Anne-Catherine Lhoumeau

Marie-Laure Arcangeli

Maria De Grandis

Marilyn Giordano

Jean-Christophe Orsoni

Frédérique Lembo

Florence Bardin

Sylvie Marchetto

Michel Aurrand-Lions

Jean-Paul Borg
email: jean-paul.borg@inserm.fr

Ptk7 deficient mice have decreased Hematopoietic Stem Cell pools as a result of deregulated proliferation and migration

Keywords: PTK7, planar cell polarity, hematopoietic stem cells, homing

Hematopoietic Stem Cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. At the top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features are controlled through key microenvironmental cues and regulatory pathways such as Wnt signaling. We have previously shown that PTK7, a tyrosine kinase receptor involved in planar cell polarity (PCP), plays a role in epithelial Wnt signaling. However, its function in hematopoiesis has remained unexplored. Here we show that PTK7 is expressed by hematopoietic stem and progenitor cells (HSPCs) with the highest protein expression level found on HSCs. Taking advantage of a Ptk7 deficient mouse strain, we demonstrate that loss of Ptk7 leads to a diminished pool of HSCs, but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlated with increased quiescence and reduced homing capacities of Ptk7 deficient HSPCs, unravelling novel and unexpected functions for planar cell polarity pathways in HSC fate.

Introduction

Hematopoiesis is a biological process consisting on the production of all blood cell types from hematopoietic stem cells (HSCs) located in the bone marrow (BM) of adult mammals. Fetal hematopoiesis occurs in different sites, particularly in the liver, during its earliest phases. Long term hematopoietic stem cells (LT-HSCs) are characterized by their capacity to provide lifelong reconstitution of all blood cell lineages after transplantation into lethally irradiated recipients, whereas short term HSCs (ST-HSCs) do so for only 8-10 weeks.

In the mouse, LT-HSCs belong to the LSK cell compartment defined by the Lineage -/low Sca1 + c-Kit + phenotype irrespectively of their fetal liver (FL) or adult bone marrow (BM) origin. Additional markers known as SLAM (Signal Lymphocytes Activating Molecules) can further subdivide this compartment (1) [START_REF] Oguro | SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors[END_REF]. Indeed, CD150 + CD48 -LSK have been shown to contain around 40% of HSCs with long-term reconstitution potential in BM as well as in FL [START_REF] Kim | Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors[END_REF].

Hematopoiesis is a highly controlled multi-step process that relies on complex interaction networks involving cell surface receptors, growth factors, and adhesion molecules expressed by HSCs and their environment (4)(5)(6) [START_REF] Ooi | The adhesion molecule esam1 is a novel hematopoietic stem cell marker[END_REF] [START_REF] Winkler | Vascular niche E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance[END_REF]. Because HSCs generate mature hematopoietic cells, including immune cells, their replacement must be adjusted to homeostatic or stress conditions such as infections, inflammation or blood loss, and their expansion must be controlled to avoid exhaustion. This control is made possible by the coordinated regulation of quiescence, self-renewal, and differentiation through appropriate signals delivered by functional BM niches [START_REF] Morrison | The bone marrow niche for haematopoietic stem cells[END_REF]. HSCs are retained in these BM niches by cell surface molecules endowed with adhesive and/or signalling functions expressed by HSCs. These receptors belong to different protein families: integrins (VLA-4) [START_REF] Papayannopoulou | The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen[END_REF], Immunoglobulin superfamily (Ig Sf) adhesion molecules [START_REF] Arcangeli | JAM-B regulates maintenance of hematopoietic stem cells in the bone marrow[END_REF] [START_REF] Hidalgo | Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells[END_REF], G Protein Coupled

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Receptors (CXCR4) [START_REF] Ara | Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny[END_REF], or tyrosine kinase receptors (TIE2, c-Kit)(13)(14) [START_REF] Barker | Sl/Sld hematopoietic progenitors are deficient in situ[END_REF] interacting with ligands present within the BM microenvironment [START_REF] Wilson | Bone-marrow haematopoietic-stem-cell niches[END_REF]. Discovery of novel molecules implicated in this multi-step program is of particular importance to embrace its complexity, develop new strategies aiming at the regeneration of damaged hematopoietic tissues and to understand hematopoietic diseases.

PTK7 is a planar cell polarity (PCP) receptor belonging to the Immunoglobulin Superfamily (Ig Sf) and playing important roles during development [START_REF] Lhoumeau | PTK7: a cell polarity receptor with multiple facets[END_REF] [START_REF] Peradziryi | The many roles of PTK7: a versatile regulator of cell-cell communication[END_REF]. PCP is controlled by a non-canonical Wnt pathway, organizes polarization of many epithelial tissues and organs within the plane and drives cell migration and cell intercalation of nonepithelial cells such as mesenchymal cells [START_REF] Angers | Proximal events in Wnt signal transduction[END_REF] [START_REF] Sebbagh | Insight into planar cell polarity[END_REF]. Ptk7 deficient mouse embryos show all signs of typical PCP defects such as severe tube neural and abdomen closure defect with secondary development abnormalities [START_REF] Lu | PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates[END_REF]. PTK7 is a type I protein composed of seven Ig loops in its extracellular region, a transmembrane domain, an intracellular region with a tyrosine kinase domain (17)(18) [START_REF] Jung | Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA[END_REF] [START_REF] Mossie | Colon carcinoma kinase-4 defines a new subclass of the receptor tyrosine kinase family[END_REF]. While PTK7 is catalytically inactive, the cytoplasmic domain containing the tyrosine kinase domain is mandatory for receptor function in vitro and in vivo [START_REF] Na | The cytosolic domain of protein-tyrosine kinase 7 (PTK7), generated from sequential cleavage by a disintegrin and metalloprotease 17 (ADAM17) and γ-secretase, enhances cell proliferation and migration in colon cancer cells[END_REF](25)(26) [START_REF] Prebet | The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome[END_REF]. Both extracellular and intracellular domains of PTK7 can be released from the plasma membrane following the sequential cleavage of the receptor by membrane type 1-matrix metalloproteinase (MT1-MMP)/ADAM-17, and γsecretase, interfering with its PCP and promigratory activities (24)(28) [START_REF] Golubkov | Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis[END_REF].

