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

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

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

The top 10 VMR clusters and their associated genes.

<p>The genes in bold text expressed at detectible level in whole blood and were selected for association testing.</p

Identification of heritable CpG methylation by mid-parent offspring (MPO) analysis.

<p>(a) An example of mid-parent offspring regression of DNA methylation at the CpG site chr1∶146549909. (b,c) DNA methylation level of heritable CpG at chr1∶146549909 and the adjacent heritable CpGs on the same cluster exhibiting consistent pattern of DNA methylation between parents and their offspring on the two trios from the same family.</p

Mapping of CpG sites identified in MPO and mQTL analyses.

<p>(a) Venn diagrams showing overlap between non-SNP CpG sites significant in mQTL on 5,257,772 imputed SNPs and heritable CpGs. (b, c) Distribution of heritable CpGs and non-heritable CpGs and associated SNP pair distance within 500kb and their corresponding p-values from mQTL analysis on imputed SNPs.</p

Genotype effects on the mean and variance of DNA methylation (a) Heatmap and line plot showing the association between rs4950357 SNP and the mean methylation of heritable CpGs cluster on chromosome 1 (chr1: 146548425-146555855).

<p>(b) The association of rs2833839 vSNP and the variance of methylation on VMR (chr21∶34405506-34405661).</p