Pericytes are organ-specific regulators of tissue morphogenesis

Pericytes are organ-specific regulators of tissue morphogenesis | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Pericytes are organ-specific regulators of tissue morphogenesis Ralf Adams, Seyed Javad Rasouli, Kai Kruse, Rodrigo Diéguez-Hurtado, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5787386/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 May, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Blood vessels provide a versatile and adaptable transport system, but recent work has established that endothelial cells, which form the innermost lining of the vascular network, are also a source of molecular signals controlling the behavior of other cell types in the surrounding tissue. Pericytes are another essential component of the vessel wall, but comparably little is known about their signaling interactions with other cell populations during organ growth and patterning. Here, we have used tissue-specific and inducible mouse genetics, high-resolution imaging, single-cell RNA sequencing and cell culture experiments to address the function of three pericyte-derived growth factors in the postnatal development of two model organs, namely lung and brain. We found that Pdgfrb-CreERT2 -controlled inactivation of the gene for hepatocyte growth factor (HGF) causes no overt alterations in the postnatal brain but impairs alveologenesis in the lung due to defective interaction with AT2 epithelial cells. Likewise, expression of brain-derived neurotrophic factor (BDNF) by pericytes is not required in the postnatal brain but controls lung development through interactions with the receptor tyrosine kinase TrkB in the pulmonary endothelium. Conversely, pericyte expression of the TGFβ family growth factor Nodal is not required for lung morphogenesis but regulates blood vessel growth and barrier function in the postnatal brain, which we attribute to signaling interactions with endothelial cells, astrocytes and microglia. Taken together, our findings establish that pericytes are a critical source of angiocrine signals that control morphogenetic processes in an organ-specific fashion. Biological sciences/Developmental biology/Angiogenesis Biological sciences/Developmental biology/Morphogenesis Biological sciences/Developmental biology/Organogenesis pericytes mural cells lung brain angiocrine signaling HGF BDNF Nodal Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Blood vessels form a highly elaborate, hierarchical network that reaches, with a few exceptions, into all parts of the vertebrate body. In addition to the essential transport function of the vasculature, endothelial cells provide critical molecular signals acting on other cell populations in their vicinity. This paracrine (also termed ‘angiocrine’) function of endothelial cells regulates morphogenesis, homeostasis and regeneration of different organs including lung, liver, heart, and bone 1 , 2 . Endothelial cells (ECs) are also a critical component of stem cell niches and thereby play key roles in hematopoiesis, bone formation, and neurogenesis 3 , 4 . Thus, ECs have emerged as important signaling centers that acquire organ-specific specialization, coordinate regeneration and help to prevent deregulated, disease-promoting processes. Even though the range of EC-derived signals has been explored only partially, it is evident that the angiocrine function of capillary EC is reflected by specialized expression of certain genes, distinguishing different organs but also local environments within the same organ 5 – 8 . Pericytes, capillary-associated supporting cells, are another essential component of the vessel wall and help to preserve vascular integrity 9 , 10 . Pericytes but also the other mural cell population, vascular smooth muscle cells (vSMCs), are fairly heterogeneous with regard to their morphology but also developmental origin. Pericytes and vSMCs in the developing heart, for example, can either arise from epicardial mesothelial cells or, as we have shown, via endothelial-to-mesenchymal transition from the embryonic endocardium 11 , 12 . Neural crest cells give rise to pericytes in the retina, brain, thymus, and the head region 13 – 16 . In gut, lung and liver, the mesothelium, a single-layer squamous epithelium, is a source of mural cells 17 , 18 . Distinct developmental origins and tissue environments may also explain the expression of specific molecular markers by pericytes in different organs, including a range of secreted molecules, as was uncovered by single cell RNA-sequencing (scRNA-seq) and other approaches 19 , 20 . Likewise, scRNA-seq has revealed substantial organ-specific gene expression profiles of pericytes and other vessel-associated cells in adult brain and lung 21 . Our own previous work has established that pulmonary pericytes control the proliferation of epithelial cells and are thereby indispensable for alveologenesis during postnatal lung development 22 . Stimulated by the findings above, we have utilized scRNA-seq in combination with mouse genetics and cell culture approaches to explore whether pericytes act as organ-specific regulators of tissue morphogenesis during postnatal development. We chose lung and brain as representative model organs and explored the function of several pericyte-derived, potentially angiocrine-acting factors, namely brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and Nodal, a TGFβ family protein. Our findings establish that pericytes are indeed functionally specialized in an organ-specific fashion and regulate the behavior of other cell types in their vicinity through the secretion of angiocrine signals. Results In vivo characterization of lung and brain pericytes. To investigate pericyte morphology in lung and brain, we employed a genetic labeling strategy involving Pdgfrb-CreERT2 transgenic mice 12 , 23 in combination with the R26-mT/mG Cre reporter 24 . Following the administration of a low dose of 4-hydroxytamoxifen (4-OHT) at postnatal day (P) 1, Cre-mediated recombination and irreversible expression of green fluorescent protein (GFP) labelled a limited number of mural cells in different postnatal organs. Analysis of GFP + cells by confocal microscopy at high resolution (Fig. 1 a-c) revealed substantial differences in pericyte abundance and morphology. GFP + pericytes in the postnatal brain cortex at P12 extend numerous short cellular protrusions and are densely covering capillaries (Fig. 1 a). Moreover, Pdgfrb-CreERT2 -labeled brain pericytes are located in close proximity to astrocytes expressing Glial fibrillary acidic protein (GFAP) and microglia expressing Allograft inflammatory factor 1 (AIF1) (Fig. 1 b). In contrast, lung pericytes exhibit a different morphology, are comparably sparse and extend long cellular processes contacting ECs (Fig. 1 c). In both brain and lung, there is good colocalization between GFP signal and PDGFRβ immunostaining, which is most prominent on the cell body (Fig. 1 a, c). In the lung, very little GFP signal is seen outside of the vasculature, consistent with previous work showing that activation of Pdgfrb-CreERT2 -mediated recombination with tamoxifen administration at P1–P3 does not lead to labeling of PDGFRα + fibroblasts, alpha smooth muscle actin (αSMA) + bronchial smooth muscle cells or myofibroblasts 22 . In addition, PDGFRβ immunostaining of pericytes is predominantly confined to the alveolar septum, the tissue between apposing alveolar walls (Fig. 1 d, f). Immunostaining indicates that alveolar type 2 (AT2) epithelial cells expressing Prosurfactant Protein C (proSP-C), which act as stem cells in the pulmonary epithelium and give rise to terminally differentiated AT1 cells 25 , 26 , are also enriched in the septum region (Fig. 1 e, f). As previous work has uncovered evidence for the expression of organ-specific markers both in mice and humans 19 , 27 , 28 , we aimed at the identification of pericyte-derived, paracrine-acting factors that might potentially control morphogenetic processes during postnatal development. Comparing a public scRNA-seq resource for mouse lung provided by the Thébaud laboratory 29 with our own scRNA-seq data, which is introduced later in the course of this article, indicates that pericytes from wild-type postnatal lung and brain express the expected general mural cell markers, namely Pdgfrb (encoding Platelet-derived growth factor receptor β, PDGFRβ), Cspg4 (Chondroitin Sulfate Proteoglycan 4), and Notch3 , which encodes a Notch family receptor (Fig. 1 g). In addition, pericytes from lung and brain show organ-specific gene expression. In particular, we identified several differentially expressed transcripts for secreted factors. Hgf (encoding hepatocyte growth factor) and Bdnf (brain-derived neurotrophic factor), a regulator of axon guidance through its receptor TrkB/Ntrk2 30,31 , are found in pericytes of the lung but are not detectable in brain (Fig. 1 g). Angpt1 (Angiopoietin 1) an important regulator of vascular growth and integrity, shows a similar distribution pattern of higher expression in lung relative to brain pericytes, which is consistent with previous work 22 and published scRNA-seq results from adult lung and brain 21 , 32 , 33 . In turn, Nodal , a member of the TGFβ superfamily, is enriched in brain pericytes relative to lung (Fig. 1 g). Based on these results, we used an inducible genetic strategy involving the Pdgfrb-CreERT2 line in combination with loxP-flanked alleles for three candidate genes, namely Hgf , Bdnf and Nodal , to investigate the role of pericyte-derived signals in organ development. Pericyte-derived BDNF controls pulmonary angiogenesis and development To investigate the function of Bdnf in pulmonary pericytes in mice, conditional Bdnf alleles were introduced into the Pdgfrb-CreERT2 background. Following tamoxifen administration at P1-3, ICAM2 immunostaining shows that vascularization is reduced in P21 lungs of the resulting Bdnf iPCKO mutants relative to littermate controls (Fig. 2 a, f). The number of ECs visualized by nuclear ERG immunostaining is decreased (Fig. 2 b, g), whereas the ratio of ERG + to PDGFRβ + cells in Bdnf iPCKO lungs is not significantly changed (Fig. 2 c, h). Immunostaining for NKX2.1 (a transcription factor expressed by both AT1 and AT2 cells) and LAMP3 (an AT2 cell marker) shows that number of AT1 and AT2 epithelial cells is also decreased in P21 Bdnf iPCKO lungs (Fig. 2 d, i). Likewise, lung immunostaining for the proliferation marker Ki67 antibody shows a significant reduction of dividing cells in Bdnf iPCKO lungs compared to controls ( Fig. 2 e, j ) . Bioinformatic analysis of scRNA-seq data from P21 Bdnf iPCKO and littermate control lungs shows significant changes in gene expression in the mutant endothelium (Supplementary Fig. 1a-c). Analysis of scRNA-seq results reveals reduced expression of multiple endothelial markers including Plvap , (encoding plasmalemma vesicle associated protein, associated with EC permeability), Gja5 (encoding Gap Junction Protein Alpha 5 protein), Itga1 (encoding Integrin Subunit Alpha 1), and Aplnr (Apelin receptor) together with a reduction in the proliferation markers Ccnd1 (Cyclin D1), Ccna2 (Cyclin A2) and Mki67 (Ki67) in mutant lungs (Supplementary Fig. 1c, d) . Arguing that pericyte-derived BDNF might act on ECs, the receptor tyrosine kinase TrkB, a high affinity receptor of BDNF encoded by the gene Ntrk2 34 , is expressed by ICAM2 + ECs in P21 lungs (Fig. 2 k). Similarly, scRNA-seq data shows that Ntrk2 is predominantly expressed by general capillary (gCap) ECs but also by mesenchymal cells in P21 lung (Fig. 2 l, m and Supplementary Fig. 1e ). TrkB phosphorylation at tyrosine residue 515 (Tyr515), an indicator of receptor activation, in substantially reduced in lysates from P21 Bdnf iPCKO lungs relative to control samples (Fig. 2 n). Given that vascularization is a prerequisite for normal pulmonary epithelial morphogenesis 35 and because of the endothelial Ntrk2 expression (Fig. 2 k, m), we used a conditional gene targeting strategy to study the function of the BDNF receptor in ECs. Cdh5-CreERT2- controlled inactivation of Ntrk2 , induced by tamoxifen administration at P1-3, leads to the reduction of ICAM2 + area, ERG + cells (Fig. 2 o, q, r) and proliferative cells (Fig. 2 p, s) in the resulting Ntrk2 iΔEC mutant lungs at P21. However, the ratio ECs to pericytes is comparable in Ntrk2 iΔEC and littermate control lungs (Fig. 2 t), consistent with the findings in Bdnf iPCKO mutants. Overall, these data indicate that pericyte-derived BDNF controls TrkB signaling in ECs and thereby pulmonary angiogenesis, which, in turn, is indispensable for lung development. These findings are consistent with earlier studies proposing roles of BDNF-TrkB signaling in the regulation of integrin or PI3K/Akt-mediated EC migration as well as cell survival 34 , 36 , 37 . Pericyte-derived BDNF or HGF are dispensable in the postnatal brain Quantitative analysis of the ICAM2 + vascular area and number of proliferating ECs (ICAM2+, EdU+) in the P12 Bdnf iPCKO brain shows no significant differences relative to littermate controls ( Supplementary Fig. 2a-c ). In addition, pericyte coverage, visualized as vessel-associated PDGFRβ + immunostaining, remains unaltered in the postnatal Bdnf iPCKO brain ( Supplementary Fig. 2d, e ). We also did not observe any changes in collagen IV expression, which is a major constituent of the vascular basement membrane 38 ( Supplementary Fig. 2f ). Impairment of the blood-brain barrier (BBB) can induce neuronal injury and neuroinflammation 39 . Double immunostaining for blood-derived immunglobulin G (IgG) shows that signal is absent within the brain parenchyma, whereas IgG is readily detectable inside the vasculature ( Supplementary Fig. 2g ). Moreover, confocal images of ICAM2 + cortical blood vessels show no obvious defects in Aquaporin-4-expressing (AQP4+) astrocyte endfeet, indicating that this key component of the neurovascular unit is maintained in the Bdnf iPCKO brain vasculature ( Supplementary Fig. 2h ). Reactive astrogliosis, a typical response to CNS injury 40 , is also absent, as indicated by immunostaining for GFAP. Likewise, immunostaining for AIF1 is comparable in Bdnf iPCKO and littermate controls, indicating that there is no activation of microglia in mutant brain ( Supplementary Fig. 2i, j ). Overall, these data show that Pdgfrb-CreERT2 -controlled inactivation of the Bdnf gene causes no overt alterations in the postnatal brain. A similar immunostaining analysis of brain sections from P12 Hgf iPCKO mutants, generated with the Pdgfrb-CreERT2 line and postnatal tamoxifen administration from P1-3, shows no obvious differences to control littermates. Quantitative analyses of vascular area, pericyte coverage and collagen type IV expression are comparable in the Hgf iPCKO and control brain vasculature ( Supplementary Fig. 3a-d ). Immunostaining also shows no noticeable differences in AQP4 + glial endfeet, GFAP + astrocytes or AIF1 expression by microglia ( Supplementary Fig. 3e-g ). Taken together, these results indicate that Pdgfrb-CreERT2 -controlled inactivation of Hgf or Bdnf leads to no detectable alterations in the postnatal brain, which is consistent with the absent or low expression of these two growth factors in brain pericytes. Alveolarization and lung morphogenesis requires pericyte-derived HGF Given the importance of HGF signaling and its c-Met receptor for lung development 22 , 41 – 43 and based on the expression of Hgf in pulmonary pericytes (Fig. 1 g), we investigated the role of pericyte-derived HGF. Immunostaining of P21 lung sections detects c-Met protein expression in LAMP3 + AT2 cells (Fig. 3 a). This is further supported by analysis of our scRNA-seq data, which shows enrichment of Met transcripts in AT2 cell clusters ( Supplementary Fig. 4a-f ). Following tamoxifen administration after birth, Pdgfrb-CreERT2- controlled inactivation of Hgf impairs alveologenesis in the resulting Hgf iPCKO mutants (Fig. 3 b). This phenotype is accompanied by significant reduction of AT2 epithelial cells as well as reduced AT2 cell proliferation, whereas apoptosis is not increased in Hgf iPCKO mutant lungs (Fig. 3 c-j). Interestingly, we observed hotspots with high levels of staining for RAGE (receptor for advanced glycation end products), a transmembrane pattern recognition receptor that has been linked to inflammatory processes in the lung 44 . The overall RAGE + AT1 epithelial area, however, is reduced in Hgf iPCKO lungs compared to littermate controls (Fig. 3 k, l), reflecting that the AT2 population serves as a progenitor pool for AT1 cells 25 . To investigate the cellular and molecular alterations in Hgf iPCKO animals in greater detail, we performed scRNA-seq analysis of P21 mutant and littermate control lung ( Supplementary Fig. 4a-g ). Bioinformatic analysis of the resulting data confirmed a reduction of AT2 cells expressing Met , and Lamp3 , Abca , Sftbp , and Tinag in Hgf iPCKO lungs (Fig. 3 m and Supplementary Fig. 4g ). Supporting our characterization of the mutant phenotype, Hgf iPCKO AT2 cells show reduced expression of genes associated with cell proliferation (Fig. 3 n). Furthermore, markers associated with differentiation into AT1 epithelium are also affected in the AT1-AT2 transitory population (Fig. 3 o). In addition to the epithelial defects, mice lacking pericyte-derived HGF show a significant decrease of PECAM1 + pulmonary endothelium relative to controls (Fig. 3 p, r), whereas the ratio of ECs to pericytes, measured by combined ERG and PDGFRβ staining, remains unchanged (Fig. 3 q, s). Given the absent or low expression of Met in endothelial cells ( Supplementary Fig. 4c ), the reduced vascularization could be secondary to the defects in alveolar epithelium observed in postnatal Hgf iPCKO lung. These data together with our previous study 22 confirm the importance of pericytes for AT2 epithelial cell proliferation and lung development. Pericyte-derived Nodal is not essential for postnatal lung development Postnatal Nodal iPCKO mutants were generated with Pdgfrb-CreERT2 mice using the same strategy as outlined above for Hgf and Bdnf . Confocal microscopy and PECAM1 immunostaining show no significant changes in the postnatal Nodal iPCKO pulmonary vasculature ( Supplementary Fig. 5a, f ). Likewise, combined ERG, PDGFRβ and DAPI staining indicate that the number of ECs, pericytes and the ratio of endothelial cells (ERG+) to pericytes (PDGFRβ+) are comparable in mutants and the corresponding littermate controls ( Supplementary Fig. 5b, g, h ). In addition, the lung epithelium, stained with RAGE and NKX2.1 ( Supplementary Fig. 5c, d, i ), is not altered in Nodal iPCKO mutants. The percentage of AT2 cells as well as the number of proliferating cells among total cells are not different in Nodal iPCKO and control littermate lungs ( Supplementary Fig. 5d, e, j, k ). Overall, these data indicate that Pdgfrb-CreERT2 -controlled inactivation of Nodal leads to no overt alterations in the postnatal lung, which is consistent with the absent or very low expression of this growth factor in pulmonary pericytes. Postnatal Nodal iPCKO mutants show reduced brain vascularization Nodal transcripts are prominently expressed of in brain pericytes (Fig. 1 g, Supplementary Fig. 6a-c ), which is further supported using a public scRNA-seq resource for adolescent mouse brain ( Supplementary Fig. 6d, e ) provided by the Linnarsson laboratory (mousebrain.org) 33 . Inducible inactivation of Nodal mediated by Pdgfrb-CreERT2 results in a reduced vascular network in the brain by P12 (Fig. 4 a, c). Microscopic and quantitative analysis reveals a decrease in vascular area together with a reduction of endothelial cells in Nodal iPCKO brains relative to littermate controls (Fig. 4 b, f). PDGFRβ + pericytes are present and associated with the brain capillary endothelium, and the ratio of ECs to pericytes remains unchanged (Fig. 4 d, g). Consistent with the reduction of Nodal iPCKO brain capillaries, EdU administration reveals a significant decrease in EC proliferation (Fig. 4 e, h). In addition, confocal images of the Nodal iPCKO brain cortex reveal small, isolated areas of dextran 70 kDa, serum Immunoglobulin G (IgG) and red blood cells (Ter119+, red) extravasation ( Supplementary Fig. 7a-e ). Moreover, transmission electron microscopy of mutant samples shows the emission of intraluminal protrusions from endothelial cells, enlargement of the sub-endothelial basement membrane and increased perivascular spaces (Fig. 4 i). Notably, vascular ensheathment by AQP4 + astrocyte endfeet is maintained in the Nodal iPCKO mutant cortex ( Supplementary Fig. 7b ). Together, these data indicate that pericyte-derived Nodal controls vessel growth and integrity in the postnatal brain. Previous work has indicated that Nodal is a positive regulator of tumor angiogenesis and can promote tube-formation by cultured human umbilical vein endothelial cells (HUVECs) 45 , 46 . To gain insight into the regulation of EC behavior by Nodal, we stimulated murine immortalized brain endothelial (b.End3) cells with recombinant rhNodal and analyzed the phosphorylation of the downstream signal transducer SMAD2 by Western blotting. Treatment with rhNodal increases the level of phosphorylated SMAD2 (p-SMAD2) in b.End3 lysates in a dose-dependent fashion without altering total SMAD2 (Fig. 4 j), which is prevented by addition of SB431542, an inhibitor of TGFβ type I receptors. Similarly, rhNodal stimulates b.End3 cell proliferation and migration in a scratch wound assay and, again, these effects are suppressed by SB431542 ( Fig. k-n ). Together, these data support that Nodal can regulate features of vascular growth directly through the stimulation of ECs. Reactive astrogliosis is negatively modulated by Nodal signaling The analysis of brain sections by immunostaining for GFAP and confocal microscopy revealed signs of reactive astrogliosis, which are most evident in the Nodal iPCKO cortex and brainstem (Fig. 5 a). Reactive astrogliosis, a typical response to CNS injury and a range of diseases 40 , involves a spectrum of molecular, cellular and functional changes in astrocytes. Remarkably, a highly increased number of reactive astrocytes, characterized by hypertrophy with an increase of GFAP + protrusions, can be detected in both sagittal (Fig. 5 a, b, d) and coronal (Fig. 5 c) sections of the Nodal iPCKO brain cortex. In addition, transmission electron microscopy of mutant and littermate control brain cortex confirm the enrichment of glycogen granules and intermediate filaments in mutant astrocytes (Fig. 5 e), which are two features of reactive astrocytes in pathologic conditions 47 . Confocal microscopy shows that reactive GFAP + astrocytes are present in regions of vascular leakage, as indicated by TER119 or IgG extravasation ( Supplementary Fig. 7c, d ), which raises the possibility that reactive astrogliosis may occur secondary to vascular defects. However, GFAP + astrocytes are also present in regions without IgG extravasation or vascular leakage ( Supplementary Fig. 7e ). For a more detailed investigation of the cellular and molecular alterations in Nodal iPCKO mutants, we performed scRNA-seq analysis of the P12 brain cortex from mutants and littermate controls (Fig. 5 f, g and Supplementary Fig. 6a-c ). Bioinformatic analysis of the resulting data confirms the increase of reactive astrocytes in Nodal iPCKO mutants relative to littermate controls (Fig. 5 f). Moreover, our scRNA-seq data shows the upregulated expression of genes associated with reactive astrocytes, including transcripts for glial fibrillary acidic protein ( Gfap ), AP-1 family transcription factors ( Jun , Junb , Fos , Fosb ), the amino acid transporter solute carrier family 7 member 5 ( Slc7a5 ), the Wnt pathway protein Axin2 ( Axin2 ), nuclear receptor 4A1 ( Nr4a1 ), the transcription factors EGR-1, Sox2 and Sox9 ( Egr1 , Sox2 , Sox9 ) and the cell cycle regulator Cyclin D1 ( Ccnd1 ) in mutant relative to control brain (Fig. 5 g and Supplementary Fig. 8a ). Analysis of immunostained brain sections by confocal microscopy confirms that the transcriptional regulators SOX2, SOX9 ( Supplementary Fig. 8b, c ) as well as FOS ( Supplementary Fig. 9a ) are enriched in Nodal iPCKO GFAP + astrocytes. In vitro experiments confirm that cultured mouse primary astrocytes are responsive to recombinant rhNodal protein. Western