We have recently found that PTK7 expression is not restricted to epithelial cells.

Indeed, PTK7 is expressed at the cell surface of human hematopoietic progenitors and acute myeloid leukemia (AML) patient blast cells. Overexpression of the PCP protein leads to increased cell survival and cell migration of leukemic cells and confers a poor prognosis to patients independently of other risk factors [START_REF] Prebet | The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome[END_REF]. Similar conclusions were recently drawn

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from studies done in chronic lymphocytic leukemia in which a set of PCP proteins is upregulated [START_REF] Kaucká | The planar cell polarity pathway drives pathogenesis of chronic lymphocytic leukemia by the regulation of B-lymphocyte migration[END_REF].

Although functions of canonical and non-canonical Wnt pathways have been extensively studied in hematopoiesis (for review see (31)(32)(33)), the role of PTK7 has never been explored in this process. In the present study, we have thus addressed the function of PTK7 in physiological hematopoiesis using a mouse model deficient for ptk7 expression. Using flow cytometry, we first mapped expression of PTK7 on HSCs and progenitors. As PTK7 deficient mice die perinatally, we then studied function of PTK7 in fetal hematopoiesis and revealed a decreased number of HSPCs in FL of Ptk7 deficient mice. Although HSC differentiation was not affected, we found a dramatic decrease in proliferation and migration properties of Ptk7 deficient HSPCs that reveal novel unexpected functions for this planar cell polarity molecule.

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Methods

Mice

C57BL/6 and C57BL/6-CD45.1 were purchased from Janvier Laboratory and Charles River Laboratories, respectively. All mice used for transplantation experiments are 6 to 12 weeks old. Lethal irradiation was done with an X-ray irradiator (X-RAD® 160 X-Ray, PXi) using a single dose of 8Gy.

PTK7 gene-trap mice were obtained by injection of gene-trap ES XST087 (Bays Genomics, USA) in E3.5 blastocyst from C57BL/6 mouse. Chimeric mice were back-crossed with B6 mice for seven generations to obtain homogenous genetic background for experiments. All mice were housed and maintained under specific pathogen-free condition. E14.5 and 15.5

were used in all FL experiments. All animal experiments were performed in agreement with the French Guidelines for Animal Handling.

Genotyping

Genotyping of adult mice and embryos was performed by genomic PCR using the following pairs of primers: 5'-ACGTCACTGGAGAGGAAGTCCGCAG-3' and 5'

GATCGAAGCAGGTCCTGTGGTCCT-3' for wild type allele and 5'-TTGTGAAGTGGAGGCTCCGGACC-3' and 5'-CGATCTTCCTGAGGCCGATACTGTC-3' for gene trap insertion allele.

Flow cytometry and cell sorting

For the analysis of hematopoietic stem/progenitor cells in FLs, FLs were dissected from E14.5 embryos and single cell suspension was made in phosphate-buffered saline (PBS)-2% Fetal Calf Serum (FCS).

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For analysis of hematopoietic stem/progenitor cells in BM, thymus or blood cells obtained from adult mice were suspended in PBS-2% FCS. For BM and thymus, cells were filtered using 70 µm cell strainer (BDFalcon) and lysis of red blood cells was done using ACK lysis buffer (0.15 M NH4Cl, 10mM KHCO3, 0.1mM Na2EDTA, Life Technology) for 2 min at room temperature. Cells were spun and resuspended in PBS-2%FCS +/-0.125mM EDTA for staining. For blood samples, red blood cell lysis was done after staining using BD FACS Lysing buffer solution (BD Biosciences) following manufacturer instructions.

For flow cytometry analysis and cell sorting, cells were stained with monoclonal antibodies purchased from Ebiosciences or Biolegend against the following molecules: B220 (clone All experiments were done by multicolour parametrics analysis using FITC, PE, APC, PE-Cy7, APC-Cy7, PerCPCy5.

Transplantation assays

For LSK sorting, FL cells (CD45.2+) were labelled for lineage positive (lin+) cells with following biotinylated primary rat antibodies (lineage mix): CD3, CD4, CD8, CD19, B220, DX5, TER-119, GR-1, CD11c. Lin+ cells were depleted by magnetic separation using antirat Ig coated magnetic beads (DynaBeads, Lifetechnology). Lin Neg cells were also stained and sorted by FACS according to the phenotype Lin Neg , CD45.2 + , Kit + and Sca-1 + . CD45.1 mice were lethally irradiated and grafted with 1000 or 400 CD45.2 + LSK cells through the retro-orbital vein along with 1.10 5 total BM recipient cells to allow short term reconstitution and mice survival. Recipient mice survival was followed and BM engraftment was examined by monitoring donor (CD45.2 + cells) and host cell (CD45.1 + cells) frequency in

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the blood. After lethal irradiation, mice were maintained on water containing neomycin sulfate 1g/500mL for 2 weeks.

