A Multimodal Cell Census and Atlas of the Mammalian Primary Motor Cortex

Resources for "A Multimodal Cell Census and Atlas of the Mammalian Primary Motor Cortex", 2021

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. The 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. This page provides a guide to access accessing data and tools associated with this work

Companion Manuscripts

A series of companion studies supported the findings in this manuscript.

• An integrated transcriptomic and epigenomic atlas of mouse primary motor cortex cell types, Yao et al., 2020
• Evolution of cellular diversity in primary motor cortex of human, marmoset monkey, and mouse, Bakken et al., 2020
• Molecular, spatial and projection diversity of neurons in primary motor cortex revealed by in situ single-cell transcriptomics, Zhang et al, 2020
• Phenotypic variation of transcriptomic cell types in mouse motor cortex, Scala et al, 2020
• Cellular Anatomy of the Mouse Primary Motor Cortex, Munoz-Casteneda, 2020
• Genetic dissection of the glutamatergic neuron system in cerebral cortex, Matho et al,2020
• DNA Methylation Atlas of the Mouse Brain at Single-Cell Resolution, Liu et al., 2020
• Epigenomic Diversity of Cortical Projection Neurons in the Mouse Brain, Zhang et al., 2020
• An atlas of gene regulatory elements in Adult Mouse Cerebrum, Li et al, 2020
• Human cortical expansion involves diversification and specialization of supragranular intratelencephalic-projecting neurons, Berg et al,2020
• Brain-wide single neuron reconstruction reveals morphological diversity in molecularly defined striatal, thalamic, cortical and claustral neuron types, Peng et al,2020

Main Figures

Data and supporting code related to analyses in figures.

1. Figure 1: Summary of experimental and computational approaches taken and as well as community resources generated by BICCN.
2. Figure 2: MOp consensus cell type taxonomy.
3. Figure 3: In situ cell-type identification, spatial mapping and projection mapping of individual cells in the MOp by MERFISH
4. Figure 4: Correspondence between transcriptomic and morpho-electrical properties of mouse MOp neurons by Patch-seq, and cross-species comparison of L5 ET neurons.
5. Figure 5: Epi-Retro-Seq links molecular cell types with distal projection targets.
6. Figure 6: Genetic tools for targeting cortical glutamatergic projection neuron types.
7. Figure 7: Global wiring diagram and anatomical characterization of MOp-ul neuron types.
8. Figure 8: Existence of L4 excitatory neurons in MOp.
9. Figure 9: Two distinct L5 ET projection neuron types in MOp.
10. Figure 10: An integrated multimodal census and atlas of cell types in the primary motor cortex of mouse, marmoset and human.

Temporarily, links to BIL-hosted datasets will direct one to the raw data directories at BIL. As BIL creates landing pages for these datasets, these links will be replaced with links to the landing pages, which will include a description of the dataset, the generating lab, contact info, and downloading info.

Figure 1: Summary of experimental and computational approaches taken and as well as community resources generated by BICCN

Primary Data Resources

All primary data is accessible through the Brain Cell Data Center and data archives.

• Brain Cell Data Center (BCDC), Overall BICCN organization and data, www.biccn.org

BRAIN Initiative Data Archives

• Neuroscience Multi-omic Data Archive (NeMO), RRID:SCR_016152

• Brain Image Library (BIL), RRID:SCR_017272

• Distributed Archives for Neurophysiology Data Integration (DANDI), RRID:SCR_017571

Analysis platform for BICCN -omics datasets

• NeMO Analytics

Techniques and methods

The following papers are referenced techniques other than those in the companion papers.

