Poster#1: Integrative Single-Cell and Epigenomic Profiling with Spatial Transcriptomics to identify Cell Types and cis-Regulatory Elements in the Human Habenula

April 10th, 2025
Event. Single Cell and Spatial Omics Symposium at JHU.
Abstract:
The human habenula (Hb) is a small, evolutionarily conserved brain structure involved in mood regulation and reward processing. It has been implicated in neuropsychiatric disorders such as major depressive disorder (MDD) and substance use disorder (SUD), yet its molecular architecture remains poorly defined due to its small size and anatomical complexity. To address this gap, we applied a multi-omic approach combining single-nucleus RNA and ATAC sequencing with Visium spatial transcriptomics to generate a comprehensive molecular atlas of the human Hb.
Integration of multi-ome data with single-cell data confirmed the presence of conserved Hb sub-domains. Furthermore, spatial transcriptomics consistently confirmed anatomically distinct domains across the Hb. Several multi-ome clusters aligned with both single-cell and spatial domains, enabling us to begin identifying cis-regulatory elements (CREs) associated with spatially distinct cell types.

Transcript

1. 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 OBJECTIVES METHOD CONCLUSION ACKNOWLEDGEMENTS HABENULA VISIUM RESULTS The human habenula (Hb) is a small, evolutionarily conserved brain structure involved in mood regulation and reward processing. It has been implicated in neuropsychiatric disorders such as major depressive disorder (MDD) and substance use disorder (SUD), yet its molecular architecture remains poorly defined due to its small size and anatomical complexity. To address this gap, we applied a multi-omic approach combining single-nucleus RNA and ATAC sequencing with Visium spatial transcriptomics to generate a comprehensive molecular atlas of the human Hb. Integration of multi-ome data with single-cell data confirmed the presence of conserved Hb sub-domains. Furthermore, spatial transcriptomics consistently confirmed anatomically distinct domains across the Hb. Several multi-ome clusters aligned with both single-cell and spatial domains, enabling us to begin identifying cis-regulatory elements (CREs) associated with spatially distinct cell types. ❖ Generate a multi-omic single-cell atlas of the human habenula by integrating snRNA-seq and snATAC-seq data to define cellular composition and regulatory landscapes. ❖ Map the molecular anatomy of the habenula using spatial transcriptomics (Visium) to identify spatially organized cell populations. ❖ Integrate spatial and multi-omic datasets to link transcriptional identities with anatomical localization and infer spatially resolved cis- regulatory elements. EXPERIMENTAL DESIGN DATA INTEGRATION Ongoing analyses aim to link these CREs to genetic variants associated with MDD, SUD, and related neuropsychiatric conditions. Collectively, these data provide new insight into the molecular organization of the human habenula and offer a framework for investigating its role in mental health and disease. FUTURE DIRECTIONS We identified 42 multi-ome clusters from 55,702 high-quality nuclei, with 13 clusters (31%) expressing habenula neuron markers and containing 17,827 cells (32%). Nine of ten fine-resolution clusters from Yalcinbas et al., 2024 were registered to our multi-ome clusters, confirming the reproducibility of Hb sub-domain architecture across donors. Integration with spatial transcriptomics revealed conserved sub-domains and enabled mapping of cis- regulatory elements to spatially distinct cell types. These findings provide a molecular framework for exploring the habenula’s role in MDD, OUD, and related 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. 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 MULTIOME RESULTS Data correction Expression of Medial Habenula GPR151 Outlier cells detected with scran and SpotSweeper Thanks to Abby Primack and Lisa Johnson from 10x Genomics for their technical support EXPLORE OUR DATA Habenula anatomical region Gene expression projection of Hb multiome Cluster.11 Cell recovery with secondary analysis “Cell Ranger ARC reanalyze” Barcodes recovery in “RNA” and “ATAC” modalities “RNA” and “ATAC” outlier detection Habenula Cluster Profiles Pre-selection of Habenula Clusters Consistency Across Datasets and Data Types in the Habenula Clusters Habenula probe panel - - TAC3 - medial habenula - GPR151 - lateral habenula - MBP - white matter - POU4F1 - general habenula Gene and Peak Profiles in Habenula Cluster 11 E Join our Learning Resources on Youtube Leonardo Collado-Torres, Ph.D. Investigator in Data-Sciences at the Lieber Institute for Brain Development. Kristen Maynard Ph.D. Investigator in the Molecular Neuroanatomy Division at the Lieber Institute for Brain Development. Join me on LinkedIn Cynthia S. Cardinault S04 S05 S06 S10 S11 S12 S03 S07 S08 S09 Kelsey Montgomery Nicholas J. Eagles S04 S05 S06 S10 S11 S12 S03 S07 S08 S09 Sarah Maguire Ryan A. Miller Svitlana Bach Louise A. Huuki-Myers https://libd.shinyapps.io/Habenula_Visium/ Does Subcellular Resolution Enhance the Anatomical Mapping of Sub-Habenula Domains?"