Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain

Cell.

2018 Aug 09

Saunders A, Macosko EZ, Wysoker A, Goldman M, Krienen FM, de Rivera H, Bien E, Baum M, Bortolin L, Wang S, Goeva A, Nemesh J, Kamitaki N, Brumbaugh S, Kulp D, McCarroll SA.
PMID: 30096299 | DOI: 10.1016/j.cell.2018.07.028

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.

Identification of visual cortex cell types and species differences using single-cell RNA sequencing

Nature communications

2022 Nov 12

Wei, JR;Hao, ZZ;Xu, C;Huang, M;Tang, L;Xu, N;Liu, R;Shen, Y;Teichmann, SA;Miao, Z;Liu, S;
PMID: 36371428 | DOI: 10.1038/s41467-022-34590-1

The primate neocortex exerts high cognitive ability and strong information processing capacity. Here, we establish a single-cell RNA sequencing dataset of 133,454 macaque visual cortical cells. It covers major cortical cell classes including 25 excitatory neuron types, 37 inhibitory neuron types and all glial cell types. We identified layer-specific markers including HPCAL1 and NXPH4, and also identified two cell types, an NPY-expressing excitatory neuron type that expresses the dopamine receptor D3 gene; and a primate specific activity-dependent OSTN + sensory neuron type. Comparisons of our dataset with humans and mice show that the gene expression profiles differ between species in relation to genes that are implicated in the synaptic plasticity and neuromodulation of excitatory neurons. The comparisons also revealed that glutamatergic neurons may be more diverse across species than GABAergic neurons and non-neuronal cells. These findings pave the way for understanding how the primary cortex fulfills the high-cognitive functions.

A harmonized atlas of mouse spinal cord cell types and their spatial organization

Nature communications

2021 Sep 29

Russ, DE;Cross, RBP;Li, L;Koch, SC;Matson, KJE;Yadav, A;Alkaslasi, MR;Lee, DI;Le Pichon, CE;Menon, V;Levine, AJ;
PMID: 34588430 | DOI: 10.1038/s41467-021-25125-1

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

Genetically Distinct Parallel Pathways in the Entopeduncular Nucleus for Limbic and Sensorimotor Output of the Basal Ganglia

Neuron

2017 Apr 05

Wallace ML, Saunders A, Huang KW, Philson AC, Goldman M, Macosko EZ, McCarroll SA, Sabatini BL.
PMID: 28384468 | DOI: 10.1016/j.neuron.2017.03.017

The basal ganglia (BG) integrate inputs from diverse sensorimotor, limbic, and associative regions to guide action-selection and goal-directed behaviors. The entopeduncular nucleus (EP) is a major BG output nucleus and has been suggested to channel signals from distinct BG nuclei to target regions involved in diverse functions. Here we use single-cell transcriptional and molecular analyses to demonstrate that the EP contains at least three classes of projection neurons-glutamate/GABA co-releasing somatostatin neurons, glutamatergic parvalbumin neurons, and GABAergic parvalbumin neurons. These classes comprise functionally and anatomically distinct output pathways that differentially affect EP target regions, such as the lateral habenula (LHb) and thalamus. Furthermore, LHb- and thalamic-projecting EP neurons are differentially innervated by subclasses of striatal and pallidal neurons. Therefore, we identify previously unknown subdivisions within the EP and reveal the existence of cascading, molecularly distinct projections through striatum and globus pallidus to EP targets within epithalamus and thalamus.

Mapping of clonal lineages across developmental stages in human neural differentiation

Cell stem cell

2023 Mar 14

You, Z;Wang, L;He, H;Wu, Z;Zhang, X;Xue, S;Xu, P;Hong, Y;Xiong, M;Wei, W;Chen, Y;
PMID: 36933556 | DOI: 10.1016/j.stem.2023.02.007

The cell lineages across developmental stages remain to be elucidated. Here, we developed single-cell split barcoding (SISBAR) that allows clonal tracking of single-cell transcriptomes across stages in an in vitro model of human ventral midbrain-hindbrain differentiation. We developed "potential-spective" and "origin-spective" analyses to investigate the cross-stage lineage relationships and mapped a multi-level clonal lineage landscape depicting the whole differentiation process. We uncovered many previously uncharacterized converging and diverging trajectories. Furthermore, we demonstrate that a transcriptome-defined cell type can arise from distinct lineages that leave molecular imprints on their progenies, and the multilineage fates of a progenitor cell-type represent the collective results of distinct rather than similar clonal fates of individual progenitors, each with distinct molecular signatures. Specifically, we uncovered a ventral midbrain progenitor cluster as the common clonal origin of midbrain dopaminergic (mDA) neurons, midbrain glutamatergic neurons, and vascular and leptomeningeal cells and identified a surface marker that can improve graft outcomes.

