Nathalie Claire

Marie-Lise Laura

Dr Marie-Ange Renault

Béatrice Desmoures

Nicolas Fritsch

Vincent Jecko

Keywords: PDZRN3, Wnt/ Frizzled, endocytose, ubiquitinylation, angiogenèse, permeabilité, developpement, barrière hémato-encéphalique. Characterization PDZRN3, Wnt/ Frizzled, endocytosis, ubiquitinylation, angiogenesis, permeability, development, blood brain barrier

Characterisation De L'ubiquitine Ligase PDZRN3 En Tant Que Nouvel Acteur Des Voies Wnt Dans La Morphogenese Et L'integrite Vasculaire Résumé : Parmi les récepteurs Frizzled, Frizzled 4 est le seul à avoir un phénotype vasculaire fort. Par criblage, nous avons identifié l'ubiquitine ligase PDZRN3 en tant que nouveau partenaire de la protéine adaptatrice Dvl3 qui agit en aval de Fzd4. En utilisant des modèles murins inductibles, nous montrons que la délétion de PDZRN3 induit une létalité embryonnaire suite à des défauts de vascularisation du sac amniotique ; et que PDZRN3 est requis pour une vascularisation normale de la rétine. De par son activité d'ubiquitine ligase, PDZRN3 induit la prise en charge du complexe Fzd4/ Dvl3 par les vésicules d'endocytose ce qui permet la transduction du signal après fixation du ligand Wnt5a sur le récepteur Fzd4. PDZRN3 régule également le maintien des jonctions des cellules endothéliales et l'intégrité de la barrière hémato-encéphalique. La délétion de PDZRN3 stabilise les microvaisseaux après ischémie cérébrale. PDZRN3 induit la disruption des jonctions serrées et la rupture de la barrière hématoencéphalique en ubiquitinant la protéine d'échafaudage MUPP1.

 12) Statistical analysis ............................................................................................................ ..................................................................................................... 2) Chemical reagents ............................................................................................................ 3) Equipments ..................................................................................................................... .............................................................................................................................. e) Summary ............................................................................................................................ ......................................................................................... e) PDZRN3 in other tissues ...................................................................................................
LIST OF FIGURES

Figure 69: a-Syx /RhoA /Amot /MUPP1 and Rab13 colocalize in response to VEGF.A , as detected 2mins after treatment in vesicles and 30mins after treatment at the leading edge of migrating cells; b -Recycling vesicles are essential for polarized migration as depletion of Rab13 dirupts polarity of migrating cells [START_REF] Wu | Rab13-dependent trafficking of RhoA is required for directional migration and angiogenesis[END_REF]) ........

AVANT-PROPOS

INTRODUCTION Chapter I. Wnt signaling and Angiogenesis 1) The Wnt pathways

Wnt signaling plays a key role in many biological processes as it regulates cell proliferation, migration and polarity establishment. Wnts are essential during development as they guide body axis specification [START_REF] Barolo | Transgenic Wnt/TCF pathway reporters: all you need is Lef?[END_REF] The canonical pathway is implicated in proliferation as transcriptional activation of Cyclin D1 is a hallmark of the activation of the pathway upon β -catenin stabilization. C-myc / axin2 / VEGF/ endothelin and claudin 5 are also transcriptional targets of the pathway [START_REF] Zeng | Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions[END_REF].

ii) Non canonical / Planar cell polarity (PCP) pathway

The non-canonical pathways do not require β-Catenin or LRP5/6. Their activation usually leads to inhibition of the canonical pathway [START_REF] Grumolato | Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors[END_REF]. One of the non canonical pathways is the planar cell polarity signaling. PCP is distinct from apical/ basal polarity and can be defined as the The current consensus is that for activation of the PCP pathways, Wnt ligand would bind to their receptor (Fzd with Ror2 or Ryk) and recruit Dishevelled, which then forms a complex with Disheveledassociated activator of morphogenesis 1, Daam1. Activation of Dvl leads to phosphorylation/ relocalization to the nucleus of c-JUN, where it acts as a transcription factor with c-FOS and to activation of the small G-protein Rac, leading to activation of JNK [START_REF] Gan | Nuclear Dvl, c-Jun, beta-catenin, and TCF form a complex leading to stabilization of beta-catenin-TCF interaction[END_REF]. In parallel, Daam1

has been identified as a regulator of cell migration as it activates the small G-protein Rho, leading to activation of ROCK (Rho-associated kinase), which is one of the major regulators of the remodeling of the actin cytoskeleton (Ju et al. 2010[START_REF] Tsuji | Involvement of p114-RhoGEF and Lfc in Wnt-3a-and dishevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells[END_REF]. Most of the proteins involved in planar cell polarity in drosophila wing formation are conserved and used in vertebrates during regulation of cell movements during events such as gastrulation. Both processes imply control of actin filaments by the small G-proteins Rho and/ or Rac (van Amerongen and Nusse 2009). Other mechanisms are activated by PCP such as inflammation; phosphorylation of mitogen activated protein kinase p-38 would be induced by the non canonical pathway but not the canonical pathway, leading to inflammation [START_REF] Verkaar | Inhibition of Wnt/beta-catenin signaling by p38 MAP kinase inhibitors is explained by cross-reactivity with casein kinase Idelta/varepsilon[END_REF]. Interestingly, the PDZ protein GIPC is also a regulator of the localization of Vangl2 and of PCP in general [START_REF] Wang | A PDZ protein regulates the distribution of the transmembrane semaphorin, M-SemF[END_REF][START_REF] Giese | Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear[END_REF].

iii) Other non canonical pathways

In the Wnt/Calcium pathway, Wnt5a and Frizzled regulate intracellular calcium levels. Wnt ligand binding to Frizzled receptor causes coupled G-protein to activate mechanisms that lead to an increase of intracellular calcium concentration. This increase activates Cdc42 (cell division control protein 42)

through PKC (phosphokinase C), and calcineurin through CamKII (calcium/calmodulin-dependent kinase II). Calcineurin then induces activation of the transcription factor NFAT, which regulates ventral patterning. CamKII can also activate TAK1 and NLK kinase, which inhibit the canonical pathway [START_REF] Kelly | Extracellular Calcium-Sensing Receptor Inhibition of Intestinal EpithelialTNF Signaling Requires CaSR-Mediated Wnt5a/Ror2 Interaction[END_REF]. iii) Wnt3a (wingless-type MMTV integration site family, member 3A) Wnt3a can bind Fzd1, Fzd2, Fzd4 and LRP6 directly. WIF1 and SFRP1 bind to Wnt3a and inhibit its activity (Dufourcq et al. 2008). Wnt3a is able to induce the canonical pathway by promoting caveolin-dependent internalization of LRP6/ Fzd after binding of LRP6 [START_REF] Yamamoto | Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of beta-catenin signaling[END_REF].

Gene

c) Fzd receptors i) Overview of the Frizzled protein family

'Frizzled' refers to the phenotype of the frizzled mutant in Drosophila that has irregular patterned and curled hairs on the thorax, wings, and feet (Bridges and Brehme, 1944). Frizzled receptors family is a family of G protein-that act as receptors in the Wnt signaling pathway. These proteins are formed of seven-transmembrane domains, an extracellular N terminus, and an intracellular C terminus domain.

Their basic architecture is reminiscent of conventional G protein-coupled receptor. Phylogenetic analysis reveals that Fzd form 4 subfamilies indicating potential redundancy of function between Fzd1,2 and 7; Fzd 3 and 6;Fzd4,9 and 10 and between Fzd5 and 8 repsectively (Fig 8). The extracellular region (Fig 9) of all Frizzled receptors consists of an N-terminal signal sequence (amino acid 1 to 36 in human FZD4) that is important for the insertion of the protein in the membrane. This is followed by a cysteine-rich domain (CRD; aa 40-161 in hFZD 4), which constitutes the binding site for Wnt ligands and Norrin [START_REF] Schulte | International Union of Basic and Clinical Pharmacology. LXXX. The class Frizzled receptors[END_REF]. It is also hypothesized that Fzd receptors could form dimers via the CRD domain. The Fzd dimers would be sufficient to activate Wnt/ -catenin signaling.

Homology analysis indicates that Fzds share only 20 to 40% of amino acid identity, but the ten cysteines that form the five disulfide bonds in the CRD domain are highly conserved between the Fzd isoforms, indicating a strong pressure of selection on these positions. The CRD domain is followed by seven transmembrane regions that form three intracellular loops, three extracellular loops, and a C-terminus of variable length. These intracellular domains are essential because they mediate various protein-protein interactions and post-translational processing through phosphorylation, ubiquitinylation, nitrosylation, or hydroxylation. The K-T-x-x-x-W domain that is very close to the membrane is highly conserved in the family. This sequence serves as the domain for interaction with the DEP domain of Disheveled. Norrin (Ndp) mutant mice were very similar. By showing that interaction between Fzd4 and Norrin was specific and that FEVR-Associated Fz4 mutants bind Norrin but were unable to transduce signal, they were able to place Norrin as one of the activator of Fzd4 during retinal vascularization (Xu et al. 2004).

Furthermore, Ye et al found that endothelial specific deletion of fzd4 lead to retardation of vascular development of the retina, reduction in retinal capillaries and alteration of vessels of the vitreal surface that penetrate the retina that formed end in ball-like structures (Ye et al. 2010). Inhibition of Fzd4 using a blocking antibody would also lead to abnormal vascularization of the retina [START_REF] Paes | Frizzled 4 is required for retinal angiogenesis and maintenance of the bloodretina barrier[END_REF], confirming the role of the protein in the process of retinal vaculature. Fzd4 is also important in other organs. Loss of Fzd4 is also associated to cerebellar, auditory, and esophageal dysfunction [START_REF] Wang | Progressive cerebellar, auditory, and esophageal dysfunction caused by targeted disruption of the frizzled-4 gene[END_REF]. In a similar manner, it was demonstrated recently that Fz4 is required for blood barrier maintenance and plasticity in the central nervous system, as loss of Fzd4 leads to leakage in the cerebellum (Wang et al. 2012). These evidence shows that Fzd4 is vital for angiogenesis. That's why our laboratory has focused part of its research on this protein. LRP5 and 6 are highly homologous proteins. LRP5 contains a large extracellular domain containing four beta-propeller motifs and four epidermal growth factor-like repeats that form the binding sites for extracellular ligands. These domains are followed by three LDLR type A domains. The intracellular domain of LRP5 contains 5 PPPSP motifs. These motifs are phosphorylated upon binding of Wnt ligands and lead to binding of Ror2 to Axin, leading to activation of the canonical pathway [START_REF] Grumolato | Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors[END_REF]). Zeng et al proposed a similar model in which Wnt-induced Fz-Lrp5/6 complex formation would lead to recruitment of the axin-Gsk3 complex, Gsk3 would then induce Lrp5/6 phosphorylation to initiate B-catenin signaling [START_REF] Zeng | Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions[END_REF]. Interestingly, the phosphorylated forms of LRP5/6 have been shown to be internalized in clathrin-dependant vesicles with Dvl/ Fzd upon activation of the Wnt pathway, suggesting that LRP5/6 has a role for signal transduction that initiate in the early endosome [START_REF] Bilic | Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation[END_REF][START_REF] Yamamoto | Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of beta-catenin signaling[END_REF]. LRP5/6 is essential for angiogenesis as loss of the protein can also cause familial exudative vitreoretinopathy in humans, independently of loss of Fzd4 or Norrin (Toomes et al. 2004). As LRP5 knockout mice display incomplete vasculature in the deeper plexus of the retina while FZD4 and Norrin knockout mice develop severe defects in both the superficial and deep plexus of the retina, Xia et al hypothesised that LRP5 would mediate Wnt signaling in Muller cells present in the deep plexus to regulate vascularization of this layer of the retina (Xia 2010). This demonstration correlates with recent findings showing that Wnt signaling is the main signaling pathway implicated in the formation of the deep retinal plexus.

d) Coreceptors of Fzd proteins

ii) Ror2 functional. The tyrosine kinase activity would be essential for signal transduction in the non canonical pathway, as activation of c-Jun/ Dishevelled would be dependant on this activity, but no targets have been found yet [START_REF] Mikels | Ror2 receptor requires tyrosine kinase activity to mediate Wnt5A signaling[END_REF][START_REF] Ford | The dual role of the novel Wnt receptor tyrosine kinase, ROR2, in human carcinogenesis[END_REF]). Ror2 is phosphorylated on serine/threonine residues in its c-Terminal cystein rich domain upon stimulation of cultured cells with Wnt5a, but not with Wnt3a. Phosphorylation of Ror2 would be GSK3 dependant as blockade of GSK3 would block Ror2 induced migration of endothelial cells [START_REF] Yamamoto | Wnt5a modulates glycogen synthase kinase 3 to induce phosphorylation of receptor tyrosine kinase Ror2[END_REF]). Phosphorylation of Ror2 on this domain would depend on various kinases such as Casein Kinase 1-ϵ (CKIϵ) or Phosphokinase C PKC-Casein Kinase 1 would be directly responsible for phosphorylation of Ror2 while PKC- would activate kinases upstream of Casein Kinase 1 [START_REF] Kani | The receptor tyrosine kinase Ror2 associates with and is activated by casein kinase Iepsilon[END_REF]). Interestingly, use of the PKC- pseudosubstrate that blocks the kinase activity of PKC-blocks the phosphorylation of Ror2 and the activation of non canonical Wnt pathway [START_REF] Nomachi | Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A[END_REF]. Using the PKC- pseudosubstrate and ror2 targetting siRNA, it was shown that Ror2 activity was essential for formation of the lamellipodia and reorientation of microtubule-organizing center in Wnt5a-induced cell migration. The proposed model is that Ror2 would associate with the actin-binding protein filamin A to regulate reorganization of the actin cytoskeleton to induce migration of endothelial cell; and would be required for c-Jun / JNK activation that is essential for activation of the small GTPases Rho and Rac [START_REF] Nomachi | Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin A[END_REF].

iii) Ryk Ryk is an atypical member of the family of growth factor receptor protein tyrosine kinases, whose activity is not regulated by phosphorylation. The protein has a leucine-rich extracellular domain with a WIF-type Wnt binding region, a single transmembrane domain, and an intracellular tyrosine kinase domain. This protein is involved in stimulating Wnt signaling in processes such as the regulation of axon pathfinding. Ryk functions as a coreceptor with Fzd and binds Dvl to activate the canonical Wnt pathway (Lu et al. 2004). More recently, it was shown that the E3 ubiquitin ligase Mindbomb1 would reduce cell surface levels of Ryk leading to activation of the Wnt canonical pathway, indicating that Ryk would activate the non canonical pathway [START_REF] Berndt | Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/beta-catenin signaling[END_REF]).

e) Downstream effectors of Wnt signaling i) Dishevelled adaptor proteins

Dvl is the cytoplasmic link between Frizzled and downstream components of the Wnt signaling pathway. Dvl has no known enzymatic activity and functions as a scaffold protein bridging the receptors and downstream signaling components. The DIX domain is essential for -catenin activation, the DEP domain is implicated in the activation of the non canonical pathway, while the PDZ domain is required for both. Dvl can bind Axin2 via its DIX domainblocking Axin-induced degradation of β-catenin [START_REF] Fiedler | Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating betacatenin[END_REF]. The PDZ domain is required for the interaction with the C-terminal regions of Vangl1 and 2. Dvl interacts with Fzd4 (Chen et al. 2003) via both the PDZ and DEP domains (Fig 17). In presence of Wnt5a, cytoplasmic Dvl is recruited to the membrane via endocytic vesicles where it is phosphorylated [START_REF] Bryja | Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism[END_REF]).

Human and murine orthologues share more than 95% sequence identity and are each 40-50% identical to Drosophila Dvl where a single member of the family is found. Sequence similarity is concentrated around the DIX domain, the PDZ domain and the DEP domain, indicating that the three domains are essential. Three members of the Dishevelled family exist in mammalians (Dvl-1 to 3). Dvl2 is strongly expressed in majority of mammalian cells, while levels of Dvl1 and 3 are much lower. Dvl1 and 3 play the major role for the activation of both Wnt pathways. -catenin, protein composed of armadillo repeats was first identified as a scaffold protein and is one of the key components of the canonical Wnt signaling pathway. In the absence of the Wnt signal, βcatenin levels are down-regulated by a complex, composed of Axin/ CKI-α (casein kinase 1-α) / APC(Adenomatous Polyposis Coli) and GSK-3 β (glycogen synthase kinase 3) (Fig20 -1). Axin and APC act as scaffold protein bringing GSK3 and its substrate (β-catenin) into close physical proximity . This positions the N-terminus of β-catenin near CK1 and GSK3. CK1 phosphorylates β-catenin at Ser45 and GSK3 phosphorylates β-catenin at Thr41, Ser37 and Ser33 (Fig20 -3and4). The E3 ubiquitin ligase -TRCP1 then binds the phosphorylated N-terminus of β -catenin, causing its degradation . Transcription of β -catenin target genes is then repressed as the transcription factors LEF/TCF form a complex with groucho, inducing histone deacetylation of the target genes of βcatenin [START_REF] Saadeddin | The links between transcription, beta-catenin/JNK signaling, and carcinogenesis[END_REF]). In the presence of Wnt ligand, Fzd recruits Dvl which in turn brings to the membrane the axin-GSK3 complex that promotes phosphorylation of LRP5/6 by GSK3 and CK1 [START_REF] Zeng | Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions[END_REF] [START_REF] Balda | Epithelial cell adhesion and the regulation of gene expression[END_REF]) that stabilizes junctions when located at the adherens junction and act as proproliferative transcription factor when located to the nucleus. 1999[START_REF] Schreiber | Placental vascularisation requires the AP-1 component Fra1[END_REF]. Behrens et al. 1999 demonstrated that phosphorylation of Jun on ser63 is required for apoptosis. Jun can induce oncogenic transformation and was also found to be highly expressed in breast cancers (Johnson et al. 1999). Jun is also one of the first proteins to be upregulated after stroke, when the blood brain barrier is disrupted [START_REF] Mccoll | Systemic inflammation alters the kinetics of cerebrovascular tight junction disruption after experimental stroke in mice[END_REF].

iv) RhoA

The small guanosine triphosphatase (GTP) Ras homolog family member A (Rho A) is a major regulator of actin remodeling during cell morphogenesis and motility. Rho activates its effector ROCK1, which phosphorylates and activates LIM kinase, which in turn, phosphorylates cofilin, inhibiting its Wnt-5a is strongly expressed in the limb bud during development and its expression gradually fades (Yamaguchi et al., 1999). In the Wnt-5a KO embryos, the fingers are missing, due to massive activation of the canonical pathway, detected as an increase of β-catenin protein level, indicating that Wnt-5a would regulate the balance between the Wnt pathways (Fig 21) [START_REF] Topol | Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation[END_REF].

 Yolk sac and placenta vascularization Wnt signaling is also important for sac yolk angiogenesis. Yolk sac angiogenesis occurs between E8.5 and E10.5. Fzd5 is strongly expressed in vessels of the yolk sac at E9. Interestingly, the non canonical pathway is essential for yolk sac angiogenesis as deletion of jun or other members of the AP1 pathway such as c-Fos lead to vascular defects of the yolk sac and placenta leading to growth retardation of the embryo as from E7.5dpc [START_REF] Schorpp-Kistner | JunB is essential for mammalian placentation[END_REF][START_REF] Schreiber | Placental vascularisation requires the AP-1 component Fra1[END_REF].

This body of evidence shows that the PCP pathway is required for angiogenesis in the placenta and yolk sac, implying that the pathway is essential for embryonic development.

 Brain vascularization

Vascularization of the central nervous system starts at embryonic day 9 via angiogenesis from a previously developed primitive vascular plexus [START_REF] Paolinelli | The molecular basis of the blood brain barrier differentiation and maintenance. Is it still a mystery?[END_REF]. The newly formed vessels acquire specific characteristics in the brain as they establish unique barrier characteristics called bloodbrain barrier. The blood brain barrier (BBB) has a protective role as it prevents the flow of molecules from the blood to the tissue via expression of tight junction proteins that seal the space between endothelial cells. (Wang et al. 2012). Endothelial specific inactivation of -catenin also leads to vascular leakage in the embryonic brain (Liebner et al. 2008). The proposed model by the group of Liebner is that activation of the canonical pathway would activate transcription of claudin 3 and PDGF. These proteins are required for formation of tight junctions and recruitment of pericytes respectively. Both processes are required for induce vessel stabilization during brain development (Fig24, left) [START_REF] Reis | Wnt signaling in the vasculature[END_REF]. ECs lose their capacity to maintain the integrity of the blood retinal barrier and would be selectively eliminated, independently of their high level of proliferation (Wang et al. 2012).

 Role of myeloid cells [START_REF] Daneman | Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis[END_REF][START_REF] Liebner | Wnt/beta-catenin signaling controls development of the blood-brain barrier[END_REF]. These ligands would promote blood vessel branching as deletion of Wnt5a in retinal myeloid cells is sufficient to alter vessel structure of the deep plexus. This confirms that the non canonical Wnt pathway is essential for retinal angiogenesis at the level of the deep plexus (Stefater et al. 2011). Using these evidences, we can propose that retinal angiogenesis in the deep plexus would be regulated by the Wnt5a ligand secreted by myeloid cells that would activate endothelial cells via Fzd4/ LRP5, as deletion of Wnt5a in myeloid cells and Fzd4 / Lrp5 in endothelial cells both lead to disorganization of the retinal deeper plexus (Ye et al. 2010).

iii)

In adult: Hindlimb Ischemia Barandon et al. have shown that the competitor of Fzd, secreted frizzled-related protein-1 (sFRP-1)

would regulate response to myocardial infarction. Overexpression of sFRP-1 reduces neutrophil infiltration in ischemic tissue, by switching the balance of pro-and antiinflammatory cytokine expression, leading to a reduction in scar formation [START_REF] Barandon | Secreted frizzled-related protein-1 improves postinfarction scar formation through a modulation of inflammatory response[END_REF]. Many works show that the Wnt canonical pathway controls neovascularization after ischemia, using notably the hindlimb ischemia model (Oses et al. 2009). Ezan et al. showed that blockade of Wnt signaling by injection of virus expressing Secreted frizzled related proteins 1 (sFRP1) induced degradation of -catenin and reduces neoangiogenesis in ischemic muscle (Ezan et al. 2004). In a similar manner, the Wnt canonical pathway antagonist Dickkopf-1 blocked neoangiogenesis [START_REF] Smadja | The Wnt antagonist Dickkopf-1 increases endothelial progenitor cell angiogenic potential[END_REF], while expression of -catenin favored capillary formation after muscle ischemia by inducing recruitment of angiogenic progenitors and promoting proliferation of endothelial cell [START_REF] Kim | Beta-catenin overexpression augments angiogenesis and skeletal muscle regeneration through dual mechanism of vascular endothelial growth factor-mediated endothelial cell proliferation and progenitor cell mobilization[END_REF]. Another proposed mechanism is that Wnt5a would induce binding of Dvl to APC that interacts directly with microtubule-plus-end binding protein, FAK and paxillin. Both proteins are involved in the stabilization of microtubules at the cell periphery, which is important for the disassembly of the focal adhesion to stimulate cell migration (Matsumoto et al. 2010) (Fig 33). Additionally, Wong et al (2003) showed that Fzd2 and integrin 1 co-localize at the leading edge of polarized migrating cells, and that they would compete for binding with Paxillin. Taken together, these observations suggest that Wnt5a signaling through Fzd / Dvl and APC complex regulates adhesion negatively to promote cell migration (Fig33). Dvl proteins 1 and 3 can regulate cell migration by promoting remodeling of the actin cytoskeleton (Gao and Chen 2010). Dvl can associate with Daam1 that interacts with the Rho guanine nucleotide exchange factor WGEF (weak-similarity GEF), leading to the formation of the Rho-GTP complex, which activates the ROCK kinase and leads to remodeling of the cytoskeleton [START_REF] Habas | Wnt/Frizzled activation of Rho regulates vertebrate gastrulation and requires a novel Formin homology protein Daam1[END_REF].

Additionally, Daam1 can interact directly with the actin-binding protein Profilin1 that can control directional movement of the cell by promoting reorganization of the actin cytoskeleton (Sato et al 2006).

Other partners of Dvl can also control migration. In Xenopus, Dvl can interact with XRNF185 that induces paxillin destabilization, leading to cytoskeleton rearrangement and migration (Lioka et al 2007).

Dvl can also activate another small GTPase of the Rho family, Rac that can directly regulate migration and cell polarity / movement (Habas et al 2003). reasons (Ju et al. 2010). In ciliated cells, Dvl is essential for apical docking and planar polarization of basal bodies, a short cylindrical array of microtubules that serve as a core for the growth of cilium axoneme. Silencing of Dvl causes failure to localize basal bodies to the apical surface of ciliated cells, leading to defects in ciliogenesis (Park et al 2008). Other proteins interacting with Dvl such as Vangl2 also regulate position of the cilium [START_REF] Giese | Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear[END_REF][START_REF] Ezan | Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton[END_REF] iii

) Interplay between Wnt and Notch signaling during sprouting retinal angiogenesis

Sprouting angiogenesis in the retina would be dependent on two types of cells: the tip and stalk cells. Notch signaling would be central to the establishment of these identities (Fig 36). translocates to the nucleus to regulate transcription of genes such as Hair/Enhancer of Split genes [START_REF] Tung | Tips, stalks, tubes: notch-mediated cell fate determination and mechanisms of tubulogenesis during angiogenesis[END_REF]. The formation of a tip cell is induced when extracellular VEGF-A binds VEGF receptor 2 (VEGFR-2) causing up-regulation of the Notch ligand Delta-like 4 (Dll4). Membrane-bound Dll4 then interacts with Notch1 receptors on the adjacent endothelial cells. This leads to the down-regulation of VEGFR-2 and up-regulation of VEGFR-1 in these cells, which is a high-affinity/ low-activity receptor of VEGF-A. This creates a situation in which one cell expresses high VEGFR-2 levels and its neighbor cell is less sensitive to VEGF-A signaling [START_REF] Liu | Regulation of Notch1 and Dll4 by Vascular Endothelial Growth Factor in Arterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis[END_REF][START_REF] Hellstrom | Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis[END_REF]). The cell expressing VEGFR-2 and Dll4 becomes a "tip" cell by producing filopodia that extend in the direction of the angiogenic factors. The neighbor of the tip cell becomes a "stalk" cells that proliferate and contribute to the elongation of the sprout (Fig36) (Herbert and Stainier 2011).

Chapter II. Regulation of Wnt signaling by endocytosis and ubiquitinylation 1) Endocytosis as a regulator of Wnt signaling

Endocytosis has recently emerged as a regulator of various pathways including Wnt signaling.

Endocytosis would regulate availability of Fzd receptors on the cell surface and would be essential for signal transduction after binding of Wnt ligands of the Fzd receptors. Endocytosis can mediate cell signaling in different ways. Endocytosis can downregulate signaling by promoting degradation of receptorsas it is the case for some small G protein-coupled proteins that are internalized and fused with the lysosome for degradation (Fig 38a). Endocytosis can also maintain signaling complex after their formation at the plasma membraneas it is the case for activation of the mitogen activated protein kinase (MAPK) that leads to formation of the RAS-RAF-MAPK-ERK complex in endosome and activation of ERK (Fig 38b). Endocytosis can also generate signalas in the case of transforming growth factor-B receptor that is internalized into endosomes leading to recruitement and phosphorylation of SMAD2 leading to signal transduction (Fig 38c) [START_REF] Grant | Pathways and mechanisms of endocytic recycling[END_REF][START_REF] Sadowski | Signaling from endosomes: location makes a difference[END_REF][START_REF] Dobrowolski | Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles[END_REF].

a) Role of the early endosome in signal transduction

b) Role of the early endosome in Wnt signaling

In The vegetal pole of the egg would contain 'maternal determinants' such as Wnt-containing early endosomes that would be incorporated into multivesicular bodies (MVB) on the dorsal side of the egg.

These MVB would sequester GSK3, making Wnt induced embryonic differentiation possible, leading notably to formation of the Nieuwkoop signaling center. The vegetal pole is transported to the opposite of the site of entry of the sperm, forming the dorsal side (Fig 41). This hypothesis is clearly relevant as injection in the egg of interfering RNAs targeted against Wnt key proteins coding RNAs doesn't block development of the embryo, even if Wnt signaling is essential for the process (as shown using KO strategy). This model would also explain why injection of Wnt inhibitors, such as dkk1 or sfrps cannot inhibit Wnt signaling in the early embryo: as the Wnt signaling complex would be protected inside early endosomes at this stage of embryonic development [START_REF] Leyns | Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer[END_REF][START_REF] Kemp | Expression of Frizzled5, Frizzled7, and Frizzled10 during early mouse development and interactions with canonical Wnt signaling[END_REF]).

