Comparative cellular analysis of motor cortex in human, marmoset and mouse

Article Comparative cellular analysis of motor cortex in human, marmoset and mouse https://doi.org/10.1038/s41586-021-03465-8 Received: 31 March 2020 Accepted: 17 March 2021 Published online: 6 October 2021 Open access Check for updates A list of authors and affiliations appears at the end of the paper. The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations. Single-cell transcriptomic and epigenomic methods have been effective in elucidating the cellular makeup of complex brain tissues from patterns of gene expression and underlying regulatory mechanisms2–6. In the mouse and human neocortex, diverse neuronal and non-neuronal cell types can be defined2,3,5,7 by their distinct transcriptional profiles and regions of accessible chromatin or of DNA methylation (DNAm)4,8, and can be aligned between species3,9–11 on the basis of these profiles. Studies such as these have shown the feasibility of quantitatively studying the evolution of cell types, but have limitations: different cortical regions have been profiled in humans and mice; different sets of transcripts have been captured with single-cell and single-nucleus assays; and transcriptomic and epigenomic studies have mostly been carried out independently. The primary motor cortex (M1, also known as MOp in mice) is an ideal region with which to address questions about cellular evolution in rodents and primates. M1 is essential for fine-motor control and is functionally conserved across mammals1. The layer 5 (L5) region of carnivore and primate M1 contains specialized ‘giganto-cellular’ corticospinal neurons (Betz cells in primates12–16) with distinctive action-potential properties that support a high conduction velocity17–19. Some Betz cells synapse directly onto spinal motor neurons, unlike rodent corticospinal neurons, which synapse indirectly via spinal interneurons20. These observations suggest that Betz cells possess species-adapted intrinsic mechanisms to support rapid communication that should be reflected in their molecular signatures. To explore the evolutionary conservation and divergence of M1 cell types and their underlying molecular regulatory mechanisms, we analysed single-nucleus transcriptomic and epigenomic data from mouse, marmoset, macaque and human M1. Multi-omic taxonomies of cell types To characterize the molecular diversity of M1 neurons and non-neuronal cells, we applied single-nucleus transcriptomic assays (plate-based SMART-seq v4 (SSv4) and droplet-based Chromium v3 (Cv3) RNA sequencing) and epigenomic assays (single-nucleus methylcytosine sequencing 2 (snmC-seq2) and single-nucleus chromatin accessibility and messenger RNA expression sequencing (SNARE–seq2)) to isolated M1 samples from human, marmoset and mouse brains (Extended Data Fig. 1a–d); we also applied Cv3 to M1 L5 from macaque brains. Single nuclei were dissociated from all layers combined or from individual layers (in the case of human SSv4 assays), and sorted using the neuronal marker NeuN to enrich cellular input to roughly 90% neurons and 10% non-neuronal cells (Extended Data Fig. 1e). Datasets from mice are reported in a companion paper5. The median detection of neuronal genes in humans was higher when we used SSv4 (7,296 genes) as compared with Cv3 (5,657 genes), partially because of the 20-fold greater read depth, and detection was lower in marmosets (4,211) and mice (5,046) when using Cv3 (Extended Data Fig. 1f–m). For each species, we defined a diverse set of neuronal and non-neuronal clusters of cell types on the basis of unsupervised clustering of snRNA-seq datasets (Extended Data Fig. 1n–r and Supplementary Tables 1, 2). We organized cell types into hierarchical taxonomies Nature | Vol 598 | 7 October 2021 | 111 Article Human Neuronal GABAergic CGE-derived Neuronal proportion 0.09 0.18 Dissected layer AC cluster DNAm cluster Lamp5 / Sncg Vip dl Lamp5 Sncg IT Sst IT Neuronal GABAergic CGE-derived MGE-derived 0.25 0.3 * * 0.2 * 0.1 IT 0.6 V t C ip ho dl Ss Pv t al b M ei s2 Ss p5 Sn cg m La AB A er gi c at Glutamatergic neurons Human Marmoset Mouse 0.4 * 0.2 * * G lu ta m G * 0 0 0 ET CT L6b Pvalb GABAergic neurons 0.4 Subclass proportion * Sst /3 IT L6 L5 IT IT C ar 3 L6 IT L5 E L5 T /6 N P L6 C T L6 b d Neurons * Vip dl ho tC Ss Nonneuronal proportion 0.28 0.56 Glutamatergic Subclass proportion Neuronal proportion 0.09 0.18 AC cluster DNAm cluster Lamp5 Sncg 0.50 Nonneuronal proportion 0.28 0.56 Non-neuronal Mouse 0.75 Consensus M1 taxonomy across species Non-neuronal Glutamatergic Pvalb Fig. 1 | Molecular taxonomy of cell types in the primary motor cortex (M1) of humans, marmosets and mice. a–c, Dendrograms showing cell-type clusters defined by RNA sequencing (RNA-seq; using Cv3) for humans (a), marmosets (b) and mice (c), annotated with the cluster proportions of total neuronal or non-neuronal cells and (for humans) with dissected layers (L1–L6). RNA-seq clusters mapped to clusters of accessible chromatin (AC) and DNAm. d, Relative proportions of some neuronal cell types were significantly different between species, based on analysis of variance (ANOVA) followed by Tukey’s HSD two-sided tests (degrees of freedom = 13; *P < 0.05 (Bonferonni-corrected)). Data in d are means ± s.d., and points represent individual donor specimens for humans (n = 2), marmosets (n = 2), and (...truncated)