Transcription

• Shared and distinct transcriptomic cell types across neocortical areas, Tasic et al., 2018
• Conserved cell types with divergent features in human versus mouse cortex, Hodge et al., 2019
• Innovations present in the primate interneuron repertoire, Krienen et al., 2020
• A transcriptomic atlas of the mouse cerebellum reveals regional specializations and novel cell types, Kozareva et al., 2020
• A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation, Yao et al., 2020

DNA Methylation

• Robust single-cell DNA methylome profiling with snmC-seq2, Luo et al., 2018
• Spatiotemporal DNA methylome dynamics of the developing mouse fetus, He et al., 2018

Open Chromatin

• Single-nucleus analysis of accessible chromatin in developing mouse forebrain reveals cell-type-specific transcriptional regulation, Preissl et al., 2018

Combined transcription/Open chromatin

• High-throughput sequencing of the transcriptome and chromatin accessibility in the same cel, Chen et al., 2019

Spatial transcriptomics

• RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells, Chen et al., 2015
• Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region, Moffitt et al., 2019

Multimodal and cross-species integration

• Comprehensive integration of single cell data, Stuart et al., 2019
• Single-Cell Multi-omic Integration Compares and Contrasts Features of Brain Cell Identity, Welch et al., 2019
• Characterizing the replicability of cell types defined by single cell RNA-sequencing data using MetaNeighbor. Crow et al., 2018

Electrophysiology, cellular morphology and transcriptomics

• Electrophysiological, transcriptomic and morphologic profiling of single neurons using Patch-seq, Cadwell et al., 2016
• Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq, Cadwell et al., 2017

Cellular morphology

• High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level, Gong et al., 2016
• Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain, Winnubst et al. 2019
• An open repository for single-cell reconstructions of the brain forest, Akram et al, 2018

Inter-areal circuit

• Neural networks of the mouse cortex, Zingg et al., 2014
• AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors, Zingg et al., 2017

Genetic tools

• Genetic dissection of glutamatergic neuron subpopulations and developmental trajectories in the cerebral cortex, Matho et al., 2020

Cellular census quantification

• Operations Research Methods for Estimating the Population Size of Neuron Types, Attili et al.,2020

Other Key Resources

Resource	Description	Link	RRID
NeMO Analytics	Companion resources	NeMO	RRID:SCR_018164
CATlas	Atlas of Cis-elements gene regulatory elements	CATlas	RRID:SCR_018690
LIGER	Integrating multiple single cell datasets	LIGER	RRID:SCR_018100
Neuromorpho.org	Inventory of digital reconstructed neurons	Neuromorpho	RRID:SCR_002145
MetaNeighbor	Characterizing cell type replicability	MetaNeighbor	RRID:SCR_016727
Patch-Seq	Companion resources	Mini-atlas PatchSeq	RRID:SCR_021115
Allen CCF	Allen Mouse Brain Common Coordinate Framework	Atlas viewer	RRID:SCR_020999
MORF Reporter	Reporter mouse resource page	MORF	RRID:SCR_021125
	Individual Mice from MORF		RRID:IMSR_JAX:035403, RRID:IMSR_JAX:035400, RRID:IMSR_JAX:035404
CCN	Common Cell Type Nomenclature	CCN	RRID:SCR_021124

Note, individual tools created by or used by the BICCN consortium are accessible via the resource registry.

Figure 2: MOp consensus cell type taxonomy

High-resolution primary data

Panel	Description of data collection	BICCN Project Summary Page	Repository Link
2a	NEMO Publication Release Bundle for MOp MiniAtlas		NEMO
2b-g	10x v3 sequencing data from macaque		NEMO
2b-g	Transcriptomic characterization of cell types in human brain: 10x nuclei v3 (10X159-1 through 10x160-8)	Project Summary	NEMO
2b-g	A Molecular and Cellular Atlas of the Marmoset Brain - 10x v3 (bi005_m1, bi006_m1)	Project Summary	NEMO
2b-g	Transcriptomic cell types in the mouse brain: 10x nuclei v3 (files with prefix pBICCNsMMrMOp)	Project Summary	NEMO

Analysis and code for main figure

Panel 2a

Panel 2b: Dendrogram reproduced from companion paper (Bakken et al. 2020)

Panel 2c

Panels 2d, 2e

Panels 2f, 2g: Reproduced from companion paper (Bakken et al. 2020)

Panel 2h

Panel 2i: Custom UCSC browser of all M1 tracks

Extended or supplementary data figures

Panel 2j: Browser

Extended Data Figure 1

Figure 3: In situ cell type identification spatial mapping and projection mapping of individual cells in the MOp by MERFISH