2) Ubiquitinylation as a regulator of Wnt signaling

Ubiquitinylation has emerged as a key process for regulation of endocytosis and Wnt pathway, by acting as a targeting system of proteins to vesicular systems, such as the endosome or lysosome.

a) Role of ubiquitylation in protein stability or function

Ubiquitinylation can control trafficking/ degradation / localization or function of signaling components depending on the type of ubiquitinchains attached to the cargo [START_REF] Tauriello | Loss of the tumor suppressor CYLD enhances Wnt/beta-catenin signaling through K63-linked ubiquitination of Dvl[END_REF].

Following addition of an ubiquitin molecule to a substrate, further ubiquitin molecules can be added, leading to formation of a polyubiquitin chain. In addition, some substrates are modified by addition of ubiquitin molecules to multiple lysines residues in a process termed multiubiquitination (Fig 42a ).

b) Ubiquitinylation of Fzd proteins

3) PDZRN3 E3 ubiquitin ligase

During my PhD, a large part of my work focused on the ubiquitin ligase PDZRN3 that we have sorted out using a yeast two hybrid screening using Dvl3 as bait.

a) Identification of PDZRN3 PDZRN3 possesses a PDZ binding domain. These are usually composed of Thr -X -Val -COOH [START_REF] Nie | A novel PTB-PDZ domain interaction mediates isoform-specific ubiquitylation of mammalian Numb[END_REF], Valiente et al. 2005). The last 150 amino acid region of PDZRN3, including the PDZ binding domain (Fig 52) are highly conserved across diverse species, suggesting that this domain is essential for the function of the protein [START_REF] Flynn | Molecular evolution of the LNX gene family[END_REF].

iii) Endocytic scaffolding

Proteins carrying PDZ and PDZ binding domains can form scaffolds. It has been suggested that because these proteins interact with junctions, they could be implicated in formation of 'endocytic scaffold'. These structures would promote endocytosis of transmembrane proteins after binding of a ligand. Kandansku suggested in 2006 that a paralogue of PDZRN3, ligand of Numb X (LNX) would provide an endocytic scaffold for JAM4, after binding of the Notch antagonist NUMB, leading to reorganization of cell junctions. [START_REF] Kansaku | Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4[END_REF]). PDZRN3 could have similar functions.

iv) Homology with GIPC / Synectin

PDZRN3 shares homology of sequence with SEMACAP 1 and 2 (GIPC 1 and 2 or Synectin). GIPC are small proteins with a single C-terminal PDZ domain, implicated in clathrin-mediated endocytosis [START_REF] Djiane | The Drosophila GIPC homologue can modulate myosin based processes and planar cell polarity but is not essential for development[END_REF]. GIPC1 also interacts with Endoglin that in turn phosphorylates Smad1 /Smad5 /Smad8, leading to inhibition of endothelial cell migration. The GIPC-null mice has reduced numbers and altered pattern of arteries, whereas the venous system is normal (Lee et al 2008).

c) PDZRN3 is a member of the LNX family of protein

Chapter III. Wnt signaling and vascular integrity 1) Wnt signaling regulates endothelial permeability in the Blood brain barrier

Wnt signaling is required for differentiation of brain vessels during development, during which it is essential for formation of TJ or recruitment of pericytes. It is also required for maintenance of the blood brain barrier (BBB) integrity in adulthood. Phosphorylation of Foxo1 is also used to assess the integrity of the BBB [START_REF] Wang | Elevated expression of forkhead box protein O1 (FoxO1) in alcohol-induced intestinal barrier dysfunction[END_REF]. (cytotoxic edema) [START_REF] Sandoval | Blood-brain barrier tight junction permeability and ischemic stroke[END_REF]. Non canonical/ Wnt5a dependant Wnt signaling is also regulated positively by Calcium influx (Liu et al. 2008[START_REF] Kelly | Extracellular Calcium-Sensing Receptor Inhibition of Intestinal EpithelialTNF Signaling Requires CaSR-Mediated Wnt5a/Ror2 Interaction[END_REF]. As calcium flux occurs early in stroke, it could be related to the observed activation of the pathway.

a) Wnt signaling as a regulator of brain vessels permeability

Brain vessels are also altered in Alzheimer disease. Endothelial inflamation and increase of blood flow have been observed in patients suffering from the disease [START_REF] Rn | Small vessel disease and alzheimer's dementia: Pathological consideration[END_REF][START_REF] Marchesi | Alzheimer's dementia begins as a disease of small blood vessels, damaged by oxidativeinduced inflammation and dysregulated amyloid metabolism: implications for early detection and therapy[END_REF]. Increases of permeability of capillaries and leakage have also been described [START_REF] Biron | Amyloid triggers extensive cerebral angiogenesis causing blood brain barrier permeability and hypervascularity in Alzheimer's disease[END_REF]. Interestingly, modulation of the canonical Wnt pathway has been observed in neurons of Alzheimer affected subjects.

This pathway would be downregulated in such subjects and restoring the pathway using chemical agents would decrease the severity of the pathology [START_REF] Kitazawa | Blocking IL-1 signaling rescues cognition, attenuates tau pathology, and restores neuronal betacatenin pathway function in an Alzheimer's disease model[END_REF], Wang et al. 2011). More recently, it was shown that Wnt5a and Fzd-5 were up-regulated in brain of mice suffering from Alzheimer and that the constituent of amyloid plaques, beta-amyloid peptide stimulated Wnt5a and Fz5 expression in cortical cultures. The Wnt5a activated pathway would be able to regulate neuroinflammation and would aggravate the pathology (Bei Li. 2011).

Parkinson disease is also a pathology where brain vessel function is altered. Increase of blood flow in subjects affected by the disease has been described [START_REF] Kortekaas | Blood-brain barrier dysfunction in parkinsonian midbrain in vivo[END_REF][START_REF] Rite | Blood-brain barrier disruption induces in vivo degeneration of nigral dopaminergic neurons[END_REF], Hutton. 2010). Several proteins have been shown to have a role in the process such as Glucose transporters;

ApoE or PKC [START_REF] Nassia Methia | ApoE Deficiency Compromises the Blood Brain Barrier Especially After Injury[END_REF][START_REF] Youdim | The essentiality of Bcl-2, PKC and proteasome-ubiquitin complex activations in the neuroprotective-antiapoptotic action of the anti-Parkinson drug, rasagiline[END_REF]. However, no direct links between Wnt signaling and Parkinson disease have been described for the moment. Interestingly, various ubiquitin ligases have been linked to the disease; Parkin that gave the name to the disease [START_REF] Shimura | Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase[END_REF] but also various other proteins linked to the proteasome [START_REF] Fornai | Parkinson-like syndrome induced by continuous MPTP infusion: convergent roles of the ubiquitin-proteasome system and alphasynuclein[END_REF], Li et al. 2006[START_REF] Meray | Reversible monoubiquitination regulates the Parkinson diseaseassociated ubiquitin hydrolase UCH-L1[END_REF][START_REF] Fishman-Jacob | A sporadic Parkinson disease model via silencing of the ubiquitin-proteasome/E3 ligase component SKP1A[END_REF][START_REF] Ullrich | Ubiquitin enzymes, ubiquitin and proteasome activity in blood mononuclear cells of MCI, Alzheimer and Parkinson patients[END_REF]). These processes have been described in the neuronal cell but not in the endothelial cell.

Finally, Wnt signaling has been linked to formation of neuroblastoma. Wnt3a and Dvl would favor RhoA signaling and neural retractation in these cells [START_REF] Tsuji | Involvement of p114-RhoGEF and Lfc in Wnt-3a-and dishevelled-induced RhoA activation and neurite retraction in N1E-115 mouse neuroblastoma cells[END_REF]. Proapoptic stimuli would induce GSK3 activation in these cells [START_REF] Bijur | Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta[END_REF], while the Wnt5a/Ror2 pathway would be upregulated in these cancer cells [START_REF] Rebagay | ROR1 and ROR2 in Human Malignancies: Potentials for Targeted Therapy[END_REF]. Augmentation of blood flow and pressure has also been described in this disease [START_REF] Wong | Acute changes in blood pressure in patients with neuroblastoma treated with (1)(3)(1)I-metaiodobenzylguanidine (MIBG)[END_REF])

2) Molecular mechanisms of regulation of vascular permeability by

Wnt signaling a) Structure of the tight junctions

The TJ first known as 'zonula occludens'(ZO) are essential for maintenance of the integrity of the The three MAGUK proteins of the TJ are phosphorylated. Initially, it was observed that ZO-1 from a low-transepithelial electrical resistance (TER) strain of MDCK cells contained almost twice as much phosphate as that from the high-TER strain. This suggests that tyrosine phosphorylation of ZO proteins would weaken junctional sealing [START_REF] Gonzalez-Mariscal | MAGUK proteins: structure and role in the tight junction[END_REF]. In a similar manner, the phosphorylation level of ZO-2 is significantly higher in cells with disassembled TJ via Calcium chelation when compared with confluent monolayers. This is due to the action of both PKC and cAMPdependent protein kinase. This suggests that the phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex [START_REF] Avila-Flores | Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C[END_REF].

b) Interplay between catenin and ZO1 ZO2 detaches from the TJliberating c-Jun, thus promoting the non canonical Wnt pathway [START_REF] Betanzos | The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells[END_REF].

3) MUPP1 acts as a bridge between TJ and Wnt signaling

MUPP1 (multiple PDZ domain containing protein-1) is a member of the LNX family of protein that is present at the TJ. In silico predictions indicate that PDZRN3 could interact with MUPP1, indicating that the protein could be a link between PDZRN3 and TJ. MUPP1 is a member of the LNX protein family that shares a high degree of similarity with LNX1/2 and its paralogue PATJ. MUPP1 contains an amino terminal L27 domain and 13 PDZ domains, while PATJ has the same architecture but with 'only' 10 PDZ domains. MUPP1 can form homodimers or dimers with LNX1 and 2 via their PDZ domains [START_REF] Flynn | Molecular evolution of the LNX gene family[END_REF]; suggesting that PDZRN3 would also be able to interact with MUPP1. AH109 cells expressing GAL4-Dvl3DIX were combined with Y187 cells expressing the mouse E11 embryo cDNA library (Clontech, 638868). The mating mixture was plated on SD/ -TRY, -LEU, -HIS, -ADE plates. From colonies obtained after transformation, more of 500 colonies tested were His positive/LacZ positive. The library plasmids from those colonies were rescued, amplified by PCR, and sequenced.

2) Expression constructs

3) Analysis of mRNA expression by quantitative RT-PCR

For RT-PCR analysis, 1µg of total RNA from mouse tissue or cell extracts was reverse-transcribed:

PCR was performed using IQ SYBR Green supermix (Bio-Rad). An MJ Research Opticon and the following parameters were used for real-time PCR: 95°C for 5 minutes followed by 34 cycles of 95°C for 30 seconds, 60°C for 1 minute, 72°C for 30 seconds, 76°C for 1 minute and 80°C for 1 minute.

Negative controls without RT were prepared in parallel for each RNA sample. All experiments were performed in triplicate and differences in cDNA input were compensated by normalization to expression of B-actin. Selected primers indicated below:

Forward primer (5' to 3') Reverse primer (5' to 3')

m beta-actin NM_007393 ATTTCTTTTGACTTGCGGGC GGAGGAGCTGGAAGCAGCC m PDZRN3 NM_018884.2 CTGACTCTTGTCCTGCATCGGGACTC ATGGGCTCCTTGGCTGTCTTGAAAGC h beta-actin NM_001101 ATTTCTTTTGACTTGCGGGC GGAGGAGCTGGAAGCAGCC h PDZRN3 NM_015009.1 ATTATTGAGGTCAACGGCAG AGGGCCATGATATGTTCAAAG-

4) Cell culture

HEK293T were cultured in DMEM supplemented with 10% FBS (Hyclone) and penicillinstreptomycin. Hela cells were cultured in RPMI supplemented with 10% FBS and penicillinstreptomycin. Human umbilical vein endothelial cells (HUVEC) were cultured in EGM-2 media (Lonza, CC-3156) supplemented with EGM-2 Single Quots (Lonza, CC-4176), and plated on 1% gelatin-coated dish. MS1 (mouse pancreatic islet endothelial cell line) were cultured in RPMI supplemented with 10%

FBS and penicillin-streptomycin, and plated on 0,2% gelatin-coated dish. All these cells were serum starved with DMEM plus 1% FBS/0.25%BSA.

a) Wnt conditioned media production

Wnt3a and Wnt5a producing mouse L cells (American Type Culture Collection ATCC) were cultured in DMEM medium, 10% fetal bovine serum (FBS) and penicillin-streptomycin supplemented with G418 (0,4 and 0,6mg/ml respectively). Conditioned medium (CM) was harvested as described in ATCC protocol. Wnt5a CM was produced in DMEM medium, 0.5% FBS and penicillin-streptomycin, while Wnt3a CM was produced in DMEM medium, 10% FBS and penicillin-streptomycin.

b) Cell transfection / transduction

Plasmids were transfected using Jetprime (Polyplus) following the manufacturer's protocol (final concentration of 1.5 µg/ml for HEK293, 0.5µg/ml for Hela). Lentivirus were tranduced using Protamin sulfate (4µg/ml) at a multipilicity of infection of 20 (MOI). SiRNA were transfected using Interferin

e) Luciferase reporter assay

In order to study the canonical pathway, HELA cells grown in 48-well culture plates were transiently transfected at 60-70% confluence with 0.2 µg/ml of 8XTOPflash reporter (Nusse lab), 0.1 µg/ml of b galactosidase reporter and 0,2 µg/ml of various plasmids. The total quantity of DNA in the transfection mixes was adjusted to a total of 0,5µg/ml using an empty vector. 24 after transfection, cells were activated overnight with Wnt3a conditioned medium as indicated. Luciferase activities were measured using the Luciferase Reporter Assay system (Promega) according to the manufacture's specifications. The firefly luciferase activity was normalized to b galactosidase activity. In order to analyze the non canonical pathway, HELA cells were transiently transfected with 0.2 µg of AP1 reporter, 0.1 µg of b galactosidase reporter (to evaluate efficiency of transfection) and various plasmids as indicated for individual experiments.

5) Protein analysis a) Western Blot

For Western Blot analysis, organ tissues or cells were lysed for 30 minutes in RIPA buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate, 50mmol/L Tris, pH 8.0, 2µg/ml aprotinin, 10µg/ml leupeptin, 200µM AEBSF, 1mM benzamidine, 1µM orthovonadate, 1nM okadaic acid). Cell lysates were cleared by centrifugation at 14 000 g for 10 min. The various fractions were dosed using BCA quantification kit (Thermo scientific). 25µg of each fraction were then resolved by 7,5% SDS-PAGE (1mm) and probed with indicated antibodies. Binding of antibodies to the blots was detected using Odyssey Infrared Imaging System (LI-COR Biosciences).

b) Immunoprecipitation assay

Immunoprecipitation assays were done on HEK293T after transfection or tissue homogenates.

Lysates were incubated 1hour with primary antibody at +4°C with gentle agitation. After this 25 µl of protein G-Sepharose slurry were added and samples were incubated overnight at +4°C. Beads were washed three times and subsequently resuspended and boiled in 60µl of loading buffer for SDS-PAGE (H2R).

c) Cell fractionation

For Cell fractionation, cell lysates or tissues lysates are resuspended in BH Buffer (Pipes pH6.8, KCl 10mM, MgCl2 1,5mM, Sucrose 250mM, EGTA 1mM, EDTA 1mM) and potterized 20times on ice.

After a centrifugation at 800g for 5mins, the supernatant is collected and spinned at 100.000g for 1hour.

The pellet is then resupended in BH buffer + Triton1% + SDS 1% and identified as the 'cell membrane fraction'. The supernatant is collected and corresponds to the 'cytoplasmic fraction'. The pellet obtained after the 800g centrifugation is washed in BH buffer and spinned at 2700g for 5mins 3 times. The cleaned pellet is then resuspended, placed as a cushion on top of a sucrose buffer (Sucrose 1M) and spinned at 2700g for 5mins. The pellet is then resuspended in a Tris buffer (Tris pH 7.4, NaCl 10mM, MgCl2 3mM, EGTA 1mM, NP40 0.05%) and spinned at 2700g for 20mins. The pellet in resuspended in a HEPES buffer (Hepes pH 7.9, NaCl 300mM, MgCl2 1,5mM, EDTA 2mM) that lyses the nuclear membrane. After a 20 min incubation, the lysate is spinned at 10000g for 20mins. The pellet is identified as the 'nuclear membrane fraction' and the cytoplasm as the 'soluble nuclear fraction'. The cytoplasmic fraction is normalized using tubulin, the nucleic fractions with Histone H1 and the membrane fraction using Ve-cadherin.

d) Ubiquitinylation assay

For ubiquitinylation assay, pRK5-HA-UB-Wt (17608, Adgene), pRK5-HA-UB K48 (17605, Addgene) that codes for lysine 48-only-ubiquitin and prK5-HA-UB K63 (17606, Addgene) that codes for lysine-63-only ubiquitin were purchased at Addgene. HEK293T were transfected with Dvl3-myc (0,5µg/ml), one of the ubiquitin coding plasmids (0,5µg/ml) and PDZRN3-V5 OR empty plasmid (0,5µg/ml). Immunoprecipitation was then realized as described before using a myc antibody.

6) Immunocytochemistry a) Immunocytochemistry

Immunofluorescence analyses were realized on HELA cells or Huvecs. The cells were fixed in Formalin 4% for 20mins and permeabilized using Triton 0,2% for 2mins before 30min of saturation in PBS+5% BSA. The cells were incubated with the primary antibody (diluted in PBS +1% BSA) for 1 hour at 37°C and with the secondary antibody for 30mins at room temperature.

b) Internalization assay

For Internalization assay, the cells were transfected with the protein we wanted to follow. After activation, the cells were fixed and incubated with a first primary/ secondary antibody directed against the target diluted in PBS + 1% BSA. After a step of permeabilization (Triton 0,1% for 5mins), the cells are incubated with a second couple of primary / secondary antiboday directed against the target. For example, we could use a rabbitpolyclonal antibody revealed with a Alexa 568 anti rabbit before permeabilization, and a mousemonoclonal antibody revealed with Alexa 488 anti mouse. The first antibody reveals the membrane located target (Alexa 568in red) and the second antibody reveals the membrane located and endocyted target (Alexa 488in green).

7) Immunohistochemistry a) Paraffin sections

Tissues are fixed in Methanol / Methanol80% + DMSO 20% or Paraformaldehyde 4%. The samples are then dehydrated with successive ethanol/ xylene baths and then included in a paraffin block. 7µM sections are then realized. Cells are colored in blue using Hemalun for 2mins. Red blood cells were stained in pink using Eosin (Sigma) for 30secs.

b) Cryosections

Tissues are fixed in paraformaldehyde 4% and transferred in Sucrose 30%. Once tissue has sunk in sucrose solution, it is embedded in OCT embedding solution. 10µM sections are then realized.

c) Vibratome sections

Tissues are fixed in paraformaldehyde 4% and included in agarose 4%. 100µM slices are realized using a vibratome (Leica).

d) In toto embryo staining

Embryos (10.5dpc maximun) are dissected and fixed in 4% paraformaldehyde overnight (or Methanol 80%+DMSO 20% for 2 hours). Once the embryo sunk to the bottom of the solutions, it is permeabilized in 0,5% Triton for 2hours. Embryos are then blocked in milk 5%+ Triton 0,5% for 2hours. Embryos are then incubated with antibody for 24-48h in milk 5%+Triton 0,5%. Embryos are then placed in 100% glycerol for 48h. Once the embryo sunk to the bottom of the solution, it is transfered in 75%glycerol+ 25% gelatin and finally transferred in 50% glycerol before being mounted.

The embryos were imaged using a confocal microscope (Olympus FSV 100).

e) Dab staining

Endogenous peroxydases are saturated by incubated the samples in PBS + 3% H202 for 20mins. The cells were incubated with the primary antibody (diluted in PBS +1% Horse serum and 0,5% Triton) overnight at 4°C, with the secondary antibody and then peroxydase for 2h at room temperature respectively. The DAB revelation Kit is then used for the revelation of the peroxydase.

f) Immunofluorescence

Immunofluorescence analyses were realized on various tissue samples. The samples were incubated with the primary antibody (diluted in PBS +1% HS+0,5% Triton) for 1 hour at 37°C or ON at 4°C and with the secondary antibody for 1 to 4 hours at room temperature. Alexa 488/ 568 / 647 goat-anti mouse/ rabbit and donkey anti-goat were used as secondary antibody. The DNA was counterstained by 4',6-diamidino-2-phenylindole (DAPI) or Hoechst.

8) Image analysis

Cells were imaged using a ZEISS microscope at 63X and analyzed using AXIOVISION. For colocalization studies, cells were analyzed with a Olympus laser scanning confocal microscope using multichannel scanning using a 63x/1.4 ApoPlan oil immersion objective. Three-dimensional projections were deconvoluted using Autoquant(Mediacybernetics) and digitally reconstituted from stacks of confocal optical slices by Imaris software (Bitplane).

9) In vitro assays on endothelial cells a) 2D tubulogenesis assay

The Capillary-like tube formation assay was carried out by plating Growth factor-reduced Matrigel® (Becton Dickinson) plated on the bottom of µ-slide angiogenesis plate (Ibidi), and allowing it to gel at 37 °C for 1 hr. 150,000 cells/well, were seeded on Matrigel and incubated at 37 °C, in growth media with 20 ng/ml purified recombinant human VEGF165 (RandD Systems). After 6 h at 37 °C, formation of tube-like structures was examined under an inverted light microscope (Olympus) at 4.2× magnification. Two images were taken for each well. The number of lumen per field, the length/number of branches per branch point and cell covered area were quantified (Axiovision, Cell profiler).

b) Wound assay

The assay was carried out by plating endothelial cells on each well of culture-inserts (Ibidi) and allowing them to reach confluence. After a 4h serum starvation, culture-inserts were removed, and cells were stimulated with control or Wnt3a conditioned medium (diluted 1/20). The subsequent gaps were imaged at baseline and 8 hr later. The area of the gaps was quantified using Axiovision.

c) 3D tubulogenesis (cytodex) assay

For 3D tubulogenesis assay, HMVECs were transduced with a GFP expressing lentivirus and transfected with various siRNAs. Gelatin-coated microcarrier beads (Cytodex 3; Sigma) were incubated with the HMVECs. When the cells reached confluence on the beads, equal numbers of HMVECs-coated beads were embedded in fibrin gels in a µ-slide angiogenesis plate (Ibidi). For preparation of fibrin gels, bovine fibrinogen (2.5 mg/mL; Sigma) was dissolved in Dulbecco's modified eagle medium

f) Junctions disruption by calcium chelation

Treating cells with PBS supplemented with the calcium chelator EDTA (2,5nM) induces disruption of the junctions maintaining cell monolayer progressively. Junctions in Huvecs cells monolayers are disrupted totally after 30mins. This process was imaged by time-lapse videomicroscopy each 30sec under an inverted microscope (Zeiss). The movies were analyzed using Imaris (Bitplane).

10) Experimental animals

a) Cre -Lox system

b) Tetracyclin activated system

We have generated mice that had integrated the gene coding for Pdzrn3 followed by the sequence coding for a V5 cassette under the control of a transactivator. These mice were subsequently backcrossed on C57BL6/J background. Interbred, homozygous Pdzrn3 -V5 mice exhibit apparently normal development, and are viable and fertile. For endothelial cell-specific overexpression, Tie2tta (Tet-off) transgenics were bred in a background of Pdzrn3-V5 mice. Tie2 -tta male were mated with Pdzrn3-V5 females. Tails of pups were genotyped by PCR using the P1/P2 primer set for the PDZRN3-V5 coding gene: 5' CAGCTTGAGGATAAGGCGCT-3'; P2: 5'-CTTCGAGCTGGACCGCTTC-3'.

c) Injections

Intraocular injection was performed using the injector from World precision instruments. 0.5µl of Dynasore (200µM in 6%DMSO; 94 % Ethanol) was injected in one eye of the animal at the speed of 50nl/sec; the other eye was injected with a solution of 6%DMSO; 94% Ethanol.

11) Surgery a) Retinal vascularization analysis

For immunofluorescence of mouse retinas, eyes from mice sacrificed at P5 were fixed in 4% paraformaldehyde at 4 °C for 1 h and dissected, before being incubated at 4 °C overnight with FITC labeled -isolectin B4 (Sigma Aldrich) diluted in PBS buffer with 0.5% Triton X-100, and 5% horse Cerebral infarcts were produced by 90mins occlusion of the middle cerebral artery (MCAO) followed by reperfusion. A silicone-coated 8-0 monofilament (Doccol) was introduced into the internal carotid artery and advanced of a distance of about 10mm to occlude the middle cerebral artery under anesthesia (isoflurane). After 90mins, the animals were anesthetized again and the filament withdrawn.

Animals are monitored 1, 3 and 24 hours after MCAO using these criterias. For Evans blue extraction, both brain hemispheres were separated, weighed and stored at -80°C.

The tissues were lysed in 1ml of PBS using a tissue-lyser and spinned at 15000g for 30mins. To 500µl of the supernatant, 500µl of trichloroacetic acid was addedand the mixture stored at 4°C overnight.

After a centrifugation at 15000g for 30mins, the supernatant is collected and optical density measured at 610 and 690nm. The quantity of Evans blue per gram of tissue is then calculated using a standard curve.

For evaluation of Dextran extravasation, 500µl of a solution of FITC -Dextran 70kDa (Sigma) was injected in the ventricle. After 5mins of circulation, the mouse was decapitated and the brain prepared for OCT embedding and cryosection [START_REF] Hoffmann | High and Low Molecular Weight Fluorescein Isothiocyanate (FITC)-Dextrans to Assess Blood-Brain Barrier Disruption: Technical Considerations[END_REF][START_REF] Bouleti | Protective effects of angiopoietinlike 4 on cerebrovascular and functional damages in ischaemic stroke[END_REF].

MRI imaging was done in collaboration with the RMSB (www.rmsb.u-bordeaux2.fr). Diffusion, volume of edema and anisotropy were measured. A strategy of diffusion tensor imaging was also put in place. Interestingly, MRI strategies have shown that volume of the edema was reduced in some knock out mice [START_REF] Bouleti | Protective effects of angiopoietinlike 4 on cerebrovascular and functional damages in ischaemic stroke[END_REF], while only diffusion was modified in other cases [START_REF] Badaut | Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference[END_REF].

c) Aortic ring assay

Dorsal aortas from from mice sacrificed at P5 were surgically removed, cut in several rings. A 10-μL drop of Matrigel® (Becton Dickinson) was added to a well of a µslide angiogenesis plate (Ibidi).

Aortic explants were put on top of the gel, covered with a 10-μL drop of gel, and incubated 15 min to solidify. 40µl of EGM-2 media (Promocell) was then added and the explants were cultured for 72h.

d) Evaluation of vascular leakage in embryo and pups

Pregnant mice were anesthetized and 15.5dpc embryos were extracted from the yolk sac with the umbilical chord still attached. 5µl of a 4mg/ ml solution of Rhodamin -Dextran (Invitrogen) was injected in the kidney; after 5mins, the embryo was killed by decapitation and the head embedded in OCT for cryosection. In pups, 10µl of the same solution was injected in the ventricle [START_REF] Choi | Minimally invasive molecular delivery into the brain using optical modulation of vascular permeability[END_REF]). The liver, heart and the hind limb vasculature were imaged with a high-resolution micro-CT imaging system (GE eXplore Locus SP), set to a 0.08-mm effective detector pixel size. The apparatus used for imaging the vessels was the eXplore Locus Micro-CT scanner from General Electric Healthcare® with spatial resolutions of 36 to 7 μm, and used with the Scan Control® and Reconstruction Utility® programs. Data were acquired in an axial mode as described before [START_REF] Tirziu | Delayed arteriogenesis in hypercholesterolemic mice[END_REF]. The quantification parameters were obtained using the Microview ABA® program.

e) Micro computer tomography

f) Isolation of primary endothelial cells from mouse kidney

Kidneys were removed in PBS-containing CaCl2 and MgCl2 (Gibco), minced and incubated for 45 min at 37 ° C in PBS supplemented with collagenase type I (Sigma). The suspensions were triturated with a cannula, filtered through a 70 microns (BD) and 30 microns filter (Miltenyi). Cells were cultured in MEM medium supplemented with D-valine for 5 days. After trypsinization, the cell suspension was incubated with anti-CD31 (Pharmingen) and anti-endoglin (Santacruz) antibodies for 30 min, placed in the presence of microbeads coupled with rat IgG (Miltenyi) for 15 min and passaged through a magnetic column (MACS MS columns, Miltenyi), the endothelial cell preparation was recolted. Brain or retina were frozen at -20°C. The tissue is potterized 20times on ice in PBS. After a 10min / 1000g centrifugation, the pellet is resuspended in PBS and placed on top of a PBS / Dextran 17,5% gradient. After a 10min / 4400g centrifugation, the pellet is resuspended and filtered via a 40µm filter (BD). The fraction retained on the filtered in then eluted with PBS by inversion of the filter. For optimal extraction of vessels, the vessel fraction can be loaded on a column containing glass beads. After washing with PBS, the purified vessel fraction is recolted by gentle agitation. This fraction is collected as is for western blotting. For immunostaining, after a 10min/13000g centrifugation, the pellet is collected in a paraformaldehyde 4% -Sucrose 2% solution and spinned 30mins at 1300rpmon a polylysine 0,01% coatedglass slide. After this operation, the capillaries are adherent to the slide.

g) Vessel extraction

12) Statistical analysis

Results were expressed as means ± SD. All analyses were performed with appropriate software (GraphPAD Prism). Comparison of continuous variables between two groups was performed by an unpaired t-test and subsequently, if statistical significance was observed, by a non parametric Mann-Whitney test. A value of p <0.05 was considered to be statistically significant.