Serial transplantation assay

Lethally irradiated (8 Gy) mice (C57Bl/6, CD45.1) received 3.10 6 total fetal liver cells (first transplantation) or BM total cells (next transplantations). BM engraftment was monitored as described above.

Competition assay

Lethally irradiated (8 Gy) mice (C57Bl/6, CD45.

Cell cycle analysis and apoptosis analysis

FL progenitors were enriched as previously described and stained with cKit-APC-eFluor750, CD150-APC, Sca-1-PerCP-Cy5.5, and CD48-PECy7. The biotin-conjugated lineage combination was revealed with AlexaFluor 594-streptavidin (Invitrogen). For cell cycle analysis, after extracellular staining, cells were permeabilized, fixed, and stained with anti-Ki67-FITC MoAb (BD Biosciences) and DAPI as previously described [START_REF] Wilson | Dormant and self-renewing hematopoietic stem cells and their niches[END_REF]. For apoptosis analysis, cells were stained with PE-annexine V to evaluate annexine V externalization (BD biosciences) and DAPI.

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Adhesion assays

MS5 stromal cells were plated on 96-well flat bottom plates, at a concentration of 5.10 3 cells/well 1 day before the assay. MS5 is a stromal cell line that has been generated by irradiating adherent cells in long-term BM culture. Lin Neg FL cells (5.10 5 ) obtained as previously described were cocultured for 2 hours with MS5 stromal cells. Plates were washed 3 times with PBS and adherent cells were removed by strong pipetting in PBS 1.25mM EDTA 2% FCS. The phenotype of adherent cells was determined using anti-CD117, Sca-1, CD150, CD48, and FITC-lineage mix to exclude any residual mature cells contaminating the Lin Neg preparation. Absolute numbers were measured using the HTS coupled to the BD-LSR2 SORP (BD Bioscience).

Migration assays

Lin Neg FL cells were enriched as previously described and suspended in Iscove modified Dulbecco medium (IMDM) containing 2% of bovine serum albumine. About 2.10 5 cells in

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chamber was filled with Serum free Media supplemented with the chemoattracting murine stromal-derived factor-1 (mSDF-1α, 100 ng/mL, Peprotech). The same number of cells was plated in distinct wells to be used as control for migration. Plates were then incubated 4 hours at 37°C, 5% CO 2 . All experiments were performed in duplicates. Cells were also stained using anti-CD117, Sca-1 and streptavidin coupled to Pacific Blue as described above. Absolute numbers were measured using the HTS coupled to the BD-LSR2 SORP (BD Bioscience) and the migration ratio was defined as the number of migrating cells between migration wells and control wells.

TF1 cell differentiation

Growth of the human erythroid/macrophage progenitor cell line TF1 is dependent on GM-CSF or IL3 and can be induced to differentiate in the presence of growth factors. TF1 cells were grown in IMDM medium supplemented with 5 ng/ml GM-CSF and supplemented with EPO (2 UI/ml) for erythroid differentiation. TF1 cells were infected by viral particles containing shRNA against PTK7 as previously described [START_REF] Prebet | The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome[END_REF]. A scramble non-targeting shRNA was used as a control. shRNA efficiency for PTK7 downregulation was controlled by flow cytometry before and at the end of experiments. TFI cells were then differentiated in IMDM with 2 IU/ml EPO and without GM-CSF for a total of seven days as previously described [START_REF] Wang | Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation[END_REF]. Erythroblastic differentiation was evaluated by glycophorin A expression.

Statistical analysis

Statistical significance was determined with nonparametric Mann-Withney U test using Prism Version 5 software (Graphpad Software Inc).

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Results

PTK7 is expressed on HSCs, myeloid progenitors, lymphoid and thymic progenitors

In a first approach, we studied PTK7 pattern of expression in adult murine hematopoietic cells. To this end, a rabbit polyclonal antibody generated against the extracellular region of PTK7 was used. This antibody recognizes mouse PTK7 endogenously expressed on endothelial and hematopoietic cells by FACS analysis in wild type but not ptk7 deficient mice (Supplementary Fig. S1). Analysis of PTK7 expression in peripheral blood and in splenocytes revealed that none of the mature mouse leukocytes subsets (granulocytes, monocytes, B cells and T cells) expressed PTK7 to detectable level (Fig. 1A and data not shown). We next analysed PTK7 expression in murine BM hematopoietic progenitors using multiparametric flow cytometry and gating strategies described in Figure 1B. When LSK subsets were examined, PTK7 expression was found to be inversely correlated to differentiation stages with the highest expression on HSCs and multipotent progenitors (MPP) type 1 HSC-MPP1 (RFI=40), and intermediate expression on MPP2 (RFI=20) and MPP3-MPP4 (RFI=15) (Fig. 1C). More committed progenitors including common myeloid progenitors (CMP) and common lymphoid progenitors (CLP) expressed PTK7 at lower levels while the protein was almost undetectable on the surface of granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) (Fig. 1D). A similar phenotypic analysis was conducted on differentiating T cells isolated from the thymus. T cell progenitors do not express CD4 and CD8 markers and are called double negative (DN) cells before differentiation into double positive (DP) and single positive (SP) cells. Within the DN compartment, four thymic progenitor populations can be identified according to CD44 and CD25 expression from the most immature DN1 to the most mature DN4 subsets.

PTK7 is expressed by the CD44+CD25-(DN1) population and expression is conserved

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13 during thymic differentiation within the DN2, DN3 and DN4 subpopulations (Supplementary Fig. S2). PTK7 expression is also detected on DP thymocytes before becoming progressively lost at the SP stage. Altogether, these phenotypic analyses reveal that PTK7 expression is inversely correlated to hematopoietic differentiation with the highest expression found in HSCs. This suggests that PTK7 may play a role at this early stage of hematopoietic differentiation.

