Marielle Balzano

Maria De Grandis

Thien-Phong Vu Manh

Lionel Chasson

Florence Bardin

Anne Farina

Arnauld Sergé

Ghislain Bidaut

Pierre Charbord

Léonard Hérault

Anne-Laure Bailly

Amandine Cartier-Michaud

Annie Boned

Marc Dalod

Estelle Duprez

Paul Genever

Mark Coles

Marc Bajenoff

Luc Xerri

Michel Aurrand-Lions
email: michel.aurrand-lions@inserm.fr

Claudine Schiff

Stéphane J C Mancini
email: stephane.mancini@inserm.fr

Nidogen-1 Contributes to the Interaction Network Involved in Pro-B Cell Retention in the Peri-sinusoidal Hematopoietic Stem Cell Niche

Keywords: B cell development, hematopoietic stem cell, stromal cell niche, interaction network, bone marrow

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The influence of bone marrow (BM) mesenchymal stromal cells on the differentiation program of mouse hematopoietic cells has become increasingly clear over the last few years. A number of stromal cell niches have been described, not only for hematopoietic stem cells (HSC), but also for B cell progenitors [START_REF] Aurrand-Lions | Murine Bone Marrow Niches from Hematopoietic Stem Cells to B Cells[END_REF]. Stromal cells supporting HSC maintenance have been separated into perivascular and endosteal/peri-arteriolar niches. Indeed, recent results indicate that HSC maintenance relies on peri-sinusoidal stromal (PSS) cells which secrete CXCL12 and stem cell factor (SCF) [START_REF] Ding | Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches[END_REF][START_REF] Ding | Endothelial and perivascular cells maintain haematopoietic stem cells[END_REF][START_REF] Mendez-Ferrer | Mesenchymal and haematopoietic stem cells form a unique bone marrow niche[END_REF][START_REF] Sugiyama | Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches[END_REF], while arteriole-associated pericytes expressing Nestin have been shown to control HSC quiescence [START_REF] Kunisaki | Arteriolar niches maintain haematopoietic stem cell quiescence[END_REF]. Furthermore, arteriolar cells close to the bone surface as well as sinusoidal BM endothelial cells (sBMEC) also contribute to HSC maintenance and retention in the BM [START_REF] Ding | Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches[END_REF][START_REF] Ding | Endothelial and perivascular cells maintain haematopoietic stem cells[END_REF][START_REF] Itkin | Distinct bone marrow blood vessels differentially regulate haematopoiesis[END_REF]. After commitment to the B cell lineage, progenitor development depends on CXCL12 and interleukin-7 (IL7). Mice deficient for CXCL12 or CXCR4 present B cell differentiation and retention defects that start at the earliest prepro-B cell stages [START_REF] Egawa | The earliest stages of B cell development require a chemokine stromal cellderived factor/pre-B cell growth-stimulating factor[END_REF][START_REF] Ma | Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4-and SDF-1-deficient mice[END_REF][START_REF] Ma | The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment[END_REF]. IL7 controls the commitment of lymphoid progenitors to the B cell lineage and is required for pro-B and pre-B cell proliferation [START_REF] Dias | Interleukin-7 is necessary to maintain the B cell potential in common lymphoid progenitors[END_REF][START_REF] Marshall | Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression[END_REF].

In support of these results, pre-pro-B cells have been localized near BM stromal cells expressing CXCL12, before re-localization in the vicinity of IL7-expressing stromal cells at the pro-B cell stage [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF]. Most of the stromal cells expressing CXCL12 or IL7 were localized in perisinusoidal areas of the BM [START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF][START_REF] Sugiyama | Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches[END_REF]. However, other studies have suggested that osteoblasts (OBs) play a central role in early B cell development [START_REF] Wu | Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}dependent signaling pathways[END_REF][START_REF] Zhu | Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells[END_REF]. Finally, IL7 depletion from CD31 + BM endothelial cells (BMEC) causes a drop in B cell numbers [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF], suggesting that endothelial sources of IL7 are required for B cell differentiation. More recently, it has been demonstrated that the large majority of pro-B and pre-B cells are located in the same niche, but are respectively static and motile [START_REF] Fistonich | Cell circuits between B cell progenitors and IL-7(+) mesenchymal progenitor cells control B cell development[END_REF]. This has been attributed to cross-circuitry between IL7R and pre-BCR signaling which positively and negatively control α4β1 integrin mediated adhesion, suggesting that high level of IL7R signaling in sessile pro-B cells is due to increased exposure to IL7 while pre-BCR signaling turn the motility on.