High-resolution primary data

Panel	Description of data collection	BICCN Project Summary Page	Repository Link
3	A molecularly defined and spatially resolved cell atlas using spatial transcriptomics; MERFISH raw data and processed	Project Summary	BIL

Analysis and code for main figure

MERFISH data processing pipeline

Panels 3a, b, e

Figure 4: Correspondence between transcriptomic and morpho-electrical properties of mouse MOp neurons by Patch-seq, and cross-species comparison of L5 ET neurons

High-resolution primary data

Panel	Description of data collection	BICCN Project Summary Page	Repository Link
4a	Patch-seq of mouse motor cortex: FASTQ files	Project Summary	NEMO
4a	Transcriptomic cell types in the mouse brain: 10x v2 cells (FASTQ)	Project Summary	NEMO
4a	Transcriptomic cell types in the mouse brain: 10x v3 cells (FASTQ)	Project Summary	NEMO
4a	Transcriptomic cell types in the mouse brain: SMART-seq cells (FASTQ)	Project Summary	NEMO
4a	Transcriptomic cell types in the mouse brain: 10x nuclei	Project Summary	NEMO
4a	Transcriptomic cell types in the mouse brain: 10x nuclei	Project Summary	NEMO
4a	Transcriptomic cell types in the mouse brain: SMART-seq nuclei (FASTQ)	Project Summary	NEMO
4b-g	Morphological reconstructions in SWC		BIL
4b-g	Patch-seq of mouse motor cortex: electrophysiological traces in NWB format	Project Summary	DANDI
4h	10x v3 sequencing data from macaque (FASTQ)		NEMO
4h	Transcriptomic characterization of cell types in human brain: 10x nuclei v3	Project Summary	NEMO
4h	A Molecular and Cellular Atlas of the Marmoset Brain - 10x v3	Project Summary	NEMO
4h	Transcriptomic cell types in the mouse brain: 10x nuclei (v3)		NEMO
4i-k	Electrophysiological traces in NWB format		DANDI
4i-k	Morphological reconstructions in SWC format		BIL

Analysis and code for main figure

Panel h:

• 10x 4 species integration -Code

Panels a-g:

• Code

Figure 5: Epi-Retro-Seq links molecular cell types with distal projection targets

High-resolution primary data

Panel	Description of data collection	BICCN Project Summary Page	Repository Link
5	Regional DNA methylation profiling of individual projection neurons isolated from the mouse brain	Project Summary	NEMO
5	Gene Expression Omnibus data		GEO

Analysis and code for main figure

Code

Figure 6: Genetic tools for targeting cortical glutamatergic projection neuron types

High-resolution primary data

Panel c

Label in Fig.	Full Descriptive ID	Induction date	Harvest date	Tamoxifen dose	Originating Lab
Lhx2	Lhx2-CreER; Ai14	E12.5	E13.5	2 mg/kg body weight	Huang
Lhx2	Lhx2-CreER; Ai14	E12.5	P30	100 mg/kg body weight	Huang
Fezf2	Fezf2-CreER; Ai14	E12.5	E13.5	2 mg/kg body weight	Huang
Fezf2	Fezf2-CreER; Ai14	E12.5	P30	2 mg/kg body weight	Huang

Panels d-k

Label in Fig.	Full Descriptive ID	Experiment ID	Originating Lab	Brain Architecture Viewer	BIL link
PlxnD1	PlxnD1-CreER;LSL-Flp	180722	Huang	Viewer	BIL
PlxnD1	PlxnD1-CreER;LSL-Flp	180730	Huang	Viewer	BIL
Fezf2	Fezf2-CreER;LSL-Flp	180830	Huang	Viewer	BIL
Fezf2	Fezf2-CreER;LSL-Flp	190903	Huang	Viewer	BIL
Tle4	Tle4-CreER;LSL-Flp	180605	Huang	Viewer	BIL
Tle4	Tle4-CreER;LSL-Flp	180816	Huang	Viewer	BIL
Foxp2	Foxp2-Cre	190915	Huang	Viewer	TBD
Foxp2	Foxp2-Cre	191209	Huang	Viewer	BIL

