Junb Regulates Angiogenesis and Neurovascular Parallel Alignment in Mouse Embryonic Skin

RESEARCH ARTICLE JunB regulates angiogenesis and neurovascular parallel alignment in mouse embryonic skin Yasuo Yoshitomi1, Takayuki Ikeda1, Hidehito Saito1, Yoshino Yoshitake1, Yasuhito Ishigaki2, Toshihisa Hatta3, Nobuo Kato4 and Hideto Yonekura1,*

ABSTRACT (reviewed in Carmeliet and Tessier-Lavigne, 2005) and since then, Blood vessels and nerve fibers are often closely arranged in parallel the presence of neurovascular parallelism has been widely throughout the body. Therefore, neurovascular interactions have recognized. Accordingly, morphological and genetic evidence been suggested to be important for the development of vascular suggests that interaction between vascular and nervous systems is networks. However, the molecular mechanisms and genes regulating critical to appropriate patterning and branching of vascular this process remain unclear. In the present study, we investigated the networks (Carmeliet, 2003). genes that are activated in endothelial cells (ECs) following In recent studies, numerous molecular cues have been associated interactions with neurons during vascular development. Microarray with guidance of EC tip cells and axonal growth cones, and these analyses of human primary microvascular ECs co-cultured with have been categorized as attractive and repulsive cues for both EC mouse primary dorsal root ganglion cells showed that JunB is strongly tip cells and axonal growth cones (reviewed in Wälchli et al., 2015). upregulated in ECs by neurovascular interactions. Furthermore, the Hence, vascular patterning seems to depend on the combination forced expression of JunB in ECs stimulated a tip-like cell formation of attractive and repulsive cues that control sites and times of and angiogenesis in vitro and induced vascular endothelial growth developmental processes. In particular, arterial-specific ephrin-B2 factor A (VEGFA) and the pro-angiogenic integrin subunit ITGB3 (EFNB2) and venous-specific EphB4 regulate the establishment of – expression. Moreover, in vivo knockdown of JunB in ECs from the arterial venous plexus and form distinct boundaries between developing mouse limb skin considerably decreased the parallel these vessels through repulsive effects (Klein, 2004; Pasquale, alignments of blood vessels and nerve fibers. Taken together, the 2005; Poliakov et al., 2004). In contrast, attraction between arteries present data demonstrates for the first time that JunB plays an and veins has been associated with apelin and its receptor APJ important role in the formation of embryonic vascular networks. (also known as APLNR) signaling (Kidoya et al., 2015). More These results contribute to the molecular understanding of specifically, apelin is secreted from arterial ECs, stimulates receptor neurovascular interactions during embryonic vascular development. APJ-expressing venous ECs and acts as an attractive cue for venous migration toward arteries. Hence, both attractive and repulsive cues KEY WORDS: JunB, AP-1 transcription factor, Neurovascular contribute to arterial–venous juxtapositional alignments (Kidoya interaction, Vascular remodeling et al., 2015). Guidance cues from the extracellular matrix (ECM) have also INTRODUCTION been reported, and behaviors of endothelial tip cells and axonal Blood vessels form vast and highly structured networks that pervade growth cones are reportedly regulated by direct interactions with the all organs in vertebrates and deliver oxygen, nutrition, biologically ECM (Gerhardt et al., 2003; Ruhrberg et al., 2002). Moreover, active substances and immune cells. These highly structured endothelial tip cell selection is reportedly regulated by laminin– vascular networks are established by endothelial cells (ECs) and integrin signaling, which induces the expression of Dll4 in ECs the cell–matrix and cell–cell interactions between various cell types, (Estrach et al., 2011; Stenzel et al., 2011). including pericytes, smooth muscle cells and neurons. The ensuing During neurovascular patterning and parallelism, local signals cell–matrix and cell–cell interactions are believed to function as from peripheral nerves reportedly control arterial differentiation and guidance cues for EC differentiation and vascular development for patterning of vascular networks in embryonic mouse limb skin establishing vascular networks. The loss of these guidance cues (Mukouyama et al., 2002). Moreover, peripheral nerve-derived reportedly leads to abnormal vascularization, which causes several VEGFA promotes arterial blood vessel differentiation via NRP1- vascular diseases (Dorrell and Friedlander, 2006). mediated positive feedback (Mukouyama et al., 2005), although Andreas Vesalius (1514–1564) was the first to describe parallel altered vascular patterning did not affect nerve patterns (Bates et al., alignments of blood vessels and nerves throughout the body 2003). Hence, cues for vascular patterning that are mediated by neurovascular interactions (i.e. interaction between the nervous and 1Department of Biochemistry, Kanazawa Medical University School of Medicine, vascular system) and may only operate from nerves to ECs. 1-1 Daigaku, Uchinada, Kahoku-gun, Ishikawa 920-0293, Japan. 2Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku- Recently, neurovascular alignment was shown to be dependent gun, Ishikawa 920-0293, Japan. 3Department of Anatomy, Kanazawa Medical on the activity of Cxcl12–Cxcr4 chemokine signaling. Specifically, University School of Medicine, 1-1 Daigaku, Uchinada, Kahoku-gun, Ishikawa peripheral neuron-derived Cxcl12 stimulates EC migration as an 920-0293, Japan. 4Department of Physiology, Kanazawa Medical University School of Medicine, 1-1 Daigaku, Uchinada, Kahoku-gun, Ishikawa 920-0293, Japan. attractive cue for the recruitment and alignment of distant vessels with nerves (Li et al., 2013). Hence, several of the neuron-derived *Author for correspondence ([email protected]) factors that are involved in neurovascular interactions have been H.Y., 0000-0002-0010-1237 described, although the ensuing EC-mediated intracellular signaling pathways that lead to neurovascular parallelism have not yet been

Received 2 August 2016; Accepted 10 January 2017 fully elucidated. Journal of Cell Science