Ptk7 -/-embryos have a quantitative defect of hematopoietic stem cells

We next questioned the function of PTK7 in hematopoiesis. To this end, we generated a Ptk7 deficient mouse strain using a gene-trapped ES cell available through the International Gene Trap Consortium. As expected from the previous description of Ptk7 deficient strains, we observed a perinatal lethality due to profound developmental defects [START_REF] Lu | PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates[END_REF]. Ptk7 -/- embryos harbour a severe phenotype with cranial neural tube closure defect, gastroschisis, kidney and forelimb abnormalities (Fig. 2A,2B). Considering this dramatic phenotype, it was impossible to study adult hematopoiesis in these mice and we thus decided to study FL hematopoiesis. In FL, as well as in the BM, HSCs are comprised within the LSK cell compartment. Using the same gating strategy as in Figure 1, we analysed the consequences of PTK7 deficiency in the different LSK cell populations (Fig. 2C). We observed a 25% decrease of total cellularity at E14.5 in PTK7 deficient FL compared to wild type FL (Fig. 2D). However proportion of leucocytes (e.g. CD45+ cells) is equivalent between deficient and wild type FL (Fig. 2E) suggesting that the decrease in FL cellularity in Ptk7 -/-embryos is likely due to a global effect of ptk7 deficiency on FL size. We next examined the hematopoietic progenitor compartment. A decrease of about 50% of LSK cell frequency was observed in Ptk7 deficient FL compared to control FL (1.33% Ptk7 +/+ and +/-LSK cells versus 0.67% Ptk7 -/-LSK cells) (Fig. 2F). In the different LSK cell populations, the

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most affected one was the HSC-MPP1 subset (Fig. 2C and2G). No significant difference in the frequency of MMP2 and MPP3-MPP4 subsets was observed in Ptk7 deficient FL compared to wild type or heterozygous FL. When we examined the absolute number of the different subsets of hematopoietic progenitors, we noticed that all LSK subsets were affected by PTK7 deficiency. Indeed, there was a 68% decrease in the LSK cell absolute number in Ptk7 deficient embryos compared to control embryos (Fig. 2H). The most dramatic decrease was observed in the HSC compartment. Indeed, there was up to 75%

decrease in HSC number in Ptk7 -/-embryos compared to control one whereas there was around a 50% decrease in MPP2 and MPP3/4 cell numbers (Fig. 2I)

To assess the frequency of functional HSC in FL, we transplanted 1000 and 400 sorted LSK cells from Ptk7 +/+ or Ptk7 -/-FL bearing the CD45.2 allele into two groups of lethally irradiated mice bearing the CD45.1 allele. As expected, all mice included in the group injected with 1000 LSK cells survived. However, with 400 injected cells, Ptk7 +/+ FL LSK cells were able to reconstitute recipient mice between 2 and 12 weeks after injection whereas all mice except one injected with Ptk7 -/-LSK cells died within 6 weeks (Fig. 2J).

We concluded that loss of PTK7 leads to a defect in functional HSC frequency. In vitro clonogenicity assays confirmed the defect in HSCs and early precursor frequency as we observed a twofold reduction in colony-forming units starting from 3.10 4 total FL cells.

(Fig. 2K).

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PTK7 deficient fetal liver cells are more quiescent than wild type fetal liver cells

To determine if the absence of PTK7 could alter HSC quiescence, we performed cell cycle phenotypic analysis of LSK FL cells using an antibody directed against Ki-67, a nuclear marker to measure cell cycle, and DAPI to evaluate the DNA content (Fig. 3A). We found that a greater proportion of HSCs and early progenitors were in G0 phase in Ptk7 deficient embryos as compared to control. Indeed, 56% of HSC-MPP1 cells were quiescent in Ptk7 deficient embryos with respect to 46% in control embryos (Fig. 3B). This significant increase in quiescent cells was maintained throughout hematopoietic differentiation with respectively 46% versus 39% and 19% versus 14% of the cells in G0 phase at the MMP2 and MPP3-MPP4 stages (Fig. 3B). To exclude that these unbalanced proportion of quiescent cells was not due to selective apoptosis of cycling Ptk7 deficient cells, we analyzed the percentage of apoptotic cells in Ptk7 deficient and control FL cells. We did not observed a difference of apoptosis as measured by Annexin V staining between wild type and mutant mice in total FL cells and LSK cells (Fig. 3C). These results demonstrate that PTK7 delivers proliferative signals to HSCs but does not affect HSC survival.

PTK7 deficient cells are able to reconstitute definitive hematopoiesis in recipient mice

To investigate the functional capacity of Ptk7 deficient HSCs, we generated chimeric mice using FL cell transplantation. CD45.1 C57Bl/6 mice were lethally irradiated (8 Gy) and then reconstituted with 3.10 6 total FL cells from Ptk7 deficient or wild type FL embryos (CD45.2). Reconstitution and chimerism were followed over time by monitoring the frequency of donor cells (CD45.2) versus recipient cells (CD45.1) in the peripheral blood of recipient mice (Fig. 4A). As expected, all recipient mice injected with control FL cells were reconstituted between 2 and 12 weeks. The same results were obtained with PTK7 deficient FL cells demonstrating that in absence of any competition, Ptk7 deficient HSCs