blot analysis of cell lysates from rhNodal-treated astrocytes reveals a significantly increased phosphorylation of the downstream signal transducer SMAD2 (p-SMAD2), which is abolished by SB431542 (Fig. 5 h). These data support that pericyte-derived Nodal might directly regulate astrocyte behavior in the postnatal brain. Cell culture experiments also show that SB431542 administration leads to an increase in GFAP + astrocytes ( Supplementary Fig. 8d and Supplementary Fig. 9c ), which is consistent with the known role of TGFβ as a negative or, depending on context, positive regulator of astrocyte reactivity 48 . Supporting our findings in the scRNA-seq analysis, Nodal stimulation reduces the nuclear immunostaining of FOS and FOSB ( Supplementary Fig. 9b, c ). Conversely, SB431542 treatment of astrocytes increases nuclear localization of SOX9, FOS and FOSB ( Supplementary Fig. 8d and Supplementary Fig. 9b, c ). Taken together, these findings support that the loss of pericyte-derived Nodal might induce reactive astrogliosis directly, which could be enhanced by vascular leakage. Activation of microglia is negatively controlled by Nodal Microglial cells are important for immune surveillance in the brain and undergo morphological changes, from ramified to amoeboid-like morphology, in response to injury or pathogens 49 – 51 . Visualization of microglia by immunostaining and confocal microscopy shows that that the expression of AIF1 and CD68 is strongly increased in P12 Nodal iPCKO brain sections relative to littermate controls (Fig. 6 a). Upregulated expression of AIF1 and CD68 in Nodal iPCKO brain lysates is confirmed by Western blot analysis (Fig. 6 b). Furthermore, Nodal iPCKO AIF1 + microglial cells acquire a highly ramified morphology, consistent with an exposure to acute stress 52 (Fig. 6 c). The increased number of activated microglial cells in Nodal iPCKO mutant brains relative to littermate controls is confirmed by the analysis of our scRNA-seq data (Fig. 6 d, e). Notably, multiple markers associated with microglial activation, including transcripts for the chemokines tumor necrosis factor ( Tnf ), Interleukin-1 alpha ( Il1a ), CD52 ( Cd52 ), CD74 ( Cd74 ), colony stimulating factor 1 ( Csf1 ), C-X-C motif chemokine ligand 10 ( Cxcl10 ) and 16 ( Cxcl16 ), C-C motif chemokine 12 ( Ccl12 ) and 4 ( Ccl4 ), are upregulated in Nodal iPCKO microglia (Fig. 6 f and Supplementary Fig. 10a ). Other markers of activated microglia, namely the AXL receptor tyrosine kinase ( Axl ) and the intermediate filament protein Nestin ( Nes ), are also increased after loss of pericyte-derived Nodal (Fig. 6 f and Supplementary Fig. 10a ). Immunostaining confirms the increase of Nestin, which is known to be elevated in microglia during inflammation 53 , in Nodal iPCKO brain sections relative to littermate controls ( Supplementary Fig. 10b, e ). For more detailed investigation of the role of Nodal, we treated cultured primary murine brain-derived microglial cells with recombinant rhNodal. This treatment significantly increases the phosphorylation of SMAD2, which is blocked by administration of SB431542 (Fig. 6 g). Cell stimulation experiments also confirm the suppression of Nestin expression by rhNodal in cultured human microglia cells, whereas SB431542 treatment leads to strongly enhanced Nestin immunostaining ( Supplementary Fig. 10c, f ). Expression of the chemokine CXCL10, which has been linked to microglial activation and migration 54 – 56 , is also reduced by rhNodal treatment and strongly increased by SB431542 ( Supplementary Fig. 10d, g ). Microglia can be classified into two main types: M1 microglia, which is pro-inflammatory and neurotoxic, and M2 microglia with anti-inflammatory and neuroprotective roles 57 – 59 . Computational analyses of our scRNA-seq data suggests that the loss of pericyte-derived Nodal signaling shifts microglial polarization towards the M1 type, indicating that pericyte-derived Nodal may have both anti-inflammatory and neuroprotective effects (Fig. 6 h). However, this simple classification does not entirely capture the complexity of microglial responses in various neurodegenerative conditions 58 , 60 . Taken together, our findings support that microglia can be directly activated by Nodal, but alterations in the brain microenvironment, such as elevated vascular permeability or changes in ECs or astrocytes, might contribute to the activation of microglia after loss of pericyte-derived Nodal. Discussion Research on angiocrine signaling has uncovered very important roles of endothelial cells or specific EC subpopulations in organ growth and regeneration 1 , 2 , whereas comparably little attention has been given to the potential role of pericytes as a source of secreted factors acting on cell populations in the surrounding tissue. In contrast, it is very well established that pericytes are critical for blood vessel integrity and the prevention of excessive vascular leakage. The latter is most evident in the blood-brain barrier where pericytes together with specialized ECs and astrocyte endfeet protect the brain against the entry of potentially harmful substances and cells from the blood stream 61 , 62 . In the lung, pericytes have been proposed to be a source of myofibroblasts driving tissue fibrosis 63 , 64 but have also been implicated in the regulation of leukocyte trafficking and cytokine signaling during inflammatory responses 65 . Historically, the reliable identification of pericytes has long been hampered by the lack of unique markers that are not shared by vascular smooth muscle cells, fibroblasts or other cell populations 66 , 67 . Accordingly, morphological criteria, in particular the close association with the endothelial monolayer, have been indispensable for the characterization of pericytes. These limitations are, at least to a great part, overcome by single cell or single nucleus RNA-seq approaches 19 , 27 , 28 , which provide detailed molecular signatures and enable important insights into organ-specific and intra-organ heterogeneity of cells. Stimulated by the availability of scRNA-seq data hinting at organ-specific angiocrine roles of pericytes, we have explored the role of three differentially expressed pericyte-derived candidate regulators, HGF, BDNF and Nodel, in postnatal lung and brain, which were selected as representative model organs. In the lung, our data show that pericyte-derived HGF promotes the expansion of c-Met-expressing AT2 cells and thereby controls alveologenesis (Fig. 7 a). This finding is consistent with our own previous work showing that the YAP1 and TAZ, transcriptional regulators in the Hippo pathway, regulate the expression of HGF and angiopoietin-1 in pericytes 22 . In the current study, we directly establish that pulmonary alveologenesis is indeed direct controlled by pericyte-derived HGF, which is consistent with previous work reporting an important role of HGF/c-Met signaling in lung epithelial morphogenesis during development and regeneration 22 , 42 , 68 . As AT2 cell-derived alveolar type 1 epithelial cells are an important source of vascular endothelial growth factor A (VEGF-A), a master regulator of angiogenic blood vessel growth, during lung development 69 , it is feasible that vascular defects in Hgf iPCKO mutants are indirect due to impaired epithelial morphogenesis. We also identify pericyte-derived BDNF as an important regulator of postnatal lung morphogenesis. The neurotrophin and its receptor, the Trk family receptor tyrosine kinase TrkB, are best known role in the formation and function of the nervous system 70 . However, more recent work has identified a specialized population of AT2 cells during lung regeneration in the adult mouse as a critical source of the neurotrophin. BDNF signals to TrkB + mesenchymal alveolar niche cells, which have been shown to promote epithelial self-renewal and myofibrogenesis in response to lung injury 71 , 72 . BDNF signaling has been also linked to various aspects of blood vessel growth, EC migration and survival 34 , 36 , 37 . Our cell type-specific genetic experiments now demonstrate that interactions via pericyte BDNF and endothelial TrkB directly contribute to postnatal lung development. Notably, Pdgfrb-CreERT2 -controlled inactivation of the Hgf or Bdnf genes causes no notable alterations in the postnatal brain, whereas the third factor investigated in our study, the TGFβ family ligand Nodal, is not expressed by pulmonary pericytes, explaining the absence of defects in Nodal iPCKO lungs. In contrast, we find that the loss of pericyte-derived Nodal impairs EC proliferation, decreases the vascularization of the postnatal brain and causes microhemorrhaging. Previous studies have reported that Nodal promotes vascularization in breast cancer 73 , and its inhibition suppresses angiogenesis and the progression of human gliomas 45 . Nodal iPCKO brains show signs of reactive astrogliosis and microglia activation. Although astrocytes are known to respond to defects in endothelial cells or compromised BBB integrity 74 – 76 , the presence of GFAP + astrocytes in mutant brain regions lacking detectable vascular leakage and cell culture experiments argue that Nodal can directly influence astrocyte reactivity. As reactive astrocytes are typically induced by neuroinflammation or brain injury and ischemia 77 – 79 , future work might address the expression and function of Nodal in pathophysiological settings. Given that TGF-β treatment has been shown to reduce the expression of reactive astrocyte-associated genes in culture 79 , potentially redundant roles of other TGF-β superfamily members need to be considered. Moreover, reactive astrocytes can be induced by cytokines secreted by reactive microglia, including Il-1α, TNFα, and C1q 79 , which generates a scenario of molecular crosstalk between multiple cell populations in and around the brain vasculature. The crosstalk between microglia and other components of the BBB, including pericytes, is essential for CNS homeostasis and maintaining a healthy brain environment 79 – 82 . Disruption of this communication can lead to neuroinflammation and contribute to various neuropathological conditions 58 , 79 , 83 . It is therefore important that Nodal negatively regulates the enrichment of factors associated with microglial activation 56 , 57 , 79 , 84 , 85 . In summary, our study identifies pericytes as central regulators of tissue morphogenesis and microenvironmental homeostasis during postnatal development. We demonstrate that pericytes are vital sources of growth factors and reveal that their signaling is organ-specific, enabling tailored functional roles aligned with distinct morphogenetic processes and tissue-specific environments. Declarations Author Contributions S.J.R. and R.H.A. designed experiments and interpreted results. S.J.R. performed the vast majority of the experiments. S.J.R. and R.D.-H performed isolation of brain cells isolation for the single-cell sequencing, and prepared samples for Transmission Electron Microscopy. S.J.R., A.A. and M.E.P. performed isolation of lung cells isolation for the single-cell sequencing. S.J.R. and K.K. performed the bioinformatic transcriptomic analysis. S.J.R and P.G. conducted the migration assay and EdU incorporation assay using bEnd.3 cells. S.J.R. and R.H.A. wrote the manuscript. Acknowledgements The authors thank S. Volkery and M. Stasch for experts advise with confocal microscopy; D. Zeuschner and K. Mildner for electron microscopy; H.-W. Jeong and K. Mueller for help with RNA-seq experiments; R. Klein for generously providing TrkB mice; D. Vestweber for kindly supplying bEnd.3 cells; E. Watson for the help with Dextran injection. This work was supported by the Max Planck Society and the German Research Foundation (CRC 1366, project no. 394046768). Disclosures The authors declare no competing interests Data Availability The scRNA-seq data generated in this study have been deposited in the Gene Expression Omnibus under accession number (GSE285933, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE285933). The mouse reference genome GRCm39 with GENCODE M26 annotation (https://www.gencodegenes.org/mouse/release_M26.html) was used for mapping the reads in this study. All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files. All individual mouse lines used in this study are commercially available at The Jackson Laboratory or through the lead author. All other biological materials described in this article are available through commercial suppliers as indicated. Code availability Custom code for scRNA-seq analysis, based on existing packages and own contributions, is available at https://keeper.mpdl.mpg.de/d/f5d1546ae3c84296b921/. 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 81-02.04.2020.A471) granted by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) of North Rhine-Westphalia, Germany. Animals were combined in groups for experiments irrespective of their sex. C57BL/6J mice were used for the analysis of wild-type lung and brain. In vivo labeling of pericytes was performed by mating Pdgfrb(BAC)-CreERT2 12 and Rosa26 mT/mG reporter mice 24 . Cre activity was induced in pups resulting from this mating by intraperitoneal injection of pups at postnatal day 1 (P1) with a single dose of 50 μg 4-hydroxy tamoxifen (4-OHT) (H7904, Sigma) in ethanol-peanut oil (P52144, Sigma). For inducible genetic experiments employing a mural cell-specific loss-of-function approach, Pdgfrb(BAC)-CreERT2 transgenics were interbred with mice carrying loxP-flanked alleles for Bdnf ( Bdnf lox/lox ) 86 , Hgf ( Hgf lox/lox ) 87 , or Nodal ( Nodal lox/lox ) 88 in separate crosses. To inactivate Ntrk2 in the postnatal endothelium, Ntrk2 lox/lox mice 31 and Cdh5(PAC)-CreERT2 +/T transgenic mice 89 were interbred. Cre activity was induced by three consecutive intraperitoneal injection of 50 μg tamoxifen (T5648, Sigma) in ethanol-peanut oil (P52144, Sigma) from P1 to P3. Lung sample preparation and immunohistochemistry. Lung samples were prepared as previously described 22 . For immunohistochemical analysis of mouse lungs, pups at P21 were anaesthetized by intraperitoneal injection of xylazine (Bayer, Rompun 2%; 10mg/kg) and ketamine (Zoetis, Ketavet 100mg/ml; 100mg/kg) dissolved in PBS. The chest cavity of each terminally anesthetized pup was opened to access the heart and lungs. A warm (37°C) solution of 6% gelatin (G1890, Sigma) in PBS was gently perfused through the right ventricle using manual pressure. To allow the gelatin to solidify, an ice-cold tissue paper was placed over the exposed heart and lungs for 15 minutes. Subsequently, the ventral trachea was cannulated using an intravenous catheter (BD Insyte, 381212), which was secured with a suture. The lungs were then inflated to full capacity by gently injecting warm (37°C) 1% low-gelling agarose (A4018, Sigma) in PBS. The agarose-inflated lungs were further chilled by placing an ice-cold tissue paper on them for 20 minutes. Afterward, the lungs were excised and placed in a 2% paraformaldehyde (PFA; Sigma, P6148) solution in PBS at 4°C for 30 minutes. Following this initial fixation, the lungs were incubated in cold PBS for 30 minutes. After washing with cold PBS, the lung lobes were sliced into 150 µm sections using a vibrating blade microtome (VT1200, Leica) and then fixed in 4% PFA at 4 °C for 1 hour. After the second fixation, the lung samples were washed thoroughly by incubating them twice in PBS for 30 minutes at room temperature (RT). Lung slices were subsequently blocked in a blocking solution composed of 5% donkey serum and 0.5% Triton X-100 in PBS for a minimum of 2 hours at RT or overnight (O/N) at 4°C. Following blocking, the vibratome sections were treated with primary antibodies diluted in the blocking solution overnight at 4°C. The sections were washed once in 0.5% Triton X-100 in PBS (PBST) for 20 minutes and then three times in PBS for 10 minutes each at RT. After washing, the sections were incubated with secondary antibodies diluted in blocking solution for 2 hours at RT or O/N at 4°C. Nuclei were counterstained with DAPI (D9542, Sigma, 2 µg/ml). After four wash steps with PBS, the sections were mounted using FluoroMount-G (Southern Biotech, 0100-01) and covered with cover slips. The mounted samples were stored at 4°C. The following primary antibodies were used for lung staining: rat anti-RAGE (1:200, R&D Systems, MAB1179), rabbit anti-Aquaporin 5 (1:200, Millipore, 178615), goat anti CD31/PECAM1 (1:200, R&D Systems, AF3628), rat anti-PDGFRβ (1:100, eBioscience, 14-1402), goat anti-PDGFRβ (1:100, R&D Systems, AF1042), rabbit anti-Prosurfactant Protein C (1:200, Millipore, AB3786), chicken anti-GFP (1:300, 2BScientific Ltd., GFP-1010), mouse anti-αSMA-Cy3 (1:300, Sigma C6198), rat anti-DC-LAMP/CD208 (1:200, Novus Biologicals/Dendritics, DDX0192P-100), rabbit anti-NKX2.1/TTF1 (1:200, abcam, ab76013), rat anti-ICAM2/CD102 (1:100, BD Pharmingen, 553326), goat anti-HGFR/c-Met (1:100, R&D Systems, AF527), rabbit anti ERG (1:100, Abcam, ab110639), rabbit anti-Cleaved Caspase-3 (1:100, Cell Signaling, #9664), Goat anti-TrkB (1:50, Biotechne; AF1494), and rabbit anti-Ki67 (1:100, Abcam; ab15580). Brain sample preparation and immunohistochemistry. Brain samples were prepared as previously described 90 . For immunohistochemical analysis of mouse brains, P12 were anaesthetized by intraperitoneal injection of xylazine (Bayer, Rompun 2%; 10mg/kg) and ketamine (Zoetis, Ketavet 100mg/ml; 100mg/kg) dissolved in PBS. Following terminal anesthesia, the chest cavity was surgically opened to expose the heart. To clear the circulatory system of blood, a puncture was made in the right atrium, and 10 ml of ice-cold PBS was perfused through the left ventricle using a peristaltic pump (Pump P-1, GE Healthcare). Tissue fixation was initiated immediately afterward by perfusing 10 ml of ice-cold 1% paraformaldehyde (PFA; Sigma, P6148) through the same route. Once perfusion was complete, brains were carefully dissected from the skull and post-fixed by immersion in 4% PFA at 4°C O/N. The fixed brains were subsequently washed four times (15 minutes each) in PBS. For sectioning, the brains were cut either along the sagittal midline or into 2-mm thick coronal sections using an acrylic brain matrix designed for mice (RBMA-200C, World Precision Instruments). The brain hemispheres or coronal sections were embedded in 4% low-gelling-temperature agarose (Sigma, A9414) dissolved in PBS at 40°C. After embedding, the samples were rapidly cooled on ice to solidify the agarose. Once the agarose had solidified, the blocks were trimmed and mounted onto a specimen holder using cyanoacrylate adhesive (UHU GmbH & Co. KG). Sections with a thickness of 100μm were then cut using a vibratome (VT 1200S, Leica). Vibratome sections were blocked and permeabilized O/N at 4°C in a solution containing 1% bovine serum albumin (BSA; Sigma, P6148), 2% normal donkey serum (Abcam, ab7475), and 0.5% Triton-X-100 (Sigma, T8787) in PBS. Primary antibodies were diluted in freshly prepared blocking solution and incubated O/N at 4°C. Following primary antibody incubation, the sections were washed once with 0.5% Triton-X-100 in PBS, followed by three washes with PBS (20 minutes each at 4°C). The sections were then incubated overnight with species-specific Alexa Fluor-conjugated secondary antibodies (Invitrogen), diluted 1:500 in the blocking buffer. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, D9542; 2µg/mL). Following secondary antibody incubation for 2 hours at RT or O/N at 4°C, the sections were washed with PBS as described and mounted using Fluoromount G (Southern Biotech, 0100-01). The following primary antibodies were used for brain immunostaining: rabbit anti- AQP4 (1:100, Sigma HPA014784), mouse anti-αSMA-Cy3 (1:200, Sigma C6198), goat anti CD31/PECAM1 (1:200, R&D Systems, AF3628), rabbit anti ERG (1:50, Abcam, ab110639), rabbit anti-GFAP (1:200, DAKO, Z0334), goat anti-GFAP (1:200, Novus Biologicals, NB100-53809), rat anti-CD68 (1:100, Abcam, ab53444), chicken anti-GFP (1:200, 2BScientific Ltd., GFP-1010), rabbit anti-GLUT1 (1:100, Millipore, 07-1401), goat anti-AIF1 (1:100, Novus Biologicals, NB100-1028), rat anti-ICAM2/CD102 (1:100, BD Pharmingen, 553326), rat anti-Nestin (1:200, Santa Cruz, sc101541), rat anti-PDGFRβ (1:100, eBioscience, 14-1402), goat anti-PDGFRβ (1:100, R&D Systems, AF1042), rat anti-TER-119 (1:200, R&D Systems, MAB1125), goat anti-Collagen IV (1:100, Millipore; AB769), Rabbit anti-SOX2 (1:100, Abcam, ab97959), goat anti-SOX9 (1:100, R&D Systems, AF3075), rat anti-Nestin (1:100, Cosmo Bio, BAM-73-100-EX), rabbit anti-FOSB (1:100, Cell Signaling, #2251), rabbit anti-FOS (1:100, Abcam, ab190289), goat anti-CXCL10 (1:100, Biotechne, AF466-SP), mouse anti-Nestin (1:100, Santa Cruz, sc-23927) and Isolectin B4 Alexa Fluor-488 (1:50, Invitrogen; I21411). The following donkey-raised secondary antibodies (all in 1:500 dilution) were used for immunostaining of brain and lung samples: anti-rabbit IgG conjugated to Alexa Fluor (AF) 488 (Thermo Fisher Scientific, A21206), anti-chicken IgY AF488 (Jackson ImmunoResearch, 703-545-155), anti-rat IgG AF488 (Thermo Fisher Scientific, A21208), anti-goat IgG AF488 (Invitrogen, A-11055), anti-mouse IgG AF546 (Thermo Fisher Scientific, A10036), anti-rat IgG AF594 (Thermo Fisher, A21209), anti-rabbit IgG AF594 (Thermo Fisher Scientific, A21207), anti-goat IgG AF594 (Thermo Fisher Scientific, A-11058), anti-rabbit IgG AF647 (Thermo Fisher Scientific, A-31573), anti-rat IgG AF647 (Jackson ImmunoResearch, 712-605-153), anti-goat IgG AF647 (Thermo Fisher Scientific, A-21447), and anti-mouse IgG AF647 (Thermo Fisher Scientific, A-31571). Nuclei were counterstained with DAPI (1μg/ml) together with secondary antibodies. Cell culture Mouse brain endothelial cells (b.End3) were cultured in DMEM (Sigma, D6546) supplemented with penicillin/streptomycin (PAA, P11-010) and 10% FCS, and kept in a humidified incubator at 37 °C, 10% CO2. Cells were seeded into six-well plates coated with 0.1% gelatin for protein extraction. or into µ-Slide 24 well (Ibidi, 82426) for immunostaining. Mouse C57 mixed astrocytes (Lonza, M-AsM-330) were cultured in Astrocyte Growth Medium BulletKit™ (AGM TM BulletKit TM , CC-3186), and kept in a humidified incubator at 37 °C, 5% CO2. Cells were seeded into six-well plates coated with poly-L-lysin (2 ug/cm 2 ,Sciencell, 0403) for protein extraction, or into µ-Slide 24 well (Ibidi, 82426) for immunostaining. Mouse microglia (Sciencell, M1900) were cultured in Microglia Medium (Sciencell, #1901), which consists of 500 ml of basal medium, supplemented with 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of microglia growth supplement (MGS, Cat. No. 1952) and 5 ml of antibiotic solution (P/S, Cat. No. 0503). Microglia were seeded into six-well plates coated with poly-L-lysin (2 ug/cm 2 , Sciencell, 0403) for protein extraction, or into µ-Slide 24 well (Ibidi, 82426) for immunostaining, kept in a humidified incubator at 37°C. Stimulation and inhibitor treatment of cultured cells bEnd.3 cells in a 6-well plate were starved in basal medium for 1 hour at 37°C, then treated with basal medium containing Nodal (100 or 200ng/ml, R&D Systems, 3218-ND-025) for 30 minutes, with or without the SB431542 inhibitor (10 µM, Selleckchem, S1067). Following stimulation, cells were processed for protein isolation. For immunostaining, cells were seeded in µ-Slide 24-well plates (Ibidi, 82426) and treated under the same conditions for 16 hours. Mouse astrocytes and microglia, cultured separately in 6-well plates at 37°C, were treated with Nodal (200 ng/ml) or SB431542 inhibitor (10 µM) for 30 minutes. Inhibitor treatment was also performed simultaneously with Nodal stimulation. After treatment, the cells were harvested for protein isolation. DMSO was used as a control treatment. For immunostaining, cells were seeded in µ-Slide 24-well plates (Ibidi, 82426) and treated under the same conditions for 16 hours. Protein isolation and Western blotting For immunoblotting, cells were washed twice with ice-cold PBS containing 1mM PMSF, then lysed on ice in a lysis buffer (20mM Tris-HCl, pH 8.0, 150mM NaCl, 0.5% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 2 mM EDTA) supplemented with Halt Protease Inhibitor Cocktail (Thermo Scientific, 78429) and Phosphatase Inhibitor Cocktail Set V (EMD Millipore, 524629). The lysates were incubated for 20 minutes at 4°C. Following vortexing, cell lysates were centrifuged for 10 min at 4°C, and protein concentrations in the supernatants were measured using the BCA Protein Assay Kit (Pierce, 23225). Lysates were combined with 2x sample loading buffer in a 1:1 ratio and heated at 95°C for 5 minutes. 