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In this study, we investigated EC gene regulatory networks that (Fig. 1A), and following nerve fiber innervation at E13.5, the are triggered by neurovascular interactions during embryonic total branch lengths of blood vessels were increased (Fig. 1B) with vascular network formation, and identified JunB as a novel decreases in individual branch lengths of blood vessels (Fig. 1C). angiogenic regulator of blood vessel patterning and the alignment These data indicate that following nerve fiber innervation, with nerve fibers during vascular development. vascular growth and branch compaction progress, and mature vascular networks are formed until E17.5. In association with RESULTS these vascular developmental stages, the numbers of Neurovascular interactions in developing embryonic mouse neurovascular cell–cell contacts increased transiently at E15.5, limb skin and then decreased by E17.5 (Fig. 1D). Moreover, the numbers of Initially, we characterized interactions of ECs with nerve fibers at neurovascular parallel branches increased from E13.5 to E17.5 various stages of mouse embryonic limb skin development, and (Fig. 1E), suggesting that neurovascular juxtapositional alignment examined vascular and nerve network formation from embryonic is triggered by direct contact of ECs with neurons (Fig. 1A–E). day (E)9.5 to E17.5 using whole-mount immunohistochemical To investigate the molecular mechanisms underlying staining (Fig. 1A). In a previous study of neurovascular neurovascular juxtapositional alignment, we analyzed gene remodeling, angiogenesis was observed in the primary capillary regulation in ECs during neurovascular interactions with co- plexus on E9.5 and E13.5, and peripheral nerve migration was cultured dorsal root ganglion cells (DRGs). Neurons produced observed on E15.5–17.5 (Mukouyama et al., 2002). In the present elongated axons in the surrounding spaces, and sufficient numbers study, nerve fibers innervated tissues at E13.5, when remodeling of ECs were concomitantly attached to neuronal axons (Fig. 1F). of the pre-existing primary capillary commenced. Subsequently, After microarray analyses of gene expression in the ECs, we neurovascular juxtapositional alignments were established selected 69 candidate genes that had a >1.2-fold expression change

Fig. 1. Interactions and juxtapositional alignment of ECs with peripheral nerve fibers during the development of mouse skin vasculature. (A) Vasculature and nerve fibers of wild-type C57BL/6 mouse limb skin from embryonic day 9.5 (E9.5), E13.5, E15.5 and E17.5 embryos were analyzed by whole-mount immunohistochemistry with antibodies against mouse CD31 (red) and neurofilament-M (NF, green), and with the nuclear stain DAPI (blue). Scale bars: 100 µm. Positions of visible cell–cell contacts between ECs and neurons are indicated with arrowheads. Closed juxtapositional alignments of blood vessels and nerve fibers are shown with dotted lines. Total branch lengths (B), individual branch lengths (C), numbers of neurovascular contacts (D), and numbers of parallel branches between blood vessels and nerve fibers (E) were quantified as described in the Materials and Methods. After nerve fiber migration at E13.5, numbers of neurovascular contacts transiently increased and numbers of juxtapositional blood vessels with nerves increased throughout development. (F) Co-cultures of human primary microvascular endothelial cells (HMVECs; ECs) and mouse dorsal root ganglion cells (DRGs). Scale bars: 100 µm. (G) Gene expression profiling of ECs in co-cultures with mouse DRGs were analyzed by performing a microarray experiment. Upregulated and downregulated genes in ECs co- cultured with DRGs were categorized according to their functions using Gene Ontology analyses. Heat maps show genes upregulated and downregulated in magenta and cyan, respectively. Boxes indicate the results of two independent experiments. Asterisks indicate genes with previously reported roles in angiogenesis (angiogenesis-related genes). Genes that were analyzed and discussed in this study are highlighted in yellow. Quantitative data in B–E show the mean with individual data points (three independent experiments). Journal of Cell Science

917 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 916-926 doi:10.1242/jcs.196303 in two independent neurovascular co-culture experiments. osteoblasts (Guo et al., 2014) and skeletal muscle cells (Raffaello Candidate genes were then categorized and compared with a list et al., 2010). Moreover, mice that are homozygous null for JunB die of genes reportedly involved in angiogenesis (Fig. 1G), and 59% of between E8.5 and E10 due to impaired placental development, and these were matched with angiogenesis-related genes (Fig. 1G, although cell proliferation is normal in JunB-null embryos, retarded indicated with asterisks). In agreement, gene ontology (GO) embryo growth may reflect failure to establish maternal circulation analyses demonstrated a high representation of angiogenesis and (Schorpp-Kistner et al., 1999). Thus, in further experiments, we vascular development GO categories among candidate genes examined the roles of JunB in vascular network formation and (Table S2). Among these, CXCR4 was increased by the presence neurovascular parallelism. of neurovascular interactions (Fig. 1G), and was induced by neurovascular interactions as with a CXCL12 receptor in a previous JunB expression in ECs during embryonic vascular study (Li et al., 2013). Neuron-derived VEGFA reportedly development and in vitro angiogenesis stimulates arterial differentiation of ECs via neurovascular Herein, we determined JunB expression in ECs during co-culture interaction (Mukouyama et al., 2005). In agreement with this, the with DRGs and confirmed induction by neurovascular interactions arterial differentiation marker EFNB2 was upregulated during by performing reverse transcription followed by quantitative real- neurovascular interactions in the present study (Fig. 1G), and strong time PCR (RT-qPCR) (Fig. 2A). In addition, we confirmed the VEGFA induction in ECs was observed (Fig. 1G), potentially expression of other AP-1 family members, including JUN, JUND, offering synergenic contributions to arterial differentiation and FOS, FOSL1, FOSL2, ATF2, ATF3 and ATF7, which reportedly angiogenesis. Furthermore, ITGB3, which encodes the β3 subunit form heterodimers with JunB (van Dam and Castellazzi, 2001). No of integrin αVβ3 and is known to be involved in angiogenesis upregulation among the AP-1 family, except JunB, was observed in (Davis and Senger, 2005; Hayashi et al., 2008), was also ECs co-cultured with DRGs (Fig. 2A). In agreement with the significantly upregulated (Fig. 1G). RT-qPCR results, JunB promoter activity was upregulated by In further analyses of central gene regulators in ECs during neurovascular interactions in luciferase reporter assays, as neurovascular juxtapositional alignment, the AP-1 transcription determined using by a NanoLuc luciferase construct containing factor JunB was the most strongly and consistently upregulated the human JunB promoter (Fig. 2B). JunB expression in DRGs was transcription factor (Fig. 2A,B). Consistent with this finding, JunB unchanged by co-culture with ECs (Fig. 2C). Moreover, whole- has been associated with cell lineage specification and mount immunohistochemical analyses of mouse embryonic limb differentiation in several cell types, including in hematopoietic skin demonstrated that JunB is expressed in ECs, particularly stem cells (Santaguida et al., 2009), T cells (Son et al., 2011), in those attached to neurons, in contrast to Fos staining (Fig. 2D).

