A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.

Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior.

The ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) contains ∼4,000 neurons that project to multiple targets and control innate social behaviors including aggression and mounting. However, the number of cell types in VMHvl and their relationship to connectivity and behavioral function are unknown. We performed single-cell RNA sequencing using two independent platforms-SMART-seq (∼4,500 neurons) and 10x (∼78,000 neurons)-and investigated correspondence between transcriptomic identity and axonal projections or behavioral activation, respectively. Canonical correlation analysis (CCA) identified 17 transcriptomic types (T-types), including several sexually dimorphic clusters, the majority of which were validated by seqFISH. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.

A Suite of Transgenic Driver and Reporter Mouse Lines with Enhanced Brain-Cell-Type Targeting and Functionality.

Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Molecularly defined and spatially resolved cell atlas of the whole mouse brain.

In mammalian brains, millions to billions of cells form complex interaction networks to enable a wide range of functions. The enormous diversity and intricate organization of cells have impeded our understanding of the molecular and cellular basis of brain function. Recent advances in spatially resolved single-cell transcriptomics have enabled systematic mapping of the spatial organization of molecularly defined cell types in complex tissues1-3, including several brain regions (for example, refs. 1-11). However, a comprehensive cell atlas of the whole brain is still missing. Here we imaged a panel of more than 1,100 genes in approximately 10 million cells across the entire adult mouse brains using multiplexed error-robust fluorescence in situ hybridization12 and performed spatially resolved, single-cell expression profiling at the whole-transcriptome scale by integrating multiplexed error-robust fluorescence in situ hybridization and single-cell RNA sequencing data. Using this approach, we generated a comprehensive cell atlas of more than 5,000 transcriptionally distinct cell clusters, belonging to more than 300 major cell types, in the whole mouse brain with high molecular and spatial resolution. Registration of this atlas to the mouse brain common coordinate framework allowed systematic quantifications of the cell-type composition and organization in individual brain regions. We further identified spatial modules characterized by distinct cell-type compositions and spatial gradients featuring gradual changes of cells. Finally, this high-resolution spatial map of cells, each with a transcriptome-wide expression profile, allowed us to infer cell-type-specific interactions between hundreds of cell-type pairs and predict molecular (ligand-receptor) basis and functional implications of these cell-cell interactions. These results provide rich insights into the molecular and cellular architecture of the brain and a foundation for functional investigations of neural circuits and their dysfunction in health and disease.

Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

A transcriptomic taxonomy of mouse brain-wide spinal projecting neurons.

The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.

Single-cell analysis of chromatin accessibility in the adult mouse brain.

Recent advances in single-cell technologies have led to the discovery of thousands of brain cell types; however, our understanding of the gene regulatory programs in these cell types is far from complete1-4. Here we report a comprehensive atlas of candidate cis-regulatory DNA elements (cCREs) in the adult mouse brain, generated by analysing chromatin accessibility in 2.3 million individual brain cells from 117 anatomical dissections. The atlas includes approximately 1 million cCREs and their chromatin accessibility across 1,482 distinct brain cell populations, adding over 446,000 cCREs to the most recent such annotation in the mouse genome. The mouse brain cCREs are moderately conserved in the human brain. The mouse-specific cCREs-specifically, those identified from a subset of cortical excitatory neurons-are strongly enriched for transposable elements, suggesting a potential role for transposable elements in the emergence of new regulatory programs and neuronal diversity. Finally, we infer the gene regulatory networks in over 260 subclasses of mouse brain cells and develop deep-learning models to predict the activities of gene regulatory elements in different brain cell types from the DNA sequence alone. Our results provide a resource for the analysis of cell-type-specific gene regulation programs in both mouse and human brains.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures.

Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ∼1,000 genes important for neural functions by in situ hybridization at a cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene's expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, and in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain.

Conserved cell types with divergent features in human versus mouse cortex.

Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we used single-nucleus RNA-sequencing analysis to perform a comprehensive study of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuron types that are mostly sparse, with excitatory types being less layer-restricted than expected. Comparison to similar mouse cortex single-cell RNA-sequencing datasets revealed a surprisingly well-conserved cellular architecture that enables matching of homologous types and predictions of properties of human cell types. Despite this general conservation, we also found extensive differences between homologous human and mouse cell types, including marked alterations in proportions, laminar distributions, gene expression and morphology. These species-specific features emphasize the importance of directly studying human brain.

Proteinuria as an Endpoint in Clinical Trials of Focal Segmental Glomerulosclerosis.

Focal Segmental Glomerulosclerosis (FSGS) is a characteristic histopathological lesion that is indicative of underlying glomerular dysfunction. It is not a single disease entity but rather a heterogeneous disorder that is an important cause of nephrotic syndrome and kidney failure in children and adults. The aim of this Kidney Health Initiative project was to evaluate potential endpoints for clinical trials in FSGS. This paper focuses on the data supporting proteinuria as a surrogate endpoint. Available data support the use of complete remission of proteinuria in patients with heavy proteinuria as a surrogate endpoint for progression to kidney failure. While substantial treatment effects on proteinuria that are short of a complete remission may also predict the effect of a treatment on progression to kidney failure, further work is needed to determine how such an endpoint should be defined. Fortunately, efforts are underway to bring together patient-level data from randomized controlled trials, observational studies, and registries to address this issue.