2 µg of total proteins were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P Polyvinylidene fluoride (PVDF) membranes, which were briefly treated with 100% methanol for 20 seconds. Membranes were blocked in either 1-4% BSA/TBST or 0.3% skim milk/TBST for 1 hour before being incubated with primary antibodies in blocking buffer O/N at 4°C with gentle agitation. After three washes with TBST, membranes were exposed to peroxidase-conjugated secondary antibodies diluted in either 1% BSA/TBST or 0.3% skim milk/TBST buffer for 1 hour at room temperature with gentle agitation. The membranes were then washed and developed using the ECL Prime detection kit (GE Healthcare, RPN2232). For tissue protein isolation, mouse brain cortices and lungs were perfused with cold PBS containing PhosSTOP (Roche) through the left and right ventricle, respectively. The perfused tissues were dissected before snap freezing in liquid nitrogen. Tissues were then homogenized in lysis buffer using a Pestle (Argos) and centrifuged for 20 minutes at 4°C to clarify the lysates. Total protein concentrations were determined with the BCA Protein Assay Kit (Pierce, 23225). 20 µg of total protein from the lysates was separated by SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies, followed by washing and detection using horseradish peroxidase-conjugated secondary antibodies and the ECL Prime detection kit. The following antibodies were used for immunoblotting: mouse anti-β-ACTN (1:6000, Invitrogen, AM4302), mouse anti-α-Tubulin (1:6000, Sigma, T5168), rabbit monoclonal anti-SMAD2 (1:5000), Cell Signaling, 5339), rabbit monoclonal anti-Phospho-SMAD2 (1:500, Cell Signaling, 3108), goat anti-AIF1 (1:5000, Novus Biologicals, NB100-1028), rat anti-CD68 (1:3000, Abcam, ab53444), goat anti-Rabbit IgG, HRP-linked whole Ab (1:5000, Cell Signaling, 7074), sheep anti-Mouse IgG, HRP-linked whole Ab (1:40000, HG-Healthcare, NA931), Peroxidase AffiniPure Bovine anti-Goat IgG (H + L) (1:20000, Jackson ImmunoResearch, 805-035-180) and goat anti-rat HRP-linked whole Ab (1:20000, Amersham, NA935) Cell Immunostaining Cells were cultured in their respective media within µ-Slide 24-well plates (Ibidi, 82426). The cells were fixed using 4% PFA for 10 minutes, followed by permeabilization with ice-cold 0.1% Triton X-100 in PBS for 5 minutes at 4°C. After washing with PBS, cells were blocked with a solution containing 4% donkey serum and 2% BSA in PBS for 1 hour at RT.Following blocking, the cells were incubated with primary antibodies diluted in the blocking buffer for 1 hour at RT. After another washing step, secondary antibodies, also diluted in the blocking buffer, were applied for an additional hour. Finally, after washing steps with PBS, 250μL of Fluoromount-G was added to each well of the µ-Slide for mounting. EdU incorporation assay in vivo and in vitro EC proliferation was evaluated by administering a single intraperitoneal injection of EdU (150 µg/50 µl, Invitrogen, A10044) to P12 pups. After 2 hours, the brains were dissected and processed as described above. EdU-positive cells were visualized using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific, C10340). To assess bEnd.3 proliferation in culture, cells were treated with Nodal and SB431542 inhibitor as indicated, were incubated with EdU (10 µM) in an old medium for 60 min at 37 °C. EdU-positive cells were visualized using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific, C10340). Scratch wound assay A scratch wound assay was performed on cultured bEnd.3 cells. Scratches were created using a 10 μL pipette tip positioned perpendicular to the bottom of the well near the well wall. The pipette tip was gently dragged across the well under light pressure to generate uniform scratches. Following scratch formation, the culture medium was replaced with low-serum medium (2% FBS) supplemented with either Nodal (200ng/mL) or a combination of Nodal (200ng/mL) and SB431542 inhibitor (10μM). In case of the latter, DMSO was added as vehicle control. Tile-scan bright-field images of the scratch areas were captured using a Zeiss AxioObserver Z1. Image analysis was conducted using ZEN Blue software and Fiji. Dextran injection A 5 mg/ml solution of 70 kDa Dextran (Texas Red Lysin fixable, Thermofisher, D1864) was prepared in sterile PBS. Pups were anesthetized before administering 50μl of the dextran solution into the bloodstream via retro-orbital injection. Image acquisition and analysis Confocal image acquisition was performed using a Zeiss confocal microscope LSM780 and LSM880 equipped with the following objective lenses: 10× Plan Apochromat (APO), Numerical Apertrue (NA) 0.45, 20× Plan APO NA 0.8, water immersion 40× LD C-APO NA 1.20, and oil immersion 63× Plan APO NA 1.40. The confocal data were then processed with the ZEN 2.3 SP1 FP3 software (black edition) and Fiji. Statistical analysis Data were processed with the Prism10 software. Values are presented as mean ± standard error of the mean (s.e.m.). P values were calculated using Student’s t-test to determine statistical significance when comparing two independent groups; For analysis of statistical significance in comparisons involving more than two groups with normal distribution, ordinary one-way ANOVA with Tukey’s (when comparing the mean of each group with the mean of every other group), Sidak’s (for comparing the means of preselected pairs of groups). Transmission Electron Microscopy For electron microscopy, mice were anesthetized as previously described and transcardially perfused with 10mL of PBS and 40 mL of 2% PFA and 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). The brain was removed and further fixed by immersion in the above-mentioned solution for 3h at RT. An acrylic matrix for mouse brains (World Precision Instruments Cat. No. RBMA-200C) was used to section the brain in coronal slices from which smaller pieces belonging to the cortex were collected and further fixed in reduced 1% osmium tetroxide containing 1.5% potassium hexacyanoferrate. Next, the tissue was dehydrated and embedded in epon. Ultrathin 60 nm-sections were cut on an ultramicrotome (Leica UC6) and counterstained with uranyl and lead. Images were taken using an electron microscope (Tecnai 12 Biotwin TEM, FEI) and representative pictures were documented in imaging plates (Ditabis, Pforzheim). Single cell RNA-sequencing For the single cell sequencing of postnatal lung, two Hgf lox/lox control miceand two Hgf iPCKO P21 old pups or two Bdnf lox/lox control miceand two Bdnf iPCKO P21 old pups were analyzed, respectively. Lung lobes were dissected, finely minced using sharp scissors and transferred into enzymatic digestion solution containing 40U/ml collagenase I (Gibco, 170100-017), 40U/ml Collagenase IV (Gibco, 170100-019), 4U/ml Dispase II (Thermo Fisher, 17105041) and 2 U/µl DNase I (Worthington, LK003170) prepared in DMEM (Gibco, 31053028) medium. Digestion was performed in a water bath, at 37°C for 30 min. During this time, the tissue was also mechanically dissociated by passing it through a needle-syringe for several times. Enzymatic digestion was stopped by addition of FACS buffer (2% FBS in DMEM) and the lysate was passed through 70 µm cell strainer (Falcon, 352350). The filtrate was centrifuged for 5 min at 4°C and the pellet was resuspended in RBC lysis solution (eBioscience, 00-4333-57) and further incubated for 10 min on ice. Post resuspension in FACS buffer, the cell lysis was pass through a 50 um Cell Trics filter (Sysmex, 04-0042-2317). Cells were centrifuged for 5 min at 4°C and the pellet was resuspended in cold MACS depletion buffer (autoMACS Running Buffer-MACS Separation Buffer, Miltenyi Biotec, 130-091-221). To proceed with magnetic separation and depletion of CD45+ cells and erythrocytes from the cell suspension, CD45 microbeads (Miltenyi Biotec, 130-052-301) and Ter119 microbeads (Miltenyi Biotec, 130-049-901) were added to the cells resuspended in MACS depletion buffer and incubated for 20 min at 4°C. After washing, the cell solutions were passed through MACS MS column (Miltenyi Biotec, 130-042-201) attached to a MACS separator placed on a magnetic Multistand. Filtrate solution consisting of CD45-/Ter119- cells was collected and cell number was counted using Luna Automated Cell Counter (L10001). For each mouse genotype, 40000 cells were prepared for further sequencing from the two mouse littermates, using equal cell number of cells per sample. Cells were loaded into a BD Rhapsody Cartridge (BD Bioscience, 633733) and captured on the BD Rhapsody Express Single-Cell Analysis System (BD Bioscience). Single-cell mRNA whole transcriptome (WTA) libraries were created using the BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification Kit (BD Bioscience, 633801) and DNA sequencing was performed on a NextSeq500 (Illumina) using 2 x 75 bp paired end reads with an 8 bp single index. For the single cell sequencing of postnatal cerebral cortex, two P12 Nodal lox/lox control and two Nodal iPCKO mice were anesthetized by intraperitoneal injection of Xylazine (16mg/Kg) and Ketamine (100mg/Kg) and transcardially perfused with 10 mL of ice-cold PBS supplemented with Heparin (25 U/mL). Brains were collected and the meningeal layers removed. Next, the cerebral cortex of each hemisphere was dissected and transferred to ice-cold DMEM supplemented with Penicillin/Streptomycin, Glutamine (GlutaMAX, Gibco) and 25 mM HEPES (PAA, Cat. No. S11-001), hereafter dissection media. The cortices belonging to mice from the same genotype were pooled together and minced using scalpels. The resulting paste was resuspended in 1 mL of enzyme blend containing Papain (25 U/mL, Worthington Cat. No. LK003176), DNAseI (113 U/mL, Worthington Cat. No. LK003170), and Liberase DH (100 µg/mL, Roche Cat. No. 5401054) dissolved in pre-warmed dissection media and incubated 30 min at 37 o C. During this time, the tissue was further homogenized by repeatedly pipetting the solution up and down every 10 minutes using filtered 1mL tips. After the first 15 min of incubation, 0.5 mL of the described enzyme blend were further added. Next, the tissue homogenate was filtered through a 70µm nylon mesh into a 50mL Falcon tube, the filter was washed with 1 mL of pre-warmed dissection media and the final volume obtained was measured. In order to remove debris and myelin, the cell suspension was mixed with 1.7x volume 22% BSA (Carl Roth Cat. No. 8076.2) dissolved in PBS and centrifuged at 1000 g for 12 min and RT. The supernatant was aspirated and the remaining cell pellet resuspended in 1mL of the described enzyme blend for a final 20min incubation step at 37 o C with pipetting every 5min. The resulting single-cell suspension was filtered through a 40µm cell strainer and diluted by addition of 8mL of pre-warmed dissection media. After centrifugation at 300 g for 5min, the supernatant was discarded and the cell pellet resuspended in 1ml of Red Blood Cell Lysis Buffer (Sigma Cat. No. R7757). After a 1 min incubation at RT, 20ml of ice-cold PBS supplemented with 2% fetal calf serum were added and the whole suspension centrifuged at 300 g for 5 min at 4 o C. Next, the supernatant was discarded and the cell pellet resuspended in 1ml of filter-sterilized endothelial cell buffer (15 mM HEPES, 153 mM NaCl, 5.6 mM KCl, 1.7 mM CaCl2, 1.2 mM MgCl2 and 10% BSA, pH 7.4) 91 . Cell concentration was assessed using an automated cell counter and 4 x 10 4 cells from each genotype were loaded into BD Rhapsody Cartridge (BD Biosciences Cat. No. 633733) for cell capture. scRNA-seq data analysis Preprocessing We used STAR version 2.7.10a 92 to generate a reference genome index for GRCm39, with Gencode annotations vM33. FASTQ reads were mapped against the reference genome index using STAR with the settings “ --soloType CB_UMI_Complex --soloUMIlen 8 --soloCellFilter None --outSAMtype BAM SortedByCoordinate --soloFeatures Gene --runRNGseed 1 --soloMultiMappers EM --readFilesCommand zcat --outSAMattributes NH HI AS nM NM MD jM jI MC ch CB UB GX GN sS CR CY UR UY”. Libraries using standard BD Rhapsody beads were mapped using the adapter parameters “--soloAdapterSequence NNNNNNNNNACTGGCCTGCGANNNNNNNNNGGTAGCGGTGACA --soloCBposition 2_0_2_8 2_21_2_29 3_1_3_9 --soloUMIposition 3_10_3_17 --soloCBwhitelist BD_CLS1.txt BD_CLS2.txt BD_CLS3.txt”, libraries with BD Rhapsody enhanced beads with --soloAdapterSequence NNNNNNNNNGTGANNNNNNNNNGACA --soloCBposition 2_0_2_8 2_13_2_21 3_1_3_9 --soloUMIposition 3_10_3_17 --soloCBwhitelist BD_CLS1_v2_draft.txt BD_CLS2_v2_draft.txt BD_CLS3_v2_draft.txt”. Raw counts were imported as AnnData () objects. We removed low complexity barcodes with the knee plot method, and further filtered out cells with a high mitochondrial mRNA content, as well as unusually high total and gene counts using manually determined cutoffs for each sample. Doublets were scored with scrublet 0.2.3 (). Finally, each sample’s gene expression matrix was normalized using scran (1.22.1, ) with Leiden clustering (https://doi.org/10.1038/s41598-019-41695-z) input at resolution 0.5. G2M and S phase scores were assigned to each cell using gene lists from and the scanpy (1.9.6, ) sc.tl.score_genes_cell_cycle function. Embedding, clustering and annotation The normalised expression matrix was subset to the 3,000 most highly variable genes (HVG, sc.pp.highly_variable_genes, flavor “seurat”).For some analyses, expression values were cell-cycle regressed using scanpy.pp.regress_out on G2M and S-phase scores. The top 100 principal components (PCs) were calculated, and batch-corrected using Harmony (0.0.5, ). The PCs served as basis for k-nearest neighbor calculation (sc.pp.neighbors, n_neighbors=30), which were used as input for UMAP () layout (sc.tl.umap, min_dist=0.3). Cells were clustered using scanpy.tl.leiden, and a suitable resolution was chosen in each sample for the main celltype annotation. Cluster marker genes were calculated using a pseudobulk approach, comparing aggregate counts with 2 pseudoreplicates for each cluster to all remaining cells (pyDeSEQ2 0.4.8). Finally, expression of select marker genes was plotted using Matplotlib (3.8.4; https://doi.org/10.1109/MCSE.2007.55) “imshow”, and clusters were annotated accordingly. Sample and celltype-specific subsets were subclustered using the top 2000 HVGs and 30PCs. Clusters were annotated at suitable Leiden resolutions using known and calculated celltype markers. Differential expression analysis Differentially expressed genes were calculated using a pseudobulk approach, comparing aggregate counts with 2 pseudoreplicates for each condition (pyDeSEQ2 0.4.8). Comparison to lung P14 pericytes We downloaded raw sequencing data and cell annotations by Hurskainen et al. 29 . 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Nat Commun 10:2817 Czupalla CJ, Yousef H, Wyss-Coray T, Butcher EC (2018) Collagenase-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry. Bio Protoc 8 Dobin A et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21 Additional Declarations There is NO Competing Interest. Supplementary Files RasouliSupplementaryFigures.pdf Supplementary figure legends Supplementary Figure 1. scRNA-seq analysis of P21 Bdnf iPCKO and control lungs. (a) Heatmap of top marker genes cell populations based on computational analysis of scRNA-seq data. (b) UMAP plot of annotated cell types from lung together with corresponding cell counts. Indicated are AT1 epithelial cells, AT1-AT2 transitional/intermediate cells, Lyz1 + AT2 and Lyz1 - AT2 epithelial cells, Lymphatic endothelial cells, Matrix fibroblast, Myofibroblast. (c) Volcano plot showing the differential expression analysis in P21 Bdnf iPCKO and littermate control pulmonary ECs. Average log2-fold change > 0.6, adjusted p-value < 0.05). (d) Violin plots showing expression of multiple endothelial markers ( Plvap , Gja5 , Aplnr and Itga1), and proliferation markers ( Ccnd1 and Mki67 ) in pulmonary ECs. (e) Violin plot of Ntrk2 expression in endothelial, epithelial, immune, and mesenchymal cells (including fibroblasts) from lung. Supplementary Figure 2. Analysis of the P12 Bdnf iPCKO brain vasculature. (a) Confocal images of brain vasculature stained with ICAM2 (green) and EdU (white). (b, c) Graphs showing the percentage of ICAM2+ area (b) and number of EdU+ ECs (c) in Bdnf iPCKO and littermate control brain cortex. Data represents mean ± s.e.m. (n=6 controls and 8 mutants in b, n=4 controls and 5 mutants in c); P-values, unpaired two tailed student t-test. (d) PDGFRb immunostaining (green) showing comparable pericyte coverage in Bdnf iPCKO and control cortex. (e) Graph showing percentage of pericyte coverage in P12 brain cortex. Data represents mean ± s.e.m. (n=4 controls and 5 mutants); P-value, unpaired two tailed student t-test. (f) Bdnf iPCKO and control brain vasculature stained for ICAM2 (green) and Collagen IV (grey), as indicated. (g) Confocal images of Bdnf iPCKO and littermate controls cortex stained for ICAM2 (green) and mouse IgG (red). (h) Astrocyte endfeet covering ICAM2+ endothelial cells (green) are visualized with AQP4 (grey). (i, j) Confocal images of brain cortex showing no change in the activation of GFAP+ astrocytes (grey) (i) or AIF+ microglia (magenta) (j). Supplementary Figure 3. Analysis of the P12 Hgf iPCKO brain vasculature. (a) Confocal images showing GLUT1+ (red) brain endothelium and PDGFRb+ (white) pericytes in P12 cortex. (b,c) Graphs showing the percentage of ICAM2+ area (b) and pericyte coverage (c) in brain cortex. Data represents mean ± s.e.m. (n=6 in b and 3 in c); P-values, unpaired two tailed student t-test. (d) Hgf iPCKO and littermate control brain endothelium stained by isolectin B4 (IB4, green) together with Collagen IV (red) immunostaining. (e) Confocal images of brain cortex showing ICAM2+ vasculature (green) and AQP4+ astrocyte endfeet coverage (red) (e), GFAP expression (white) (f), the microglia-specific marker AIF1 (white) (g). Supplementary Figure 4. scRNA-seq analysis of P21 Hgf iPCKO and control lungs. (a, b) Heatmap of top marker genes within lung cell types based on scRNA-seq results (a) and UMAP plot of annotated lung cell types with the corresponding cell counts (b). Indicated are AT1 epithelial cells, AT1-AT2 transitional/intermediate cells, Lyz1 + AT2 and Lyz1 - AT2 epithelial cells, Lymphatic endothelial cells, Matrix fibroblast, Myofibroblast. (c) Violin plot showing that Met expression dominates in epithelial cells relative to endothelial, immune and mesenchymal cells from P21 lung. (d) UMAP plot of annotated alveolar cell types. Indicated are AT1 epithelial cells, AT1-AT2 transitional/intermediate cells, Lyz1 + AT2 and Lyz1 - AT2 cells. (e) UMAP plot showing Lyz1 expression in a subcluster of AT2 epithelial cells. (f) UMAP plots showing Met expression in AT1-AT2, Lyz1 + AT2 and Lyz1 - AT2 cells from Hgf iPCKO and littermate control lungs. (g) Volcano plot showing the differential expression analysis of endothelial-specific responses in Hgf iPCKO and littermate control lungs. Average log2-fold change > 0.6, adjusted p-value < 0.05). Supplementary Figure 5. Normal lung development in Nodal iPCKO mutants. (a, b) Confocal images of endothelium (PECAM1+, grey) (a), EC nuclei (ERG+, green) and pericytes (PDGFRb+, white) (b), which show pulmonary vascular development is not affected in P21 Nodal iPCKO mutants. (c, d) Maximum intensity projections of Nodal iPCKO and control lung sections showing AT1 cells (RAGE+, grey) (c), nuclei of AT1 and AT2 cells (NKX2.1+, green), and type 2 alveolar epithelial cells (LAMP3+, white) (d). (e) Confocal image of proliferating cells (Ki67+, red) and DAPI-stained nuclei (blue) in Nodal iPCKO and littermate control lungs. (f-i) Graphs showing the percentage of PECAM1+ area (f), percentage of ERG+ cells in total cells (g), ratio of ERG+ to PDGFRb+ cells (h), percentage of AT1 (RAGE+) area (i), percentage of LAMP3+ AT2 cells (j) and percentage of proliferating cells (Ki67+) in total cells (k). Data represent mean ± s.e.m. (n=4 controls and 7 mutants in f, n=4 controls and 5 mutants in g-k); P-values, unpaired two tailed student t-test. Supplementary Figure 6. scRNA-seq analysis of Nodal expression in brain. (a) Heatmap of top marker genes within brain cell types using based on scRNA-seq data. (b) UMAP plot of annotated brain cell types. Each cell type is represented by distinct clusters and the corresponding cell counts are indicated. PVMs, perivascular macrophages. (c) UMAP plots show enriched expression of Nodal in control brain pericytes relative to other cell types and loss of expression in Nodal iPCKO pericytes. (d) The expression of Nodal , Pdgfrb and transcripts for TGFβ family receptors in published scRNA-seq data of postnatal mouse brain. Dendrogram (obtained from mousebrain.org) displaying the taxonomy of all identified cell types from postnatal mouse brain and expression of the indicated genes. Note presence of TGFβ family receptor transcripts in many cell types. (e) Circle plots of scRNA-seq data of adolescent mouse brain 33 showing the expression of Nodal , Pdgfrb, Myh11 and TGFβ family receptor transcripts in mural cell populations. Supplementary Figure 7. Characterization of Nodal iPCKO brain capillaries. (a) Confocal images of P12 brain cortex stained for mouse immunoglobulin G (IgG, white) from Nodal iPCKO and control littermates injected with Texas Red-dextran (70 kDa) (red). Green arrows indicate local leakage. b) Maximum intensity projections showing comparable coverage of Aquaporin-4+ (AQP4+) astrocyte endfeet (grey) of ICAM2+ endothelium (green) in Nodal iPCKO and control brain cortex. (c) Confocal images of GLUT1+ (green) capillaries in brain cortex. Red blood cells (Ter119, red) are confined to vessels in control but leakage (arrows, white arrows) into the brain parenchyma and GFAP+ astrocytes (white, yellow arrowheads) can be seen in Nodal iPCKO mutants. (d, e) Confocal images of CD31+ (green) brain capillaries with area of extravasated IgG (red, white arrows) in Nodal iPCKO mutant surrounded by GFAP+ astrocytes (white, yellow arrows) in brain cortex (d). However, reactive astrocytes (yellow arrows) can also be observed in areas of Nodal iPCKO cortex lacking extravasated IgG (asterisks) (e). Supplementary Figure 8. Nodal-dependent expression of reactive astrocyte markers. (a) Volcano plot showing differential gene expression in astrocytes from Nodal iPCKO and littermate control cortex. Average log2-fold change > 0.6, adjusted p-value < 0.05). (b, c) Confocal images of P12 Nodal iPCKO and littermate control brain cortex. Mutant GFAP+ (red) astrocytes show nuclear enrichment of Sox2 (green, white arrows) (b) as well as Sox9 (green, blue arrows) compared to control astrocytes (c). (d) Expression of Sox9 (green) and GFAP (red) in cultured mouse primary astrocytes treated with recombinant human Nodal (rhNodal) and SB431542 inhibitor. Inhibition of TGFβ signaling increases nuclear SOX9 (yellow arrows). Supplementary Figure 9. TGFβ signaling controls levels of nuclear FOS and FOSB. (a) Confocal images of brain cortex stained for FOS (green) and GFAP (red). White arrows indicate nuclear enrichment of FOS in Nodal iPCKO astrocytes. (b, c) Inhibition of TGFβ signaling with SB431542 increases nuclear FOS (green, yellow arrows) (b) and FOSB (green, orange arrows) (c) in cultured mouse primary astrocytes. Supplementary Figure 10. Marker genes of activated microglia are negative regulated by Nodal signaling. (a) Volcano plot showing the differential expression of genes in Nodal iPCKO microglia relative to control cells. Average log2-fold change > 0.6, adjusted p-value < 0.05). (b) Increased expression of Allograft inflammatory factor-1 (AIF1, red) and Nestin (green) in Nodal iPCKO microglia compared to control. (c, d) Confocal images showing Nestin (c) and CXCL10 (d) expression in primary microglia treated with Nodal and SB431542 inhibitor. (e) Graph showing the increased expression of Nestin in Nodal iPCKO sections of brain cortex compared to control littermate. Data represents mean ± s.e.m. (n=3); P-value, unpaired two tailed student t-test. (f, g) Graphs showing that Nodal signaling reduces the expression of Nestin (f) and CXCL10 (g) in cultured mouse primary microglia, whereas SB431542 inhibitor has the opposite effect. Data represents mean ± s.e.m. (n=3 in f, g); P-value, one-way ANOVA with Tukey’s test in f; one-way ANOVA with Sidak’s test in g. Cite Share Download PDF Status: Published Journal Publication published 12 May, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5787386","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":403045451,"identity":"d11b3f14-c689-4399-be62-acfd42ff2595","order_by":0,"name":"Ralf Adams","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-3031-7677","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":true,"prefix":"","firstName":"Ralf","middleName":"","lastName":"Adams","suffix":""},{"id":403045452,"identity":"fd51fd13-768a-4fe0-ba2c-c634724d07c7","order_by":1,"name":"Seyed Javad Rasouli","email":"","orcid":"","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Javad","lastName":"Rasouli","suffix":""},{"id":403045453,"identity":"6b78bb36-8546-44d2-9bb7-62d1b88fdaf0","order_by":2,"name":"Kai Kruse","email":"","orcid":"https://orcid.org/0000-0002-7951-7357","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Kruse","suffix":""},{"id":403045454,"identity":"19af62af-f77a-4919-a353-11075a6882cf","order_by":3,"name":"Rodrigo Diéguez-Hurtado","email":"","orcid":"https://orcid.org/0000-0002-2055-599X","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Diéguez-Hurtado","suffix":""},{"id":403045455,"identity":"de782810-f5f6-4945-903c-6055c47bd5e9","order_by":4,"name":"Parisa Ghanbari","email":"","orcid":"","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Parisa","middleName":"","lastName":"Ghanbari","suffix":""},{"id":403045456,"identity":"2517ab1c-272b-4275-bffd-9569e86d6183","order_by":5,"name":"Anusha Aravamudhan","email":"","orcid":"","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Anusha","middleName":"","lastName":"Aravamudhan","suffix":""},{"id":403045457,"identity":"5f3921c5-a75e-400f-9176-48001cc50fce","order_by":6,"name":"Mara Pitulescu","email":"","orcid":"https://orcid.org/0000-0001-5322-8146","institution":"Max Planck Institute for Molecular Biomedicine","correspondingAuthor":false,"prefix":"","firstName":"Mara","middleName":"","lastName":"Pitulescu","suffix":""}],"badges":[],"createdAt":"2025-01-08 09:05:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5787386/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5787386/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-71643-1","type":"published","date":"2026-05-12T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":74680086,"identity":"60a0c19d-ab75-44f4-aa72-deb6b61b9467","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2064000,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of lung and brain pericytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eLabeling of pericytes in the P12 brain cortex with the help of \u003cem\u003ePdgfrb(BAC)-CreERT2\u003c/em\u003e-mediated, tamoxifen-induced GFP expression (green). Confocal images also show PDGFRβ (gray), a pericyte marker, and ICAM2, labeling ECs (red). Bottom panels show higher magnification of the yellow dashed boxes in top row.