Fig. 2. JunB is upregulated during neurovascular interactions in vivo, and during in vitro 3D-collagen angiogenesis. (A) Expression of JunB and its heterodimer counterparts in ECs co-cultured with DRGs were examined using RT-qPCR. The data were normalized to the expression of Gapdh and are presented as fold changes compared to separate control cultures. (B) Mouse JunB promoter activities in ECs were analyzed in monocultures and in co- cultures with DRGs. (C) JunB expression in DRGs co-cultured with ECs was examined using RT- qPCR. Quantitative data in A–C show the mean±s.d. (three independent experiments). (D) Whole-mount immunohistochemistry of EC-tdTomato mouse embryonic limb skin at E15.5 and E17.5 using antibodies against JunB (green), and tubulin βIII (Cyan); JunB expression was observed in ECs that were in contact with neurons (arrow heads). Scale bars: 100 µm. (E) JunB induction was observed in tip cell-like ECs at angiognenic frontiers in in vitro 3D-collagen matrices. Journal of Cell Science

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In agreement with these results, in vitro 3D angiogenesis assays and GFP only in Cre-expressing cells, and when injected into VE- showed strong JunB induction in tip cell-like ECs at invading cadherin-Cre transgenic mice, the loxp-STOP-loxp stuffer sequence angiogenic frontiers (Fig. 2D). is removed by Cre-mediated recombination only in cells expressing the vascular EC marker VE-cadherin. JunB expression in ECs affects EC shapes and the formation During nerve and vascular development in embryonic mouse of tip cell-like protrusions, and promotes angiogenesis limb skin, the capillary primary plexus is formed at around E9.5, in vitro and peripheral nerves (PNs) migrate onto the capillary plexus at To elucidate JunB functions in ECs, we tested the effects of JunB around E13.5. After innervations of PNs onto the capillary plexus, overexpression and suppression using shRNA gene silencing mature vascular networks are formed through neurovascular methods. In these experiments, basal JunB expression in ECs was interactions until E17.5. Accordingly, we administered not detectable under these conditions (Fig. 3A, left panel). lentiviruses via intra-uterine injections into embryo limbs at Subsequently, knockdown of JunB using an shJunB vector was E12.5, immediately before the onset of neurovascular interactions, confirmed in JunB-overexpressing HEK293 cells (Fig. 3A, right and examined limb skin vasculature after embryo growth until panel). Overexpression and suppression of JunB strongly affected E17.5 (Fig. S4B). Following injection of shJunB lentiviruses (Fig. EC morphology (Fig. 3B). Specifically, JunB-overexpressing ECs S4A, upper panel) into mice, lentivirus-infected and shRNA- exhibited elongated cell shapes with tip cell-like filopodia and expressing cells were identified as GFP positive (Fig. S4C). In ruffling membrane formations, whereas JunB-suppressed ECs subsequent whole-mount immunohistochemical analyses, mosaic showed polygonal and less-elongated cell shapes (Fig. 3C). patterns of shJunB expressing and GFP-positive cells were observed Annexin V staining confirmed that JunB knockdown does not in CD31-positive ECs (CD31 is also known as PECAM1) and in induce apoptosis (Fig. S2). In addition, cell widths were other cell types (Fig. S4D). GFP-negative and CD31-positive ECs significantly decreased following JunB overexpression and were were also observed in the fields (Fig. S4E). Thus, these mosaic increased by JunB suppression (Fig. 3C,D). Accordingly, spheroid patterns of infection were used to analyze EC-specific JunB outgrowth assays showed that the formation of tip cell-like ECs was functions in comparison with GFP-negative (non-infected) ECs. dependent on JunB expression (Fig. 3E). Mixing spheroid We used EC-tdTomato mice for lentivirus injection. EC-tdTomato outgrowth assays using control cells and JunB-overexpressing mice were generated by crossbreeding of VE-cadherin-Cre mice with cells with different fluorescent markers showed that JunB- B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mice. EC-tdTomato overexpressing ECs preferentially produce tip-like cells compared mice expressed tdTomato fluorescent protein specifically in CD31- to control cells (Fig. 3F). Moreover, in vitro 3D angiogenesis assays positive ECs (Fig. S1), allowing identification of vascular ECs showed that overexpression of JunB significantly increased the according to tdTomato expression (Fig. 4A). Subsequently, we numbers of cells that migrated into collagen matrices compared with analyzed blood vessel formation and neurovascular alignment of limb those transduced with the vector control, and JunB knockdown skin vasculature in shJunB lentivirus-injected embryos (Fig. 4). suppressed EC migration into collagen matrices (Fig. 3G). Taken Although no significant differences in lengths of non-parallel together, these data show that JunB controls EC cell elongation and branches were observed between GFP-positive and -negative blood cell motility, and positively regulates angiogenesis in collagen vessels (Fig. 4B), lengths of GFP-positive (JunB knockdown) parallel matrices. In contrast, tube formation on Matrigel surfaces was branches were significantly less than those of non-GFP parallel indistinguishable between JunB-overexpressing and JunB- branches (Fig. 4C). These results indicate that parallel neurovascular suppressed ECs, with no significant differences in tube lengths or alignment was inhibited in shJunB-expressing vasculature. In numbers of branches (data not shown), suggesting that JunB- agreement, the numbers of parallel GFP-positive JunB knockdown mediated angiogenesis is accompanied by cell migration and branches were decreased in these animals (see data points in Fig. 4C). degradation of 3D matrices. Non-target control shRNA injection did not show any effect on branch length or the proportion of parallel branches, similar to no In vivo JunB knockdown in ECs from developing mouse limb injection (data not shown). skin abrogates neurovascular alignment In further studies, we compared the number of parallel branches To validate our in vitro data, we examined the effects of in vivo JunB to the total number of branches between GFP-positive and -negative knockdown in ECs from embryonic limb skin vasculature. Because vessels, and found that percentage of parallel branches as a JunB knockout (KO) mice die by E9.5 due to placental proportion the total number of branches was significantly decreased malformation (Schorpp-Kistner et al., 1999), analyses of the in GFP-positive (JunB knockdown) vessels (Fig. 4D). In further roles of JunB in vascular network formation at E9.5–17.5 cannot analyses of neurovascular parallelisms in larger blood vessels be achieved in both conventional and EC-specific conditional (>6.6 µm in diameter), the percentage of parallel branches in GFP- JunB KO mice. Therefore, we developed a novel strategy for positive (JunB knockdown) vessels were dramatically decreased to developmental stage-, position- and cell-specific JunB knockdown 20%, whereas those in GFP-negative vessels were 60% of the total using a combination of in utero lentivirus injections and number of branches (Fig. 4E). Similar results were obtained in mice Cre-mediated cell-specific gene knockdown. injected with EC-specific shRNA (EC-spe-shJunB; Fig. 4F–H), These experiments were performed using a shJunB lentivirus indicating that downregulation of JunB expression disrupts the vector (Fig. S4A, upper panel) that expresses shRNA and GFP in all balance of EC cell migration and differentiation that is necessary for infected cells, allowing positive identification of lentivirus-infected the development of vasculature and the morphogenesis required for cells according to GFP expression. In addition, we administered an neurovascular juxtaposition. EC-specific shJunB lentivirus vector (EC-spe-shJunB; Fig. S4A, lower panel), which contains a loxp-STOP-loxp stuffer sequence Correlations of EC gene regulation with JunB overexpression between the EF1 promoter and the GFP-miR-E-backbone-shRNA- and neurovascular interactions JunB-coding sequence (Fellmann et al., 2013; see Materials and To investigate pathways downstream of JunB, we performed

