Synapse formation by each hair cell of the zebrafish's lateral line occurs during a particular interval after the cell's birth. During the same period, projections emerge from the cellular soma, extending toward a specific subpopulation of mature hair cells and interacting with polarity-specific afferent nerve terminals. The terminals then extend along the projections to reach appropriately matched presynaptic sites, after which the projections recede. Our results suggest that presynaptic projections act as transient scaffolds for short-range partner matching, a mechanism that may occur elsewhere in the nervous system.

Daple coordinates organ-wide and cell-intrinsic polarity to pattern inner-ear hair bundles.

The establishment of planar polarization by mammalian cells necessitates the integration of diverse signaling pathways. In the inner ear, at least two systems regulate the planar polarity of sensory hair bundles. The core planar cell polarity (PCP) proteins coordinate the orientations of hair cells across the epithelial plane. The cell-intrinsic patterning of hair bundles is implemented independently by the G protein complex classically known for orienting the mitotic spindle. Although the primary cilium also participates in each of these pathways, its role and the integration of the two systems are poorly understood. We show that Dishevelled-associating protein with a high frequency of leucine residues (Daple) interacts with PCP and cell-intrinsic signals. Regulated by the cell-intrinsic pathway, Daple is required to maintain the polarized distribution of the core PCP protein Dishevelled and to position the primary cilium at the abneural edge of the apical surface. Our results suggest that the primary cilium or an associated structure influences the domain of cell-intrinsic signals that shape the hair bundle. Daple is therefore essential to orient and pattern sensory hair bundles.

GABAergic neuronal lineage development determines clinically actionable targets in diffuse hemispheric glioma, H3G34-mutant.

Diffuse hemispheric gliomas, H3G34R/V-mutant (DHG-H3G34), are lethal brain tumors lacking targeted therapies. They originate from interneuronal precursors; however, leveraging this origin for therapeutic insights remains unexplored. Here, we delineate a cellular hierarchy along the interneuron lineage development continuum, revealing that DHG-H3G34 mirror spatial patterns of progenitor streams surrounding interneuron nests, as seen during human brain development. Integrating these findings with genome-wide CRISPR-Cas9 screens identifies genes upregulated in interneuron lineage progenitors as major dependencies. Among these, CDK6 emerges as a targetable vulnerability: DHG-H3G34 tumor cells show enhanced sensitivity to CDK4/6 inhibitors and a CDK6-specific degrader, promoting a shift toward more mature interneuron-like states, reducing tumor growth, and prolonging xenograft survival. Notably, a patient with progressive DHG-H3G34 treated with a CDK4/6 inhibitor achieved 17 months of stable disease. This study underscores interneuronal progenitor-like states, organized in characteristic niches, as a distinct vulnerability in DHG-H3G34, highlighting CDK6 as a promising clinically actionable target.

Single-cell genomics reveals region-specific developmental trajectories underlying neuronal diversity in the human hypothalamus.

The development and diversity of neuronal subtypes in the human hypothalamus has been insufficiently characterized. To address this, we integrated transcriptomic data from 241,096 cells (126,840 newly generated) in the prenatal and adult human hypothalamus to reveal a temporal trajectory from proliferative stem cell populations to mature hypothalamic cell types. Iterative clustering of the adult neurons identified 108 robust transcriptionally distinct neuronal subtypes representing 10 hypothalamic nuclei. Pseudotime trajectories provided insights into the genes driving formation of these nuclei. Comparisons to single-cell transcriptomic data from the mouse hypothalamus suggested extensive conservation of neuronal subtypes despite certain differences in species-enriched gene expression. The uniqueness of hypothalamic neuronal lineages was examined developmentally by comparing excitatory lineages present in cortex and inhibitory lineages in ganglionic eminence, revealing both distinct and shared drivers of neuronal maturation across the human forebrain. These results provide a comprehensive transcriptomic view of human hypothalamus development through gestation and adulthood at cellular resolution.

Transcriptomic diversity of cell types across the adult human brain.