B) Reagents 1) List of used antibodies

a) State of art

Fzd4 is a receptor for Wnt proteins. Binding of the Wnt ligands to the Fzd receptor lead to activation of disheveled proteins and activation of Wnt target genes. Fzd4 can activate both the canonical and the non canonical pathway depending of receptor context. In the canonical pathway, it promotes accumulation of -catenin and stimulation of LEF/TCF-mediated transcriptional program. In the non canonical pathway, it induces activation of c-jun in a process that depends on PKC [START_REF] Schulte | beta-Arrestins -scaffolds and signalling elements essential for WNT/Frizzled signalling pathways?[END_REF].

The protein also plays a key role in retinal vascularization where it interacts with norrin. The exudative vitreoretinopathy is caused by mutations affecting the fzd4 gene. This disease is characterized by a cessation of growth of retinal capillaries, leading to formation of an avascular region in the periphery of the retina. This leads to compensatory retinal neovascularization, which induces in some cases exudation and bleeding / or retinal detachment. Similar defects have been found in the fzd4 knockout mice where the retinal vasculature is massively reduced.

b) Hypothesis

c) Summary

This study identifies Fzd4 and Dvl3 as essential actors of vascular morphogonesis and remodeling after ischemia. The fact that both proteins modulate activation of the Wnt / non canonical pathway and are essential for the orientation of the endothelial cell in vitro; and for neovascularization to occur correctly, identifies for the first time the link between non canonical Wnt pathway and orientation/ function of the endothelial cell. During development, blood vessel formation ensures tissue Fzd4 is linked to sterility. 10 We have previously demonstrated growth and organ function in the entire organism. Fzd4 impairs vascular cell proliferation and migration and performs this function through a noncanonical Wnt/PCP-dependent pathway altering golgi organization and tubulin acetylation. Among the Dvl members, we identified Dvl3 as a preferential target of Fzd4, because Fzd4 relocalizes and activates Dvl3 on the apical cell membrane. We report that Fzd4 expression is polarized in cells and organizes with Dvl3 to create a signaling platform favoring -tubulin recruitment and reorganization. Moreover, in postischemic neoangiogenesis, fzd4 deletion impairs the angiogenic process. In control mice, after surgery, an arterial network is generated that extends directionally toward the extremity of the leg during the revascularization process, where as depletion of fzd4 induces formation of a disorganized nondirectional and nonfunctional arterial network.

Results

Impairment of Arterial and Arteriolar Formation in the Absence of Fzd4

To elucidate the function of Fzd4 in postnatal development, we studied its vascular expression pattern in fzd4 /LacZ mice, which allows visualization of Fzd4-expressing cells using the -galactosidase reporter driven by the fzd4 promoter. Our results demonstrated differential expression in the adult vasculature with expression in the aorta, carotid artery, and other large arteries, and no expression in large veins (online Figure I). Frequent fzd4 promoter activity was detected in the endothelial cell (EC) and smooth muscle cell (SMC) layers of small arterioles and in various vascular beds, including the heart, kidney, and skeletal muscle (online Figure I).

Microcomputed tomography three-dimensional quantitative analysis of the cardiac and renal vasculature was performed in fzd / mice. Comparison between fzd4 / and fzd4 / adult mice revealed a striking arterial phenotype with a quantitative reduction in arterial branching and density (Figure 1A,B). In the heart of fzd4 / mice, large coronary arteries enclose the heart and divide to give rise to an arterial network toward the apex that penetrates and perfuses the heart wall. In fzd4 / mice, the large coronary arteries were present, but the arterial network was severely reduced, most notably in the apex and interventricle septum. In the kidney of fzd4 / mice, the main renal artery courses in the renal hilum, dividing into segmental arteries that branch into the lobar arteries, supplying a dense capillary network in the cortex. In contrast, the kidney of fzd4 / mice showed a reduction of the smaller arteries, particularly in the renal cortex while the primary arterial architecture in the renal hilum was conserved. Quantitative polymerase chain reaction analysis of heart and kidney from fzd4 / and fzd4 / pups demonstrated a significant decrease in the arterial marker Ephrin B2 mRNA expression in both the heart (0.370.24-fold) and kidney (0.350.20-fold) of fzd4 / compared to fzd4 / pups (P0.05), whereas mRNA for the venous marker Eph B4 was not statistically different between these grou ps (Figure 1C). Western blot analysis of kidney and heart tissues also confirmed a significant decrease in expression of Ephrin B2 (Figure 1D). These data support the notion that Fzd4 is required for the development of arterioles and capillaries, but is not required for the formation of larger vessels.

Because the loss of fzd4 induced an impressive vascular phenotype described, we next examined the functional impact of fzd4 / on cardiac and renal function. No difference in diastolic or systolic heart function was observed in any group (Tau and contractile index dP/dt minimum and maximum under basal or dobutamine treatment), and we did not identify any water, acid-base, or electrolyte imbalance when mice were studied under basal conditions (online Table I).

Loss of fzd4 Altered Vascular Cell Proliferation and Migration

To gain further insight into the cell-autonomous function of Fzd4, we isolated ECs, SMCs, and murine embryonic fibroblasts (MEFs) from wild type and fzd4 / mice (Figure 2A, online Figure II). Primary fzd4 / EC, SMC, and MEF proliferation, analyzed by counting cells positive for BrdUrd, was significantly reduced compared to fzd4 / EC and MEF, respectively (Figure 2B,C,D). Consistent with these findings, fzd4 knockdown (kDa) of fzd4 / MEF and human microvascular EC reduced their proliferation as early as 2 days after culture, confirming an important impact of Fzd4 on EC and MEF proliferation (online Figure III). We next investigated the branching capability of fzd4 / EC in an in vitro three-dimensional matrigel assay. Consistent with results of the proliferation assay, fzd4 / EC had decreased ability to form branches in matrigel assay (Figure 2C). Additional comparisons showed that fzd4 / EC had a reduced migration compared to control EC using scratch and transwell assays under serum, vascular endothelial growth factor A, or fetal growth factor 2 activation (Figure 2F). These data suggest that Fzd4 controls cell migration under indistinct stimuli. Similarly, wounding assays demonstrated that fzd4 / SMC and MEF have a significantly reduced capacity to migrate compared to, respectively, that of fzd4 / SMC and MEF (Figure 2G,H).

Loss of fzd4 Altered Microtubule Stabilization and Cell Polarization During Migration

Because cell proliferation and migration are affected by fzd4 depletion, we analyzed the level of acetylated tubulin in MEF. Cell extracts were examined by Western blotting. The absence of fzd4 resulted in decreased -tubulin acetylation without a change in the protein level of -tubulin (Figure 3A). Because cell migration requires cell polarization, we directly measured cell polarity when stimulated by a directional stimulus by evaluating microtubule organization and by calculating the organization and volume of the Golgi in EC, SMC, and MEF migrating out of a wounded monolayer. The fzd4 / MEF display a filamentous network of -tubulin oriented toward the wound healing. Suppression of Fzd4 decreased and altered the organization of the tubulin in migrating MEF (Figure 3B). Quantitative analysis of fixed immunostained samples showed that in migrating cells, Golgi assembled in front of the nucleus in a dense and compact structure (Figure 3C). We estimated the average labeling intensity of the Golgi along the line crossing the center of the nucleus with the center of the Golgi and showed that the Golgi labeling intensity was reduced in the front of the nucleus in fzd4 / EC, SMC, and MEF compared to control wildtype cells (Figure 3C). Moreover,infzd4 / cells,thevolume of the Golgi was increased compared to control cells, revealing that the Golgi was less organized and largest at the front of the nucleus in fzd4 / cells compared to control cells (Figure 3).

Cooperation of Fzd4 With Dvl3 Is Required for Noncanonical Signaling Pathway

Because Dvl has been shown to play a role in cell polarity, we next investigated whether Fzd4 may shuttle Dvl away from the Wnt/-catenin pathway to favor a noncanonical pathway. We found that in fzd4 / compared to fzd4 / MEF, the basal level in the active -catenin fraction (ABC, the form of -catenin dephosphorylated on Ser37 and Thr41) 15 was significantly increased; under Wnt3a activation, both fzd4 / and fzd4 / MEF were able to induce the active catenin fraction (Figure 4A). To determine if this increase in active -catenin is associated with transcriptional activity, we transfected the cells with the topflash -catenin construct. A significant increase in reporter activity was found in fzd4 / compared to fzd4 / MEF under basal condition or under Wnt3a activation (Figure 4B). These data suggest that Fzd4 might repress the canonical Wnt/-catenin signaling pathway. To test if Fzd4 can control the Wnt canonical pathway, we analyzed the modulation by Fzd4 of the top reporter activity. In HEK293T cells, we show that expression of Fzd4 alone represses Wnt3A activation of the topflash reporter. Dvl3 overexpression induces strong top reporter activity. Coexpression of Fzd4 and Dvl3 blocks Dvl3-dependent activation of -catenin transcriptional activity (Figure 4C).

We next addressed the requirement of Fzd4 to induce noncanonical signaling. We have previously demonstrated that in EC, sFRP1 via Fzd4 was able to induce a noncanonical pathway via a dependent activation of downstream components of the Wnt-PCP pathway such as GSK3 and Rac1. 2 Because it was reported that in Wnt/PCP signaling, Dvl activates JNK via a Rac-dependant pathway, 16 we analyzed JNK signaling in fzd4 / and fzd4 / MEF transfected with an AP1-dependent transcriptional luciferase reporter and analyzed its activation under Wnt5A activation. We found an increase in AP1 reporter activity in fzd4 / MEF under Wnt5A stimulation after 6 hours but not in fzd4 / MEF (Figure 4D). Because Dvl proteins are central in the control of the PCP pathway, we sought to **P0.01. B, The fzd4 / and fzd4 / MEFs were transiently cotransfected with 8X top flash and -galactosidase reporter vectors. After culture in the presence of Wnt3a CM during 3 hours and 6 hours, activity of the -catenin signaling pathway was quantified by measuring the relative luciferase activity (RLU) normalized to -galactosidase activity. Relative luciferase values are shown. *P0.05. C, Fzd4 reduces -catenin-induced activity. HEK293T cells were cotransfected with 8X top flash and -galactosidase reporter plasmids with an empty vector (pcDNA3), with one plasmid encoding for either Fzd4-tagged HA (Fzd4-HA) or Dvl3-tagged green (Dvl3G), or with both the Fzd4 and Dvl3 plasmids. After culture in the presence of Wnt3a CM for 16 hours, cells were harvested for luciferasebased top flash assay of Wnt signaling. *P0.05, **P0.01. Fzd4 favors AP1 activity. D, Loss of fzd4 inhibits AP1 activity. The fzd4 / and fzd4 / MEF were transiently cotransfected with AP1 and -galactosidase reporter vectors. After culture in the presence of Wnt5a CM for 1, 3, and 6 hours, AP1 signaling pathway activity was quantified by measuring the relative firefly luciferase activity normalized to -galactosidase activity. **P0.01. E, Fzd4 in presence of Dvl1 or Dvl3 induces a robust AP1 activity. AP1 and -galactosidase reporter plasmid transfected HEK293T cells were cotransfected with plasmid coding for either Dvl1, Dvl2, or Dvl3 tagged green (respectively, Dvl1G, Dvl2G, or Dvl3G) either with an empty vector (pcDNA3) or with a plasmid encoding Fzd4 (Fzd4-HA). AP1 signaling pathway activity was quantified by measuring the relative firefly luciferase activity normalized to -galactosidase activity (arbitrary units). *P0.05.

interrogate the ability of Fzd4 to interact preferentially with one of the Dvl isoforms to induce the Wnt noncanonical pathway. We transfected HEK293T cells with the AP1 luciferase reporter and measured activity of cells transfected with Dvl1, Dvl2, and Dvl3 in the absence or presence of Fzd4 (Figure 4E). Transfections with plasmid coding for either Dvl isoforms or Fzd4 alone do not activate the reporter. However, cotransfection with Fzd4 and Dvl1 or Fzd4 and Dvl3 lead to an activation of the AP1 reporter, which is not found when Fzd4 and Dvl2 are cotransfected.

Fzd4 Preferentially Directs Relocalization and Activation of Dvl3 and, to a Lesser Extent, Dvl1

We examined, on a cell-by-cell basis, colocalization of Dvl isoforms with Fzd4. Transient expression of Dvl1, Dvl2, and Dvl3 tagged with green epitope (respectively, Dvl3G, Dvl2G, and Dvl3G) showed punctate staining were transiently transfected either with an empty vector () or with a plasmid coding for Fzd4 with a Tag GFP (Fzd4-GFP). Cell lysates were immunoprecipitated with anti-GFP antibody. The samples were analyzed by Western blotting with the indicated antibodies. Fzd4 induces Dvl3 activation. C, HEK293T cells were transiently cotransfected with a plasmid coding for Dvl3 tagged with a green epitope (Dvl3G) either with an empty vector () or with plasmids coding for Fzd4-HA or for Fzd4-CRD. Cell lysates were analyzed by Western blotting with the indicated antibodies. D, HEK293T cells were transiently transfected either with an empty vector () or with plasmid coding for Fzd4 (Fzd4-GFP). After cell fractionation, Western blotting was performed on membrane (Mbne) and cytosolic (Cyto) fractions with indicated antibodies. Open arrow indicates Dvl3 unphosphorylated forms (78 and 80 kDa) and black arrow shows the phoshorylated (82 kDa) shifted form. Fzd4 polarizes Dvl3 expression at the edge of the cell with -tubulin. E, NIH3T3 cells were transfected with plasmids coding for Dvl3-tagged green (Dvl3G; green) alone or in the presence of a plasmid coding for Fzd4 with a tag HA (Fzd4HA). Cells were immunostained for Fzd4 with anti-HA (purple) and for -tubulin with anti--tubulin (red). Note that Dvl3 alone is expressed in large punctates, whereas, in the presence of Fzd4, it is redistributed and colocalized at the plasma membrane with -tubulin. F, NIH 3T3 cells were transfected either with Fzd4-GFP or with Dvl3G plasmid. Lysates were immunoprecipitated with anti-GFP antibody and then analyzed by Western blotting with the indicated antibodies. throughout the cytoplasm (Figure 5A), which correlates with large multiprotein complexes, as previously reported. 17,18 Fzd4 tagged with the HA epitope (Fzd4-HA) showed preferential localization at the apical side of the cytoplasmic membrane. Cotransfection with Fzd4-HA revealed a major change in the distribution of Dvl3G and Dvl1G with relocalization to the cytoplasmic membrane and an even appearance throughout the cytoplasm in 95% and 70% of the cells, respectively. We did not find relocalization of Dvl2G when cotransfected with Fzd4-HA (Figure 5A). Quantification of Fzd4-induced relocalization of Dvl to the cytoplasmic membrane was performed by merging images and using Imaris software confocal image analysis. Through this method, we established that Fzd4-HA was able to preferentially relocalize Dvl3G to the membrane as compared to Dvl1G (28.4% of Dvl3G vs 2.7% of Dvl1G). Together, these results imply that Fzd4 interacts preferentially with Dvl3 and, to a lesser extent, with Dvl1, but not with Dvl2 to induce the noncanonical Wnt pathway.

We confirmed the ability of Fzd4 to associate with the endogenous Dvl1 and Dvl3 isoforms from mammalian cell lysates by immunoprecipitation and immunoblotting assays. Cell extracts were prepared from HEK293T cells transfected with Fzd4-GFP fusion protein and immunoprecipitated with antibodies against GFP. Immunoprecipitation of Fzd4-GFP led to coprecipitation of endogenous Dvl1 and Dvl3 (Figure 5B). We next examined the capacity of Fzd4 to activate Dvl3 and found that cotransfection of HEK293T with Fzd4-HA and Dvl3G led to strong phosphorylation of Dvl3G, as shown by a mobility shift of the protein on SDS-PAGE (Figure 5C). To confirm that the observed signaling is specific to Fzd4, cells were cotransfected with a plasmid coding for the CRD domain of Fzd4 (Fzd4 CRD). 8 Fzd4 CRD overexpression did not induce exogenous Dvl3G phosphorylation. Because we show that Fzd4 affects Dvl3 localization and interacts physically with Dvl3, we next wanted to confirm that the activation of Dvl3 was localized at the cytoplasmic membrane. HEK293T were transfected with a plasmid coding for Fzd4GFP; then, membrane and cytoplasmic fractions were separated using ultracentrifugation steps. Western blot analysis revealed that Fzd4-GFP was exclusively detected in the membrane fraction and not in the cytosolic fraction, although endogenous Dvl3 is evenly distributed in both fractions. These results indicate that Fzd4 leads to an endogenous Dvl3 shift in Fzd4 transfected cells, but not in control membrane fractions. In the cytosolic fractions, we did not detect any modification of endogenous Dvl3 profile both in Fzd4-GFP transfected and control conditions (Figure 5D).

We found that Fzd4 accumulates in a polarized way at the cytoplasmic membrane on the top of the cell (Figure 5A). When cells were transfected with Fzd4-GFP, we found an increase in -tubulin in membrane fractions, although the overall level of -tubulin detected in the cytoplasm was not modified in Fzd4 transfected versus control conditions (Figure 5D). Because Fzd4 affects tubulin organization (Figure 3B), we then investigated whether Fzd4-based molecular signals might drive the selective localization of Dvl3 and -tubulin in polarizing migrating cells. When we overexpressed Fzd4-HA with DVL3G and then visualized it with immunofluorescence, we found that both colocalized in an oriented manner driven partially by -tubulin at the cytoplasmic membrane (Figure 5E). Using immunoprecipitation, we found that Fzd4-GFP interacts with -tubulin, and when Fzd4-GFP and Dvl3G were cotransfected, Fzd4 and Dvl3G form a complex that enhances -tubulin recruitment (Figure 5F).

We have performed experiments to analyze whether reexpression of Fzd4 with Dvl3 in sifzd4-treated EC might restore their ability to proliferate and form tubes. We show that double expression of Fzd4 along with Dvl3 was able to significantly rescue the impact of fzd4 deletion on EC proliferation, but expression of Fzd4 or Dvl3 alone did not have this effect (Figure 6A). Tube formation assays revealed that sihfzd4 treatment reduced the ability of cells to form capillary networks and that Dvl3 with Fzd4 was able to rescue the fzd4 deletion phenotype, significantly increasing branch lengths and branch numbers per branch point (Figure 6B). It is noteworthy that Dvl3 alone is able to significantly increase capillary network formation. Collectively, these results suggest that Fzd4 and Dvl3 interact to promote the Wnt-dependent pathway in vascular cells.

Our genetic analysis together with a biochemical analysis have identified Fzd4 with Dvl3, and to a lesser extent Dvl1, as crucial partners for PCP pathway induction in vascular cells. These results confirmed that Fzd4 expression is polarized in cells and organizes a platform with Dvl3 to enhance -tubulin recruitment.

Dvl3 DEP Domain Is Crucial for Fzd4-Induced Relocalization

Next, we sought to identify the domain of Dvl3 that interacts with Fzd4. It has been previously shown that Dvlmediated activation of the Wnt/-catenin or the Wnt/PCP signaling pathway is dependent of the availability of specific domains DIX, PDZ, and DEP. 19,20 Although the DIX and PDZ domains are necessary for Wnt canonical signaling, the DEP domain is essential for PCP signaling 21-23 and linked to Rac 1 activation. 20,24 To identify the Dvl domain necessary for Fzd4 interaction, we used myc-tagged truncated Dvl constructs lacking the DIX (DIX Dvl3), the PDZ (PDZ Dvl3), the DEP (DEP Dvl3), and a construct containing DEP domain (DEP). Distribution of the constructs was examined after transfection of HEK293T cells. Any one of the myc-tagged Dvl3 deleted constructs were detected in the cytoplasm in a punctate pattern mimicking the Dvl3-tagged myc pattern. It should be noted that the DEP Dvl3 construct also was detected in the nucleus (data not shown). The constructs were then cotransfected with a plasmid coding for HA-tagged Fzd4 in HEK293T cells. Only deletion of DEP domain impeded Dvl3 relocalization at the membrane. It is worth noting that DEP mutants strongly accumulate and colocalize with Fzd4 into the cytoplasmic membrane, confirming the major role of the DEP domain for interaction with Fzd4 (online Figure IV). In combination, these results indicate that Fzd4 regulates the phosphorylation and polarized cellular localization of Dvl3, and that Fzd4/Dvl3 interaction functions in noncanonical Wnt signaling.

Fzd4 Deletion Induces Arterial Disorganization in Ischemia-Induced Angiogenesis

To understand the importance of Fzd4 during vascular formation, we evaluated in vivo whether Fzd4 gene disruption impairs adult angiogenesis. It is noteworthy that under nonischemic conditions, quantitative analysis of the arterial network by microcomputed tomography showed a reduction in both arterial density and branching in the hind limb of fzd4 / compared to control mice (Figure 7 A,C,D). These results were confirmed with a lower capillary density in fzd4 / versus control mice, evaluated by immunohistochemistry (online Table I). We then explored the capacity of fzd4 / mice to restore a functional arterial network after ischemia using a femoral artery ligation model. Blood flow recovery in the hind limb was reduced in fzd4 / compared to control mice after 2 weeks (Figure 7B). This was consistent with a reduction in both the arterial density and branching in fzd4 / mice after ischemia, evaluated by quantitative analysis of microcomputed tomography images (Figure 7C,D). Interestingly, three-dimensional images revealed that in fzd4 / mice compared to controls, neovasculature structures were severely disorganized. In control mice, angiogenesis induces the formation of vascular structures oriented directionally toward the extremity of the hind limb. In fzd4 / mice, we found a modification of the organization with the formation of few clusters of arterioles with a radial organization (Figure 7A). These data demonstrate that Fzd4 deletion impairs arterial formation and vesseloriented branching in peripheral organ. These data support the concept that Fzd4 plays a role major in vessel organization via Wnt-dependent PCP polarity pathway.

Discussion

Fzd4 is expressed in different vascular beds throughout the organism. Genetic defects of fzd4 have been linked to familial exudative vitreoretinopathy, a progressive retinal vascular disease. 5 The fzd4 deletion has been shown in mice to impair fertility, 10 to alter vessel formation in the cerebellum, 9 and to interact with Norrin to form retinal vessel network. 8 In addition, our group has previously demonstrated that Fzd4 can interact with sFRP1, a potent angiogenic factor. 2 All these data taken together indicate that Fzd4 signaling may play an important role in vascular formation. In this article, we extend and strengthen this notion by providing evidence that Fzd4 is required for formation and branching of the vascular arterial network in peripheral organs (with regard to their vascular system) through a noncanonical Wnt/PCPdependent mechanism.

Fzd4 Deletion Selectively Impairs Small Artery and Capillary Formation in Peripheral Organs

We have demonstrated that Fzd4 is expressed throughout the vasculature, and in EC and SMC of arteries, small arterioles, and capillaries, but not in large veins, and that fzd4 depletion results in a drastic reduction in the number of small-caliber arteries and in their branching. This defective vessel development is restricted to the arterial system consistent with the observation of reduced arterial, but not venous, marker expressions. Collectively, these data suggest the importance of properly regulating Fzd4 activity during arterial development because Fzd4 controls induction and branching of small arterioles and capillary beds at the distal end of tissue. Despite these Figure 7. The fzd4 deletion impairs the angiogenic response. A, Reconstructed microcomputed tomography images representative in fzd4 / (/) and fzd4 / (/) hind limb mice before (NI) and after ischemia at D15. B, Quantitative analysis of hind limb blood flow was performed by calculating ischemic/normal limb perfusions ratios after the induction of hind limb ischemia. Note a decrease (C) in vessels number and (D) in branching/connectivity in fzd4 / (/) and fzd4 / (/) hind limb in nonischemic (NI) conditions and 15 days after ischemia (D15). *P0.05. marked arterial defects and a reduction in body size, other aspects of heart and kidney vascular development, such as formation and branching of large arteries, are not affected.

The formation of new vessels requires both vascular cell proliferation and migration to enable sprout growth and the formation of a branched network. We have demonstrated that reducing expression of Fzd4 mitigated EC and SMC proliferation, migration, and EC tube formation. In a wound model, the Golgi is oriented in front of the nucleus 25 around the centrosome, the organizing center for cytoplasmic microtubules. 26 In this model, we showed that fzd4 deletion impairs both tubulin and Golgi reorganization and capillary-like network formation ex vivo, which support a critical role of Fzd4 in vessel branching. Thus, we hypothesize that Fzd4 can control vascular formation by a noncanonical-dependent mechanism in peripheral organs. How Is the Wnt/Frizzled Signaling Pathway Involved in the Vascular System? In vitro studies have demonstrated a functional role of the canonical -catenin-dependent pathway in EC. 27 It has been proposed that during development, the Wnt/-catenin pathway was only functional during angiogenesis in the central nervous system but not in peripheral tissue as in heart and kidney 28 ; after birth, the activation of the Lef/Tcf Wnt reporter system is only restricted to a few vascular beds in the central nervous system. 29 Analysis of -catenin conditional depletion demonstrated a major role of -catenin in blood-brain barrier maturation. 30 Consistent with these findings, in responder top gal transgenic mice, we could not detect any -gal-positive EC, outside of the central nervous system, in heart and kidney at postnatal day 5 or in adults (data non shown). Additionally, we have demonstrated that sFRP1 was also able to activate a noncanonical Rac1-dependant signaling pathway in EC. 2 Recent studies demonstrated a role of PCP in endothelial cells through Daam1-forming family members. Daam1 has been shown to be involved in polarity of embryonic development in Xenopus and Drosophila acting downstream of frizzled receptors via an interaction with Dvl. 23 In EC, activated Daam1 would control proliferation and migration, 3 affecting both cell polarity and cell movement, 31 regulating the microtubule network. 32 Our data corroborate the role of PCP in EC and disclose the role of Fzd4 in vascular cells in Wnt/PCP signaling pathway, because we showed that Fzd4 regulates microtubule network. Using in vitro assays, we report that Fzd4 was able to repress the Wnt canonical pathway and activate the noncanonical pathway. These results demonstrate that Fzd4 is localized in a polarized manner on the top of the cell membrane, and it is able to recruit and preferentially activate DVl3. Three isoforms of Dvl proteins have been identified and share high-sequence homology. Genetic approaches have previously shown that Dvl has been involved in PCP signaling during gastrulation and that it has a functionally redundant role during development. 33,34 Interestingly, Dvl3 mutants exhibited craniorachischisis, which is often associated with PCP signaling disruption. Our data further demonstrate that Fzd4 is one of the major receptors of the Wnt/PCP pathway in angiogenesis and facilitates cell polarization through tubulin network reorganization.

Fzd4 and Pathogenesis

Our results suggest that absence of Fzd4 leads to impaired vascular cell polarization. To test the impact of Fzd4 deletion on angiogenesis, we used a mouse model of vessel formation after hind limb ischemia. These data revealed that the absence of Fzd4 impairs adult neoangiogenesis and results in the formation of a disorganized network. Given the fact that the noncanonical Wnt cascade is involved in the growth and guidance of neurons, 35 it may be speculated that the directional growth of the peripheral vascular network is a feature of PCP signaling involving Fzd4 receptors.

This study extends our understanding of the role of Fzd4 in adult peripheral organs and during postischemic angiogenesis, and implicates Fzd4 as an important regulator in the directional development and branching morphogenesis of small-caliber arteries and arterioles through a Wnt/PCP pathway in vascular cells.

DETAILED METHODS

Experimental Animals fzd4 +/LacZ heterozygous knock-in-mice were bred in C57BL6 ( 8backcross) and CBA (7 backcross) background and then interbred to generate fzd4 -/-and control (wild-type) littermate mice. Genotyping of fzd4 mutant mice were performed as previously described 1 . A description of the hindlimb ischemia model and laser Doppler perfusion imaging can be found in the Supplemental section.fzd4

We produced fzd4 +/-hets during the breeding as fzd4 -/-mice are sterile and could only be generated using fzd4 +/-mice crossbreeding. The fzd4 +/-mice are healthy and fertile and are grossly indistinguishable from their littermate wild-type. They displayed no gross alteration in the morphology (there is no significative differences in weight and size compared to wild type littermate). However a careful examination showed that fzd4 +/-mice have a vascular phenotypic alteration of the arterial network in the hindlimb after ischemia, compared to wildtype littermates but not likely at the fzd4 +/-level.