Methods section). This EC-spe-shJunB vector expresses shRNA microarray gene expression analyses in JunB-overexpressing ECs, Journal of Cell Science

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Fig. 3. JunB promotes tip cell-like EC formation and in vitro 3D-collagen angiogenesis. (A) JunB was overexpressed or suppressed in ECs through lentivirus vectors, and expression was confirmed by western blotting with an anti-JunB antibody; Cont, vector control; JunBOE, JunB overexpression; shCont, non-target shRNA control vector; shJunB, JunB-targeted shRNA. Suppression of JunB by shJunB was confirmed in JunB-overexpressing HEK293 cells (right panel). (B–D) ECs were transduced with lentiviruses expressing human JunB or shJunB and morphological changes were monitored. (B) Phase contrast images. (C) Longitudinal and lateral lengths of cells were measured and plotted. The bar indicates the regression line as calculated by the software R. (D) Quantification of the lateral lengths of JunB-overexpressing and JunB-suppressed ECs (n=5, three independent experiments). (E) EC spheroid sprouting assays. Overexpression of JunB in ECs stimulated the formation of angiogenic tip cell-like protrusions in 3D-collagen angiogenesis assays, whereas suppression of JunB expression inhibited the formation of tip cells. Results are mean±s.d. (three independent experiments). (F) EC spheroid sprouting assays of control and JunB-overexpressing ECs. Mixed cultures with equal amounts of mCherry-expressing control ECs (Cont, in red) and GFP-expressing control ECs (Cont, in green), or GFP-expressing-JunB-overexpressing ECs (JunBOE, in green) were subjected to the tip-cell assay, and the ratio of the number of tip cells for the green cells to the red cells was quantified (right panel, mean±s.d., *P<0.01 mCherry-expressing-EC with GFP-expressing-EC vs mCherry-expressing-EC with JunBOE- EC). (G) In vitro 3D angiogenesis assays represented in inverted images of DAPI staining. White lines indicate the surface of collagen matrices and dotted lines indicate average migration distance of ECs; overexpression of JunB significantly stimulated angiogenesis into 3D collagen matrices and suppression of JunB resulted in dramatic inhibition of 3D angiogenesis. (n=5, three independent experiments). For the plots in D and G, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the maximum and minimum values (three independent experiments). Scale bars: 100 µm (B,E,F). Journal of Cell Science

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Fig. 4. JunB knockdown in ECs reduces juxtapositional alignment of blood vessels with neurons in vivo. Vascular and neuronal network formations were analyzed with or without JunB knockdown using in utero lentiviral infection. (A) Two typical immunohistochemistry images of mouse limb skin at E17.5 following in utero lentivirus injections of shJunB to visualize blood vessels (red), neurons (cyan), and shRNA-JunB-expressing and GFP-positive blood vessels (green) are shown. Immunohistochemistry (IHC), skeletonized images, neurovascular parallel branches, and merged skeletonized images are shown (left through right). Parallel alignments of blood vessels with nerve fibers are blue, and shRNA-JunB positive blood vessels are orange. (B–E) Lengths of non-parallel branches (B), lengths of parallel branches and nerve fiber alignments (C), the percentage of parallel branch numbers as a proportion of total branch numbers (D), and the percentage of parallel branch numbers as a proportion of total branch numbers among larger vessels (E) were quantified and compared between GFP-positive [GFP(+), JunB-knockdown, green dots], and GFP-negative [GFP(−), wild-type, red dots] vessels. Quantification criteria are listed in Fig. S2. N.S., not significant. (F–H) EC-specific JunB knockdown in limb skin following in utero injection of EC-spe-shJunB lentivirus in VE- cadherin-Cre mice. Two typical images of immunohistochemistry and skeletonized images (F) and quantifications (G,H) are shown. Scale bars: 100 µm. Knockdown of JunB in ECs decreased juxtapositional alignment of blood vessels with nerve fibers. Quantitative data in C–E,G and H show the mean with individual data points (four independent experiments). For the plots in B, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the maximum and minimum values (four independent experiments).

and analyzed the expression of the 69 genes that were identified in Accordingly, we also observed the induction of VEGFA, EFNB2 neurovascular interaction experiments (Fig. 1G). Among these 69 and HES1 in JunB-overexpressing ECs (Fig. 5B). In addition, genes, 34 gene expression changes were consistent with those ITGB3 knockdown in JunB-overexpressing cells significantly accompanying neurovascular interactions (Fig. 5A), and were likely reduced angiogenesis activity in 3D collagen matrices (Fig. 6E,F). downstream of JunB signaling pathways that are triggered by direct The integrin αVβ3-specific inhibitor Cyclo (RGDfC) also caused neurovascular interactions. significant suppression of JunB-induced angiogenesis (Fig. 6F). Among candidate molecules, the integrin subunit β3 (encoded by ITGB3) was previously shown to promote angiogenesis (Davis and Involvement of JunB in known neurovascular interaction Senger, 2005; Hayashi et al., 2008). In agreement, the present signaling pathways analyses showed a significant upregulation of ITGB3 in JunB- Finally, we investigated relationships between JunB activation and overexpressing ECs (Fig. 5B), indicating that ITGB3 expression other known signaling pathways during neurovascular interactions. is activated by JunB following neurovascular interactions. A previous study showed that Cxcl12 is secreted by peripheral Journal of Cell Science