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Confocal images showing the proximity of GFP+ pericytes (green), GFAP+ astrocytes (magenta) and AIF+ microglia (grey) in the P12 brain cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e \u003cem\u003ePdgfrb(BAC)-CreERT2\u003c/em\u003e-mediated labeling of pericytes (GFP, green) in the P21 lung. Confocal images also show PDGFRβ (grey) and ICAM2 (red).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Maximum intensity projections showing PDGFRb+ pericytes (grey) and ICAM2+ ECs (red) in the lung alveolus. Red arrows indicate pulmonary pericytes in the alveolar septum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Confocal images showing the localization of Prosurfactant Protein C (proSP-C)-expressing type 2 alveolar epithelial cells (green) and Receptor for Advanced Glycation End Products (RAGE)-positive type 1 alveolar epithelial cells (grey).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e Graphs showing the number (per area / 82 x 82 x 6 µm) of pericytes and type 2 alveolar epithelial cells in the septal and alveolar regions of lung alveoli. Data represents mean ± s.e.m. (n=5); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g) \u003c/strong\u003eHeatmap of differentially-expressed ligands (padj\u0026lt;0.01) in pericytes from postnatal lung\u003csup\u003e29\u003c/sup\u003e versus brain.\u003c/p\u003e","description":"","filename":"RasouliMainFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/fce8154b1d05a952d4dd4dd0.png"},{"id":74680088,"identity":"d21098e1-a048-4314-b7eb-87a1226895cc","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1725700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePulmonary vascular development is regulated by BDNF-TrkB signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b)\u003c/strong\u003e Confocal images of \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs stained for ICAM2 (green) \u003cstrong\u003e(a)\u003c/strong\u003e or ERG (green) and DAPI (blue) \u003cstrong\u003e(b)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Confocal images of \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs showing PDGFRβ-labeled pericytes (green) and DAPI-stained nuclei (blue).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Confocal images of NKX2.1-stained alveolar epithelial cells (grey) and LAMP3+ type 2 alveolar epithelial cells (green) in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Confocal images of \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs showing Ki67-stained proliferating cells (red). Nuclei, DAPI (blue).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f-j)\u003c/strong\u003e Graphs showing the percentage of ICAM2+ EC surface \u003cstrong\u003e(f)\u003c/strong\u003e, ratio of ERG+ EC nuclei to total cells \u003cstrong\u003e(g)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eratio of ERG+ cells to PDGFRb+ cells \u003cstrong\u003e(h)\u003c/strong\u003e, ratio of AT2 epithelial cells to total cells \u003cstrong\u003e(i),\u003c/strong\u003e and ratio of proliferating cells to total cells \u003cstrong\u003e(j) \u003c/strong\u003ein P12 \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs. Data represents mean ± s.e.m. (n=4 (\u003cstrong\u003ef\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e), n=6 (\u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e and \u003cstrong\u003ej\u003c/strong\u003e); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(k)\u003c/strong\u003e Confocal images showing TrkB immunostaining (grey) in ICAM2+ (red) ECs in pulmonary capillaries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i-m)\u003c/strong\u003e Validation of expression of \u003cem\u003eNtrk2\u003c/em\u003e (encoding TrkB) in\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eECs by scRNA-seq analysis. UMAP plot showing color-coded EC subclusters, namely arterial, venous, general capillary (gCap) and aerocytes (aCap), in P21 lung \u003cstrong\u003e(i)\u003c/strong\u003e. \u003cem\u003eNtrk2\u003c/em\u003e expression in gCap endothelial cells \u003cstrong\u003e(m)\u003c/strong\u003e. \u003cstrong\u003e(n) \u003c/strong\u003eWestern blot showing TrkB and Phospho-TrkB (p-TrkB) in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control total lung lysates (n=4 control and 3 mutants). Molecular weight marker (kDa) is indicated. β-Actin is shown as loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(o-p)\u003c/strong\u003e Confocal images of ICAM2+ (red) and ERG+ (green) endothelial cells in EC-specific \u003cem\u003eNtrk2\u003c/em\u003e\u003csup\u003eiDEC\u003c/sup\u003e loss-of-function mutant and littermate control lungs \u003cstrong\u003e(o)\u003c/strong\u003e. Confocal images of \u003cem\u003eNtrk2\u003c/em\u003e\u003csup\u003eiDEC\u003c/sup\u003e and control lungs showing Ki67+ proliferating cells (red). Nuclei, DAPI (blue) \u003cstrong\u003e(p)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(q-s) \u003c/strong\u003eGraphs showing the percentage of ICAM2+ surface \u003cstrong\u003e(q)\u003c/strong\u003e, ratio of ERG+ cells to total cells \u003cstrong\u003e(r)\u003c/strong\u003e, ratio of proliferating cells to total cells \u003cstrong\u003e(s)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eratio of ERG+ cells to PDGFRb+ cells \u003cstrong\u003e(t)\u003c/strong\u003e, in \u003cem\u003eNtrk2\u003c/em\u003e\u003csup\u003eiDEC\u003c/sup\u003e and littermate control lungs. Data represents mean ± s.e.m. (n= 4); P-values, unpaired two tailed student t-test).\u003c/p\u003e","description":"","filename":"RasouliMainFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/e3440f7b5d9c13dcb4e428f4.png"},{"id":74680089,"identity":"9fe395dc-cbfb-4212-8b78-df313fe0506c","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1939736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePericyte-derived HGF promotes lung alveolarization.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Confocal images of P21 lung section showing proSP-C+ AT2 cells (red) and co-staining by c-Met (green). \u003cstrong\u003e(b)\u003c/strong\u003e Tile scan confocal view of RAGE+ AT1 cells (green) in PDGFRβ cell-specific \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e loss-of-function and littermate control lungs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c-e)\u003c/strong\u003e Confocal images of NKX2.1+ alveolar epithelial cells (grey) and LAMP3+ AT2 cells (red) \u003cstrong\u003e(c)\u003c/strong\u003e, c-Met (green) and LAMP3 (red) co-staining \u003cstrong\u003e(d),\u003c/strong\u003e and LAMP3+ AT2 cells (green) together with DAPI (blue) \u003cstrong\u003e(e)\u003c/strong\u003e in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs. \u003cstrong\u003e(f-h)\u003c/strong\u003e Graphs showing the percentage of LAMP3 positive cells in total cells \u003cstrong\u003e(f)\u003c/strong\u003e, the percentage of proliferative LAMP3+ cells \u003cstrong\u003e(g)\u003c/strong\u003e, and the percentage of apoptotic cells \u003cstrong\u003e(h)\u003c/strong\u003e in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs. Data represents mean ± s.e.m. (n= 4); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i-k)\u003c/strong\u003e Confocal images of \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control lungs stained for LAMP3 (green) and Ki67 (red) \u003cstrong\u003e(i)\u003c/strong\u003e, c-Caspase3 (grey) and nuclei (DAPI, blue) \u003cstrong\u003e(j)\u003c/strong\u003e, RAGE (red) and Aqp5 (green) \u003cstrong\u003e(k)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(l)\u003c/strong\u003e Graph showing the percentage of RAGE positive area. Data represents mean ± s.e.m. (n= 6); P-value, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(m-o)\u003c/strong\u003e Violin plots showing expression of AT2 cell-specific markers \u003cstrong\u003e(m)\u003c/strong\u003e, proliferation markers in AT2 cells \u003cstrong\u003e(n),\u003c/strong\u003e and expression of type 1 and 2 alveolar epithelial cell-specific markers \u003cstrong\u003e(o)\u003c/strong\u003e in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(p, q)\u003c/strong\u003e Confocal images of PECAM1+ pulmonary endothelium (grey) \u003cstrong\u003e(p)\u003c/strong\u003e and ERG+ ECs (red) together with PDGFRb+ pericytes (grey) in P21 \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control lungs \u003cstrong\u003e(q)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(r, s)\u003c/strong\u003e Graphs showing the PECAM1+ endothelial area\u003cstrong\u003e (r)\u003c/strong\u003e and ratio of ERG+ cells to PDGFRb+ cells in P21 \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control lungs \u003cstrong\u003e(s)\u003c/strong\u003e. Data represents mean ± s.e.m. (n=7 for controls, n=9 for mutants in \u003cstrong\u003er\u003c/strong\u003e, and n=4 in \u003cstrong\u003es\u003c/strong\u003e); P-values, unpaired two tailed student t-test.\u003c/p\u003e","description":"","filename":"RasouliMainFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/f6c69f0f5bd535f36a619f19.png"},{"id":74680091,"identity":"89d071ba-60af-45f6-9ec2-26954bb79088","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2301180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePericyte-derived Nodal is required for brain vascular development.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTile scan of coronal sections through P12 \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain cortex immunostained for ICAM2 (green).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Confocal images of ICAM2+ (green) and ERG+ (red) endothelial cells in \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Graph showing the percentage of ICAM2 positive area. Data represents mean ± s.e.m. (n=8); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d, e)\u003c/strong\u003e Maximum intensity projections of P12 \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain sections. Images show PDGFRb+ pericytes (grey) and ERG+ (red) ECs \u003cstrong\u003e(d)\u003c/strong\u003e, and EdU+ (grey) proliferating cells together ICAM2+ (green) ECs (\u003cstrong\u003ee\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f-h)\u003c/strong\u003e Graphs showing the number of ERG+ cells per area (212 x 212 x 32 µm) \u003cstrong\u003e(f)\u003c/strong\u003e, the ratio of ERG+ to PDGFRb+ cells \u003cstrong\u003e(g),\u003c/strong\u003e and the number of EdU+ ECs per area (283 x 283 x 22 µm) \u003cstrong\u003e(h)\u003c/strong\u003e. Data represents mean ± s.e.m. (n=6 in \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(i) \u003c/strong\u003eElectron micrographs of brain cortex showing the tight association of ECs (red arrows) and pericytes (white arrows) in control. Note irregular protrusions emerging from \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e ECs (blue arrows) and enlargement of the sub-endothelial basement membrane (yellow arrows).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(j)\u003c/strong\u003e Western blot analysis of total and phosphorylated SMAD2 (p-SMAD2) in lysates of mouse brain endothelial cells (bEnd.3) treated with Nodal and SB431542 inhibitor, as indicated. Molecular weight marker (kDa) is indicated. β-Actin is shown as loading control.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ek) \u003c/strong\u003eConfocal images of VE-cadherin+ (CDH5, green) and ERG+ (red) bEnd.3 cells treated with Nodal in the presence/absence of SB431542 inhibitor.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003el) \u003c/strong\u003eBright field images showing that Nodal increases bEnd.3 cell migration \u003cem\u003ein vitro\u003c/em\u003e in a scratch wound assay, which is blocked by SB431542. Left column shows start of assay (0h) and red lines indicate the edges of scratch wounds, whereas images on the right are taken after 26h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(m-n) \u003c/strong\u003eGraphs showing the impact of Nodal on bEnd.3 cell proliferation \u003cstrong\u003e(m)\u003c/strong\u003e and migration \u003cstrong\u003e(n)\u003c/strong\u003e. Data represents mean ± s.e.m. (n=6); P-values, one-way ANOVA with Sidak’s test in\u003cstrong\u003e m\u003c/strong\u003e; one-way ANOVA with Tukey’s test in \u003cstrong\u003en\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"RasouliMainFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/0b656bcd929a20e7401d335e.png"},{"id":74680095,"identity":"caf8fdec-f175-497c-aebe-4929967a3b71","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1465428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNodal regulates astrocyte reactivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Tile scan of sagittal sections through\u003cstrong\u003e \u003c/strong\u003ebrains stained for GFAP (white). Yellow arrows indicate increase in GFAP+ reactive astrocytes in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e cortex relative to littermate control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Higher magnification confocal images of GFAP+ astrocytes in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO \u003c/sup\u003ebrain cortex relative to control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Tile scan confocal view of P12 \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and control coronal brain sections immunostained for GFAP (white). Panels on the right show higher magnification of areas in red boxes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e Quantitation of GFAP+ area in the \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain cortex. Data represents mean ± s.e.m. (n=6); P-value, unpaired two tailed student t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Electron micrographs showing astrocytes of mouse brain cortex reactive astrocytes in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e. Red arrows indicate glycogen granules and blue arrows mark intermediate filaments in mutant astrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e UMAP projections showing reactive astrocytes (red dots) versus resting astrocytes (blue dots). Ratio of the two populations is displayed in bar graph on the right.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e Violin plots showing the upregulation of markers of reactive astrocytes in scRNA-seq samples from \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e relative to control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(j)\u003c/strong\u003e Western blot analysis of SMAD2 and phosphorylated SMAD2 (p-SMAD2) in lysates from cultured mouse primary astrocytes treated with Nodal and SB431542 inhibitor, as indicated. Quantitation of p-SMAD2/SMAD2 ratio is shown in graph on the right. Data represents mean ± s.e.m. (n=6); P-values, one-way ANOVA with Sidak’s test.\u003c/p\u003e","description":"","filename":"RasouliMainFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/1bdbc5e15659777ef9154e5d.png"},{"id":74680090,"identity":"8dc65744-32af-4fee-ad4a-e2c3f0435e23","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":887569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia activation is controlled by pericyte-derived Nodal.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e P12 Brain cortex of \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO \u003c/sup\u003eand control littermates stained for AIF1 (red) and CD68 (green).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Western blot analysis of AIF1 and CD68 proteins in brain cortex lysates from P12 \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and control littermates. Molecular weight marker (kDa) is indicated. β-Actin and α-Tubulin are shown as loading controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Confocal images showing AIF1 (magenta) immunostaining of \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO \u003c/sup\u003eand control brain cortex sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e UMAP projections showing activated microglia (red dots) versus resting microglia (blue dots) in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO \u003c/sup\u003eand control scRNA-seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Graph showing the ratio of resting, activated and proliferating microglia in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO \u003c/sup\u003eand control brain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e Violin plots showing increased expression of markers of activated microglia in P12 \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e brain cortex relative to control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e UMAP plots showing the gene set score (scanpy score_genes) for type 1 (M1) and 2 (M2) microglia\u003cstrong\u003e \u003c/strong\u003ein \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e versus control brain cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(h)\u003c/strong\u003e Western blot analysis of total SMAD2 and phosphorylated SMAD2 (p-SMAD2) in lysates from cultured mouse primary microglia treated with rhNodal and SB431542 inhibitor, as indicated. Molecular weight marker (kDa) is indicated. β-Actin is shown as loading control. Graph on the right shows quantitation of p-SMAD2/SMAD2 ratio. Data represents mean ± s.e.m. (n=3); P-values, one-way ANOVA with Sidak’s test.\u003c/p\u003e","description":"","filename":"RasouliMainFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/b01c272382e3495a0dbe25ad.png"},{"id":74680092,"identity":"401b7c26-57bd-4642-a1a8-17e4b8460d07","added_by":"auto","created_at":"2025-01-24 15:42:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":377059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of findings.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a-b)\u003c/strong\u003e Schematic representation of the modulation of tissue microenvironments in lung \u003cstrong\u003e(a)\u003c/strong\u003e and brain \u003cstrong\u003e(b)\u003c/strong\u003e by pericyte-derived factors.\u003c/p\u003e","description":"","filename":"RasouliMainFigures7.png","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/3cf9b565f1b9508c2dbe1d23.png"},{"id":109158052,"identity":"2acd45bc-b082-40e5-9f42-29b18dd6c405","added_by":"auto","created_at":"2026-05-13 07:05:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11831268,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/cc034cd8-d90d-40e7-9536-d6b8980708e7.pdf"},{"id":74680096,"identity":"b29d68b7-97f0-432e-813f-e03fedade3cd","added_by":"auto","created_at":"2025-01-24 15:42:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":227048288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. scRNA-seq analysis of P21 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBdnf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eiPCKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and control lungs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Heatmap of top marker genes cell populations based on computational analysis of scRNA-seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e UMAP plot of annotated cell types from lung together with corresponding cell counts.\u003c/p\u003e\n\u003cp\u003eIndicated are AT1 epithelial cells, AT1-AT2 transitional/intermediate cells, \u003cem\u003eLyz1\u003c/em\u003e+ AT2 and \u003cem\u003eLyz1\u003c/em\u003e- AT2 epithelial cells, Lymphatic endothelial cells, Matrix fibroblast, Myofibroblast.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Volcano plot showing the differential expression analysis in P21 \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control pulmonary ECs.\u0026nbsp; Average log2-fold change \u0026gt; 0.6, adjusted p-value \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d) \u003c/strong\u003eViolin plots showing expression of multiple endothelial markers (\u003cem\u003ePlvap\u003c/em\u003e, \u003cem\u003eGja5\u003c/em\u003e, \u003cem\u003eAplnr\u003c/em\u003e and Itga1), and proliferation markers (\u003cem\u003eCcnd1\u003c/em\u003e and \u003cem\u003eMki67\u003c/em\u003e) in pulmonary ECs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Violin plot of \u003cem\u003eNtrk2 \u003c/em\u003eexpression in endothelial, epithelial, immune, and mesenchymal cells (including fibroblasts) from lung.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. Analysis of the P12 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBdnf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eiPCKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e brain vasculature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Confocal images of brain vasculature stained with ICAM2 (green) and EdU (white).