A transcriptomic and epigenomic cell atlas of the mouse primary motor cortex.

Single-cell transcriptomics can provide quantitative molecular signatures for large, unbiased samples of the diverse cell types in the brain1-3. With the proliferation of multi-omics datasets, a major challenge is to validate and integrate results into a biological understanding of cell-type organization. Here we generated transcriptomes and epigenomes from more than 500,000 individual cells in the mouse primary motor cortex, a structure that has an evolutionarily conserved role in locomotion. We developed computational and statistical methods to integrate multimodal data and quantitatively validate cell-type reproducibility. The resulting reference atlas-containing over 56 neuronal cell types that are highly replicable across analysis methods, sequencing technologies and modalities-is a comprehensive molecular and genomic account of the diverse neuronal and non-neuronal cell types in the mouse primary motor cortex. The atlas includes a population of excitatory neurons that resemble pyramidal cells in layer 4 in other cortical regions4. We further discovered thousands of concordant marker genes and gene regulatory elements for these cell types. Our results highlight the complex molecular regulation of cell types in the brain and will directly enable the design of reagents to target specific cell types in the mouse primary motor cortex for functional analysis

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

Binding and neutralizing potentials of MAbs isolated from the NLGS-3 Core immunized animals.

<p>(A) Binding (<i>top</i>) and neutralizing (<i>bottom</i>) titers of the indicated macaque MAbs against the indicated Envs and viruses. (B) Antibody-binding competition. Top panels: The binding of the indicated macaque MAbs to the 426c NLGS-3 Core gp140 protein was determined in the absence of competing antibody <i>(left)</i>, or following the pre-incubation of 426c NLGS-3 Core gp140 with saturating concentrations of VRC01 <i>(middle left)</i>, CD4-IgG <i>(middle right)</i>, or b12 <i>(right)</i>. Bottom panels: Control experiments were performed with the indicated human MAbs. Direct binding of the indicated human MAbs to the 426c NLGS-3 Core gp140 protein (<i>left panel</i>), following the pre-incubation of Env with VRC01 (<i>second from left panel</i>), CD4-IgG (<i>second from right panel</i>), or b12 (<i>right panel</i>).</p

Changes in IGH, IGK and IGL frequencies upon WT and NLGS-3 Core immunization.

<p>(A) Number of genes in each V gene family with a significant positive Log Fold Change (LogFC) value for WT (<i>blue</i>) and NLGS-3 Core (<i>red</i>) immunization groups. Positive LogFC was calculated and compiled between the pre-immunization and post DNA/Protein Boost 2 for IgHV <i>(left)</i>, IgKV <i>(middle)</i>, and IgLV <i>(right)</i> gene families. All LogFC have a false discovery rate of <0.05. (B) Total number of V genes with a positive LogFC and FDR < 0.05 for WT and NLGS-3 Core immunization groups between pre-immunization and post DNA Prime or post DNA/Protein Boost 2 for IgHV <i>(left)</i>, IgKV <i>(middle)</i>, and IgLV <i>(right)</i> V alleles. Bars in A and B represent the total number of genes with significant LogFC. Bars at baseline indicate no genes scored a significant LogFC.</p

Binding and neutralizing serum activities.

<p>(A) Sera collected from the WT <i>(left)</i> and NLGS-3 Core <i>(right)</i> prior to immunization <i>(blue)</i>, following the DNA/Protein Boost 1 <i>(orange)</i>, and following the DNA/Protein Boost 2 <i>(red)</i> were tested for antibody reactivity to autologous rEnv immunogens. * <i>indicates p value < 0</i>.<i>01</i>, ** <i>p value < 0</i>.<i>001</i>, or *** <i>p value < 0</i>.<i>0001</i>. (B) Reciprocal IC50 serum neutralization titers following the DNA/Protein Boost 1 immunization (Post-DP1) and the Post DNA/Protein Boost 2 immunization (Post-DP2). Neutralizing activities were determined against viruses expressing WT 426c envelope and 426c envelope NLGS variants with mutations in Loop D and V5. NLGS-1 lacks the NLGS at position 276 in Loop D, NLGS-2 lacks two NLGS in V5 at positions 460 and 463, while NLGS-3 lacks all three NLGS.</p

B cell clonal lineage alterations upon recombinant HIV-1 envelope immunization of rhesus macaques

<div><p>Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs, but is also targeted by binding, non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus, our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here, in addition to serological analysis, we employed high-throughput BCR sequence analysis from the periphery, lymph nodes and bone marrow, as well as B cell- and antibody-isolation and characterization methods, to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1, 2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic, but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003–0.02%) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors.</p></div

<i>Ex vivo</i> production of neutralizing antibodies from B cells isolated animals immunized with the NLGS-3 Core immunogen.

<p>IgG+ B cells were sorted from PBMC and cultured as discussed in the Materials and Methods section. B cell supernatants were tested for neutralizing activity against the WT 426c virus (426c WT; left panels) or a variant lacking three NLGS at positions N276, N460 and N463 (426c NLGS-3; right panels). Results from supernatants from B cells isolated from animals immunized with the WT immunogen are shown in the top two panels, while results from the NLGS-3 Core immunized animals are shown in the bottom two panels. Each dot corresponds to a single well (which contained approximately 1,000 B cells).</p