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Fig. 5. Gene expression analysis of JunB-overexpressing ECs. (A) Gene expression changes in JunB-overexpressing ECs were analyzed by performing microarray experiments. Heat maps represent upregulated (magenta) and downregulated (cyan) genes in JunB-overexpressing ECs, and are presented with the categorized list of genes associated with neurovascular interactions (see Fig. 1G). Genes that were upregulated and downregulated by neurovascular interactions (see Fig. 1H) are indicated by ‘up’ and ‘down’, respectively. Genes are categorized according to their functions. Genes that were analyzed and discussed in this study are highlighted in yellow. (B) Expression levels of JUNB, VEGFA, EFNB2, HES1 and ITGB3 in JunB-overexpressing (JunBOE) ECs were analyzed using RT-qPCR. Data show the mean±s.d. (three independent experiments).

neurons and Schwann cells, and stimulates angiogenesis and transcription factor, indicating that it had roles in neurovascular vascular remodeling via the Cxcl12 receptor Cxcr4 during the interactions during vascular network formation (Figs 1G and 2A). formation of embryonic vascular networks (Li et al., 2013). Thus, Further whole-mount immunohistochemistry analyses of mouse we examined the involvement of JunB in Cxcl12-induced 3D embryonic limb skin confirmed increased expression of JunB in ECs angiogenesis in vitro (Koh et al., 2008) as described in Fig. 3D. In that were attached to neurons at E13.5 and E17.5 (Fig. 2C), and these experiments, the presence of Cxcl12 significantly activated strong JunB induction was observed in tip cell-like ECs at invading angiogenesis as previously reported (Fig. 6A), but did not induce angiogenic frontiers in the in vitro 3D angiogenesis assays (Fig. 2D). JunB expression in EC cultures (Fig. 6B), suggesting that JunB is Additionally, EC morphologies were found to be dependent on not regulated downstream of the Cxcl12 pathway. JunB expression levels (Fig. 3), indicating that JunB controls EC Subsequently, we determined whether neurovascular interactions shape and motility. Accordingly, in vitro 3D angiogenesis was in this system were mediated by soluble factors or by direct cell–cell dependent on JunB expression levels (Fig. 3), further indicating that interactions using analyses of the effects of conditioned medium from JunB positively regulates angiogenesis. DRGs on JunB expression in ECs. However, JunB expression was not In accordance with the present in vitro data, in vivo JunB affected by the addition of DRG conditioned medium in RT-qPCR knockdown in the ECs of embryonic limb skin vasculature just (Fig. 6C) or JunB promoter assays (Fig. 6D). These results indicate before the onset of neurovascular interactions significantly that induction of JunB during neurovascular interactions of ECs is decreased the numbers and lengths of parallel branches with directly triggered by cell–cell or cell–ECM interaction. neurons (Fig. 4). Moreover, the percentage of parallel branches as a proportion of the total branches was significantly decreased in DISCUSSION JunB-knockdown vasculature (Fig. 4), and these effects were In the present study, we demonstrated that JunB is induced in ECs by greater in blood vessels of over 6.6 µm in diameter (Fig. 4E). Taken neurovascular interactions, and that it regulates neurovascular together, these data demonstrate the necessity of JunB for blood juxtapositional alignments during embryonic vascular development. vessel formation and neurovascular juxtaposition. The present whole-mount immunohistochemical analyses showed that Further microarray analyses showed that interactions with DRGs neurovascular interactions started at E13.5, and neurovascular induced arterial differentiation of ECs, as indicated by upregulation juxtapositional alignments were established during E13.5–17.5 of EFNB2 (Fig. 1G), suggesting that neuron-derived VEGFA (Fig. 1). Moreover, neurovascular juxtapositional alignments were induces arterial differentiation of ECs, as reported previously (Li triggered by the direct contact of ECs with neurons (Fig. 1A). Thus, to et al., 2013). In the present study, neurovascular interactions model in vivo neurovascular interactions and investigate the underlying induced VEGFA expression in ECs (Fig. 1G), indicating self- molecular mechanisms, we analyzed changes in EC gene expression activation of arterial differentiation and angiogenesis following during co-culture with DRG neurons. The ensuing microarray analyses neurovascular interactions. VEGF induction was also observed in identified 69 genes that were significantly regulated under these JunB-overexpressing ECs (Fig. 5), further implicating JunB in the conditions, and JunB was the most differentially expressed induction of VEGFA in ECs during neurovascular interactions. Journal of Cell Science

