Artery formation in intestinal wall and mesentery by intestine-derived Esm1+ endothelial cells

Artery formation in intestinal wall and mesentery by intestine-derived Esm1+ endothelial cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Artery formation in intestinal wall and mesentery by intestine-derived Esm1+ endothelial cells Ralf Adams, Esther Bovay, Kai Kruse, Emma Watson, Vishal Mohanakrishnan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5411147/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Arterial blood transport into peripheral organs is indispensable for developmental growth, homeostasis and tissue repair. While it is appreciated that defective formation or compromised function of arteries is associated with a range of human diseases, the cellular and molecular mechanisms mediating arterial development remain little understood for most organs. Here, we show with genetic approaches that a small subpopulation of endothelial cells inside the intestinal villi of the embryonic mouse, characterized by the expression of endothelial cell-specific molecule 1 (Esm1/endocan), gives rise to arterial endothelium in the intestinal wall but also in the distant mesenteric vasculature. This involves cell migration but also substantial changes in morphology and gene expression. Immunohistochemistry and single cell RNA-sequencing confirm that intestinal Esm1 + cells have a distinct molecular profile and the capacity to undergo arterial differentiation. Genetic approaches establish that artery formation by the progeny of Esm1 + cells requires integrin β1 and signaling by the growth factor VEGF-C and its receptor VEGFR3. The sum of these findings demonstrates that Esm1 + cells inside the villus capillary network contribute to the formation of intestinal and mesenteric arteries during development. Biological sciences/Developmental biology/Angiogenesis Biological sciences/Developmental biology/Experimental organisms/Model vertebrates Biological sciences/Developmental biology/Embryogenesis Endothelial cells intestine mesentery artery development single-cell RNA sequencing. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Arteries are the conduit for blood transport into peripheral organs and thereby are crucial for the supply of essential nutrients and oxygen. Research in early mouse, zebrafish and avian embryos have provided a good understanding of the processes mediating the vasculogenic formation of the dorsal aorta and the cardinal vein, the two large axial vessels 1 – 3 . However, comparably little is known about the formation of the majority of arteries during the angiogenic expansion of the vasculature in the embryonic and postnatal organism. In the developing heart, it was shown that coronary artery growth involves the reprogramming of venous (sinus venosus-derived) endothelial cells (ECs) to an arterial fate 4 . However, endocardial cells, which form the innermost lining of the heart and share many features with arterial ECs, have been also shown to contribute to coronary artery development 5 – 8 . Genetic fate tracking has established that venous ECs, which exhibit comparably high rates of proliferation, give rise to arterial endothelium in retina and brain 9 , 10 . In the developing and regenerating zebrafish tail fin, dynamic live imaging has shown that vein-derived endothelial tip cells give rise to arterial ECs 11 . Strikingly, genetic alterations affecting guided EC migration impair normal arterial patterning, suggesting that defects in this process might be a cause of arteriovenous malformations 10 , 11 . In the postnatal murine eye, tip cells at the distal end of endothelial sprouts, which express high levels of endothelial cell-specific molecule 1 (Esm1/endocan), are important for the angiogenic expansion of the developing retinal vasculature. Genetic fate tracking with Esm1-CreERT2 transgenic mice has established that tip cell progeny contributes to the arterial but not the venous branch of the vasculature, which involves signaling interactions via the Notch pathway, the chemokine receptor CXCR4 and its ligand CXCL12, and interactions between ephrin-B2 and its receptor EphB4 11–13 . Arterial differentiation involves arrested proliferation and endothelial cell cycle state has been shown to generate a fate bias during arterial-venous specification 14 , 15 . Apart from molecular regulators, blood flow and fluid shear stress are important factors controlling cell cycle status and the migration of ECs against the direction of blood flow 14 , 16 – 19 . In the developing intestinal system of the mouse, arteriogenesis is initiated during gut rotation, at around embryonic stage E10.0, forming a first arterial connection between the intestinal vascular plexus with the dorsal aorta 20 . It also has been established that high signaling by vascular endothelial growth factor A (VEGF-A) and its receptor VEGFR2 upregulates the expression of Esm1 in the villus apex, which has relevance for nutrient uptake, blood vessel remodeling and normal arteriovenous patterning 19 , 21 , 22 . Despite of these insights, our understanding of the heterogeneity and functional specialization of ECs in the gastrointestinal system remains limited and the processes controlling artery formation in the intestine and adjacent mesentery are unknown. Our new findings reveal that a small subpopulation of ECs in the capillary network of the villus gives rise to arterial endothelium in the adjacent intestinal wall but also in the distant mesenteric vasculature. Genetic labeling, immunohistochemistry and single cell RNA-sequencing (scRNA-seq) show that these prearterial cells express Esm1 and other tip cell markers despite being located inside a patent capillary network. Mechanistically, Esm1-CreERT2 -mediated inactivation of the Itgb1 gene encoding integrin β1 in the mouse embryo results in the loss of arterial progenitors and a subsequent reduction in the diameter of large mesenteric arteries. Additionally, we show that the VEGF-C/VEGFR3 signaling pathway, which is a primary lymphatic growth factor and tyrosine kinase receptor 23 , 24 , participates in the migration of Esm1 + cells into the mesenteric arterial network and expansion of the arterial network. Taken together, these findings establish fundamental principles of developmental artery formation in the intestine and mesentery. Results Identification of arterial progenitors in the embryonic intestine Stratification of the intestinal epithelium begins around embryonic day (E) 14.0 25–27 . However, the formation of the initial intestinal vascular network occurs earlier, beginning as early as E10.5 20 . We investigated whether the blood capillary network at these early stages already expresses Esm1. We stained E12.5 mesenteries and observed ESM1 protein expression within the capillary network surrounding the epithelium (Supplementary Fig. 1a). We further used Esm1-CreERT2 transgenic mice in the Rosa26-mTmG Cre reporter background 28 , 29 to detect Esm1 + cells and their progeny. After tamoxifen treatment initiated at E10.5, recombined GFP + ECs appear in the SOX17 + intestinal and mesenteric vascular network at E13.5 (Supplementary Fig. 1b). We further investigated whether the early mesenteric vasculature already shows segregation of arteries and veins. To analyze known arterial and venous markers, we used Aplnr-CreERT2 and Bmx-CreERT2 transgenic mice in combination with the Rosa26-mTmG Cre reporter 28 , 30 , 31 . After daily tamoxifen administration from E10.5 onward, Bmx-CreERT2 -controlled GFP expression was observed in the Endomucin-negative arterial network of E13.5 mesenteric tissues (Supplementary Fig. 1c). Conversely, 24-hour Aplnr-CreERT2 induction with 4-hydroxytamoxifen (4-OHT) resulted in widespread GFP expression in E12.5 mesenteric and intestinal vessels. However, SOX17 + vessels connected to the cranial mesenteric artery (CMA) remained GFP low (Supplementary Fig. 1d). These findings indicate that Esm1 + cell progeny contributes to arteries during an early stage of intestinal and mesenteric development. Furthermore, it is evident that acute Aplnr-CreERT2 -mediated recombination in the E11.5 embryo is prominent in capillaries but spares BMX + SOX17 + mesenteric arteries. We next examined Esm1 expression at later stages. Whole-mount staining of embryonic tissues at E18.0 revealed strong ESM1 protein expression in the intestinal villi and mesenteric capillary sprouting cells (Fig. 1 a). To assess the long-term contribution of Esm1 + cells, we treated Esm1-CreERT2 transgenic mice in the Rosa26-mTmG Cre reporter background from E10.5 to E15.5 (Fig. 1 b). GFP + ECs were abundant in large mesenteric arteries and smaller arterial branches near the intestinal wall at E16.5 (Fig. 1 b) but absent from veins or lymphatic vessels. To gain a better insight into the spatial distribution and dynamic behavior of Esm1 + cells, we conducted multiple genetic fate tracking experiments (Fig. 1 c). Short-term (24 hours) induction with 4-hydroxytamoxifen (4-OHT) leads to robust GFP signal in the embryonic intestine, while the mesentery exhibits minimal presence of GFP + ECs, except for mesenteric sprouting capillaries (Fig. 1 d-f). Analysis of intestinal villi at E18.5 revealed that GFP signal is predominantly localized to the upper portion of the villus vascular network, reflecting heightened VEGF signaling at the apex of the villus 22 (Fig. 1 g). Analysis of embryos at the same stage but after 3 and 5 days post-induction shows lower GFP signal within the intestine but an increased presence of GFP + cells within large mesenteric arteries (Fig. 1 d-f). While the extent of arterial labeling is limited in these short-term genetic fate tracking experiments, the results indicate that Esm1-CreERT2 -labeled cells are incorporated into mesenteric arteries but not into nearby veins or lymphatic vessels (Supplementary Fig. 1e). Careful analysis of GFP + cells over time indicates morphological changes during the transition from the intestine into the mesentery. Whereas GFP + ECs in the intestine display irregular shapes with numerous protrusions, cells in the mesentery exhibit the typical slender and elongated morphology of arterial ECs (Fig. 1 e, g). With increasing time between 4-OHT administration and analysis, more elongated arterial GFP + cells are observed, and these cells are found deeper inside the mesenteric arterial tree (Fig. 1 e, f). Further analysis of intestines confirms the presence of GFP signal in the upper portion of the villus vascular network at 24 hours post-induction (Fig. 1 g, left panels), whereas GFP + cells contribute to the SOX17 + villus capillaries and submucosal arteries over time (Fig. 1 g-i). These results reveal a progressive contribution of the progeny of Esm1 + ECs to the submucosal and mesenteric arterial network (Fig. 1 d-i, Supplementary Fig. 1e). The sum of our genetic tracking data also indicates that Esm1 + cells continue to be induced throughout development of the embryonic intestinal vasculature from midgestation to birth, which implies that a pulse of tamoxifen/4-OHT will lead to Esm1-CreERT2 -mediated recombination only in a fraction of these cells. Intestinal arterial progenitors contribute to the villus capillary expansion after birth To investigate whether progeny from Esm1 + ECs continues to contribute to arteries after birth, lineage tracing experiments were initiated from postnatal day (P) 1 (Supplementary Fig. 2a). Analysis at P8 reveals an abundance of GFP + ECs within the intestine, whereas the mesentery lacks GFP-expressing cells in large arteries. GFP + cells are located in the expanding capillary network surrounding mesenteric arteries and veins, but lack a direct connection to these large vessels (Supplementary Fig. 2b). Further examination of the P8 and P21 intestines confirms the presence of GFP + cells in the SOX17 + region of the villus capillary network (Supplementary Fig. 2c, d). These results indicate the continued presence of Esm1 + cells in the intestinal vasculature after birth, but these cells no longer contribute to large mesenteric arteries. Previous work has shown that high VEGF signaling at the villus apex, indicated by elevated expression of the VEGF-responsive genes Flt4 (encoding VEGFR3) and Esm1 , facilitates the reorganization of EC junctions to enhance nutrient uptake in the adult mouse intestine 22 . Consistent with these findings, whole-mount analysis of the adult mouse intestine shows Esm1 immunostaining in the more arterial part of the villus capillary network, which is characterized by pronounced anti-Caveolin 1 signal and absent Endomucin expression (Supplementary Fig. 3a, b). Lineage tracing experiments conducted over 24 hours and 2 weeks in 20 to 24-week-old male mice reveal only minimal presence of GFP + ECs in the retina of these animals (Supplementary Fig. 3c-e). In contrast, robust GFP signal is seen inside the capillary network of intestinal villi, with a significant enrichment in Endomucin - areas (Supplementary Fig. 3f-h). Altogether, these findings show that the expression of Esm1 in the intestinal vascular network commences during embryogenesis and persists into postnatal and adult stages. However, the contribution of Esm1 + EC-derived arterial progenitors to the formation of mesenteric arteries is confined to embryonic development. Fate and properties of intestinal arterial progenitors at single cell resolution To validate the developmental fate of intestinal Esm1 + ECs and gain deeper insight into their molecular properties, we isolated cells from E18.0 embryos 24 hours after genetic lineage tracing with Esm1-CreERT2 (Fig. 2 a, Supplementary Fig. 4a, b). We first individually isolated all cells from the intestine and mesentery, followed by single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody system. To augment our population of blood vessel ECs, we conducted an additional 24 hours lineage tracing experiment with ECs enriched in GFP + cells by fluorescence-activated cell sorting (FACS) (Supplementary Fig. 4a, b). In total, we acquired 34518 cells, among which 7122 were blood vessel ECs (Supplementary Fig. 4b, right panels). These datasets were combined and visualized as Uniform Manifold Approximation and Projection (UMAP) plots (Fig. 2 b, c). Based on established markers from the literature, we identified most of the known intestinal and mesenteric cell types, including ECs from blood and lymphatic vessels, epithelial, mesothelial, immune, neural and various mesenchymal cells (Fig. 2 d, e). We then focused on blood vessel ECs, which segregate into 8 subgroups (after exclusion of contaminating red blood cells) (Fig. 2 c, e). ECs expressing higher levels of venous markers, such as Nrp2, Madcam1, Aplnr and Nr2f2 (Coup-TFII) , can be seen on the left side of the UMAP plot, subdivided into proliferative (G2/M and S phase) and two non-proliferative groups (Venous EC1 and Venous EC2). ECs with higher levels of arterial markers, namely Unc5b , Hey1, Gja4 , Igfbp3 , Gja5 , Dll4 and Sox17 , cluster on the right (Arterial EC1, Arterial EC2, and Arterial EC3). Venous EC1 and Arterial EC3 cells are enriched in ECs from large mesenteric veins and arteries, expressing markers such as Adamts18 , Fam174b and Bmx , respectively (Fig. 2 e, f, Supplementary Fig. 4b), which indicates that these two populations are most mature with respect to arteriovenous specification. Interestingly, an intermediate subgroup of ECs is located in the center between the areas expressing venous or arterial markers. This population shows high levels of markers characteristic for endothelial tip cells ( Esm1 , Flt4 , Lamb1 , Nid2 ) and represents Esm1 + ECs (Fig. 2 c, e). We further corroborated the distribution of venous and arterial markers at the protein level by immunohistochemistry (Fig. 2 g-j, Supplementary Fig. 5a-d). Staining of E17.0-E19.0 embryonic intestines confirms the expression of MADCAM1 and DLL4 by the “venous" and "arterial" side of the villus capillary network, respectively (Fig. 2 g). To validate the expression of Aplnr as a marker of the venous domain, we used the Aplnr-CreERT2 line in combination with the Rosa26-mTmG Cre reporter 28 , 30 . At 24 hours after 4-OHT administration, expression of GFP is detected in most of the villus capillary network but is excluded from the SOX17 + (i.e., arterial) branch (Fig. 2 h). In the mesenteric tissue, as expected, Aplnr-CreERT2 -controlled GFP expression is found specifically in large mesenteric veins and capillaries, but is excluded from SOX17 + arteries and lymphatic vessels (Supplementary Fig. 5a-d). Analysis of a Hey1-eGFP knock-in line carrying an insertion of a GFP cassette in the Notch pathway gene Hey1 (Hairy/enhancer-of-split related with YRPW motif protein 1) is more prominent in the arterial side of the villus capillary network and in large mesenteric arteries, reflecting the known role of Notch signaling in arterial specification (Fig. 2 i, j). Consistent with previous descriptions, Hey1-eGFP expression is also detected in some epithelial cells and DLL4 + ECs 32–35 . Analysis of the mesentery confirmed robust GFP signal in the SOX17 + mesenteric arteries. Taken together, these findings validate our scRNA-seq data and confirm arteriovenous zonation within the developing villus vasculature, which gradually extends into the large veins and arteries of the mesenteric vascular network. Transient cell cycle arrest and arterial differentiation characterize venous-derived Esm1 cells In the mouse retina and embryonic heart, venous cells actively divide and give rise to all other EC subpopulations including tip cells and committed arterial ECs that exhibit cell cycle arrest 4 , 14 , 15 , 36 , 37 . To investigate the origin of Esm1 + cells and the cells generating upstream arteries, we performed lineage-tracing of Aplnr + ECs (Supplementary Fig. 5e-k). At 4 to 6 days after 4-OHT treatment, Aplnr + EC-derived cells contribute strongly to the villus capillary network, SOX17 + submucosal arteries, and large upstream mesenteric arteries (Supplementary Fig. 5e-k). Notably, lineage tracing from E13.5 leads to labeling of large veins, capillaries, and arteries closer to the intestine, while the majority of lymphatic vessels lacks GFP expression. However, when 4-OHT treatment is initiated at E11.5, nearly all vessels, including lymphatic vessels, are GFP + (Supplementary Fig. 5e-j). These findings are consistent with previous research indicating a large proportion of venous origin for mesenteric lymphatic vessels starting in the developmental window around E10 to E12.5 in the mouse 38 , 39 and confirm that Esm1 + cells and arterial ECs are also derived from Aplnr + ECs. Consistent with this and previous studies, the venous EC subclusters in our scRNA-seq analysis predominantly harbor proliferative cells in the G2/M or S phase (Fig. 3 a, b). To exclude clustering effects due to dominant cell cycle expression patterns, we regressed out the expression of known G2/M and S phase genes, re-clustered the cells, and annotated the new clusters to match the original EC type annotation as closely as possible (Supplementary Fig. 6a). We then determined the proportion of cycling cells in each EC population, by counting the number of cells expressing G1, G2/M, and S phase genes (Fig. 3 b, Supplementary Fig. 6b), which confirmed the high proliferation of venous ECs. To validate these observations, we conducted a Partition-based Graph Abstraction (PAGA) Trajectory analysis, facilitating diffusion pseudotime calculation 40 (Supplementary Fig. 6c). For this analysis, the two proliferative subgroups, i.e. Prolif EC (G2/M phase) and Prolif EC (S phase), were excluded. Pseudotime results are consistent with a trajectory along the vein-to-artery axis, extending from the venous side through the Esm1 + population into the arterial domain (Supplementary Fig. 6d). As expected, the Venous EC1 and Venous EC2 subgroups show low expression of arterial markers but high levels of cell cycle genes relative to other EC subpopulations (Fig. 3 c, d). Conversely, Esm1 + ECs, Arterial EC1 and Arterial EC2 exhibit higher expression of arterial markers but low transcript levels for cell cycle genes, confirming previous observations 4 , 14 , 15 , 36 , 37 , 41 . The Arterial EC3 subgroup, which is enriched in the mesenteric sample and therefore represents ECs from larger arteries, surprisingly shows higher levels of cell cycle genes compared to other arterial subpopulations (Fig. 3 b-d, Supplementary Fig. 6b). We validated these findings with EdU staining to assess EC proliferation in the intestine and mesentery. The majority of EdU + ERG + ECs in villi lack SOX17 expression, reflecting the expected low proliferation of arterial progenitors undergoing differentiation (Fig. 3 e-g). In large mesenteric arteries, however, over 10% of ERG + elongated arterial nuclei are EdU + . Taken together, these results indicate that Esm1 + cells are derived from venous ECs in the developing intestine. Esm1 + cell progeny shows the expected downregulation of cell cycle genes during arterial specification. In contrast, a fraction of fully differentiated mesenteric arterial ECs express cell cycle genes, suggesting that local proliferation of these cells contributes to the size increase of larger arteries during development. Comparison of Esm1 in the embryonic villus and postnatal retina As previous work has shown that Esm1 + ECs in the postnatal retina contribute to the expanding arterial network 13 , 42 , we compared the molecular properties of these cells to the Esm1 + population from embryonic intestine. To this end, we reanalyzed a previously published scRNA-seq dataset of wildtype P6 retinal endothelial tip cells 43 and compared the Esm1 + population to the Esm1 + cluster in our study using pseudobulk differential expression analysis (DEA) (Supplementary Fig. 7a). While Esm1 + cells from the retina and embryonic intestine share expression of signature genes such as Esm1 , Flt4 (encoding the receptor tyrosine kinase VEGFR3) or Lama4 (laminin α4), the two populations show substantial differences in gene expression. One example is the Fabp4 gene and its product fatty acid binding protein 4, which are highly expressed by Esm1 + ECs in the distal villus but absent in retinal tip cells 44 , 45 (Supplementary Fig. 7b-d). Whole-mount immunostaining shows substantial morphological differences between Esm1 + cells in the retina and intestine (Supplementary Fig. 7c-f). While intestinal Esm1 + cells are an integral part of the villus capillary network, Esm1 + cells in the retina are sprouting tip cells that extend filopodia into the avascular tissue. Furthermore, Caveolin-1 (encoded by the Cav1 gene), is differentially expressed between the intestinal and retinal Esm1 + populations by scRNA-seq (Supplementary Fig. 7b). Immunostaining confirms low expression of Caveolin-1 in sprouting retinal tip cells, whereas Esm1 + cells in the capillary network of intestinal villi are prominently labeled (Supplementary Fig. 7e, f). Whole-mount immunostaining of the embryonic intestine at E18.0/E18.5 confirms that endogenous ESM1 protein expression is enriched in the upper portion of the villus, in contrast to ubiquitous CD31 expression by all ECs (Fig. 4 a, b). Esm1-CreERT2 -mediated lineage tracing for 24 hours in combination with immunostaining shows that Esm1 expression precedes the upregulation of SOX17, thus affirming the role of Esm1 + ECs as arterial progenitors (Fig. 4 c). Analysis of genetic lineage tracing over 24 hours by scRNA-seq provides further proof of the contribution of Esm1-CreERT2 -labeled ( GFP + ) progeny from the central Esm1 + population to the arterial branch of the intestinal vasculature (Fig. 4 d). We then performed immunostaining for the markers laminin α4 (encoded by the gene Lama4 ), collagen IV and VEGFR3 (Fig. 4 e-g). Indeed, all three gene products were found to decorate the capillaries in the distal villus, confirming that the microenvironment at the apex of the embryonic villus facilitates the emergence of a specialized, Esm1 + EC population with a role in artery formation (Supplementary Fig. 7g). Integrin β1 in Esm1 + ECs controls cell shape and mesenteric artery diameter Gene Ontology (GO) enrichment analysis of genes exhibiting a similar expression pattern as Esm1 revealed a significant enrichment of genes involved in the biological processes “angiogenesis” and “cell migration”. Likewise, “Focal adhesion” and “Actin filament” were among the top hits for cellular components (Supplementary Fig. 8a). To address a potential role of cell migration and cell-matrix interactions, we used the Esm1-CreERT2 line to inactivate the Itgb1 gene, which encodes the integrin β1 subunit (Fig. 5 a). Integrins are heterodimeric transmembrane receptors composed of an α and β subunit 46 – 49 . Integrin β1 plays a crucial role in the development and maintenance of the blood vascular network. Pan-endothelial deletion of Itgb1 was shown to result in vascular leakage and the disruption of arterial EC polarity 50 – 52 . Esm1-CreERT2 -mediated loss of Itgb1 has no impact on embryonic weight, mesenteric artery density, or organization of the intestinal villus vasculature, despite the confirmed loss of integrin β1 protein in GFP + cells (Fig. 5 b, c, Supplementary Fig. 6b-d). However, Itgb1 i Esm 1 KO embryos show a reduction of mesenteric artery diameter relative to control or heterozygous mutant ( Itgb1 i Esm 1 HET ) littermates (Fig. 5 b, c). High-magnification analysis of the large mesenteric arteries reveals no change in the density of EC nuclei but confirms the decrease in vascular diameter (Fig. 5 d-f). The inclusion of a R26-mTmG reporter allele shows that GFP + ECs in Itgb1 i Esm 1 KO mesenteric arteries acquire a rounded shape, which is distinct from the strongly elongated appearance of control arterial ECs (Fig. 5 d-f). Similar alterations in GFP + cell morphology are evident in arteries of the intestinal submucosa (Fig. 5 g). These findings support that arterial progenitor cells generated within the intestine functionally contribute to the formation of the large mesenteric arteries. Accordingly, the loss of Itgb1 in Esm1 + ECs results in the reduction of arterial diameter. Artery-derived VEGF-C promotes artery development In the adult intestine, the VEGFR3 ligand VEGF-C is expressed by vascular and intestinal smooth muscle cells (SMCs), macrophages, and a subset of villus fibroblasts 53 – 55 . Recent work, supported by scRNA-seq analysis, has identified mesenteric arteries as a major source of VEGF-C with a crucial role in the development of a secondary postnatal lymphatic capillary network 56 , 57 . In our scRNA-seq data, Flt4/Vegfr3 is enriched in lymphatic ECs but also in Esm1 + capillary ECs, whereas Vegfc is strongly expressed in the arterial endothelium, which is supported by pseudotime analysis (Fig. 6 a, b). RNAscope analysis on mesenteric and intestinal cryosections confirms the pattern of Vegfc mRNA expression. Despite strong autofluorescence from red blood cells (RBCs), Vegfc transcripts are predominantly detected in the mesenteric and submucosal arteries as well as in SOX17 + villus capillaries (Fig. 6 c, d). In the intestine, Vegfc mRNA is detected throughout the mucosal tissue, suggesting that various embryonic intestinal cell types express this growth factor (Fig. 6 d). To specifically target arterial ECs, we used the tamoxifen-inducible Bmx-CreERT2 mouse line 31 in combination with the R26-mTmG reporter. In lineage tracing experiments with the common end point at E18.5, GFP signal labels large mesenteric arteries and intestinal submucosal SOX17 + arteries (Supplementary Fig. 9a-d). Over longer time periods, namely after 4-OHT induction at E13.5 or E15.5, GFP-negative ECs replace the Bmx-CreERT2 -labeled GFP + cells in submucosal and smaller (distal) mesenteric arteries so that recombined ECs reside only in the larger caliber arteries (Supplementary Fig. 9b). These results strongly support the notion that the continuous migration of arterial progenitors from the intestine contributes to the expansion of embryonic mesenteric arteries (Supplementary Fig. 9a-d). To address whether arterial VEGF-C might facilitate the recruitment of Esm1 + cell progeny into the mesenteric vasculature, we introduced the Bmx-CreERT2 line into a background of mice carrying conditional Vegfc lox/lox alleles 58 (Supplementary Fig. 9e). Given the immature state of arteries observed at E13.5 (Supplementary Fig. 1c), we performed short-term 4-OHT-induced Vegfc inactivation starting from E14.5. After 4 days of Vegfc deletion from arteries, the lymphatic vessel area is significantly reduced in Vegfc i Bmx KO mutants compared to control littermate embryos (Fig. 6 e-h). These results confirm prior studies indicating that arteries are a key source of Vegfc for mesenteric lymphangiogenesis 57 . While large mesenteric arteries remain unaffected by the loss of VEGF-C, Vegfc i Bmx KO arteries in the intestinal mucosa are significantly thinner (Fig. 6 f-h), indicating that arteries closest to the intestine are most affected. RNAscope analysis confirms the loss of Vegfc mRNA expression in large Vegfc i Bmx KO mesenteric and submucosal arteries (Supplementary Fig. 9f, g). However, residual Vegfc mRNA is still detected in the intestinal mucosa and SOX17 + ECs in Vegfc i Bmx KO intestinal villi, which may reflect incomplete Bmx-CreERT2 -mediated recombination or Vegfc expression prior to upregulation of Bmx . The latter is consistent with the pseudotime analysis of our scRNA-seq data, showing that Vegfc expression is initiated in Esm1 + ECs during early steps of arterial specification and prior to Bmx expression (Supplementary Fig. 9h). Nevertheless, analysis of intestines shows that Vegfc i Bmx KO villus capillaries show an increase in CD31 + ERG + ECs area but significantly thinner SOX17 + arterial branches (Fig. 6 i, j). These results suggest that arterial Vegfc expression promotes the translocation of arterial progenitor cells from intestinal villi into the adjacent submucosal arteries. Accordingly, EC density is increased in Vegfc i Bmx KO villi, whereas the diameter of the submucosal arterial network is decreased (Fig. 6 g-j). VEGFR3 signaling promotes the translocation of Esm1 cell progeny into arteries Based on the results above, we hypothesized that signaling by VEGF-C and VEGFR3 enhances the efficient translocation of intestinal arterial progenitor cells into the large mesenteric arteries. To examine the role of the VEGFR3 tyrosine kinase receptor, we inhibited its activity by administering MAZ51 to pregnant females during a short lineage tracing experiment of Esm1 + cell progeny (Fig. 7 a). MAZ51 is a potent inhibitor of VEGFR3 kinase activity, which only weakly affects the related receptor tyrosine kinase VEGFR2 59,60 . After MAZ51 treatment, growing lymphatic capillaries in the intestine stained for LYVE1 are significantly reduced compared to vehicle controls (Fig. 7 b-d), indicating efficient inhibition of VEGFR3 activity (Fig. 7 d). In contrast, MAZ51 treatment does not affect the villus capillary network, Esm1-CreERT2 -controlled GFP expression, or SOX17 signal (Fig. 7 e, f). Strikingly, the contribution of GFP + Esm1 cell progeny to the submucosal and large mesenteric arteries is significantly reduced by MAZ51 relative to vehicle controls (Fig. 7 g-i). These findings support that inhibiting VEGFR3 activity in arterial progenitor cells impairs their translocation into submucosal and mesenteric arteries. To test this hypothesis further, we generated mutant embryos carrying loxP-flanked versions of Flt4 ( Flt4 lox/lox ) 61 together with Esm1 - CreERT2 and Rosa26-mTmG alleles. Following tamoxifen administration from E10.5 onward, the appearances of control, Flt4 i Esm 1 HET , and Flt4 i Esm 1 KO embryos are indistinguishable at E18.0 (Supplementary Fig. 10a, data not shown). Immunostaining confirms the expected reduction of VEGFR3 expression in the Flt4 i Esm 1 HET and, more completely, in the Flt4 i Esm 1 KO capillary network at the villus apex compared to control littermates (Supplementary Fig. 10b). Quantification of VEGFR3 revealed a strong residual VEGFR3 signal in the Flt4 i Esm 1 HET and Flt4 i Esm 1 KO villus capillary networks, but with significant loss in GFP + areas (Supplementary Fig. 10b, c). Despite the substantial deletion of VEGFR3 in the GFP + ECs in Flt4 i Esm 1 KO samples, these results reflect that the villus apex is not entirely composed of Esm1-CreERT2 -targeted ECs, so that only a subset of ECs in the villus apex loses VEGFR3 (Supplementary Fig. 10b, c). Unlike MAZ51 treatment, targeted Flt4 deletion in Esm1-derived cells does not impact lacteal length, suggesting that this approach does not interfere with lymphatic growth (Supplementary Fig. 10b, d). Flt4 i Esm 1 KO mutants exhibit a slight increase in blood vessel width relative to control littermates (Supplementary Fig. 10b, d). Despite the incomplete deletion of VEGFR3 in the villus vasculature, Flt4 i Esm 1 KO mesenteric samples relative to Flt4 i Esm 1 HET littermates show reduced incorporation of GFP + cells into submucosal arteries and large mesenteric arteries, wherease control ( Flt4 i Esm 1 WT ) samples show significantly higher incorporation of GFP + cells (Supplementary Fig. 10e, f). To inactivate Flt4 more efficiently in villus ECs, we made use of the finding that Aplnr-CreERT2 -labeled cells give rise to Esm1 + ECs and, subsequently, arterial endothelium. We therefore introduced the Aplnr-CreERT2 line into the Flt4 lox/lox background. To avoid lymphatic involvement (Supplementary Fig. 5h-j), treatment was initiated at E13.5 (Fig. 8 a) prior to development of the lymphatic vasculature. By E18.0, we observed significant loss of VEGFR3 specifically in Flt4 i Aplnr KO blood vessels without affecting the intestinal (lymphatic) lacteals (Fig. 8 b, c). Interestingly, the Flt4 i Aplnr KO villus blood vessel network is reduced, whereas VEGFR3 high lacteals grow longer, which may reflect higher availability of VEGF-C due to the lack of VEGFR3 in blood vessel ECs. Upstream of the villus, submucosal arteries are notably thinner than in control littermates (Fig. 8 d, e), mirroring the phenotype observed in Vegfc i Bmx KO mutants (Figs. 6 g, h). Together with the effects seen following MAZ51 treatment and Vegfc and Flt4 inactivation, these results indicate that VEGFR3 promotes the recruitment of Esm1 + ECs from villus capillaries into upstream arteries. Discussion Arteries are an indispensable part of the vascular tree and arterial malfunction is causally linked to detrimental, sometimes life-threatening human diseases. Mesenteric arteries carry blood from the abdominal aorta to the gastrointestinal tract and are thereby indispensable for the function of this organ. Accordingly, the blockade or narrowing of mesenteric arteries, which occurs in human patients due to the build-up of atherosclerotic plaques or other reasons, can lead to severe abdominal pain, intestinal damage and the need for surgical intervention 62 . Our study provides fundamental insights into the development of arteries of the mesentery and intestinal wall. In particular, we show that a small population of Esm1 + cells, located inside intestinal villi, gives rise to arterial ECs both in the intestine and mesentery. Accordingly, the formation of these arteries requires EC migration over substantial distances in addition to arterial specification. Previous work has established that Esm1 + tip cells in the postnatal retina, which are located at the distal end of endothelial sprouts and thereby at the border to avascular tissue, generate arterial but also capillary ECs 12 , 13 . Remarkably, Esm1 + cells in the embryonic intestine express typical tip cell markers even though they are part of patent capillary tubes and do not show sprouting behavior. Thus, Esm1/endocan expression in this setting is likely to reflect elevated levels of growth factor signaling, especially of VEGF-A and VEGF-C, as has been shown previously 63 , 64 . Mechanistically, Esm1 has been shown to bind to fibronectin and displace fibronectin-bound VEGF-A, thereby increasing the bioavailability and signaling capacity of the growth factor 29 . As a result, Esm1 enhances endothelial sprouting activity as well as vascular permeability 29 , 65 . This role of Esm1 might be also relevant in the apex of the villus vasculature where VEGF-A modulates EC junctions to enhance nutrient uptake in the adult murine intestine 22 . In the context of arterial development, Esm1 might facilitate the migration of prearterial ECs into the arterial walls and further into mesenteric arteries. Apart from VEGF-A, which is highly expressed in the distal villus, the related ligand VEGF-C is also relevant. As previously described for the murine postnatal and adult intestine 22 , 56 , 57 , VEGFR3 expression marks Esm1 + cells in the embryonic intestine, whereas its ligand, VEGF-C, is provided by submucosal and mesenteric arteries. Functionally, the migration and arterial incorporation of Esm1 + (and VEGFR3 + ) ECs is compromised after genetic or pharmacological disruption of signaling by VEGF-C and its receptor VEGFR3. The function of VEGFR3 and VEGF-C is known to be crucial for the lymphatic vessel development and maintenance, but also modulates sprouting angiogenesis and blood vessel growth in mouse and zebrafish models 66 – 72 . Our new findings reveal an additional and unexpected function of this ligand-receptor pair, namely the guidance of Esm1 + ECs into the growing arterial vasculature. Our genetic fate tracking experiments show that Esm1 + cell progeny inside arteries acquires the typical elongated shape characteristic of arterial ECs 73 . Esm1-CreERT2 -mediated inactivation of integrin β1, an important subunit of many integrin receptor heterodimers, leads to the rounding of Esm1 + cell progeny and reduces the diameter of embryonic mesenteric arteries. This rounding phenotype is reminiscent of previous findings showing that pan-endothelial (but incomplete and therefore mosaic) inactivation of the Itgb1 gene impairs arteriolar lumen formation due to defective EC polarization 52 . Other studies showed that integrin β1 is also required for endothelial sprouting in the postnatal retina, arteriole formation in the ischemic heart but also for the barrier function of the endothelium and the prevention of vascular leakage 51 , 74 , 75 . All these aspects might be of relevance for the formation and function of the arterial network in the intestine and mesentery. Our findings also raise the question whether the recruitment of Esm1 + cell progeny is the predominant or even sole process responsible for mesenteric artery development in the embryo. The existence of alternative, redundantly acting mechanisms might contribute to biological robustness and resilience. In the developing heart, ECs derived both from the sinus venosus and the endocardium contribute to coronary blood vessel formation, and both pools of arterial progenitors can compensate for each other 6 , 76 – 78 . Similarly, multiple non-venous derived sources of lymphatic ECs have been identified in various organs, including the mesentery 38 , 79 . Our own findings show that fully differentiated mesenteric arterial ECs overcome the cell cycle arrest that is characteristic for ECs undergoing arterial differentiation 13 – 15 and exhibit some level of proliferation. Similarly, EC proliferation in situ contributes to the regeneration of damaged aortic endothelium in the adult mouse 80 . Thus, it is feasible that the expansion of mature arterial ECs in the mesentery and intestinal wall might be able to compensate for insufficient specification of prearterial cells. Alternatively, processes such as vessel remodeling and pruning 18 , 81 , which play important roles during the angiogenic expansion of the vasculature, might help to ensure or restore sufficient arterial blood flow. Taken together, our findings establish a fundamental framework for artery development in the intestine and mesentery. We propose that intestinal villi represent a niche microenvironment for the induction of Esm1 + arterial progenitors, which will integrate into growing arteries, but not into veins or lymphatic vessels, and thereby contribute to the expansion of the arterial network. Materials and Methods Mouse models All animal experiments were performed according to the institutional guidelines and laws, approved by local animal ethical committee and were conducted at the Max Planck Institute for Molecular Biomedicine with necessary permissions (Az 81-02.04.2019.A114) granted by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) of North Rhine-Westphalia, Germany. Esm1-CreERT2 29 , Bmx-CreERT2 31 , Aplnr-CreERT2 30 , Itgb1 lox/lox 82 , Flt4 lox/lox 61 , Vegfc lox/lox 58 , R26-mTmG 28 , Ai14 83 , Cdh5-mTnG 84 and Hey1-eGFP ( Hey1-GFP (Tg(Hey1-EGFP)ID40Gsat; http://www.gensat.org ) were previously described. Embryonic, postnatal and adult mouse treatments For embryo experiments, vaginal plugs were verified in the morning. Embryonic age (E) was determined according to the day of the vaginal plug (E0.5). For lineage tracing embryo experiments, one injection of 2mg total 4-hydroxytamoxifen (4-OHT; Sigma, #H7904) in oil was injected intraperitoneally (i.p.) in pregnant mice. For lineage tracing experiments with VEGFR3 tyrosine kinase inhibition, MAZ51 (2% DMSO in PBS, #HY-116624) was injected subcutaneously (s.c.) at E14.5 and E17.0. One dose of 2mg of 4-OHT in oil was injected i.p. in pregnant mice at E16.0. Embryos were collected 48 hours later at E18.0. Click-it EdU cell proliferation Alexa Fluor 647 kit (Life Technologies, #C10340) was used for EdU detection. When indicated, 5µg/g EdU was injected i.p. 1 hour before sacrifice. For continuous Esm1 + cell tracking, pregnant females were injected with 2.5mg tamoxifen (Sigma, #T5648) and progesterone (Sigma, #P3972) mixed in oil s.c. starting from E10.5 every day until the day before embryo collection. Esm1-CreERT2 tg/+ R26-mTmG tg/+ embryos were analyzed at E13.5 and E16.5. In loss-of-function experiments, Itgb1 i Esm 1 KO embryos ( Itgb1 lox/lox Esm1-CreERT2 tg/+ ) were compared to Itgb1 i Esm 1 HET ( Itgb1 lox/+ Esm1-CreERT2 tg/+ ) or control littermates ( Itgb1 +/+ ; Esm1-CreERT2 tg/+ or mice without the Esm1-CreERT2 transgene). All embryos carried the reporter transgene R26-mTmG ( R26-mTmG tg/+ ). In Esm1-CreERTR2 -controlled Flt4 loss-of-function experiments, Flt4 i Esm 1 KO embryos ( Flt4 lox/lox Esm1-CreERT2 tg/+ ) were compared to Flt4 i Esm 1 HET ( Flt4 lox/+ Esm1-CreERT2 tg/+ ), Flt4 i Esm 1 WT ( Flt4 +/+ Esm1-CreERT2 tg/+ ), or control littermates without the Esm1-CreERT2 transgene. All embryos carried the reporter transgene R26-mTmG ( R26-mTmG tg/+ ) and some carried the Ai14 transgene. Itgb1 and Flt4 long-term deletion experiments were induced with 2.5mg tamoxifen and progesterone mixed in oil s.c. starting from E10.5 every day until the day before embryo collection. For Aplnr-CreERTR2 -controlled Flt4 loss-of-function experiments, Flt4 i Aplnr KO ( Flt4 lox/lox Aplnr-CreERT2 tg/+ ) embryos were compared with control littermates ( Flt4 lox/lox Aplnr-CreERT2 +/+ ). Long-term deletion experiments were induced with 2.5mg tamoxifen and progesterone mixed in oil s.c. at E13.5, E14.5 and E15.5. For Bmx-CreERTR2 -controlled Vegfc loss-of-function experiments, Vegfc i Bmx KO ( Vegfc lox/lox Bmx-CreERT2 tg/+ ) embryos were compared with control littermates ( Vegfc lox/lox Bmx-CreERT2 +/+ ). Short-term deletion experiments were induced with 2mg of 4-OHT in oil injected s.c. starting from E14.5 every day until the day before embryo collection. For postnatal lineage tracing experiments, Esm1-CreERT2 tg/+ R26-mTmG tg/+ pups were injected once at P1 with 50µg 4-OHT in oil s.c. and analyzed at P8 or P21. For lineage tracing in adult mice, 20–24 week-old Esm1-CreERT2 tg/+ R26-mTmG tg/+ males were injected once with 2mg 4-OHT in oil i.p. and analyzed 24 hours or 2 weeks later. Mouse embryonic mesenteric and intestinal tissue collection, and staining procedures Embryonic gastro-intestinal tracts were collected in ice-cold PBS. Duodenum (around 1 cm at E18.0) and first part of jejunum (next 1 cm) were collected, cut open and pinned in a 12-well elastomer-coated dish and thoroughly washed with ice-cold PBS. From the remaining tissue, intestine was pinned in a circle to visualize the mesentery. Clean duodenum, jejunum and mesentery were fixed with ice-cold 4% paraformaldehyde (PFA) in PBS (Sigma, #P6148) overnight on a rotating platform at 4 o C. Samples were then washed with ice-cold PBS and incubated overnight with 10% sucrose in PBS followed by 20% sucrose and 10% glycerol in PBS. For staining, 0.5cm-long intestine pieces or mesenteric tissues were washed with PBS, permeabilized with 0.5% Triton X-100 and blocked with 5% donkey serum overnight. Tissues were incubated with primary, followed by secondary antibodies overnight on a rotating platform at 4 o C. Intestine samples were incubated overnight with Histodenz (Sigma, #D2158) at room temperature (RT). All samples were mounted in Histodenz with DAPI (Sigma, #D9542). A list of antibodies used in this study is provided in Table 1 . Mouse postnatal mesentery, postnatal and adult intestine collection and staining The protocol was adapted from previous publications 85 , 86 . Pups and adult mice were perfused with PBS then 4% PFA in PBS after anesthesia. Isolated tissues were washed in ice-cold PBS and duodenum and first jejunum parts were collected, cut open and pinned in a 6-well or 10-cm elastomer-coated dish and thoroughly washed with ice-cold PBS. The remaining postnatal jejunum and ileum were pinned down in a circle to visualize the mesentery. Clean duodenum and jejunum samples and mesenteries were fixed with ice-cold 4% PFA in PBS overnight on a rotating platform at 4 o C. Samples were then washed with ice-cold PBS and incubated overnight with 10% sucrose in PBS followed by 20% sucrose and 10% glycerol in PBS. For staining, 1cm-long intestinal pieces and mesenteries were washed with PBS, permeabilized with 0.5% Triton X-100 and blocked with 5% donkey serum overnight. Tissues were incubated with primary, followed by secondary antibodies overnight on a rotating platform at 4 o C. Intestine samples were incubated overnight with Histodenz at RT. All samples were mounted in Histodenz with DAPI. A list of antibodies used in this study is provided in Table 1 . Adult mouse retina collection and staining Tissues were fixed with 4% PFA for 1 hour at RT, washed with ice-cold PBS and permeabilized with 0.5% Triton X-100 and blocked with 5% donkey serum overnight on a rotating platform at 4 o C. Tissues were incubated with primary antibodies followed by secondary antibodies overnight on a rotating platform at 4 o C. Retinas were mounted in Fluoromount-G (Southern Biotech, #0100-01) with DAPI. Postnatal P5 mouse retina collection and staining Retina immunostaining was performed as previously described with minor modifications 87 . Tissues were collected and fixed in 4% PFA in PBS. Dissected retinas were incubated in blocking buffer (1% BSA, 0.3% Triton X-100 in PBS) for 2 hours at 4 o C, rinsed with modified Pblec buffer (1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 0.1% Triton X-100 in PBS), and incubated with primary antibodies diluted in modified Pblec buffer overnight at 4°C. After washing in blocking buffer diluted 1:1 with PBS and 3 washes in PBS, retinas were incubated for 1 hour at RT with secondary antibodies diluted in blocking buffer. Retinas were then washed, refixed with 4% PFA for 20 min at RT and washed twice with PBS prior to mounting with Fluoromount-G (Southern Biotech, #0100-01). Staining of intestine cryosections Tissues were fixed with 4% PFA in PBS overnight, washed with ice-cold PBS and incubated overnight with 30% sucrose in PBS. Intestines were embedded in OCT (Leica Biosystems, #14020108926) and kept at -80°C until sectioning. 