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b, c)\u003c/strong\u003e Graphs showing the percentage of ICAM2+ area \u003cstrong\u003e(b)\u003c/strong\u003e and number of EdU+ ECs \u003cstrong\u003e(c)\u003c/strong\u003e in \u003cem\u003eBdnf \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain cortex. Data represents mean ± s.e.m. (n=6 controls and 8 mutants in \u003cstrong\u003eb\u003c/strong\u003e, n=4 controls and 5 mutants in \u003cstrong\u003ec\u003c/strong\u003e); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e PDGFRb immunostaining (green) showing comparable pericyte coverage in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Graph showing percentage of pericyte coverage in P12 brain cortex. Data represents mean ± s.e.m. (n=4 controls and 5 mutants); P-value, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control brain vasculature stained for ICAM2 (green) and Collagen IV (grey), as indicated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e Confocal images of \u003cem\u003eBdnf \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate controls cortex stained for ICAM2 (green) and mouse IgG (red). \u003cstrong\u003e(h)\u003c/strong\u003e Astrocyte endfeet covering ICAM2+ endothelial cells (green) are visualized with AQP4 (grey). \u003cstrong\u003e(i, j)\u003c/strong\u003e Confocal images of brain cortex showing no change in the activation of GFAP+ astrocytes (grey) \u003cstrong\u003e(i)\u003c/strong\u003e or AIF+ microglia (magenta) \u003cstrong\u003e(j)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. Analysis of the P12 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHgf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eiPCKO\u003c/strong\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebrain vasculature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Confocal images showing GLUT1+ (red) brain endothelium and PDGFRb+ (white) pericytes in P12 cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b,c) \u003c/strong\u003eGraphs showing the percentage of ICAM2+ area \u003cstrong\u003e(b)\u003c/strong\u003e and pericyte coverage \u003cstrong\u003e(c) \u003c/strong\u003ein brain cortex. Data represents mean ± s.e.m. (n=6 in \u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003e3\u003c/strong\u003e in c); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain endothelium stained by isolectin B4 (IB4, green) together with Collagen IV (red) immunostaining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Confocal images of brain cortex showing ICAM2+ vasculature (green) and AQP4+ astrocyte endfeet coverage (red) (\u003cstrong\u003ee\u003c/strong\u003e), GFAP expression (white) (\u003cstrong\u003ef\u003c/strong\u003e), the microglia-specific marker AIF1 (white) (\u003cstrong\u003eg\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. scRNA-seq analysis of P21 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHgf\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eiPCKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and control lungs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b)\u003c/strong\u003e Heatmap of top marker genes within lung cell types based on scRNA-seq results (\u003cstrong\u003ea\u003c/strong\u003e) and UMAP plot of annotated lung cell types with the corresponding cell counts (\u003cstrong\u003eb\u003c/strong\u003e). Indicated are AT1 epithelial cells, AT1-AT2 transitional/intermediate cells, \u003cem\u003eLyz1\u003c/em\u003e+ AT2 and \u003cem\u003eLyz1\u003c/em\u003e- AT2 epithelial cells, Lymphatic endothelial cells, Matrix fibroblast, Myofibroblast.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) \u003c/strong\u003eViolin plot showing that \u003cem\u003eMet\u003c/em\u003e expression dominates in epithelial cells relative to endothelial, immune and mesenchymal cells from P21 lung.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e UMAP plot of annotated alveolar cell types. Indicated are AT1 epithelial cells, AT1-AT2 transitional/intermediate cells, \u003cem\u003eLyz1\u003c/em\u003e+ AT2 and \u003cem\u003eLyz1\u003c/em\u003e- AT2 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e UMAP plot showing \u003cem\u003eLyz1 \u003c/em\u003eexpression in a subcluster of AT2 epithelial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f)\u003c/strong\u003e UMAP plots showing \u003cem\u003eMet\u003c/em\u003e expression in AT1-AT2, \u003cem\u003eLyz1\u003c/em\u003e+ AT2 and \u003cem\u003eLyz1\u003c/em\u003e- AT2 cells from \u003cem\u003eHgf \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(g)\u003c/strong\u003e Volcano plot showing the differential expression analysis of endothelial-specific responses in \u003cem\u003eHgf \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs. Average log2-fold change \u0026gt; 0.6, adjusted p-value \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5. Normal lung development in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNodal \u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003eiPCKO\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mutants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b) \u003c/strong\u003eConfocal images of endothelium (PECAM1+, grey) \u003cstrong\u003e(a)\u003c/strong\u003e, EC nuclei (ERG+, green) and pericytes (PDGFRb+, white) \u003cstrong\u003e(b)\u003c/strong\u003e, which show pulmonary vascular development is not affected in P21 \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e mutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c, d)\u003c/strong\u003e Maximum intensity projections of \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and control lung sections showing AT1 cells (RAGE+, grey) \u003cstrong\u003e(c)\u003c/strong\u003e, nuclei of AT1 and AT2 cells (NKX2.1+, green), and type 2 alveolar epithelial cells (LAMP3+, white) \u003cstrong\u003e(d)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e) \u003c/strong\u003eConfocal image of proliferating cells (Ki67+, red) and DAPI-stained nuclei (blue) in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f-i) \u003c/strong\u003eGraphs showing the percentage of PECAM1+ area \u003cstrong\u003e(f)\u003c/strong\u003e, percentage of ERG+ cells in total cells \u003cstrong\u003e(g)\u003c/strong\u003e, ratio of ERG+ to PDGFRb+ cells \u003cstrong\u003e(h)\u003c/strong\u003e, percentage of AT1 (RAGE+) area \u003cstrong\u003e(i)\u003c/strong\u003e, percentage of LAMP3+ AT2 cells \u003cstrong\u003e(j)\u003c/strong\u003e and percentage of proliferating cells (Ki67+) in total cells \u003cstrong\u003e(k)\u003c/strong\u003e. Data represent mean ± s.e.m. (n=4 controls and 7 mutants in \u003cstrong\u003ef\u003c/strong\u003e, n=4 controls and 5 mutants in \u003cstrong\u003eg-k\u003c/strong\u003e); P-values, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 6. scRNA-seq analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNodal\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression in brain.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Heatmap of top marker genes within brain cell types using based on scRNA-seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e UMAP plot of annotated brain cell types. Each cell type is represented by distinct clusters and the corresponding cell counts are indicated. PVMs, perivascular macrophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) \u003c/strong\u003eUMAP plots show enriched expression of Nodal in control brain pericytes relative to other cell types and loss of expression in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e pericytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d)\u003c/strong\u003e The expression of \u003cem\u003eNodal\u003c/em\u003e, \u003cem\u003ePdgfrb \u003c/em\u003eand transcripts for TGFβ family receptors in published scRNA-seq data of postnatal mouse brain. Dendrogram (obtained from mousebrain.org) displaying the taxonomy of all identified cell types from postnatal mouse brain and expression of the indicated genes. Note presence of TGFβ family receptor transcripts in many cell types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e)\u003c/strong\u003e Circle plots of scRNA-seq data of adolescent mouse brain\u003csup\u003e33\u003c/sup\u003e showing the expression of \u003cem\u003eNodal\u003c/em\u003e, \u003cem\u003ePdgfrb, Myh11 \u003c/em\u003eand TGFβ family receptor transcripts in mural cell populations.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 7. Characterization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNodal\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eiPCKO \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003ebrain capillaries.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Confocal images of P12 brain cortex stained for mouse immunoglobulin G (IgG, white) from \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and control littermates injected with Texas Red-dextran (70 kDa) (red). Green arrows indicate local leakage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb)\u003c/strong\u003e Maximum intensity projections showing comparable coverage of Aquaporin-4+ (AQP4+) astrocyte endfeet (grey) of ICAM2+ endothelium (green) in \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and control brain cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c)\u003c/strong\u003e Confocal images of GLUT1+ (green) capillaries in brain cortex. Red blood cells (Ter119, red) are confined to vessels in control but leakage (arrows, white arrows) into the brain parenchyma and GFAP+ astrocytes (white, yellow arrowheads) can be seen in \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e mutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d, e)\u003c/strong\u003e Confocal images of CD31+ (green) brain capillaries with area of extravasated IgG (red, white arrows) in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e mutant surrounded by GFAP+ astrocytes (white, yellow arrows) in brain cortex \u003cstrong\u003e(d)\u003c/strong\u003e. However, reactive astrocytes (yellow arrows) can also be observed in areas of \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e cortex lacking extravasated IgG (asterisks) \u003cstrong\u003e(e)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 8. Nodal-dependent expression of reactive astrocyte markers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Volcano plot showing differential gene expression in astrocytes from \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control cortex. Average log2-fold change \u0026gt; 0.6, adjusted p-value \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b, c)\u003c/strong\u003e Confocal images of P12 \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e and littermate control brain cortex. Mutant GFAP+ (red) astrocytes show nuclear enrichment of Sox2 (green, white arrows) (\u003cstrong\u003eb\u003c/strong\u003e) as well as Sox9 (green, blue arrows) compared to control astrocytes (\u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(d) \u003c/strong\u003eExpression of Sox9 (green) and GFAP (red) in cultured mouse primary astrocytes\u003cstrong\u003e \u003c/strong\u003etreated with recombinant human Nodal (rhNodal) and SB431542 inhibitor. Inhibition of TGFβ signaling increases nuclear SOX9 (yellow arrows).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 9. TGFβ signaling controls levels of nuclear FOS and FOSB.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Confocal images of brain cortex stained for FOS (green) and GFAP (red). White arrows indicate nuclear enrichment of FOS in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e astrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b, c)\u003c/strong\u003e Inhibition of TGFβ signaling with SB431542 increases nuclear FOS (green, yellow arrows) (\u003cstrong\u003eb\u003c/strong\u003e) and FOSB (green, orange arrows) (\u003cstrong\u003ec\u003c/strong\u003e) in cultured mouse primary astrocytes.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 10. Marker genes of activated microglia are negative regulated by Nodal signaling.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Volcano plot showing the differential expression of genes in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e microglia relative to control cells. Average log2-fold change \u0026gt; 0.6, adjusted p-value \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b)\u003c/strong\u003e Increased\u003cstrong\u003e \u003c/strong\u003eexpression of Allograft inflammatory factor-1 (AIF1, red) and Nestin (green) in \u003cem\u003eNodal\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e microglia compared to control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c, d)\u003c/strong\u003e Confocal images showing Nestin \u003cstrong\u003e(c)\u003c/strong\u003e and CXCL10 \u003cstrong\u003e(d)\u003c/strong\u003e expression in primary microglia treated with Nodal and SB431542 inhibitor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(e) \u003c/strong\u003eGraph showing the\u003cstrong\u003e \u003c/strong\u003eincreased\u003cstrong\u003e \u003c/strong\u003eexpression of Nestin in \u003cem\u003eNodal \u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e sections of brain cortex compared to control littermate. Data represents mean ± s.e.m. (n=3); P-value, unpaired two tailed student t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(f, g)\u003c/strong\u003e Graphs showing that Nodal signaling reduces the expression of Nestin \u003cstrong\u003e(f)\u003c/strong\u003e and CXCL10 \u003cstrong\u003e(g)\u003c/strong\u003e in cultured mouse primary microglia, whereas SB431542 inhibitor has the opposite effect. Data represents mean ± s.e.m. (n=3 in \u003cstrong\u003ef, g\u003c/strong\u003e); P-value, one-way ANOVA with Tukey’s test in\u003cstrong\u003e f\u003c/strong\u003e; one-way ANOVA with Sidak’s test in \u003cstrong\u003eg\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"RasouliSupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5787386/v1/9668c562942d3bee9a1532e4.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Pericytes are organ-specific regulators of tissue morphogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBlood vessels form a highly elaborate, hierarchical network that reaches, with a few exceptions, into all parts of the vertebrate body. In addition to the essential transport function of the vasculature, endothelial cells provide critical molecular signals acting on other cell populations in their vicinity. This paracrine (also termed \u0026lsquo;angiocrine\u0026rsquo;) function of endothelial cells regulates morphogenesis, homeostasis and regeneration of different organs including lung, liver, heart, and bone\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Endothelial cells (ECs) are also a critical component of stem cell niches and thereby play key roles in hematopoiesis, bone formation, and neurogenesis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Thus, ECs have emerged as important signaling centers that acquire organ-specific specialization, coordinate regeneration and help to prevent deregulated, disease-promoting processes. Even though the range of EC-derived signals has been explored only partially, it is evident that the angiocrine function of capillary EC is reflected by specialized expression of certain genes, distinguishing different organs but also local environments within the same organ\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.\u003c/p\u003e \u003cp\u003ePericytes, capillary-associated supporting cells, are another essential component of the vessel wall and help to preserve vascular integrity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Pericytes but also the other mural cell population, vascular smooth muscle cells (vSMCs), are fairly heterogeneous with regard to their morphology but also developmental origin. Pericytes and vSMCs in the developing heart, for example, can either arise from epicardial mesothelial cells or, as we have shown, via endothelial-to-mesenchymal transition from the embryonic endocardium\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Neural crest cells give rise to pericytes in the retina, brain, thymus, and the head region\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In gut, lung and liver, the mesothelium, a single-layer squamous epithelium, is a source of mural cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Distinct developmental origins and tissue environments may also explain the expression of specific molecular markers by pericytes in different organs, including a range of secreted molecules, as was uncovered by single cell RNA-sequencing (scRNA-seq) and other approaches\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Likewise, scRNA-seq has revealed substantial organ-specific gene expression profiles of pericytes and other vessel-associated cells in adult brain and lung\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our own previous work has established that pulmonary pericytes control the proliferation of epithelial cells and are thereby indispensable for alveologenesis during postnatal lung development\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStimulated by the findings above, we have utilized scRNA-seq in combination with mouse genetics and cell culture approaches to explore whether pericytes act as organ-specific regulators of tissue morphogenesis during postnatal development. We chose lung and brain as representative model organs and explored the function of several pericyte-derived, potentially angiocrine-acting factors, namely brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF) and Nodal, a TGFβ family protein. Our findings establish that pericytes are indeed functionally specialized in an organ-specific fashion and regulate the behavior of other cell types in their vicinity through the secretion of angiocrine signals.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003echaracterization of lung and brain pericytes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate pericyte morphology in lung and brain, we employed a genetic labeling strategy involving \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e transgenic mice\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e in combination with the \u003cem\u003eR26-mT/mG\u003c/em\u003e Cre reporter\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Following the administration of a low dose of 4-hydroxytamoxifen (4-OHT) at postnatal day (P) 1, Cre-mediated recombination and irreversible expression of green fluorescent protein (GFP) labelled a limited number of mural cells in different postnatal organs. Analysis of GFP\u0026thinsp;+\u0026thinsp;cells by confocal microscopy at high resolution (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea-c) revealed substantial differences in pericyte abundance and morphology. GFP\u0026thinsp;+\u0026thinsp;pericytes in the postnatal brain cortex at P12 extend numerous short cellular protrusions and are densely covering capillaries (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). Moreover, \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-labeled brain pericytes are located in close proximity to astrocytes expressing Glial fibrillary acidic protein (GFAP) and microglia expressing Allograft inflammatory factor 1 (AIF1) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). In contrast, lung pericytes exhibit a different morphology, are comparably sparse and extend long cellular processes contacting ECs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). In both brain and lung, there is good colocalization between GFP signal and PDGFR\u0026beta; immunostaining, which is most prominent on the cell body (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, c). In the lung, very little GFP signal is seen outside of the vasculature, consistent with previous work showing that activation of \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-mediated recombination with tamoxifen administration at P1\u0026ndash;P3 does not lead to labeling of PDGFR\u0026alpha;\u0026thinsp;+\u0026thinsp;fibroblasts, alpha smooth muscle actin (\u0026alpha;SMA)\u0026thinsp;+\u0026thinsp;bronchial smooth muscle cells or myofibroblasts\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In addition, PDGFR\u0026beta; immunostaining of pericytes is predominantly confined to the alveolar septum, the tissue between apposing alveolar walls (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed, f). Immunostaining indicates that alveolar type 2 (AT2) epithelial cells expressing Prosurfactant Protein C (proSP-C), which act as stem cells in the pulmonary epithelium and give rise to terminally differentiated AT1 cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, are also enriched in the septum region (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee, f).\u003c/p\u003e\n\u003cp\u003eAs previous work has uncovered evidence for the expression of organ-specific markers both in mice and humans\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, we aimed at the identification of pericyte-derived, paracrine-acting factors that might potentially control morphogenetic processes during postnatal development. Comparing a public scRNA-seq resource for mouse lung provided by the Th\u0026eacute;baud laboratory\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e with our own scRNA-seq data, which is introduced later in the course of this article, indicates that pericytes from wild-type postnatal lung and brain express the expected general mural cell markers, namely \u003cem\u003ePdgfrb\u003c/em\u003e (encoding Platelet-derived growth factor receptor \u0026beta;, PDGFR\u0026beta;), \u003cem\u003eCspg4\u003c/em\u003e (Chondroitin Sulfate Proteoglycan 4), and \u003cem\u003eNotch3\u003c/em\u003e, which encodes a Notch family receptor (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). In addition, pericytes from lung and brain show organ-specific gene expression. In particular, we identified several differentially expressed transcripts for secreted factors. \u003cem\u003eHgf\u003c/em\u003e (encoding hepatocyte growth factor) and \u003cem\u003eBdnf\u003c/em\u003e (brain-derived neurotrophic factor), a regulator of axon guidance through its receptor TrkB/Ntrk2\u003csup\u003e30,31\u003c/sup\u003e, are found in pericytes of the lung but are not detectable in brain (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). \u003cem\u003eAngpt1\u003c/em\u003e (Angiopoietin 1) an important regulator of vascular growth and integrity, shows a similar distribution pattern of higher expression in lung relative to brain pericytes, which is consistent with previous work\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and published scRNA-seq results from adult lung and brain\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In turn, \u003cem\u003eNodal\u003c/em\u003e, a member of the TGF\u0026beta; superfamily, is enriched in brain pericytes relative to lung (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg). Based on these results, we used an inducible genetic strategy involving the \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e line in combination with loxP-flanked alleles for three candidate genes, namely \u003cem\u003eHgf\u003c/em\u003e, \u003cem\u003eBdnf\u003c/em\u003e and \u003cem\u003eNodal\u003c/em\u003e, to investigate the role of pericyte-derived signals in organ development.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePericyte-derived BDNF controls pulmonary angiogenesis and development\u003c/h2\u003e\n \u003cp\u003eTo investigate the function of \u003cem\u003eBdnf\u003c/em\u003e in pulmonary pericytes in mice, conditional \u003cem\u003eBdnf\u003c/em\u003e alleles were introduced into the \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e background. Following tamoxifen administration at P1-3, ICAM2 immunostaining shows that vascularization is reduced in P21 lungs of the resulting \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants relative to littermate controls (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, f). The number of ECs visualized by nuclear ERG immunostaining is decreased (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb, g), whereas the ratio of ERG\u0026thinsp;+\u0026thinsp;to PDGFR\u0026beta;\u0026thinsp;+\u0026thinsp;cells in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs is not significantly changed (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, h). Immunostaining for NKX2.1 (a transcription factor expressed by both AT1 and AT2 cells) and LAMP3 (an AT2 cell marker) shows that number of AT1 and AT2 epithelial cells is also decreased in P21 \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, i). Likewise, lung immunostaining for the proliferation marker Ki67 antibody shows a significant reduction of dividing cells in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs compared to controls \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, j\u003cstrong\u003e)\u003c/strong\u003e. Bioinformatic analysis of scRNA-seq data from P21 \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate control lungs shows significant changes in gene expression in the mutant endothelium \u003cstrong\u003e(Supplementary Fig.