High resolution spatial profiling of the hematopoietic landscape of the murine lung.

Our current understanding of blood cell development stems primarily from adult bone marrow and the fetal liver. However, emerging evidence highlights the lung as a previously underappreciated residence for hematopoietic cells. Spatial in situ transcriptomics enables accurate mapping of cell identities and interactions within intact tissue, providing insights not accessible by dissociated single-cell profiling. Here, we present a high-resolution spatial transcriptomic atlas of the healthy adult murine lung, emphasizing the hemato-endothelial landscape. As a case study, we developed a workflow to identify and curate rare, often multinucleated, megakaryocytes, by binning spatial enrichment of canonical markers, expert curation, and correction for segmentation artifacts. We characterized the spatial neighborhoods of megakaryocytes within vascular, stromal, and immune microenvironments. We demonstrate the utility of this dataset for hypothesis-driven signaling studies by examining ligand-receptor interactions across canonical pathways. This resource defines the lung-blood niche and advances our understanding of the organ-specific properties of blood cells.