60µm cryosections were thawed, fixed with 4% PFA in PBS for 5 min, washed in PBS, permeabilized with 0.5% Triton X-100 in PBS and blocked with 5% donkey serum for 30 min. Samples were incubated with primary antibodies in blocking buffer overnight at 4°C. Slides were then washed and incubated with secondary antibodies for 1 hour at RT. Slides were washed and mounted in Fluoromount-G (Southern Biotech, #0100-01) with DAPI. RNAscope analysis of tissue cryosections Tissues were fixed with 10% Neutral-Buffered Formalin (NBF) for 24 hours at RT, washed with ice-cold PBS and incubated overnight with 30% sucrose in PBS. Mesenteries and intestines were embedded in OCT (Leica Biosystems, #14020108926) and kept at -80°C until sectioning. For Vegfc mRNA detection, samples were processed according to the protocol provided by Advanced Cell Diagnostics (ACD, RNAcope Multiplex Fluorescent v2 Assay combined with Immunofluorescence). Briefly, 22µm cryosections were thawed, fixed with 10% NBF and dehydrated with 50%, 70% and 100% EtOH in distilled water. Samples were then treated with RNAscope hydrogen peroxide and transferred in antigen retrieval buffer before the incubation with primary antibodies overnight at 4°C. The next day, RNAscope Multiplex fluorescent v2 assay was performed using Vegfc and control probes (see Table 3 ). To detect primary antibodies, Alexa-fluor secondary antibodies diluted in co-detection antibody diluent were added for 40 min at RT. Slides were incubated with DAPI and mounted in Fluoromount-G. Image acquisition and analysis All images were captured using Leica SP8 or Zeiss LSM 980 confocal microscopes, and analyzed using Imaris, ImageJ and Photoshop softwares. All images of whole-mount mesenteric staining are shown in the same orientation, i.e. intestine at the bottom of the image. Cell isolation and scRNA-seq experiments For all scRNA-seq experiments, Esm1-CreERT2 tg/+ and R26-mTmG tg/+ E18.0 embryos (injected with 2 mg 4-OHT at E17.0) were dissected in ice-cold PBS. The small intestines and mesenteries were separated, and the pancreas and mesenteric lymph node were removed. The small intestines were cut open, and both tissues were thoroughly washed in ice-cold PBS. Mesenteries and intestines were then transferred in FACS complete medium (Phenol red-free DMEM with 5% FBS). Digestion was performed in PBS 0.1% BSA comprising 0.25 mg/ml Liberase DH (Roche, #05401054001) and 0.08 mg/ml DNaseI (Sigma, #DN25) at 37°C for 30 min for mesenteries and 1 hour for intestines. Digestion was completely stopped by adding FACS complete medium to the cell suspensions. Cells were subsequently filtered through a 40 µm cell strainer (Falcon, #352340) in FACS complete medium and centrifuged for 3 min at 4°C. For total mesenteric and small intestine cells, pellets were resuspended in red blood cell (RBC) lysis buffer (Sigma, #R7757) for 3 min at RT, and washed with FACS complete medium. Each cell suspension was directly loaded onto a microwell cartridge of the BD Rhapsody Express system (BD Biosciences, #400000847) and libraries were prepared using the BD Rhapsody WTA Reagent kit (BD Biosciences, #633802) following the manufacturer’s instructions. scRNA-seq libraries were evaluated and quantified by Agilent Bioanalyzer using High sensitivity DNA kit (#5067 − 4626) and Qubit (ThermoFisher Scientific, #Q32851). Individual libraries were pooled, diluted to 4 nM and sequenced by using NextSeq 500/550 High Output kit (150 cycle, Illumina,) with a NextSeq500 sequencer (Illumina). For fluorescence-activated cell sorting (FACS) of intestinal ECs, cell pellets were incubated with Fc block (antibody to CD16/32; BD Biosciences, #553142) for 10 min on ice and stained with conjugated antibodies in FACS buffer for 30 min (see Table 2 ). All GFP + and GFP − ECs were individually sorted using a FACSAria Fusion (BD Biosciences). GFP + ECs (around 9000 cells) were complemented with GFP − ECs to reach a total of 40000 cells and further processed for scRNA-seq library preparation and sequencing as described for total intestinal and mesenteric cells. scRNA-seq Preprocessing Raw FASTQ reads were quality and adapter trimmed using TrimGalore! (version 0.6.4 length cutoff 66, quality cutoff 20). The UMI, complex barcode, and sample tags were extracted and demultiplexed using custom scripts ( rhapsody-extract-barcode and rhapsody-demultiplex). Reads were mapped to the GRCm38 reference genome, Esm1-CreERT2 tg/+ , and R26-mTmG tg/+ construct sequences with Gencode annotations vM22, using STAR version 2.7.3a 88 (--soloType CB_UMI_Simple –soloCellFilter None –soloFeatures Gene –soloCBstart 1 –soloCBlen 27 –soloUMIstart 28 –soloUMIlen 8 –outFilterMultimapNmax 1 –soloCBwhitelist ). Raw counts were imported as AnnData 89 objects. We removed low complexity barcodes with the knee plot method, and further filtered out cells with a total contribution above 20% of reads belonging to mitochondrial mRNA. Doublets were predicted with scrublet 90 and cells with a doublet score above 0.1 have been removed. Finally, each sample’s gene expression matrix was normalized using scran (1.22.1) 91 with Leiden clustering 92 input at resolution 0.5. G2/M and S phase scores were assigned to each cell using gene lists from 93 and the scanpy (1.8.2) 94 sc.tl.score_genes_cell_cycle function. scRNA-seq Embedding, clustering and annotation At this stage, samples were merged. For 2D embedding, the expression matrix was subset to the 2,000 most highly variable genes (sc.pp.highly_variable_genes, flavor “seurat”). The top 50 principal components (PCs) were calculated, and batch-corrected using Harmony (0.0.5) 95 . The PCs served as basis for k-nearest neighbor calculation (sc.pp.neighbors, n_neighbors = 30), which were used as input for UMAP 96 layout (sc.tl.umap, min_dist = 0.3). Known marker genes were plotted using Scanpy (1.7.1) scanpy.pl.dotplot, cell populations were clustered using scanpy.tl.leiden at resolution 0.1 for annotation. The endothelial cell population was subclustered separately at Leiden resolution 0.9, and annoated using known marker genes. scRNA-seq trajectory and pseudotime analyses PAGA 40 from the Scanpy package was used to calculate non-mitotic EC population connectivities, and determine trajectories. Based on PAGA and Esm1 lineage tracing information, we calculated diffusion pseudotime 40 , 97 using scanpy.tl.dpt, choosing the Venous EC 1 population as starting cluster. Expression values for all genes with a minimum average normalised expression larger than 0.3 to reduce noise were binned into 200 bins according to pseudotime. The resulting z-transformed matrix was clustered hierarchically (scipy 1.10.0 scipy.cluster.hierarchical, Ward linkage, Euclidian distance metric) to identify gene expression patterns. Clusters were obtained with a tree distance cutoff of 60 in the dendrogram. Profiles for select clusters were plotted along binned pseudotime, celltype annotations for each bin were determined by majority vote. In profile plots for individual genes, the confidence interval of expression values is plotted as grey background (1.96*SD(expr)/sqrt(n)). Enrichr 98 was used to calculate Gene Ontoloy (BP, CC) enrichment on the expression profile cluster containing Esm1. Integration with P6 retinal endothelial tip cells For the comparison of Esm1 + cells from the intestinal villus with sprouting retinal tip cells, we reprocessed the publicly available single-cell data from Zarkada et al. 2021. Raw FASTQ files from P6 WT were processed analogously to the intestine and msentery samples above. Divergent STARsolo options were “--soloType Droplet --soloCBwhitelist 10xv3_whitelist.txt --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 10”. Mitochondrial mRNA content cutoff was set to 10%. AnnData objects were merged (outer join) with intestine and mesentery data using Scanpy concatenate. The merged data was reclustered at Leiden resolution 0.05 to identify ECs, the EC population was then further subclustered at resolution 1.0 to identify and remove contaminants and doublets. ECs from the Zarkada et al. data were subclustered at resolution 0.7, and the Esm1 + cluster was identified and annotated accordingly. Differential expression was performed on Esm1 + clusters from the original and Zarkada et al. data using a pseudobulk approach based on pyDESeq2 99 . Quantifications and statistics For whole-mount mesenteric artery and lymphatic vessel analysis, mesenteric tissue (DAPI), artery (CD31 + SOX17 + ) and lymphatic vessel (PROX1 + ) areas and artery length were quantified using Photoshop and ImageJ softwares. In lineage tracing experiments, GFP + cells in the mesenteric arteries were manually counted and in long-term loss-of-function experiments, GFP + area in mesenteric arteries was quantified using Photoshop and ImageJ. For whole-mount intestinal tissue analysis, GFP + , SOX17 + and intestinal tissue area were quantified using Photoshop and ImageJ softwares. For large mesenteric artery and intestinal submucosal artery analysis, at least 3 high magnification images were taken per sample. CD31 + SOX17 + artery area, length, GFP + area and nuclear density were quantified using Photoshop and ImageJ softwares. GFP + cells with changes in morphology (elongated vs round) were counted using Photoshop and ImageJ softwares. In the embryonic intestinal villus lineage tracing experiments, GFP + area in CD31 + SOX17 + and CD31 + SOX17 - areas were quantified using Photoshop and ImageJ softwares. For villus blood capillary length and width, images were directly quantified in 3D using Imaris software. For villus blood and lymphatic vessel, ERG + nucleus, SOX17 + nucleus and GFP + 3D volume and intensity analysis, images were quantified using Imaris software. In Itgb1 iEsm 1 KO , Itgb1 iEsm 1 HET and control littermates, 6–13 villi per sample were analyzed. In Vegfc iBmx KO and control littermates, 12–29 villi per sample were analyzed. Villus artery diameter and proportion of villus with more than one SOX17 + branch was quantified using Imaris, Photoshop and ImageJ softwares. In MAZ51-treated and DMSO control embryo experiments, 4–5 images comprising around 3 villi each were analyzed. In Flt4 iEsm 1 KO , Flt4 iEsm 1 HET and control littermates, 16–31 villi per sample were analyzed. For Esm1, LAMA4, COLIV, VEGFR3 and CD31 intensity quantifications, 7–15 villi per sample were analyzed. Each villus was divided into top and bottom villus capillary network area. In both top and bottom areas, vessel volume and staining intensities were quantified using Imaris software. For proliferation analyses, cells were double-stained for ERG and/or SOX17 and EdU. For villi from embryonic intestines, 6–9 high magnification images were analyzed. For embryonic mesenteries, 3 mesenteric artery branches per sample were imaged and analyzed. Double ERG and EdU masks were generated using Imaris software and cells were quantified using Photoshop and ImageJ softwares, counting as positive only cells in which all ERG + area was EdU + . For ESM1 + area quantification in adult villi, 5 images per animals comprising around 3 villi each were quantified. CAV1 + Esm1 + and CAV1 + EMCN + Esm1 + masks were generated using Imaris software and areas were quantified using Photoshop and ImageJ softwares. For GFP + area quantification in EMCN + and EMCN - of the adult villus, 9–12 images comprising around 3 villi each were analyzed. VEGFR2 + GFP + and VEGFR2 + EMCN + GFP + masks were generated using Imaris software and areas were quantified using Photoshop and ImageJ softwares. The number of embryos analyzed is indicated for each image in figure legends. Differences were considered statistically significant at P < 0.05. Data are shown as mean ± SD. Table 1 Primary and secondary antibodies and dyes used for stainings Antigen Reactivity Species Source Primary antibodies Caveolin-1 Human/Mouse/Rat Rabbit Cell Signaling (#3238) CD31 Human/Mouse/Rat Goat R&D Systems (#AF3628) CD31 Human/Mouse Rabbit Abcam (#ab28364) CD31 Mouse Rat BD Pharmingen (#553370) Collagen IV Mouse Rabbit Chemicon (#AB756P) Dll4 Mouse Goat R&D (#AF1389) E-cadherin Human/Mouse Rabbit Cell Signaling (#3195) Endomucin Mouse Rat Santa Cruz (#SC-65495) Erg Human/Mouse Rabbit Abcam (#ab110639) Esm1 Mouse Goat R&D (#AF1999) Fabp4 Human/Mouse Rabbit Abcam (#ab13979) GFP - Chicken 2BScientific Ltd (#GFP-1010) IB4 - Biotinylated Mouse/Rat/ Rabbit/Goat Griffonia simplicifolia Vector (#B-1205) Integrin b1 Mouse Rat BD Pharmingen (#553715) Laminin α4 Mouse Rabbit Serum 377 (Gift from L. Sorokin) 100 Lyve1 Mouse Rat R&D (#MAB2125) MAdCAM1 Mouse Rat Abcam (#ab80680) Prox1 Human Rabbit ReliaTech (#102-PA32AG) αSMA - Cy3 Human/Mouse/Rat Mouse Sigma (#C6198) αSMA - eFluor660 Human/Mouse/Rat Mouse eBioscience (#50-9760-82) Sox17 Human Goat R&D (#AF1924) Vegfr2 Mouse Goat R&D (#AF644) Vegfr3 Mouse Goat R&D (#AF743) Secondary antibodies Alexa 405-conjugated Streptavidin - - Invitrogen (#S32351) Alexa 488–conjugated Chicken Donkey Jackson Laboratories (#703-545-155) Alexa 488–conjugated Goat/Rabbit/ Rat Donkey Invitrogen (#A11055, #A21206, #A21208) Alexa 594–conjugated Goat/Rabbit/ Rat Donkey Invitrogen (#A11058, #A21207, #A21209) Alexa 647–conjugated Goat/Rabbit Donkey Invitrogen (#A21447, #A31573) Alexa 647–conjugated Rat Donkey Jackson ImmunoResearch (#712-605-153) Dyes DAPI - - Sigma (#D9542) Click-It EdU Alexa Fluor 647 - - Invitrogen (#C10340) Table 2 FACS-sorting antibodies Antigen Reactivity Specie Source FACS antibodies CD31-BV711 Mouse Rat BD Biosciences (#740680) CD45- BV421 Mouse Rat Biolegend (#103134) Epcam - PE-Cy7 Mouse Rat BioLegend (#118216) Podoplanin - eFluor660 Mouse Hamster eBioscience (#50-5381) Table 3 RNAscope reagents Reagents Source RNAscope Multiplex Fluorescent Reagent Kit v2 ACD (#323100) RNA-Protein Co-Detection Ancillary Kit ACD (#323180) RNAscope 3-plex Positive Control Probe - Mm ACD (#320881) RNAscope 3-plex Negative Control Probe - Mm ACD (#320871) RNAscope Probe - Mm - Vegfc ACD (#492701) TSA Plus Fluorescein Akoya Biosciences (#NEL741001KT) Declarations Data availability The scRNA-seq data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE275961. Acknowledgments We thank Pr. Mark L. Kahn for Flt4 lox/lox and Vegfc lox/lox mice and Dr. Martin Stehling and Dr. Bong-Ihn Koh for FACS analysis and sorting experiments. We thank Dr. Kishor K. Sivaraj for sharing Hey1-eGFP mice and Dr. Hongryeol Park for sharing Aplnr-CreERT2 mice. For sharing RNAscope protocols, reagents and discussions, we would like to thank Dr. Clé­mentine Vil­len­euve and Claudia Ortmeier from the Wickström’s department of the Max Planck Institute of Münster . We also thank Dr. Rodrigo Diéguez-Hurtado, Frank Berkenfeld and Silke Schröder for discussions and mouse colony maintenance. Animal, FACS, Genotyping, BioOptic and Bioinformatics facilities of the Max Planck Institute of Münster are gratefully acknowledged. The study was supported by the Max Planck Society (R.H.A.), the DFG Collaborative Research Center 1348 Project A10 (R.H.A., M.E.P., E.B.), the Leducq Foundation (R.H.A.) and the Cells in Motion (CiM) graduate school (V.M). Author Contributions E.B. and R.H.A. designed the study. E.B. performed the majority of the experiments. E.C.W. and E.B. performed the single-cell sequencing, and K.K. performed the bioinformatic transcriptomic analysis. V.M. generated samples for RNAscope. M.S. helped set up staining for FACS and perfomed FACS-sorting experiment. M.E.P. and F.B. generated P5 retina results. M.L.K provided critical mouse models and information for RNAscope experiments. E.B. and R.H.A. wrote the manuscript. Disclosures The authors declare no competing interests. References Chen D, Schwartz MA, Simons M (2021) Developmental Perspectives on Arterial Fate Specification. Front Cell Dev Biol 9:691335 Garriock RJ, Mikawa T (2011) Early arterial differentiation and patterning in the avian embryo model. Semin Cell Dev Biol 22:985–992 Zhong TP (2005) Zebrafish genetics and formation of embryonic vasculature. 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ESM1 (green), CD31 (red), SMA (white) and DAPI (blue). Arrowheads point at ESM1\u003csup\u003e+\u003c/sup\u003e ECs. Mes: Mesentery, Int: intestine. \u003cem\u003en\u003c/em\u003e = 4. Scale bars, 30, 50 and 200 µm. (\u003cstrong\u003eb\u003c/strong\u003e) Esm1\u003csup\u003e+\u003c/sup\u003e cell-derived GFP\u003csup\u003e+\u003c/sup\u003e (green) ECs contribute to large mesenteric arteries (SMA, red) at E16.5. DAPI (blue). Cranial (superior) mesenteric artery (CMA), mesentery (Mes) and intestine (Int) are indicated. \u003cem\u003en\u003c/em\u003e = 3. Scale bar, 150 µm. (\u003cstrong\u003ec\u003c/strong\u003e) Experimental design of Esm1\u003csup\u003e+\u003c/sup\u003e cell lineage tracing. (\u003cstrong\u003ed\u003c/strong\u003e) Whole-mount of E18.5 mesentery and intestine at 1 day, 3 days and 5 days of lineage tracing. GFP (green) and CD31 (red). Arrowheads mark GFP\u003csup\u003e+\u003c/sup\u003e arterial ECs. 1 day\u003cem\u003e n\u003c/em\u003e = 6; 3 days\u003cem\u003e n\u003c/em\u003e = 3;\u003cem\u003e \u003c/em\u003e5 days\u003cem\u003e n\u003c/em\u003e = 6. Scale bar, 200 µm. (\u003cstrong\u003ee\u003c/strong\u003e) High-magnification images of E18.5 mesentery and intestine after lineage tracing; GFP (green) and CD31 (red). Arrowheads, GFP\u003csup\u003e+\u003c/sup\u003e cells in mesenteric arteries. Scale bar, 30 µm. (\u003cstrong\u003ef\u003c/strong\u003e) Quantification of GFP\u003csup\u003e+\u003c/sup\u003e area in the intestinal tissue (µm\u003csup\u003e2\u003c/sup\u003e, normalized to tissue area) and number of arterial GFP\u003csup\u003e+\u003c/sup\u003e ECs in mesenteric tissue (normalized to tissue area). P values, 1-way ANOVA with Tukey post-hoc test; Error bars, Mean ± SD. (\u003cstrong\u003eg\u003c/strong\u003e) Whole-mount of E18.5 intestinal villi at 1 day, 3 days and 5 days of lineage tracing. GFP (green), CD31 (red), SOX17 (white) and DAPI (blue). Arrowheads mark GFP\u003csup\u003e+\u003c/sup\u003e cells descending into the SOX17\u003csup\u003e+\u003c/sup\u003e intestinal arterioles. Scale bar, 15 µm. (\u003cstrong\u003eh\u003c/strong\u003e) GFP\u003csup\u003e+\u003c/sup\u003e area in the villus capillary network (µm\u003csup\u003e2\u003c/sup\u003e, norm by vessel area). 1 day\u003cem\u003e n\u003c/em\u003e = 6; 3 days\u003cem\u003e n\u003c/em\u003e = 3;\u003cem\u003e \u003c/em\u003e5 days\u003cem\u003e n\u003c/em\u003e = 4. P values, 1-way ANOVA with Tukey post-hoc test; Error bars, Mean ± SD. (\u003cstrong\u003ei\u003c/strong\u003e) GFP\u003csup\u003e+\u003c/sup\u003e area in SOX17\u003csup\u003e+\u003c/sup\u003e and SOX17\u003csup\u003e-\u003c/sup\u003e villus capillaries (µm\u003csup\u003e2\u003c/sup\u003e, normalized to vessel area). ). 1 day\u003cem\u003e n\u003c/em\u003e = 6; 3 days\u003cem\u003e n\u003c/em\u003e = 3;\u003cem\u003e \u003c/em\u003e5 days\u003cem\u003e n\u003c/em\u003e = 4. P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test for each time point. Error bars, Mean ± SD.\u003c/p\u003e","description":"","filename":"BovayMainFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/3da715ca11cdf5a39f07b77e.png"},{"id":69024396,"identity":"95c0950a-5e9a-4a58-abe4-bb1c6f0cf4d5","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1802313,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndothelial cell zonation in the embryonic intestine and mesentery.