The heterogeneity of BM stromal cell niches remains unclear because of the use of multiple reporter mice strains and experimental set-ups. The study of CXCL12-GFP and SCF-GFP knock-in mice revealed that PSS cells are the main source of CXCL12 and SCF [START_REF] Ding | Endothelial and perivascular cells maintain haematopoietic stem cells[END_REF][START_REF] Omatsu | The essential functions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche[END_REF][START_REF] Sugiyama | Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches[END_REF]. Co-expression of these factors by LepR + PSS cells has been confirmed using CXCL12-dsRed/SCF-GFP double knock-in mice [START_REF] Ding | Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches[END_REF]. With regard to early B cell niches, it was initially postulated that distinct stromal cell subpopulations express CXCL12 and IL7 [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF]. However, we identified a BM stromal cell subpopulation co-expressing these two factors [START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF]. This population was phenotypically defined as CD45 -CD54 + CD31 - BP1 + stromal cells and morphologically similar to LepR + cells which co-express CXCL12 and SCF [START_REF] Ding | Endothelial and perivascular cells maintain haematopoietic stem cells[END_REF], suggesting that LepR + cell may co-express IL7, SCF and CXCL12. In accordance with these findings, a recent study showed that some LepR + stromal cells co-express IL7, SCF and CXCL12 [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF]. Importantly, specific deletion of CXCL12 using LepR-Cre/Cxcl12 lox/lox mice has a more profound effect on HSC maintenance than deletion using IL7-Cre/Cxcl12 lox/lox mice, suggesting that several PSS subsets specialized in HSC maintenance or IL7 secretion may exist.

In the current study, we phenotypically characterized BM stromal cell niches which were previously identified based on reporter gene expression. This analysis revealed a limited diversity of BM mesenchymal subsets, and PSS cells were found to co-express LepR, BP1, CXCL12, SCF and IL7 at the single cell level. HSC and pro-B cells were often simultaneously associated to PSS cells and respectively formed specific interaction networks involving genes implicated in HSC maintenance and lymphoid development. Furthermore, among factors expressed by PSS cells, we identified Nidogen-1 as a specific retention signal for pro-B cells in the peri-sinusoidal niche. Finally, a global gene expression analysis revealed that murine PSS cells were identical to CD317 + human BM stromal cells, and that both HSPC and pro-B cells were located close to these cells. Taken together, our results demonstrate the existence of a homogeneous BM stromal cell population present in mouse and human that has the capacity to produce niche factors involved in the maintenance and development of distinct hematopoietic subsets.

Results

Mesenchymal cell characterization in BM reveals the existence of a restricted number of subsets