Cascade diversification directs generation of neuronal diversity in the hypothalamus

Cell stem cell

2021 Apr 17

Zhang, YH;Xu, M;Shi, X;Sun, XL;Mu, W;Wu, H;Wang, J;Li, S;Su, P;Gong, L;He, M;Yao, M;Wu, QF;
PMID: 33887179 | DOI: 10.1016/j.stem.2021.03.020

The hypothalamus contains an astounding heterogeneity of neurons that regulate endocrine, autonomic, and behavioral functions. However, its molecular developmental trajectory and origin of neuronal diversity remain unclear. Here, we profile the transcriptome of 43,261 cells derived from Rax+ hypothalamic neuroepithelium to map the developmental landscape of the mouse hypothalamus and trajectory of radial glial cells (RGCs), intermediate progenitor cells (IPCs), nascent neurons, and peptidergic neurons. We show that RGCs adopt a conserved strategy for multipotential differentiation but generate Ascl1+ and Neurog2+ IPCs. Ascl1+ IPCs differ from their telencephalic counterpart by displaying fate bifurcation, and postmitotic nascent neurons resolve into multiple peptidergic neuronal subtypes. Clonal analysis further demonstrates that single RGCs can produce multiple neuronal subtypes. Our study reveals that multiple cell types along the lineage hierarchy contribute to fate diversification of hypothalamic neurons in a stepwise fashion, suggesting a cascade diversification model that deconstructs the origin of neuronal diversity.

Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly

bioRxiv : the preprint server for biology

2023 Feb 07

Ayupe, AC;Choi, JS;Beckedorff, F;Mccartan, R;Levay, K;Park, KK;
PMID: 36778361 | DOI: 10.1101/2023.02.01.526254

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates input from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment and refinement of neural connectivity. Here we performed single nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We used these data to identify Pax7 as a marker for an anatomically homogeneous population of GABAergic neurons. Lastly, we report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known to regulate axon guidance and synapse formation. Our data provide a valuable resource for interrogating the mechanisms of circuit development, and identifying markers for manipulating specific SC neuronal populations and circuits.

SCAMPR: Single-Cell Automated Multiplex Pipeline for RNA Quantification and Spatial Mapping

SSRN Electronic Journal

2022 Mar 24

Ali Marandi Ghoddousi, R;Magalong, V;Kamitakahara, A;Levitt, P;
| DOI: 10.2139/ssrn.4064105

Spatial gene expression, achieved classically through _in situ_ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for the visualization of multiple mRNAs at single-cell resolution, greatly expanding the ability to link gene expression to tissue topography. Yet, methods for efficient and accurate quantification and analysis of high dimensional _in situ_- hybridization are limited. To this end, the Single-Cell Automated Multiplex Pipeline for RNA (SCAMPR) was developed, facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low and high abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on differential gene expression by vagal neuron subtypes.

Parahippocampal latrophilin-2 (ADGRL2) expression controls topographical presubiculum to entorhinal cortex circuit connectivity

Cell reports

2021 Nov 23

Donohue, JD;Amidon, RF;Murphy, TR;Wong, AJ;Liu, ED;Saab, L;King, AJ;Pae, H;Ajayi, MT;Anderson, GR;
PMID: 34818557 | DOI: 10.1016/j.celrep.2021.110031

Brain circuits are comprised of distinct interconnected neurons that are assembled by synaptic recognition molecules presented by defined pre- and post-synaptic neurons. This cell-cell recognition process is mediated by varying cellular adhesion molecules, including the latrophilin family of adhesion G-protein-coupled receptors. Focusing on parahippocampal circuitry, we find that latrophilin-2 (Lphn2; gene symbol ADGRL2) is specifically enriched in interconnected subregions of the medial entorhinal cortex (MEC), presubiculum (PrS), and parasubiculum (PaS). Retrograde viral tracing from the Lphn2-enriched region of the MEC reveals unique topographical patterning of inputs arising from the PrS and PaS that mirrors Lphn2 expression. Using a Lphn2 conditional knockout mouse model, we find that deletion of MEC Lphn2 expression selectively impairs retrograde viral labeling of inputs arising from the ipsilateral PrS. Combined with analysis of Lphn2 expression within the MEC, this study reveals Lphn2 to be selectively expressed by defined cell types and essential for MEC-PrS circuit connectivity.

SALM4 negatively regulates NMDA receptor function and fear memory consolidation

Communications biology

2021 Sep 29

Lie, E;Yeo, Y;Lee, EJ;Shin, W;Kim, K;Han, KA;Yang, E;Choi, TY;Bae, M;Lee, S;Um, SM;Choi, SY;Kim, H;Ko, J;Kim, E;
PMID: 34588597 | DOI: 10.1038/s42003-021-02656-3

Many synaptic adhesion molecules positively regulate synapse development and function, but relatively little is known about negative regulation. SALM4/Lrfn3 (synaptic adhesion-like molecule 4/leucine rich repeat and fibronectin type III domain containing 3) inhibits synapse development by suppressing other SALM family proteins, but whether SALM4 also inhibits synaptic function and specific behaviors remains unclear. Here we show that SALM4-knockout (Lrfn3-/-) male mice display enhanced contextual fear memory consolidation (7-day post-training) but not acquisition or 1-day retention, and exhibit normal cued fear, spatial, and object-recognition memory. The Lrfn3-/- hippocampus show increased currents of GluN2B-containing N-methyl-D-aspartate (NMDA) receptors (GluN2B-NMDARs), but not α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors (AMPARs), which requires the presynaptic receptor tyrosine phosphatase PTPσ. Chronic treatment of Lrfn3-/- mice with fluoxetine, a selective serotonin reuptake inhibitor used to treat excessive fear memory that directly inhibits GluN2B-NMDARs, normalizes NMDAR function and contextual fear memory consolidation in Lrfn3-/- mice, although the GluN2B-specific NMDAR antagonist ifenprodil was not sufficient to reverse the enhanced fear memory consolidation. These results suggest that SALM4 suppresses excessive GluN2B-NMDAR (not AMPAR) function and fear memory consolidation (not acquisition).

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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