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design of the scRNA-seq experiments. Unsorted total intestinal cells, unsorted total mesenteric cells and FACS-sorted intestinal blood ECs were individually sequenced and the resulting datasets were integrated (unsorted samples, \u003cem\u003en\u003c/em\u003e = 3 pooled embryos; FACS-sorted sample, \u003cem\u003en\u003c/em\u003e = 13 pooled embryos). (\u003cstrong\u003eb, c\u003c/strong\u003e) UMAP plot of the 3 combined datasets with all identified intestinal and mesenteric cell types (\u003cstrong\u003eb\u003c/strong\u003e) or blood vessel ECs only (\u003cstrong\u003ec\u003c/strong\u003e). (\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e) Dot plots of known marker genes for each cell population. Color code indicates average expression level in each group, and the dot size indicates the percentage of cells in each group expressing the given gene. (\u003cstrong\u003ef\u003c/strong\u003e) UMAP plots showing the expression of venous and arterial markers in the blood vessel EC populations. (\u003cstrong\u003eg\u003c/strong\u003e) Whole-mount of E18.5 intestine showing that MADCAM1 (green, yellow arrowheads) and DLL4 (white, white arrowheads) immunostaining marks the venous and arterial side of the villus capillary network (CD31, red), respectively.\u003cem\u003e n\u003c/em\u003e = 4. Scale bar, 20 µm. (\u003cstrong\u003eh\u003c/strong\u003e) Whole-mount of E17.0 villus at 24 hours of \u003cem\u003eAplnr\u003c/em\u003e lineage tracing; GFP (green), CD31 (red), SOX17 (white) and DAPI (blue). Arrowheads mark SOX17\u003csup\u003e+\u003c/sup\u003e GFP\u003csup\u003e-\u003c/sup\u003e arterial ECs. \u003cem\u003en\u003c/em\u003e = 4. Scale bars, 30 and 10 µm. (\u003cstrong\u003ei\u003c/strong\u003e) Whole-mount of E18.5 \u003cem\u003eHey1-eGFP \u003c/em\u003eintestines. GFP (green), CD31 (red), DLL4 (white) and DAPI (blue). Arrowheads, GFP\u003csup\u003e+\u003c/sup\u003e DLL4\u003csup\u003e+\u003c/sup\u003e vessels. \u003cem\u003en\u003c/em\u003e = 6. Scale bar, 30 µm. (\u003cstrong\u003ej\u003c/strong\u003e) Whole-mount of E18.5 \u003cem\u003eHey1-eGFP\u003c/em\u003e embryonic mesenteries. GFP (green), CD31 (red), SOX17 (white) and DAPI (blue). Arrowheads indicate GFP\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e arteries. \u003cem\u003en\u003c/em\u003e = 6. Scale bar, 200 µm.\u003c/p\u003e","description":"","filename":"BovayMainFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/69a96a29294de7ee42625770.png"},{"id":69024726,"identity":"dd22ad93-f6b9-4b7f-aa81-7005f8f30f3f","added_by":"auto","created_at":"2024-11-14 16:47:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":862611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEC proliferation during and after arterial specification.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) UMAP plot of ECs determined to be in G2/M, S and G1 phase. (\u003cstrong\u003eb\u003c/strong\u003e) Number of cells in G2/M, S and G1 phase in each EC subgroup after cell cycle regression. (\u003cstrong\u003ec\u003c/strong\u003e) Increase of early arterial gene expression along the vein-to-artery pseudotime axis. (\u003cstrong\u003ed\u003c/strong\u003e) Downregulation of cell cycle genes in the Esm1\u003csup\u003e+ \u003c/sup\u003eEC, Arterial EC1 and Arterial EC2 subgroups. (\u003cstrong\u003ee\u003c/strong\u003e) SOX17\u003csup\u003e+\u003c/sup\u003e villus capillary ECs have lower proliferation compared to SOX17\u003csup\u003e-\u003c/sup\u003e ECs. Whole-mount of E18.5 intestine with ERG (green), SOX17 (red) and EdU (white) staining. A mask was applied to identify EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e cells (yellow). Arrowheads indicate EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e cells. \u003cem\u003en\u003c/em\u003e = 4. Scale bar, 20 µm. (\u003cstrong\u003ef\u003c/strong\u003e) Proliferation of large mesenteric arterial ERG+ ECs. Whole-mount of E18.5 mesentery with CD31 (green), ERG (red) and EdU (white) staining. A mask was applied to identify EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e cells (yellow). Arrowheads mark EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e cells. \u003cem\u003en\u003c/em\u003e = 4. Scale bar, 15 µm. (\u003cstrong\u003eg\u003c/strong\u003e) Proportion of EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e and EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e-\u003c/sup\u003e ECs in the intestine and EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e ECs in the mesenteric artery. \u003cem\u003en\u003c/em\u003e = 4 for each tissue. P values, 1-way ANOVA with Tukey post-hoc test; Error bars, Mean ± SD.\u003c/p\u003e","description":"","filename":"BovayMainFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/909600473a1ceeeebbe2e74f.png"},{"id":69024403,"identity":"20c191e1-40f3-4fb4-bc86-1a79579ca885","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2518239,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntestinal arterial progenitor cells express tip cell-like markers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Whole-mount of E18.0 intestine shows expression of Esm1 (arrowheads) in the apical part of the villus capillary network. Esm1 (white), CD31 (green) and DAPI (blue). \u003cem\u003en\u003c/em\u003e = 3. Scale bar, 20 µm. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of Esm1 and CD31 intensity (A.U.) inside villi normalized to blood vessel volume (µm\u003csup\u003e3\u003c/sup\u003e). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test. Error bars, Mean ± SD. (\u003cstrong\u003ec\u003c/strong\u003e) Genetic lineage tracing with \u003cem\u003eEsm1-CreERT2\u003c/em\u003e (24 hours) shows recombined GFP\u003csup\u003e+\u003c/sup\u003e cells in the arterial side of the villus vasculature in the E18.5 intestine. GFP (green), CD31 (red) and SOX17 (white). White arrowheads mark GFP\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e ECs. \u003cem\u003en\u003c/em\u003e = 6. Scale bars, 30 and 7 µm.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ed\u003c/strong\u003e) UMAP plot showing the expression levels of \u003cem\u003eEsm1,\u003c/em\u003e \u003cem\u003eGFP\u003c/em\u003e, \u003cem\u003eLama4, Col4a2, Flt4 \u003c/em\u003eand\u003cem\u003e Nid2\u003c/em\u003e. \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-induced \u003cem\u003eGFP \u003c/em\u003eexpression (24 hours after 4-OHT treatment) is highest in the Esm1\u003csup\u003e+\u003c/sup\u003e population and the arterial cell area. (\u003cstrong\u003ee\u003c/strong\u003e) Whole-mount \u003cem\u003eCdh5-mTnG\u003c/em\u003e intestines at E18.0 (GFP marks EC nuclei in green and mTomato EC surface in red) shows Laminin α4 (LAMA4) immunostaining (white) in the upper part of the villus vasculature. Nuclei, DAPI (blue). \u003cem\u003en\u003c/em\u003e = 4. Scale bar, 30 µm. Graph shows quantification of LAMA4 intensity (A.U.) in villi normalized to vascular volume (µm\u003csup\u003e3\u003c/sup\u003e). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e) Increased Collagen type IV (COLIV) and VEGFR3 expression in the upper part of the villus vasculature in the E18.0-E18.5 intestine. CD31 (green), COLIV or VEGFR3 (red) and DAPI (blue). Yellow arrowheads mark VEGFR3\u003csup\u003ehigh\u003c/sup\u003e blood vessels, white arrowhead indicates VEGFR3\u003csup\u003ehigh\u003c/sup\u003e lymphatic vessel. \u003cem\u003en\u003c/em\u003e = 3. Scale bars, 20 µm. Graph shows quantification of COLIV and VEGFR3 intensity (A.U.) in villi normalized to vascular volume (µm\u003csup\u003e3\u003c/sup\u003e). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD.\u003c/p\u003e","description":"","filename":"BovayMainFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/a0896c94362da5cf406c9a2f.png"},{"id":69024397,"identity":"806885be-0c07-4db6-8b12-c40c4d4b9f1d","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1555623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of integrin β1\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein Esm1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e ECs leads to thinning of mesenteric arteries.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design of \u003cem\u003eEsm1-CreERT2-\u003c/em\u003emediated \u003cem\u003eItgb1\u003c/em\u003e inactivation. (\u003cstrong\u003eb\u003c/strong\u003e)\u003cem\u003e \u003c/em\u003eWhole-mount SOX17 (white) staining of E18.5 mesenteries shows that \u003cem\u003eItgb1\u003c/em\u003e deficiency in Esm1\u003csup\u003e+\u003c/sup\u003e cells\u003cem\u003e \u003c/em\u003e(\u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO\u003c/sup\u003e) reduces average mesenteric artery diameter. Mesenteric lymph node (mLN) and intestine (Int) are indicated. \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eHET\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e = 3 and \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 3. Scale bar, 400 µm. (\u003cstrong\u003ec\u003c/strong\u003e) Graphs show quantification of embryonic weight, mesenteric artery diameter (in µm, artery area normalized to artery length) and mesenteric artery density (in µm, artery length normalized to tissue area) in control, \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO\u003c/sup\u003e mutants and \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eHET\u003c/sup\u003e heterozygous littermates. \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eHET \u003c/sup\u003egroup set to 1. Data from 3 experiments were pooled. P values, 1-way ANOVA with Tukey post-hoc test; Error bars, Mean ± SD. (\u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e) Whole-mount staining of mesenteric artery image (\u003cstrong\u003ed\u003c/strong\u003e) and high magnification longitudinal and transverse single confocal planes (\u003cstrong\u003ee\u003c/strong\u003e). GFP (green), CD31 (red), SOX17 (white) and DAPI (blue). White arrowheads indicate rounded GFP\u003csup\u003e+\u003c/sup\u003e cells in \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO\u003c/sup\u003e mutant. \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eHET\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e = 3; \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 3. Scale bars, 30 and 20 µm. (\u003cstrong\u003ef\u003c/strong\u003e) Graphs show quantification of SOX17\u003csup\u003e+\u003c/sup\u003e cells in large mesenteric arteries (normalized to artery area), average diameter of large mesenteric arteries (in µm, artery area normalized to artery length) and number of round GFP\u003csup\u003e+\u003c/sup\u003e cells in large mesenteric arteries (normalized to total number of GFP\u003csup\u003e+\u003c/sup\u003e cells). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003eg\u003c/strong\u003e) Whole-mount staining of E18.5 intestine shows that \u003cem\u003eItgb1\u003c/em\u003e inactivation in Esm1\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eECs alters the shape of GFP\u003csup\u003e+\u003c/sup\u003e cells (green) in intestinal submucosal arteries. CD31 (red), SOX17 (white) and DAPI (blue). White arrowheads indicate rounded GFP\u003csup\u003e+\u003c/sup\u003e cells.\u003cem\u003e Itgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eHET\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e = 3; \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eEsm1\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 3. Scale bar, 30 µm.\u003c/p\u003e","description":"","filename":"BovayMainFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/17652a22c66f7213c74b4442.png"},{"id":69024399,"identity":"a794e716-6a16-49c4-be40-8154efb80a25","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1896565,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of arterial \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eVegfc\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e impairs arterial and lymphatic vessel development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) UMAP plots showing the expression of \u003cem\u003eFlt4 \u003c/em\u003eand\u003cem\u003e Vegfc\u003c/em\u003e in all cells isolated from intestine and mesentery. (\u003cstrong\u003eb\u003c/strong\u003e) Pseudotime plots showing increased \u003cem\u003eFlt4\u003c/em\u003e expression level in the Esm1\u003csup\u003e+\u003c/sup\u003e EC area, whereas \u003cem\u003eVegfc \u003c/em\u003edecreases relative to the artery area. (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e) RNAscope\u003cem\u003e \u003c/em\u003edetects \u003cem\u003eVegfc \u003c/em\u003emRNAs (green, arrowheads) in SOX17\u003csup\u003e+\u003c/sup\u003e (red) mesenteric arteries (\u003cstrong\u003ec\u003c/strong\u003e) and in SOX17\u003csup\u003e+\u003c/sup\u003e intestinal arteries (\u003cstrong\u003ed\u003c/strong\u003e) at E19.0. Nuclei, DAPI (blue). Arteries (Ar) and red blood cells (RBCs), a source of background signal, are indicated. \u003cem\u003en\u003c/em\u003e = 5 each. Scale bar, 15 µm. (\u003cstrong\u003ee\u003c/strong\u003e) Loss of \u003cem\u003eVegfc\u003c/em\u003e from E14.5 in Bmx\u003csup\u003e+\u003c/sup\u003e arteries impairs mesenteric lymphatic vessel (PROX1, white) development at E19.0. Control\u003cem\u003e n\u003c/em\u003e = 12; \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eBmx\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 6. Scale bar, 250 µm. (\u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e) Loss of \u003cem\u003eVegfc\u003c/em\u003e from E14.5 in BMX\u003csup\u003e+\u003c/sup\u003e arteries does not affect large SOX17\u003csup\u003e+\u003c/sup\u003e (white) mesenteric arteries (F) but leads to thinning of arteries (arrowheads) in the intestinal mucosa (\u003cstrong\u003eg\u003c/strong\u003e) at E19.0. CD31 (red). Control\u003cem\u003e n\u003c/em\u003e = 12; \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eBmx\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003e\u003cem\u003en\u003c/em\u003e = 6. Scale bars, 50 µm. (\u003cstrong\u003eh\u003c/strong\u003e) Quantification of mesenteric lymphatic vessel area (µm\u003csup\u003e2\u003c/sup\u003e, normalized to tissue area), large mesenteric artery (close to the intestine) and intestinal submucosal artery diameter (in µm, artery area normalized to artery length) in \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eBmx\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003eE19.0 embryos and control littermates. P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003ei\u003c/strong\u003e) Whole-mount staining showing ERG (green), CD31 (red) and SOX17 (white) staining of control\u003cem\u003e \u003c/em\u003eand \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eBmx\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO \u003c/sup\u003eE19.0 intestinal villi. Panels on right show higher magnifcations of boxed areas. A mask was applied to identify CD31\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e cells (green, left panels). Arrowheads indicate ERG\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e villus arteries. Control\u003cem\u003e n\u003c/em\u003e = 12; \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eBmx\u003c/em\u003e\u003c/sup\u003e\u003csup\u003eKO\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e = 6. Scale bars, 30 and 10 µm. (\u003cstrong\u003ej\u003c/strong\u003e) Quantification of villus blood capillary network length and width (µm), total villus vessel nuclear density (µm\u003csup\u003e3\u003c/sup\u003e, total ERG\u003csup\u003e+\u003c/sup\u003e volume normalized to CD31\u003csup\u003e+\u003c/sup\u003e volume), SOX17\u003csup\u003e+\u003c/sup\u003e nuclear density (µm\u003csup\u003e3\u003c/sup\u003e, total SOX17\u003csup\u003e+\u003c/sup\u003e volume normalized to CD31\u003csup\u003e+\u003c/sup\u003e volume), number of villi with more than one main CD31\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e vessel branch, and average villus SOX17\u003csup\u003e+\u003c/sup\u003e vessel diameter (µm). For all analysis, P values were determined by 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD.\u003c/p\u003e","description":"","filename":"BovayMainFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/7212b8aab84851ccc899106d.png"},{"id":69024727,"identity":"b469a5cc-bbfc-4eb4-a0a0-e1736ff9001a","added_by":"auto","created_at":"2024-11-14 16:47:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2732652,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVEGFR3 inhibition reduces Esm1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cell contribution to mesenteric arteries.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design of short-term 48h lineage tracing experiment with MAZ51 or vehicle treatment. (\u003cstrong\u003eb\u003c/strong\u003e) Whole-mount images of E18.0 intestine shows regression of LYVE1\u003csup\u003e+\u003c/sup\u003e (green) lymphatic vessels after MAZ51 treatment. VEGFR2 (red) and DAPI (blue). Arrowheads mark LYVE1\u003csup\u003e+\u003c/sup\u003e lacteals. Vehicle\u003cem\u003e n\u003c/em\u003e = 8; MAZ51\u003cem\u003e n\u003c/em\u003e = 6.\u003csup\u003e \u003c/sup\u003eScale bar, 20 µm. (\u003cstrong\u003ec\u003c/strong\u003e) Embryonic weight is comparable between vehicle and MAZ51 groups. P value, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of LYVE1\u003csup\u003e+\u003c/sup\u003e lymphatic capillaries in E18.0 intestine (µm\u003csup\u003e3\u003c/sup\u003e, normalized to tissue volume). P value, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003ee\u003c/strong\u003e) Whole-mount of E18.0 intestine at 48h after \u003cem\u003eEsm1-CreERT2-\u003c/em\u003emediated reporter activation. GFP (green), CD31(red), SOX17 (white) and DAPI (blue). Vehicle\u003cem\u003e n\u003c/em\u003e = 8; MAZ51\u003cem\u003e n\u003c/em\u003e = 10.\u003csup\u003e \u003c/sup\u003eScale bar, 20 µm. (\u003cstrong\u003ef\u003c/strong\u003e) Quantification of average GFP and SOX17 intensity in villus capillaries (per µm\u003csup\u003e3\u003c/sup\u003e). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e) Whole-mount of E18.0 intestines (\u003cstrong\u003eg\u003c/strong\u003e) and mesenteries (\u003cstrong\u003eh\u003c/strong\u003e) showing decreased GFP\u003csup\u003e+\u003c/sup\u003e cell contribution to arteries after MAZ51 treatment. GFP (green), CD31 (red), SOX17 (white) and DAPI (blue). Vehicle\u003cem\u003e n\u003c/em\u003e = 5; MAZ51\u003cem\u003e n\u003c/em\u003e = 8.\u003csup\u003e \u003c/sup\u003eScale bar, 80 µm and 100 µm. (\u003cstrong\u003ei\u003c/strong\u003e) Quantification of GFP\u003csup\u003e+\u003c/sup\u003e area in submucosal arteries (µm\u003csup\u003e2\u003c/sup\u003e, normalized to total arterial area) and number of GFP\u003csup\u003e+\u003c/sup\u003e cells found in mesenteric tissue (normalized to total tissue area). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD.\u003c/p\u003e","description":"","filename":"BovayMainFigures7.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/7921dccf9cb8e3785d9b5ee2.png"},{"id":69024401,"identity":"3ed4381c-e007-4f26-b5ec-500bdc73d130","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1037742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFlt4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in Aplnr\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e ECs impairs artery development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental design for Aplnr\u003csup\u003e+\u003c/sup\u003e cell-specific \u003cem\u003eFlt4 \u003c/em\u003einactivation from E13.5. (\u003cstrong\u003eb\u003c/strong\u003e) Loss of VEGFR3 (white) expression, reduced villus blood capillary length and increase lacteal length in \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eAplnrKO\u003c/em\u003e\u003c/sup\u003e mutant villi compared to control littermates at E18.0. VEGFR3 (white), CD31 (red) and DAPI (blue). Yellow arrowheads mark VEGFR3\u003csup\u003e+\u003c/sup\u003e capillaries in villus apex. Blue arrowheads mark VEGFR3\u003csup\u003e+\u003c/sup\u003e lacteals. Control\u003cem\u003e n\u003c/em\u003e = 4; \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eAplnrKO\u003c/em\u003e\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e = 4. Scale bar, 30 µm. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of VEGFR3\u003csup\u003e+\u003c/sup\u003e area in villus blood vessels (µm\u003csup\u003e2\u003c/sup\u003e, normalized to vessel area), villus capillary network length (µm) and villus lacteal length (µm). P values, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD. (\u003cstrong\u003ed\u003c/strong\u003e) Whole-mount of E18.0 intestine and mesentery showing thinner SOX17\u003csup\u003e+\u003c/sup\u003e arteries in \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eAplnrKO\u003c/em\u003e\u003c/sup\u003e mutants compared to control littermates. CD31 (red) and SOX17 (white). Control\u003cem\u003e n\u003c/em\u003e = 4; \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eAplnrKO\u003c/em\u003e\u003c/sup\u003e \u003cem\u003en\u003c/em\u003e = 4. Scale bar, 200 µm. (\u003cstrong\u003ee\u003c/strong\u003e) Embryonic weight is comparable between control\u003cem\u003e \u003c/em\u003eand \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eAplnrKO\u003c/em\u003e\u003c/sup\u003e littermates. Average artery diameter leaving the intestine in \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003c/sup\u003e\u003csup\u003e\u003cem\u003eAplnrKO\u003c/em\u003e\u003c/sup\u003e littermates is reduced compared to controls. P value, 2-tailed unpaired Student’s\u0026nbsp;\u003cem\u003et\u003c/em\u003e\u0026nbsp;test; Error bars, Mean ± SD.\u003c/p\u003e","description":"","filename":"BovayMainFigures8.png","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/846e1b6119a3262bf996dea8.png"},{"id":92235080,"identity":"1a72c55b-a15d-4c08-9e95-a756a932366a","added_by":"auto","created_at":"2025-09-26 07:09:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16766707,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/b0182f6e-3032-4053-a04a-b0e8328187ea.pdf"},{"id":69024402,"identity":"7de69a08-29f9-4171-98bc-b778f4503b2d","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1682211,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Bovaynreditorialpolicychecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/90843b8d51fe1d1dc092a8b9.pdf"},{"id":69024400,"identity":"4d3850d5-8f07-45e4-8e20-0d6e67b96a66","added_by":"auto","created_at":"2024-11-14 16:39:03","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1667693,"visible":true,"origin":"","legend":"","description":"","filename":"Bovaynrreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/6e75d5b12e0e48cff6c922f5.pdf"},{"id":69554094,"identity":"6cf906c6-930f-4d65-8237-abe3a422451c","added_by":"auto","created_at":"2024-11-21 15:07:39","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15397421,"visible":true,"origin":"","legend":"","description":"","filename":"BovaySupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5411147/v1/d467b0655ab08e59f3fd1680.