To define mesenchymal cell heterogeneity in the BM, we used multiparametric flow cytometry to simultaneously analyze the different phenotypic markers described separately in the literature. BM cells were separated into conduit or bone fractions. In the conduit fraction, CD31 + BMEC were mainly composed of Sca1 lo Endomucin (Emcn) lo L-type sBMEC and of low numbers of Sca1 + Emcn -arteriolar BMEC (aBMEC) as expected [START_REF] Kusumbe | Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone[END_REF]. Two CD51 + stromal subsets were identified (Figure 1A-B). CD54 lo NG2 + cells corresponded to pericytes and CD54 + NG2 -stromal cells expressed high levels of LepR, indicating that they were PSS cells. Furthermore, PSS cells expressed CD106, marker of CAR cells [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF] as well as BP1 and CD317, markers of IL7 + PSS cells [START_REF] James | Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes[END_REF][START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF] suggesting that they may correspond to a single subset. In contrast, the bone fraction was enriched for aBMEC, H-type (Sca1 + Emcn hi ) BMEC, few L-type sBMEC and CD51 + mesenchymal cells (Figure 1C). Few PSS cells were observed and CD51 + CD54 lo/-BP1 -cells mostly corresponded to OB as they did not express the immature mesenchymal cell marker CD105 but expressed high levels of Bglap, Col1a1, Col2a1 genes specific for mature OB (Figure 1D). These results suggest that BM mesenchymal subsets are essentially composed of MSC, PSS cells, pericytes and OBs.

Pro-B cells are in direct contact with peri-sinusoidal stromal cells

We next explored the localization of pro-B cells with respect to PSS cells. LepR + cells were clearly identified in the vicinity of sinusoidal structures (Figure 1E). In the BM, most Terminal deoxynucleotidyl transferase (TdT) positive cells corresponded to pro-B cells (Figure 1E and Figure S1). TdT + cells were detected close to LepR + PSS cells and mainly away from the endosteum (Figure 1F). Furthermore, localization of pro-B cells close to LepR + cells was specific since cells positioned randomly in the same images were found to be at a significantly greater distance whereas their positioning relative to the bone was not different. These results indicate that PSS cells form a cellular niche for pro-B cells.

IL7 is exclusively expressed by peri-sinusoidal stromal cells co-expressing high levels of CXCL12 and SCF Previous results showed that PSS cells are composed of IL7 + and IL7 -subsets (Cordeiro [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF]. In order to analyze these subsets molecularly and their relation to pro-B cells, we used IL7-Cre/Rosa-eYFP mice (hereafter IL7 reporter mice). YFP expression was only detected in PSS cells, pericytes and OBs (Figure 2A-B), and YFP + BMEC were neither detected in the conduit nor in the bone fraction (n=10). As shown earlier [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF][START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF], most of the YFP + cells in adult mice corresponded to PSS cells (Figure 2A and Figure S2A-B). Furthermore, most of the LepR + PSS cells were YFP + in contrast to pericytes and OBs (Figure 2C). YFP + stromal cells were next isolated from the conduit and bone fractions from IL7 reporter mice, and tested for gene expression by quantitative PCR. Il7 was expressed by PSS cells sorted from both fractions, but was not detected in pericytes or OBs, regardless of YFP expression status (Figure 2D).

Since YFP expression is irreversible in the Rosa-eYFP system once the Cre recombinase has been expressed, this result indicates that YFP expression may be related to lineage tracing of a progenitor cell expressing IL7. This is the case for OBs which are known to be generated from PSS cells in adult mice but not in young animals [START_REF] Mizoguchi | Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development[END_REF]. Indeed, in contrast to adult, none of the bone lining cells in 2-3-week-old IL7 reporter expressed YFP (Figure S2A-C).

