The formation and maintenance of tissue integrity requires complex, coordinated activities by thousands of genes and their encoded products. Until recently, transcript levels could only be quantified ...for a few genes in tissues, but advances in DNA sequencing, oligonucleotide synthesis and fluorescence microscopy have enabled the invention of a suite of spatial transcriptomics technologies capable of measuring the expression of many, or all, genes in situ. These technologies have evolved rapidly in sensitivity, multiplexing and throughput. As such, they have enabled the determination of the cell-type architecture of tissues, the querying of cell-cell interactions and the monitoring of molecular interactions between tissue components. The rapidly evolving spatial genomics landscape will enable generalized high-throughput genomic measurements and perturbations to be performed in the context of tissues. These advances will empower hypothesis generation and biological discovery and bridge the worlds of tissue biology and genomics.
Defining cell types requires integrating diverse single-cell measurements from multiple experiments and biological contexts. To flexibly model single-cell datasets, we developed LIGER, an algorithm ...that delineates shared and dataset-specific features of cell identity. We applied it to four diverse and challenging analyses of human and mouse brain cells. First, we defined region-specific and sexually dimorphic gene expression in the mouse bed nucleus of the stria terminalis. Second, we analyzed expression in the human substantia nigra, comparing cell states in specific donors and relating cell types to those in the mouse. Third, we integrated in situ and single-cell expression data to spatially locate fine subtypes of cells present in the mouse frontal cortex. Finally, we jointly defined mouse cortical cell types using single-cell RNA-seq and DNA methylation profiles, revealing putative mechanisms of cell-type-specific epigenomic regulation. Integrative analyses using LIGER promise to accelerate investigations of cell-type definition, gene regulation, and disease states.
Display omitted
•Shared and dataset-specific metagene factors enable single-cell data integration•LIGER reveals inter-individual differences in bed nucleus and substantia nigra cells•Integration of in situ and dissociated scRNA-seq maps cell types in space•Joint definition of cortical cell types from single-cell RNA and epigenome profiles
A platform called LIGER allows for the integration of gene expression, epigenetic regulation, and spatial relationships across single-cell datasets.
A limitation of spatial transcriptomics technologies is that individual measurements may contain contributions from multiple cells, hindering the discovery of cell-type-specific spatial patterns of ...localization and expression. Here, we develop robust cell type decomposition (RCTD), a computational method that leverages cell type profiles learned from single-cell RNA-seq to decompose cell type mixtures while correcting for differences across sequencing technologies. We demonstrate the ability of RCTD to detect mixtures and identify cell types on simulated datasets. Furthermore, RCTD accurately reproduces known cell type and subtype localization patterns in Slide-seq and Visium datasets of the mouse brain. Finally, we show how RCTD's recovery of cell type localization enables the discovery of genes within a cell type whose expression depends on spatial environment. Spatial mapping of cell types with RCTD enables the spatial components of cellular identity to be defined, uncovering new principles of cellular organization in biological tissue. RCTD is publicly available as an open-source R package at https://github.com/dmcable/RCTD .
Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting ...roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.
Display omitted
•Specific neuromodulators are necessary for long-lasting roaming and dwelling states•Serotonergic neurons induce dwelling states through an inhibitory receptor•PDF neuropeptides act through cAMP signaling to induce prolonged roaming states•Optogenetic imitation of serotonin or PDF signaling elicits behavioral changes
Two opposing neuromodulators regulate neural circuits with slow temporal dynamics that establish long-lasting alternative behavioral states—roaming and dwelling—related to foraging in C. elegans.
The mammalian brain is composed of diverse, specialized cell populations. To systematically ascertain and learn from these cellular specializations, we used Drop-seq to profile RNA expression in ...690,000 individual cells sampled from 9 regions of the adult mouse brain. We identified 565 transcriptionally distinct groups of cells using computational approaches developed to distinguish biological from technical signals. Cross-region analysis of these 565 cell populations revealed features of brain organization, including a gene-expression module for synthesizing axonal and presynaptic components, patterns in the co-deployment of voltage-gated ion channels, functional distinctions among the cells of the vasculature and specialization of glutamatergic neurons across cortical regions. Systematic neuronal classifications for two complex basal ganglia nuclei and the striatum revealed a rare population of spiny projection neurons. This adult mouse brain cell atlas, accessible through interactive online software (DropViz), serves as a reference for development, disease, and evolution.
Display omitted
•690,000 individual cells analyzed from 9 regions of adult mouse brain•RNA expression patterns corresponding to cell types, states, and locations•Transcriptional programs supporting axonal function and neuronal specializations•Online data and analysis resource “DropViz”
Sampling across multiple brain regions identifies hundreds of transcriptionally distinct groups of cells and reveals large-scale features of brain organization and neuronal diversity.
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have ...prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution.
Display omitted
Display omitted
•Drop-seq enables highly parallel analysis of individual cells by RNA-seq•Drop-seq encapsulates cells in nanoliter droplets together with DNA-barcoded beads•Systematic evaluation of Drop-seq library quality using species mixing experiments•Drop-seq analysis of 44,808 cells identifies 39 cell populations in the retina
Capturing single cells along with sets of uniquely barcoded primer beads together in tiny droplets enables large-scale, highly parallel single-cell transcriptomics. Applying this analysis to cells in mouse retinal tissue revealed transcriptionally distinct cell populations along with molecular markers of each type.
Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, ...harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.
Display omitted
•Unsupervised clustering of 25,000 single-cell transcriptomes reveals 15 bipolar types•Molecularly defined types correspond 1:1 to morphologically defined types•One previously undescribed bipolar cell type has hybrid bipolar-amacrine features•Shallow sequencing of large cell numbers facilitates comprehensive classification
Single-cell transcriptome sequencing of retinal bipolar cells reveals known and new types including one with a non-canonical morphology.
Many neuron types consist of populations with continuously varying molecular properties. Here, we show a continuum of postsynaptic molecular properties in three types of neurons and assess the ...functional correlates in cerebellar unipolar brush cells (UBCs). While UBCs are generally thought to form discrete functional subtypes, with mossy fiber (MF) activation increasing firing in ON-UBCs and suppressing firing in OFF-UBCs, recent work also points to a heterogeneity of response profiles. Indeed, we find a continuum of response profiles that reflect the graded and inversely correlated expression of excitatory mGluR1 and inhibitory mGluR2/3 pathways. MFs coactivate mGluR2/3 and mGluR1 in many UBCs, leading to sequential inhibition-excitation because mGluR2/3-currents are faster. Additionally, we show that DAG-kinase controls mGluR1 response duration, and that graded DAG kinase levels correlate with systematic variation of response duration over two orders of magnitude. These results demonstrate that continuous variations in metabotropic signaling can generate a stable cell-autonomous basis for temporal integration and learning over multiple time scales.
In vitro models of the developing brain such as three-dimensional brain organoids offer an unprecedented opportunity to study aspects of human brain development and disease. However, the cells ...generated within organoids and the extent to which they recapitulate the regional complexity, cellular diversity and circuit functionality of the brain remain undefined. Here we analyse gene expression in over 80,000 individual cells isolated from 31 human brain organoids. We find that organoids can generate a broad diversity of cells, which are related to endogenous classes, including cells from the cerebral cortex and the retina. Organoids could be developed over extended periods (more than 9 months), allowing for the establishment of relatively mature features, including the formation of dendritic spines and spontaneously active neuronal networks. Finally, neuronal activity within organoids could be controlled using light stimulation of photosensitive cells, which may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli.
PDF