The human brain directs complex behaviors, ranging from fine motor skills to abstract intelligence, but the diversity of cell types that support these skills has not been fully described. In this work, we used single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain. We sampled more than three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain in three postmortem donors. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins and revealing high diversity in midbrain and hindbrain neurons. Astrocytes and oligodendrocyte-lineage cells also exhibited regional diversity at multiple scales. The transcriptomic census of the entire human brain presented in this work provides a resource for understanding the molecular diversity of the human brain in health and disease.

A comparative atlas of single-cell chromatin accessibility in the human brain.

Recent advances in single-cell transcriptomics have illuminated the diverse neuronal and glial cell types within the human brain. However, the regulatory programs governing cell identity and function remain unclear. Using a single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq), we explored open chromatin landscapes across 1.1 million cells in 42 brain regions from three adults. Integrating this data unveiled 107 distinct cell types and their specific utilization of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly a third of the cCREs demonstrated conservation and chromatin accessibility in the mouse brain cells. We reveal strong links between specific brain cell types and neuropsychiatric disorders including schizophrenia, bipolar disorder, Alzheimer's disease (AD), and major depression, and have developed deep learning models to predict the regulatory roles of noncoding risk variants in these disorders.

Single-cell DNA methylation and 3D genome architecture in the human brain.

Delineating the gene-regulatory programs underlying complex cell types is fundamental for understanding brain function in health and disease. Here, we comprehensively examined human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in 517 thousand cells (399 thousand neurons and 118 thousand non-neurons) from 46 regions of three adult male brains. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. We further developed single-cell methylation barcodes that reliably predict brain cell types using the methylation status of select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell-type-specific gene regulation in adult human brains.

Comparative transcriptomics reveals human-specific cortical features.

The cognitive abilities of humans are distinctive among primates, but their molecular and cellular substrates are poorly understood. We used comparative single-nucleus transcriptomics to analyze samples of the middle temporal gyrus (MTG) from adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets to understand human-specific features of the neocortex. Human, chimpanzee, and gorilla MTG showed highly similar cell-type composition and laminar organization as well as a large shift in proportions of deep-layer intratelencephalic-projecting neurons compared with macaque and marmoset MTG. Microglia, astrocytes, and oligodendrocytes had more-divergent expression across species compared with neurons or oligodendrocyte precursor cells, and neuronal expression diverged more rapidly on the human lineage. Only a few hundred genes showed human-specific patterning, suggesting that relatively few cellular and molecular changes distinctively define adult human cortical structure.

Transcriptomic cytoarchitecture reveals principles of human neocortex organization.

Variation in cytoarchitecture is the basis for the histological definition of cortical areas. We used single cell transcriptomics and performed cellular characterization of the human cortex to better understand cortical areal specialization. Single-nucleus RNA-sequencing of 8 areas spanning cortical structural variation showed a highly consistent cellular makeup for 24 cell subclasses. However, proportions of excitatory neuron subclasses varied substantially, likely reflecting differences in connectivity across primary sensorimotor and association cortices. Laminar organization of astrocytes and oligodendrocytes also differed across areas. Primary visual cortex showed characteristic organization with major changes in the excitatory to inhibitory neuron ratio, expansion of layer 4 excitatory neurons, and specialized inhibitory neurons. These results lay the groundwork for a refined cellular and molecular characterization of human cortical cytoarchitecture and areal specialization.

Single nucleus multi-omics identifies human cortical cell regulatory genome diversity.

Single-cell technologies measure unique cellular signatures but are typically limited to a single modality. Computational approaches allow the fusion of diverse single-cell data types, but their efficacy is difficult to validate in the absence of authentic multi-omic measurements. To comprehensively assess the molecular phenotypes of single cells, we devised single-nucleus methylcytosine, chromatin accessibility, and transcriptome sequencing (snmCAT-seq) and applied it to postmortem human frontal cortex tissue. We developed a cross-validation approach using multi-modal information to validate fine-grained cell types and assessed the effectiveness of computational data fusion methods. Correlation analysis in individual cells revealed distinct relations between methylation and gene expression. Our integrative approach enabled joint analyses of the methylome, transcriptome, chromatin accessibility, and conformation for 63 human cortical cell types. We reconstructed regulatory lineages for cortical cell populations and found specific enrichment of genetic risk for neuropsychiatric traits, enabling the prediction of cell types that are associated with diseases.

