Poster#2: Integrative Single-Cell and Epigenomic Profiling with Spatial Transcriptomics to identify Cell Types and cis-Regulatory Elements in the Human Habenula

JHU. April 10th.
Event: Single Cell and Spatial Omics Symposium
Abstract:
The habenula (Hb) is a bilateral midline structure in the brain containing a unique distribution of cell types implicated in reward processing. Due to its small size, few studies have investigated the molecular anatomy of the human Hb and little is known about the molecular signatures of spatially organized cell types in this region. Here we generated a spatiomolecular map of the human Hb using 10X Genomics Multi-ome and Visium platforms. We performed combined single nucleus RNA and ATAC sequencing on post-mortem human Hb from 10 adult neurotypical control donors to identify 13 transcriptomic signatures for medial and lateral Hb cell types and cis-regulatory elements associated with these spatially distinct cell types. Using Visium, we also identified data-driven spatial domains enriched in Hb cell types across the anterior-posterior axis of the human Hb. In summary, we present an integrated single cell multi-omic and spatial transcriptomic atlas of the human Hb.

Transcript

1. Gene Expression SLC5A7 Integrative Single-Cell and Epigenomic Profiling with Spatial Transcriptomics to identify Cell Types and cis-Regulatory Elements in the Human Habenula Kelsey D. Montgomery*, Cynthia S. Cardinault*, Svitlana Bach, Sarah Maguire, Nicholas J. Eagles, Chunyu Liu, Heena R. Divecha, Ruth Zhang, Atharv Chandra, James Tooke, Amy Deep-Soboslay, Ryan A. Miller, Louise A. Huuki-Myers, Ege A. Yalcinbas, Manisha Barse, Joel E. Kleinman, Thomas M. Hyde, Brion Maher, Rahul A. Bharadwaj, Leonardo Collado-Torres, Kristen R. Maynard Lieber Institute for Brain Development, Johns Hopkins Medical Campus, Baltimore, MD, 21205, USA ABSTRACT CONCLUSIONS ACKNOWLEDGEMENTS HABENULA BACKGROUND, VISIUM PROCESSING, AND SPATIAL DOMAIN IDENTIFICATION EXPERIMENTAL DESIGN • High-resolution multi-omic profiling identified 42 clusters from 55,702 high-quality nuclei. • 13 clusters (31%) expressed habenula (Hb) neuron markers, comprising 17,827 nuclei (32%). • 9 out of 10 fine-resolution snRNA-seq clusters from Yalcinbas et al., bioRxiv, 2024 mapped to our clusters, confirming reproducibility of Hb sub-domain architecture. • Hb multi-ome clusters showed strong correspondence with independent snRNA-seq data, supporting cross-platform consistency. • Spatial transcriptomics integration revealed conserved sub-domains and enabled mapping of cis-regulatory elements to spatially distinct cell types. • Findings establish a molecular framework to study the role of the Hb in depression, addiction, and related neuropsychiatric conditions. We sincerely thank the families and individuals whose donations made this research possible. We also appreciate the Neuropathology team at the Lieber Institute for their assistance with tissue processing. We thank the 10x Genomics team for their support in preparing the data for customized secondary analysis. This work was supported by NIH grant 1R01DA055823 (awarded to Dr. Kristen Maynard) and the Lieber Institute for Brain Development. The authors declare no conflicts of interest. HABENULA MULTI-OME PROCESSING Expression of Hb gene marker GPR151 on Visium data INTERACTIVELY EXPLORE THIS DATA (A) Schematic Workflow (B) Strategy for cell recovery with secondary analysis Barcodes recovery in both modalities Spatial Registration: Consistency Across Multi-ome and Visium HD Kelsey Montgomery, BS, HTL(ASCP) Cynthia S. Cardinault, Ph.D Leonardo Collado-Torres, Ph.D. Investigator in Data Science at the Lieber Institute for Brain Development. Kristen Maynard, Ph.D. Investigator in the Molecular Neuroanatomy Division at the Lieber Institute for Brain Development. S04 S05 S06 S10 S11 S12 S03 S07 S08 S09 S04 S05 S06 S10 S11 S12 S03 S07 S08 S09 Does Subcellular Resolution Enhance the Anatomical Mapping of Hb Subdomains? Spatial Registration of Visium with snRNA-seq at “Broad resolution” EXPLORING DGE ACROSS HABENULA ANTERIOR AND POSTERIOR AXIS SCHEMATIC INTEGRATION OF SPATIAL DATA AND MULTI-OME DATA The habenula (Hb) is a bilateral midline structure in the brain containing a unique distribution of cell types implicated in reward processing. Due to its small size, few studies have investigated the molecular anatomy of the human Hb and little is known about the molecular signatures of spatially organized cell types in this region. Here we generated a spatiomolecular map of the human Hb using 10X Genomics Multi-ome and Visium platforms. We performed combined single nucleus RNA and ATAC sequencing on post-mortem human Hb from 10 adult neurotypical control donors to identify 13 transcriptomic signatures for medial and lateral Hb cell types and cis-regulatory elements associated with these spatially distinct cell types. Using Visium, we also identified data-driven spatial domains enriched in Hb cell types across the anterior-posterior axis of the human Hb. In summary, we present an integrated single cell multi-omic and spatial transcriptomic atlas of the human Hb. *Secondary analysis acts on: clustering, dimensionality_reduction, feature_linkage, tf_analysis (A) The spatial (BS-K13) habenula domain 11 demonstrates strong concordance with snRNA-seq independent data. (B) The table highlights consistent habenula signals identified across snRNA-seq and Visium 10x independent data, at both broad and fine resolution levels. MULTI-OME QUALITY CONTROL AND CLUSTER IDENTIFICATION Expression of Habenula Marker Genes Spatial Registration of Multiome with snRNA-seq at “Fine resolution” “Fine Resolution” “RNA” and “ATAC” outlier detection Locating the Habenula Image shows clusters identified in one Visium sample. Habenula cluster six matches with Habenula regions confirmed by RNAScope. 1 cm Anatomic Verification via smFISH Utilization of the Entire Habenula snRNAseq and ATACseq (Multi-ome) Data Generation (A) Visium Data Generation (B) Visium HD Data Generation (A) (B) POU4F1 GPR151 TAC3 POU4F1 GPR151 TAC3 Explore Visium data: libd.shinyapps.io/Habenula_Visium/ Peak Profile for SLC5A7: NeuN Enrichment Multi-ome → Visium HD → Multi-ome → snRNA-seq Fine → Visium → ← snRNA-seq (Yalcinbas, 2024) Spatial Localization of Gene Expression Yalcinbas bioRxiv Hb Level Clustering of Human Epithalamic Cell Types SPATIAL DATA INTEGRATION WITH MULTI-OME