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are able to reconstitute all mature hematopoietic lineages of lethally irradiated recipients (Fig. 4B-C). To investigate whether Ptk7 deficiency could lead to more subtle hematopoietic defects, BM of chimeric mice was analyzed after twelve weeks. No significant difference was found in frequencies of mature and progenitor cells in the BM (Fig. 4E-F). In agreement with reconstitution experiments, in vitro differentiation assays showed that Ptk7 deficient FL cells are able to give rise to all subtypes of colony forming units in the same proportion than control FL cells (Fig. 4G, left panel and supplementary Fig. S4). However, we observed fewer colonies generated by Ptk7 deficient HSPC than by control cells (Fig. 4G, right panel). This is likely due to decreased cell cycle activity of Ptk7 deficient HSPCs while the potential of differentiation is not affected. Since quiescence of HSCs could be correlated to loss of self-renewal (36), we then tested self-renewal capacities of Ptk7 deficient HSCs by serial transplantation assays. Recipient CD45.1 mice were lethally irradiated and reconstituted with CD45.2 donor cells from wild type and mutant mice. The first injection was performed with 3.10 6 total FL cells. After 12 weeks, BM cells were harvested and 3.10 6 cells were then injected to a second lethally irradiated recipient.

The same protocol was applied for the following transplantations. We monitored chimerism by flow cytometry analysis and obtained a very good reconstitution with about 95 to 100% of donor cells in BM for all transplantations (Fig. 4H). We also investigated erythroblastic reconstitution in mice and did not find any differences in term of hemoglobin level and red blood cell count (Supplementary Fig. S3A andS3B). In vitro differentiation using TF1 cells that are able to differentiate in response to EPO did not reveal any difference in the presence or absence of PTK7 (Supplementary Fig. S3C andS3D). We thus concluded that PTK7 is not required for hematopoietic differentiation.

To get further insights into PTK7 function in early hematopoiesis, we performed mixed chimera competition experiments. CD45.1 C57Bl/6 mice were lethally irradiated (8 Gy)

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and then reconstituted with 2.10 6 total FL cells from Ptk7 deficient (CD45.2) and 2.10 6 total FL cells from wild type FL embryos (CD45.2xCD45.1). Reconstitution and chimerism were followed over time by monitoring the frequency of PTK7 -/-cells (CD45.2) and PTK7 +/+ (CD45.2 + CD45.1 + ) versus recipient cells (CD45.1) in the peripheral blood of recipient mice. We did not observe significant differences at D30 and D60, but observed an advantage of PTK7 -/-cells at long term (>120 jours) (Fig. 5A). Since maintenance of quiescence is a stem cell property, we tested cell cycle activity of adult HSC in chimeric mice. Recipient CD45.1 mice were lethally irradiated and reconstituted with CD45.2 donor cells from wild type and mutant FL mice as previously described. We performed cell cycle analysis on hematopoietic progenitors more than 6 months post transplantation, when stable hematopoietic reconstitution is obtained. We confirmed that hematopoietic progenitors isolated from mice reconstituted with PTK7 deficient cells are more quiescent than wild type counterparts (Fig. 5B and5C). Clonogenic experiments confirmed that fewer colonies are generated by Ptk7 deficient HSPC than by control from chimeric mice (Fig. 5D). These results suggest that PTK7 is essential for quiescence independently of the microenvironment.

Homing and migration defects of Ptk7 deficient cells

Since we have previously described a pro-migratory function of PTK7 in leukemic cell lines and because increased HSC quiescence may not be the unique cause of the functional defect of Ptk7 -/-HSCs (Fig. 2J), we decided to investigate the role of PTK7 in hematopoietic homing. FL cells from several Ptk7 -/-or Ptk7 +/+ embryos were pooled and stained separately with two different fluorescent trackers. Cells were injected according to a 1/1 ratio and mice were sacrificed 12h after injection. Analysis of homing was performed by flow cytometry (Fig. 6A-C). Although we observed comparable levels of residual

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18 circulating cells regardless the PTK7 status in peripheral blood, Ptk7 deficient cells had a very poor capacity to home to the BM, spleen, liver, and thymus as compared to control (Fig. 6D). Since homing defect can be explained by loss of cell adhesion to BM stromal cells and/or by a defect of cell migration toward a chemoattractant, we tested these two hypotheses. Adhesion of Ptk7 deficient LSK cells on the murine MS5 stromal cell line was not significantly different from control LSK cells (Fig. 7A). In contrast, we found a decrease in spontaneous transmigration of Ptk7 deficient FL cells as compared to control cells in Boyden chamber assay (8% versus 5%, Fig. 7B). No effect was observed with heterozygous FL cells. We also observed significant differences when the chemokine SDF1α was used as a chemoattractive molecule. Indeed, whereas 12% of control FL cells migrated toward SDF1α, only 8% of PTK7 deficient FL cells were able to do so (Fig. 7C).

Without chemoattractant, less than 5% of LSK cells migrated across the transwell regardless the PTK7 status (Fig. 7D). When SDF1α was used as a chemoattrative molecule, a twofold decrease in LSK transmigration was observed for Ptk7 deficient cells as compared to control cells (20% versus 40%, Fig. 7E).

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Discussion

Discovery of novel molecular pathways implicated in HSC maintenance is of particular interest to develop new strategies aiming at the regeneration of damaged hematopoietic tissues and to understand hematopoietic diseases such as leukemia. We have previously identified the cell polarity protein, PTK7, as a novel tyrosine kinase receptor overexpressed in acute myeloid leukemia (AML). High expression of PTK7 is correlated to poor prognosis and resistance to chemotherapeutic regimens [START_REF] Prebet | The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid leukemia and impairs clinical outcome[END_REF]. Recently, another study revealed that PTK7 is also upregulated in T acute lymphocytic leukemia (T-ALL) [START_REF] Jiang | PTK7: a new biomarker for immunophenotypic characterization of maturing T cells and T cell acute lymphoblastic leukemia[END_REF]. In humans, we and others have evidenced that PTK7 expression is mostly restricted to normal early progenitors committed to myeloid and T lymphoid lineages.

