ARTICLE https://doi.org/10.1038/s41467-020-17291-5 OPEN A molecular map of murine lymph node blood vascular endothelium at single cell resolution Kevin Brulois1,13, Anusha Rajaraman1,2,3,13, Agata Szade 1,4,13,Sofia Nordling1,13, Ania Bogoslowski 5,6, Denis Dermadi 1, Milladur Rahman 1, Helena Kiefel1, Edward O’Hara1, Jasper J. Koning3, Hiroto Kawashima7, Bin Zhou 8, Dietmar Vestweber 9, Kristy Red-Horse10, Reina E. Mebius3, Ralf H. Adams 11, ✉ Paul Kubes 5,6, Junliang Pan1,2 & Eugene C. Butcher1,2,12 1234567890():,; Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicro- scopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis. 1 Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. 2 Palo Alto Veterans Institute for Research, Palo Alto, CA, USA. 3 Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands. 4 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. 5 Calvin, Phoebe & Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada. 6 Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 4N1, Canada. 7 Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Hoshi University, Tokyo, Japan. 8 The State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 200031 Beijing, China. 9 Department Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany. 10 Department of Biology, Stanford University, Stanford, CA, USA. 11 Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, University of Münster, Faculty of Medicine, Münster, Germany. 12 The Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA. 13These authors contributed equally: ✉ Kevin Brulois, Anusha Rajaraman, Agata Szade, Sofia Nordling. email: [email protected] NATURE COMMUNICATIONS | (2020) 11:3798 | https://doi.org/10.1038/s41467-020-17291-5 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-17291-5 he vascular endothelium lining blood vessels regulates male and female mice and by the independently processed sam- Texchange of oxygen and metabolites between the blood ples (Supplementary Fig. 1b–d). Gene expression signatures were vascular compartment and tissues. In lymph nodes (LN), generated for each subset using the combined scRNAseq datasets additionally, the endothelium plays essential roles in controlling (Methods; Supplementary Data 1). Correlation in mean gene immune cell access. The organization of the vasculature in LN is expression profiles of the identified subsets across the cohorts is well characterized: Arteries entering at the hilus lead to capillary shown in Supplementary Fig. 1e. Expression of marker genes arcades in the LN cortex; capillaries link to ‘high endothelial depicted in Fig. 1f showed overall consistency across all biological venules’ (HEV), the post capillary venules that recruit lympho- replicates (Supplementary Fig. 2). cytes from the blood1; and the vasculature exits the lymph node at the hilus. Upon immunization, lymph nodes can increase in volume 10-fold or more within days, and the vascular endothe- Characterization of the arterial and venous subsets. The arterial lium expands roughly proportionally from local precursors1,2. cluster among the profiled BEC was identified by expression of Vessel expansion involves extensive proliferation of both capillary Gja5 and Gja4 (encoding connexin 37 and 40, respectively) and and high endothelial cells (HEC)3, without contribution from Bmx (Fig. 1f). Consistent with prior reports describing Gkn3 as a blood borne progenitors4; and an increase in vessel numbers marker for mature arteries8, it is selectively expressed in mature through intussusceptive (splitting) angiogenesis5. However, the Art, and absent in pre-Art, which lay closer to the capillary nature and extent of endothelial cell diversity within LN remains subsets in trajectory space (Fig. 1e). In contrast, Depp1 is pre- incompletely understood. ferentially expressed in pre-Art, consistent with a prior study Single-cell RNA profiling is a transformative technology for the showing its transient expression in developing arterial endothelial identification of cell diversity and elucidation of developmental cells and subsequent downregulation in mature vessels11 (Fig. 1f). and physical relationships. Here we provide a survey of blood Pre-Art and Art express Klf2 and Klf4, genes induced by laminar vessel endothelial cells (BEC) from mouse peripheral lymph shear flow and preferentially associated with linear segments nodes (PLN). We confirm known features of high endothelium6,7, compared to branched vessels9,12. identify endothelial subsets, describe unexpected diversity among Venous EC, comprising HEC and non-HEC (Vn) subsets, capillary cells and demonstrate a distinctive role of medullary share expression of the vein-specifying transcription factor Nr2f2 veins in selective myeloid cell recruitment. We define gene sig- (Coup-TFII)13, and the vein-associated chemokine interceptor natures and transcription regulatory factors for these subsets, and Ackr1 (DARC14; Fig. 1f). HEC express genes required for map key subsets to the vasculature. We also identify a primed lymphocyte recruitment including Chst4 and Glycam115, with capillary resident regenerative population (CRP) that displays more pronounced expression on HEC distal in tSpace (late HEC; the angiogenic endothelial marker Apln, is enriched in cells Fig. 1e, f). Consistent with their large size and plump morphology undergoing cell division, and possesses stem cell and migratory and with prior whole-genome expression studies of sorted HEC6, gene signatures. Genetic lineage tracing suggests that CRP their gene signature is enriched for glycoprotein synthesis function as progenitors, contributing to neogenesis of the blood (Supplementary Data 1), and they have uniquely high numbers vascular endothelium including high endothelium in immune of transcripts per cell (Fig. 1g). angiogenesis. Cells of the Vn subset branch prominently from proximal HEC and TrEC in tSpace projection (Fig. 1e). To identify these cells in the LN vasculature, we imaged whole LN removed shortly after Results i.v. delivery of fluorescently labeled antibodies (Methods): anti- Single-cell profiling of lymph node blood endothelial cells.We Ly6c, specific for arteries and capillaries; MECA79 to the performed single-cell RNA sequencing (scRNAseq, 10× Chro- Peripheral Node vascular Addressin (PNAd) defining HEV; and mium) of sorted BEC from PLN of adult mice (Fig. 1a). Four anti-PLVAP, which stains capillary and venous EC but not cohorts were analyzed including a group of male and female arteries (Fig. 2a). Subset markers allow visualization of arterial Balb/c mice (PLN1) processed together and resolved in silico entry from the LN hilus, linking to capillary arbors in the cortex (PLN1_m and PLN1_f), an additional group of female Balb/c which in turn connect to HEC. We identified PNAd− veins mice (PLN2), and one of female mice of a mixed background downstream of and as a continuation of HEV in the lymph node (PLN3) (Supplementary Fig. 1a). Unsupervised analyses (Meth- medulla (Fig. 2a): they bound injected antibodies to VE-cadherin, ods) defined 8 BEC subsets with distinct gene expression: arterial PLVAP, and ICAM1 (Fig. 2b) but were negative for capillary EC (Art); 2 venous EC subsets, high endothelial cells (HEC) and markers Ly6c and podocalyxin (PODXL; Supplementary Fig. 4), non-HEC veins (Vn); and five capillary phenotype EC subsets, as predicted by gene expression (Fig. 2c). including a transitional phenotype capillary EC (TrEC) and The Vn gene signature includes genes associated with primed EC comprising a capillary resident regenerative popula- regulation of neutrophil activation (GO:1902563; Fig. 3a) and tion (CRP) (Fig. 1b–d). Annotations of the arterial and venous platelet degranulation (GO:0002576; Supplementary Fig. 3). Vn populations were guided by known gene markers6,8,9. Nearest express Von Willebrand Factor (Vwf), which is stored in Weibel- neighbor alignments, which can model developmental relation- Palade bodies and released during inflammation