Distinct descending motor cortex pathways and their roles in movement.

Activity in the motor cortex predicts movements, seconds before they are initiated. This preparatory activity has been observed across cortical layers, including in descending pyramidal tract neurons in layer 5. A key question is how preparatory activity is maintained without causing movement, and is ultimately converted to a motor command to trigger appropriate movements. Here, using single-cell transcriptional profiling and axonal reconstructions, we identify two types of pyramidal tract neuron. Both types project to several targets in the basal ganglia and brainstem. One type projects to thalamic regions that connect back to motor cortex; populations of these neurons produced early preparatory activity that persisted until the movement was initiated. The second type projects to motor centres in the medulla and mainly produced late preparatory activity and motor commands. These results indicate that two types of motor cortex output neurons have specialized roles in motor control.

Shared and distinct transcriptomic cell types across neocortical areas.

The neocortex contains a multitude of cell types that are segregated into layers and functionally distinct areas. To investigate the diversity of cell types across the mouse neocortex, here we analysed 23,822 cells from two areas at distant poles of the mouse neocortex: the primary visual cortex and the anterior lateral motor cortex. We define 133 transcriptomic cell types by deep, single-cell RNA sequencing. Nearly all types of GABA (γ-aminobutyric acid)-containing neurons are shared across both areas, whereas most types of glutamatergic neurons were found in one of the two areas. By combining single-cell RNA sequencing and retrograde labelling, we match transcriptomic types of glutamatergic neurons to their long-range projection specificity. Our study establishes a combined transcriptomic and projectional taxonomy of cortical cell types from functionally distinct areas of the adult mouse cortex.

A comprehensive transcriptional map of primate brain development.

The transcriptional underpinnings of brain development remain poorly understood, particularly in humans and closely related non-human primates. We describe a high-resolution transcriptional atlas of rhesus monkey (Macaca mulatta) brain development that combines dense temporal sampling of prenatal and postnatal periods with fine anatomical division of cortical and subcortical regions associated with human neuropsychiatric disease. Gene expression changes more rapidly before birth, both in progenitor cells and maturing neurons. Cortical layers and areas acquire adult-like molecular profiles surprisingly late in postnatal development. Disparate cell populations exhibit distinct developmental timing of gene expression, but also unexpected synchrony of processes underlying neural circuit construction including cell projection and adhesion. Candidate risk genes for neurodevelopmental disorders including primary microcephaly, autism spectrum disorder, intellectual disability, and schizophrenia show disease-specific spatiotemporal enrichment within developing neocortex. Human developmental expression trajectories are more similar to monkey than rodent, although approximately 9% of genes show human-specific regulation with evidence for prolonged maturation or neoteny compared to monkey.

Role of Hypoxia-Inducible Factors in Regulating Right Ventricular Function and Remodeling during Chronic Hypoxia-induced Pulmonary Hypertension.

Pulmonary hypertension (PH) and right ventricular (RV) hypertrophy frequently develop in patients with hypoxic lung disease. Chronic alveolar hypoxia (CH) promotes sustained pulmonary vasoconstriction and pulmonary artery (PA) remodeling by acting on lung cells, resulting in the development of PH. RV hypertrophy develops in response to PH, but coronary arterial hypoxemia in CH may influence that response by activating HIF-1α (hypoxia-inducible factor 1α) and/or HIF-2α in cardiomyocytes. Indeed, other studies show that the attenuation of PH in CH fails to prevent RV remodeling, suggesting that PH-independent factors regulate RV hypertrophy. Therefore, we examined the role of HIFs in RV remodeling in CH-induced PH. We deleted HIF-1α and/or HIF-2α in hearts of adult mice that were then housed under normoxia or CH (10% O2) for 4 weeks. RNA-sequencing analysis of the RV revealed that HIF-1α and HIF-2α regulate the transcription of largely distinct gene sets during CH. RV systolic pressure increased, and RV hypertrophy developed in CH. The deletion of HIF-1α in smooth muscle attenuated the CH-induced increases in RV systolic pressure but did not decrease hypertrophy. The deletion of HIF-1α in cardiomyocytes amplified RV remodeling; this was abrogated by the simultaneous loss of HIF-2α. CH decreased stroke volume and cardiac output in wild-type but not in HIF-1α-deficient hearts, suggesting that CH may cause cardiac dysfunction via HIF-dependent signaling. Collectively, these data reveal that HIF-1 and HIF-2 act together in RV cardiomyocytes to orchestrate RV remodeling in CH, with HIF-1 playing a protective role rather than driving hypertrophy.

Transcriptional landscape of the prenatal human brain.