Since YFP + PSS cells represented only 59.4% of total PSS cells (Figure 2C), their gene expression signature was compared to PSS cells contributing to the HSC niche. Gene expression profiles obtained from PSS cells isolated from WT mice as well as YFP + and YFP -PSS cells from IL7 reporter mice were compared to public datasets of PSS sorted from CXCL12-GFP + , CXCL12-dsRed + , Nestin-GFP lo and SCF-GFP + stromal cells. Microarrays from total BM were used as a reference control between datasets and gene expression profiles for BMEC, osteoblasts and MSC as unrelated datasets. Unsupervised Principal Component Analysis (PCA) showed that the stromal cell subpopulations independently sorted from CXCL12, SCF, Nestin, and IL7 reporter mice clustered together (Figure 3A), and were distant from BMEC, OB and MSC. Technical bias most probably account for the spreading of the PSS cluster along the first dimension. Indeed, SCF-GFP + cells as well as total BM samples from the same dataset were distant to a similar extent from our PSS cell and total BM samples respectively. Furthermore, spreading was also observed for PSS cells sorted from two different CXCL12 reporter mouse models using respectively GFP and dsRed [START_REF] Decker | Leptinreceptor-expressing bone marrow stromal cells are myofibroblasts in primary myelofibrosis[END_REF][START_REF] Greenbaum | CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance[END_REF]. Interestingly, PSS cells isolated from WT mice were identical to YFP -PSS cells from IL7 reporter mice and both populations clustered with the other PSS cell datasets, indicating that Cre/lox recombination is only partial in IL7 reporter mice, and that PSS cells may represent a homogeneous population. This result was confirmed by qPCR showing Il7 expression in YFP -PSS cells (Figure 2D). Unsupervised hierarchical clustering revealed that the PSS cells from the different datasets expressed both HSC and B cell niche genes (Figure 3B). Notably, Il7, Cxcl12 and Kitl (encoding SCF) were co-expressed in PSS cells irrespective of dataset origin and of the reporter system used and this result was confirmed by single-cell qPCR analysis on PSS cells isolated from WT mice and compared to pericytes (Figure 3C). Finally, single cell RNA sequencing (scRNAseq) of BM stromal cells showed that PSS cells formed a single cluster based on the significant up-and downregulation of 443 and 371 genes respectively, as compared to MSC, OB, BMEC or pericytes (Figure 3D, Figure S3 and Table S1). Altogether, these results indicate that Il7, Cxcl12 and Kitl are coexpressed by CD54 + BP1 + PSS cells, and that these cells are identical to peri-sinusoidal CAR, Nestin-GFP lo and SCF + LepR + cells.

Peri-sinusoidal stromal cells simultaneously accommodate HSC and early B cells, forming specific interaction networks with them

We next examined the relative localization of HSC and pro-B cells with respect to PSS cells. HSC were defined as Lineage negative (Lin -)CD41 -CD48 -cells expressing CD150 [START_REF] Ding | Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches[END_REF] cells and were frequently seen in contact with the same LepR + stromal cell (Figure 4A and video S1).

Furthermore, the distribution analysis for HSC compared to their closest pro-B cell neighbors in 3D revealed that 15.2% were at a distance of less than 16 µm (two-cell distance) and 50% at less than 41.7 µm (5-cell distance) (Figure 4B). Simulations of pro-B cell random positioning in the image produced a significantly greater average distance between pro-B cells and HSC. These results demonstrate that HSC and pro-B cells are in the vicinity of PSS cells and that migration of pro-B cell differentiating from HSC occurs over a relatively restricted area.