For microCT scanning

The vasculature was imaged with a high-resolution micro-CT imaging system (GE eXplore Locus SP), set to a 0.016-mm effective detector pixel size as previously described 7 . The apparatus used for imaging the vessels was the eXplore Locus Micro-CT scanner from General Electric Healthcare® with spatial resolutions of 36 to 7 μm, and used with the 3D image reconstruction software (Digisens®). Data were acquired in an axial mode covering a single hindlimb as described 8 . The quantification parameters were obtained using the Microview ABA® program.

Bones of the hindlimb were delimited and digitally deleted and the density of contrast medium was used as a reference for the program.

Immunohistochemistry

Staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) was performed with heart, kidney, skeletal muscle and large arteries and veins as described 2 . Tissues were then embedded in paraffin and sectioned. Nuclear counterstaining was carried out with nuclear red.

Five-micron, paraffin-embedded sections were cut transversely from the mouse muscle. After immunolabelling with antibodies against CD-31 (BMA biomedical), the capillary density was recorded as the number of CD31 positive capillaries per mm 2 as described 7 . A minimum of 30 randomly chosen pictures were recorded at 40X magnification under a microscope for each animal at each time point with a camera connected to a PC. Positive cells were manually counted on captured images and the data were analyzed with the help of Sigma Plot software.

All analysis was performed by an investigator blinded to treatment groups.

Cell isolation from mutant and wild-type mice:

Primary endothelial cells (EC) and smooth muscle cells (SMC) from adult mouse kidneys and aorta, were isolated as previously described 3 4 . MEF were isolated from 12.5 day post coïtum mouse embryo 5 .

-Primary Renal Endothelial Cell Isolation

Mice were sacrificed, and kidneys were removed, minced in slices and incubated with collagenase (Type I, 2mg/ml; Sigma) for 45 min at 37°C with agitation. The suspensions were triturated with a canule and filtered with 70µm filters (BD) followed by a second filtration with 30µm filters (Miltenyi). After centrifugation and washings, the pellets were resuspended and plated in growth media (MEM supplemented with D-valine). After five days, cells were trypsinized and incubated with 12.5µg/ml of anti-CD31 antibody (Pharmingen) and 5µg/ml of anti-endoglin antibody (Santacruz) for 30min at room temperature. Next, the cell solution was incubated with 20µl of Goat anti-rat IgG microbeads (Miltenyi) for 15min, at 4°C. After washing, magnetic separation with MS MACS columns (Miltenyi) was performed and isolated cells were plated in growth media (EBM-2). Greater than 85% of the cells stained positively for CD31.

-Primary Aortic Smooth Muscle Cells Isolation

Aortas were removed and dissected away from fat and extraveneous tissue in sterile, ice-cold, phosphate-buffered saline (PBS) plus CaCl2/MgCl2 (Gibco). Each aorta was incubated in dissecting media consisting of PBS plus CaCl2/MgCl2, 1mg/ml collagenase (Type IV, Sigma), and 125 µg/ml elastase (Sigma), for 30 min at 37°C. After removal of the adventitia, aortas were minced and shaken gently at 37°C for 2h in dissecting media. The dissociated tissues were subsequently rinsed with PBS plus CaCl2/MgCl2, centrifuged, resuspended and plated in growth media. Greater than 95% of the cells stained positively for -smooth muscle actin, and myosin heavy chain. All assays were performed with cells before passage four.

-Mouse Embryonic Fibroblast (MEF) Isolation

Primary MEFs were derived from 13.5-day-old embryos. Following removal of the head and organs, embryos were rinsed with PBS, minced, and digested with trypsin (0.05% solution containing 25µg/ml DNase) for 90 min at 37°C. Trypsin was inactivated by addition of 100% FBS and cells were plated in growth media. All assays were performed with cells before passage five.

Proliferation assay

Standard procedures were employed. The bromodeoxyuridine (BrdU) assay was conducted as follows, cells were grown on glass slides (Labtek-II system, Nalge Nunc International Corp) and incubated with a BrdU solution (10µM, Sigma) for 30 min, and revealed with an antiBrdU antibody (Oxford Biotechnologies) after incubation with hydrochloric acid and neutralization using sodium tetraborate as described 6 . Nuclei cells were stained with Vectashield mounting medium with DAPI (Vector). BrdU labelling cells were counted, and a ratio BrdU labelling /total cell per field was expressed.

Migration (Wound) Assay

To study cell migration, we used Culture-Inserts (Ibidi). Cells were plated on each well of Culture-Inserts and allowed to reach confluence. Cells were serum starved for 24h, then Culture-Inserts were gently removed, and cells were stimulated or not with FGF2 (20ng/ml).

The subsequent gaps were imaged at baseline and 20 hr later.

Chemotaxis Assay

Transwell cell culture chambers containing polycarbonate membrane with 8-μm pore size (Becton Dickinson.) were coated with 0.2% gelatin for MEF and 2% for endothelial cells. Primary and siRNA treated cells were serum starved for 24 hr then trypsinized and plated at 10 5 cells/insert in a Transwell chamber. After 2 hr, the bottom compartment was filled with 800 μl DMEM plus 1% or 10% FBS, or DMEM plus FGF2 (20ng/ml) or VEGF-A (40ng/ml), and the assembly was incubated at 37°C for 16 h, to allow cell migration. In the negative controls, bottom chambers were filled with DMEM serum-free. After incubation, the membranes were washed with PBS, and cells were fixed in 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained with Mayer's Hematoxylin solution (Merck) according to the manufacturer's protocols. Cells, that did not migrate, were gently removed from the top surface, and cells, that had migrated to the bottom of the membrane, were counted in seven random fields using a Nikon inverted microscope.

Capillary-like tube formation on Matrigel

Tube formation assays were set up using -slide angiogenesis (Ibidi) following manufacturer instructions. Human Umbilical Vein cells (HUVEC), purchased from Lonza, were transfected with scramble siRNA or anti-hfzd4 siRNA (si hfzd4) (30 nM) and transfected either with control plasmid or with plasmids coding for Dvl3 tagged myc or Fzd4 tagged HA. 48 hours after transfection, 15,000 cells per well were plated on top of Matrigel® (Becton Dickinson) in growth medium (EBM-2). After 6 hr at 37 °C, tube formation was examined under an inverted light microscope (Olympus,). Branch lengths were measured and branch numbers per branch point were counted. siRNAs were transfected using Interferin (Ozyme) and plasmid with jetprime (Ozyme), following the manufacturer's protocol. The oligonucleotides used were either a validated negative control (Eurogentec) or designed by Qiagen for hfzd4: 5'-GAA UGA UAG UGC CUU UAA Att -3' (#SI02639105) and Ambion for mfzd4: 5'-GGA UGU GCA AUA AUU UUC Utt -3' (#61777). The plasmids encoding for Dvl3 tagged myc were generated in the laboratory and for Fzd4 tagged HA were kindly provided by Tomas Kirchhausen (CBR Institute for biomedical research, Boston).

Golgi polarization assay

This assay was conducted in a migration assay using Culture-Inserts (Ibidi) (see above). 24hrs before experiments, confluent monolayers were serum starved and then activated with FGF2 (20ng/ml). Anti-Golgin97 antibody (Invitrogen), pericentrine and FITC phalloidin (Sigma) were used to stain the Golgi, Microtubule Organizing Center (MTOC) and actin cytoskeleton, respectively.

For the analysis, all images were taken with the same intensity parameters and only cells located in the first row of the wound edge were measured. For determination of the Golgi profile, a line was drawn along the cell and passing by the nucleus, in the direction of the migration edge. For each fluorochrome, the intensity profile was measured along this line using the Axiovision 4.7 program. To quantify Golgi volume, golgi and nucleus surface reconstructions were assigned and cell surface area/cell volume analysis was performed using the Surpass program (Imaris) after 3D deconvolution with autoQuant deconvolution (Mediacybernetics). For each cellular type, at least 50 cells were examined.

Cell culture

Wnt3a and Wnt5a producing mouse L cells (American Type Culture Collection ATCC) were cultured in DMEM medium, 10% fetal bovine serum (FBS) and penicillin-streptomycin.

Conditioned medium was harvested as described in ATCC protocol. Primary renal endothelial cells were cultured in MEM supplemented with 100µg/ml Heparin, 50µg/ml ECGF, 3.5g/L Glucose, 50mg/L D-valine, 20% FBS (Hyclone) and penicillin-streptomycin, and plated on a 0.2% gelatin-coated dish. After purification, they were cultured in EBM-2 complete medium with EGM2 single coat (Clonetics), and plated on a 5µg/ml fibronectin-coated dish. Mouse smooth muscle cells, embryonic fibroblasts (MEF), NIH3T3 and HEK293T were cultured in DMEM supplemented with 10% FBS (Hyclone) and penicillin-streptomycin, and plated on a 0.1% gelatin-coated dish. Human microvascular endothelial cells (HMEC-1) were cultured in EBM complete medium with EGM-MV single coat (Clonetics), and plated on a 2% gelatincoated dish. All cells were serum starved with DMEM plus 1% FBS/0.25%BSA.

Analysis of mRNA expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)

For RT-PCR analysis 1µg of total RNA from mouse tissue or cell extracts was reversetranscribed: PCR was performed using IQ SYBR Green supermix (Bio-Rad) 2 . An MJ Research Opticon and the following parameters were used for real-time PCR: 95°C for 5 minutes followed by 34 cycles of 95°C for 30 seconds, 60°C for 1 minute, 72°C for 30 seconds, 76°C for 1 minute and 80°C for 1 minute. Negative controls without RT were prepared in parallel for each RNA sample. All experiments were performed in triplicate and differences in cDNA input were compensated by normalization to expression of P0 oractin.

qPCR Primer list

Primers used are as follows: P0 forward primer: 5′-GCGACCTGGAAGTCCAACT-3′, and P0 reverse primer: 5′-CCATCAGCACCACAGCCTTC-3′, beta-actin forward primer: 5′

Western Blot analysis and immunoprecipitation

Organ tissues or cells were lysed for 30 minutes in RIPA buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulphate, 50mmol/L Tris, pH 8.0, 2µg/ml aprotinin, 10µg/ml leupeptin, 200µM AEBSF, 1mM benzamidine, 1µM orthovonadate). Cell lysates were cleared by centrifugation at 14,000 g for 20 min. Supernatant fractions were used for Western blot or immunoprecipitation. For Western blot analysis, cell lysates were resolved by SDS-PAGE and probed with indicated antibodies. For immunoprecipitation analysis, 500 g of lysates were incubated overnight with the primary antibody at +4°C with gentle agitation. After this 20 l of protein G-Sepharose slurry was added and samples were incubated for 1 hr at +4°C. Beads were washed three times and subsequently resuspended and boiled in 20 l of loading buffer for SDS-PAGE, followed by western blotting.

Antibodies used were: anti EphrinB2 and EphB4 antibodies from Santa Cruz, anti phosphorylated catenin (anti-ABC) clone 8E7 monoclonal antibody from Upstate, anti catenin, acetylated tubulin and tubulin from sigma, anti Dvl3 and Dvl1 from Santa Cruz, anti GFP from Molecular probes and anti HA from Roche. Binding of antibodies to the blots was detected using Odyssey Infrared Imaging System (LI-COR Biosciences).

Luciferase reporter assay

For the canonical pathway, MEF and HEK 293T cells grown in 24-well culture plates were transiently transfected at 60-70% confluence with 0.2 g of 8XTOPflash reporter, 0.1 g of galactosidase reporter (to evaluate efficiency of transfection) and various plasmids as indicated for individual experiments. The total quantity of DNA in the transfection mixes was adjusted to equal amounts with empty vector. 24 hr after transfection, cells were activated with Wnt3a or Wnt5a conditioned medium as indicated. Luciferase activity was measured using the Luciferase Reporter Assay system (Promega) and a luminometer according to the manufacture's specifications. The firefly luciferase activity was normalized to galactosidase activity.

For the non canonical pathway, MEF and HEK 293T cells were transiently transfected with 0.2 g of AP1 reporter, 0.1 g of galactosidase reporter (to evaluate efficiency of transfection) and various plasmids as indicated for individual experiments.

Immunofluoresence microscopy

For tubulin staining, cells were fixed in and stained with monoclonal anti tubulin. For Fzd4 and Dvl3 co-localization, HA-tagged Frizzled 4 was detected using anti-HA antibodies (Roche) followed by Alexa Fluor 568 conjugated goat anti-mouse IgG. The DNA was counterstained by 4',6-diamidino-2-phenylindole (DAPI). For co-localization studies, cells were analyzed with a Nikon laser scanning confocal microscope using multichannel scanning (Nikon PCM 2000) using a 60x/1.4 ApoPlan oil immersion objective, and EZ200 software (Nikon). Single XY scans had an optical slice thickness of 0.4 m. Three-dimensional projections were digitally reconstituted from stacks of confocal optical slices by Imaris software (Bitplane).

Mouse model of unilateral hind limb ischemia

Unilateral hind limb ischemia was operatively induced as previously described with the excision of the femoral and saphenous arteries 6 . This study was conducted in accordance with both institutional guidelines and those in force in the European community for experimental animal use (L358-86/609/EEC). Seven days after the surgery, perfusion using Laser Doppler perfusion imaging (LPDI) vascular density was used to record serial blood flow measurements, and connectivity was analyzed using microCT analysis on serial tissue sections.

Statistical analysis

Results are expressed as means ± SD. All analyses were performed with appropriate software (Statview 5-1, Abacus). Comparison of continuous variables between two groups was performed by a one-way ANOVA and subsequently, if statistical significance was observed, by a two-sided paired t test. A value of p <0.05 was considered to be statistically significant.

b) Hypothesis

We hypothesize that Dvl interacts via its PDZ/ DEP domains with partners involved in the non canonical pathway.

c) Strategy

As the DIX domain of Dvl is essential for signal transduction in the canonical pathway, a Dvl3 construct deleted from the DIX domain was chosen as bait for the yeast two hybrid. A bank of cDNAs from mouse embryo day post coïtum 12.5 was chosen as prey, because Wnt signaling is important

Jun-B oncogene (Junb)

Figure 89 A few of the proteins found to be interacting with Dvl3 Dix using the screening A few proteins were chosen for characterization. We performed quick assays on each candidate (Larp1b; Lmo4; Kif26b; Pdzrn3; Dram2). In this screening, we evaluated whether the candidate was expressed in endothelial cells, regulated properties of EC such as migration or tubulogenesis and finally, whether they modulated Wnt pathway. The E3 ubiquitin ligase PDZRN3 was one of the more interesting candidates. The protein is composed of a RING domain essential for its activity of ubiquitin ligase, two PDZ domains and one PDZ binding motif (thr-thr-valine motif)(Fig88). This domain is rare and is required for binding of proteins containing PDZ domains.

To study PDZRN3, we asked for the plasmid coding for the protein to Dr. Yamamato that was tagged with a V5 cassette (Ji-Ae Ko 2006, Honda et al. 2010). We expressed the plasmid in Hela cells and assessed the localization of the tagged protein. PDZRN3 -V5 was found in cytoplasmic puncta.

e) Summary

This work identifies PDZRN3 as a switch between Wnt non canonical and canonical pathways.PDZRN3 ubiquitinylates Dvl3. This promotes clathrin -mediated endocytosis of the Fzd4/ Dvl3 complex; and enables signal transduction from the early endosome, a mechanism similar to the one described with TLR signaling [START_REF] Dobrowolski | Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles[END_REF]. This process takes off the Fzd/ Dvl complex from the canonical Wnt pathway signal transduction that probably uses caveolin-mediated endocytosis for signal transduction. Non canonical pathway is essential for vascular morphogenesis (migration of EC, remodeling) while canonical is required for proliferation and differentiation. PDZRN3

would activate angiogenesis by inducing non canonical pathway and blocking the canonical pathway.

Introduction

During development or tissue regeneration, vascular growth is a complex process that can be schematically divided in two dynamic steps. First a highly unstable vascular plexus is developed, with vessels expanding and collapsing at a high rate. Then, the newly formed vascular system is remodeled to ensure the transition toward a stable and functional network (Herbert and Stainier, 2011). Evolution toward a structured vascular system is characterized by the formation of a 3 dimensional (3D)

hierarchically branched and organized vascular network (Fruttiger, 2007), suggesting that vascular cells or groups of vascular cell, are instructed to be oriented within stable functional tubes. Recently, several groups, including our own, have provided evidence to underscore the importance of Wnt/Frizzled/planar cell polarity (Wnt/PCP) signaling in vascular system morphogenesis and remodeling (Cirone et al., 2008;Ju et al., 2010).

Evidence has previously linked the Wnt canonical pathway to vascular formation. Mutations of Norrin (Ndp), Frizzled4 (Fzd4), Tetraspanin-12 (TSPAN12) or LRP5, led to ocular syndromes, such as exudative vitreoretinopathy (EVR) [START_REF] Junge | TSPAN12 regulates retinal vascular development by promoting Norrin-but not Wnt-induced FZD4/beta-catenin signaling[END_REF]Nikopoulos et al., 2010;[START_REF] Poulter | Mutations in TSPAN12 cause autosomal-dominant familial exudative vitreoretinopathy[END_REF]Xu et al., 2004;Ye et al., 2010). In ndp, fzd4, lrp5 and tspan12 loss-of-function mice, development of the superficial retinal plexus has been shown to be altered, with a lack of the deep and intermediate vascular plexuses [START_REF] Junge | TSPAN12 regulates retinal vascular development by promoting Norrin-but not Wnt-induced FZD4/beta-catenin signaling[END_REF]Xia et al., 2010;Xu et al., 2004;[START_REF] Zuercher | Norrin stimulates cell proliferation in the superficial retinal vascular plexus and is pivotal for the recruitment of mural cells[END_REF], linking the Wnt canonical signaling complex to vascular formation in the retina. However, the role of the non-canonical PCP pathway is not yet well understood during vessel development and the mechanisms by which the endothelial Wnt/PCP pathway regulates vessel morphogenesis remain elusive.

The conserved Wnt/PCP signaling is involved in patterning, by instructing cell polarity within a tissue [START_REF] Wu | A quest for the mechanism regulating global planar cell polarity of tissues[END_REF]. First observed in insects, where it regulates the orientation of hair on wings and facets on the eye, Fzd/PCP is now known to be involved in regulating gastrulation and sensory cell orientation in vertebrates (Wang and Nathans, 2007). PCP signaling controls basic mechanisms including cell migration, axonal guidance, asymmetric cell division, as well as hair and cilia orientation [START_REF] Devenport | Planar polarization in embryonic epidermis orchestrates global asymmetric morphogenesis of hair follicles[END_REF][START_REF] Seifert | Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility[END_REF]. PCP signaling in insects and vertebrates, shares a core pathway that includes members of the dishevelled (Dvl1-3 members) (Etheridge et al., 2008) and fzd gene families, but is distinct from the -catenin-dependent canonical Wnt pathway (Montcouquiol et al., 2006). Dvl proteins are the relays between the Fzd receptor and Wnt downstream signaling pathways (Gao and Chen, 2010). To regulate all of these functions, Dvl has to associate with different protein partners and induce PCP signaling cascades that involve activation of c-jun, a hallmark for PCP signaling and the small GTPAses Rho and Rac [START_REF] Park | Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos[END_REF].

Interestingly, among Dvl members, Dvl3 could be an important relay for Wnt/PCP signaling. Indeed, Dvl3 has a unique function compared to that of the other isoforms in analyzing genetic Dvl1-3 mutant phenotypes (Etheridge et al., 2008). In vitro studies showed that Dvl3 compared to Dvl1 and Dvl2 potentiated non canonical Dab2-dependent activity [START_REF] Hocevar | Regulation of the Wnt signaling pathway by disabled-2 (Dab2)[END_REF]. Recently, our group has identified an important role for Dvl3 in PCP signaling induction in endothelial cells (Descamps et al., 2012). To further elucidate the molecular mechanisms regulating the activity of Dvl3 in the PCP pathway, we performed a yeast-two hybrid screening to search for novel Dvl3-interacting proteins. Among these we identified the E3 ubiquitin ligase PDZ domain-containing RING finger protein 3 (PDZRN3). Whereas previously published studies have revealed the role of PDZRN3 in skeletal muscle (Ko et al., 2006) and osteoblast differentiation acting as an inhibitor of Wnt/b catenin signaling (Honda et al., 2010), its role in Wnt signaling is poorly understood.

We report that PDZRN3 is expressed in endothelium and using a gene-targeting strategy, we explored the in vivo role of Pdzrn3 during angiogenesis. First, we deleted Pdzrn3 during angiogenic gestational steps from E7.5, taking advantage of the inducible Ubc Cre ERT2 mice. We report that embryos died very early in utero at E11.5, exhibiting defects in large vitelline vessel morphogenesis in the yolk sac, and impairment of embryonic capillary invasion in the placenta leading, to severe growth retardation. Taken together, our data reveals a novel and crucial role of Wnt PCP signaling via the ubiquitin ligase PDZRN3 during vascular plexus directional extension and planar organization.

Results

were absent. Comparatively, in wild-type embryos, fetal vessels were evenly distributed throughout the labyrinth (Fig 1d). Ubc-Cre ERT2 Pdzrn3 f/f embryo vasculature, revealed by whole mount PECAM staining, appeared altered with reduction of the brain vasculature (Fig 1e). Taken together, our results suggest that severe growth retardation and embryonic death, at mid gestation in Ubc-Cre ERT2 + Pdzrn3 f/f mice, could be caused by defective yolk sac circulation and/or placenta function.

Furthermore, we created mice with targeted deletions of Pdzrn3, by crossing a LoxP-flanked Pdzrn3 gene with Cre expression driven by a Pdgf tamoxifen-inducible promoter which targets ECs (Claxton et al., 2008). We then analyzed Pdgf icre Pdzrn3 f/f embryo mutants (iEC KO) between E9.5 and E11.5 versus Pdzrn3f/f embryo controls (iEC WT) (Supplementary Fig. 2). We observed an altered pattern of vessel organization in the yolk sacs of the iEC KO embryos similar to that reported for the Ubc-CreERT2 + Pdzrn3f/f mutants (Supplementary Fig. 2a). Moreover, histological analysis of mutant placenta at E10.5, revealed a statistical decrease in the number of embryonic vessels that invade the labyrinth layer (Supplementary Fig. 2b). However, it is noteworthy that the alterations of the embryo vascular network, either in yolk sacs or placenta, were milder that the one found under Ubc-Cre ERT2 depletion and only 25% of the iEC KO embryos had growth retarded at E11.5. In conclusion, this complementary genetic approach strongly supports a direct role for Pdzrn3 on vascular yolk sac organization and the development of the embryonic vascular labyrinth structure in placenta.

PDZRN3 gene deletion in ECs induces vascular defects

Next, we analyzed the effects of PDZRN3 deficiency on vessel morphogenesis postnatally, in the mouse retina. Western blots on wild-type whole retinal lysates from P3 to P21 showed that PDZRN3 is expressed in EC, with peak expression at P12, which coincides with intense vascular remodeling (Fig 2a

PDZRN3 silencing impairs directional migration and favors sprout regression in vitro

We then explored whether the observed angiogenic phenotype in Pdzrn3 mutants was due to EC

Pdzrn3 regulates Dvl3 ubiquitinylation

We then explored mechanistically, the role of PDZRN3 in endothelial morphogenetic events.

First, we confirmed that at endogeneous levels Dvl interacts with PDZRN3. In Pdzrn3 iECWT lung lysates, co-immunoprecipitation of endogenous Dvl3 using anti Dvl3 antibody, co-precipitated PDZRN3, which included several ubiquitin chains, whereas in Pdzrn3 iECKO lung extracts, the Dvl3-PDZRN3 interactions and Dvl3 ubiquitinylation were markedly reduced (Fig 4a).

We further investigated whether Dvl3 was a substrate of PDZRN3-mediated ubiquitinylation. We co-expressed either Ub WT-HA or Ub K48only -HA and Ub K63only -HA, in which all the lysine residues are mutated to arginine except Lys48 or Lys63, respectively, with Dvl3-myc. PDZRN3-V5 was able to induce Dvl3 ubiquitinylation in the presence of either Ub WT-HA or Ub K63only -HA, but not with HA-Ub K48only . As Lys63 is the major site for PDZRN3 mediated polyubiquitinylation of Dvl3, our data suggests that ubiquitinylation of Dvl3 by PDZRN3 is an intracellular signal promoting Dvl3 trafficking (Fig 4b). Since Dvl proteins are known to be involved in endocytosis during Wnt/PCP signaling (Chen et al., 2003;[START_REF] Kim | Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements[END_REF][START_REF] Kishida | Dvl regulates endo-and exocytotic processes through binding to synaptotagmin[END_REF]Yu et al., 2007), we hypothesized that PDZRN3 may facilitate endocytosis of the Dvl3 complex, in order to induce non-canonical PCP signaling. To test this

PDZRN3 promotes the non-canonical Wnt/PCP pathway while repressing Wnt canonical signaling in endothelial cells ex vivo and in vitro

We investigated whether PDZRN3 could regulate the Wnt signaling pathway using Western blot.

We evaluated active beta catenin (ABC) and p-c jun levels, as markers of canonical and PCP Wnt pathways, respectively (Fig 6a). There was significant up regulation of ABC expression with a down regulation of p-c jun in EC isolated from Pdzrn3 iECWT versus Pdzrn3 iECKO lungs at P7, suggesting that PDZRN3 is required to attenuate the Wnt/ catenin canonical signal transduction and to promote the Wnt/PCP pathway in postnatal EC, confirming previous work on cultured osteoblast differentiation (Honda et al., 2010). To test this hypothesis, we followed  catenin activation in cultured EC under

Endocytosis blockade delays endothelial sprouting in vivo and ex vivo

Next, we asked whether PDZRN3-induced endocytosis is a mechanism that drives angiogenic morphogenetic events. We tested endocytosis blockade, for its effect in vivo and ex vivo on sprouting

Discussion

Understanding the molecular mechanisms required for coordinating and orienting cell movements to shape vascular networks in a precise and tightly regulated pattern, is an important question in developmental biology. In this study, we used mouse loss-of-function experiments to demonstrate that the loss of E3 ubiquitin ligase Pdzrn3 activity, leads to disorganization of the vascular vitelline network during embryogenesis and defects in retinal vascular branching. We have also identified Dvl ubiquitinylation as a physiological target of PDZRN3 signaling as it is strongly reduced in the absence of Pdzrn3 in vivo and in vitro. We further demonstrate an important and unexpected role for PDZRN3, inducing PCP signaling and directing endothelial cell movements, in an autonomous Fzd/Dvl dependent manner.

We then designed an in vivo study, to conditionally block Pdzrn3 expression within a narrow time window, when earlier vasculogenesis would not be affected. We induced Pdzrn3 excision after the formation of the primary vascular plexus, by injection of tamoxifen in pregnant mice at E7.5 and 8.5. By E9.5, vascular remodeling has already started in the yolk sac with large vitelline vessels and at E10.5, an intricate, orderly arrangement of large vitelline arteries and veins branching in a fractal pattern to smaller vessels can be seen. Deletion of Pdzrn3 did not impair vitelline vessel formation but did effect the pattern of hierarchically organized, vitelline vessel architecture throughout the yolk sac. At E10.5 the mutant vascular network degenerated with numerous small disorganized vitelline vessels, evenly distributed throughout the yolk sac. Moreover, embryonic vessels did not invade into the labyrinth placenta layer.

Based on this data, we conclude that mutant embryos may die from failure to establish a functional placenta, which is required for embryo nutrition. The identification of PDZRN3 as a physiological target of Wnt signaling is consistent with previous loss-of-function analyses of Wnt signaling components in mice, reporting their involvement in different steps of placental development [START_REF] Aoki | R-spondin3 is required for mouse placental development[END_REF][START_REF] Cross | Branching morphogenesis during development of placental villi[END_REF][START_REF] Galceran | Wnt3a-/--like phenotype and limb deficiency in Lef1(-/-)Tcf1(-/-) mice[END_REF][START_REF] Parr | Wnt7b regulates placental development in mice[END_REF]. Interestingly, embryos deficient in Wnt2 and fzd5 share similar vascular defects. Wnt2 mutants have poorly developed labyrinth layers with reduced fetal vasculature [START_REF] Monkley | Targeted disruption of the Wnt2 gene results in placentation defects[END_REF], while fzd5 deficient embryos die in utero with early defects in the yolk sac, and placental angiogenesis with degeneration of large vitelline vessels [START_REF] Ishikawa | Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis[END_REF].