Notch-Mediated Determination of Hair-Bundle Polarity in Mechanosensory Hair Cells of the Zebrafish Lateral Line.

The development of mechanosensory epithelia, such as those of the auditory and vestibular systems, results in the precise orientation of mechanosensory hair cells. After division of a precursor cell in the zebrafish's lateral line, the daughter hair cells differentiate with opposite mechanical sensitivity. Through a combination of theoretical and experimental approaches, we show that Notch1a-mediated lateral inhibition produces a bistable switch that reliably gives rise to cell pairs of opposite polarity. Using a mathematical model of the process, we predict the outcome of several genetic and chemical alterations to the system, which we then confirm experimentally. We show that Notch1a downregulates the expression of Emx2, a transcription factor known to be involved in polarity specification, and acts in parallel with the planar-cell-polarity system to determine the orientation of hair bundles. By analyzing the effect of simultaneous genetic perturbations to Notch1a and Emx2, we infer that the gene-regulatory network determining cell polarity includes an undiscovered polarity effector.

Connectomics of the zebrafish's lateral-line neuromast reveals wiring and miswiring in a simple microcircuit.

The lateral-line neuromast of the zebrafish displays a restricted, consistent pattern of innervation that facilitates the comparison of microcircuits across individuals, developmental stages, and genotypes. We used serial blockface scanning electron microscopy to determine from multiple specimens the neuromast connectome, a comprehensive set of connections between hair cells and afferent and efferent nerve fibers. This analysis delineated a complex but consistent wiring pattern with three striking characteristics: each nerve terminal is highly specific in receiving innervation from hair cells of a single directional sensitivity; the innervation is redundant; and the terminals manifest a hierarchy of dominance. Mutation of the canonical planar-cell-polarity gene vangl2, which decouples the asymmetric phenotypes of sibling hair-cell pairs, results in randomly positioned, randomly oriented sibling cells that nonetheless retain specific wiring. Because larvae that overexpress Notch exhibit uniformly oriented, uniformly innervating hair-cell siblings, wiring specificity is mediated by the Notch signaling pathway.

Roop Mallik: From machines to molecular motors.

Roop Mallik studies how molecular motors and lipids interact to drive intracellular transport.

An In Toto Approach to Dissecting Cellular Interactions in Complex Tissues.

Single-cell measurements have broadened our understanding of heterogeneity in biology, yet have been limited to mostly observational studies of normal or globally perturbed systems. Typically, perturbations are utilized in an open-ended approach wherein an endpoint is assayed during or after the biological event of interest. Here we describe ShootingStar, a platform for perturbation analysis in vivo, which combines live imaging, real-time image analysis, and automated optical perturbations. ShootingStar builds a quantitative record of the state of the sample being analyzed, which is used to automate the identification of target cells for perturbation, as well as to validate the impacts of the perturbation. We used ShootingStar to dissect the cellular basis of development, morphogenesis, and polarity in the lateral line of Danio rerio and the embryo of Caenorhabditis elegans. ShootingStar can be extended to diverse optical manipulations and enables more robust and informative single-cell perturbations in complex tissues.

Conserved microsatellites in ants enable population genetic and colony pedigree studies across a wide range of species.

Broadly applicable polymorphic genetic markers are essential tools for population genetics, and different types of markers have been developed for this purpose. Microsatellites have been employed as particularly polymorphic markers for over 20 years. However, PCR primers for microsatellite loci are often not useful outside the species for which they were designed. This implies that a new set of loci has to be identified and primers developed for every new study species. To overcome this constraint, we identified 45 conserved microsatellite loci based on the eight currently available ant genomes and designed primers for PCR amplification. Among these loci, we chose 24 for in-depth study in six species covering six different ant subfamilies. On average, 11.16 of these 24 loci were polymorphic and in Hardy-Weinberg equilibrium in any given species. The average number of alleles for these polymorphic loci within single populations of the different species was 4.59. This set of genetic markers will thus be useful for population genetic and colony pedigree studies across a wide range of ant species, supplementing the markers available for previously studied species and greatly facilitating the study of the many ant species lacking genetic markers. Our study shows that it is possible to develop microsatellite loci that are both conserved over a broad range of taxa, yet polymorphic within species. This should encourage researchers to develop similar tools for other large taxonomic groups.