The anatomical and functional architecture of the human brain is mainly determined by prenatal transcriptional processes. We describe an anatomically comprehensive atlas of the mid-gestational human brain, including de novo reference atlases, in situ hybridization, ultra-high-resolution magnetic resonance imaging (MRI) and microarray analysis on highly discrete laser-microdissected brain regions. In developing cerebral cortex, transcriptional differences are found between different proliferative and post-mitotic layers, wherein laminar signatures reflect cellular composition and developmental processes. Cytoarchitectural differences between human and mouse have molecular correlates, including species differences in gene expression in subplate, although surprisingly we find minimal differences between the inner and outer subventricular zones even though the outer zone is expanded in humans. Both germinal and post-mitotic cortical layers exhibit fronto-temporal gradients, with particular enrichment in the frontal lobe. Finally, many neurodevelopmental disorder and human-evolution-related genes show patterned expression, potentially underlying unique features of human cortical formation. These data provide a rich, freely-accessible resource for understanding human brain development.

Sensors and signals: the role of reactive oxygen species in hypoxic pulmonary vasoconstriction.

When lung cells experience hypoxia, the functional response, termed hypoxic pulmonary vasoconstriction, activates a multitude of pathways with the goal of optimizing gas exchange. While previously controversial, overwhelming evidence now suggests that increased reactive oxygen species - produced at complex III of the mitochondrial electron transport chain and released into the intermembrane space - is the cellular oxygen signal responsible for triggering hypoxic pulmonary vasoconstriction. The increased reactive oxygen species (ROS) activate many downstream targets that ultimately lead to increased intracellular ionized calcium concentration and contraction of pulmonary arterial smooth muscle cells. While the specific targets of ROS signals are not completely understood, it is clear that this signalling pathway is critical for development and for normal lung function in newborns and adults.

Addressing the Need for Clinical Trial End Points in Autosomal Dominant Polycystic Kidney Disease: A Report From the Polycystic Kidney Disease Outcomes Consortium (PKDOC).

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease. Expansion of multiple cysts throughout both kidneys is thought to lead to progressive loss of kidney function and kidney failure in some patients. In recent years, much has been learned about the pathophysiology of ADPKD. However, to date, only one therapy has been approved in the United States and in other regions for the treatment of ADPKD. Feasible end points and a clear regulatory pathway may stimulate further development in this area and ultimately lead to more treatments for ADPKD successfully reaching the market. In July 2016, the PKD Outcomes Consortium under the auspices of the Critical Path Institute and the PKD Foundation convened a PKD Summit to facilitate a discussion among patients, regulators, pharmaceutical sponsors, and academic clinical trialists regarding trial end points and the regulatory path to approval of new drugs for ADPKD. Following the summit, participants continued the dialogue using regularly scheduled teleconferences. This article addresses key considerations related to the design of clinical trials in ADPKD based on these discussions.

An anatomically comprehensive atlas of the adult human brain transcriptome.

Neuroanatomically precise, genome-wide maps of transcript distributions are critical resources to complement genomic sequence data and to correlate functional and genetic brain architecture. Here we describe the generation and analysis of a transcriptional atlas of the adult human brain, comprising extensive histological analysis and comprehensive microarray profiling of ∼900 neuroanatomically precise subdivisions in two individuals. Transcriptional regulation varies enormously by anatomical location, with different regions and their constituent cell types displaying robust molecular signatures that are highly conserved between individuals. Analysis of differential gene expression and gene co-expression relationships demonstrates that brain-wide variation strongly reflects the distributions of major cell classes such as neurons, oligodendrocytes, astrocytes and microglia. Local neighbourhood relationships between fine anatomical subdivisions are associated with discrete neuronal subtypes and genes involved with synaptic transmission. The neocortex displays a relatively homogeneous transcriptional pattern, but with distinct features associated selectively with primary sensorimotor cortices and with enriched frontal lobe expression. Notably, the spatial topography of the neocortex is strongly reflected in its molecular topography-the closer two cortical regions, the more similar their transcriptomes. This freely accessible online data resource forms a high-resolution transcriptional baseline for neurogenetic studies of normal and abnormal human brain function.

Spatiotemporal dynamics of the postnatal developing primate brain transcriptome.

Developmental changes in the temporal and spatial regulation of gene expression drive the emergence of normal mature brain function, while disruptions in these processes underlie many neurodevelopmental abnormalities. To solidify our foundational knowledge of such changes in a primate brain with an extended period of postnatal maturation like in human, we investigated the whole-genome transcriptional profiles of rhesus monkey brains from birth to adulthood. We found that gene expression dynamics are largest from birth through infancy, after which gene expression profiles transition to a relatively stable state by young adulthood. Biological pathway enrichment analysis revealed that genes more highly expressed at birth are associated with cell adhesion and neuron differentiation, while genes more highly expressed in juveniles and adults are associated with cell death. Neocortex showed significantly greater differential expression over time than subcortical structures, and this trend likely reflects the protracted postnatal development of the cortex. Using network analysis, we identified 27 co-expression modules containing genes with highly correlated expression patterns that are associated with specific brain regions, ages or both. In particular, one module with high expression in neonatal cortex and striatum that decreases during infancy and juvenile development was significantly enriched for autism spectrum disorder (ASD)-related genes. This network was enriched for genes associated with axon guidance and interneuron differentiation, consistent with a disruption in the formation of functional cortical circuitry in ASD.