Recently, GCM1, a key transcription factor involved in placental branching morphogenesis, was identified as a novel target of Wnt- catenin signaling. Its expression was decreased in BCL9-Like, fzd5

and R-spondin3 deficient mice, which all display placental defects [START_REF] Matsuura | Identification of a link between Wnt/beta-catenin signalling and the cell fusion pathway[END_REF].

To confirm a functional role of PDZRN3 in angiogenesis, we specifically deleted Pdzrn3 in EC using a Pdgf icre mutant. Mutants had an increase in vascular regression/remodeling of the superficial plexus, but no effect on tip cells, and a planar mis-orientation of the intermediate plexus in the plane orthogonal to the axis of the apical plexus, which leads to an incomplete and disorganized deep plexus.

The Wnt signaling components and canonical signaling pathway have been involved in the control of hyaloid regression and primary plexus extension [START_REF] Junge | TSPAN12 regulates retinal vascular development by promoting Norrin-but not Wnt-induced FZD4/beta-catenin signaling[END_REF][START_REF] Phng | Nrarp coordinates endothelial Notch and Wnt signaling to control vessel density in angiogenesis[END_REF][START_REF] Richter | Retinal vasculature changes in Norrie disease mice[END_REF]Xia et al., 2010;Xu et al., 2004) et al., 2011). Here, we propose a novel, autonomous role of Wnt non canonical signaling to regulate endothelial cell morphogenetic movements mainly for deeper plexus architecture.

These in vivo data demonstrate that PDZRN3 signaling is required for the oriented branching architecture in vessels. Vascular maturation is a tightly regulated process, controlled by a balance of angiogenic signaling. Several recent reports have involved Wnt PCP in vascular morphogenesis (Cirone et al., 2008;Ju et al., 2010;[START_REF] Tatin | Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis[END_REF]. Wnt PCP signaling, controls large mechanisms such as; cells migration, axonal guidance, asymmetric cell division, hair or cilia orientation [START_REF] Seifert | Frizzled/PCP signalling: a conserved mechanism regulating cell polarity and directed motility[END_REF][START_REF] Wu | A quest for the mechanism regulating global planar cell polarity of tissues[END_REF][START_REF] Zallen | Planar polarity and tissue morphogenesis[END_REF] as well as mammalian wound healing repair [START_REF] Caddy | Epidermal wound repair is regulated by the planar cell polarity signaling pathway[END_REF]. Manifestation of planar polarity features, depends on tissue context, and as such we sought to examine the role of the PCP pathway in control of endothelial cell migration and polarity and in fine vessel morphogenesis. In vertebrate, the PCP pathway was shown to require a core of conserved proteins, including Frizzled receptors, disheveled (Dvl) 1/2/3, prickle1/2, diversin, Celsr1/2/3, Vangl2 and Daam1 (Ju et al., 2010;Montcouquiol et al., 2006). Here, we identified Pdzrn3 as a novel PCP gene required for EC directional migration, using loss and gain of function approaches in in vitro and ex vivo models. It is not yet clear how Wnt activates the PCP pathway after binding to the Fzd receptor, to impart information to EC during vascular remodeling. Fzd4 has attracted considerable attention, due to its major role in retinal vascular plexus formation, through Wnt canonical signaling involving the receptor complex Tetraspanin-12 and LRP5 [START_REF] Junge | TSPAN12 regulates retinal vascular development by promoting Norrin-but not Wnt-induced FZD4/beta-catenin signaling[END_REF]Wang et al., 2012). We recently reported that Fzd4 was also involved in the Wnt/PCP pathway and proposed its role in vascular morphogenesis in fzd4 KO mice (Descamps et al., 2012). Endocytic mechanisms have emerged to control either  catenin Wnt-dependent signaling [START_REF] Blitzer | A critical role for endocytosis in Wnt signaling[END_REF][START_REF] Jiang | Disabled-2 (Dab2) inhibits Wnt/beta-catenin signalling by binding LRP6 and promoting its internalization through clathrin[END_REF][START_REF] Seto | Internalization is required for proper Wingless signaling in Drosophila melanogaster[END_REF] or PCP signaling (Chen et al., 2003;[START_REF] Kim | Essential role for beta-arrestin 2 in the regulation of Xenopus convergent extension movements[END_REF]Yu et al., 2007). Importantly, endothelial cell depletion of Pdzrn3, modified the Wnt canonical versus non canonical balance, as we found an ABC accumulation, coupled with a blockade of c-jun phosphorylation, in mutant versus control EC ex vivo and in vitro under Wnt5a

stimulation. Similarly, it has been reported that Wnt5a signals through the receptor Ror2 to inhibit the  catenin dependent canonical Wnt signaling pathway and activate c-Jun N-terminal kinase (JNK) in vitro (Mikels and Nusse, 2006;[START_REF] Oishi | The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway[END_REF]. Our demonstration that Dvl ubiquitinylation is a consequence of PDZRN3 activity, raises the possibility that the ubiquitinylated form of Dvl could be a molecular switch to specify non canonical versus canonical functionality. However, we cannot exclude the possibility that PDZRN3 may have other still unknown substrates, which may participate in the vascular function of PDZRN3. This, then raises the possibility that part of the vascular phenotype induced under Pdzrn3 depletion may be independent of Dvl3 trafficking.

It has been proposed that Wnt responsiveness is controlled by Fzd receptor levels at the cell surface via interplay between E3 ubiquitin and deubiquitine ligase [START_REF] Hao | ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner[END_REF]Mukai et al., 2010).

Here we proposed that Dvl3-K63 polyubiquitnation could act as a cargo selection signal required to induce Fzd/Dvl complex internalization and activation of the Wnt/PCP signaling. Indeed we report that PDZRN3 would preferentially adds K63 ubiquitin chain to Dvl3 versus K48 ubiquitin.. A recent study combining genetic and quantitative proteomic approaches proposed that K63 Ub linkages is a nondegratative form of ubiquitination [START_REF] Xu | Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation[END_REF] and as such, could allow various proteasomeindependent biological outcomes. For example, in a yeast model K63 ubiquitination is involved in DNA repair [START_REF] Spence | A ubiquitin mutant with specific defects in DNA repair and multiubiquitination[END_REF]; and parkin-mediated K63 ubiquitination is involved in NFκB signaling [START_REF] Henn | Parkin mediates neuroprotection through activation of IkappaB kinase/nuclear factor-kappaB signaling[END_REF]. As it was proposed that the routing of receptor in intracellular organelles could be an active process for signaling transduction [START_REF] Dobrowolski | Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles[END_REF], here we provide evidence that internalization of active Fzd receptor is required for signal transduction, bringing active Fzd/Dvl complex to the intracellular compartment.

Given its expression in distinct tissues, and the presence of PDZ domains that mediate protein clustering to regulate the signaling network, this indicates a broader role of PDZRN3. As a large number of cancers have been linked to deregulation of the Wnt pathway, we searched for PDZRN3 expression in high-throughput cancer profiling databases. In a human breast cancer microarray database, PDZRN3 was co-expressed with other Wnt genes such as DKK3, TCF4, netrin receptor Unc5B that is involved in EC guidance (Lu et al., 2004) and Col4A1 that is associated with autosomal dominant hereditary angiopathy [START_REF] Lanfranconi | COL4A1 mutations as a monogenic cause of cerebral small vessel disease: a systematic review[END_REF]) (Oncomine TM , Compendia Bioscence, Ann Arbor, MI). In a multi cancer database, PDZRN3 expression was linked with a large number of EC markers, suggesting that PDZRN3 could be a novel Wnt signaling regulator in human cancer pathologies linked to vascular disorders.

This study provides a novel molecular partner that could fine-tune Wnt complex signaling pathways to control of EC behavior. In collectively regulating EC migration and polarity, PDZRN3

induces PCP signaling which is critical for vascular morphogenesis.

Methods

Author contributions

Mouse retina analysis

Mouse eyes at postnatal stage were dissected and stained with Isolectin B4 (Sigma, L2895, 1/100) or

Collagen IV (Santa Cruz, sc-113360, 1/100) in PBS supplemented with 5% BSA . Retinal vasculatures were imaged with a fluorescent microscope (Apo-observer, Zeiss) and vascularized retinal areas quantified with axiovision software (Zeiss). The number of branch points was quantified after skeletonization (Cell profiler) in an area of 0,25mm 2 (8 fields per retina). To assess blood vessel regression, the ratio of the number collagen IV-positive/Isolectin-negative to the total number of capillary segments was assessed (8 fields per retina). The retinal vascularization at P12 was imaged with a confocal microscope (Olympus FV 1000) and analyzed using Imaris (Bitplane). The organization of the secondary plexus was analyzed using Image J (tracking plugin) and the Chemotaxis and Migration tool (Ibidi). BrdU staining of retina was performed as described before 5 . Counts of BrdU+ nuclei on or near vessels per field were normalized to the densities of the vessels of the field. Intraocular injection was performed using the injector from World precision instruments. 0.5µl of Dynasore (200 µM in 6% DMSO; 94 % Ethanol) was injected in one eye of the animal at the speed of 50nl/sec; the other control eye was injected with a solution of 6% DMSO; 94% Ethanol. (Lonza,. All these cells were serum starved with DMEM plus 1% FBS/0.25%BSA.

Conditioned medium (CM) from Wnt3a and Wnt5a producing mouse L cells (American Type Culture

Collection ATCC) was harvested as described in ATCC. Cells were activated either with Wnt3a CM, and with Wnt5a CM.

Plasmids were transfected using jetPRIME TM (Polyplus) following the manufacturer's protocol (final concentration of 1.5 µg/ml for HEK293 cells, 0.5µg/ml for HeLa cells). SiRNA were transfected using

INTERFERin TM (Polyplus) at a final concentration of 30nM. The oligonucleotides used were designed by Origene for h-pdzrn3: SR307944A SR307944B and SR307944C. Lentivirus were tranduced at a multiplicity of infection of 20 (MOI). Ly.72632 (Sigma) was used at 10µM (Sigma). Dynasore (Santa Cruz) was used at 40µM in vitro and ex vivo.

In vitro assays on endothelial cells

Chemotaxis assay was carried out by plating Hoechst pre-treated HUVEC cells in µ-Slide Chemotaxis 2D slides (Ibidi). After 6 hours, 90 µl of control CM was added to one of the chemotaxic chamber and 90 µl of Wnt5a CM (diluted 1/20) to the other chamber, creating a gradient of Wnt factors in the capillary. The capillary was then filled with fresh medium and the slide placed for 6 hours under an inverted microscope (AxioObserver, Zeiss). Nuclei of each cell were tracked during time and surfaced using Imaris software (Bitplane).

For 3D tubulogenesis assay, HUVECs were transduced with control or Pdzrn3 expressing lentivirus and transfected with various siRNAs and nuclei labeled by supplementation with Hoechst (5µg/ml) for 30mins. Cells were then incubated with Gelatin-coated microcarrier beads (Cytodex 3; Sigma) for 5 to 6 hours. When the cells reached confluence, equal numbers of HUVECs-coated beads were embedded in fibrin gels in a µ-slide angiogenesis plate (Ibidi). The fibrin gel was prepared by dissolving bovine fibrinogen (2.5 mg/mL; Sigma) in EBM2 medium supplemented with Aprotinin (0.05 mg/mL; Sigma).

Clotting was induced by the addition of thrombin (1.2 U/mL) (Sigma). After 60 min, control or Wnt5a transfected human fibroblasts were plated on the top of gel and EBM2 medium added. The formation of tube-like structure was imaged by time-lapse videomicroscopy (in fluorescence) for 12 h under an inverted microscope (AxioObserver, Zeiss) 48h after plating for quantification. The number of sprouts per bead, length of sprout and number of nuclei per sprout was quantified at 96h after plating.

Luciferase reporter assay

Transactivation analysis of TopFlash and AP1 reporter plasmids were performed in HUVEC cells.

Briefly, the cells grown in 48-well culture plates were transiently transduced at 60-70% confluence with microscope (Olympus FV 1000). Visualisation was obtained after digitized image multichannel deconvolution by Autodeblur (Media Cybernetics Inc., MD 20910 USA) and three-dimensional projections were digitally reconstituted from stacks of confocal optical slices by Imaris software (Bitplane) (Bitplane AG, Zurich, Swizerland).

Aortic endothelial cell sprouting assay

Thoracic aorta were removed from mice P7 (males) and cut into 1mm rings. The aortic rings were embedded in Growth factor-reduced Matrigel® (Becton Dickinson) on a µ-slide angiogenesis plate (Ibidi), incubated in EBM2 medium for 72h and imaged using an Olympus inverted microscope after incorporation of Hoechst (5µg/ml in medium for 30mins). The aorta rings were transduced with Pdzrn3 lentivirus or control lentivirus mixed in matrigel (10 6 particles / 100µl of gel). The sprouting area was then quantified after surfacing using Imaris (Bitplane).

Isolation of primary endothelial cells from mouse kidney or lung

Lungs were removed in PBS-containing CaCl2 and MgCl2 (Gibco), and endothelial cells were isolated after incubation with anti-CD31 (Pharmingen) and anti-endoglin (Santacruz) antibodies using microbeads (Miltenyi) as reported 1 .

Statistical analysis

Results were expressed as mean ± SD or mean ± min to max. All analyses were performed with appropriate software (Graphpad PRISM). Comparison of continuous variables between two groups was performed by a one-way ANOVA and subsequently, if statistical significance was observed, by an unpaired t test. A value of p <0.05 was considered to be statistically significant.

This article is not in final form and represents a work in progress.

a) State of art

PDZRN3 is remarkable as it possesses PDZ domains/ PDZ binding domains and a RING domain.

The RING domain is functional and identifies PDZRN3 as an ubiquitin ligase, a protein that gives the specificity to the reaction of ubiquitinylation. The PDZ binding domain of PDZRN3 indicates that it potentially interacts with proteins containing PDZ domains such as components of Tight Junctions.

b) Hypothesis

Our hypothesis is that PDZRN3 could interact with junctions via PDZ interactions and regulate TJ function.

c) Preliminary Results

Preliminary results indicated a role of Pdzrn3 signaling in endothelial junction maintenance.

Transwell assay indicated that depletion of Pdzrn3 reduced permeability of the endothelial layer. We

d) Methods

To test our hypothesis, we developed transgenic models to delete PDZRN3 in EC and analyze its impacts on EC TJ functions. Experiments in this article were carried out on PDGF icre; pdzrn3 f,f mice.

Vessels preparation was realized from non infarcted and infarcted hemispheres from iEC WT and iEC KO mice after cerebral artery occlusion, and subjected to quantitative RT PCR. Enrichment of the preparations was assessed by measuring expression of Ve-cadherin and Tie2. Vessel fractions strongly We also developed transgenic models to over-express PDZRN3 in endothelial cells by taking advantage of the tetracyclin activated system. Mice having integrated the gene coding for PDZRN3-V5

were crossed with mice expressing the transactivator (tta) under the control of the Tie2 promotor. The gene over-expression was conditional as in the presence of doxycycline, the transactivator could not interact with its target (tet off system). A β-Gal reporter cassette was also under the control of the Tie2tta promoter, making it possible to reveal cell specific expression of the transactivator. Ainsi, bloquer la voie Wnt via PDZRN3 pourrait réduire l'impact délètere de l'AVC sur les vaissseaux cérébraux en permettant une meilleure résistance à la montée de pression de la BHE.

Conclusion et Perspectives

1) Proposed Model

This work identifies PDZRN3 as a novel regulator of Wnt signalling and tight junctions regulation.

The protein was identified using a Yeast Two Hybrid screening using the adaptor protein Dvl3 as bait.

We were able to confirm that Dvl3 was a substrate of PDZRN3 in vitro and in vivo, and that this interaction triggers induction of the c-Jun dependant Wnt pathway.

Our proposed model is that endogenously PDZRN3 is located in many subcellular compartments.

It can interact with membrane proteins or vesicular proteins via its PDZ binding domain. When realizing immunostaining on cells culture, PDZRN3 was found in cytoplasmic puncta that were identified as Rab positive vesicles but also at the cytoplasmic membrane or the nucleus. This indicates that many pools of the protein exist, which might have different functions.

Endogenous Dvl3 also form cytoplasmic puncta.

When Fzd4 is tranfected, we were able to detect a massive relocalization of Dvl3 and PDZRN3 to the cytoplasmic membrane. This suggests that Fzd4 is able to trigger cell signalling cues leading to the recruitment of both proteins to the membrane. Interestingly, a form of PDZRN3 deleted from its interaction domain with Dvl3 was not recruited to the membrane, indicating that the interaction between PDZRN3 and Dvl3 is a prerequisite for the relocalization of PDZRN3.

To confirm that the Fzd4/ Dvl complex was recruited to endocytic vesicles after Wnt activation, we used a strategy of immunostaining on Fzd4-transfected cells. Fzd4 was found at the membrane in control cells

2) Prospects a) Expression of PDZRN3 in pathologies

This work identifies PDZRN3 as a regulator of vital vessel functions. However, many points have to be resolved. Expression of PDZRN3 was found to be up-regulated in many cancers such as Carcinoma or Melanoma in a micro-array database, Oncomine (Fig 100). This is interesting as Wnt signalling has been shown to be upregulated in cancers. The link between PDZRN3 and transformation of normal cells into tumor cells would be an interesting topic, as this process also implicates translocation of ZO1 and c-JUN to the nucleus [START_REF] Polette | Beta-catenin and ZO-1: shuttle molecules involved in tumor invasion-associated epithelial-mesenchymal transition processes[END_REF].

b) Interaction of PDZRN3 with MUPP1

This work identifies PDZRN3 as a partner of the TJ associated protein MUPP1. MUPP1 has a dual function as it participates in the Crumbs complex. This complex regulates targeting of RhoA to the leading edge of migrating cells. It would be interesting to assess whether loss of PDZRN3 affects the activity of the Crumbs complex positively or negatively. It would also important to confirm the direct interaction between MUPP1 and PDZRN3 using another method such as precipitation of a HIS-tag protein using a Cobalt agarose resin column.

c) Phosphorylation status of PDZRN3

We were able to identify a phosphorylation site of PDZRN3. It would be interesting to see whether this phosphorylation modifies the 3D structure of the protein and accessibility of the PDZ binding domain, using crystallography. This would confirm that this phosphorylation is vital for activity of the

e) PDZRN3 in other tissues

PDZRN3 was first identified at the neuromuscular junction where it regulates integrity of the junction by ubiquitinylating MUSK1. We were able to show that PDZRN3 was expressed in the endothelium in the brain/ retina/ yolk sac and placenta. Interestingly, PDZRN3 was also found to be expressed in the cardiomyocytes in the heart. As overexpression of Dvl1 induces a cardiac hypertrophy and dysfunction and ubiquitin signalling have been linked to similar pathologies, it would be interesting to test whether loss or over-expression of PDZRN3 would affect similar functions [START_REF] Malekar | Wnt signaling is critical for maladaptive cardiac hypertrophy and accelerates myocardial remodeling[END_REF][START_REF] Powell | The ubiquitin-proteasome system and cardiovascular disease[END_REF]).

  d) Evaluation of vascular leakage in embryo and pups ....................................................... e) Micro computer tomography .......................................................................................... f) Isolation of primary endothelial cells from mouse kidney ............................................. g) Vessel extraction .............................................................................................................

  .. B) REAGENTS ............................................................................................................................. 1) List of used antibodies .

ARTICLE 1 .

 1 FRIZZLED 4 REGULATES ARTERIAL NETWORK ORGANIZATION THROUGH NONCANONICAL WNT/PLANAR CELL POLARITY SIGNALING .................................................................. a) State of art ....

  .. CONCLUSION AND PROSPECTS ............................................................................................. 1) PROPOSED MODEL ................................................................................................................ 2) PROSPECTS ............................................................................................................................ a) Expression of PDZRN3 in pathologies ............................................................................... b) Interaction of PDZRN3 with MUPP1 ................................................................................. c) Phosphorylation status of PDZRN3 .................................................................................... d) Chemical inhibition of PDZRN3 .

Figure 1 :

 1 Figure 1: The Wnt canonical and non canonical pathways (adapted from Saadeddin et al 2009) ..........................

Figure 2 :

 2 Figure 2: Transcriptional targets of the Wnt/ canonical pathway (Wnt homepage, Stanford) ................................

Figure 3 :

 3 Figure 3: PCP induces asymmetrical organization of Fzd/ Dvl and Vangl2/ Pk (Schulte 2010) ............................

Figure 4 :

 4 Figure 4: Distribution of Wnt Genes in Metazoans. X indicate that no member of the Wnt subfamily found in the genome of the organism; the color is different for each Wnt; two blocks indicate presence of paralogue in the specie (adapted from Prud'homme et al. 2002) .......................................................................................................

Figure 5 :

 5 Figure 5: Phylogenetic tree of the Wnt family (adapted from Prud'homme et al. 2002) .........................................

Figure 6 :

 6 Figure 6: List of Wnt genes (Wnt homepage, Stanford)..........................................................................................

Figure 7 :

 7 Figure 7: Schematic presentation of Wnt/ Fzd signaling kinetics (Schulte and Shenoy 2011) ...............................

Figure 8 :

 8 Figure 8: Phylogenetic tree of the human Frizzled receptors FZD (Schulte and Shenoy 2011) ............................

Figure 9 :

 9 Figure 9: Schematic representation of Frizzled protein (binding sites for Dvl/ PDZ and G proteins indicated) (Schulte and Shenoy 2011) ......................................................................................................................................

Figure 10 :

 10 Figure 10: Conservation of the PDZ binding domain (TXV-COOH) in the Fzd family; functional and non functional PDZ binding domain is noted as (+) or (-) respectively (Schulte and Shenoy 2011) .............................

Figure 11 :

 11 Figure 11: List of Frizzled receptors (Wnt homepage, Stanford) ............................................................................

Figure 12 :

 12 Figure 12: Diagram of Fzd-4 showing mutations associated to the disease (Robitaille et al. 2002, Robitaille et al. 2011) ........................................................................................................................................................................

Figure 13 :

 13 Figure 13: Competition between Wnt ligands would regulate activation of Wnt pathways by modulating phosphorylation of LRP5/6 and Ror2 (adapted from Grumolato et al 2010) ..........................................................

Figure 14 :

 14 Figure 14: Structure of LRP5 (He et al 2005) ........................................................................................................

Figure 15 :

 15 Figure 15: Structure of the Ror2 protein (adapted from Scopus) ............................................................................

Figure 16 :

 16 Figure 16: Structure of the Ryk protein ...................................................................................................................

Figure 17 :

 17 Figure 17: Structure of Dvl (SCOPUS) ...................................................................................................................

Figure 18 :

 18 Figure 18: List of Dvl proteins (adapted from Wnt homepage, Stanford)...............................................................

Figure 19 :

 19 Figure 19: Positive (left) and negative (right) regulators of Dvl activity (Gao et al 2010) .....................................

Figure 20 :

 20 Figure 20: The -catenin destruction complex (Kimelman and Xu 2006) ..............................................................

Figure 21 :

 21 Figure 21: Canonical Wnt signaling is up-regulated after Wnt5a KO (Topol et al 2003) .......................................

Figure 22 :

 22 Figure 22: a-Expression of various components of Wnt signaling in placenta and yolk sac; b-Placenta and Yolk Sac of Fzd5 -/-embryos are not vascularized correctly; Vascular pattern is normal in Fzd5-/-embryo but vasculature remains superficial in placenta of Fzd5-/-embryos (Tomo et al 2001). .............................................

Figure 23 :

 23 Figure 23: a-The yolk sac of the junB KO embryo at E9.5 is translucid and shows no vessels; b-Morphological phenotypes of JunB deficient embryos (Schorpp-Kistner 1999). ............................................................................

Figure 24 :

 24 Figure 24: a-Wnts and Fzd4 are mediators of Brain angiogenesis (right) and differentiation of the Blood brain barrier (left) (Paolinelli et al 2011); b-Lrp5/6 and -catenin are also essential for vessel formation in the hindbrain as KD of both proteins lead to vascular leakage (in white) (Zhou et al. 2014) .......................................

Figure 25 :

 25 Figure 25: Schematic of retina during its vascularization (adapted from Franco et al 2009) ..................................

Figure 26 :

 26 Figure 26: a-Neutralizing FZD4 alters development of the intermediate and deep plexus; b-leading to leakage as shown by fluorescein extravasation and PLVAP expression (Ye et al 2010) .........................................................

Figure 27 :

 27 Figure 27: a -Fzd4 deletion leads to expression of PLVAP; the cells expressing PLVAP are progressively lost, indicating that endothelial cells deleted for Fzd4 are eliminated; b-Proliferation is increased in Fzd4-/-cells as indicated by Edu staining. .......................................................................................................................................

Figure 28 :

 28 Figure 28: Retinal myeloid cells (in red) interact with descending vertical sprouts (in green) ...............................

Figure 29 :

 29 Figure 29: Retinal myeloid cells flow sorted from the deep plexus are subjected to PCR for Wnt pathway components (Stefater, Lewkowich et al. 2011) .......................................................................................................

Figure 30 :

 30 Figure 30: a-c micro-CT images of control and stem cells injected hindlimb after ischemia; d -quantification of vessel density and connectivity; e-expression of Wnt factors as detected in ischemic muscle after injection of normal/ hypoxic stem cell (adapted from Leroux et al.) ..........................................................................................

Figure 31 :

 31 Figure 31: Events that occur after cerebral ischemia (Sandoval 2009) ...................................................................

Figure 32 :

 32 Figure 32: Wnt5a induces migration by relocalizing c-Jun to the leading edge (Nomachi 2008) ..........................

Figure 33 :

 33 Figure 33: Cell migration and adhesion regulated by the Wnt5a pathway (Kikuchi et al. 2012) ............................

Figure 34 :

 34 Figure 34: Regulation of migration via the actin skeleton by Dvl and Daam (Chen et al 2003).............................

Figure 35 :

 35 Figure 35: Regulation of migration via microtubules by Dvl and Daam (Chen et al 2003) ....................................

Figure 36 :

 36 Figure 36: Sprouting endothelial cells are hierarchically organized into tips cells and stalk cells during retinal angiogenesis. Tip cell formation is induced by VEGF signaling, while stalk cell are determined by Notch signaling (Herbert et al. 2011) .................................................................................................................................

Figure 37 :

 37 Figure 37: Different effects of Wnt/-catenin and Notch signaling during sprouting angiogenesis (Gerhardt et al. 2009) ........................................................................................................................................................................

Figure 38 :

 38 Figure 38: Endosome regulates signaling by different manner (Dobrowolski et al 2011) ......................................

Figure 39 :

 39 Figure 39: Endocytosis of Wnt receptors can lead to signal transduction / recycling or signal termination depending on context (Gagliardi et al 2008) ...........................................................................................................

Figure 40 :

 40 Figure 40: Endosome and Wnt signal transduction (adapted from Drobowolski et al 2011) ..................................

Figure 41 :

 41 Figure 41: The dorsal determinants of the Xenopus egg may be Wnt endosomal components (adapted from Drobowolski et al 2011) ..........................................................................................................................................

Figure 42 :

 42 Figure 42: a -different types of ubiquitin chains, b-3D structure of Lysine 63 linked diubiquitin, c-3D structure of Lysine 48 linked diubiquitin (Ikeda, Dikic 2008) ...............................................................................................

Figure 43 :

 43 Figure 43: a-position of the different lysines residues on intracellular domains of Fzd4; ubiquitinylation of Fzd4 (smear) is mediated by Wnt3a and UBPY (Mukai et al 2010) ................................................................................

Figure 44 :

 44 Figure 44: UBPY deubiquitin ligase controls availability of FZD4 for Wnts (Mukai et al 2010) ..........................

Figure 45 :

 45 Figure 45: Different regulators of Dvl ubiquitinylation (adapted from Tauriello 2010) .........................................

Figure 46 :

 46 Figure 46: a-Cyld mediated K63 dependant ubiquitinylation of Dvl (detected as a smear); b-model of regulation of Dvl activity by CYLD (Tauriello et al. 2010) .....................................................................................................

Figure 47 :

 47 Figure 47: Inversin regulation of Dvl (adapted from Simons 2006) .......................................................................

Figure 48 :

 48 Figure 48: Ubiquitin mediated regulation of core Wnt proteins such as catenin (adapted from Tauriello et al. 2010) ........................................................................................................................................................................

Figure 49 :

 49 Figure 49: Orthologues of PDZRN3 (Gene Ontology) ...........................................................................................

Figure 50 :

 50 Figure 50: Structure of the protein (Scopus, EMBL) ..............................................................................................

Figure 51 :

 51 Figure 51: Process of ubiquitinylation (Dikic et al. 2012) ......................................................................................

Figure 52 :

 52 Figure 52: ClustalX alignment of the last amino acids of murine PDZRN3 (mm LNX3), PDZRN3 4 (mm LNX4) as well as orthologues from invertebrate species(Flynn et al. 2011). ......................................................................