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Artery formation in intestinal wall and mesentery by intestine-derived Esm1+ endothelial cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArteries are the conduit for blood transport into peripheral organs and thereby are crucial for the supply of essential nutrients and oxygen. Research in early mouse, zebrafish and avian embryos have provided a good understanding of the processes mediating the vasculogenic formation of the dorsal aorta and the cardinal vein, the two large axial vessels\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, comparably little is known about the formation of the majority of arteries during the angiogenic expansion of the vasculature in the embryonic and postnatal organism. In the developing heart, it was shown that coronary artery growth involves the reprogramming of venous (sinus venosus-derived) endothelial cells (ECs) to an arterial fate\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, endocardial cells, which form the innermost lining of the heart and share many features with arterial ECs, have been also shown to contribute to coronary artery development\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Genetic fate tracking has established that venous ECs, which exhibit comparably high rates of proliferation, give rise to arterial endothelium in retina and brain\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the developing and regenerating zebrafish tail fin, dynamic live imaging has shown that vein-derived endothelial tip cells give rise to arterial ECs\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Strikingly, genetic alterations affecting guided EC migration impair normal arterial patterning, suggesting that defects in this process might be a cause of arteriovenous malformations\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the postnatal murine eye, tip cells at the distal end of endothelial sprouts, which express high levels of endothelial cell-specific molecule 1 (Esm1/endocan), are important for the angiogenic expansion of the developing retinal vasculature. Genetic fate tracking with \u003cem\u003eEsm1-CreERT2\u003c/em\u003e transgenic mice has established that tip cell progeny contributes to the arterial but not the venous branch of the vasculature, which involves signaling interactions via the Notch pathway, the chemokine receptor CXCR4 and its ligand CXCL12, and interactions between ephrin-B2 and its receptor EphB4\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e. Arterial differentiation involves arrested proliferation and endothelial cell cycle state has been shown to generate a fate bias during arterial-venous specification\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Apart from molecular regulators, blood flow and fluid shear stress are important factors controlling cell cycle status and the migration of ECs against the direction of blood flow\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the developing intestinal system of the mouse, arteriogenesis is initiated during gut rotation, at around embryonic stage E10.0, forming a first arterial connection between the intestinal vascular plexus with the dorsal aorta\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. It also has been established that high signaling by vascular endothelial growth factor A (VEGF-A) and its receptor VEGFR2 upregulates the expression of Esm1 in the villus apex, which has relevance for nutrient uptake, blood vessel remodeling and normal arteriovenous patterning\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Despite of these insights, our understanding of the heterogeneity and functional specialization of ECs in the gastrointestinal system remains limited and the processes controlling artery formation in the intestine and adjacent mesentery are unknown. Our new findings reveal that a small subpopulation of ECs in the capillary network of the villus gives rise to arterial endothelium in the adjacent intestinal wall but also in the distant mesenteric vasculature. Genetic labeling, immunohistochemistry and single cell RNA-sequencing (scRNA-seq) show that these prearterial cells express Esm1 and other tip cell markers despite being located inside a patent capillary network. Mechanistically, \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-mediated inactivation of the \u003cem\u003eItgb1\u003c/em\u003e gene encoding integrin β1 in the mouse embryo results in the loss of arterial progenitors and a subsequent reduction in the diameter of large mesenteric arteries. Additionally, we show that the VEGF-C/VEGFR3 signaling pathway, which is a primary lymphatic growth factor and tyrosine kinase receptor\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, participates in the migration of Esm1\u003csup\u003e+\u003c/sup\u003e cells into the mesenteric arterial network and expansion of the arterial network. Taken together, these findings establish fundamental principles of developmental artery formation in the intestine and mesentery.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of arterial progenitors in the embryonic intestine\u003c/h2\u003e \u003cp\u003eStratification of the intestinal epithelium begins around embryonic day (E) 14.0\u003csup\u003e25\u0026ndash;27\u003c/sup\u003e. However, the formation of the initial intestinal vascular network occurs earlier, beginning as early as E10.5\u003csup\u003e20\u003c/sup\u003e. We investigated whether the blood capillary network at these early stages already expresses Esm1. We stained E12.5 mesenteries and observed ESM1 protein expression within the capillary network surrounding the epithelium (Supplementary Fig.\u0026nbsp;1a). We further used \u003cem\u003eEsm1-CreERT2\u003c/em\u003e transgenic mice in the \u003cem\u003eRosa26-mTmG\u003c/em\u003e Cre reporter background\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e to detect Esm1\u003csup\u003e+\u003c/sup\u003e cells and their progeny. After tamoxifen treatment initiated at E10.5, recombined GFP\u003csup\u003e+\u003c/sup\u003e ECs appear in the SOX17\u003csup\u003e+\u003c/sup\u003e intestinal and mesenteric vascular network at E13.5 (Supplementary Fig.\u0026nbsp;1b).\u003c/p\u003e \u003cp\u003eWe further investigated whether the early mesenteric vasculature already shows segregation of arteries and veins. To analyze known arterial and venous markers, we used \u003cem\u003eAplnr-CreERT2\u003c/em\u003e and \u003cem\u003eBmx-CreERT2\u003c/em\u003e transgenic mice in combination with the \u003cem\u003eRosa26-mTmG\u003c/em\u003e Cre reporter\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. After daily tamoxifen administration from E10.5 onward, \u003cem\u003eBmx-CreERT2\u003c/em\u003e-controlled GFP expression was observed in the Endomucin-negative arterial network of E13.5 mesenteric tissues (Supplementary Fig.\u0026nbsp;1c). Conversely, 24-hour \u003cem\u003eAplnr-CreERT2\u003c/em\u003e induction with 4-hydroxytamoxifen (4-OHT) resulted in widespread GFP expression in E12.5 mesenteric and intestinal vessels. However, SOX17\u003csup\u003e+\u003c/sup\u003e vessels connected to the cranial mesenteric artery (CMA) remained GFP\u003csup\u003elow\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;1d). These findings indicate that Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny contributes to arteries during an early stage of intestinal and mesenteric development. Furthermore, it is evident that acute \u003cem\u003eAplnr-CreERT2\u003c/em\u003e-mediated recombination in the E11.5 embryo is prominent in capillaries but spares BMX\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e mesenteric arteries.\u003c/p\u003e \u003cp\u003eWe next examined Esm1 expression at later stages. Whole-mount staining of embryonic tissues at E18.0 revealed strong ESM1 protein expression in the intestinal villi and mesenteric capillary sprouting cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). To assess the long-term contribution of Esm1\u003csup\u003e+\u003c/sup\u003e cells, we treated \u003cem\u003eEsm1-CreERT2\u003c/em\u003e transgenic mice in the \u003cem\u003eRosa26-mTmG\u003c/em\u003e Cre reporter background from E10.5 to E15.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). GFP\u003csup\u003e+\u003c/sup\u003e ECs were abundant in large mesenteric arteries and smaller arterial branches near the intestinal wall at E16.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) but absent from veins or lymphatic vessels. To gain a better insight into the spatial distribution and dynamic behavior of Esm1\u003csup\u003e+\u003c/sup\u003e cells, we conducted multiple genetic fate tracking experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Short-term (24 hours) induction with 4-hydroxytamoxifen (4-OHT) leads to robust GFP signal in the embryonic intestine, while the mesentery exhibits minimal presence of GFP\u003csup\u003e+\u003c/sup\u003e ECs, except for mesenteric sprouting capillaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f). Analysis of intestinal villi at E18.5 revealed that GFP signal is predominantly localized to the upper portion of the villus vascular network, reflecting heightened VEGF signaling at the apex of the villus\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Analysis of embryos at the same stage but after 3 and 5 days post-induction shows lower GFP signal within the intestine but an increased presence of GFP\u003csup\u003e+\u003c/sup\u003e cells within large mesenteric arteries (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-f). While the extent of arterial labeling is limited in these short-term genetic fate tracking experiments, the results indicate that \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-labeled cells are incorporated into mesenteric arteries but not into nearby veins or lymphatic vessels (Supplementary Fig.\u0026nbsp;1e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCareful analysis of GFP\u003csup\u003e+\u003c/sup\u003e cells over time indicates morphological changes during the transition from the intestine into the mesentery. Whereas GFP\u003csup\u003e+\u003c/sup\u003e ECs in the intestine display irregular shapes with numerous protrusions, cells in the mesentery exhibit the typical slender and elongated morphology of arterial ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, g). With increasing time between 4-OHT administration and analysis, more elongated arterial GFP\u003csup\u003e+\u003c/sup\u003e cells are observed, and these cells are found deeper inside the mesenteric arterial tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Further analysis of intestines confirms the presence of GFP signal in the upper portion of the villus vascular network at 24 hours post-induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, left panels), whereas GFP\u003csup\u003e+\u003c/sup\u003e cells contribute to the SOX17\u003csup\u003e+\u003c/sup\u003e villus capillaries and submucosal arteries over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-i). These results reveal a progressive contribution of the progeny of Esm1\u003csup\u003e+\u003c/sup\u003e ECs to the submucosal and mesenteric arterial network (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-i, Supplementary Fig.\u0026nbsp;1e). The sum of our genetic tracking data also indicates that Esm1\u003csup\u003e+\u003c/sup\u003e cells continue to be induced throughout development of the embryonic intestinal vasculature from midgestation to birth, which implies that a pulse of tamoxifen/4-OHT will lead to \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-mediated recombination only in a fraction of these cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIntestinal arterial progenitors contribute to the villus capillary expansion after birth\u003c/h3\u003e\n\u003cp\u003eTo investigate whether progeny from Esm1\u003csup\u003e+\u003c/sup\u003e ECs continues to contribute to arteries after birth, lineage tracing experiments were initiated from postnatal day (P) 1 (Supplementary Fig.\u0026nbsp;2a). Analysis at P8 reveals an abundance of GFP\u003csup\u003e+\u003c/sup\u003e ECs within the intestine, whereas the mesentery lacks GFP-expressing cells in large arteries. GFP\u003csup\u003e+\u003c/sup\u003e cells are located in the expanding capillary network surrounding mesenteric arteries and veins, but lack a direct connection to these large vessels (Supplementary Fig.\u0026nbsp;2b). Further examination of the P8 and P21 intestines confirms the presence of GFP\u003csup\u003e+\u003c/sup\u003e cells in the SOX17\u003csup\u003e+\u003c/sup\u003e region of the villus capillary network (Supplementary Fig.\u0026nbsp;2c, d). These results indicate the continued presence of Esm1\u003csup\u003e+\u003c/sup\u003e cells in the intestinal vasculature after birth, but these cells no longer contribute to large mesenteric arteries.\u003c/p\u003e \u003cp\u003ePrevious work has shown that high VEGF signaling at the villus apex, indicated by elevated expression of the VEGF-responsive genes \u003cem\u003eFlt4\u003c/em\u003e (encoding VEGFR3) and \u003cem\u003eEsm1\u003c/em\u003e, facilitates the reorganization of EC junctions to enhance nutrient uptake in the adult mouse intestine\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, whole-mount analysis of the adult mouse intestine shows Esm1 immunostaining in the more arterial part of the villus capillary network, which is characterized by pronounced anti-Caveolin 1 signal and absent Endomucin expression (Supplementary Fig.\u0026nbsp;3a, b). Lineage tracing experiments conducted over 24 hours and 2 weeks in 20 to 24-week-old male mice reveal only minimal presence of GFP\u003csup\u003e+\u003c/sup\u003e ECs in the retina of these animals (Supplementary Fig.\u0026nbsp;3c-e). In contrast, robust GFP signal is seen inside the capillary network of intestinal villi, with a significant enrichment in Endomucin\u003csup\u003e-\u003c/sup\u003e areas (Supplementary Fig.\u0026nbsp;3f-h). Altogether, these findings show that the expression of Esm1 in the intestinal vascular network commences during embryogenesis and persists into postnatal and adult stages. However, the contribution of Esm1\u003csup\u003e+\u003c/sup\u003e EC-derived arterial progenitors to the formation of mesenteric arteries is confined to embryonic development.\u003c/p\u003e\n\u003ch3\u003eFate and properties of intestinal arterial progenitors at single cell resolution\u003c/h3\u003e\n\u003cp\u003eTo validate the developmental fate of intestinal Esm1\u003csup\u003e+\u003c/sup\u003e ECs and gain deeper insight into their molecular properties, we isolated cells from E18.0 embryos 24 hours after genetic lineage tracing with \u003cem\u003eEsm1-CreERT2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;4a, b). We first individually isolated all cells from the intestine and mesentery, followed by single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody system. To augment our population of blood vessel ECs, we conducted an additional 24 hours lineage tracing experiment with ECs enriched in GFP\u003csup\u003e+\u003c/sup\u003e cells by fluorescence-activated cell sorting (FACS) (Supplementary Fig.\u0026nbsp;4a, b). In total, we acquired 34518 cells, among which 7122 were blood vessel ECs (Supplementary Fig.\u0026nbsp;4b, right panels). These datasets were combined and visualized as Uniform Manifold Approximation and Projection (UMAP) plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on established markers from the literature, we identified most of the known intestinal and mesenteric cell types, including ECs from blood and lymphatic vessels, epithelial, mesothelial, immune, neural and various mesenchymal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). We then focused on blood vessel ECs, which segregate into 8 subgroups (after exclusion of contaminating red blood cells) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, e). ECs expressing higher levels of venous markers, such as \u003cem\u003eNrp2, Madcam1, Aplnr\u003c/em\u003e and \u003cem\u003eNr2f2 (Coup-TFII)\u003c/em\u003e, can be seen on the left side of the UMAP plot, subdivided into proliferative (G2/M and S phase) and two non-proliferative groups (Venous EC1 and Venous EC2). ECs with higher levels of arterial markers, namely \u003cem\u003eUnc5b\u003c/em\u003e, \u003cem\u003eHey1, Gja4\u003c/em\u003e, \u003cem\u003eIgfbp3\u003c/em\u003e, \u003cem\u003eGja5\u003c/em\u003e, \u003cem\u003eDll4\u003c/em\u003e and \u003cem\u003eSox17\u003c/em\u003e, cluster on the right (Arterial EC1, Arterial EC2, and Arterial EC3). Venous EC1 and Arterial EC3 cells are enriched in ECs from large mesenteric veins and arteries, expressing markers such as \u003cem\u003eAdamts18\u003c/em\u003e, \u003cem\u003eFam174b\u003c/em\u003e and \u003cem\u003eBmx\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, f, Supplementary Fig.\u0026nbsp;4b), which indicates that these two populations are most mature with respect to arteriovenous specification. Interestingly, an intermediate subgroup of ECs is located in the center between the areas expressing venous or arterial markers. This population shows high levels of markers characteristic for endothelial tip cells (\u003cem\u003eEsm1\u003c/em\u003e, \u003cem\u003eFlt4\u003c/em\u003e, \u003cem\u003eLamb1\u003c/em\u003e, \u003cem\u003eNid2\u003c/em\u003e) and represents Esm1\u003csup\u003e+\u003c/sup\u003e ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, e).\u003c/p\u003e \u003cp\u003eWe further corroborated the distribution of venous and arterial markers at the protein level by immunohistochemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg-j, Supplementary Fig.\u0026nbsp;5a-d). Staining of E17.0-E19.0 embryonic intestines confirms the expression of MADCAM1 and DLL4 by the \u0026ldquo;venous\" and \"arterial\" side of the villus capillary network, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). To validate the expression of \u003cem\u003eAplnr\u003c/em\u003e as a marker of the venous domain, we used the \u003cem\u003eAplnr-CreERT2\u003c/em\u003e line in combination with the \u003cem\u003eRosa26-mTmG\u003c/em\u003e Cre reporter\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. At 24 hours after 4-OHT administration, expression of GFP is detected in most of the villus capillary network but is excluded from the SOX17\u003csup\u003e+\u003c/sup\u003e (i.e., arterial) branch (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). In the mesenteric tissue, as expected, \u003cem\u003eAplnr-CreERT2\u003c/em\u003e-controlled GFP expression is found specifically in large mesenteric veins and capillaries, but is excluded from SOX17\u003csup\u003e+\u003c/sup\u003e arteries and lymphatic vessels (Supplementary Fig.\u0026nbsp;5a-d). Analysis of a \u003cem\u003eHey1-eGFP\u003c/em\u003e knock-in line carrying an insertion of a \u003cem\u003eGFP\u003c/em\u003e cassette in the Notch pathway gene \u003cem\u003eHey1\u003c/em\u003e (Hairy/enhancer-of-split related with YRPW motif protein 1) is more prominent in the arterial side of the villus capillary network and in large mesenteric arteries, reflecting the known role of Notch signaling in arterial specification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, j). Consistent with previous descriptions, \u003cem\u003eHey1-eGFP\u003c/em\u003e expression is also detected in some epithelial cells and DLL4\u003csup\u003e+\u003c/sup\u003e ECs\u003csup\u003e32\u0026ndash;35\u003c/sup\u003e. Analysis of the mesentery confirmed robust GFP signal in the SOX17\u003csup\u003e+\u003c/sup\u003e mesenteric arteries. Taken together, these findings validate our scRNA-seq data and confirm arteriovenous zonation within the developing villus vasculature, which gradually extends into the large veins and arteries of the mesenteric vascular network.\u003c/p\u003e\n\u003ch3\u003eTransient cell cycle arrest and arterial differentiation characterize venous-derived Esm1 cells\u003c/h3\u003e\n\u003cp\u003eIn the mouse retina and embryonic heart, venous cells actively divide and give rise to all other EC subpopulations including tip cells and committed arterial ECs that exhibit cell cycle arrest\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. To investigate the origin of Esm1\u003csup\u003e+\u003c/sup\u003e cells and the cells generating upstream arteries, we performed lineage-tracing of Aplnr\u003csup\u003e+\u003c/sup\u003e ECs (Supplementary Fig.\u0026nbsp;5e-k). At 4 to 6 days after 4-OHT treatment, Aplnr\u003csup\u003e+\u003c/sup\u003e EC-derived cells contribute strongly to the villus capillary network, SOX17\u003csup\u003e+\u003c/sup\u003e submucosal arteries, and large upstream mesenteric arteries (Supplementary Fig.\u0026nbsp;5e-k). Notably, lineage tracing from E13.5 leads to labeling of large veins, capillaries, and arteries closer to the intestine, while the majority of lymphatic vessels lacks GFP expression. However, when 4-OHT treatment is initiated at E11.5, nearly all vessels, including lymphatic vessels, are GFP\u003csup\u003e+\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;5e-j). These findings are consistent with previous research indicating a large proportion of venous origin for mesenteric lymphatic vessels starting in the developmental window around E10 to E12.5 in the mouse\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and confirm that Esm1\u003csup\u003e+\u003c/sup\u003e cells and arterial ECs are also derived from Aplnr\u003csup\u003e+\u003c/sup\u003e ECs.\u003c/p\u003e \u003cp\u003eConsistent with this and previous studies, the venous EC subclusters in our scRNA-seq analysis predominantly harbor proliferative cells in the G2/M or S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). To exclude clustering effects due to dominant cell cycle expression patterns, we regressed out the expression of known G2/M and S phase genes, re-clustered the cells, and annotated the new clusters to match the original EC type annotation as closely as possible (Supplementary Fig.\u0026nbsp;6a). We then determined the proportion of cycling cells in each EC population, by counting the number of cells expressing G1, G2/M, and S phase genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Supplementary Fig.\u0026nbsp;6b), which confirmed the high proliferation of venous ECs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate these observations, we conducted a Partition-based Graph Abstraction (PAGA) Trajectory analysis, facilitating diffusion pseudotime calculation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;6c). For this analysis, the two proliferative subgroups, i.e. Prolif EC (G2/M phase) and Prolif EC (S phase), were excluded. Pseudotime results are consistent with a trajectory along the vein-to-artery axis, extending from the venous side through the Esm1\u003csup\u003e+\u003c/sup\u003e population into the arterial domain (Supplementary Fig.