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JunB-null mice die prior to E9.5 due to placental dysplasia (Schorpp-Kistner et al., 1999). Hence, JunB KO may cause vascular malformation and abnormal placental vasculogenesis, leading to embryonic lethality. Accordingly, embryonic lethality was three times more frequent in shJunB lentivirus-injected embryos than in control shRNA-injected embryos (data not shown). In addition, limb malformation and shortness was often observed following shJunB injections, likely reflecting failure of vascular development (data not shown). Roles for JunB in vascular development have been reported previously, and JunB is known to activate Foxo1 by inducing miR- 182, contributing to lymphatic vascular development in zebrafish. In agreement, loss of JunB in zebrafish resulted in the formation of parachordal lymphangioblasts (Kiesow et al., 2015), and Foxo1 expression reportedly effected EC migration, sprouting and tube formation (Potente et al., 2005). However, the present microarray analysis showed no upregulation of Foxo1 during neurovascular interactions (data not shown), suggesting that the JunB–miR-182– Foxo1 axis is not involved in JunB-mediated neurovascular juxtapositional alignment. Gene expression analyses of JunB-overexpressing ECs co- identified about half of those that were regulated by neurovascular interactions (Fig. 5), indicating considerable involvement of JunB signaling in neurovascular interactions. Integrins play important roles in cell adhesion and migration (Desgrosellier and Cheresh, 2010), and ECs express several integrin heterodimers, including αVβ3, α5β1 and αvβ5 (Plow et al., 2000). In particular, integrin αVβ3 is a critical angiogenic regulator for neovascularization that acts by co-operating with VEGFR2 (Somanath et al., 2009). Integrin αVβ3 is expressed at lower levels in quiescent ECs, but it is significantly upregulated in angiogenesis (Leu et al., 2002). Functional blocking of αVβ3 results in defects pertaining to neovasculature without affecting the normal pre-existing vasculature (Brooks et al., 1994a,b). Thus, integrin αVβ3isakey regulator of angiogenesis. In the present experiment, we observed the upregulation of ITGB3, which encodes the β3 subunit of integrin αVβ3, during neurovascular interaction (Fig. 1G). We also observed the upregulation of ITGB3 in JunB-overexpressing ECs (Fig. 5), indicating that the upregulation of ITGB3 during neurovascular Fig. 6. JunB functions in known pathways of neurovascular interaction interaction is mediated by JunB. Moreover, ITGB3 knockdown and induced angiogenesis. (A) Effects of Cxcl12 on 3D-collagen integrin αVβ3-specific inhibition significantly suppressed JunB- angiogenesis (n=5). (B) RT-qPCR expression analysis of JunB following induced 3D angiogenesis (Fig. 5D), suggesting that integrin αVβ3 Cxcl12-induced angiogenesis. (C,D) Effects of conditioned medium from is one of the downstream effector molecules in JunB signaling. The DRG cell cultures on JunB expression (C) and JunB promoter activity (D) in ECs; soluble factors in DRG conditioned medium did not activate JunB notch signaling protein HES1 reportedly regulates vascular expression in ECs. (E,F) Effects of ITGB3 knockdown in JunB- remodeling and arterial specification of ECs during the overexpressing ECs and the integrin αVβ3-specific inhibitor Cyclo (RGDfC) development of brain vasculature (Kitagawa et al., 2013). HES1 were analyzed in 3D-collagen angiogenesis. Quantitative data in B–Eshow was reportedly regulated by JunB via direct binding to the HES1 the mean±s.d. (three independent experiments). For the plots in A and F, promoter (Santaguida et al., 2009). These results suggest that – the box represents the 25 75th percentiles, and the median is indicated. vascular remodeling and formation of neurovascular parallelism are The whiskers show the maximum and minimum values (three independent experiments). mediated in part by integrin and/or notch signaling pathways. Neuron-derived Cxcl12 is known to stimulate angiogenesis and vascular remodeling following binding of its receptor Cxcr4 during Li et al. reported that neurovascular interactions activate VEGFA the formation of embryonic vascular networks (Li et al., 2013). and Cxcl12 production in neurons, and that neuron-derived VEGFA However, the present data do not indicate that JunB is regulated induces EC differentiation, whereas Cxcl12 induces angiogenesis and downstream of the Cxcl12 pathway during neurovascular vascular remodeling (Li et al., 2013). Similarly, the present data show interactions (Fig. 6A,B), and instead suggest angiogenic roles for that neurovascular interactions activate VEGFA expression in ECs JunB that are independent of the Cxcl12 pathway under these (Fig. 1G), likely contributing synergistically to EC differentiation, conditions. Cxcl12 was upregulated in ECs upon JunB vascular remodeling and activation of angiogenesis. In addition, overexpression (Fig. 5B), and was downregulated by VEGFA was upregulated in JunB-overexpressing ECs (Fig. 5), neurovascular interaction in ECs (Fig. 1G). We need to clarify the suggesting that VEGFA is induced by JunB during neurovascular mechanisms for these differences in gene regulation of Cxcl12 interactions. between ECs and neurons in future studies. Journal of Cell Science