\u0026nbsp;1a-c).\u003c/strong\u003e Analysis of scRNA-seq results reveals reduced expression of multiple endothelial markers including \u003cem\u003ePlvap\u003c/em\u003e, (encoding plasmalemma vesicle associated protein, associated with EC permeability), \u003cem\u003eGja5\u003c/em\u003e (encoding Gap Junction Protein Alpha 5 protein), \u003cem\u003eItga1\u003c/em\u003e(encoding Integrin Subunit Alpha 1), and \u003cem\u003eAplnr\u003c/em\u003e (Apelin receptor) together with a reduction in the proliferation markers \u003cem\u003eCcnd1\u003c/em\u003e (Cyclin D1), \u003cem\u003eCcna2\u003c/em\u003e (Cyclin A2) and \u003cem\u003eMki67\u003c/em\u003e (Ki67) in mutant lungs \u003cstrong\u003e(Supplementary Fig.\u0026nbsp;1c, d)\u003c/strong\u003e. Arguing that pericyte-derived BDNF might act on ECs, the receptor tyrosine kinase TrkB, a high affinity receptor of BDNF encoded by the gene \u003cem\u003eNtrk2\u003c/em\u003e \u003csup\u003e34\u003c/sup\u003e, is expressed by ICAM2\u0026thinsp;+\u0026thinsp;ECs in P21 lungs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ek). Similarly, scRNA-seq data shows that \u003cem\u003eNtrk2\u003c/em\u003e is predominantly expressed by general capillary (gCap) ECs but also by mesenchymal cells in P21 lung (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003el, m and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;1e\u003c/strong\u003e). TrkB phosphorylation at tyrosine residue 515 (Tyr515), an indicator of receptor activation, in substantially reduced in lysates from P21 \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs relative to control samples (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003en).\u003c/p\u003e\n \u003cp\u003eGiven that vascularization is a prerequisite for normal pulmonary epithelial morphogenesis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and because of the endothelial \u003cem\u003eNtrk2\u003c/em\u003e expression (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ek, m), we used a conditional gene targeting strategy to study the function of the BDNF receptor in ECs. \u003cem\u003eCdh5-CreERT2-\u003c/em\u003econtrolled inactivation of \u003cem\u003eNtrk2\u003c/em\u003e, induced by tamoxifen administration at P1-3, leads to the reduction of ICAM2\u0026thinsp;+\u0026thinsp;area, ERG\u0026thinsp;+\u0026thinsp;cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eo, q, r) and proliferative cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ep, s) in the resulting \u003cem\u003eNtrk2\u003c/em\u003e\u003csup\u003ei\u0026Delta;EC\u003c/sup\u003e mutant lungs at P21. However, the ratio ECs to pericytes is comparable in \u003cem\u003eNtrk2\u003c/em\u003e\u003csup\u003ei\u0026Delta;EC\u003c/sup\u003e and littermate control lungs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003et), consistent with the findings in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants. Overall, these data indicate that pericyte-derived BDNF controls TrkB signaling in ECs and thereby pulmonary angiogenesis, which, in turn, is indispensable for lung development. These findings are consistent with earlier studies proposing roles of BDNF-TrkB signaling in the regulation of integrin or PI3K/Akt-mediated EC migration as well as cell survival\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePericyte-derived BDNF or HGF are dispensable in the postnatal brain\u003c/h3\u003e\n\u003cp\u003eQuantitative analysis of the ICAM2\u0026thinsp;+\u0026thinsp;vascular area and number of proliferating ECs (ICAM2+, EdU+) in the P12 \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain shows no significant differences relative to littermate controls (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2a-c\u003c/strong\u003e). In addition, pericyte coverage, visualized as vessel-associated PDGFR\u0026beta;\u0026thinsp;+\u0026thinsp;immunostaining, remains unaltered in the postnatal \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2d, e\u003c/strong\u003e). We also did not observe any changes in collagen IV expression, which is a major constituent of the vascular basement membrane\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2f\u003c/strong\u003e). Impairment of the blood-brain barrier (BBB) can induce neuronal injury and neuroinflammation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Double immunostaining for blood-derived immunglobulin G (IgG) shows that signal is absent within the brain parenchyma, whereas IgG is readily detectable inside the vasculature (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2g\u003c/strong\u003e). Moreover, confocal images of ICAM2\u0026thinsp;+\u0026thinsp;cortical blood vessels show no obvious defects in Aquaporin-4-expressing (AQP4+) astrocyte endfeet, indicating that this key component of the neurovascular unit is maintained in the \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain vasculature (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2h\u003c/strong\u003e). Reactive astrogliosis, a typical response to CNS injury\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, is also absent, as indicated by immunostaining for GFAP. Likewise, immunostaining for AIF1 is comparable in \u003cem\u003eBdnf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and littermate controls, indicating that there is no activation of microglia in mutant brain (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2i, j\u003c/strong\u003e). Overall, these data show that \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-controlled inactivation of the \u003cem\u003eBdnf\u003c/em\u003e gene causes no overt alterations in the postnatal brain.\u003c/p\u003e\n\u003cp\u003eA similar immunostaining analysis of brain sections from P12 \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants, generated with the \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e line and postnatal tamoxifen administration from P1-3, shows no obvious differences to control littermates. Quantitative analyses of vascular area, pericyte coverage and collagen type IV expression are comparable in the \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control brain vasculature (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3a-d\u003c/strong\u003e). Immunostaining also shows no noticeable differences in AQP4\u0026thinsp;+\u0026thinsp;glial endfeet, GFAP\u0026thinsp;+\u0026thinsp;astrocytes or AIF1 expression by microglia (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3e-g\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTaken together, these results indicate that \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-controlled inactivation of \u003cem\u003eHgf\u003c/em\u003e or \u003cem\u003eBdnf\u003c/em\u003e leads to no detectable alterations in the postnatal brain, which is consistent with the absent or low expression of these two growth factors in brain pericytes.\u003c/p\u003e\n\u003ch3\u003eAlveolarization and lung morphogenesis requires pericyte-derived HGF\u003c/h3\u003e\n\u003cp\u003eGiven the importance of HGF signaling and its c-Met receptor for lung development\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e and based on the expression of \u003cem\u003eHgf\u003c/em\u003e in pulmonary pericytes (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg), we investigated the role of pericyte-derived HGF. Immunostaining of P21 lung sections detects c-Met protein expression in LAMP3\u0026thinsp;+\u0026thinsp;AT2 cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). This is further supported by analysis of our scRNA-seq data, which shows enrichment of \u003cem\u003eMet\u003c/em\u003e transcripts in AT2 cell clusters (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4a-f\u003c/strong\u003e). Following tamoxifen administration after birth, \u003cem\u003ePdgfrb-CreERT2-\u003c/em\u003econtrolled inactivation of \u003cem\u003eHgf\u003c/em\u003e impairs alveologenesis in the resulting \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). This phenotype is accompanied by significant reduction of AT2 epithelial cells as well as reduced AT2 cell proliferation, whereas apoptosis is not increased in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutant lungs (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec-j). Interestingly, we observed hotspots with high levels of staining for RAGE (receptor for advanced glycation end products), a transmembrane pattern recognition receptor that has been linked to inflammatory processes in the lung\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The overall RAGE\u0026thinsp;+\u0026thinsp;AT1 epithelial area, however, is reduced in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs compared to littermate controls (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ek, l), reflecting that the AT2 population serves as a progenitor pool for AT1 cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To investigate the cellular and molecular alterations in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e animals in greater detail, we performed scRNA-seq analysis of P21 mutant and littermate control lung (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4a-g\u003c/strong\u003e). Bioinformatic analysis of the resulting data confirmed a reduction of AT2 cells expressing \u003cem\u003eMet\u003c/em\u003e, and \u003cem\u003eLamp3\u003c/em\u003e, \u003cem\u003eAbca\u003c/em\u003e, \u003cem\u003eSftbp\u003c/em\u003e, and \u003cem\u003eTinag\u003c/em\u003e in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003em and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;4g\u003c/strong\u003e). Supporting our characterization of the mutant phenotype, \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e AT2 cells show reduced expression of genes associated with cell proliferation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003en). Furthermore, markers associated with differentiation into AT1 epithelium are also affected in the AT1-AT2 transitory population (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eo). In addition to the epithelial defects, mice lacking pericyte-derived HGF show a significant decrease of PECAM1\u0026thinsp;+\u0026thinsp;pulmonary endothelium relative to controls (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ep, r), whereas the ratio of ECs to pericytes, measured by combined ERG and PDGFR\u0026beta; staining, remains unchanged (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eq, s). Given the absent or low expression of \u003cem\u003eMet\u003c/em\u003e in endothelial cells (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4c\u003c/strong\u003e), the reduced vascularization could be secondary to the defects in alveolar epithelium observed in postnatal \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lung. These data together with our previous study\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e confirm the importance of pericytes for AT2 epithelial cell proliferation and lung development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePericyte-derived\u003c/strong\u003e \u003cstrong\u003eNodal\u003c/strong\u003e \u003cstrong\u003eis not essential for postnatal lung development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePostnatal \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants were generated with \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e mice using the same strategy as outlined above for \u003cem\u003eHgf\u003c/em\u003e and \u003cem\u003eBdnf\u003c/em\u003e. Confocal microscopy and PECAM1 immunostaining show no significant changes in the postnatal \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e pulmonary vasculature (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5a, f\u003c/strong\u003e). Likewise, combined ERG, PDGFR\u0026beta; and DAPI staining indicate that the number of ECs, pericytes and the ratio of endothelial cells (ERG+) to pericytes (PDGFR\u0026beta;+) are comparable in mutants and the corresponding littermate controls (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5b, g, h\u003c/strong\u003e). In addition, the lung epithelium, stained with RAGE and NKX2.1 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5c, d, i\u003c/strong\u003e), is not altered in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants. The percentage of AT2 cells as well as the number of proliferating cells among total cells are not different in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e and control littermate lungs (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;5d, e, j, k\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eOverall, these data indicate that \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-controlled inactivation of \u003cem\u003eNodal\u003c/em\u003e leads to no overt alterations in the postnatal lung, which is consistent with the absent or very low expression of this growth factor in pulmonary pericytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePostnatal\u003c/strong\u003e \u003cstrong\u003eNodal\u003c/strong\u003e \u003csup\u003e\u003cstrong\u003eiPCKO\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003emutants show reduced brain vascularization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNodal\u003c/em\u003e transcripts are prominently expressed of in brain pericytes (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;6a-c\u003c/strong\u003e), which is further supported using a public scRNA-seq resource for adolescent mouse brain (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;6d, e\u003c/strong\u003e) provided by the Linnarsson laboratory (mousebrain.org)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Inducible inactivation of \u003cem\u003eNodal\u003c/em\u003e mediated by \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e results in a reduced vascular network in the brain by P12 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, c). Microscopic and quantitative analysis reveals a decrease in vascular area together with a reduction of endothelial cells in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brains relative to littermate controls (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb, f). PDGFR\u0026beta;\u0026thinsp;+\u0026thinsp;pericytes are present and associated with the brain capillary endothelium, and the ratio of ECs to pericytes remains unchanged (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed, g). Consistent with the reduction of \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain capillaries, EdU administration reveals a significant decrease in EC proliferation (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee, h). In addition, confocal images of the \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain cortex reveal small, isolated areas of dextran 70 kDa, serum Immunoglobulin G (IgG) and red blood cells (Ter119+, red) extravasation (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;7a-e\u003c/strong\u003e). Moreover, transmission electron microscopy of mutant samples shows the emission of intraluminal protrusions from endothelial cells, enlargement of the sub-endothelial basement membrane and increased perivascular spaces (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ei). Notably, vascular ensheathment by AQP4\u0026thinsp;+\u0026thinsp;astrocyte endfeet is maintained in the \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutant cortex (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;7b\u003c/strong\u003e). Together, these data indicate that pericyte-derived Nodal controls vessel growth and integrity in the postnatal brain.\u003c/p\u003e\n\u003cp\u003ePrevious work has indicated that Nodal is a positive regulator of tumor angiogenesis and can promote tube-formation by cultured human umbilical vein endothelial cells (HUVECs)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. To gain insight into the regulation of EC behavior by Nodal, we stimulated murine immortalized brain endothelial (b.End3) cells with recombinant rhNodal and analyzed the phosphorylation of the downstream signal transducer SMAD2 by Western blotting. Treatment with rhNodal increases the level of phosphorylated SMAD2 (p-SMAD2) in b.End3 lysates in a dose-dependent fashion without altering total SMAD2 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ej), which is prevented by addition of SB431542, an inhibitor of TGF\u0026beta; type I receptors. Similarly, rhNodal stimulates b.End3 cell proliferation and migration in a scratch wound assay and, again, these effects are suppressed by SB431542 (\u003cstrong\u003eFig. k-n\u003c/strong\u003e). Together, these data support that Nodal can regulate features of vascular growth directly through the stimulation of ECs.\u003c/p\u003e\n\u003ch3\u003eReactive astrogliosis is negatively modulated by Nodal signaling\u003c/h3\u003e\n\u003cp\u003eThe analysis of brain sections by immunostaining for GFAP and confocal microscopy revealed signs of reactive astrogliosis, which are most evident in the \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e cortex and brainstem (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Reactive astrogliosis, a typical response to CNS injury and a range of diseases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, involves a spectrum of molecular, cellular and functional changes in astrocytes. Remarkably, a highly increased number of reactive astrocytes, characterized by hypertrophy with an increase of GFAP\u0026thinsp;+\u0026thinsp;protrusions, can be detected in both sagittal (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, d) and coronal (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec) sections of the \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain cortex. In addition, transmission electron microscopy of mutant and littermate control brain cortex confirm the enrichment of glycogen granules and intermediate filaments in mutant astrocytes (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee), which are two features of reactive astrocytes in pathologic conditions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eConfocal microscopy shows that reactive GFAP\u0026thinsp;+\u0026thinsp;astrocytes are present in regions of vascular leakage, as indicated by TER119 or IgG extravasation (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;7c, d\u003c/strong\u003e), which raises the possibility that reactive astrogliosis may occur secondary to vascular defects. However, GFAP\u0026thinsp;+\u0026thinsp;astrocytes are also present in regions without IgG extravasation or vascular leakage (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;7e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFor a more detailed investigation of the cellular and molecular alterations in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants, we performed scRNA-seq analysis of the P12 brain cortex from mutants and littermate controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef, g and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;6a-c\u003c/strong\u003e). Bioinformatic analysis of the resulting data confirms the increase of reactive astrocytes in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants relative to littermate controls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ef). Moreover, our scRNA-seq data shows the upregulated expression of genes associated with reactive astrocytes, including transcripts for glial fibrillary acidic protein (\u003cem\u003eGfap\u003c/em\u003e), AP-1 family transcription factors (\u003cem\u003eJun\u003c/em\u003e, \u003cem\u003eJunb\u003c/em\u003e, \u003cem\u003eFos\u003c/em\u003e, \u003cem\u003eFosb\u003c/em\u003e), the amino acid transporter solute carrier family 7 member 5 (\u003cem\u003eSlc7a5\u003c/em\u003e), the Wnt pathway protein Axin2 (\u003cem\u003eAxin2\u003c/em\u003e), nuclear receptor 4A1 (\u003cem\u003eNr4a1\u003c/em\u003e), the transcription factors EGR-1, Sox2 and Sox9 (\u003cem\u003eEgr1\u003c/em\u003e, \u003cem\u003eSox2\u003c/em\u003e, \u003cem\u003eSox9\u003c/em\u003e) and the cell cycle regulator Cyclin D1 (\u003cem\u003eCcnd1\u003c/em\u003e) in mutant relative to control brain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;8a\u003c/strong\u003e). Analysis of immunostained brain sections by confocal microscopy confirms that the transcriptional regulators SOX2, SOX9 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8b, c\u003c/strong\u003e) as well as FOS (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;9a\u003c/strong\u003e) are enriched in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e GFAP\u0026thinsp;+\u0026thinsp;astrocytes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e experiments confirm that cultured mouse primary astrocytes are responsive to recombinant rhNodal protein. Western blot analysis of cell lysates from rhNodal-treated astrocytes reveals a significantly increased phosphorylation of the downstream signal transducer SMAD2 (p-SMAD2), which is abolished by SB431542 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh). These data support that pericyte-derived Nodal might directly regulate astrocyte behavior in the postnatal brain. Cell culture experiments also show that SB431542 administration leads to an increase in GFAP\u0026thinsp;+\u0026thinsp;astrocytes (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8d\u003c/strong\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;9c\u003c/strong\u003e), which is consistent with the known role of TGF\u0026beta; as a negative or, depending on context, positive regulator of astrocyte reactivity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Supporting our findings in the scRNA-seq analysis, Nodal stimulation reduces the nuclear immunostaining of FOS and FOSB (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;9b, c\u003c/strong\u003e). Conversely, SB431542 treatment of astrocytes increases nuclear localization of SOX9, FOS and FOSB (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8d\u003c/strong\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;9b, c\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTaken together, these findings support that the loss of pericyte-derived Nodal might induce reactive astrogliosis directly, which could be enhanced by vascular leakage.