\u0026nbsp;6d). As expected, the Venous EC1 and Venous EC2 subgroups show low expression of arterial markers but high levels of cell cycle genes relative to other EC subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). Conversely, Esm1\u003csup\u003e+\u003c/sup\u003e ECs, Arterial EC1 and Arterial EC2 exhibit higher expression of arterial markers but low transcript levels for cell cycle genes, confirming previous observations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The Arterial EC3 subgroup, which is enriched in the mesenteric sample and therefore represents ECs from larger arteries, surprisingly shows higher levels of cell cycle genes compared to other arterial subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-d, Supplementary Fig.\u0026nbsp;6b). We validated these findings with EdU staining to assess EC proliferation in the intestine and mesentery. The majority of EdU\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e ECs in villi lack SOX17 expression, reflecting the expected low proliferation of arterial progenitors undergoing differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g). In large mesenteric arteries, however, over 10% of ERG\u003csup\u003e+\u003c/sup\u003e elongated arterial nuclei are EdU\u003csup\u003e+\u003c/sup\u003e. Taken together, these results indicate that Esm1\u003csup\u003e+\u003c/sup\u003e cells are derived from venous ECs in the developing intestine. Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny shows the expected downregulation of cell cycle genes during arterial specification. In contrast, a fraction of fully differentiated mesenteric arterial ECs express cell cycle genes, suggesting that local proliferation of these cells contributes to the size increase of larger arteries during development.\u003c/p\u003e\n\u003ch3\u003eComparison of Esm1 in the embryonic villus and postnatal retina\u003c/h3\u003e\n\u003cp\u003eAs previous work has shown that Esm1\u003csup\u003e+\u003c/sup\u003e ECs in the postnatal retina contribute to the expanding arterial network\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, we compared the molecular properties of these cells to the Esm1\u003csup\u003e+\u003c/sup\u003e population from embryonic intestine. To this end, we reanalyzed a previously published scRNA-seq dataset of wildtype P6 retinal endothelial tip cells\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and compared the Esm1\u003csup\u003e+\u003c/sup\u003e population to the Esm1\u003csup\u003e+\u003c/sup\u003e cluster in our study using pseudobulk differential expression analysis (DEA) (Supplementary Fig.\u0026nbsp;7a). While Esm1\u003csup\u003e+\u003c/sup\u003e cells from the retina and embryonic intestine share expression of signature genes such as \u003cem\u003eEsm1\u003c/em\u003e, \u003cem\u003eFlt4\u003c/em\u003e (encoding the receptor tyrosine kinase VEGFR3) or \u003cem\u003eLama4\u003c/em\u003e (laminin α4), the two populations show substantial differences in gene expression. One example is the \u003cem\u003eFabp4\u003c/em\u003e gene and its product fatty acid binding protein 4, which are highly expressed by Esm1\u003csup\u003e+\u003c/sup\u003e ECs in the distal villus but absent in retinal tip cells\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;7b-d). Whole-mount immunostaining shows substantial morphological differences between Esm1\u003csup\u003e+\u003c/sup\u003e cells in the retina and intestine (Supplementary Fig.\u0026nbsp;7c-f). While intestinal Esm1\u003csup\u003e+\u003c/sup\u003e cells are an integral part of the villus capillary network, Esm1\u003csup\u003e+\u003c/sup\u003e cells in the retina are sprouting tip cells that extend filopodia into the avascular tissue. Furthermore, Caveolin-1 (encoded by the \u003cem\u003eCav1\u003c/em\u003e gene), is differentially expressed between the intestinal and retinal Esm1\u003csup\u003e+\u003c/sup\u003e populations by scRNA-seq (Supplementary Fig.\u0026nbsp;7b). Immunostaining confirms low expression of Caveolin-1 in sprouting retinal tip cells, whereas Esm1\u003csup\u003e+\u003c/sup\u003e cells in the capillary network of intestinal villi are prominently labeled (Supplementary Fig.\u0026nbsp;7e, f).\u003c/p\u003e \u003cp\u003eWhole-mount immunostaining of the embryonic intestine at E18.0/E18.5 confirms that endogenous ESM1 protein expression is enriched in the upper portion of the villus, in contrast to ubiquitous CD31 expression by all ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-mediated lineage tracing for 24 hours in combination with immunostaining shows that \u003cem\u003eEsm1\u003c/em\u003e expression precedes the upregulation of SOX17, thus affirming the role of Esm1\u003csup\u003e+\u003c/sup\u003e ECs as arterial progenitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Analysis of genetic lineage tracing over 24 hours by scRNA-seq provides further proof of the contribution of \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-labeled (\u003cem\u003eGFP\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e) progeny from the central Esm1\u003csup\u003e+\u003c/sup\u003e population to the arterial branch of the intestinal vasculature (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). We then performed immunostaining for the markers laminin α4 (encoded by the gene \u003cem\u003eLama4\u003c/em\u003e), collagen IV and VEGFR3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee-g). Indeed, all three gene products were found to decorate the capillaries in the distal villus, confirming that the microenvironment at the apex of the embryonic villus facilitates the emergence of a specialized, Esm1\u003csup\u003e+\u003c/sup\u003e EC population with a role in artery formation (Supplementary Fig.\u0026nbsp;7g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntegrin β1 in Esm1\u003csup\u003e+\u003c/sup\u003e ECs controls cell shape and mesenteric artery diameter\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) enrichment analysis of genes exhibiting a similar expression pattern as \u003cem\u003eEsm1\u003c/em\u003e revealed a significant enrichment of genes involved in the biological processes \u0026ldquo;angiogenesis\u0026rdquo; and \u0026ldquo;cell migration\u0026rdquo;. Likewise, \u0026ldquo;Focal adhesion\u0026rdquo; and \u0026ldquo;Actin filament\u0026rdquo; were among the top hits for cellular components (Supplementary Fig.\u0026nbsp;8a). To address a potential role of cell migration and cell-matrix interactions, we used the \u003cem\u003eEsm1-CreERT2\u003c/em\u003e line to inactivate the \u003cem\u003eItgb1\u003c/em\u003e gene, which encodes the integrin β1 subunit (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Integrins are heterodimeric transmembrane receptors composed of an α and β subunit\u003csup\u003e\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Integrin β1 plays a crucial role in the development and maintenance of the blood vascular network. Pan-endothelial deletion of \u003cem\u003eItgb1\u003c/em\u003e was shown to result in vascular leakage and the disruption of arterial EC polarity\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-mediated loss of \u003cem\u003eItgb1\u003c/em\u003e has no impact on embryonic weight, mesenteric artery density, or organization of the intestinal villus vasculature, despite the confirmed loss of integrin β1 protein in GFP\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c, Supplementary Fig.\u0026nbsp;6b-d). However, \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e embryos show a reduction of mesenteric artery diameter relative to control or heterozygous mutant (\u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e) littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, c). High-magnification analysis of the large mesenteric arteries reveals no change in the density of EC nuclei but confirms the decrease in vascular diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f). The inclusion of a \u003cem\u003eR26-mTmG\u003c/em\u003e reporter allele shows that GFP\u003csup\u003e+\u003c/sup\u003e ECs in \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e mesenteric arteries acquire a rounded shape, which is distinct from the strongly elongated appearance of control arterial ECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-f). Similar alterations in GFP\u003csup\u003e+\u003c/sup\u003e cell morphology are evident in arteries of the intestinal submucosa (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). These findings support that arterial progenitor cells generated within the intestine functionally contribute to the formation of the large mesenteric arteries. Accordingly, the loss of \u003cem\u003eItgb1\u003c/em\u003e in Esm1\u003csup\u003e+\u003c/sup\u003e ECs results in the reduction of arterial diameter.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eArtery-derived VEGF-C promotes artery development\u003c/h3\u003e\n\u003cp\u003eIn the adult intestine, the VEGFR3 ligand VEGF-C is expressed by vascular and intestinal smooth muscle cells (SMCs), macrophages, and a subset of villus fibroblasts\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Recent work, supported by scRNA-seq analysis, has identified mesenteric arteries as a major source of VEGF-C with a crucial role in the development of a secondary postnatal lymphatic capillary network\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In our scRNA-seq data, \u003cem\u003eFlt4/Vegfr3\u003c/em\u003e is enriched in lymphatic ECs but also in Esm1\u003csup\u003e+\u003c/sup\u003e capillary ECs, whereas \u003cem\u003eVegfc\u003c/em\u003e is strongly expressed in the arterial endothelium, which is supported by pseudotime analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). RNAscope analysis on mesenteric and intestinal cryosections confirms the pattern of \u003cem\u003eVegfc\u003c/em\u003e mRNA expression. Despite strong autofluorescence from red blood cells (RBCs), \u003cem\u003eVegfc\u003c/em\u003e transcripts are predominantly detected in the mesenteric and submucosal arteries as well as in SOX17\u003csup\u003e+\u003c/sup\u003e villus capillaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). In the intestine, \u003cem\u003eVegfc\u003c/em\u003e mRNA is detected throughout the mucosal tissue, suggesting that various embryonic intestinal cell types express this growth factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo specifically target arterial ECs, we used the tamoxifen-inducible \u003cem\u003eBmx-CreERT2\u003c/em\u003e mouse line \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e in combination with the \u003cem\u003eR26-mTmG\u003c/em\u003e reporter. In lineage tracing experiments with the common end point at E18.5, GFP signal labels large mesenteric arteries and intestinal submucosal SOX17\u003csup\u003e+\u003c/sup\u003e arteries (Supplementary Fig.\u0026nbsp;9a-d). Over longer time periods, namely after 4-OHT induction at E13.5 or E15.5, GFP-negative ECs replace the \u003cem\u003eBmx-CreERT2\u003c/em\u003e-labeled GFP\u003csup\u003e+\u003c/sup\u003e cells in submucosal and smaller (distal) mesenteric arteries so that recombined ECs reside only in the larger caliber arteries (Supplementary Fig.\u0026nbsp;9b). These results strongly support the notion that the continuous migration of arterial progenitors from the intestine contributes to the expansion of embryonic mesenteric arteries (Supplementary Fig.\u0026nbsp;9a-d).\u003c/p\u003e \u003cp\u003eTo address whether arterial VEGF-C might facilitate the recruitment of Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny into the mesenteric vasculature, we introduced the \u003cem\u003eBmx-CreERT2\u003c/em\u003e line into a background of mice carrying conditional \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e alleles\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;9e). Given the immature state of arteries observed at E13.5 (Supplementary Fig.\u0026nbsp;1c), we performed short-term 4-OHT-induced \u003cem\u003eVegfc\u003c/em\u003e inactivation starting from E14.5. After 4 days of \u003cem\u003eVegfc\u003c/em\u003e deletion from arteries, the lymphatic vessel area is significantly reduced in \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e mutants compared to control littermate embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-h). These results confirm prior studies indicating that arteries are a key source of \u003cem\u003eVegfc\u003c/em\u003e for mesenteric lymphangiogenesis\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. While large mesenteric arteries remain unaffected by the loss of VEGF-C, \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e arteries in the intestinal mucosa are significantly thinner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-h), indicating that arteries closest to the intestine are most affected.\u003c/p\u003e \u003cp\u003eRNAscope analysis confirms the loss of \u003cem\u003eVegfc\u003c/em\u003e mRNA expression in large \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e mesenteric and submucosal arteries (Supplementary Fig.\u0026nbsp;9f, g). However, residual \u003cem\u003eVegfc\u003c/em\u003e mRNA is still detected in the intestinal mucosa and SOX17\u003csup\u003e+\u003c/sup\u003e ECs in \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e intestinal villi, which may reflect incomplete \u003cem\u003eBmx-CreERT2\u003c/em\u003e-mediated recombination or \u003cem\u003eVegfc\u003c/em\u003e expression prior to upregulation of \u003cem\u003eBmx\u003c/em\u003e. The latter is consistent with the pseudotime analysis of our scRNA-seq data, showing that \u003cem\u003eVegfc\u003c/em\u003e expression is initiated in Esm1\u003csup\u003e+\u003c/sup\u003e ECs during early steps of arterial specification and prior to \u003cem\u003eBmx\u003c/em\u003e expression (Supplementary Fig.\u0026nbsp;9h). Nevertheless, analysis of intestines shows that \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e villus capillaries show an increase in CD31\u003csup\u003e+\u003c/sup\u003e ERG\u003csup\u003e+\u003c/sup\u003e ECs area but significantly thinner SOX17\u003csup\u003e+\u003c/sup\u003e arterial branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, j). These results suggest that arterial \u003cem\u003eVegfc\u003c/em\u003e expression promotes the translocation of arterial progenitor cells from intestinal villi into the adjacent submucosal arteries. Accordingly, EC density is increased in \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e villi, whereas the diameter of the submucosal arterial network is decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-j).\u003c/p\u003e\n\u003ch3\u003eVEGFR3 signaling promotes the translocation of Esm1 cell progeny into arteries\u003c/h3\u003e\n\u003cp\u003eBased on the results above, we hypothesized that signaling by VEGF-C and VEGFR3 enhances the efficient translocation of intestinal arterial progenitor cells into the large mesenteric arteries. To examine the role of the VEGFR3 tyrosine kinase receptor, we inhibited its activity by administering MAZ51 to pregnant females during a short lineage tracing experiment of Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). MAZ51 is a potent inhibitor of VEGFR3 kinase activity, which only weakly affects the related receptor tyrosine kinase VEGFR2\u003csup\u003e59,60\u003c/sup\u003e. After MAZ51 treatment, growing lymphatic capillaries in the intestine stained for LYVE1 are significantly reduced compared to vehicle controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d), indicating efficient inhibition of VEGFR3 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). In contrast, MAZ51 treatment does not affect the villus capillary network, \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-controlled GFP expression, or SOX17 signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). Strikingly, the contribution of GFP\u003csup\u003e+\u003c/sup\u003e Esm1 cell progeny to the submucosal and large mesenteric arteries is significantly reduced by MAZ51 relative to vehicle controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg-i). These findings support that inhibiting VEGFR3 activity in arterial progenitor cells impairs their translocation into submucosal and mesenteric arteries.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test this hypothesis further, we generated mutant embryos carrying loxP-flanked versions of \u003cem\u003eFlt4\u003c/em\u003e (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e)\u003csup\u003e61\u003c/sup\u003e together with \u003cem\u003eEsm1\u003c/em\u003e-\u003cem\u003eCreERT2\u003c/em\u003e and \u003cem\u003eRosa26-mTmG\u003c/em\u003e alleles. Following tamoxifen administration from E10.5 onward, the appearances of control, \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e, and \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e embryos are indistinguishable at E18.0 (Supplementary Fig.\u0026nbsp;10a, data not shown). Immunostaining confirms the expected reduction of VEGFR3 expression in the \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e and, more completely, in the \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e capillary network at the villus apex compared to control littermates (Supplementary Fig.\u0026nbsp;10b). Quantification of VEGFR3 revealed a strong residual VEGFR3 signal in the \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e and \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e villus capillary networks, but with significant loss in GFP\u003csup\u003e+\u003c/sup\u003e areas (Supplementary Fig.\u0026nbsp;10b, c). Despite the substantial deletion of VEGFR3 in the GFP\u003csup\u003e+\u003c/sup\u003e ECs in \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e samples, these results reflect that the villus apex is not entirely composed of \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-targeted ECs, so that only a subset of ECs in the villus apex loses VEGFR3 (Supplementary Fig.\u0026nbsp;10b, c).\u003c/p\u003e \u003cp\u003eUnlike MAZ51 treatment, targeted \u003cem\u003eFlt4\u003c/em\u003e deletion in Esm1-derived cells does not impact lacteal length, suggesting that this approach does not interfere with lymphatic growth (Supplementary Fig.\u0026nbsp;10b, d). \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e mutants exhibit a slight increase in blood vessel width relative to control littermates (Supplementary Fig.\u0026nbsp;10b, d). Despite the incomplete deletion of VEGFR3 in the villus vasculature, \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e mesenteric samples relative to \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e littermates show reduced incorporation of GFP\u003csup\u003e+\u003c/sup\u003e cells into submucosal arteries and large mesenteric arteries, wherease control (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eWT\u003c/sup\u003e) samples show significantly higher incorporation of GFP\u003csup\u003e+\u003c/sup\u003e cells (Supplementary Fig.\u0026nbsp;10e, f).\u003c/p\u003e \u003cp\u003eTo inactivate \u003cem\u003eFlt4\u003c/em\u003e more efficiently in villus ECs, we made use of the finding that \u003cem\u003eAplnr-CreERT2\u003c/em\u003e-labeled cells give rise to Esm1\u003csup\u003e+\u003c/sup\u003e ECs and, subsequently, arterial endothelium. We therefore introduced the \u003cem\u003eAplnr-CreERT2\u003c/em\u003e line into the \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e background. To avoid lymphatic involvement (Supplementary Fig.\u0026nbsp;5h-j), treatment was initiated at E13.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea) prior to development of the lymphatic vasculature. By E18.0, we observed significant loss of VEGFR3 specifically in \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eAplnr\u003c/em\u003eKO\u003c/sup\u003e blood vessels without affecting the intestinal (lymphatic) lacteals (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, c). Interestingly, the \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eAplnr\u003c/em\u003eKO\u003c/sup\u003e villus blood vessel network is reduced, whereas VEGFR3\u003csup\u003ehigh\u003c/sup\u003e lacteals grow longer, which may reflect higher availability of VEGF-C due to the lack of VEGFR3 in blood vessel ECs. Upstream of the villus, submucosal arteries are notably thinner than in control littermates (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, e), mirroring the phenotype observed in \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e mutants (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h). Together with the effects seen following MAZ51 treatment and \u003cem\u003eVegfc\u003c/em\u003e and \u003cem\u003eFlt4\u003c/em\u003e inactivation, these results indicate that VEGFR3 promotes the recruitment of Esm1\u003csup\u003e+\u003c/sup\u003e ECs from villus capillaries into upstream arteries.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eArteries are an indispensable part of the vascular tree and arterial malfunction is causally linked to detrimental, sometimes life-threatening human diseases. Mesenteric arteries carry blood from the abdominal aorta to the gastrointestinal tract and are thereby indispensable for the function of this organ. Accordingly, the blockade or narrowing of mesenteric arteries, which occurs in human patients due to the build-up of atherosclerotic plaques or other reasons, can lead to severe abdominal pain, intestinal damage and the need for surgical intervention\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Our study provides fundamental insights into the development of arteries of the mesentery and intestinal wall. In particular, we show that a small population of Esm1\u003csup\u003e+\u003c/sup\u003e cells, located inside intestinal villi, gives rise to arterial ECs both in the intestine and mesentery. Accordingly, the formation of these arteries requires EC migration over substantial distances in addition to arterial specification. Previous work has established that Esm1\u003csup\u003e+\u003c/sup\u003e tip cells in the postnatal retina, which are located at the distal end of endothelial sprouts and thereby at the border to avascular tissue, generate arterial but also capillary ECs\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Remarkably, Esm1\u003csup\u003e+\u003c/sup\u003e cells in the embryonic intestine express typical tip cell markers even though they are part of patent capillary tubes and do not show sprouting behavior. Thus, Esm1/endocan expression in this setting is likely to reflect elevated levels of growth factor signaling, especially of VEGF-A and VEGF-C, as has been shown previously\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Mechanistically, Esm1 has been shown to bind to fibronectin and displace fibronectin-bound VEGF-A, thereby increasing the bioavailability and signaling capacity of the growth factor\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. As a result, Esm1 enhances endothelial sprouting activity as well as vascular permeability\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. This role of Esm1 might be also relevant in the apex of the villus vasculature where VEGF-A modulates EC junctions to enhance nutrient uptake in the adult murine intestine\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the context of arterial development, Esm1 might facilitate the migration of prearterial ECs into the arterial walls and further into mesenteric arteries. Apart from VEGF-A, which is highly expressed in the distal villus, the related ligand VEGF-C is also relevant. As previously described for the murine postnatal and adult intestine\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, VEGFR3 expression marks Esm1\u003csup\u003e+\u003c/sup\u003e cells in the embryonic intestine, whereas its ligand, VEGF-C, is provided by submucosal and mesenteric arteries. Functionally, the migration and arterial incorporation of Esm1\u003csup\u003e+\u003c/sup\u003e (and VEGFR3\u003csup\u003e+\u003c/sup\u003e) ECs is compromised after genetic or pharmacological disruption of signaling by VEGF-C and its receptor VEGFR3. The function of VEGFR3 and VEGF-C is known to be crucial for the lymphatic vessel development and maintenance, but also modulates sprouting angiogenesis and blood vessel growth in mouse and zebrafish models\u003csup\u003e\u003cspan additionalcitationids=\"CR67 CR68 CR69 CR70 CR71\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Our new findings reveal an additional and unexpected function of this ligand-receptor pair, namely the guidance of Esm1\u003csup\u003e+\u003c/sup\u003e ECs into the growing arterial vasculature.