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In summary, we present several lines of evidence for a novel synthetic oligonucleotides against human and mouse JunB antisense mechanism involving the induction of the transcription factor JunB sequences (shJunB1, 5′-AGGCTCGGTTTCAGGAGTTTGTAGTCGTG- during neurovascular interactions, and subsequent regulation of 3′ or shJunB2, 5′-TTGAGCGTCTTCACCTTGTCCTCCAGGCG-3′) and human ITGB3 antisense sequences (shITGB3-1, 5′-TACTGGAATCTG- angiogenesis toward arterial differentiation and neurovascular ′ ′ juxtapositional alignment. Specifically, neurovascular interactions ACGACACAGTCATCCTC-3 or shITGB3-2, 5 -AAGACAGGTCCATC- AAGTAGTAGATGTCC-3′) with loop 5′-TCAAGAG-3′. A synthetic induced JunB expression in ECs of the primary capillary plexus, and DNA containing the non-target sequence 5′-GCACTACCAGAGCTAAC- the resulting activation of pro-angiogenic genes, such as VEGFA TCAGATAGTACT-3′ carrying the same loop was used as the negative and ITGB3, led to the morphological changes of ECs, with control (sh-Vec). Tissue-specific JunB knockdown in mice was achieved by formation of tip cell-like cells and migration toward nerve fibers, using the lentivirus (EC-spe-shJunB) containing the modified miR30- and establishment of mature neurovascular aligned blood vessels. (miR-E-) miroRNA backbone shRNA (Fellmann et al., 2013) with Although the present experiments reveal novel aspects of loxp-stop-loxp stuffer sequence. In brief, the mouse JunB antisense neurovascular interactions during the formation of vascular sequence 5′-TTTCAGGAGTTTGTAGTCGTG-3′ with a 5′-TAGTGAA- networks, further studies are necessary to confirm the GCCACAGATGTA-3′ loop for shRNA-JunB was inserted into the miR-E- relationships between JunB and other signaling pathways known microRNA backbone (miR-E-shJunB). miR-E-shJunB was cloned into the to be associated with neurovascular interactions, and to define the pCDH vector downstream of the GFP open reading frame (ORF). The loxp- stop-loxp fragment was then inserted between EF1 promoter and the GFP mechanisms of JunB induction. Nonetheless, the present study transcriptional start site to allow induction of GFP and shRNA expression provides clues for future studies regarding the regulatory upon Cre-mediated recombination in Cre expressing cells. Lentivirus mechanisms involved in embryonic vascular development. particles were then produced in HEK293TN cells (System Biosciences) following transfection of the above constructs, the psPAX2 packaging MATERIALS AND METHODS vector and the pMD2.G envelope vector [deposited by Didier Trono, Cell culture and animals Laboratory of Virology and Genetics, École Polytechnique Fédérale de Human primary microvascular ECs (HMVECs) were isolated from newborn Lausanne, Switzerland; Addgene plasmid #12259 and #12259 respectively, foreskins (Lifeline Cell Technology, Frederick, MD) and were maintained in Addgene, MA]. Supernatants from 48 h culture media were then collected HuMedia-EB2 medium supplemented with 5% (v/v) fetal bovine serum and lentiviral particles were concentrated using PEG-it solution (System (FBS), 5 ng/ml basic fibroblast growth factor (bFGF), 10 μg/ml heparin, Biosciences). Lentivirus infectious units (IFU) were determined according 10 ng/ml epidermal growth factor (EGF), 1 μg/ml hydrocortisone, and to the number of GFP-positive ECs as assessed with serial dilutions of 39.3 μg/ml dibutyryl cAMP according to the manufacturer’s instructions lentivirus from cells at 24 h post transduction. (Kurabo, Tokyo Japan). Telomerase immortalized HMVECs (TIME cells; #CRL4025, ATCC, Manassas, VA) were maintained in M131 medium Microarray analysis supplemented with MVGF growth supplements (Invitrogen, Carlsbad, CA). Total RNA was extracted using TRIzol reagent (Invitrogen) and purified Dorsal root ganglion cells (DRGs) were isolated from C57BL/6 mice on using RNeasy Mini kits (Qiagen, Valencia, CA). RNA quality was postnatal day 3 and were dissociated for 10 min at 37°C using 1-mg/ml assessed according to 28S/18S ratios of rRNA bands on electrophoresis collagenase and 5-mg/ml Dispase in HBSS, and were then cultured on poly- gels under denaturing conditions. Subsequently, 100-ng aliquots of total D-lysine-coated plates. Cultures were grown in neurobasal medium RNA were labeled according to the manufacturer’s instructions for the supplemented with B27 and 10-ng/ml NGF (BD Biosciences, Franklin GeneChiP WT Sense Target Labeling kit (Affymetrix, Santa Clara, CA). Lakes, NJ), 100-ng/ml penicillin, and 100-U/ml streptomycin. Prior to EC Fragmented and labeled cDNAs were then hybridized onto Affymetrix and DRG co-culture experiments, DRGs were grown in six-well plates, and Genechip Human Gene 1.0 ST or human U133 plus 2.0 arrays. Arrays detached HMVECs were inoculated onto DRG cultures in EC medium at a were then washed and stained using the GeneChip Fluidics Station 450 EC:DRG ratio of 10:1. Co-cultures were then incubated for 8 h and RNA and scanned using a 3000 7G GeneChip Scanner (Affymetrix). All was extracted. This in vitro co-culture may mimic the initial attachment of arrays passed the quality control criteria of the Expression Console ECs with neurons, which causes gene expression changes in ECs. For software (Affymetrix). Raw data CEL files were then normalized using separate cultures, ECs and DRGs were cultured separately in EC medium the RMA algorithm and the data exported using Expression Console or and incubated for 8 h in parallel with neurovascular co-cultures. After 8 h, Gene Spring software (Agilent Technologies, Santa Clara, CA). Genes RNA was extracted separately from ECs and DRGs. RNA corresponding to with a >1.2-fold change in transcription in two independent experiments the number of each cell type in the co-culture was mixed in the same amount were selected, and non-annotated genes and functionally unknown genes by weight, and used as a separate control culture sample in the microarray were excluded. Gene ontology (GO) analyses of genes with significant analysis. Conditioned medium from DRGs or NIH-3T3 fibroblasts was transcriptional changes during neurovascular interactions were prepared by culturing cells for 24 h in serum-free M131 medium. performed using the human reference list in the PANTHER v10.0 An EC-specific tdTomato fluorescent protein-expressing mouse (EC- Enrichment analysis program provided by the Gene Ontology tdTomato mouse) was generated by crossing VE-cadherin-Cre mice from consortium (http://pantherdb.org/). the National Institute of Biomedical Innovation (NIBIO), JAPAN (repository ID: NIBIO132; Kogata et al., 2006) with B6.Cg-Gt(ROSA) RT-qPCR 26Sortm9(CAG-tdTomato)Hze/J mouse from the Jackson Laboratory, ME Total RNA was extracted from cells as described above. Subsequently, 1-µg (repository ID :007909; Madisen et al., 2010). Subsequently, tdTomato aliquots of total RNA samples were reverse transcribed using PrimeScript expression in ECs was confirmed in CD31-positive ECs by FACS analysis RT reagent Kit (TaKaRa, Otsu, Japan). qPCR was performed using the with APC-labeled anti-mouse-CD31 antibody (MEC13.3, BioLegend, San resulting cDNAs with an initial denaturation at 95°C for 30 s, followed by Diego, CA; Fig. S1). Animal procedures were performed in accordance with 45 cycles of 95°C for 5 s and 60°C for 30 s using the SYBR Green Premix the Kanazawa Medical University Animal Guidelines under the authority of (Takara) with the primers listed in Table S1 in a StepOnePlus Realtime-PCR project license #2016-43. Animals were analyzed regardless of gender. System (Applied Biosystems, CA).

Plasmid construction and lentivirus production Western blotting To prepare lentiviral constructs, the coding region of human JunB (RefSeq Cells were suspended in Laemmli-sample buffer containing 5% NM_002229) was amplified by RT-PCR using total RNA from HMVECs 2-mercaptoethanol and were sonicated for 30 s and heated at 98°C for and was cloned into BamHI and NotI sites of the pCDH vector (System 5 min. The resulting protein samples were subjected to SDS-PAGE using Biosciences, Palo Alto, CA), and the sequences were then verified. shRNA 4–12% polyacrylamide gels (Wako, Osaka Japan). After electrophoresis, vectors were constructed in the pSIH vector (System Biosciences) using proteins were transferred onto PVDF membranes (Invitrogen) and blocked Journal of Cell Science