In the present study, we show that PTK7 expression is similar between mouse and human systems. PTK7 is absent in mature blood cells and its expression in BM hematopoietic progenitors is inversely correlated with differentiation. The highest expression of PTK7 is found in HSC-MPP1 compartment. PTK7 expression is also conserved between mice and human when the thymic compartment is considered. PTK7 is easily detectable at the cell surface of CLP and immature DN and DP populations whereas its expression is lost in single positive CD4 or CD8 lymphocytes. These data correlate with results from other groups that demonstrated expression of PTK7 by recent thymic emigrants cells and thymocytes (38) [START_REF] Jiang | PTK7: a new biomarker for immunophenotypic characterization of maturing T cells and T cell acute lymphoblastic leukemia[END_REF].

In order to study the role of PTK7 in hematopoiesis, we generated Ptk7 deficient mice using available gene-trapped ES cells. Our Ptk7 deficient strain exhibited expected planar cell polarity defects as compared to another strain of mice issued from the same ES clone [START_REF] Lu | PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates[END_REF].

Since these mutant mice cannot reach adulthood, functions of PTK7 were investigated in FL hematopoietic cells. The major hematopoietic phenotype of Ptk7 deficient mice consists in increased quiescence of HSPCs and shrinkage of the FL HSPC pool. Although these

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20 phenotypes may sound logical if one considers that less proliferation produces less cells, they are difficult to reconciliate with loss of functional reconstitution observed when recipient mice are engrafted with limited numbers of LSK cells (Fig. 2J). Indeed, PTK7 is implicated in canonical and non-canonical Wnt pathway (17) [START_REF] Bin-Nun | PTK7 modulates Wnt signaling activity via LRP6[END_REF], which are thought to be the guardians of the balance between HSC self-renewal and HSC differentiation [START_REF] Malhotra | Wnt-related molecules and signaling pathway equilibrium in hematopoiesis[END_REF]. We have recently shown that PTK7 plays a role in canonical pathway downstream of Wnt3a

suggesting that deficiency in PTK7 may mirror deficiency in Wnt3a ( 25). This would be consistent with results showing that Wnt3a deficiency leads to a reduction in the numbers of HSPCs in the FL but would not fit with the reduced reconstitution capacity observed in secondary transplantation assays of Wnt3a deficient cells which is not observed with Ptk7 deficient cells [START_REF] Luis | Wnt3a nonredundantly controls hematopoietic stem cell function and its deficiency results in complete absence of canonical Wnt signaling[END_REF]. Alternatively, it may well be that PTK7 controls non-canonical pathway through interaction with Wnt ligands (41) [START_REF] Martinez | The PTK7 and ROR2 Protein Receptors Interact in the Vertebrate WNT/Planar Cell Polarity (PCP) Pathway[END_REF]. In this way, Wnt5a antagonizes Wnt3amediated canonical signaling and increases quiescence of HSCs [START_REF] Nemeth | Wnt5a inhibits canonical Wnt signaling in hematopoietic stem cells and enhances repopulation[END_REF]. This occurs through activation of the small Rho GTPase Cdc42 that increases HSC polarity (44) [START_REF] Schlessinger | Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity[END_REF]. Such a function of PTK7 in Wnt5a-mediated non-canonical pathway would fit with the planar cell polarity function of PTK7 and with altered homing and migrating properties of Ptk7 deficient cells that likely depend on small Rho GTPase activation. Some studies show that PTK7 regulates the balance between Wnt canonical and non-canonical pathways by interacting with Dishevelled (26) [START_REF] Wehner | RACK1 is a novel interaction partner of PTK7 that is required for neural tube closure[END_REF], ROR2 (42)(47), β-catenin (25) and LRP6 [START_REF] Bin-Nun | PTK7 modulates Wnt signaling activity via LRP6[END_REF]. We unsuccessfully tried to identify downstream pathways that could be regulated by Ptk7 in HSCPs using a candidate gene/protein approach. We also looked at gene expression differences between Ptk7 knock-out and control hematopoietic cells using unbiased gene expression profiling. Although we identified differences (data not shown), we could not find obvious Gene Ontology enrichments or pathways that could be experimentally tested.

Further studies will be needed to unravel how Ptk7 functions in HSPCs.

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21

In our model of FL hematopoietic cells, we confirmed the role of PTK7 in cell migration and found a new role of this protein in cell homing to hematopoietic organs and in the control of cell proliferation. This observation confirms our previous data obtained from human leukemia samples ( 27) can be linked with already described functions of PTK7 in cell migration and in metastatic dissemination. Indeed, we and others have found that PTK7 expression on various cancer cells is linked with higher metastasis potential (27)(29)(48) [START_REF] Jin | High expression of protein tyrosine kinase 7 significantly associates with invasiveness and poor prognosis in intrahepatic cholangiocarcinoma[END_REF]. Recently, we also described a poor prognosis of PTK7 expression in colon cancer cells linked with higher metastatic events [START_REF] Lhoumeau | Overexpression of the Promigratory and Prometastatic PTK7 Receptor Is Associated with an Adverse Clinical Outcome in Colorectal Cancer[END_REF]. In addition, our data show for the first time that PTK7 is involved in the regulation of hematopoietic and stem and progenitors cell (HSPC) cycle. Lack of PTK7 expression increases quiescence and