Figure 53 :

 53 Figure 53: Molecular evolution of the LNX family (Flynn et al 2011) ...................................................................

Figure 55 :

 55 Figure55: VE-cadherin traps FoxO1 and β-catenin at the membrane. In the nucleus, both would block claudin-5 transcription. In acute permeability condition, VE-cadherin adhesion is destabilized and FoxO1 and β-catenin are phosphorylated and translocate to the nucleus where they block claudin-5 transcription[START_REF] Gavard | VE-cadherin and claudin-5: it takes two to tango[END_REF] .......

Figure 56 :

 56 Figure 56: a-Transcription of claudin 3 is activated by-catenin (Liebner et al 2008); b-Conditional loss of catenin leads to vascular leakage during development (Catellino et al 2003) .........................................................

Figure 57 :

 57 Figure 57: a/b-Injection of Evans Blue of Fz4 deficient in EC mice shows leakage in the cerebellum and retina; c-Leakage arises from the fact that vessels are fenestrated as indicated by Biotin / Mouse igG extravazation(adapted from Wang et al 2013/ Zhou et al 2014 ) ............................................................................

Figure 58 :

 58 Figure 58: Response to stroke (adapted from Kahle et al. 2014) ............................................................................

Figure 59 :

 59 Figure 59: Structure of the tight junctions (adapted from González-Mariscal et al 2000) ......................................

Figure 60 :

 60 Figure 60: Structure of MAGUK Proteins (adapted from González-Mariscal et al 2000) ......................................

Figure 61 :

 61 Figure 61: Proposed model for the formation of TJ (Rajasekaran et al 1996) ........................................................

Figure 62 :

 62 Figure 62: Differential expression and localization of -catenin and ZO-1 in migratory versus stationary MCF10A human breast cells (adapted from Polette et al 2007) ..............................................................................

Figure 63 :

 63 Figure 63: Interaction of ZO1/catenin during EMT transition (Polette et al 2007) ...........................................

Figure 64 :

 64 Figure 64: In subconfluent cultures, ZO-2 and Jun are found at the nuclei and cell borders, while in the confluent condition, they are only detected at the cell membrane (Betanzos et al 2007) ........................................................

Figure 65 :

 65 Figure 65: In subconfluent cultures, ZO-2 and Jun are found at the nuclear fraction, while in confluent cultures, they are only detected in the membrane fraction as indicated by cell fractionation (Betanzos et al 2007) .............

Figure 66 :

 66 Figure 66: ZO-2 immunoprecipitation realized from membrane (a) and nuclear extracts (b) from sparse and confluent cell cultures (Betanzos et al. 2004) ..........................................................................................................

Figure 67 :

 67 Figure 67: a -Interaction of claudins with scaffolding proteins ZOs and MUPP1; b-Western blot showing expression of MUPP1 in brain tissues (adapted from Soini et al 2011 and Sitek et al 2003) .................................

Figure 68

 68 Figure 68: a-Interactions of MUPP1/ Patj (first 10 PDZ domains are identical in MUPP1 and Patj); b-Relocalization RhoA to Leading edge by the Crumbs complex (Ernkvist et al. 2009) ..........................................

Figure 70 :

 70 Figure 70: a -Depletion of MUPP1 and Syx disrupts junctional integrity as shown by ZO1 stainin and Transmission electron micrographs of sections of ventricle myocardium; b-Model of regulation of Junctional stability via MUPP1 (Ngok et al. 2012) ..................................................................................................................

Figure 71 :

 71 Figure 71: Dvl3 sequence used for cloning in each corresponding plasmids in red ................................................

Figure 72 :

 72 Figure 72: PDZRN3 sequence used for cloning in each corresponding plasmids in red.........................................

Figure 73 :

 73 Figure 73: Formation of tube-like structures after cytodex plating (control condition) ..........................................

Figure 74 :

 74 Figure 74: Floxed PDZRN3 mice construct ............................................................................................................

Figure 75 :

 75 Figure 75: Mating of PDGF-icre Pdzrn3fl/fl males with Pdzrn3 fl/fl females ........................................................

Figure 76 :

 76 Figure 76: Validation of the PDGF icre recombinase system using the mT/mG reporter mice ..............................

Figure 77 :

 77 Figure 77: Quantification of Vascular area/ Regression and Proliferation ..............................................................

Figure 78 :

 78 Figure 78: Region of retina imaged for quantification ; Representative view of the superficial plexus; a plane of the intermediate plexus and the deep plexus ...................................................................................................................

Figure 79 :

 79 Figure 79: View of the module of Tracking (Image J) and data analysis (Chemotaxis and migration tool Ibidi) ..

Figure 80 :

 80 Figure 80: Scheme of the cerebral artery occlusion procedure (Rousselet et al 2012) ............................................

Figure 81 :

 81 Figure 81: Evans Blue diffusion after MCAO: a-whole brain; b-100µm vibratome slice observed under white light; c-observed at 545nm .....................................................................................................................................

Figure 82 :

 82 Figure 82: Scheme indicating position of the x-ray lamp, sample and camera in the microCT ..............................

Figure 83 :

 83 Figure 83: Vessel extracts from adult -PDGF cre+ mT/mG mice injected with tamoxifen (vessels fluoresce in green; Hoechst staining of nuclei in blue) ...............................................................................................................

Figure 84 :

 84 Figure 84: Phenotype of Fzd4-/-mice ...................................................................................................................

Figure 85 :

 85 Figure 85: Effet of deletion of Fzd4 on vessel density ..........................................................................................

Figure 86 :

 86 Figure 86: Interaction of Fzd4 and Dvl favor Wnt non canonical pathway to favor angiogenesis .......................

Figure 87 :

 87 Figure 87: Alignment of the Dvl1/ 2 and 3 sequences ..........................................................................................

Figure 88 :

 88 Figure 88: Yeast two Hybrid Strategy and Structure of the PDZRN3 protein ......................................................

Figure 91

 91 Figure 91 Depletion of Pdzrn3 using two siRNAs reduces migration of endothelial cells ...................................

Figure 92

 92 Figure 92 Loss of PDZRN3 modifies the ability of endothelial cells to form tubes .............................................

Figure 93 :

 93 Figure 93: Role of PDZRN3 for transduction of Wnt signaling and vascular morphogenesis ............................

Figure 94 :

 94 Figure 94: EDTA treatment induces disruption of cell to cell junctions in control Huvecs; a delay is observed in Pdzrn3 depleted cells .............................................................................................................................................

Figure 95 :

 95 Figure 95: Huvec cells are orientated in a flow of 10, 20 or 30dyn/cm2; Pdzrn3 depleted cells are unable to align to the flow and reorganize their junctions accordingly..........................................................................................

Figure 96 :

 96 Figure 96: a -validation of the PDGF Cre activation using a mt/mg reporter; b-vessel extracts were purified from pdgf cre mt/mg mice and subjected to confocal microscopy ........................................................................

Figure 97 :

 97 Figure 97: Validation of the enrichment of the brain vessel fraction by q RT PCR ..............................................

Figure 98 :

 98 Figure 98: Expression of PDZRN3 in vessel / wash fraction ................................................................................

Figure 99 :

 99 Figure 99: Proposed Model ...................................................................................................................................

Figure 100 :

 100 Figure 100: Expression of the Pdzrn3 in Carcinoma (Oncomine) .........................................................................

Figure 101 :

 101 Figure 101: Correlation of expression of PDZRN3 with other genes in Carcinomainset represent the scaling colour (Oncomine) .................................................................................................................................................

Figure 102 :

 102 Figure 102: Phosphorylation of PSD-95 on Y397 makes access to the 3rd PDZ domain of PSD 95 possible (Zhang et al. 2011).................................................................................................................................................

Figure 103 :

 103 Figure 103: Interaction of compound 3289-8625 (blue) and Dpr peptide(brown) with the PDZ domain of Dvl (Grandy et al. 2009) ...............................................................................................................................................

  . Ectopic placement of Wnts in Xenopus eggs during early gastrulation leads to formation of a secondary body axis and head, while inhibition of Wnt signaling results in a lack of dorsal structures in the frog embryo. Wnts are produced from specific sites during development, such as the edge of the developing fly wing or the dorsal region of the neural tube in vertebrates. Failure of closure of the neural tube is characteristic of knock-out mice of proteins of the Wnt pathway(Wang et al. 2006). Dysregulation of Wnt signaling is associated with cancer development[START_REF] Saadi-Kheddouci | Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene[END_REF] as alteration of proteins implicated in the pathway such as Axin or Ror2 are linked to carcinogenesis[START_REF] Saadeddin | The links between transcription, beta-catenin/JNK signaling, and carcinogenesis[END_REF][START_REF] Ford | The dual role of the novel Wnt receptor tyrosine kinase, ROR2, in human carcinogenesis[END_REF]. ……..

Figure 1 :

 1 Figure 1: The Wnt canonical and non canonical pathways (adapted from Saadeddin et al 2009)

Figure 2 :

 2 Figure 2: Transcriptional targets of the Wnt/ canonical pathway (Wnt homepage, Stanford)

  alignment of cells within a cell sheet (Fig 2). PCP signaling creates an orientation of the cell by producing asymmetrical organization of Wnt signaling components: Fzd/Dvl are present at the distal side of the cell and Vangl2/ Prickle at the proximal side (Veeman et al 2003)(Montcouquiol et al. 2006). Both complex exclude each other: Prickle antagonizes Fzd/ Dvl by inducing removal of Fzd/ Dvl from the cell membrane by endocytosis while Dvl recruits E3 ubiquitin ligase Smurf to Prickle, leading to its loss (Narimatsu et al 2007).

Figure 3 :

 3 Figure 3: PCP induces asymmetrical organization of Fzd/ Dvl and Vangl2/ Pk (Schulte 2010)

Finally, in

  the Wnt/GSK3 pathway, Wnt inhibition of GSK-3 activates mTOR without involvement of -Catenin. It's why the mTOR inhibitor, Rapamycin inhibits Wnt-induced cell growth and cancer formation independently of -Catenin. The Wnt/ m-Tor pathway regulates cell cycle and is important in carcinogenesis (van Amerongen and Nusse 2009).b) Wnt (wingless-type MMTV integration site family) Factors i) Overview of the Wnt family of proteinsMouse mammary tumor virus (MMTV) is a retrovirus that utilizes an insertional mutagenesis strategy to infect mammalian cells, leading to malignant transformation only in mammary epithelium, having a potent role in breast cancer formation. MMTV activates aberrant expression of number of developmental genes, via proviral insertion, leading to oncogenetic transformation. These normally silent genes activated by the MMTV have been named as ints. The association of MMTV with the Wingless family of proteins (drosophila) began with int-1 that shares 54% identity with Drosophila Wng, with a conservation of 20 cysteine residues. Thus, an amalgam was created out of the winglessint-1 similarity (Wnt) that served for naming of the family. The Wnt family includes 19 members in mammals. Ligands such as Wnt1, Wnt3a, and Wnt8 interact with Fzd and LRP5/6 to activate the canonical pathway, while Wnt4, Wnt5a, Wnt7a and Wnt11 activate non canonical pathways.

Figure 4 :

 4 Figure 4: Distribution of Wnt Genes in Metazoans. X indicate that no member of the Wnt subfamily found in the genome of the organism; the color is different for each Wnt; two blocks indicate presence of paralogue in the specie (adapted from Prud'homme et al. 2002)

Figure 5 :

 5 Figure 5: Phylogenetic tree of the Wnt family (adapted from Prud'homme et al. 2002)

  iv) Wnt5a (wingless-type MMTV integration site family, member 5A) Wnt5a can interact with Fzd1, Fzd2, Fzd4, Fzd7 or Ror2. Wnt5a can activate or inhibit canonical Wnt signaling, depending on receptor context. It can activate catenin signaling by inducing caveolin-mediated endocytosis of Fzd receptors or inhibit the canonical Wnt pathway by promoting clathrin-mediated endocytosis of Fzd / Ror2(Grumolato et al. 2010). Wnt5a is required during embryogenesis for extension of the primary anterior-posterior axis and for gastrulation.

Figure 7 :

 7 Figure 7: Schematic presentation of Wnt/ Fzd signaling kinetics (Schulte and Shenoy 2011)

Figure 8 :Figure 9 :

 89 Figure 8: Phylogenetic tree of the human Frizzled receptors FZD (Schulte and Shenoy 2011)

Figure 10 :

 10 Figure 10: Conservation of the PDZ binding domain (TXV-COOH) in the Fzd family; functional and non functional PDZ binding domain is noted as (+) or (-) respectively (Schulte and Shenoy 2011)

Figure 12 :

 12 Figure 12: Diagram of Fzd-4 showing mutations associated to vitreoretinopathy (Robitaille et

Figure 13 :

 13 Figure 13: Competition between Wnt ligands would regulate activation of Wnt pathways by

i) LRP5/ 6 Figure 14 :

 614 Figure 14: Structure of LRP5 (He et al 2005)

Figure 15 :

 15 Figure 15: Structure of the Ror2 protein (adapted from Scopus)

Figure 16 :

 16 Figure 16: Structure of the Ryk protein (adapted from Scopus)

Figure 17 :

 17 Figure 17: Structure of Dvl (adapted from Scopus)

Figure 18 :Figure 19 :

 1819 Figure 18: List of Dvl proteins (adapted from Wnt homepage, Stanford)

Figure 20 :

 20 Figure 20: The -catenin destruction complex (Kimelman and Xu 2006)

2 )Figure 21 :

 221 Figure 21: Canonical Wnt signaling is up-regulated after Wnt5a KO (Topol et al 2003)

Figure 22 :

 22 Figure 22: a-Expression of various components of Wnt signaling in placenta and yolk sac; b-

5 (

 5 Fig 22a) and Fzd5-/embryo die in utero around E10.5, due to defects in yolk sac angiogenesis, as indicated by a poorly developed vascular bed when compared to Wt mice (Fig 22b). Vasculogenesis in the placenta is also defective as embryonic vessels do not invade the placental labyrinth in Fzd5-/-mouse (Fig 22c). Because Wnt5a and Wnt10b co-localize with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for Fzd5-dependent signaling for endothelial growth in the yolk sac (Fig 22a) (Tomo-o Ishikawa and Shin-ichi Nishikawa 2001).

Figure 23 :

 23 Figure 23: a-The yolk sac of the junB KO embryo at E9.5 is translucid and shows no vessels;

aFigure 24 :

 24 Figure 24: a-Wnts and Fzd4 are mediators of Brain angiogenesis (right) and differentiation of

Figure 25 :

 25 Figure 25: Schematic of retina during its vascularization; the superficial plexus is formed

Figure 26 :

 26 Figure 26: a-Neutralizing Fzd4 alters development of the intermediate and deep plexus; b-

Figure 27 :

 27 Figure 27: a -Fzd4 deletion leads to expression of PLVAP; the cells expressing PLVAP are

Figure 28 :Figure 29 :

 2829 Figure 28: Retinal myeloid cells (in red) interact with descending vertical sprouts (in green)

Figure 30 :

 30 Figure 30: a-c micro-CT images of control and stem cells injected hindlimb after ischemia; dquantification of vessel density and connectivity; e-expression of Wnt factors as detected in ischemic muscle after injection of normal/ hypoxic stem cell (adapted from Leroux et al.)

Finally,

  Leroux et al showed that injection of hypoxia pretreated mesenchymal stem cells would promote neoangiogenesis after ischemia (Fig 30 a to d). The stem cells after hypoxia strongly expressed Wnt4 (Fig 30e); suggesting a pivotal role of Wnt4 in promoting capillary formation (Leroux et al. 2010).

Figure 31 :Figure 32 :

 3132 Figure 31: Events that occur after cerebral ischemia (Sandoval 2009)

Figure 33 :

 33 Figure 33: Cell migration and adhesion regulated by the Wnt5a pathway (Kikuchi et al. 2012)

Figure 34 :

 34 Figure 34: Regulation of migration via the actin skeleton by Dvl and Daam (Chen et al 2003)

Figure 35 :

 35 Figure 35: Regulation of migration via microtubules by Dvl and Daam (Chen et al 2003)

Figure 36 :

 36 Figure 36: Sprouting endothelial cells are hierarchically organized into tips cells and stalk cells

Figure 37 :

 37 Figure 37: Different effects of Wnt/-catenin and Notch signaling during sprouting

Figure 38 :

 38 Figure 38: Endosome regulates signaling by different manner (Dobrowolski et al 2011)

  the case of Wnt signaling, endocytosis is required at different levels. Early endosomes would sequester GSK3- in vesicles, leading to protection of its cytosolic substrates ( catenin or c-Jun) from phosphorylation, stabilizing newly translated β-catenin protein. Interestingly, GSK3- would be recruited to the multivesicular endosome after binding to the Dvl/Axin-2 complex in the cytosol(Jibin Zhou 2005).

Figure 39 :

 39 Figure 39: Endocytosis of Wnt receptors can lead to signal transduction / recycling or signal

Figure 40 :

 40 Figure 40: Endosome and Wnt signal transduction (adapted from Drobowolski et al 2011)

Figure 41 :

 41 Figure 41: The dorsal determinants of the Xenopus egg may be Wnt endosomal components

Figure 42 :

 42 Figure 42: a -different types of ubiquitin chains, b-3D structure of Lysine 63 linked diubiquitin, c-3D structure of Lysine 48 linked diubiquitin (Ikeda, Dikic 2008)

Figure 43 :

 43 Figure 43: a-position of the different lysines residues on intracellular domains of Fzd4;

Figure 44 :Figure 45 :

 4445 Figure 44: UBPY deubiquitin ligase controls availability of FZD4 for Wnts (Mukai et al 2010)

Figure 46 :

 46 Figure 46: a-Cyld mediated K63 dependant ubiquitinylation of Dvl (detected as a smear); b-

Figure 47 :

 47 Figure 47: Inversin regulation of Dvl (adapted from Simons 2006)

  Figure 48: Ubiquitin mediated regulation of core Wnt proteins such as -catenin (adapted

  b) Structure and Function of PDZRN3 E3 ubiquitin ligaseThe 1066 amino acid protein is composed of a RING domain, a depreciated zf-TRAF domain, two PDZ domains and a C-terminal PDZ binding domain.

Figure 50 :

 50 Figure 50: Structure of the protein (Scopus, EMBL)

Figure 51 :

 51 Figure 51: Process of ubiquitinylation (Dikic et al. 2012)

Figure 52 :

 52 Figure 52: ClustalX alignment of the last amino acids of murine PDZRN3 (mm LNX3),

Figure 53 :

 53 Figure 53: Molecular evolution of the LNX family (Flynn et al 2011)

Fgure 54 :

 54 Brain capillaries under normal and pathological conditions (Paolinelli et al 2011)Permeability of the BBB depends on the stability of adherens and tight junctions (TJ). In normal conditions, cerebral endothelial cells form complex junctions that block the passage of large molecules. Nutrients such as amino acids or glucose are able cross the barrier through specific transporters, while large molecules (such as antibodies, lipoproteins, peptides) can be transferred by vesicular-based systems. In pathological condition (such as ischemia), TJ become leaky and vesicular transport strongly increased. This results in the entry of fluids in the brain leading to edema and other pathological reactions[START_REF] Paolinelli | The molecular basis of the blood brain barrier differentiation and maintenance. Is it still a mystery?[END_REF]. Wnt signaling would be one of the signaling pathways essential for maintenance of blood brain barrier integrity.

Figure 55 :

 55 Figure 55: VE-cadherin traps FoxO1 and β-catenin at the membrane. In the nucleus, both

Figure 56

 56 Figure 56: a-Transcription of claudin 3 is activated by B-catenin (Liebner et al 2008); b-

Figure 57 Figure 58 :

 5758 Figure 57: a/b-Injection of Evans Blue of Fz4 deficient in EC mice shows leakage in the

  blood brain barrier and of consist of transmembrane proteins named claudins and occludins, as well as TJ-associated proteins, ZOs. Claudins form a family of over 16 transmembrane proteins; Claudin 3 and 5 iare present at TJ of endothelial cells. Occludin incorporates itself into claudin polymers, while the ZOs proteins act as linkers between occluding/ claudin and the actin-based cytoskeleton (Fig 59).

Figure 59 :

 59 Figure 59: Structure of the tight junctions (adapted from González-Mariscal et al 2000)

Figure 60 :

 60 Figure 60: Structure of MAGUK Proteins (adapted from González-Mariscal et al 2000)

Figure 61 :

 61 Figure 61: Proposed model for the formation of TJ (Rajasekaran et al 1996)

Figure 62 :

 62 Figure 62: Differential expression and localization of B-catenin and ZO-1 in migratory versus

Figure 63 :

 63 Figure 63: Interaction of ZO1/ catenin during EMT transition (Polette et al 2007)

Figure 65 :

 65 Figure 65: In subconfluent cultures, ZO-2 and Jun are found at the nuclear fraction, while in

Figure 66 :

 66 Figure 66: ZO-2 immunoprecipitation realized from membrane (a) and nuclear extracts (b)

  a) MUPP1 is important for tight junctions stability MUPP1 was first identified as an interacting partner of the serotonin receptor type 2C. Hamazaki et al. showed that MUPP1 is located at tight junctions of epithelial and endothelial cells, where it binds claudins and acts as a scaffolding protein, anchoring these proteins to the F-actin cytoskeleton.

Figure 67 :

 67 Figure 67: a -Interaction of claudins with scaffolding proteins ZOs and MUPP1; b-Western

Figure 69 :

 69 Figure 69: a-Syx /RhoA /Amot /MUPP1 and Rab13 colocalize in response to VEGF.A , as

  DIX domain (amino acid 106 to 716) was subcloned into pGBTK7 in-frame with the DNA binding domain of galactosidase-4 (GAL4). Yeast two-hybrid screening and assays were performed as described in the Clontech protocol (MatchMaker system3, Clontech, K1612-1).

Figure 71 :

 71 Figure 71: Dvl3 sequence used for cloning in each corresponding plasmids in red

Figure 72 :

 72 Figure 72: PDZRN3 sequence used for cloning in each corresponding plasmids in red

(

  Polyplus) at a final concentration of 30nM. Selected siRNAs sequence are detailed below Reference / Produced by Sequence h PDZRN3 NM_015009.1 SR307944A (Origene) CGGCACTAGAGTATACAATTCCTTC h PDZRN3 NM_015009.1 SR307944B (Origene) CGATCCAAATGACTACATTGGAGAC h PDZRN3 NM_015009.1 SR307944C (Origene) GCATGTACAGGAAAGTTGTGGATG c) Chemical treatments Ly.72632 (sigma) was used at 10µm (sigma). Phorbol myristate acetate(pma) (sigma) was used at 1µg/ml (YU ET AL. 2007). Dynasore (Santa Cruz) was used at 0,25M (MIKELS AND NUSSE 2006).d) Proliferation (MTT) assayProliferation was assessed 0, 24, 48, 72 hours after cell seeding. MTT(3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide) (0,5mg/ml) was added to cell media for 4hours. The MTT is incorporated into living cells and reduced to formazan. Addition of DMSO induces solubilization of formazan into a purple soluble solution that can be quantified by fluorescence at 570nm. The measured optical density correlates with the number of living cells.

(Figure 73 :

 73 Figure 73: Formation of tube-like structures after cytodex plating (control condition)

Figure 74 :

 74 Figure 74: Floxed PDZRN3 mice construct

Figure 75 :

 75 Figure 75: Mating of PDGF-icre Pdzrn3fl/fl males with Pdzrn3 fl/fl females

Figure 76 :

 76 Figure 76: Validation of the PDGF icre recombinase system using the mT/mG reporter mice

  serum. Ratio of Vascular area was quantified by delineating the area where vessels were present and related to the total area of the retina. The number of branch points per field was quantified after Isolectin staining of the retina. The number of regressing branches was quantified by counting the number of collagen positive/ isolectin negative branches. Proliferation was quantified by counting the number of Brdu positive nuclei in the vessels and related to the isolectin stained area per field (Fig 77).

Figure 77 :

 77 Figure 77: Quantification of Vascular area/ Regression and Proliferation

Figure 78 :Figure 79 :

 7879 Figure 78: Region of retina imaged for quantification ; Representative view of the superficial plexus; a plane of the intermediate plexus and the deep plexus

  On a scale of 1 to 6, 1 denotes animals showing a normal comportement; 2 denotes animals showing rotation of the ischemic side; 3 -constant rotation on the ischemic side; 4 -barelling; 5 -unconsciousness; 6 -death.

Figure 81 :

 81 Figure 81: Evans Blue diffusion after MCAO: a-whole brain; b-100µm vibratome slice observed under white light; c-observed at 545nm

Figure 82 :

 82 Figure 82: Scheme indicating position of the x-ray lamp, sample and camera in the microCT

Figure 83 :

 83 Figure 83: Vessel extracts from adult -PDGF cre+ mT/mG mice injected with tamoxifen

Figure 84 :

 84 Figure 84: Phenotype of fzd4-/-mice

Figure 85 :

 85 Figure 85: Effet of deletion of Fzd4 on vessel density

Figure 86 :Frizzled 4

 864 Figure 86: Interaction of Fzd4 and Dvl favor Wnt non canonical pathway to favor angiogenesis

Figure 1 .

 1 Figure 1. Loss of fzd4 results in decrease in density and branching of small arteries in heart and kidney. Reconstructed microcomputed tomography images of fzd4 / and fzd4 / heart (A) and kidney (B). Bar graphs show arterial density and branching in heart (fzd4 / , n16; fzd4 / , n10; A) and in kidney (fzd4 / , n9; fzd4 / , n5; B). ***P0.001. Loss of fzd4 reduces EphrinB2 expression. Fold difference (C) in expression (quantitative polymerase chain reaction) of EphrinB2 and EphB4 expressions in fzd4 / compared to fzd4 / heart and kidney. Values are meanSD (n4). *P0.05 vs fzd4 / . D, Representative Western blot analysis of EphrinB2 expression in two different samples of, respectively, wild-type control (fzd4 / ) vs mutant (fzd4 / ) heart and kidney. Alpha tubulin served as loading control. The graphs represent pooled data from three distinct experiments. Values are fold increase SD; n3. *P0.05 and ***P0.001 vs fzd4 / .

Figure 2

 2 Quantitative polymerase chain reaction analysis of fzd4 expression in primary endothelial cells (ECs), smooth muscle cells (SMCs), and murine embryonic fibroblasts (MEFs) from fzd4 / and fzd4 / mice revealed by electrophoresis gel. Values represent control SD; n7. ND indicates not detected. Proliferation of primary fzd4 / compared to fzd4 / EC (n4) (B), SMC (n3; C), and MEF (n4; D) was analyzed respectively by BrdUrd incorporation assay or by cell count number at indicated times vs fzd4 / and fzd4 / compared to fzd4 / cells *P0.05, **P0.005, and ***P0.001 vs fzd4 / . NS indicates nonsignificant. E, Evaluation of fzd4 / vs fzd4 / EC differentiation in Matrigel. Branch numbers were quantified (phase contrast) at 6 hours after plating in the presence of vascular endothelial growth factor A (VEGFA) (right; n3). *P0.05 vs control. Branch numbers per branch point and mean length of branches (m 2 ) were evaluated. **P0.01. F, Images represent chemotactic transwell migration of fzd4 / ECs in the presence in the lower chamber of 10% serum, VEGFA, or fetal growth factor 2 (FGF2) after 16 hours and quantification of cell number (%) in comparison to fzd4 / ECs (right; n4). *P0.05 and ***P0.001 vs fzd4 / and fzd4 / vs fzd4 / . G, SMC and MEF migration was quantified in a wound healing assay under FGF2 stimulation during 20 hours. Values represented in graph correspond to the percentage of migration per fieldSD; n3. ***P0.001 vs fzd4 / .

Figure 3 .

 3 Figure 3. Loss of fzd4 impairs vascular cell polarization. The fzd4 deletion leads to decreased acetyl tubulin expression and organization. A, The fzd4 / and fzd4 / murine embryonic fibroblast (MEF) extracts were prepared and analyzed by Western blotting. The membranes were probed with antibodies recognizing -tubulin either indiscriminately (-tubulin) or only when in acetylated form (acetyl tubulin), and relative expression of acetyl tubulin was reported on the graph. **P0.01. B, Wound-edge fzd4 / and fzd4 / MEFs were stained with anti--tubulin to visualize the microtubule network. Loss of fzd4 revealed an alteration of oriented tubulin organization. C, Golgi organization in migrating fzd4 / primary endothelial cells (ECs), smooth muscle cells (SMCs) and MEFs was established by measuring the fluorescence labeling of the Golgi (red) and the nucleus (DAPI; blue) along a line drawn through the nucleus toward the migrating edge and the volume in each cell (as shown in fzd4 / and fzd4 /

Figure 4 .