924 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 916-926 doi:10.1242/jcs.196303 with 5% skimmed milk in Tris-buffered saline. Anti-JunB (G53, cat. no. Whole-mount immunohistochemistry 3746, 1:1000; Cell Signaling Technology, Danvers, MA) and anti-β-actin Whole-mount immunohistochemistry analyses of mouse limb skin from (cat. no. A2228, 1:4000; Sigma-Aldrich) antibodies were used as primary embryos was performed according to a previously described procedure (Li and antibodies, and anti-rabbit IgG-HRP (Amasham GE, MA) was used as a Mukouyama, 2011) with some modifications. Briefly, mouse limbs from E9.5, secondary antibody. Protein signals were detected using ECL Plus E13.5 and E17.5 embryos were dissected and fixed in 4% paraformaldehyde Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). (PFA) overnight with gentle agitation. After three washes with phosphate- buffered saline (PBS) containing 0.2% Triton X-100 (PBS-T), dermal tissues In vitro 3D collagen matrix angiogenesis assays were separated from muscles and bones, and skin vasculature and neural fibers Angiogenic activities of ECs were analyzed using three-dimensional (3D) in dermal tissues were blocked with 10% goat serum in PBS-T, and were then collagen matrix angiogenesis assays as described previously (Koh et al., 2008). stained with anti-mouse-CD31 (MEC13.3, 1:200; BD Biosciences), anti- Briefly, type I collagen from rat tails (Millipore, Billerica, MA) was Neurofilament M (MAB1621, 1:200; Millipore), anti-JunB (ab28838 and reconstituted in M199 medium supplemented with 1-µM sphingosine 1- ab128878, 1:100 and 1:250, respectively; Abcam), eFluor-660-conjugated- phosphate (S1P; Avanti Polar Lipids, AL) with or without 200 ng/ml anti-β tubulin class III (2G10-TB3, cat. no. 50-4510, 1:250 eBioscience, San recombinant human Cxcl12 (R&D Systems, Minneapolis, MN) and was then Diego, CA) and DAPI (Invitrogen). Alexa Fluor conjugates of anti-rat, anti- placed in the bottom chamber of a µ-Slide (ibidi, Martinsried, Germany) and mouse, and anti-rabbit IgG (Invitrogen) were then used as secondary was transformed into a gel by incubation at 37°C in a CO2 incubator. Detached antibodies and series of fluorescent Z-stack images of dermal tissue ECs were then counted and re-suspended in M199medium containing reduced vasculature and nerve fiber networks were generated by using a LSM 710 serum supplement II at a dilution of 1:250, 40 ng/ml FGF-2, 50 µg/ml ascorbic confocal microscope (Carl Zeiss). acid and 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA). To analyze the involvement of integrin αVβ3 in JunB-induced 3D collagen angiogenesis, Apoptosis assay Cyclo (RGDfC) peptide (AnaSpec, CA), an inhibitor for integrin αVβ3- An apoptosis assay was performed using the Annexin-V Apoptosis mediated cell attachment, was added in collagen gel matrices at aconcentration Detection kit (BioVision, CA). ECs infected with the control shRNA of 5 µM. Subsequently, 2×104 cells were plated on the upper layer of the vector or the shJunB vector, both containing a GFP marker, were detached – µ-Slide and were incubated in a CO2 incubator for 48 h. Cells were fixed and and re-suspended in binding buffer containing 5 µl of Annexin-V biotin then stained with anti-human CD31 (WM59, cat no. 303101, 1:200; solution and incubated for 5 min. After washing, the cells were incubated BioLegend), anti-JunB (ab28838, 1:100; Abcam, Cambridge, MA) with avidin–APC. Annexin-V-positive and GFP-expressing cells were antibodies and DAPI (Invitrogen). Tubes with cells that invaded collagen analyzed by confocal microscopy (Carl Zeiss). matrices were observed using a LSM 710 confocal microscope (Carl Zeiss, Germany) and angiogenic activities were assessed according to numbers of Statistical and image analyses ECs that had migrated to a depth >100 µm. Data are expressed as mean±s.d. or s.e.m. Statistical analyses were performed using the R package (version 3.3.0; http://www.r-project.org). Pairwise EC spheroid sprouting assays differences between homoscedastic samples were identified using Student’s Thin Matrigel (BD Biosciences) matrices were formed by adding 10 µl t-tests, and those between heteroscedastic samples were identified using of Matrigel to the bottom chambers of µ-Slide Angiogenesis plates Welch’s t-test. Image analysis and quantification were performed with the Fiji (ibidi). Subsequently, detached ECs were inoculated at high density package of ImageJ (http://fiji.sc/), and Z-stacks were flattened through a (4×104 cells/well) in HuMedia-EB2 medium supplemented 1% FBS and maximum intensity projections. Vasculature formation analyses were were incubated until multicellular spheroids formed. After 24 h culture, performed by counting numbers of neurovascular contacts and blood vessel spheroid cells were visualized using a ZEISS Axiovert A1 microscope (Carl branches, and measuring branch lengths according to the criteria described in Zeiss) at 10× magnification, and the numbers of migrated cells per 20 Fig. S2. The images presented are representative of the analyzed data. surface cells were counted on at least six spheroids. Acknowledgements We thank Kazumi Tanaka (Kanazawa Medical University) for her help and Analyses of human JunB promoter activity experimental assistance. A human JunB promoter region containing 2 kb of sequence upstream of the start site was PCR amplified using genomic DNA from HMVECs and the Competing interests following primers: 5′-GAAGATCTAATTTCTGGCAGACATGTCT-3′ The authors declare no competing or financial interests. and 5′-CCCAAGCTTCGCTGCGGTGACCGGACTGG-3′, and were cloned into pNL1.2 Nano-Luc-PEST vectors (Promega, Madison, WI). Author contributions Y. Yoshitomi and H.Y. conceived and designed the experiments; Y.Yoshitomi and JunB promoter-Nano-Luc-PEST fragments were then cloned into pCDH H.Y. performed the majority of the experiments and analyzed the data with some vectors for lentivirus production. Subsequently, ECs were transduced with contributions from Y.I., T.I., H.S. and Y. Yoshitake. N.K. and T.H. assisted with DRG JunB promoter-Nano-Luc lentivirus and JunB promoter activity was culture and in utero injection, respectively. Y. Yoshitomi and H.Y. interpreted the analyzed by determining the NanoLuc luciferase activity using a Nano data. Y. Yoshitomi and H.Y. wrote the manuscript. Glo system (Promega). Funding This work was supported in part by the grants-in-aid from the Ministry of Education, In utero microinjections into embryonic limbs of mice Culture, Sports, Science and Technology (24790297 to Y.Y., 20590290 to H.Y.), and EC-tdTomato mouse embryos were accessed via the uterus under controlled Strategic Research Project grants from the Kanazawa Medical University and anesthesia on E12.5. Abdominal cavities were then cut open with a scalpel Ministry of Education, Culture, Sports, Science and Technology, Japan (H2012-16 and uterine horns were carefully extracted using ring forceps. Subsequently, [S1201022] to H.Y.). 2-µl lentiviral solutions (IFU=1.6×105 cells) were administered to forelimbs of embryos via in utero injections using 100-nm needles (Altair Data availability Corporation, Yokohama, Japan). The injection rate was controlled at The JunB overexpression effect on microvascular endothelial cell culture microarray data are available in Gene Expression Omnibus (GEO) under accession number 0.2 µl/s using a syringe pump (YSP-201, YMC, Tokyo, Japan) that was GSE93616. Microarray data for gene expression in microvascular endothelial cells co- controlled by a foot switch. After injection, uterine horns were carefully cultured with dorsal root ganglion cells are available under accession number replaced in the abdominal cavities and muscle and skin incisions were GSE93696. sutured separately. Mice were then rested on a thermal support device and recovery was monitored. At E17.5, embryos were harvested and limb skin Supplementary information vasculature was analyzed using whole-mount immunohistochemistry as Supplementary information available online at described below. http://jcs.biologists.org/lookup/doi/10.1242/jcs.196303.supplemental Journal of Cell Science

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