\u003c/p\u003e\n\u003ch3\u003eActivation of microglia is negatively controlled by Nodal\u003c/h3\u003e\n\u003cp\u003eMicroglial cells are important for immune surveillance in the brain and undergo morphological changes, from ramified to amoeboid-like morphology, in response to injury or pathogens\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Visualization of microglia by immunostaining and confocal microscopy shows that that the expression of AIF1 and CD68 is strongly increased in P12 \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain sections relative to littermate controls (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Upregulated expression of AIF1 and CD68 in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain lysates is confirmed by Western blot analysis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Furthermore, \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e AIF1\u0026thinsp;+\u0026thinsp;microglial cells acquire a highly ramified morphology, consistent with an exposure to acute stress\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003eThe increased number of activated microglial cells in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutant brains relative to littermate controls is confirmed by the analysis of our scRNA-seq data (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). Notably, multiple markers associated with microglial activation, including transcripts for the chemokines tumor necrosis factor (\u003cem\u003eTnf\u003c/em\u003e), Interleukin-1 alpha (\u003cem\u003eIl1a\u003c/em\u003e), CD52 (\u003cem\u003eCd52\u003c/em\u003e), CD74 (\u003cem\u003eCd74\u003c/em\u003e), colony stimulating factor 1 (\u003cem\u003eCsf1\u003c/em\u003e), C-X-C motif chemokine ligand 10 (\u003cem\u003eCxcl10\u003c/em\u003e) and 16 (\u003cem\u003eCxcl16\u003c/em\u003e), C-C motif chemokine 12 (\u003cem\u003eCcl12\u003c/em\u003e) and 4 (\u003cem\u003eCcl4\u003c/em\u003e), are upregulated in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e microglia (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;10a\u003c/strong\u003e). Other markers of activated microglia, namely the AXL receptor tyrosine kinase (\u003cem\u003eAxl\u003c/em\u003e) and the intermediate filament protein Nestin (\u003cem\u003eNes\u003c/em\u003e), are also increased after loss of pericyte-derived Nodal (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;10a\u003c/strong\u003e). Immunostaining confirms the increase of Nestin, which is known to be elevated in microglia during inflammation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brain sections relative to littermate controls (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10b, e\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFor more detailed investigation of the role of Nodal, we treated cultured primary murine brain-derived microglial cells with recombinant rhNodal. This treatment significantly increases the phosphorylation of SMAD2, which is blocked by administration of SB431542 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg). Cell stimulation experiments also confirm the suppression of Nestin expression by rhNodal in cultured human microglia cells, whereas SB431542 treatment leads to strongly enhanced Nestin immunostaining (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10c, f\u003c/strong\u003e). Expression of the chemokine CXCL10, which has been linked to microglial activation and migration\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, is also reduced by rhNodal treatment and strongly increased by SB431542 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10d, g\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eMicroglia can be classified into two main types: M1 microglia, which is pro-inflammatory and neurotoxic, and M2 microglia with anti-inflammatory and neuroprotective roles\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Computational analyses of our scRNA-seq data suggests that the loss of pericyte-derived Nodal signaling shifts microglial polarization towards the M1 type, indicating that pericyte-derived Nodal may have both anti-inflammatory and neuroprotective effects (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eh). However, this simple classification does not entirely capture the complexity of microglial responses in various neurodegenerative conditions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTaken together, our findings support that microglia can be directly activated by Nodal, but alterations in the brain microenvironment, such as elevated vascular permeability or changes in ECs or astrocytes, might contribute to the activation of microglia after loss of pericyte-derived Nodal.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eResearch on angiocrine signaling has uncovered very important roles of endothelial cells or specific EC subpopulations in organ growth and regeneration\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, whereas comparably little attention has been given to the potential role of pericytes as a source of secreted factors acting on cell populations in the surrounding tissue. In contrast, it is very well established that pericytes are critical for blood vessel integrity and the prevention of excessive vascular leakage. The latter is most evident in the blood-brain barrier where pericytes together with specialized ECs and astrocyte endfeet protect the brain against the entry of potentially harmful substances and cells from the blood stream\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In the lung, pericytes have been proposed to be a source of myofibroblasts driving tissue fibrosis\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e but have also been implicated in the regulation of leukocyte trafficking and cytokine signaling during inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Historically, the reliable identification of pericytes has long been hampered by the lack of unique markers that are not shared by vascular smooth muscle cells, fibroblasts or other cell populations\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Accordingly, morphological criteria, in particular the close association with the endothelial monolayer, have been indispensable for the characterization of pericytes. These limitations are, at least to a great part, overcome by single cell or single nucleus RNA-seq approaches\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, which provide detailed molecular signatures and enable important insights into organ-specific and intra-organ heterogeneity of cells.\u003c/p\u003e \u003cp\u003eStimulated by the availability of scRNA-seq data hinting at organ-specific angiocrine roles of pericytes, we have explored the role of three differentially expressed pericyte-derived candidate regulators, HGF, BDNF and Nodel, in postnatal lung and brain, which were selected as representative model organs. In the lung, our data show that pericyte-derived HGF promotes the expansion of c-Met-expressing AT2 cells and thereby controls alveologenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). This finding is consistent with our own previous work showing that the YAP1 and TAZ, transcriptional regulators in the Hippo pathway, regulate the expression of HGF and angiopoietin-1 in pericytes\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the current study, we directly establish that pulmonary alveologenesis is indeed direct controlled by pericyte-derived HGF, which is consistent with previous work reporting an important role of HGF/c-Met signaling in lung epithelial morphogenesis during development and regeneration\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. As AT2 cell-derived alveolar type 1 epithelial cells are an important source of vascular endothelial growth factor A (VEGF-A), a master regulator of angiogenic blood vessel growth, during lung development\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, it is feasible that vascular defects in \u003cem\u003eHgf\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e mutants are indirect due to impaired epithelial morphogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also identify pericyte-derived BDNF as an important regulator of postnatal lung morphogenesis. The neurotrophin and its receptor, the Trk family receptor tyrosine kinase TrkB, are best known role in the formation and function of the nervous system\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. However, more recent work has identified a specialized population of AT2 cells during lung regeneration in the adult mouse as a critical source of the neurotrophin. BDNF signals to TrkB\u0026thinsp;+\u0026thinsp;mesenchymal alveolar niche cells, which have been shown to promote epithelial self-renewal and myofibrogenesis in response to lung injury\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. BDNF signaling has been also linked to various aspects of blood vessel growth, EC migration and survival\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Our cell type-specific genetic experiments now demonstrate that interactions via pericyte BDNF and endothelial TrkB directly contribute to postnatal lung development.\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-controlled inactivation of the \u003cem\u003eHgf\u003c/em\u003e or \u003cem\u003eBdnf\u003c/em\u003e genes causes no notable alterations in the postnatal brain, whereas the third factor investigated in our study, the TGFβ family ligand Nodal, is not expressed by pulmonary pericytes, explaining the absence of defects in \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e lungs. In contrast, we find that the loss of pericyte-derived Nodal impairs EC proliferation, decreases the vascularization of the postnatal brain and causes microhemorrhaging. Previous studies have reported that Nodal promotes vascularization in breast cancer\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, and its inhibition suppresses angiogenesis and the progression of human gliomas\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eNodal\u003c/em\u003e \u003csup\u003eiPCKO\u003c/sup\u003e brains show signs of reactive astrogliosis and microglia activation. Although astrocytes are known to respond to defects in endothelial cells or compromised BBB integrity\u003csup\u003e\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, the presence of GFAP\u0026thinsp;+\u0026thinsp;astrocytes in mutant brain regions lacking detectable vascular leakage and cell culture experiments argue that Nodal can directly influence astrocyte reactivity. As reactive astrocytes are typically induced by neuroinflammation or brain injury and ischemia\u003csup\u003e\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, future work might address the expression and function of Nodal in pathophysiological settings. Given that TGF-β treatment has been shown to reduce the expression of reactive astrocyte-associated genes in culture\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, potentially redundant roles of other TGF-β superfamily members need to be considered. Moreover, reactive astrocytes can be induced by cytokines secreted by reactive microglia, including Il-1α, TNFα, and C1q\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, which generates a scenario of molecular crosstalk between multiple cell populations in and around the brain vasculature. The crosstalk between microglia and other components of the BBB, including pericytes, is essential for CNS homeostasis and maintaining a healthy brain environment\u003csup\u003e\u003cspan additionalcitationids=\"CR80 CR81\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Disruption of this communication can lead to neuroinflammation and contribute to various neuropathological conditions\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e,\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. It is therefore important that Nodal negatively regulates the enrichment of factors associated with microglial activation\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e,\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e,\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our study identifies pericytes as central regulators of tissue morphogenesis and microenvironmental homeostasis during postnatal development. We demonstrate that pericytes are vital sources of growth factors and reveal that their signaling is organ-specific, enabling tailored functional roles aligned with distinct morphogenetic processes and tissue-specific environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.J.R. and R.H.A. designed experiments and interpreted results. S.J.R. performed the vast majority of the experiments. S.J.R. and R.D.-H performed isolation of brain cells isolation for the single-cell sequencing, and prepared samples for Transmission Electron Microscopy. S.J.R., A.A. and M.E.P. performed isolation of lung cells isolation for the single-cell sequencing. S.J.R. and K.K. performed the bioinformatic transcriptomic analysis. S.J.R and P.G. conducted the migration assay and EdU incorporation assay using bEnd.3 cells. S.J.R. and R.H.A. wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank S. Volkery and M. Stasch for experts advise with confocal microscopy; D. Zeuschner and K. Mildner for electron microscopy; H.-W. Jeong and K. Mueller for help with RNA-seq experiments; R. Klein for generously providing TrkB mice; D. Vestweber for kindly supplying bEnd.3 cells; E. Watson for the help with Dextran injection.\u0026nbsp;This work was supported by the Max Planck Society and the German Research Foundation (CRC 1366, project no. 394046768).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\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 under accession number (GSE285933, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE285933). The mouse reference genome GRCm39 with GENCODE M26 annotation (https://www.gencodegenes.org/mouse/release_M26.html) was used for mapping the reads in this study. All other relevant data supporting the key findings of this study are available within the article and its Supplementary Information files.\u003c/p\u003e\n\u003cp\u003eAll individual mouse lines used in this study are commercially available at The Jackson Laboratory or through the lead author. All other biological materials described in this article are available through commercial suppliers as indicated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCustom code for scRNA-seq analysis, based on existing packages and own contributions, is available at https://keeper.mpdl.mpg.de/d/f5d1546ae3c84296b921/.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMouse models\u003c/strong\u003e\u003c/p\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 81-02.04.2020.A471) granted by the Landesamt f\u0026uuml;r Natur, Umwelt und Verbraucherschutz (LANUV) of North Rhine-Westphalia, Germany. Animals were combined in groups for experiments irrespective of their sex.\u003c/p\u003e\n\u003cp\u003eC57BL/6J mice were used for the analysis of wild-type lung and brain. \u003cem\u003eIn vivo\u003c/em\u003e labeling of pericytes was performed by mating \u003cem\u003ePdgfrb(BAC)-CreERT2\u003c/em\u003e \u003csup\u003e12\u003c/sup\u003e and \u003cem\u003eRosa26\u0026nbsp;\u003c/em\u003e\u003csup\u003emT/mG\u003c/sup\u003e reporter mice\u003csup\u003e24\u003c/sup\u003e. Cre activity was induced in pups resulting from this mating by intraperitoneal injection of pups at postnatal day 1 (P1) with a single dose of 50 \u0026mu;g 4-hydroxy tamoxifen (4-OHT) (H7904, Sigma) in ethanol-peanut oil (P52144, Sigma). For inducible genetic experiments employing a mural cell-specific loss-of-function approach, \u003cem\u003ePdgfrb(BAC)-CreERT2\u003c/em\u003e transgenics were interbred with mice carrying loxP-flanked alleles for \u003cem\u003eBdnf\u003c/em\u003e (\u003cem\u003eBdnf\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox\u003c/sup\u003e)\u003csup\u003e86\u003c/sup\u003e, \u003cem\u003eHgf\u0026nbsp;\u003c/em\u003e(\u003cem\u003eHgf\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox\u003c/sup\u003e)\u003csup\u003e87\u003c/sup\u003e, or \u0026nbsp;\u003cem\u003eNodal\u0026nbsp;\u003c/em\u003e(\u003cem\u003eNodal\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox\u003c/sup\u003e) \u003csup\u003e88\u003c/sup\u003ein separate crosses. To inactivate \u003cem\u003eNtrk2\u003c/em\u003e in the postnatal endothelium, \u003cem\u003eNtrk2\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox\u003c/sup\u003e mice\u003csup\u003e31\u003c/sup\u003eand \u003cem\u003eCdh5(PAC)-CreERT2\u003c/em\u003e\u003csup\u003e+/T\u003c/sup\u003e transgenic mice\u003csup\u003e89\u003c/sup\u003e were interbred. Cre activity was induced by three consecutive intraperitoneal injection of 50 \u0026mu;g tamoxifen (T5648, Sigma) in ethanol-peanut oil (P52144, Sigma) from P1 to P3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLung sample preparation and immunohistochemistry.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLung samples were prepared as previously described\u003csup\u003e22\u003c/sup\u003e. For immunohistochemical analysis of mouse lungs, pups at P21 were anaesthetized by intraperitoneal injection of xylazine (Bayer, Rompun 2%; 10mg/kg) and ketamine (Zoetis, Ketavet 100mg/ml; 100mg/kg) dissolved in PBS. The chest cavity of each terminally anesthetized pup was opened to access the heart and lungs. A warm (37\u0026deg;C) solution of 6% gelatin (G1890, Sigma) in PBS was gently perfused through the right ventricle using manual pressure. To allow the gelatin to solidify, an ice-cold tissue paper was placed over the exposed heart and lungs for 15 minutes. Subsequently, the ventral trachea was cannulated using an intravenous catheter (BD Insyte, 381212), which was secured with a suture. The lungs were then inflated to full capacity by gently injecting warm (37\u0026deg;C) 1% low-gelling agarose (A4018, Sigma) in PBS. The agarose-inflated lungs were further chilled by placing an ice-cold tissue paper on them for 20 minutes. Afterward, the lungs were excised and placed in a 2% paraformaldehyde (PFA; Sigma, P6148) solution in PBS at 4\u0026deg;C for 30 minutes. Following this initial fixation, the lungs were incubated in cold PBS for 30 minutes. After washing with cold PBS, the lung lobes were sliced into 150 \u0026micro;m sections using a vibrating blade microtome (VT1200, Leica) and then fixed in 4% PFA at 4 \u0026deg;C for 1 hour. After the second fixation, the lung samples were washed thoroughly by incubating them twice in PBS for 30 minutes at room temperature (RT). Lung slices were subsequently blocked in a blocking solution composed of 5% donkey serum and 0.5% Triton X-100 in PBS for a minimum of 2 hours at RT or overnight (O/N) at 4\u0026deg;C. Following blocking, the vibratome sections were treated with primary antibodies diluted in the blocking solution overnight at 4\u0026deg;C. The sections were washed once in 0.5% Triton X-100 in PBS (PBST) for 20 minutes and then three times in PBS for 10 minutes each at RT. After washing, the sections were incubated with secondary antibodies diluted in blocking solution for 2 hours at RT or O/N at 4\u0026deg;C. Nuclei were counterstained with DAPI (D9542, Sigma, 2 \u0026micro;g/ml). After four wash steps with PBS, the sections were mounted using FluoroMount-G (Southern Biotech, 0100-01) and covered with cover slips. The mounted samples were stored at 4\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe following primary antibodies were used for lung staining: rat anti-RAGE (1:200, R\u0026amp;D Systems, MAB1179), rabbit anti-Aquaporin 5 (1:200, Millipore, 178615), goat anti CD31/PECAM1 (1:200, R\u0026amp;D Systems, AF3628), rat anti-PDGFR\u0026beta; (1:100, eBioscience, 14-1402), goat anti-PDGFR\u0026beta; (1:100, R\u0026amp;D Systems, AF1042), rabbit anti-Prosurfactant Protein C (1:200, Millipore, AB3786), chicken anti-GFP (1:300, 2BScientific Ltd., GFP-1010), mouse anti-\u0026alpha;SMA-Cy3 (1:300, Sigma C6198), rat anti-DC-LAMP/CD208 (1:200, Novus Biologicals/Dendritics, DDX0192P-100), rabbit anti-NKX2.1/TTF1 (1:200, abcam, ab76013), rat anti-ICAM2/CD102 (1:100, BD Pharmingen, 553326), goat anti-HGFR/c-Met (1:100, R\u0026amp;D Systems, AF527), rabbit anti ERG (1:100, Abcam, ab110639), rabbit anti-Cleaved Caspase-3 (1:100, Cell Signaling, #9664), Goat anti-TrkB (1:50, Biotechne; AF1494), and rabbit anti-Ki67 (1:100, Abcam; ab15580).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBrain sample preparation and immunohistochemistry.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBrain samples were prepared as previously described\u003csup\u003e90\u003c/sup\u003e. For immunohistochemical analysis of mouse brains, P12 were anaesthetized by intraperitoneal injection of xylazine (Bayer, Rompun 2%; 10mg/kg) and ketamine (Zoetis, Ketavet 100mg/ml; 100mg/kg) dissolved in PBS. Following terminal anesthesia, the chest cavity was surgically opened to expose the heart. To clear the circulatory system of blood, a puncture was made in the right atrium, and 10 ml of ice-cold PBS was perfused through the left ventricle using a peristaltic pump (Pump P-1, GE Healthcare). Tissue fixation was initiated immediately afterward by perfusing 10 ml of ice-cold 1% paraformaldehyde (PFA; Sigma, P6148) through the same route. Once perfusion was complete, brains were carefully dissected from the skull and post-fixed by immersion in 4% PFA at 4\u0026deg;C O/N. The fixed brains were subsequently washed four times (15 minutes each) in PBS. For sectioning, the brains were cut either along the sagittal midline or into 2-mm thick coronal sections using an acrylic brain matrix designed for mice (RBMA-200C, World Precision Instruments). The brain hemispheres or coronal sections were embedded in 4% low-gelling-temperature agarose (Sigma, A9414) dissolved in PBS at 40\u0026deg;C. After embedding, the samples were rapidly cooled on ice to solidify the agarose. Once the agarose had solidified, the blocks were trimmed and mounted onto a specimen holder using cyanoacrylate adhesive (UHU GmbH \u0026amp; Co. KG). Sections with a thickness of 100\u0026mu;m were then cut using a vibratome (VT 1200S, Leica). Vibratome sections were blocked and permeabilized O/N at 4\u0026deg;C in a solution containing 1% bovine serum albumin (BSA; Sigma, P6148), 2% normal donkey serum (Abcam, ab7475), and 0.5% Triton-X-100 (Sigma, T8787) in PBS. Primary antibodies were diluted in freshly prepared blocking solution and incubated O/N at 4\u0026deg;C. Following primary antibody incubation, the sections were washed once with 0.5% Triton-X-100 in PBS, followed by three washes with PBS (20 minutes each at 4\u0026deg;C). The sections were then incubated overnight with species-specific Alexa Fluor-conjugated secondary antibodies (Invitrogen), diluted 1:500 in the blocking buffer. Nuclei were counterstained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) (Sigma, D9542; 2\u0026micro;g/mL). Following secondary antibody incubation for 2 hours at RT or O/N at 4\u0026deg;C, the sections were washed with PBS as described and mounted using Fluoromount G (Southern Biotech, 0100-01).\u003c/p\u003e\n\u003cp\u003eThe following primary antibodies were used for brain immunostaining: rabbit anti-\u003c/p\u003e\n\u003cp\u003eAQP4 (1:100, Sigma HPA014784), mouse anti-\u0026alpha;SMA-Cy3 (1:200, Sigma C6198), goat anti CD31/PECAM1 (1:200, R\u0026amp;D Systems, AF3628), rabbit anti ERG (1:50, Abcam, ab110639), rabbit anti-GFAP (1:200, DAKO, Z0334), goat anti-GFAP (1:200, Novus Biologicals, NB100-53809), rat anti-CD68 (1:100, Abcam, ab53444), chicken anti-GFP (1:200, 2BScientific Ltd., GFP-1010), rabbit anti-GLUT1 (1:100, Millipore, 07-1401), goat anti-AIF1 (1:100, Novus Biologicals, NB100-1028), rat anti-ICAM2/CD102 (1:100, BD Pharmingen, 553326), rat anti-Nestin (1:200, Santa Cruz, sc101541), rat anti-PDGFR\u0026beta; (1:100, eBioscience, 14-1402), goat anti-PDGFR\u0026beta; (1:100, R\u0026amp;D Systems, AF1042), rat anti-TER-119 (1:200, R\u0026amp;D Systems, MAB1125), goat anti-Collagen IV (1:100, Millipore; AB769), Rabbit anti-SOX2 (1:100, Abcam, ab97959), goat anti-SOX9 (1:100, R\u0026amp;D Systems, AF3075), rat anti-Nestin (1:100, Cosmo Bio, BAM-73-100-EX), rabbit anti-FOSB (1:100, Cell Signaling, #2251), rabbit anti-FOS (1:100, Abcam, ab190289), goat anti-CXCL10 (1:100, Biotechne, AF466-SP), mouse anti-Nestin (1:100, Santa Cruz, sc-23927) and Isolectin B4 Alexa Fluor-488 (1:50, Invitrogen; I21411).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following donkey-raised secondary antibodies (all in 1:500 dilution) were used for immunostaining of brain and lung samples: anti-rabbit IgG conjugated to Alexa Fluor (AF) 488 (Thermo Fisher Scientific, A21206), anti-chicken IgY AF488 (Jackson ImmunoResearch, 703-545-155), anti-rat IgG AF488 (Thermo Fisher Scientific, A21208), anti-goat IgG AF488 (Invitrogen, A-11055), anti-mouse IgG AF546 (Thermo Fisher Scientific, A10036), anti-rat IgG AF594 (Thermo Fisher, A21209), anti-rabbit IgG AF594 (Thermo Fisher Scientific, A21207), anti-goat IgG AF594 (Thermo Fisher Scientific, A-11058), anti-rabbit IgG AF647 (Thermo Fisher Scientific, A-31573), anti-rat IgG AF647 (Jackson ImmunoResearch, 712-605-153), anti-goat IgG AF647 (Thermo Fisher Scientific, A-21447), and anti-mouse IgG AF647 (Thermo Fisher Scientific, A-31571). Nuclei were counterstained with DAPI (1\u0026mu;g/ml) together with secondary antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse brain endothelial cells (b.End3) were cultured in DMEM (Sigma, D6546) supplemented with penicillin/streptomycin (PAA, P11-010) and 10% FCS, and kept in a humidified incubator at 37\u0026thinsp;\u0026deg;C, 10% CO2. Cells were seeded into six-well plates coated with 0.1% gelatin for protein extraction. or into \u0026micro;-Slide 24 well (Ibidi, 82426) for immunostaining.