\u003c/p\u003e \u003cp\u003eOur genetic fate tracking experiments show that Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny inside arteries acquires the typical elongated shape characteristic of arterial ECs\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eEsm1-CreERT2\u003c/em\u003e-mediated inactivation of integrin β1, an important subunit of many integrin receptor heterodimers, leads to the rounding of Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny and reduces the diameter of embryonic mesenteric arteries. This rounding phenotype is reminiscent of previous findings showing that pan-endothelial (but incomplete and therefore mosaic) inactivation of the \u003cem\u003eItgb1\u003c/em\u003e gene impairs arteriolar lumen formation due to defective EC polarization\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Other studies showed that integrin β1 is also required for endothelial sprouting in the postnatal retina, arteriole formation in the ischemic heart but also for the barrier function of the endothelium and the prevention of vascular leakage\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. All these aspects might be of relevance for the formation and function of the arterial network in the intestine and mesentery.\u003c/p\u003e \u003cp\u003eOur findings also raise the question whether the recruitment of Esm1\u003csup\u003e+\u003c/sup\u003e cell progeny is the predominant or even sole process responsible for mesenteric artery development in the embryo. The existence of alternative, redundantly acting mechanisms might contribute to biological robustness and resilience. In the developing heart, ECs derived both from the sinus venosus and the endocardium contribute to coronary blood vessel formation, and both pools of arterial progenitors can compensate for each other\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Similarly, multiple non-venous derived sources of lymphatic ECs have been identified in various organs, including the mesentery\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Our own findings show that fully differentiated mesenteric arterial ECs overcome the cell cycle arrest that is characteristic for ECs undergoing arterial differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e and exhibit some level of proliferation. Similarly, EC proliferation in situ contributes to the regeneration of damaged aortic endothelium in the adult mouse\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Thus, it is feasible that the expansion of mature arterial ECs in the mesentery and intestinal wall might be able to compensate for insufficient specification of prearterial cells. Alternatively, processes such as vessel remodeling and pruning\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e, which play important roles during the angiogenic expansion of the vasculature, might help to ensure or restore sufficient arterial blood flow. Taken together, our findings establish a fundamental framework for artery development in the intestine and mesentery. We propose that intestinal villi represent a niche microenvironment for the induction of Esm1\u003csup\u003e+\u003c/sup\u003e arterial progenitors, which will integrate into growing arteries, but not into veins or lymphatic vessels, and thereby contribute to the expansion of the arterial network.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMouse models\u003c/h2\u003e\n \u003cp\u003eAll animal experiments were performed according to the institutional guidelines and laws, approved by local animal ethical committee and were conducted at the Max Planck Institute for Molecular Biomedicine with necessary permissions (Az 81-02.04.2019.A114) granted by the Landesamt f\u0026uuml;r Natur, Umwelt und Verbraucherschutz (LANUV) of North Rhine-Westphalia, Germany.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eEsm1-CreERT2\u003c/em\u003e \u003csup\u003e29\u003c/sup\u003e, \u003cem\u003eBmx-CreERT2\u003c/em\u003e \u003csup\u003e31\u003c/sup\u003e, \u003cem\u003eAplnr-CreERT2\u003c/em\u003e \u003csup\u003e30\u003c/sup\u003e, \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e \u003cspan class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eFlt4\u003c/em\u003e \u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e \u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eVegfc\u003c/em\u003e \u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e \u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eR26-mTmG\u003c/em\u003e \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eAi14\u003c/em\u003e \u003csup\u003e83\u003c/sup\u003e, \u003cem\u003eCdh5-mTnG\u003c/em\u003e\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eHey1-eGFP\u003c/em\u003e (\u003cem\u003eHey1-GFP (Tg(Hey1-EGFP)ID40Gsat;\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.gensat.org\u003c/span\u003e\u003c/span\u003e) were previously described.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eEmbryonic, postnatal and adult mouse treatments\u003c/h2\u003e\n \u003cp\u003eFor embryo experiments, vaginal plugs were verified in the morning. Embryonic age (E) was determined according to the day of the vaginal plug (E0.5). For lineage tracing embryo experiments, one injection of 2mg total 4-hydroxytamoxifen (4-OHT; Sigma, #H7904) in oil was injected intraperitoneally (i.p.) in pregnant mice. For lineage tracing experiments with VEGFR3 tyrosine kinase inhibition, MAZ51 (2% DMSO in PBS, #HY-116624) was injected subcutaneously (s.c.) at E14.5 and E17.0. One dose of 2mg of 4-OHT in oil was injected i.p. in pregnant mice at E16.0. Embryos were collected 48 hours later at E18.0. Click-it EdU cell proliferation Alexa Fluor 647 kit (Life Technologies, #C10340) was used for EdU detection. When indicated, 5\u0026micro;g/g EdU was injected i.p. 1 hour before sacrifice.\u003c/p\u003e\n \u003cp\u003eFor continuous Esm1\u003csup\u003e+\u003c/sup\u003e cell tracking, pregnant females were injected with 2.5mg tamoxifen (Sigma, #T5648) and progesterone (Sigma, #P3972) mixed in oil s.c. starting from E10.5 every day until the day before embryo collection. \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e \u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e embryos were analyzed at E13.5 and E16.5.\u003c/p\u003e\n \u003cp\u003eIn loss-of-function experiments, \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e embryos (\u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e) were compared to \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e (\u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e) or control littermates (\u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e; \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e or mice without the \u003cem\u003eEsm1-CreERT2\u003c/em\u003e transgene). All embryos carried the reporter transgene \u003cem\u003eR26-mTmG\u003c/em\u003e (\u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003e\u003cem\u003etg/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e).\u003c/em\u003e In \u003cem\u003eEsm1-CreERTR2\u003c/em\u003e-controlled \u003cem\u003eFlt4\u003c/em\u003e loss-of-function experiments, \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e embryos (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e) were compared to \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e), \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eWT\u003c/sup\u003e (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e), or control littermates without the \u003cem\u003eEsm1-CreERT2\u003c/em\u003e transgene. All embryos carried the reporter transgene \u003cem\u003eR26-mTmG\u003c/em\u003e (\u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003e\u003cem\u003etg/+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e and some carried the \u003cem\u003eAi14\u003c/em\u003e transgene. \u003cem\u003eItgb1\u003c/em\u003e and \u003cem\u003eFlt4\u003c/em\u003e long-term deletion experiments were induced with 2.5mg tamoxifen and progesterone mixed in oil s.c. starting from E10.5 every day until the day before embryo collection. For \u003cem\u003eAplnr-CreERTR2\u003c/em\u003e-controlled \u003cem\u003eFlt4\u003c/em\u003e loss-of-function experiments, \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003ei\u003cem\u003eAplnr\u003c/em\u003eKO\u003c/sup\u003e (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAplnr-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e) embryos were compared with control littermates (\u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eAplnr-CreERT2\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e). Long-term deletion experiments were induced with 2.5mg tamoxifen and progesterone mixed in oil s.c. at E13.5, E14.5 and E15.5. For \u003cem\u003eBmx-CreERTR2\u003c/em\u003e-controlled \u003cem\u003eVegfc\u003c/em\u003e loss-of-function experiments, \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003ei\u003cem\u003eBmx\u003c/em\u003eKO\u003c/sup\u003e (\u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBmx-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e) embryos were compared with control littermates (\u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003e\u003cem\u003elox/lox\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eBmx-CreERT2\u003c/em\u003e\u003csup\u003e+/+\u003c/sup\u003e). Short-term deletion experiments were induced with 2mg of 4-OHT in oil injected s.c. starting from E14.5 every day until the day before embryo collection.\u003c/p\u003e\n \u003cp\u003eFor postnatal lineage tracing experiments, \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e \u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e pups were injected once at P1 with 50\u0026micro;g 4-OHT in oil s.c. and analyzed at P8 or P21. For lineage tracing in adult mice, 20\u0026ndash;24 week-old \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e \u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e males were injected once with 2mg 4-OHT in oil i.p. and analyzed 24 hours or 2 weeks later.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eMouse embryonic mesenteric and intestinal tissue collection, and staining procedures\u003c/h2\u003e\n \u003cp\u003eEmbryonic gastro-intestinal tracts were collected in ice-cold PBS. Duodenum (around 1 cm at E18.0) and first part of jejunum (next 1 cm) were collected, cut open and pinned in a 12-well elastomer-coated dish and thoroughly washed with ice-cold PBS. From the remaining tissue, intestine was pinned in a circle to visualize the mesentery. Clean duodenum, jejunum and mesentery were fixed with ice-cold 4% paraformaldehyde (PFA) in PBS (Sigma, #P6148) overnight on a rotating platform at 4\u003csup\u003eo\u003c/sup\u003eC. Samples were then washed with ice-cold PBS and incubated overnight with 10% sucrose in PBS followed by 20% sucrose and 10% glycerol in PBS. For staining, 0.5cm-long intestine pieces or mesenteric tissues were washed with PBS, permeabilized with 0.5% Triton X-100 and blocked with 5% donkey serum overnight. Tissues were incubated with primary, followed by secondary antibodies overnight on a rotating platform at 4\u003csup\u003eo\u003c/sup\u003eC. Intestine samples were incubated overnight with Histodenz (Sigma, #D2158) at room temperature (RT). All samples were mounted in Histodenz with DAPI (Sigma, #D9542). A list of antibodies used in this study is provided in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eMouse postnatal mesentery, postnatal and adult intestine collection and staining\u003c/h2\u003e\n \u003cp\u003eThe protocol was adapted from previous publications\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e85\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Pups and adult mice were perfused with PBS then 4% PFA in PBS after anesthesia. Isolated tissues were washed in ice-cold PBS and duodenum and first jejunum parts were collected, cut open and pinned in a 6-well or 10-cm elastomer-coated dish and thoroughly washed with ice-cold PBS. The remaining postnatal jejunum and ileum were pinned down in a circle to visualize the mesentery. Clean duodenum and jejunum samples and mesenteries were fixed with ice-cold 4% PFA in PBS overnight on a rotating platform at 4\u003csup\u003eo\u003c/sup\u003eC. Samples were then washed with ice-cold PBS and incubated overnight with 10% sucrose in PBS followed by 20% sucrose and 10% glycerol in PBS. For staining, 1cm-long intestinal pieces and mesenteries were washed with PBS, permeabilized with 0.5% Triton X-100 and blocked with 5% donkey serum overnight. Tissues were incubated with primary, followed by secondary antibodies overnight on a rotating platform at 4\u003csup\u003eo\u003c/sup\u003eC. Intestine samples were incubated overnight with Histodenz at RT. All samples were mounted in Histodenz with DAPI. A list of antibodies used in this study is provided in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eAdult mouse retina collection and staining\u003c/h2\u003e\n \u003cp\u003eTissues were fixed with 4% PFA for 1 hour at RT, washed with ice-cold PBS and permeabilized with 0.5% Triton X-100 and blocked with 5% donkey serum overnight on a rotating platform at 4\u003csup\u003eo\u003c/sup\u003eC. Tissues were incubated with primary antibodies followed by secondary antibodies overnight on a rotating platform at 4\u003csup\u003eo\u003c/sup\u003eC. Retinas were mounted in Fluoromount-G (Southern Biotech, #0100-01) with DAPI.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003ePostnatal P5 mouse retina collection and staining\u003c/h2\u003e\n \u003cp\u003eRetina immunostaining was performed as previously described with minor modifications\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. Tissues were collected and fixed in 4% PFA in PBS. Dissected retinas were incubated in blocking buffer (1% BSA, 0.3% Triton X-100 in PBS) for 2 hours at 4\u003csup\u003eo\u003c/sup\u003eC, rinsed with modified Pblec buffer (1 mM CaCl2, 1 mM MgCl2, 0.1 mM MnCl2, 0.1% Triton X-100 in PBS), and incubated with primary antibodies diluted in modified Pblec buffer overnight at 4\u0026deg;C. After washing in blocking buffer diluted 1:1 with PBS and 3 washes in PBS, retinas were incubated for 1 hour at RT with secondary antibodies diluted in blocking buffer. Retinas were then washed, refixed with 4% PFA for 20 min at RT and washed twice with PBS prior to mounting with Fluoromount-G (Southern Biotech, #0100-01).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eStaining of intestine cryosections\u003c/h2\u003e\n \u003cp\u003eTissues were fixed with 4% PFA in PBS overnight, washed with ice-cold PBS and incubated overnight with 30% sucrose in PBS. Intestines were embedded in OCT (Leica Biosystems, #14020108926) and kept at -80\u0026deg;C until sectioning. 60\u0026micro;m cryosections were thawed, fixed with 4% PFA in PBS for 5 min, washed in PBS, permeabilized with 0.5% Triton X-100 in PBS and blocked with 5% donkey serum for 30 min. Samples were incubated with primary antibodies in blocking buffer overnight at 4\u0026deg;C. Slides were then washed and incubated with secondary antibodies for 1 hour at RT. Slides were washed and mounted in Fluoromount-G (Southern Biotech, #0100-01) with DAPI.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eRNAscope analysis of tissue cryosections\u003c/h2\u003e\n \u003cp\u003eTissues were fixed with 10% Neutral-Buffered Formalin (NBF) for 24 hours at RT, washed with ice-cold PBS and incubated overnight with 30% sucrose in PBS. Mesenteries and intestines were embedded in OCT (Leica Biosystems, #14020108926) and kept at -80\u0026deg;C until sectioning. For \u003cem\u003eVegfc\u003c/em\u003e mRNA detection, samples were processed according to the protocol provided by Advanced Cell Diagnostics (ACD, RNAcope Multiplex Fluorescent v2 Assay combined with Immunofluorescence). Briefly, 22\u0026micro;m cryosections were thawed, fixed with 10% NBF and dehydrated with 50%, 70% and 100% EtOH in distilled water. Samples were then treated with RNAscope hydrogen peroxide and transferred in antigen retrieval buffer before the incubation with primary antibodies overnight at 4\u0026deg;C. The next day, RNAscope Multiplex fluorescent v2 assay was performed using \u003cem\u003eVegfc\u003c/em\u003e and control probes (see Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). To detect primary antibodies, Alexa-fluor secondary antibodies diluted in co-detection antibody diluent were added for 40 min at RT. Slides were incubated with DAPI and mounted in Fluoromount-G.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eImage acquisition and analysis\u003c/h2\u003e\n \u003cp\u003eAll images were captured using Leica SP8 or Zeiss LSM 980 confocal microscopes, and analyzed using Imaris, ImageJ and Photoshop softwares. All images of whole-mount mesenteric staining are shown in the same orientation, i.e. intestine at the bottom of the image.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eCell isolation and scRNA-seq experiments\u003c/h2\u003e\n \u003cp\u003eFor all scRNA-seq experiments, \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e and \u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e E18.0 embryos (injected with 2 mg 4-OHT at E17.0) were dissected in ice-cold PBS. The small intestines and mesenteries were separated, and the pancreas and mesenteric lymph node were removed. The small intestines were cut open, and both tissues were thoroughly washed in ice-cold PBS. Mesenteries and intestines were then transferred in FACS complete medium (Phenol red-free DMEM with 5% FBS). Digestion was performed in PBS 0.1% BSA comprising 0.25 mg/ml Liberase DH (Roche, #05401054001) and 0.08 mg/ml DNaseI (Sigma, #DN25) at 37\u0026deg;C for 30 min for mesenteries and 1 hour for intestines. Digestion was completely stopped by adding FACS complete medium to the cell suspensions. Cells were subsequently filtered through a 40 \u0026micro;m cell strainer (Falcon, #352340) in FACS complete medium and centrifuged for 3 min at 4\u0026deg;C.\u003c/p\u003e\n \u003cp\u003eFor total mesenteric and small intestine cells, pellets were resuspended in red blood cell (RBC) lysis buffer (Sigma, #R7757) for 3 min at RT, and washed with FACS complete medium. Each cell suspension was directly loaded onto a microwell cartridge of the BD Rhapsody Express system (BD Biosciences, #400000847) and libraries were prepared using the BD Rhapsody WTA Reagent kit (BD Biosciences, #633802) following the manufacturer\u0026rsquo;s instructions. scRNA-seq libraries were evaluated and quantified by Agilent Bioanalyzer using High sensitivity DNA kit (#5067\u0026thinsp;\u0026minus;\u0026thinsp;4626) and Qubit (ThermoFisher Scientific, #Q32851). Individual libraries were pooled, diluted to 4 nM and sequenced by using NextSeq 500/550 High Output kit (150 cycle, Illumina,) with a NextSeq500 sequencer (Illumina).\u003c/p\u003e\n \u003cp\u003eFor fluorescence-activated cell sorting (FACS) of intestinal ECs, cell pellets were incubated with Fc block (antibody to CD16/32; BD Biosciences, #553142) for 10 min on ice and stained with conjugated antibodies in FACS buffer for 30 min (see Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). All GFP\u003csup\u003e+\u003c/sup\u003e and GFP\u003csup\u003e\u0026minus;\u003c/sup\u003e ECs were individually sorted using a FACSAria Fusion (BD Biosciences). GFP\u003csup\u003e+\u003c/sup\u003e ECs (around 9000 cells) were complemented with GFP\u003csup\u003e\u0026minus;\u003c/sup\u003e ECs to reach a total of 40000 cells and further processed for scRNA-seq library preparation and sequencing as described for total intestinal and mesenteric cells.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003escRNA-seq Preprocessing\u003c/h2\u003e\n \u003cp\u003eRaw FASTQ reads were quality and adapter trimmed using TrimGalore! (version 0.6.4 length cutoff 66, quality cutoff 20). The UMI, complex barcode, and sample tags were extracted and demultiplexed using custom scripts (\u0026lt;\u0026thinsp;TODO add Git repo\u0026thinsp;\u0026gt;\u0026thinsp;rhapsody-extract-barcode and rhapsody-demultiplex).\u003c/p\u003e\n \u003cp\u003eReads were mapped to the GRCm38 reference genome, \u003cem\u003eEsm1-CreERT2\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e, and \u003cem\u003eR26-mTmG\u003c/em\u003e\u003csup\u003etg/+\u003c/sup\u003e construct sequences with Gencode annotations vM22, using STAR version 2.7.3a\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e (--soloType CB_UMI_Simple \u0026ndash;soloCellFilter None \u0026ndash;soloFeatures Gene \u0026ndash;soloCBstart 1 \u0026ndash;soloCBlen 27 \u0026ndash;soloUMIstart 28 \u0026ndash;soloUMIlen 8 \u0026ndash;outFilterMultimapNmax 1 \u0026ndash;soloCBwhitelist\u0026thinsp;\u0026lt;\u0026thinsp;TODO rhapsody_whitelist.txt\u0026gt;).