enhances the long term repopulating potential after six months, while a trend toward decreased chimerism is observed after one or two months. This could reflect the dual function of PTK7 loss of expression in decreasing short term homing to the bone marrow and increasing long term establishment of hematopoiesis. Alternatively, it may well be that homing assay performed with total liver cells reveals a constitutive promigratory function of PTK7, in contrast to long term engraftment reflecting bone marrow micro-environmental signaling through PTK7 in HSCs. Similar complex mechanisms of cell cycle regulation and differential homing of mature cells versus HSPCs homing to the bone marrow have also been attributed to CXCR4 (51) (52) (53) [START_REF] Nie | CXCR4 is required for the quiescence of primitive hematopoietic cells[END_REF]. Whether PTK7 and CXCR4 signaling share more in common remains to be addressed. In conclusion, PTK7 overexpression in immature blasts cells of acute myeloid leukemia has been associated to bad prognosis, early relapse and chemotherapeutic resistance and a recent study shows that PTK7 can be used as a stem cell marker of colon cancer stem cells (27) [START_REF] Jung | Isolation of Human Colon Stem Cells Using Surface Expression of PTK7[END_REF]. Taken together, all these data form a set of arguments to involve PTK7 expression in stem cells including cancer stem cells but also to prove a functional role of PTK7 in stem cell biology. We think that these new data

F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

on PTK7 involvement on cell cycle are very promising to understand molecular mechanisms of leukemia/cancer development.

Altogether, our results report a novel unexpected function for the planar cell polarity molecule PTK7 in the crosstalk between expansion of HSPC compartment and HSPC migration. This opens new avenue for manipulation of HSC fate using agonists or antagonists of PTK7. A.

F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e . F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e . F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e . F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e . F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e . F o r P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

  6B2), CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), CD11b/Mac-1 (M1/70), CD11c (N418), CD16/32 (93), CD19 (6D5), CD24 (M1/69), CD34 (RAM34), CD45 (30F11), CD45.1/Ly5.1 (A20), CD45.2/Ly5.2 (104), CD48 (HM48-1), CD49b (DX5), CD49d (R1-2), CD117/c-Kit (2B8 and/or ACK2), CD135 (A2F10), CD150 (TC15-12F12.2), F4/80 (BM8), Gr1 (RB6-8C5), Sca-1 (D7), Ter-119. Antibody human PTK7 and glycophorin A (11E4B-7-6) were purchased from Miltenyi biotech and Beckman Coulter, respectively.

Figure 1 :

 1 Figure 1: PTK7 expression in wild type bone marrow. (A) Expression of PTK7 in blood mature cells. Granulocytes are defined as Gr1 + CD11b + cells. Monocytes are defined as F4/80 + CD11b + CD115 + cells. Lymphocytes are gated using B220, CD19, CD3, CD4 and CD8 combination. MFI ratios are indicated in dots plots. (B) Strategy of bone marrow progenitors gating. First panel: Dot plot showing the LSK + gate within Lin -bone marrow cells. Second panel: dot plot showing early progenitors gating using SLAM markers. Hematopoietic stem cells (HSC) are defined as CD150 + CD48 -cells. More mature multipotents progenitors (MPP) are defined as CD150 + CD48 + (MPP2) and then CD150 - CD48 + (MPP3/4). Third panel: dot plot showing engaged progenitors within c-Kit + Sca-1 - Lin -cells. CMP (Common Myeloid Progenitor) is defined as CD16/32 lo CD34 + . GMP (Granulocyte-Macrophage Progenitor) is defined as CD34 + CD16/32 hi . MEP (Megakaryocyte-Erythroid Progenitor) is defined as CD16/32 -CD34 -. Fourth panel: Gating of CLP (Common Lymphoid Progenitor) defined as Lin -IL7Ra + B220 -CD24 lo CD117 lo . (C) Overlays showing expression of PTK7 during differentiation. Black line: PTK7, gray histogram: isotypic control (pre-immune rabbit antiserum). (D) Quantitative comparison of expression levels between different progenitor populations: normalization of PTK7 mean of fluorescence (MFO) with CD117 MFO on LSK subpopulations and progenitors (CD117 + Sca-1 -cells). Statistical significance was calculated by nonparametric Mann-Whitney test. All data are shown as the mean ± s.d. *p<0.05. **p<0.01.***p<0.001.

Figure 2 :F 29 litter

 229 Figure 2: Phenotype of PTK7 deficient mouse and analysis of fetal liver at E14.5. (A) PTK7 mutants embryos showing severe planar cell polarity and developmental abnormalities. (B) Western blot of murine embryonic fibroblasts (MEFs) obtained from a

Figure 3 : 5 F

 35 Figure 3: Cell cycle and apoptosis analysis of LSK FL cells. (A) Representative dot plots of DNA content (DAPI) plotted versus Ki-67 nuclear antigen staining on LSK cells. The cell-cycle phases were defined as G0 (Ki-67 -and 2n DNA), G1(Ki-67 + and 2n DNA) and S-G2-M (Ki-67 + and DNA >2n). (B) Percentage of LSK FL cells in cell-cycle phases using SLAM markers. Data are pooled from 3 separate experiments and embryos (Ptk7 -/-N=10; Ptk7 +/+ N=11) from 7 different litters. *p<0.05. **p<0.01. (C) Determination of percentage of apoptotic cells by staining with annexin V and DAPI. Apoptotic cells were defined as annexin V + DAPI -. Results are representative of 8 Ptk7 -/-and 6 WT embryos from 5