Panel j

Label in Fig.	Full Descriptive ID	Experiment ID	Originating Lab	Brain Architecture Viewer	BIL link
PlxnD1-Tbr2	Tbr2-CreER;PlxnD1-Flp;dtTA TM E17.5	210203	Huang	Viewer	TBD
PlxnD1-Tbr2	Tbr2-CreER;PlxnD1-Flp;dtTA TM E17.5	210211	Huang	Viewer	TBD
PlxnD1-Tbr2	Tbr2-CreER;PlxnD1-Flp;dtTA TM E17.5	210301	Huang	Viewer	TBD
PlxnD1-Tbr2	Tbr2-CreER;PlxnD1-Flp;dtTA TM E17.5	210216	Huang	Viewer	TBD
PlxnD1-Tbr2	Tbr2-CreER;PlxnD1-Flp;dtTA TM E17.5	210222	Huang	Viewer	TBD
PlxnD1-Tbr2	Tbr2-CreER;PlxnD1-Flp;dtTA TM E13.5	210302	Huang	Viewer	TBD

Figure 7: Global wiring diagram and anatomical characterization of MOp-ul neuron types

Panel c

Label in Fig.	Full Descriptive ID	Experiment ID	Originating Lab	SWC (single cells) or 25 um grid (tracer)
Rabies	Tlx3-660759241	660759241	Allen	Data link
AAV	C57BL/6J-127084296	127084296	Allen	Data link
Cux2 L2/3/4 IT	Cux2-IRES-Cre-947242021	947242021	Allen	Data link
Nr5a1 L4 IT	Nr5a1-Cre-882407664	882407664	Allen	Data link
Tlx3 L5 IT	Tlx3-Cre_PL56-880719308	880719308	Allen	Data link
Rbp4 L5 IT+ET	Rbp4-Cre_KL100-948130129	948130129	Allen	Data link
Sim1 L5 ET	Sim1-Cre_KJ18-297711339	297711339	Allen	Data link
Ntsr1 L6 CT	Ntsr1-Cre_GN220-159651060	159651060	Allen	Data link
IT projections	L2/3_Cux2-CreERT2-18453_3456_x24161_y6646	18453_3456_x24161_y6646	Allen	Data link
IT projections	L4_Cux2-CreERT2-18864_3338_x3396_y21865	18864_3338_x3396_y21865	Allen	Data link
IT projections	L5_AA0271 [A]	AA0271 [A]	MouseLight	Data link
ET projections	L5_+MY+TH_Fezf2-CreERT2-182725_3762_x5563_y19178	182725_3762_x5563_y19178	Allen	Data link
ET projections	L5_no MY_Fezf2-CreERT2_182725_4080_x6576_y11407	182725_4080_x6576_y11407	Allen	Data link
CT projections	L6_AA0649 [A]	AA0649 [A]	MouseLight	Data link
CT projections	L6_AA0898 [A]	AA0898 [A]	MouseLight	Data link

Panels d-i

Label in Fig.	Full Descriptive ID	Experiment ID	Originating Lab	Brain Architecture viewer
PlxnD1	PlxnD1-CreER;LSL-Flp	180722	Huang	Viewer
PlxnD1	PlxnD1-CreER;LSL-Flp	180730	Huang	Viewer
Tle4	Tle4-CreER;LSL-Flp	180605	Huang	Viewer
Tle4	Tle4-CreER;LSL-Flp	180816	Huang	Viewer

Analysis and code for main figure

Panel c Rendering of registered data in 3D

Figure 8: Existence of L4 excitatory neurons in MOp

Analysis and code for main figure

Panels 8a,c,e

Panel 8b

Figure 9: Two distinct L5 ET projection neuron types in MOp

Analysis and code for main figure

Panel 9d

Panel 9e

Figure 10: An integrated multimodal census and atlas of cell types in the primary motor cortex of mouse, marmoset and human

Analysis and code for main figure

snATAC-seq Utilites

Extended or supplementary data figures

snATAC-seq Utilites

Extended data figure 3