\u003c/p\u003e\n \u003cp\u003eRaw counts were imported as AnnData\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e objects. We removed low complexity barcodes with the knee plot method, and further filtered out cells with a total contribution above 20% of reads belonging to mitochondrial mRNA. Doublets were predicted with scrublet\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e and cells with a doublet score above 0.1 have been removed. Finally, each sample\u0026rsquo;s gene expression matrix was normalized using scran (1.22.1)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e with Leiden clustering\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e input at resolution 0.5.\u003c/p\u003e\n \u003cp\u003eG2/M and S phase scores were assigned to each cell using gene lists from\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e and the scanpy (1.8.2)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e sc.tl.score_genes_cell_cycle function.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003escRNA-seq Embedding, clustering and annotation\u003c/h2\u003e\n \u003cp\u003eAt this stage, samples were merged. For 2D embedding, the expression matrix was subset to the 2,000 most highly variable genes (sc.pp.highly_variable_genes, flavor \u0026ldquo;seurat\u0026rdquo;). The top 50 principal components (PCs) were calculated, and batch-corrected using Harmony (0.0.5)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. The PCs served as basis for k-nearest neighbor calculation (sc.pp.neighbors, n_neighbors\u0026thinsp;=\u0026thinsp;30), which were used as input for UMAP\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e layout (sc.tl.umap, min_dist\u0026thinsp;=\u0026thinsp;0.3).\u003c/p\u003e\n \u003cp\u003eKnown marker genes were plotted using Scanpy (1.7.1) scanpy.pl.dotplot, cell populations were clustered using scanpy.tl.leiden at resolution 0.1 for annotation. The endothelial cell population was subclustered separately at Leiden resolution 0.9, and annoated using known marker genes.\u003c/p\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003escRNA-seq trajectory and pseudotime analyses\u003c/h2\u003e\n \u003cp\u003ePAGA\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e from the Scanpy package was used to calculate non-mitotic EC population connectivities, and determine trajectories. Based on PAGA and Esm1 lineage tracing information, we calculated diffusion pseudotime\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e using scanpy.tl.dpt, choosing the Venous EC 1 population as starting cluster.\u003c/p\u003e\n \u003cp\u003eExpression values for all genes with a minimum average normalised expression larger than 0.3 to reduce noise were binned into 200 bins according to pseudotime. The resulting z-transformed matrix was clustered hierarchically (scipy 1.10.0 scipy.cluster.hierarchical, Ward linkage, Euclidian distance metric) to identify gene expression patterns. Clusters were obtained with a tree distance cutoff of 60 in the dendrogram. Profiles for select clusters were plotted along binned pseudotime, celltype annotations for each bin were determined by majority vote. In profile plots for individual genes, the confidence interval of expression values is plotted as grey background (1.96*SD(expr)/sqrt(n)).\u003c/p\u003e\n \u003cp\u003eEnrichr\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e98\u003c/span\u003e\u003c/sup\u003e was used to calculate Gene Ontoloy (BP, CC) enrichment on the expression profile cluster containing Esm1.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003eIntegration with P6 retinal endothelial tip cells\u003c/h2\u003e\n \u003cp\u003eFor the comparison of Esm1\u003csup\u003e+\u003c/sup\u003e cells from the intestinal villus with sprouting retinal tip cells, we reprocessed the publicly available single-cell data from Zarkada et al. 2021. Raw FASTQ files from P6 WT were processed analogously to the intestine and msentery samples above. Divergent STARsolo options were \u0026ldquo;--soloType Droplet --soloCBwhitelist 10xv3_whitelist.txt --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 10\u0026rdquo;. Mitochondrial mRNA content cutoff was set to 10%. AnnData objects were merged (outer join) with intestine and mesentery data using Scanpy concatenate. The merged data was reclustered at Leiden resolution 0.05 to identify ECs, the EC population was then further subclustered at resolution 1.0 to identify and remove contaminants and doublets. ECs from the Zarkada et al. data were subclustered at resolution 0.7, and the Esm1\u003csup\u003e+\u003c/sup\u003e cluster was identified and annotated accordingly. Differential expression was performed on Esm1\u003csup\u003e+\u003c/sup\u003e clusters from the original and Zarkada et al. data using a pseudobulk approach based on pyDESeq2\u003csup\u003e99\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003eQuantifications and statistics\u003c/h2\u003e\n \u003cp\u003eFor whole-mount mesenteric artery and lymphatic vessel analysis, mesenteric tissue (DAPI), artery (CD31\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e) and lymphatic vessel (PROX1\u003csup\u003e+\u003c/sup\u003e) areas and artery length were quantified using Photoshop and ImageJ softwares. In lineage tracing experiments, GFP\u003csup\u003e+\u003c/sup\u003e cells in the mesenteric arteries were manually counted and in long-term loss-of-function experiments, GFP\u003csup\u003e+\u003c/sup\u003e area in mesenteric arteries was quantified using Photoshop and ImageJ. For whole-mount intestinal tissue analysis, GFP\u003csup\u003e+\u003c/sup\u003e, SOX17\u003csup\u003e+\u003c/sup\u003e and intestinal tissue area were quantified using Photoshop and ImageJ softwares. For large mesenteric artery and intestinal submucosal artery analysis, at least 3 high magnification images were taken per sample. CD31\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e artery area, length, GFP\u003csup\u003e+\u003c/sup\u003e area and nuclear density were quantified using Photoshop and ImageJ softwares. GFP\u003csup\u003e+\u003c/sup\u003e cells with changes in morphology (elongated \u003cem\u003evs\u003c/em\u003e round) were counted using Photoshop and ImageJ softwares.\u003c/p\u003e\n \u003cp\u003eIn the embryonic intestinal villus lineage tracing experiments, GFP\u003csup\u003e+\u003c/sup\u003e area in CD31\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e+\u003c/sup\u003e and CD31\u003csup\u003e+\u003c/sup\u003e SOX17\u003csup\u003e-\u003c/sup\u003e areas were quantified using Photoshop and ImageJ softwares. For villus blood capillary length and width, images were directly quantified in 3D using Imaris software. For villus blood and lymphatic vessel, ERG\u003csup\u003e+\u003c/sup\u003e nucleus, SOX17\u003csup\u003e+\u003c/sup\u003e nucleus and GFP\u003csup\u003e+\u003c/sup\u003e 3D volume and intensity analysis, images were quantified using Imaris software. In \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003e\u003cem\u003eiEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e, \u003cem\u003eItgb1\u003c/em\u003e\u003csup\u003e\u003cem\u003eiEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e and control littermates, 6\u0026ndash;13 villi per sample were analyzed. In \u003cem\u003eVegfc\u003c/em\u003e\u003csup\u003e\u003cem\u003eiBmx\u003c/em\u003eKO\u003c/sup\u003e and control littermates, 12\u0026ndash;29 villi per sample were analyzed. Villus artery diameter and proportion of villus with more than one SOX17\u003csup\u003e+\u003c/sup\u003e branch was quantified using Imaris, Photoshop and ImageJ softwares. In MAZ51-treated and DMSO control embryo experiments, 4\u0026ndash;5 images comprising around 3 villi each were analyzed. In \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003eiEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eKO\u003c/sup\u003e, \u003cem\u003eFlt4\u003c/em\u003e\u003csup\u003e\u003cem\u003eiEsm\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/em\u003eHET\u003c/sup\u003e and control littermates, 16\u0026ndash;31 villi per sample were analyzed. For Esm1, LAMA4, COLIV, VEGFR3 and CD31 intensity quantifications, 7\u0026ndash;15 villi per sample were analyzed. Each villus was divided into top and bottom villus capillary network area. In both top and bottom areas, vessel volume and staining intensities were quantified using Imaris software. For proliferation analyses, cells were double-stained for ERG and/or SOX17 and EdU. For villi from embryonic intestines, 6\u0026ndash;9 high magnification images were analyzed. For embryonic mesenteries, 3 mesenteric artery branches per sample were imaged and analyzed. Double ERG and EdU masks were generated using Imaris software and cells were quantified using Photoshop and ImageJ softwares, counting as positive only cells in which all ERG\u003csup\u003e+\u003c/sup\u003e area was EdU\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eFor ESM1\u003csup\u003e+\u003c/sup\u003e area quantification in adult villi, 5 images per animals comprising around 3 villi each were quantified. CAV1\u003csup\u003e+\u003c/sup\u003e Esm1\u003csup\u003e+\u003c/sup\u003e and CAV1\u003csup\u003e+\u003c/sup\u003e EMCN\u003csup\u003e+\u003c/sup\u003e Esm1\u003csup\u003e+\u003c/sup\u003e masks were generated using Imaris software and areas were quantified using Photoshop and ImageJ softwares. For GFP\u003csup\u003e+\u003c/sup\u003e area quantification in EMCN\u003csup\u003e+\u003c/sup\u003e and EMCN\u003csup\u003e-\u003c/sup\u003e of the adult villus, 9\u0026ndash;12 images comprising around 3 villi each were analyzed. VEGFR2\u003csup\u003e+\u003c/sup\u003e GFP\u003csup\u003e+\u003c/sup\u003e and VEGFR2\u003csup\u003e+\u003c/sup\u003e EMCN\u003csup\u003e+\u003c/sup\u003e GFP\u003csup\u003e+\u003c/sup\u003e masks were generated using Imaris software and areas were quantified using Photoshop and ImageJ softwares.\u003c/p\u003e\n \u003cp\u003eThe number of embryos analyzed is indicated for each image in figure legends. Differences were considered statistically significant at\u0026nbsp;\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePrimary and secondary antibodies and dyes used for stainings\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAntigen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReactivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003ePrimary antibodies\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCaveolin-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse/Rat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell Signaling (#3238)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse/Rat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D Systems (#AF3628)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbcam (#ab28364)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBD Pharmingen (#553370)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCollagen IV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChemicon (#AB756P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDll4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D (#AF1389)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eE-cadherin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCell Signaling (#3195)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEndomucin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSanta Cruz (#SC-65495)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eErg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbcam (#ab110639)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEsm1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D (#AF1999)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFabp4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbcam (#ab13979)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChicken\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2BScientific Ltd (#GFP-1010)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIB4 - Biotinylated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse/Rat/\u003c/p\u003e\n \u003cp\u003eRabbit/Goat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGriffonia simplicifolia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVector (#B-1205)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIntegrin b1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBD Pharmingen (#553715)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLaminin \u0026alpha;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSerum 377 (Gift from L. Sorokin)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLyve1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D (#MAB2125)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMAdCAM1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAbcam (#ab80680)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProx1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eReliaTech (#102-PA32AG)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026alpha;SMA - Cy3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse/Rat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSigma (#C6198)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026alpha;SMA - eFluor660\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman/Mouse/Rat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eeBioscience (#50-9760-82)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSox17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D (#AF1924)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVegfr2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D (#AF644)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVegfr3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u0026amp;D (#AF743)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eSecondary antibodies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlexa 405-conjugated\u003c/p\u003e\n \u003cp\u003eStreptavidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvitrogen (#S32351)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlexa 488\u0026ndash;conjugated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChicken\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonkey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJackson Laboratories (#703-545-155)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlexa 488\u0026ndash;conjugated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat/Rabbit/\u003c/p\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonkey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvitrogen (#A11055, #A21206, #A21208)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlexa 594\u0026ndash;conjugated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat/Rabbit/\u003c/p\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonkey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvitrogen (#A11058, #A21207, #A21209)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlexa 647\u0026ndash;conjugated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGoat/Rabbit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonkey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvitrogen (#A21447, #A31573)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlexa 647\u0026ndash;conjugated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDonkey\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eJackson ImmunoResearch (#712-605-153)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eDyes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSigma (#D9542)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClick-It EdU Alexa Fluor 647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eInvitrogen (#C10340)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eFACS-sorting antibodies\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAntigen\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReactivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecie\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eFACS antibodies\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD31-BV711\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBD Biosciences (#740680)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCD45- BV421\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiolegend (#103134)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEpcam - PE-Cy7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBioLegend (#118216)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePodoplanin - eFluor660\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMouse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHamster\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eeBioscience (#50-5381)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eRNAscope reagents\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReagents\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eRNAscope Multiplex Fluorescent Reagent Kit v2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACD (#323100)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eRNA-Protein Co-Detection Ancillary Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACD (#323180)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eRNAscope 3-plex Positive Control Probe - Mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACD (#320881)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eRNAscope 3-plex Negative Control Probe - Mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACD (#320871)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eRNAscope Probe - Mm - Vegfc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eACD (#492701)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eTSA Plus Fluorescein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAkoya Biosciences (#NEL741001KT)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scRNA-seq data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession no. GSE275961.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Pr. Mark L. Kahn for \u003cem\u003eFlt4\u003csup\u003e\u0026nbsp;lox/lox\u003c/sup\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Vegfc\u003csup\u003e\u0026nbsp;lox/lox\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emice and Dr. Martin Stehling and Dr. Bong-Ihn Koh\u0026nbsp;for FACS analysis and sorting experiments. We thank Dr. Kishor K. Sivaraj for sharing \u003cem\u003eHey1-eGFP\u003c/em\u003e mice and Dr. Hongryeol Park for sharing \u003cem\u003eAplnr-CreERT2\u003c/em\u003e mice. For sharing RNAscope protocols, reagents and discussions, we would like to thank Dr. Cl\u0026eacute;\u0026shy;mentine Vil\u0026shy;len\u0026shy;euve and Claudia Ortmeier from the Wickstr\u0026ouml;m\u0026rsquo;s department of the Max Planck Institute of M\u0026uuml;nster\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eWe also thank Dr. Rodrigo Di\u0026eacute;guez-Hurtado, Frank Berkenfeld and Silke Schr\u0026ouml;der for discussions and mouse colony maintenance. Animal, FACS, Genotyping, BioOptic and Bioinformatics facilities of the Max Planck Institute of M\u0026uuml;nster are gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Max Planck Society (R.H.A.), the DFG Collaborative Research Center 1348 Project A10 (R.H.A., M.E.P., E.B.), the Leducq Foundation (R.H.A.) and the Cells in Motion (CiM) graduate school (V.M).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.B. and R.H.A. designed the study. E.B. performed the majority of the experiments. E.C.W. and E.B. performed the single-cell sequencing, and K.K. performed the bioinformatic transcriptomic analysis. V.M. generated samples for RNAscope. M.S. helped set up staining for FACS and perfomed FACS-sorting experiment. M.E.P. and F.B. generated P5 retina results. M.L.K provided critical mouse models and information for RNAscope experiments. 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Exp Cell Res 246:165\u0026ndash;182\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Endothelial cells, intestine, mesentery, artery development, single-cell RNA sequencing.","lastPublishedDoi":"10.21203/rs.3.rs-5411147/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5411147/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArterial blood transport into peripheral organs is indispensable for developmental growth, homeostasis and tissue repair. While it is appreciated that defective formation or compromised function of arteries is associated with a range of human diseases, the cellular and molecular mechanisms mediating arterial development remain little understood for most organs. Here, we show with genetic approaches that a small subpopulation of endothelial cells inside the intestinal villi of the embryonic mouse, characterized by the expression of endothelial cell-specific molecule 1 (Esm1/endocan), gives rise to arterial endothelium in the intestinal wall but also in the distant mesenteric vasculature. This involves cell migration but also substantial changes in morphology and gene expression. Immunohistochemistry and single cell RNA-sequencing confirm that intestinal Esm1\u003csup\u003e+\u003c/sup\u003e cells have a distinct molecular profile and the capacity to undergo arterial differentiation. Genetic approaches establish that artery formation by the progeny of Esm1\u003csup\u003e+\u003c/sup\u003e cells requires integrin β1 and signaling by the growth factor VEGF-C and its receptor VEGFR3. The sum of these findings demonstrates that Esm1\u003csup\u003e+\u003c/sup\u003e cells inside the villus capillary network contribute to the formation of intestinal and mesenteric arteries during development.\u003c/p\u003e","manuscriptTitle":"Artery formation in intestinal wall and mesentery by intestine-derived Esm1+ endothelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-14 16:38:58","doi":"10.21203/rs.3.rs-5411147/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"048969bd-40ad-4909-976a-338e2e512f52","owner":[],"postedDate":"November 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40226564,"name":"Biological sciences/Developmental biology/Angiogenesis"},{"id":40226565,"name":"Biological sciences/Developmental biology/Experimental organisms/Model vertebrates"},{"id":40226566,"name":"Biological sciences/Developmental biology/Embryogenesis"}],"tags":[],"updatedAt":"2025-09-26T07:09:07+00:00","versionOfRecord":{"articleIdentity":"rs-5411147","link":"https://doi.org/10.1038/s41467-025-64047-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-09-25 04:00:00","publishedOnDateReadable":"September 25th, 2025"},"versionCreatedAt":"2024-11-14 16:38:58","video":"","vorDoi":"10.1038/s41467-025-64047-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-64047-0","workflowStages":[]},"version":"v1","identity":"rs-5411147","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5411147","identity":"rs-5411147","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}