Figure 4 :

 4 Figure 4: Hematological capacity of reconstitution and differentiation. Lethally irradiated CD45.1 mice (8 Gy) were reconstituted with 3.10 6 CD45.2 total FL cells isolated from Ptk +/+ and Ptk -/-embryos. Percentage of each hematological populations from donor (CD45.2) was monitored by flow cytometry (A). Percentage of matures cells in recipient blood (B) and in spleen (C). Percentage of BM mature cells (D) and BM progenitors (E) in recipient mice. (F) Percentage of LSK cells and LSK subpopulation in recipient BM. White histograms depict mice reconstituted with Ptk -/-FL. Black histograms depict mice reconstituted with Ptk +/+ FL. N=6 mice per group. (G) In vitro differentiation capacity of FL cells. 3.10 4 FL cells were seeded in methylcellulose. Left panel: Quantification and characterization of CFU-C. Right panel: Proportion of CFU. (H) Serial transplantation. First transplantation: Lethally irradiated mice were injected with 3.10 6 of total E14.5 FL cells (T1: transplantation 1). Next transplantations were done by injection of 3.10 6 total bone marrow cells from previously reconstituted mice in irradiated C57Bl/6 CD45.1 mice (T2 to T5). Percentage of donor cells (CD45.2) versus recipient cells (CD45.1) was monitored. Each data represents chimerism of definitive hematopoiesis 3 months after irradiation. Data are the mean ± s.d. N=6 mice per group.

Figure 5 :F

 5 Figure 5: Competition experiments and cell cycle in adult mice. (A) 2.10 6 cells from Ptk7 +/+ (CD45.1xCD45.2) and 2.10 6 cells from Ptk7 -/-(CD45.2) are injected in recipient (CD45.1) mice. Chimerism is followed in blood samples. Data are representative of 4 independent experiments (N=6 mice per group). (B-D) Recipient CD45.1 mice reconstituted with CD45.2 donor cells from wild type and mutant FL mice (B)

Figure 6 :

 6 Figure 6: Homing capacities of deficient PTK7 cells. (A) 2.10 6 of total fetal liver cells from Ptk7 -/-or Ptk7 +/+ embryos were stained with cell tracker calcein AM and calcein redorange respectively, pooled in 1/1 ratio and injected in recipient mice. (B-D) 12h after injection, mice were sacrificed and biological samples were removed and analyzed to evaluate homing capacities of each cells . (B) Bone marrow of uninjected mice was used as control for staining specificity. (C) Dot plots of stained cells removed from blood, BM, spleen, liver and thymus. (D) Quantification and statistical analysis. Data are pooled from 3 independent experiments or representative (for spleen panel). N=6 mice per group. All statistical significance was calculated by nonparametric Mann-Wihtney test. *p<0.05. **p<0.01.***p<0.001.

Figure 7 :FF

 7 Figure 7: Adhesion and migration assays (A). Adhesion assay on MS5 stromal cells. Percentage of adherent FL cells on MS5 stromal bone marrow cell line after 2 hours of incubation. Left panel: total FL cells. Right panel: LSK cells. Data are representative of 3 independent experiments. Ptk -/-N=4; Ptk +/-N=6; Ptk +/+ N=3. (B to E) Migration in Boyden chamber assay. Number of migrating cells are normalized to the number of input cells for each sample (duplicate wells). (B) Migration of lin Neg enriched FL cells without chemoattractant. (C) Migration of lin Neg enrichied FL cells with SDF1a as chemoattracting

  Figure1

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Figure 7

 7 Figure6

P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

	were used as secondary antibodies. To exclude lineage positive cells, streptavidin 405, 488, 561, and 633; BD Bioscience) or sorted by ARIA III (BD Bioscience). LSK cell populations were purified by cell sorting using a FACS-ARIA with > 95% purity. Results were analyzed using BD-DIVA Version 6.1.2 software (BD Biosciences) or FlowJo Version 7.6.2 (TreeStar) software. Homing experiments E14.5 embryos were sacrificed and were immediately genotyped. FL cells from wild type or gene trap mice were suspended, counted and stained with cell tracker calcein AM and calcein red-orange (Invitrogen), respectively, according manufacturer instructions. Cells were mixed at 1/1 ratio and 4.10 6 total cells were injected through retro-orbital vein in previously irradiated (8 Gy) mice. 12h after injection, mice were sacrificed and blood, bone F o r added in last staining in PBS solution. Stained cells were analysed by LSR II SORP (laser marrow, spleen, liver and thymus samples were analysed for fluorescents cells.
	conjugated with Alexa A594 (Invitrogen) or Pacific blue (Ebiosciences) were used.
	Irrelevant isotype-matched antibodies were used as controls. Cells were stained 20 minutes
	at 4°C and rinsed in PBS-2% FCS solution. Viability marker (vivid dye, Invitrogen) was

5, AlexaFluor700, AlexaFluor594, Pacific blue, PE-Cy5, Q-dot 605 fluorochroms or spectral equivalents. Commercial antibodies were used either directly conjugated or biotinylated. For mouse PTK7 expression, polyclonal rabbit antibody raised against recombinant PTK7-Fc was used in the experiments. For detection of interesting antigen, PE or APC-conjugated goat-anti-rabbit (Jackson Immunoresearch Laboratories)

Acknowledgments

We thank Patrick Gibier, Patrick Garzino and Annaëlle Legrand from the animal facility of CRCM.

Disclosure of potential conflicts of interest

All authors declare no conflict of interest.