\u003c/p\u003e\n\u003cp\u003eMouse C57 mixed astrocytes (Lonza, M-AsM-330) were cultured in Astrocyte Growth Medium BulletKit\u0026trade; (AGM\u003csup\u003eTM\u003c/sup\u003e BulletKit\u003csup\u003eTM\u003c/sup\u003e, CC-3186),\u0026nbsp;and kept in a humidified incubator at 37\u0026thinsp;\u0026deg;C, 5% CO2. Cells were seeded into six-well plates coated with poly-L-lysin (2 ug/cm\u003csup\u003e2\u0026nbsp;\u003c/sup\u003e,Sciencell, 0403) for protein extraction, or into \u0026micro;-Slide 24 well (Ibidi, 82426) for immunostaining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMouse microglia (Sciencell, M1900) were cultured in Microglia Medium (Sciencell, #1901), which consists of 500 ml of basal medium, supplemented with 25 ml of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of microglia growth supplement (MGS, Cat. No. 1952) and 5 ml of antibiotic solution (P/S, Cat. No. 0503). Microglia were seeded into six-well plates coated with poly-L-lysin (2 ug/cm\u003csup\u003e2\u003c/sup\u003e, Sciencell, 0403) for protein extraction, or into \u0026micro;-Slide 24 well (Ibidi, 82426) for immunostaining, kept in a humidified incubator at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStimulation and inhibitor treatment of cultured cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ebEnd.3 cells in a 6-well plate were starved in basal medium for 1 hour at 37\u0026deg;C, then treated with basal medium containing Nodal (100 or 200ng/ml, R\u0026amp;D Systems, 3218-ND-025) for 30 minutes, with or without the SB431542 inhibitor (10 \u0026micro;M, Selleckchem, S1067). Following stimulation, cells were processed for protein isolation. For immunostaining, cells were seeded in \u0026micro;-Slide 24-well plates (Ibidi, 82426) and treated under the same conditions for 16 hours.\u003c/p\u003e\n\u003cp\u003eMouse astrocytes and microglia, cultured separately in 6-well plates at 37\u0026deg;C, were treated with Nodal (200 ng/ml) or SB431542 inhibitor (10 \u0026micro;M) for 30 minutes. Inhibitor treatment was also performed simultaneously with Nodal stimulation. After treatment, the cells were harvested for protein isolation. DMSO was used as a control treatment.\u003c/p\u003e\n\u003cp\u003eFor immunostaining, cells were seeded in \u0026micro;-Slide 24-well plates (Ibidi, 82426) and treated under the same conditions for 16 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein isolation and Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor immunoblotting, cells were washed twice with ice-cold PBS containing 1mM PMSF, then lysed on ice in a lysis buffer (20mM Tris-HCl, pH 8.0, 150mM NaCl, 0.5% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 2 mM EDTA) supplemented with\u0026nbsp;Halt Protease Inhibitor Cocktail (Thermo Scientific, 78429) and Phosphatase Inhibitor Cocktail Set V (EMD Millipore, 524629). The lysates were incubated for 20 minutes at 4\u0026deg;C. Following vortexing, cell lysates were centrifuged for 10\u0026thinsp;min at 4\u0026deg;C, and protein concentrations in the supernatants were measured using the BCA Protein Assay Kit (Pierce, 23225). Lysates were combined with 2x sample loading buffer in a 1:1 ratio and heated at 95\u0026deg;C for 5 minutes. 2 \u0026micro;g of total proteins were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P Polyvinylidene fluoride (PVDF) membranes, which were briefly treated with 100% methanol for 20 seconds. Membranes were blocked in either 1-4% BSA/TBST or 0.3% skim milk/TBST for 1 hour before being incubated with primary antibodies in blocking buffer O/N at 4\u0026deg;C with gentle agitation. After three washes with TBST, membranes were exposed to peroxidase-conjugated secondary antibodies diluted in either 1% BSA/TBST or 0.3% skim milk/TBST buffer for 1 hour at room temperature with gentle agitation. The membranes were then washed and developed using the ECL Prime detection kit (GE Healthcare, RPN2232).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor tissue protein isolation, mouse brain cortices and lungs were perfused with cold PBS containing PhosSTOP (Roche) through the left and right ventricle, respectively. The perfused tissues were dissected before snap freezing in liquid nitrogen. Tissues were then homogenized in lysis buffer using a Pestle (Argos) and centrifuged for 20 minutes at 4\u0026deg;C to clarify the lysates. Total protein concentrations were determined with the BCA Protein Assay Kit\u0026nbsp;(Pierce, 23225). 20 \u0026micro;g of total protein from the lysates was separated by SDS-PAGE and transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies, followed by washing and detection using horseradish peroxidase-conjugated secondary antibodies and the ECL Prime detection kit.\u003c/p\u003e\n\u003cp\u003eThe following antibodies were used for immunoblotting: mouse anti-\u0026beta;-ACTN (1:6000, Invitrogen, AM4302), mouse anti-\u0026alpha;-Tubulin (1:6000, Sigma, T5168), rabbit monoclonal anti-SMAD2 (1:5000), Cell Signaling, 5339), rabbit monoclonal anti-Phospho-SMAD2 (1:500, Cell Signaling, 3108), goat anti-AIF1 (1:5000, Novus Biologicals, NB100-1028), rat anti-CD68 (1:3000, Abcam, ab53444), goat anti-Rabbit IgG, HRP-linked whole Ab (1:5000, Cell Signaling, 7074), sheep anti-Mouse IgG, HRP-linked whole Ab (1:40000, HG-Healthcare, NA931), Peroxidase AffiniPure Bovine anti-Goat IgG (H + L) (1:20000, Jackson ImmunoResearch, 805-035-180) and goat anti-rat HRP-linked whole Ab (1:20000, Amersham, NA935)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Immunostaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were cultured in their respective media within \u0026micro;-Slide 24-well plates (Ibidi, 82426). The cells were fixed using 4% PFA for 10 minutes, followed by permeabilization with ice-cold 0.1% Triton X-100 in PBS for 5 minutes at 4\u0026deg;C. After washing with PBS, cells were blocked with a solution containing 4% donkey serum and 2% BSA in PBS for 1 hour at RT.Following blocking, the cells were incubated with primary antibodies diluted in the blocking buffer for 1 hour at RT. After another washing step, secondary antibodies, also diluted in the blocking buffer, were applied for an additional hour. Finally, after washing steps with PBS, 250\u0026mu;L of Fluoromount-G was added to each well of the \u0026micro;-Slide for mounting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEdU incorporation assay \u003cem\u003ein vivo\u003c/em\u003e and\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEC proliferation was evaluated by administering a single intraperitoneal injection of EdU (150 \u0026micro;g/50 \u0026micro;l, Invitrogen, A10044) to P12 pups. After 2 hours, the brains were dissected and processed as described above. EdU-positive cells were visualized using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific, C10340).\u003c/p\u003e\n\u003cp\u003eTo assess bEnd.3 proliferation in culture, cells were treated with Nodal and SB431542 inhibitor as indicated, were incubated with EdU (10\u0026thinsp;\u0026micro;M) in an old medium for 60\u0026thinsp;min at 37\u0026thinsp;\u0026deg;C. EdU-positive cells were visualized using the Click-iT EdU Imaging Kit (Thermo Fisher Scientific, C10340).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScratch wound assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA scratch wound assay was performed on cultured bEnd.3 cells. Scratches were created using a 10 \u0026mu;L pipette tip positioned perpendicular to the bottom of the well near the well wall. The pipette tip was gently dragged across the well under light pressure to generate uniform scratches. Following scratch formation, the culture medium was replaced with low-serum medium (2% FBS) supplemented with either Nodal (200ng/mL) or a combination of Nodal (200ng/mL) and SB431542 inhibitor (10\u0026mu;M). In case of the latter, DMSO was added as vehicle control. Tile-scan bright-field images of the scratch areas were captured using a Zeiss AxioObserver Z1. Image analysis was conducted using ZEN Blue software and Fiji.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDextran injection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 5 mg/ml solution of 70 kDa Dextran (Texas Red Lysin fixable, Thermofisher, D1864) was prepared in sterile PBS. Pups were anesthetized before administering 50\u0026mu;l of the dextran solution into the bloodstream via retro-orbital injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage acquisition and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfocal image acquisition was performed using a Zeiss confocal microscope LSM780 and LSM880 equipped with the following objective lenses: 10\u0026times; Plan Apochromat (APO), Numerical Apertrue (NA) 0.45, 20\u0026times; Plan APO NA 0.8, water immersion 40\u0026times; LD C-APO NA 1.20, and oil immersion 63\u0026times; Plan APO NA 1.40. The confocal data were then processed with the ZEN 2.3 SP1 FP3 software (black edition) and Fiji.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData were processed with the Prism10 software. Values are presented as mean \u0026plusmn; standard error of the mean (s.e.m.). P values were calculated using Student\u0026rsquo;s t-test\u0026nbsp;to determine statistical significance when comparing two independent groups; For analysis of statistical significance in comparisons involving more than two groups with normal distribution, ordinary one-way ANOVA with Tukey\u0026rsquo;s (when comparing the mean of each group with the mean of every other group), Sidak\u0026rsquo;s (for comparing the means of preselected pairs of groups).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor electron microscopy, mice were anesthetized as previously described and transcardially perfused with 10mL of PBS and 40 mL of 2% PFA and 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.2). The brain was removed and further fixed by immersion in the above-mentioned solution for 3h at RT. An acrylic matrix for mouse brains (World Precision Instruments Cat. No. RBMA-200C) was used to section the brain in coronal slices from which smaller pieces belonging to the cortex were collected and further fixed in reduced 1% osmium tetroxide containing 1.5% potassium hexacyanoferrate. Next, the tissue was dehydrated and embedded in epon. Ultrathin 60 nm-sections were cut on an ultramicrotome (Leica UC6) and counterstained with uranyl and lead. Images were taken using an electron microscope (Tecnai 12 Biotwin TEM, FEI) and representative pictures were documented in imaging plates (Ditabis, Pforzheim).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSingle cell RNA-sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the single cell sequencing of postnatal lung, two\u003cem\u003e\u0026nbsp;Hgf\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox \u0026nbsp;\u003c/sup\u003econtrol miceand two \u003cem\u003eHgf\u0026nbsp;\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e P21 old\u0026nbsp;pups or two\u003cem\u003e\u0026nbsp;Bdnf\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox \u0026nbsp;\u003c/sup\u003econtrol miceand two \u003cem\u003eBdnf\u0026nbsp;\u003c/em\u003e\u003csup\u003eiPCKO\u0026nbsp;\u003c/sup\u003eP21 old\u0026nbsp;pups were analyzed, respectively. Lung lobes were dissected, finely minced using sharp scissors and transferred into enzymatic digestion solution containing 40U/ml collagenase I (Gibco, 170100-017), 40U/ml Collagenase IV (Gibco, 170100-019), 4U/ml Dispase II (Thermo Fisher, 17105041) and 2 U/\u0026micro;l DNase I (Worthington, LK003170) prepared in DMEM (Gibco, 31053028) medium. Digestion was performed in a water bath, at 37\u0026deg;C for 30 min. During this time, the tissue was also mechanically dissociated by passing it through a needle-syringe for several times. \u0026nbsp; Enzymatic digestion was stopped by addition of FACS buffer (2% FBS in DMEM) and the lysate was passed through 70 \u0026micro;m cell strainer (Falcon, 352350). The filtrate was centrifuged for 5 min at 4\u0026deg;C and the pellet was resuspended in RBC lysis solution (eBioscience, 00-4333-57) and further incubated for 10 min on ice. Post resuspension in FACS buffer, the cell lysis was pass through a 50 um Cell Trics filter (Sysmex, 04-0042-2317). Cells were centrifuged for 5 min at 4\u0026deg;C and the pellet was resuspended in cold MACS depletion buffer (autoMACS Running Buffer-MACS Separation Buffer, Miltenyi Biotec, 130-091-221). To proceed with magnetic separation and depletion of CD45+ cells and erythrocytes from the cell suspension, CD45 microbeads (Miltenyi Biotec, 130-052-301) and Ter119 microbeads (Miltenyi Biotec, 130-049-901) were added to the cells resuspended in MACS depletion buffer and incubated for 20 min at 4\u0026deg;C. After washing, the cell solutions were passed through MACS MS column (Miltenyi Biotec, 130-042-201) attached to a MACS separator placed on a magnetic Multistand. Filtrate solution consisting of CD45-/Ter119- cells was collected and cell number was counted using Luna Automated Cell Counter (L10001). For each mouse genotype, 40000 cells were prepared for further sequencing from the two mouse littermates, using equal cell number of cells per sample.\u0026nbsp;Cells were loaded into a BD Rhapsody Cartridge (BD Bioscience, 633733) and captured on the BD Rhapsody Express Single-Cell Analysis System (BD Bioscience). Single-cell mRNA whole transcriptome (WTA) libraries were created using the BD Rhapsody Whole Transcriptome Analysis (WTA) Amplification Kit (BD Bioscience, 633801) and DNA sequencing was performed on a NextSeq500 (Illumina) using 2 x 75 bp paired end reads with an 8 bp single index.\u003c/p\u003e\n\u003cp\u003eFor the single cell sequencing of postnatal cerebral cortex, two P12 \u003cem\u003eNodal\u0026nbsp;\u003c/em\u003e\u003csup\u003elox/lox \u0026nbsp;\u003c/sup\u003econtrol and two \u003cem\u003eNodal\u0026nbsp;\u003c/em\u003e\u003csup\u003eiPCKO\u003c/sup\u003e mice were anesthetized by intraperitoneal injection of Xylazine (16mg/Kg) and Ketamine (100mg/Kg) and transcardially perfused with 10 mL of ice-cold PBS supplemented with Heparin (25 U/mL). Brains were collected and the meningeal layers removed. Next, the cerebral cortex of each hemisphere was dissected and transferred to ice-cold DMEM supplemented with Penicillin/Streptomycin, Glutamine (GlutaMAX, Gibco) and 25 mM HEPES (PAA, Cat. No. S11-001), hereafter dissection media. The cortices belonging to mice from the same genotype were pooled together and minced using scalpels. The resulting paste was resuspended in 1 mL of enzyme blend containing Papain (25 U/mL, Worthington Cat. No. LK003176), DNAseI (113 U/mL, Worthington Cat. No. LK003170), and Liberase DH (100 \u0026micro;g/mL, Roche Cat. No. 5401054) dissolved in pre-warmed dissection media and incubated 30 min at 37\u003csup\u003eo\u003c/sup\u003eC. During this time, the tissue was further homogenized by repeatedly pipetting the solution up and down every 10 minutes using filtered 1mL tips. After the first 15 min of incubation, 0.5 mL of the described enzyme blend were further added. Next, the tissue homogenate was filtered through a 70\u0026micro;m nylon mesh into a 50mL Falcon tube, the filter was washed with 1 mL of pre-warmed dissection media and the final volume obtained was measured. In order to remove debris and myelin, the cell suspension was mixed with 1.7x volume 22% BSA (Carl Roth Cat. No. 8076.2) dissolved in PBS and centrifuged at 1000 g for 12 min and RT. The supernatant was aspirated and the remaining cell pellet resuspended in 1mL of the described enzyme blend for a final 20min incubation step at 37\u003csup\u003eo\u003c/sup\u003eC with pipetting every 5min. The resulting single-cell suspension was filtered through a 40\u0026micro;m cell strainer and diluted by addition of 8mL of pre-warmed dissection media. After centrifugation at 300 g for 5min, the supernatant was discarded and the cell pellet resuspended in 1ml of Red Blood Cell Lysis Buffer (Sigma Cat. No. R7757). After a 1 min incubation at RT, 20ml of ice-cold PBS supplemented with 2% fetal calf serum were added and the whole suspension centrifuged at 300 g for 5 min at 4\u003csup\u003eo\u003c/sup\u003eC. Next, the supernatant was discarded and the cell pellet resuspended in 1ml of filter-sterilized endothelial cell buffer (15 mM HEPES, 153 mM NaCl, 5.6 mM KCl, 1.7 mM CaCl2, 1.2 mM MgCl2 and 10% BSA, pH 7.4)\u003csup\u003e91\u003c/sup\u003e. Cell concentration was assessed using an automated cell counter and 4 x 10\u003csup\u003e4\u003c/sup\u003e cells from each genotype were loaded into BD Rhapsody Cartridge (BD Biosciences Cat. No. 633733) for cell capture.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003escRNA-seq\u003c/strong\u003e \u003cstrong\u003edata analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePreprocessing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe used STAR version 2.7.10a\u003csup\u003e92\u003c/sup\u003e to generate a reference genome index for GRCm39, with Gencode annotations vM33. FASTQ reads were mapped against the reference genome index using STAR with the settings \u0026ldquo; --soloType CB_UMI_Complex --soloUMIlen 8 --soloCellFilter None --outSAMtype BAM SortedByCoordinate --soloFeatures Gene --runRNGseed 1 --soloMultiMappers EM --readFilesCommand zcat --outSAMattributes NH HI AS nM NM MD jM jI MC ch CB UB GX GN sS CR CY UR UY\u0026rdquo;. Libraries using standard BD Rhapsody beads were mapped using the adapter parameters \u0026ldquo;--soloAdapterSequence NNNNNNNNNACTGGCCTGCGANNNNNNNNNGGTAGCGGTGACA --soloCBposition 2_0_2_8 2_21_2_29 3_1_3_9 --soloUMIposition 3_10_3_17 --soloCBwhitelist BD_CLS1.txt BD_CLS2.txt BD_CLS3.txt\u0026rdquo;, libraries with BD Rhapsody enhanced beads with --soloAdapterSequence NNNNNNNNNGTGANNNNNNNNNGACA --soloCBposition 2_0_2_8 2_13_2_21 3_1_3_9 --soloUMIposition 3_10_3_17 --soloCBwhitelist BD_CLS1_v2_draft.txt BD_CLS2_v2_draft.txt BD_CLS3_v2_draft.txt\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003eRaw counts were imported as AnnData (\u0026lt;https://doi.org/10.1101/2021.12.16.473007\u0026gt;) objects. We removed low complexity barcodes with the knee plot method, and further filtered out cells with a high mitochondrial mRNA content, as well as unusually high total and gene counts using manually determined cutoffs for each sample. Doublets were scored with scrublet 0.2.3 (\u0026lt;https://doi.org/10.1016/j.cels.2018.11.005\u0026gt;). Finally, each sample\u0026rsquo;s gene expression matrix was normalized using scran (1.22.1, \u0026lt;https://doi.org/10.1186/s13059-016-0947-7\u0026gt;) with Leiden clustering (https://doi.org/10.1038/s41598-019-41695-z) input at resolution 0.5.\u003c/p\u003e\n\u003cp\u003eG2M and S phase scores were assigned to each cell using gene lists from \u0026lt;10.1126/science.aad0501\u0026gt; and the scanpy (1.9.6, \u0026lt;https://doi.org/10.1186/s13059-017-1382-0\u0026gt;) sc.tl.score_genes_cell_cycle function.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEmbedding, clustering and annotation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe normalised expression matrix was subset to the 3,000 most highly variable genes (HVG, sc.pp.highly_variable_genes, flavor \u0026ldquo;seurat\u0026rdquo;).For some analyses, expression values were cell-cycle regressed using scanpy.pp.regress_out on G2M and S-phase scores. The top 100 principal components (PCs) were calculated, and batch-corrected using Harmony (0.0.5, \u0026lt;https://doi.org/10.1038/s41592-019-0619-0\u0026gt;). The PCs served as basis for k-nearest neighbor calculation (sc.pp.neighbors, n_neighbors=30), which were used as input for UMAP (\u0026lt;https://doi.org/10.48550/arXiv.1802.03426\u0026gt;) layout (sc.tl.umap, min_dist=0.3). Cells were clustered using scanpy.tl.leiden, and a suitable resolution was chosen in each sample for the main celltype annotation. Cluster marker genes were calculated using a pseudobulk approach, comparing aggregate counts with 2 pseudoreplicates for each cluster to all remaining cells (pyDeSEQ2 0.4.8). Finally, expression of select marker genes was plotted using Matplotlib (3.8.4; https://doi.org/10.1109/MCSE.2007.55) \u0026ldquo;imshow\u0026rdquo;, and clusters were annotated accordingly.\u003c/p\u003e\n\u003cp\u003eSample and celltype-specific subsets were subclustered using the top 2000 HVGs and 30PCs. Clusters were annotated at suitable Leiden resolutions using known and calculated celltype markers.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDifferential expression analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDifferentially expressed genes were calculated using a pseudobulk approach, comparing aggregate counts with 2 pseudoreplicates for each condition (pyDeSEQ2 0.4.8).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComparison to lung P14 pericytes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe downloaded raw sequencing data and cell annotations by Hurskainen et al. \u003csup\u003e29\u003c/sup\u003e. (\u0026lt;https://doi.org/10.1038/s41467-021-21865-2\u0026gt;) from GEO (GSE151974) and processed them analogously to the datasets generated in this study, except for using STAR settings appropriate for 10X Chromium v3 (\u0026ldquo;\u0026mdash;soloType CB_UMI_Simple \u0026ndash;soloCBlen 16 \u0026ndash;soloUMIstart 17 \u0026ndash;soloUMIlen 12 \u0026ndash;soloCBwhitelist 10xv3_whitelist.txt\u0026rdquo;).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAugustin HG, Koh GY (2017) Organotypic vasculature: From descriptive heterogeneity to functional pathophysiology. Science 357\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRafii S, Butler JM, Ding BS (2016) Angiocrine functions of organ-specific endothelial cells. Nature 529:316\u0026ndash;325\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamasamy SK et al (2016) Regulation of Hematopoiesis and Osteogenesis by Blood Vessel-Derived Signals. 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Bioinformatics 29:15\u0026ndash;21\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":"pericytes, mural cells, lung, brain, angiocrine signaling, HGF, BDNF, Nodal","lastPublishedDoi":"10.21203/rs.3.rs-5787386/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5787386/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBlood vessels provide a versatile and adaptable transport system, but recent work has established that endothelial cells, which form the innermost lining of the vascular network, are also a source of molecular signals controlling the behavior of other cell types in the surrounding tissue. Pericytes are another essential component of the vessel wall, but comparably little is known about their signaling interactions with other cell populations during organ growth and patterning. Here, we have used tissue-specific and inducible mouse genetics, high-resolution imaging, single-cell RNA sequencing and cell culture experiments to address the function of three pericyte-derived growth factors in the postnatal development of two model organs, namely lung and brain. We found that \u003cem\u003ePdgfrb-CreERT2\u003c/em\u003e-controlled inactivation of the gene for hepatocyte growth factor (HGF) causes no overt alterations in the postnatal brain but impairs alveologenesis in the lung due to defective interaction with AT2 epithelial cells. Likewise, expression of brain-derived neurotrophic factor (BDNF) by pericytes is not required in the postnatal brain but controls lung development through interactions with the receptor tyrosine kinase TrkB in the pulmonary endothelium. Conversely, pericyte expression of the TGFβ family growth factor Nodal is not required for lung morphogenesis but regulates blood vessel growth and barrier function in the postnatal brain, which we attribute to signaling interactions with endothelial cells, astrocytes and microglia. Taken together, our findings establish that pericytes are a critical source of angiocrine signals that control morphogenetic processes in an organ-specific fashion.\u003c/p\u003e","manuscriptTitle":"Pericytes are organ-specific regulators of tissue morphogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-24 15:42:04","doi":"10.21203/rs.3.rs-5787386/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":"January 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42969766,"name":"Biological sciences/Developmental biology/Angiogenesis"},{"id":42969767,"name":"Biological sciences/Developmental biology/Morphogenesis"},{"id":42969768,"name":"Biological sciences/Developmental biology/Organogenesis"}],"tags":[],"updatedAt":"2026-05-13T07:05:34+00:00","versionOfRecord":{"articleIdentity":"rs-5787386","link":"https://doi.org/10.1038/s41467-026-71643-1","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-05-12 04:00:00","publishedOnDateReadable":"May 12th, 2026"},"versionCreatedAt":"2025-01-24 15:42:04","video":"","vorDoi":"10.1038/s41467-026-71643-1","vorDoiUrl":"https://doi.org/10.1038/s41467-026-71643-1","workflowStages":[]},"version":"v1","identity":"rs-5787386","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5787386","identity":"rs-5787386","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}