 4 Figure 4. Fzd4 represses Wnt canonical pathway and favors Wnt noncanonical AP1-dependent pathway. Loss of fzd4 favors activity of -catenin. A, The fzd4 / and fzd4 / murine embryonic fibroblasts (MEFs) were cultured in the presence () or absence () of Wnt3A conditioned medium (Wnt3a CM) for 16 hours. Cell lysates were analyzed by Western blotting using an antidephosphorylated -catenin (ABC) and anti--catenin (-catenin) and relative expression of ABC was reported on the graph. Relative luciferase values are shown. Quantification of ABC levels without and after addition of Wnt3a CM was reported on graph. Error bars in this and subsequent figures shown as meanSE (n3). Probability values were determined by the two-tailed Student t test (paired).

Figure 5 .

 5 Figure 5. Fzd4 interacts with Dvl1 and Dvl3 and induces AP1 signaling pathway activation. A, Fzd4 induces a relocalization at the cytosolic membrane of Dvl1 and Dvl3. NIH 3T3 cells were cotransfected with plasmids coding for either Dvl1G, Dvl2G, or Dvl3G (green) alone or in the presence of plasmid coding for Fzd4 with a tag HA (Fzd4-HA) and immunostained for Fzd4 with an anti-HA (red). On coexpression with Fzd4, Dvl1 and Dvl3 green labeling in large punctuates in the cytoplasm was decreased and associated with a plasma membrane pattern. Meanwhile, Dvl2G labeling remained in the cytoplasm in presence of Fzd4. B, Fzd4 interacts with endogenous Dvl1 and Dvl3. HEK293Twere transiently transfected either with an empty vector () or with a plasmid coding for Fzd4 with a Tag GFP (Fzd4-GFP). Cell lysates were immunoprecipitated with anti-GFP antibody. The samples were analyzed by Western blotting with the indicated antibodies. Fzd4 induces Dvl3 activation. C, HEK293T cells were transiently cotransfected with a plasmid coding for Dvl3 tagged with a green epitope (Dvl3G) either with an empty vector () or with plasmids coding for Fzd4-HA or for Fzd4-CRD. Cell lysates were analyzed by Western blotting with the indicated antibodies. D, HEK293T cells were transiently transfected either with an empty vector () or with plasmid coding for Fzd4 (Fzd4-GFP). After cell fractionation, Western blotting was performed on membrane (Mbne) and cytosolic (Cyto) fractions with indicated antibodies. Open arrow indicates Dvl3 unphosphorylated forms (78 and 80 kDa) and black arrow shows the phoshorylated (82 kDa) shifted form. Fzd4 polarizes Dvl3 expression at the edge of the cell with -tubulin. E, NIH3T3 cells were transfected with plasmids coding for Dvl3-tagged green (Dvl3G; green) alone or in the presence of a plasmid coding for Fzd4 with a tag HA (Fzd4HA). Cells were immunostained for Fzd4 with anti-HA (purple) and for -tubulin with anti--tubulin (red). Note that Dvl3 alone is expressed in large punctates, whereas, in the presence of Fzd4, it is redistributed and colocalized at the plasma membrane with -tubulin. F, NIH 3T3 cells were transfected either with Fzd4-GFP or with Dvl3G plasmid. Lysates were immunoprecipitated with anti-GFP antibody and then analyzed by Western blotting with the indicated antibodies.

Figure 6

 6 Figure 6. Re-expression of Fzd4 with Dvl3 in sifzd4-treated endothelial cells (ECs) restored their ability to proliferate and form tube. A, ECs were transfected and after 48 hours were counted per well. The sifzd4treated ECs reduced their proliferation compared to control (siCt). Cotransfection with Fzd4-HA and Dvl3myc plasmids rescue significant sifzd4-EC proliferation compared to Fzd4 or Dvl3 plasmid transfected cells. *P0.05, n3. B, ECs were seeded on matrigel after transfection with siRNA and plasmid as indicated. After 6 hours, ECs were fixed, photographed, and mean length and branch number per branch point were counted. The sihfzd4 treatment reduced cell ability to form capillary network compared to siCt treatment. The sifzd4 EC transfection with Dvl3 plasmid alone (sifzd4Dvl3) and with Dvl3 and Fzd4 plasmids (sifzd4Dvl3Fzd4) significantly increased branch lengths and branch numbers per branch point compared to siCtl conditions. *P0.05; **P0.001, n3.

  est importante durant la morphogenèse et le remodelage vasculaire. Cependant, les partenaires en aval de cette voie dans ce processus restent méconnus. Des études récentes suggèrent que l'endocytose des récepteurs Fzd et l'ubiquitination des protéines Dvl seraient essentielles dans la transduction du signal dans les voies Wnt. Résultats Grâce à une stratégie de double Hybride, nous avons identifié des nouveaux partenaires de Dvl, dont l'E3 ubiquitine ligase, PDZRN3. Des mutants murins délétés pour le gène Pdzrn3 ont été créés et croisés avec des souris Ubc-iCre et PDGF-iCre, permettant la délétion de Pdzrn3 de façon ubiquitaire ou spécifiquement dans les cellules endothéliales respectivement. La délétion ubiquitaire de Pdzrn3 induit une létalité embryonaire précoce due à un défaut de vascularisation du placenta et du sac amniotique. La délétion de Pdzrn3 dans les cellules endothéliales avant vascularisation de la rétine induit un retard et une désorganisation des différents plexus. La répression de Pdzrn3 réduit la migration des cellules endothéliales et la formation de tubes endothéliaux dans un modèle de culture en 3D sous activation Wnt. PDZRN3 modulerait l'activité du complexe Fzd/Dvl à la surface des cellules endothéliales. PDZRN3, en induisant la prise en charge de Fzd4 par les voies d'endocytose, favoriserait l'activation de la voie Wnt dans les cellules endothéliales. Ainsi, PDZRN3 ubiquitinerait Dvl, ce qui favoriserait le recrutement de Fzd4 dans les vésicules d'endocytose. Conclusion Nous proposons un mécanisme par lequel la proteine PDZRN3 favoriserait l'activation de la voie Wnt non canonique au cours de la morphogenèse vasculaire.a) State of artPDZ domains are described in scaffolding proteins. PDZ-containing scaffolds assemble proteins in large molecular complex at defined localizations in the cell. It is the case of PSD-95 that organizes glutamate receptors in synapse. PDZ scaffolds can also modulate trafficking of cargo proteins via vesicles. PDZRN3 is remarkable as it possesses PDZ domains/ PDZ binding domains and a RING domain. The RING domain is functional and identifies PDZRN3 as an ubiquitin ligase, a protein that gives the specificity to the reaction of ubiquitinylation.As downstream effectors of Fzd4 and Dvl3 are not known well, a strategy of Yeast two hybrid was set up. Dvl proteins are composed of various domains such as the Dix domain, essential for the interaction with catenin and for activation of the canonical pathway. The PDZ and DEP domains are essential for Dvl interaction with Fzd proteins.

Figure 87 :

 87 Figure 87: Alignment of the Dvl1/ 2 and 3 sequences

  during development(Fig 88). Using this strategy, we were able to identify many targets implicated in cell cycle/ proteasome and microtubules rearrangement (Fig89).

Figure 88 :

 88 Figure 88: Yeast two Hybrid Strategy and Structure of the PDZRN3 protein

  These puncta collocalized with markers of clathrin mediated endocytosis such as Rab5 and Rab11; but also with endogenous ubiquitin (Fig90).

Figure 90

 90 Figure 90 PDZRN3 (V5) colocalizes with clathrin, Rab5 and Rab11; wth ubiquitin

Figure 91

 91 Figure 91 Depletion of Pdzrn3 using two siRNAs reduces migration of endothelial cells

Figure 93 :

 93 Figure 93: Role of PDZRN3 for transduction of Wnt signaling and vascular morphogenesis

  Next, we showed that conditional deletion of Pdzrn3 in EC, in neonates induced a defect in branch extension with excessive vessel regression of the superficial retinal vascular plexus, and ultimately a global mis-orientation of polarized capillaries in the intermediate plexus. In vitro and ex vivo gain and loss of Pdzrn3 expression experiments, revealed a role of PDZRN3 in EC coordinated extension, polarization and tubular plexus formation. Mechanistically, we present evidence that PDZRN3 acts as a new partner of Dvl3 and could promote Dvl3 ubiquitinylation, to mediate PCP signaling activation in EC.

  motility defects. First we analyzed the role of PDZRN3 on EC migration under Wnt induction. Cell tracking of migrating EC revealed that Wnt5a enhanced the distance of EC migration compared to control conditions (Fig3a). We then showed that Pdzrn3 depletion, using siRNA, impairs Wnt5a-induced EC migration and that conversely, lentiviral Pdzrn3 transduction markedly enhanced EC migration in Wnt5atreated conditions. Importantly, Pdzrn3 depleted cells lost their ability to migrate in a directional manner while Pdzrn3 lentivirus transduced EC migrated primarily in a straight path, in Wnt5a conditions(Fig 3a).Next, we assessed whether PDZRN3 is required for sprouting extension using an assay for collective EC migration, where HUVECs form sprouts in a fibrin gel(Fig 3b). In this assay, we first confirmed that Wnt5a enhanced angiogenic sprout lengths, by showing that Wnt5a-transfected fibroblasts layered on top of the gel enhanced EC tube length significantly, compared to control fibroblasts. Ninetysix hours after plating, silencing of Pdzrn3 blocked cells from leaving the beads in response to basal or Wnt5a conditions. Pdzrn3-silenced EC did not migrate collectively and could not form long, multi-nuclei branched structures, while PDZRN3 lentiviral transdution of Pdzrn3-silenced HUVECs restored 3D EC migration in response to basal or Wnt5a conditions(Fig 3b). The analysis of the duration of tube-like structure on recorded time lapse images, revealed that sprouts are dynamically formed over the course of 16 hours, while Pdzrn3-silenced EC were incapable of maintaining sprouts. This data demonstrates the role of PDZRN3 in favoring capillary like structure formation and stabilization (Fig3c).

  hypothesis, we followed Dvl3 ubiquitinylation under Wnt/PCP activation. Incubation with Wnt5a conditioned medium (CM) for 1 hour, induced ubiquitinylation of Dvl3-Myc in HEK293T cells co-interaction between PDZRN3 and Dvl3, via the PDZ domain of Dvl3, is required for recruitment of PDZRN3 toward Fzd4. Next, we examined the role of PDZRN3 in endocytosis of the Fzd4/Dvl3 complex upon activation of Wnt5a, using in vitro models. Immunoprecipitation assays demonstrated that Wnt5a treatment induces Fzd4/Dvl3 re-localization in the Rab5 positive compartment, and its effects are impaired under conditions of Pdznr3 depletion. (Fig 5a). Using cell fractionation approaches, we confirmed that Wnt5a treatment induces re-localization of the Fzd4/Dvl3 complex from the cytosolic membrane, to early endosome fractions. Treatment with either the inhibitor of dynamin, Dynasore, or depletion of Pdzrn3, blocked Fzd4/Dvl3 re-localization (Fig 5b). Using confocal imaging, we next demonstrated that Fzd4 accumulates at the cell membrane in HeLa cells transiently transfected with HA-tagged Fzd4, while 30 minutes of exposure to Wnt5a induces re-localization of Fzd4 in the intracellular compartment. PDZRN3 is required for the process, as internalization of Fzd4 was efficiently blocked when Pdzrn3 was depleted with siRNA (Fig 5c). Fzd4 was detected in intracellular Rab5-positive early endosome vesicles after incubation with Wnt5a conditioned medium, as shown by an overlapping punctate staining pattern of HA-Fzd4 with Rab5. Pdzrn3 depletion via siRNA transfection or treatment with the inhibitor of dynamin, dynasore, abolished Fzd4 co-localization with Rab5 in intracellular cargos under Wnt5a activation. Co-localization of Fzd4 with Rab5 was quantified in each condition (Fig 5c).

  Wnt3a activation. Our data showed that, under basal conditions and after Wnt3a stimulation,  catenin and ABC expression were significantly elevated in Pdzrn3-depleted EC versus control EC (Fig 6b). Interestingly, upregulation of p-c jun by Wnt5a was impaired in Pdzrn3-depleted cells (Fig 6c). Next,  catenin transcriptional activity was measured using the Top-Flash/luciferase reporter construct. Again, we observed that under basal conditions and after Wnt3a stimulation, the Top flash reporter was more robustly activated in EC depleted of Pdzrn3 with siRNA versus control, and reduced in Pdzrn3 lentiviral transduced EC (Fig 6d). Conversely, we monitored c-jun-dependent gene transcription using an AP1responsive luciferase-based reporter, and observed that Wnt5a induction of luciferase reporter gene expression was repressed in Pdzrn3-depleted EC and induced in Pdzrn3 lentiviral transduced conditions (Fig 6e). Taken together, these data support our hypothesis that PDZRN3 favors Dvl3 re-routing from the Wnt canonical pathway toward the PCP signaling pathway.

  endothelial cells. Intravitreal injection of dynasore or vehicle was performed in P3 neonatal pups to compare the extent of primary plexus at P5 in the control eye, and in the dynasore-injected eye of the same animal (Fig 7a). Radial extension of the primary plexus in dynasore injected eyes was significantly reduced and vessel regression was greater, demonstrated by an increase in the number of empty collagen IV sleeves without ECs, as compared to those injected with vehicle only (Fig 7a). Similar results were obtained when dynasore was added ex vivo to mouse aortic ring cultures. Aortic rings were obtained from either P7 Pdzrn3 iECKO or Pdzrn3 iECWT neonatal pups. Pdzrn3 iECKO and Pdzrn3 iECWT treatment with dynasore led to the formation of fewer sprouts compared to Pdzrn3 iECWT control conditions (Fig 7b). Interestingly, incubation of the Pdzrn3 iECKO aortic rings with Pdzrn3 lentivirus, restored their ability to form sprouts (Fig 7b). We then examined whether impairment of endocytosis could impair Wnt signaling by Western blot, using retina lysates. At P5, Pdzrn3 iECKO versus Pdzrn3 IECWT induced repression of p-c-jun and an increase of ABC expression (Fig 7c) while intravitreal injection of dynasore, decreased p-c jun level but did not affect ABC expression, suggesting that dynasore treatment partially mimics the Pdzrn3 deletion phenotype (Fig 7d). We can not exclude the possibility that the vascular morphogenetic impairment under dynasore treatment might reflect an accumulative effect on targets other than endothelial cells, such as myeloid cells.

Fig 1 .

 1 Fig 1. Ubc creERT2 Pdzrn3 f/f embryos displayed defects in yolk-sac and placental angiogenesis a. Level of Pdzrn3 depletion in Ubc creERT2 Pdzrn3 f/f versus Pdzrn3 f/f mice was evaluated by Western blot on E10.5 embryo brain lysates after tamoxifen treatment of pregnant mothers at E7.5. Normalization was done using the expression of  tubulin. b. Survival rate of Ubc creERT2 Pdzrn3 f/f embryos was measured after gene deletion induced by daily tamoxifen treatment (tmx) of pregnant mothers at E7.5 (red arrowhead) (n=214) or at E10.5 (blue arrowhead) (n=44). Pdzrn3 deletion at E7.5 leads to growth retardation and early embryo lethality from E11.5 to E12.5 (n=79).c. Deletion of Pdzrn3 coding gene by tamoxifen injection at E7.5 leads to severe growth retardation as observed at E9.5 and E10.5. Ubc creERT2 Pdzrn3 f/f embryos were significantly smaller and exhibited abnormal vascularization of the yolk sac (scale bar=500µm). Whole-mount visualization of Ubc creERT2 Pdzrn3 f/f versus Pdzrn3 f/f yolk sac demonstrated an organized and ordered vitelline architecture in control mice, while this network was disorganized in mutant embryos at E9.5 and E10.5. Scale bar= 500µm in the Inset and 200µm at higher magnification. d. Placental vasculature was examined in hematoxylin/Eosin-stained sections of control and mutant placentas at E9.5, revealing a reduced labyrinth layer in Ubc creERT2 Pdzrn3 f/f mutant embryos (scale bar=100µm). In a higher magnification, embryonic vessels (arrowhead), as defined by the presence of nucleated red cells are evenly distributed in the labyrinth in Pdzrn3 f/f control placenta while in mutant placentas they are missing (scale bar=50µm). Embryonic vessels are close to maternal vessels (arrow) in control embryos indicating that maternal-fetal exchanges occur in control but not in mutant embryos. MD: maternal deciduas; GT: Giant trophoblasts; L: labyrinth; YS: yolk sac.e. Whole-mount of CD31 of Ubc creERT2 Pdzrn3 f/f mutant and Pdzrn3 f/f control embryos. Vessel network of the brain, but not of the somites is altered in mutants compared to controls at E10.5 (scale bar = 500µm; 300µm in insets). The mutant embryo showed fewer vessels and branches in the brain.

Fig2

  Fig2. PDZRN3 is essential in EC for capillary extension/ stabilization and 2D directional angiogenesis during retina vascular development a.Wild-type retina protein extracts, evaluated by Western blot, revealed that PDZRN3 levels peak at P12; normalization was done usingVe-cadherin and  tubulin.

Fig6

  Fig6. PDZRN3 regulates the balance between non -canonical Wnt/PCP and Wnt canonical signaling in endothelial cells a. The Wnt canonical pathway is activated constitutively and the c-Jun dependent/PCP pathway repressed, in lung lysates from P7 mice when Pdzrn3 was silenced in endothelial cells (iEC KO) compared to lysates from control littermate mice (iEC WT) as indicated by anti-active  catenin (ABC) and anti p-c Jun immunoblot analysis. PDZRN3 expression was evaluated. Normalization was done using the expression of  tubulin. b. siRNA depletion of Pdzrn3 enhanced the accumulation of cytoplasmic  catenin in HUVEC after treatment with Wnt3a CM; quantification of cytoplasmic catenin and ABC are normalized with cytoplasmic  tubulin after cell fractionation. c. The phosphorylation of c-Jun in HUVEC is indicative of activation of the PCP pathway, as a 30min incubation with Wnt5a CM activates phosphorylation of c-Jun. Depletion of Pdzrn3 blocks Wnt mediated phosphorylation of c-Jun. quantification of c-Jun was normalized with  tubulin. d. Depletion of Pdzrn3 (si Pdzrn3) increases the activation of the TOP luciferase reporter system while lentivirus Pdzrn3 (lenti Pdzrn3) transduction blocks the activation of a Lef/Tcf luciferase reporter system (Top reporter) after incubation with Wnt3a CM in HUVEC; experiments were performed in triplicate, error bars denote standard deviation, n=3. e. Depletion of Pdzrn3 (si Pdzrn3) blocks activation of the AP1 luciferase reporter system after incubation with Wnt5a CM while lentivirus Pdzrn3 (lenti Pdzrn3) transduction activates the AP1 luciferase reporter moderately in HUVEC; experiments were performed in triplicate, error bars denote standard deviation, n=3. Unpaired t test on the average values obtained in 3 independent experiments.

Fig7.Supplementary Fig 3 :

 3 Fig7. Endocytosis is essential for activation of the PCP pathway and stability of vessels during retinal vascularization a. Blockade of endocytosis by Dynasore intraocular injection at P3, induces retinal vasculature regression quantified at P5. The number of branch points after Isolectin FITC staining, in the superficial plexus was decreased and capillary regression was increased in retinas injected with Dynasore when compared to retinas injected with DMSO (control), as quantified by the ratio of collagen IV positive/ isolectin FITC negative branches to total branches. Whiskers denote min to max, n= 6 DMSO injected; n=6 Dynasore injected. Unpaired t test. Scale bars=100µm b. Aortic rings were implanted in Matrigel and the area of sprouting was quantified 72 hrs after plating. Aortic rings from P7 Pdzrn3 iECKO (iEC KO) mice produce significantly smaller and less organized sprouts, as compared to aortic rings from Pdzrn3 iECWT (iEC WT) mice. Treatment of wild-type

.

  Cell culture/ DNA transfection and drug treatments HEK293-Tq cells (ATCC, CRL-1573) were cultured in DMEM supplemented with 10% FBS (Hyclone) and penicillin-streptomycin. HeLa and MS1 (mouse pancreatic islet endothelial cell line) cells were cultured in RPMI supplemented with 10% FBS and penicillin-streptomycin. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC)(ATCC, CRL-1730 & CRL-4025) were cultured in EGM-2 media (Lonza, CC-3156) supplemented with EGM-2 Single Quots

  also observed a delay to EDTA treatment (Calcium chelation) of Pdzrn3 depleted endothelial cells. The endothelial cells would detach more slowly than their control after EDTA treatment (Fig 94).

Figure 94 :Figure 95 :

 9495 Figure 94: EDTA treatment induces disruption of cell to cell junctions in control Huvecs; a delay is

  expressed both genes when compared to the wash fraction(Fig 97). Validation of the specificity of the PDGF icre mice was done by crossing PDGF icre mice with mT/mG mice that expresses membranetargeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) after excision. Vessels express GFP in PDGF icre mice but not in control mice, where they express Tomato only as shown on vibratome slices of the cortex and on purified vessels extract(Fig 96). Confocal imaging shows that the endothelial cells express the GFP and the mural cells only express tomato in vessel purification from PDGF icre mice.

Figure 96 :Figure 97 :

 9697 Figure 96: a -validation of the PDGF iCre activation using a mT/mG reporter; b-vessel extracts were

Figure 98 :

 98 Figure 98: Expression of PDZRN3 in vessel / wash fraction

  Pendant ce travail de thèse, nous avons pu étendre nos connaissances sur le rôle des voies Wnt/ Frizzled dans les processus d'angiogenèse au cours du développement et dans le maintien de l'intégrité de la barrière hémato-encéphalique. Ce travail s'est notamment axé sur un nouvel acteur des voies Wnt, l'ubiqutine ligase PDZRN3.Nous proposons un modèle par lequel PDZRN3 ubiquitine la protéine adaptatrice Dvl3 pour induire l'endocytose du complexe Fzd4/ Dvl3 et favoriser la transduction du signal en aval de la fixation du ligand Wnt sur le récepteur Fzd4. Ce mécanisme novateur serait essentiel pour le développement vasculaire régulé par les voies Wnt : notamment la vascularisation du sac amniotique et de la rétine. Ce rôle clé de la protéine ouvre des perspectives intéressantes pour le traitement de pathologie ischémique.Activer la voie Wnt via PDZRN3 pourrait permettre de récupérer un réseau vasculaire fonctionnel plus rapidement. Cette protéine pourrait également être ciblée dans des pathologies tumorales où l'angiogenèse est suractivée. En effet, PDZRN3 est surexprimé dans certains cancers tels que le cancer du sein (Oncomine™).Dans un second temps, nous proposons un modèle par lequel PDZRN3 régulerait la stabilité des jonctions sérrées en induisant le décrochage de la protéine associé aux jonctions ZO1 par ubiquitination de la protéine d'échaffaudage MUPP1. Ce processus serait essentiel pour le maintien de l'intégrité de la barrière hématoencéphalique au cours du développement et dans un contexte ischémique. En effet, la surexpression de PDZRN3 induit une létalité embryonnaire et la délétion de PDZRN3 stabilise les jonctions des cellules endothéliales dans un modèle d'ischémie cérébrale. Ce rôle clé de la protéine ouvre des perspectives intéressantes pour le traitement des accidents vasculaires cérébraux (AVC).

  but was found in Rab-positive structures in Wnt activated cells. This indicates that Fzd4 is endocyted via a clathrin/ rab dependant pathway after Wnt activation. Interestingly, depletion of Pdzrn3 blocked the process of internalization of the receptor, suggesting that Pdzrn3 is required for signal transduction after binding of Wnt ligands to Fzd receptors. Interestingly, PDZRN3 was also found to interact with a protein associated to tight junctions MUPP1 when tyrosinephosphorylated. MUPP1was ubiquitinylated after activation of cells with Wnt in a process that required PDZRN3. More specifically, PDZRN3 ubiquitinylated MUPP1 with ubiquitin chains linked by Lysine 48, inducing the degradation of MUPP1 as demonstrated in vitro. This degradation induced dissociation of proteins segregated to the transmembrane components of the TJ such as ZO1 or c-Jun. These proteins were then relocated to the nucleus where they would act as transcription factors to activate their respective target genes. We were able to demonstrate this relocalization by cell fractionation. C Jun is activated in the nucleus and triggers target of Wnt non canonical pathway. To confirm this hypothesis, we assessed if Wnt targets were modulated by depletion / overexpression of PDZRN3. PDZRN3 was shown to favour the non canonicalc Jun dependant pathway over the canonical --catenin pathway; as depletion of the protein blocked activation of c Jun and induced activation of catenin.

Figure 100 :

 100 Figure 100: Expression of the Pdzrn3 in Carcinoma (Oncomine)

Figure 101 :

 101 Figure 101: Correlation of expression of PDZRN3 with other genes in Carcinomainset

  protein. Similar approaches have been realized on PSD-95 and were highly succesfull (Fig 95) (Zhang et al. 2011). . It would also important to confirm the existence of the Tyrosine 972 -phosphorylation site of PDZRN3 using another method such as Mass Spectrometry.

Figure 102 :

 102 Figure 102: Phosphorylation of PSD-95 on Y397 makes access to the 3rd PDZ domain of PSD

Figure 103 :

 103 Figure 103: Interaction of compound 3289-8625 (blue) and Dpr peptide(brown) with the PDZ

  ...................................................................................................................... b) Hypothesis .......................................................................................................................... ARTICLE 2. THE UBIQUITIN LIGASE PDZRN3 IS REQUIRED FOR VASCULAR MORPHOGENESIS THROUGH WNT/PLANAR CELL POLARITY SIGNALING ........................................................................... ARTICLE 3.THE UBIQUITIN LIGASE PDZRN3 REGULATES BLOOD BRAIN BARRIER INTEGRITY BY CONTROLLING TIGHT JUNCTIONS STABILITY VIA A WNT DEPENDANT MECHANISM ..............................

c)

Summary ............................................................................................................................. a) State of art .......................................................................................................................... b) Hypothesis ........................................................................................................................... c)

Strategy ................................................................................................................................ d) Preliminary Results ............................................................................................................. e) Summary ..............................

................................................................................................ a) State of art ........................................................................................................................... b) Hypothesis ........................................................................................................................... c) Preliminary Results ............................................................................................................. d)

Methods .

  Les maladies cardiovasculaires sont la première cause de mortalité en France ; environ 170 000 décès sont recensés chaque année. Parmi les plus fréquentes, les cardiopathies ischémiques sont responsables de 27% des décès, les accidents vasculaires cérébraux de 25 % et les insuffisances cardiaques de 23 %. Les mécanismes d'adaptation à l'ischémie requièrent une adaptation vasculaire au niveau du tissu ischémique, avec le développement de nouveaux vaisseaux sanguins afin de corriger le défaut de perfusion tissulaire. Mieux comprendre les mécanismes favorisant la formation et le maintien de néo-vaisseaux fonctionnels dans un tissu ischémique est un pré-requis pour améliorer le traitement Dans le cadre de l'étude du système Wnt/Frizzled dans la formation des vaisseaux sanguins, j'ai participé à un premier travail décrivant l'implication de Frizzled 4 dans le développement vasculaire au cours du développement et dans un modèle d'ischémie du membre inférieur. Ce travail a mis en avant le rôle de Frizzled4 en tant qu'activateur des voies Wnt, requis pour la migration des cellules endothéliales.

des ces pathologies. Ma recherche s'inscrit dans l'étude des mécanismes de formation et de maintien des vaisseaux en condition physiologique et pathologique au cours de l'embryogenèse et chez l'adulte. Les protéines Wnt et leurs récepteurs Frizzled régulent la formation des vaisseaux sanguins via une signalisation intracellulaire complexe. Comme les acteurs en aval des Frizzled restent méconnus, le laboratoire a mis en place une stratégie de criblage qui a permis d'identifier de nouveaux acteurs des voies Wnt/ Frizzled. Mes travaux ont porté sur l'étude d'un d'entre eux, l'ubiquitine ligase PDZRN3 et de son rôle dans la formation des vaisseaux sanguins.

L'introduction de ce manuscrit comporte trois chapitres. J'ai tout d'abord décrit le rôle de la voie Wnt/Frizzled dans la régulation de l'angiogenèse, en décrivant les différentes voies et en présentant les acteurs du système. J'ai détaillé les données concernant le rôle du ligand Wnt5a, du récepteur Frizzled, de la protéine adaptatrice Dishevelled 3 et de l'effecteur c-Jun, des protéines retrouvées en filigrane au cours du travail, avant de détailler les différents modèles de régulation proposés dans la littérature. La 2 ème partie de l'introduction décrit comment l'endocytose permet de réguler les voies Wnt, avec différents modèles proposés qui pourraient notamment expliquer comment la balance entre les différentes voies Wnt se fait. J'ai ensuite abordé comment l'ubiquitination permet de réguler l'endocytose pour favoriser la transduction du signal dans les voies de signalisation Wnt. Dans une 3 ème partie, j'ai abordé la régulation de la perméabilité vasculaire par les voies Wnt, avant de mettre en avant les liens existant entre les voies Wnt et les jonctions serrées qui sont essentielles au maintien de l'intégrité vasculaire au niveau cérébral.

