biostudies-arrayexpressDaisuke OkuzakiMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15929Receptor activator of nuclear factor kappa B ligand (RANKL) is an essential cytokine that induces osteoclastic differentiation by monocyte-macrophage lineage precursors. Here, we showed that in addition to its conventional action, RANKL controls vascular permeability in the bone marrow, where it facilitates the mobilization of hematopoietic monocytic cells, including osteoclast precursors, and resultantly regulate bone metabolism. RANK, a cognate receptor for RANKL, is abundantly expressed in sinusoidal endothelial cells and controls vascular permeability by regulating the expression patterns of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. High RANKL expression was detected in perivascular CXCL12-abundant reticular (CAR) stromal cells. Specific deletion of RANKL expression in CAR cells abrogated the vascular leakage, suggesting that perivascular RANKL is responsible for controlling permeability. In summary, our study revealed a role for RANK/RANKL signaling as a gatekeeper of bone marrow sinusoids in vivo.biostudies-arrayexpressSample Collection - In accordance with the manufacturer’s recommendations, approximately 16,500 live cells per sample were loaded onto the Chromium controller to generate 10,000 single-cell gel-bead emulsions for library preparation and sequencing. Single cell droplets were generated by 10x Genomics Chromium Controller, and cDNAs with UMIs were generated at single-cell level. Raw FASTQ files obtained from below (Stromal cell 1, Stromal cell 2, CD45- Ter119- CD31+, and RANKL_CD45-Ter119-CD31+) were processed using the Cell Ranger pipeline (v6.0.0 or v7.1.0, 10x Genomics) for alignment to the mm10 genome, UMI collapsing, and UMI counting for each gene–cell (barcode) combination.Library Construction - Raw FASTQ files obtained from below (Stromal cell 1, Stromal cell 2, CD45- Ter119- CD31+, and RANKL_CD45-Ter119-CD31+) were processed using the Cell Ranger pipeline (v6.0.0 or v7.1.0, 10x Genomics) for alignment to the mm10 genome, UMI collapsing, and UMI counting for each gene–cell (barcode) combination.Sequencing - Sequencing was performed using two platforms: Stromal cell 1 and Stromal cell 2 (CD45- Ter119- CD31-) were sequenced on the MGI DNBSEQ-G400RS platform with a 100+100 base paired-end mode, while CD45- Ter119- CD31+ cells were sequenced on the NovaSeq 6000 platform with a 28+90 base paired-end mode.Nucleic Acid Extraction - Sinlge cell droplets were generated by 10x Genomics Chromium Controller, and cDNAs with UMIs were generated at single-cell level.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Graph-based clustering results were manually annotated using Loupe Browser. Only cells with UMI counts between 1,500 and 59,456 were retained for the analysis. Cell barcodes with > 10% mitochondrial UMIs were regarded as potential dead cells and excluded. Cells with a Tnfrsf11a UMI count of 0 (before normalization or other scaling) were defined as RANK-negative cells; those with a positive count were defined as RANK-positive cells. Expression levels of VCAM1 and ICAM1 in RANK+ and RANK– cells were compared using Seurat (version 4.3.0).MetabolomicsUnknownTranscriptomicsGenomicsProteomics10x GenomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsMus musculusThe RANK/RANKL axis controls vascular dynamics in the bone marrowDaisuke OkuzakiTakeshi Kaneko, Shinya Yari, Junichi Kikuta, Yoshiki Omatsu, Shigeto Seno, Takao Sudo, Tetsuo Hasegawa, Kosuke Muraoka, Yoshiaki Okada, Yoshiaki Kubota, Daisuke Okuzaki, Yasuhiro Kobayashi, Atsushi Kumanogoh, Nobuyuki Udagawa, Takashi Nagasawa, Josef M. Penninger, and Masaru IshiifalseThe RANK/RANKL axis controls vascular dynamics in the bone marrowReceptor activator of nuclear factor kappa B ligand (RANKL) is an essential cytokine that induces osteoclastic differentiation by monocyte-macrophage lineage precursors. Here, we showed that in addition to its conventional action, RANKL controls vascular permeability in the bone marrow, where it facilitates the mobilization of hematopoietic monocytic cells, including osteoclast precursors, and resultantly regulate bone metabolism. RANK, a cognate receptor for RANKL, is abundantly expressed in sinusoidal endothelial cells and controls vascular permeability by regulating the expression patterns of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. High RANKL expression was detected in perivascular CXCL12-abundant reticular (CAR) stromal cells. Specific deletion of RANKL expression in CAR cells abrogated the vascular leakage, suggesting that perivascular RANKL is responsible for controlling permeability. In summary, our study revealed a role for RANK/RANKL signaling as a gatekeeper of bone marrow sinusoids in vivo.2025-10-23T00:00:00Z2026-05-27T13:42:25.032Z2025-10-23T12:18:58.141ZE-MTAB-15929ERP182817EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184