Elucidating the cellular architecture of the human cerebral cortex is central to understanding our cognitive abilities and susceptibility to disease. Here we applied single nucleus RNA-sequencing to perform a comprehensive analysis of cell types in the middle temporal gyrus of human cortex. We identified a highly diverse set of excitatory and inhibitory neuronal 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 human cell type properties. Despite this general conservation, we also find extensive differences between homologous human and mouse cell types, including dramatic alterations in proportions, laminar distributions, gene expression, and morphology. These species-specific features emphasize the importance of directly studying human brain.

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.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO+ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA-sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4+ T cell compartment in the human fetal intestine. We identified 22 CD4+ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4+ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4+ T cells. Imaging-mass cytometry indicated that memory-like CD4+ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4+ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

23The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved 24 across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single 25 nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup 26 of this region, whose similarity mirrors evolutionary distance and is consistent between the 27 transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal 28 types allowed the generation of a cross-species consensus cell type classification and inference of 29 conserved cell type properties across species. Despite overall conservation, many species 30 specializations were apparent, including differences in cell type proportions, gene expression, DNA 31 methylation, and chromatin state. Few cell type marker genes were conserved across species, 32 providing a short list of candidate genes and regulatory mechanisms responsible for conserved features 33 of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic 34 classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human 35 primate and human and characterization of their highly specialized physiology and anatomy. These 36 findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and 37 point to the genes and regulatory pathways responsible for the functional identity of cell types and their 38 species-specific adaptations. 39 40 distinguished on the basis of regions of open chromatin or DNA methylation 5,9,10 . Furthermore, several 48 recent studies have shown that transcriptomically-defined cell types can be aligned across species 2,11-49 13 , indicating that these methods provide a path to quantitatively study evolutionary conservation and 50 divergence at the level of cell types. However, application of these methods has been highly 51 fragmented to date. Human and mouse comparisons have been performed in different cortical regions, 52 using single-cell (with biases in cell proportions) versus single-nucleus (with biases in transcript 53 makeup) analysis, and most single-cell transcriptomic and epigenomic studies have been performed 54 independently. 55 56The primary motor cortex (MOp in mouse, M1 in human and non-human primates, all referred to as M1 57 herein) provides an ideal cortical region to address questions about cellular evolution in rodents and 58 primates by integrating these approaches. Unlike the primary visual cortex (V1), which is highly 59 specialized in primates, or frontal and temporal association areas, whose homologues in rodents 60 remain poorly defined, M1 is essential for fine motor control and is functionally conserved across 61 placental mammals. M1 is an agranular cortex, lacking a defined L4, although neurons with L4-like 62properties have been described 14 . L5 of carnivore and primate M1 contains exceptionally large 63 "giganto-cellular" corticospinal neurons (Betz c...

When Genetic Programming is used to evolve decision trees for data classification, search spaces tend to become extremely large. We present several methods using techniques from the field of machine learning to refine and thereby reduce the search space sizes for decision tree evolvers. We will show that these refinement methods improve the classification performance of our algorithms.