A la suite de ce travail, une approche de criblage par double hybride a été menée permettant d'identifier un nouveau partenaire de la voie, l'ubiquitine ligase PDZRN3. Un premier travail a porté sur le rôle de la protéine dans l'angiogenèse. Des souris délétées pour la protéine de façon ubiquitaire ou spécifiquement dans les cellules endothéliales ont été générées. J'ai étudié le rôle de PDZRN3 dans le développement vasculaire au cours de l'embryogenèse et du développement de la rétine. J'ai ensuite utilisé des approches biochimiques et de culture cellulaire pour comprendre comment PDZRN3 jouait sur les voies Wnt pour favoriser le développement vasculaire.

Dans un dernier travail, le rôle physiologique de la voie Wnt via PDZRN3 dans le maintien des jonctions serrées des cellules endothéliales du cerveau et le contrôle de la barrière hémato-encéphalique a été analysé. J'ai notamment étudié l'impact de la surexpression de PDZRN3 dans les cellules endothéliales au cours de l'embryogenèse et qualifié les mutants délétés pour PDZRN3 dans les cellules endothéliales dans un modèle d'ischémie cérébral. Nous montrons par des approches de biochimie que PDZRN3 fait le lien entre voies Wnt et stabilité des jonctions en ubiquitinant la protéine associée aux jonctions MUPP1 et en favorisant la rupture des jonctions sérrées.

Cet ensemble de travaux montre l'importance de la voie Wnt non canonique et de PDZRN3 dans le développement vasculaire et le maintien des fonctions vasculaires chez l'adulte.

Figure 6: List of Wnt genes (Wnt homepage, Stanford)

		 Longitudinal skeletal outgrowth	2 diabetes
		 Female reproductive tract defects
		 Shortened and widened cochlea
		 Mammary gland defects
		 Prostate gland defects
		 Intestinal elongation defects
		 Required for endothelial differentiation of ES cells
	Wnt5b
	Wnt6	Phenotype of Knockouts Mouse or other functions	Associated diseases in
	Wnt7a	 Limb polarity defects	Human  Fuhrmann syndrome
	Wnt1	 Loss of midbrain and cerebellum  Infertility due to lack of Mullerian duct regression
		 With Wnt3a deletion: reduction of number of neural  Delayed maturation of synapses in Cerebellum precursors in the neural tube  Promotes neuronal differentiation  With Wnt4 deletion: reduction of number of thymocytes  With Wnt7b: required for central nervous system
	Wnt2	 Placental defects vasculaturization and maturation
	Wnt7b	 With Wnt2b : defective lung development  Placental development defects
	Wnt2b/13	 Respiratory failure due to defects in mesenchymal  Retinal cell differentiation defects proliferation, required for lung development
		 With Wnt2: defective lung development  With Wnt7a: required for central nervous system
		vasculaturization and maturation
	Wnt3	 Early gastrulation defect  Determines cortico-medullary axis in the kidney	 Tetra-Amelia
		 Hair growth defects
	Wnt8a	 Apical ectodermal ridge defects
	Wnt8b	 Medial-lateral retinotectal topography  Hippocampal neurogenesis defects  Changes in gene expression profile in neurons
	Wnt3a Wnt9a	 Somites, tailbud defects  Joint integrity defects
	Wnt9b	 With Wnt1: reduction in number of neural precursors in  Regulates of mesenchymal/ epithelial transitions the neural tube together  Regulates renal vesicle induction  Loss of hippocampus  Segmentation oscillation clock defects  Planar cell polarity defects of the kidney
	Wnt10a	 Left right asymmetry  Hematopoetic stem cell self-renewal defects	 Odonto-onycho-dermal dysplasia
	Wnt4 Wnt10b	 Kidney defects  Defects in female development due to absence Mullerian Duct, leading to ectopic testosterone synthesis  Side-branching in mammary gland  With Wnt1: decrease in the number of thymocytes  Decreased trabecular bone (Bennett 2005) and bone mass loss  Required for taste papilla development  Overexpression inhibits adipogenesis  Loss activates adipogenesis	 Mutations in Obesity  Mullerian-duct patients regression and  Split-Hand/Foot virilization  SERKAL syndrome Malformation
	Wnt11	 Repression of the migration of steroidogenic adrenal  Ureteric branching defects precursors into the gonad  Cardiogenesis defects
	Wnt5a Wnt16	 Truncated limbs and anteroposterior axis	 Associated with
		 Distal lung morphogenesis defects	Susceptibility to type

ii) Phenotypes of Knockout Mice for Fzd receptors Loss

  of Fzd receptors leads to severe phenotypes where various territories are affected. Current works indicate that Fzd4 would be the only Fzd receptor shown to be essential for angiogenesis in adult, while Fzd5 would be essential for placental angiogenesis.

	Gene	Functions	Phenotype of Knockouts Mouse	Associated diseases
	FZD1		 Viable
	FZD2		 Viable
	FZD3	 Activates PKC	 Defect in guidance of axons
		 Activates -catenin	 With Fzd6: Midbrain
			morphogenesis defect
	FZD4	 Activates PKC	 Cerebellar, Auditory, and	 Retinal
		 Becomes internalized	Esophageal Defects	angiogenesis
		after adding Wnt5a	 Impaired Corpora Lutea	in familial
		 Activates Wnt	Formation	exudative
		reporter with norrin	 Defects in vascular growth,	vitreoretinop
		 Activates Wnt	endothelial defects	athy
		reporter with LRP5	 With Fzd8: Kidney phenotype
	FZD5		 Essential for yolk sac and
			placental angiogenesis
			 Paneth cell phenotype
			 Neural potential in the
			developing Xenopus retina
			 Mammalian ocular development
	FZD6	 Activates PKC	 Hair patterning, tissue polarity
			 With Fzd3: Midbrain
			morphogenesis defect
	FZD7		 Viable
	FZD8	 Activates Wnt with	 With Fzd4: Kidney phenotype
		Respondin
	FZD9		 B cell development
			 Hippocampal defects

FZD10 Figure 11: List of Frizzled receptors (Wnt homepage, Stanford) iii) Frizzled 4

  PDZRN3 was identified in 2006 as a regulator of the differentiation of myoblast in myotubes. In another study, the protein was shown to bind and ubiquitinylate MUSK. The overexpression of PDZRN3 would induce degradation of MUSK and was shown to alter the structure of neuromuscular junctions[START_REF] Lu | Regulation of synaptic growth and maturation by a synapse-associated E3 ubiquitin ligase at the neuromuscular junction[END_REF]). In 2010, PDZRN3 was shown to negatively regulate BMP-2-induced osteoblast differentiation through inhibition of Wnt canonical signaling (Ji-AeKo 2006[START_REF] Lu | Regulation of synaptic growth and maturation by a synapse-associated E3 ubiquitin ligase at the neuromuscular junction[END_REF]

	Honda et al. 2010). PDZRN3 could also interact with Neuroligin1 and EphrinB2 (by similarity, in silico
	predictions).
		Taxonomic	Gene	Description	Human
	Organism	classification			Similarity
	mouse	Mammalia	Pdzrn3	PDZ domain containing RING finger 3	88.51(n)
	(Mus musculus)				94.54(a)
	zebrafish	Actinopterygii	Dr.5264		78.8(n)
	(Danio rerio)
	Figure 49: Orthologues of PDZRN3 (Gene Ontology)
	PDZRN3 is coded by a gene on the chromosome 3 in humans. Many Single Nucleotide
	Polymorphisms (SNPs) exist in the coding gene but none of them correlate with specific diseases. The
	protein is highly conserved as there is 88,51% of nucleotidic homology between the coding gene in
	humans and in mice (Fig49). This low mutation rate indicate a strong pressure of selection on the gene

and a key role of the protein. A paralogue for PDZRN3 exists in humans, PDZRN4 has no known function, and there doesn't seem to be redundancy between the function of the two proteins.

Chemical reagents Reagents used for q RT-PCR Enzymes used for q RT -PCR

	Aricle 1
		3) Equipments
	Reagent		Produced by	Reagent		Units		Produced by
	Antibody SYBR ® Green ® Apparatus	Reference	Produced Applied Reference	Western RNase inhibitor Immuno-	Immuno 40U/µl	-	Immuno Roche	-
	by Roche Biosystems 11583816001 BDH Ultracentrifugator Isopropanol HA mix Centrikon T2060 blot 1/1000 M-MLV	precipitation NO Reverse 200U/µl cytochemisty 1/200	histochemistry* NO Roche
	rab 5 Chloroform Spectrophotometer sc 28570	Santa cruz 1/1000 AnalaR Transcriptase (RT) NO Sigma-Taq DNA Teccan Infinite M200 PRO	1/500 200U/µl		NO Invitrogen
	v5	46-0705 Robot for immunostaining Ventana Discovery XT Invitrogen 1/3000 Aldrich polymerase	1/1000	1/1000		ND
	Myc GelRed™ PDZRN3 c-JUN 10mM Western Blot membrane 05-724 sc-99507 sc-45 dNTP imaging mix Active  catenin 05-665 TRIzol®Reagent Micro computer 1 kb DNA tomography (8E7)  catenin C2206 Agarose ladder Time lapse imaging	Milipore Biotum Santa Cruz 1/1000 1/3000 DNase Santa cruz 1/1000 Odyssey LICOR Invitrogen Reagents used for immunostaining NO 1/1000 200U/µl 1/1000 1/500 1/1000 1/200 Milipore 1/1000 ND 1/500 Invitrogen Reagent GE eXplore Locus SP Invitrogen Triton X100 Sigma 1/3000 ND 1/1000 Fisher Trichloroacetic acid Zeiss Axiobserver	Invitrogen ND 1/100 1/100 ND Produced by Sigma Sigma 1/500
	 tubulin Confocal imaging B 5-1-2	Sigma BioReagents Olympus FSV 100 1/8000	ND	ND		ND
	phosphorylated c-JUN 10x PCR buffer Bright field imaging 9261S Myc 05-419 10x RT buffer Electrophoresis generator Invitrogen Cell Nikon Microphot FXE; Nikon SM 780800 1/1000 ND ND Saccharose signaling Milipore 1/3000 1/1000 ND Roche Paraformaldehyde Bio-Rad Power Pack 300		Sigma ND Sigma ND
	GFP Glycogen Vibratome slices 118144600001 Molecular Invitrogen Leica VT1000S 1/3000 Bovine albumin probes Clathrin sc 6579 Santa cruz 1/3000 DNase 10x Invitrogen Donkey serum 1/1000 serum ND buffer Cryosections Microm cryostat microtome	ND 1/1000		Sigma ND Sigma ND
	rab11 Fzd4 Reagents used for Western blotting sc 9020 AF 194 Parrafin sections Reichert-Jung 2030 BioCut Rotary Microtome Santa cruz 1/1000 ND 1/200 RandD 1/1000 NO 1/200 Hemalun Eosin Ubiquitin U5379-1vl Sigma 1/3000 ND 1/500 Reagent Produced by Glycerol Microcentrifugator Eppendorf 5415 Microcentrifuge CD31 T-2001 BMA ND ND 1/500 Protein G plus agarose Santa Cruz Cell/ In vivo treatments Cell centrifugator Jouan GR411		ND Sigma 1/100 Sigma ND 1/100
	ZO1 Ve -cadherin RIPA lysis Cell incubator 617300 Sc 6458 buffer Phosphotyrosine 05-1050 Protein ladder Hybridization oven	Invitrogen 1/1000 Santa Cruz 1/1000 Reagent Nuaire NU-5500E Milipore 1/3000 Invitrogen Ly72632 Hybaid Shake n Stack	1/1000 ND 1/1000	1/1000 1/500 ND inhibitor Rho	kinase	1/200 Produced by 1/200 ND Santa Cruz
	c-Fos β-mercaptoethanol PCR machine Sc 52	Santa Cruz 1/1000 Sigma SP600125 VWR Uno cycler	ND	1/500 JNK inhibitor	1/100 Santa Cruz
	MUPP1 Tris Tissue homogenizer 104174	Abcam Sigma QIAGEN Tissue lyser 1/1000 Dynasore	1/1000	ND Dynamin inhibitor	I	ND Santa Cruz
	Isolectin FITC Tween Shaker	L2895	Sigma Sigma Labover rotatest; Heidolph unimax 2010 agitator ND ND 1/100 DMSO Dimethylsulfoxyde Sigma 1/100
	Rhodamin Phallodin Sodium dodecyl UV Transilluminator R415 Collagen IV Sc 113360 sulfate TEMED qPCR machine	Molecular Sigma UVP probes Santa Cruz ND ND Tamoxifen Sigma LiCl Biorad DNA engine Opticon 2 ND ND	1/1000 1/100 Lithium Chloride	1/1000 Sigma 1/100 Sigma
		Balance		FITC-Dextran Sartorius Scale LC 4201S	40kDA		Sigma

 Indicates that staining of cryosections / vibratome slices / in toto yolk sac works after paraformaldehyde 4% fixation of mouse samples ; ND = non determined 2)

Frizzled 4 regulates arterial network organization through noncanonical wnt/planar cell polarity signaling

	Rationnel	Frizzled4 est essentiel pour le développement vasculaire au niveau de la rétine et du cerveau. Ce
		travail a consisté à analyser le rôle de la protéine dans le développement vasculaire des organes
		périphériques dans un contexte physiologique et pathologique en tirant avantage d'un modèle de
		souris délétée pour fzd4. Le rôle de Fzd4 dans la régulation des fonctions endothéliales et dans la
		régulation des voies Wnt est aussi abordé.
	Résultats	La souris délétée pour fzd4 présente un phénotype vasculaire fort dans les organes périphériques.
		Finalement, dans un modèle pathologique d'ischémique du membre inférieur, nous montrons que
		la délétion de fzd4 fait que le réseau vasculaire néoformé 10 jours après ischemie est réduit et
		désorganisé.

La structure 3D du réseau artériel a été étudiée par une stratégie de tomographie assistée par ordinateur après injection d'un produit de contraste. La souris KO pour fzd4 présente une réduction du réseau artériel dans les coeurs et reins, avec perte des vaisseaux de petite taille.

La délétion de fzd4 par une stratégie de siRNA altère les fonctions de prolifération, de migration et de tubulogenèse des cellules endothéliales in vitro. Les cellules déplétées pour Fzd4 expriment moins de tubuline acétylée, et sont incapables de positionner le centre organisateur des microtubules (MTOC) correctement, ce qui explique le défaut de migration observé. La délétion de fzd4 in vitro induit une altération des voies Wnt canoniques et non canoniques, indiquant un rôle majeur de la protéine dans l'équilibre entre les deux voies. Dans le même ordre idée, nous montrons que la protéine adaptatrice Dvl3 qui est normalement cytoplasmique est recrutée et activée à la membrane dans les cellules surexprimant Fzd4. Conclusion Ces résultats mettent en évidence le rôle clé de Fzd4 dans le développement vasculaire des organes périphériques via une voie de signalisation Wnt /PCP.

d) Preliminary Results

	cell division cycle	microtubule	Niemann Pick	proteasome 26S	large tumor
	associated 8	associated	type C2 (Npc2)	subunit, non-	suppressor (Lats1)
	(Cdca8)	serine/threonine		ATPase, 8 (Psmd8)
		kinase 2 (Mast2)
	Notch gene	homeodomain	La	E2F transcription	DNA-damage
	homolog 1	interacting protein	ribonucleoprotei	factor 7 (E2f7)	regulated autophagy
	(Notch1)	kinase 1 (Hipk1)	n domain		modulator 2
			family, member		(Dram2)
			2 (Larp2)
	dapper homolog 1,	syntaxin 8 (Stx8) elastin	von Hippel-Lindau	stathmin-like 2
	antagonist of beta-		microfibril	binding protein 1	(Stmn2)
	catenin (Dact1)		interfacer 1	(Vbp1)
			(Emilin1)
	vang-like 2 (van	topoisomerase	LIM domain	kinesin family
	gogh, Drosophila)	(DNA) III beta	only 4 (Lmo4),	member 26B
	(Vangl2)	(Top3b)

  ).Pdgf icre +Pdzrn3 f/f mice. Depletion of Pdzrn3 was verified by Western blot, in Pdzrn3 iECKO versus littermate control (Pdzrn3 iECWT ) retina lysates(Fig 2b). Retina from Pdzrn3 iECKO mice had delayed vascularization of the superficial retinal plexus (SRVP) at P7 compared to control Pdzrn3 iECWT mice, with reduced vascular density and a decrease in the number of branch points, while EC proliferation was significantly increased in tip cells and in the plexus as revealed after BrdU injection. Pdzrn3 iECKO vessels appeared to be unstable, with an increase in the number of empty Collagen IV positive/ isolectin B4 negative "sleeves" in the central areas(Fig 2c). The number of tip cells was not affected. At P12, the SVRP still displayed excessive capillary regression(Fig 2d). Interestingly, in control retinas the deep capillary plexus displayed a complex hexagonal pattern, reminiscent of honey comb structures, while in mutant retinas the capillary network was abnormal and massively reduced. To test whether Pdzrn3 deletion could alter plexus formation via the PCP pathway, we analyzed the planar polarized organization of the intermediate capillary network, which forms bridges between the parent apical plexus (superficial retinal vascular plexus, SRVP) and the parallel basal deep plexus (deep retinal vascular plexus, DRVP). This intermediate network emerged in a two-dimensional sheet, orthogonal to the axis of the SRVP (Fig2d). We collected confocal images and extracted capillary skeletons in this plane, which revealed that the pattern of vessel organization from SRVP to DRVP, was altered. In mutants, capillaries that initiated from the SRVP, subsequently pointed in inappropriate directions in the deepest plexus compared to that of the well-organized polarized network in control mice(Fig 2e). These data demonstrate that Pdzrn3

	deletion alters vessel remodeling and stabilization during retinal angiogenesis, and impairs polarized
	capillary structure formation during tertiary plexus formation.
	Post natal recombination efficiency was evaluated by crossing mT/mG Pdzrn3 f/f reporter mice with

Acknowledgments

The authors thank Jerome Guignard for assistance in mouse breeding.

Sources of Funding

This work was supported by ANR grant (ANR-2010-BLAN-113501), Aquitaine Region grant (20101301039MP), Communaute´ de Travail Pyre´ne´enne, and from the Fondation pour la Recherche Me´dicale (DCV20070409258). B.D. was a recipient of a fellowship from Fondation pour la Recherche Medicale. R.S. was a recipient of a felloship from Aquitaine Region and Inserm.

Disclosures

None.

Acknowledgements

We thank Dr Jerome Ezan and Dr Martin Hagedorn for reading of the manuscript and discussions.

We thank the excellent technical support of the U1034 laboratory by Jerome Guignard for assistance in animal breeding and by Myriam Petit for assistance in placenta histological analysis . This work was supported by grants from the Agence Nationale de la Recherche (ANR-2010-Blanc-1135-01), from Region Aquitaine (20101301039MP). R.N.S is supported by grant from the Fondation pour la Recherche Médicale (FDT20130928208).

RESULTS

The ubiquitin ligase PDZRN3 is required for vascular morphogenesis through Wnt/Planar cell polarity signaling Raj N. Sewduth 1 , Béatrice Jaspard-Vinassa 1,2 , Claire Peghaire 1 , Aude Guillabert 1 , Nathalie Franzl 1 , Catherine Moreau 1 , Markus Fruttiger 3 , Pascale Dufourcq 1,2 , Thierry Couffinhal 1, 2, 4 , Cécile Duplàa 1,2 .

1 INSERM, Adaptation cardiovasculaire à l'ischémie, U1034, F-33600 Pessac, France 2 Univ. Bordeaux, Adaptation cardiovasculaire à l'ischémie, U1034, F-33600 Pessac, France 3 UCL Institute of Ophthalmology, London EC1V 9EL, UK 4 CHU de Bordeaux, Service des Maladies Cardiaques et Vasculaires, F-33000 Bordeaux, France Contact: Cécile Duplàa M : cecile.duplaa@inserm.fr

Summary

Development and stabilization of a vascular plexus requires the coordination of multiple signaling processes. Wnt Planar cell polarity (PCP) signaling is critical in vertebrates for diverse morphogenesis events, which coordinate cell orientation within a tissue specific plane. However, its functional role in vascular morphogenesis is not well understood. Here we identify PDZRN3, an ubiquitin ligase, and report that Pdzrn3 deficiency impairs embryonic angiogenic remodeling and post natal retinal vascular patterning with a loss of 2D polarized orientation of the intermediate retinal plexus. Using in vitro and ex vivo Pdzrn3 loss-of-function and gain-of-function experiments, we demonstrate a key role of PDZRN3 in endothelial cell directional and coordinated extension. PDZRN3 ubiquitinates Dishevelled 3 (Dvl3), to promote endocytosis of the Frizzled/Dvl3 complex, for PCP signal transduction. These results highlight the role of PDZRN3 to direct Wnt PCP signaling, and broadly implicate this pathway in the planar orientation and highly branched organization of vascular plexuses.

PDZRN3 gene deletion induces vascular defects in the yolk sac and placenta

A yeast two-hybrid screen was conducted on an 11.5 dpc mouse embryonic library, with Dvl3 as bait in which the DIX domain was deleted (Dvl3∆DIX) as bait. The DIX domain has been shown to favor the canonical pathway over the PCP pathway (Kishida et al., 1999;[START_REF] Liu | Molecular basis of Wnt activation via the DIX domain protein Ccd1[END_REF]Wong et al., 2004).

We identified PDZRN3, an E3 ubiquitin-protein ligase, shown to negatively regulate Wnt signaling during osteoblast differentiation. As it is widely expressed in various adult mouse tissues (Ko et al., 2006) (Supplementary Fig 1), we asked if PDZRN3 was expressed in a common structure, the endothelium. We first performed real time RT/PCR experiments on distinct mouse and human primary and cultured endothelial cells and showed that it is expressed at a high level (Supplementary Fig 1a-c). Then we showed that its expression during angiogenic processes, co-localized predominantly with CD31+ vessels in E12.5 yolk sac, E14.5 brain and, postnatally at day 7 (P7), in retinal EC and aortic EC, providing support for a potential role during angiogenesis (Supplementary Fig 1d). To investigate the role of PDZRN3 in blood vessel development, we created mice with targeted deletions of Pdzrn3, by crossing a homozygous LoxP-flanked Pdzrn3 allele with Cre expression driven by Ubc-cre ERT2 , a potent ubiquitous promoter [START_REF] Ruzankina | Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss[END_REF]. Pregnant mice were treated daily with tamoxifen injections from E7.5 to E9.5, to inactivate Pdzrn3 during earlier angiogenesis phases. We verified that PDZRN3 expression at E10.5 was significantly reduced in embryonic brain lysates by Western blot (Fig 1a). The Ubc-Cre ERT2 + Pdzrn3 f/f genotype had retarded growth from E9.5 and died between E11. . In wholemount, the pattern of vascularization of Ubc-Cre ERT2 ;Pdzrn3 f/f embryos yolk sacs from E9.5 through E10.5 appeared altered compared to that in their wild-type littermates, suggesting a defect in circulation (Fig 1c). At E9.5, all Ubc-Cre ERT2 +;Pdzrn3 f/f mutant yolk sacs showed an altered pattern of distribution compared to wild type, associated with an increase of vitelline artery diameter, while at E10.5, disorganization of the vascular network was more apparent suggesting an inability to complete angiogenic remodeling (Fig 1c). It is worth noting that tamoxifen treatment of pregnant mice from E10.5 to E12.5, did not induce lethality (Fig 1b), indicating a key role for Pdzrn3 in the early angiogenic phases.

Histological analysis of the placenta from E9.5 and E10.5 mutant embryos, revealed that while chorioallantoic fusion had occurred, the labyrinth layer was reduced and fetal capillaries invading the labyrinth with the reporter transgenic line mTmG ((ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J, Jackson Lab). For yolk sac vascularization analysis, gene inactivation was was induced by tamoxifen injection (100l of a 10 mg.ml -1 solution, corn oil) intraperitoneally in pregnant mother daily from d7.5 to d9.5 or from d10.5 to d12.5 and pups were analyzed respectively starting from d10.5 and d13.5. For pups, tamoxifen was administered by i.p. injection at 50l of a 1 mg.ml

Histology and Immunohistochemistry

For whole mount or tissue section histologic and immunohistologic analysis, embryos and yolk sacs were fixated in 80% Methanol / 20% DMSO overnight, treated with 3% H2O2 for 5 hours to block endogenous peroxidases then rehydrated in 50% Methanol/ 50% PBS for analysis. Brains from embryos (E14.5) were fixed in PFA 4%-PBS, frozen in OCT and cryosectioned. For en face aorta staining, aorta were removed from P7 pups and fixed in PFA 4%-PBS for 30 mins. Placentas were fixed in PFA 4%-PBS then paraffin embedded and sections were stained with hematoxylin/eosin for morphologic evaluation. Briefly, immunohistochemical staining was performed in PBS supplemented with 1% Donkey Serum with the following antibodies: anti CD31 (BMA Pharmingen, T-2001, 1/50), anti PDZRN3 (Santa cruz, sc-99507, 1/50).

the 8XTOPflash reporter (Nusse lab), and a  galactosidase reporter for normalization. . Cells were activated 24h after transfection with Wnt3a, Wnt5a or control CM.

Western blot, immunoprecipitation, ubiquitinylation analysis and in vitro Pull-Down

Assay

For cell fractionation, cells lysates were potterized and centrifuged at 1,000g to obtain the pellet and the supernatant. The pellet from the cell lysate was then resuspended and loaded on cushions of Sucrose 40%;

Sucrose 30%; Sucrose 20%; Sucrose 16%; after a 60,000g centrifugation, the interface between the Sucrose 30% and Sucrose 20% phases was collected, resuspended; finally after a spinning at 100,000, the pellet obtained corresponded to the early endosome. The supernatant from the cell lysate was centrifuged at 100,000g and the pellet obtained was resuspended in the TRIS buffer + 1% triton and spinned again for 10min at 13,000g. The supernatant was then collected and identified as the membrane fraction. For ubiquitinylation assay, cells were transfected with the following vectors: pRK5-HA-UB-wt, pRK5-HA-UB K48 only , which codes for lysine 48-only-ubiquitin or prK5-HA-UB K63 only which codes for lysine-63only ubiquitin (Addgene). HEK293 were cotransfected with Dvl3-myc (0.5µg/ml), one of the ubiquitin coding plasmids (0.5µg/ml) and PDZRN3-V5 or empty control plasmid (0.5µg/ml). For immunoprecipitation, lysates were incubated with primary antibody at +4°C followed by incubation with protein G-Sepharose slurry . Immunoprecipitation was carried out using the following antibodies at 2µg for 500µg of protein lysate: anti Dvl3 (Santa Cruz, sc 8027) and anti myc (9E10, Millipore, 05-724).

Proteins were then resolved by SDS-PAGE and blotted with the following antibodies: anti HA (Cell signaling, 3724, 1/1000), anti rab 5 (Santa cruz, sc-9020, 1/1000), anti V5 (Invitrogen, 46-0705, 1/3000), anti PDZRN3 (Santa Cruz, sc-99507, 1/500), anti c-JUN (Upstate, 06-225, 1/500), anti activated  catenin (anti-ABC, clone 8E7, Millipore, 05-665, 1/1000), anti  catenin (Sigma, C2206, 1/3000), tubulin (Sigma, T5168, 1/5000), anti phosphorylated c-Jun (Cell signaling, 9261S, 1/500), anti myc (9E10, Millipore, 05-724, 1/3000), anti GFP (Molecular probes, 11-814-460-001, 1/3000), anti Vecadherin (Santa Cruz, sc 6458, 1/1000), anti Ubiquitin (Sigma, U5379, 1/1000), anti Paxillin (Santa Cruz, sc-5574, 1/1000 ), and anti Dvl3 (Santa Cruz, sc 8027, 1/500). Binding of antibodies to the blots was detected using Odyssey Infrared Imaging System (LI-COR Biosciences). 2). Ror2 is then active and would phosphorylate PDZRN3 (3) on a tyrosine residue. PDZRN3 would then ubiquitinylate MUPP1 leading to its degradation (4,5). This would lead to detachment of ZO1;

Immunocytochemistry

ZO2 and c-Jun from the tight junctions and their relocalization to the nucleus (6) where they act as transcription factors. Tight junctions are disrupted leading to endothelial permeability (7). PDZRN3 would be found under an inactive form in normal conditions (where Wnt ligands are expressed in a small quantity); and activated by tyrosine phosphorylation after cerebral occlusion (where Wnt ligands are expressed strongly)leading to disruption of the blood brain barrier.

CONCLUSION and PROSPECTS

We were able to show that PDZRN3 was