Since PSS cells have the capacity to sustain the development of both HSC and pro-B cells, we looked for receptor/ligand pairs that could explain such multispecific cognate interactions. To do so, we defined the interactome between PSS cells and long term (LT)-HSC or pro-B cells. Pre-pro-B cells have been reported to be in contact with PSS cells [START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF], and were therefore used as positive controls. As negative controls, we used the closest related hematopoietic subsets localized to distinct niches: short term (ST)-HSC or pre-B cells [START_REF] Kiel | SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells[END_REF][START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF] (Figure 4C). An interactome bioinformatics pipeline was developed to confront gene expression datasets from YFP + PSS cells from IL7 reporter mice and publicly available datasets for LT-HSC, ST-HSC, prepro-B, pro-B and pre-B cells. Genes encoding for trans-interacting ligand/receptor pairs were identified (Table S2). When comparing LT-HSC to ST-HSC, a higher number of gene products expressed by LT-HSC were found to interact with gene products expressed by PSS cells (12 gene products specifically expressed by LT-HSC had a ligand expressed by PSS cells as compared to 1 for ST-HSC; Figure 4D). Similarly, higher numbers of genes specifically expressed in pre-pro-B and pro-B cells were involved in interactions with PSS cells compared to pre-B cells. Furthermore, LT-HSC, prepro-B or pro-B cell specific genes involved in interactions with PSS cells -as identified in Figure 4D were highly relevant niche genes, as demonstrated by the greater significant enrichment for Gene Ontology (GO) terms related to adhesion, secretion, differentiation or proliferation compared to ST-HSC or pre-B cells, respectively (Figure 4E and Table S3). We next compared the expression signatures of LT-HSC, pre-pro-B and pro-B cells by focusing on genes implicated in interaction with PSS cells. As expected, Cxcr4, Kit and the Il7r/Il2rg heterodimer were identified as hematopoietic genes encoding ligands for CXCL12, SCF and IL7, respectively (Figure 4F). More than 50% of the genes identified encoded proteins with known functions in HSPC maintenance or lymphoid development and migration. Among these, we found ligand/receptor pairs that have been extensively studied in HSC regulation (Tie2/Angiopoietin-1, TGFβR/TGFβ, Integrinα6β1/Laminin) or in early B cell development (α4β1/VCAM-1). Furthermore, although some overlapping gene expression was found between LT-HSC and pre-pro-B or pre-pro-B and pro-B cells, there were few overlaps between LT-HSC and pro-B cells. Finally, we found that hematopoietic cells expressed growth factors involved in the development of mesenchymal or endothelial cells indicating that reciprocal interactions between PSS and hematopoietic cells contribute to the maintenance of hematopoietic niches.

We then tested whether genes encoding niche proteins involved in hematopoietic/PSS cell interactions were also expressed by sBMEC or ALCAM + Sca1 -and ALCAM -Sca1 -OBs [START_REF] Nakamura | Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells[END_REF]. GSEA analysis revealed a significant enrichment (FDR<0.25) for niche gene expression by PSS cells as compared to sBMEC and ALCAM + Sca1 -or ALCAM -Sca1 -OBs (Figure 4G). Moreover, unsupervised hierarchical clustering performed on HSC and B cell niche genes identified earlier confirmed that the highest levels of these genes, including Cxcl12, Kitl and Il7, were found in PSS cells (Figure S4). These results show that LT-HSC, pre-pro-B and pro-B cells express a network of genes allowing preferential interactions with PSS cells rather than endothelial or osteoblastic cells.

Nidogen-1 is implicated in pro-B cell retention in their niche

Among the genes involved in the specific interaction between PSS cells and pro-B cells, we identified the ligand/receptor pair Nid1/Plxdc1. Nidogen-1 is part of the basement membrane together with laminin and is involved in matrix to cell interactions with PLXDC1 expressed at the plasma membrane [START_REF] Lee | Identification of the basement membrane protein nidogen as a candidate ligand for tumor endothelial marker 7 in vitro and in vivo[END_REF]. Plxdc1 expression was shown to be controlled by the B cell transcription factor Pax5 [START_REF] Revilla | The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis[END_REF]. Pro-B cells indeed expressed PLXDC1 and were located close to LepR + Nid1 + sinusoidal structures (Figure 5A). Importantly, expression of CD51, another cellular ligand for Nidogen-1 [START_REF] Yi | Adhesion of cultured human kidney mesangial cells to native entactin: role of integrin receptors[END_REF], was undetectable in pro-B cells (Figure S5A), suggesting that pro-B cell interaction with Nidogen-1 depends on PLXDC1. The influence of Nidogen-1 deficiency on pro-B cell differentiation was analyzed in Nid1 -/-mice. Total BM cellularity and B cell frequencies were normal in Nid1 -/-mice (Figure 5B and Figure S5B). However, the frequency of pre-BI cells was specifically decreased, revealing impaired pro-B to pre-BI cell transition. The decrease in pre-BI cells in Nid1 -/- mice had repercussions on the proportion of the later large pre-BII cells, and thus on the most immature BP1 + pre-BII cells, even though the overall frequency of pre-BII cells was not affected (Figure 5B and Figure S5B). As a consequence, immature and recirculating B cells were also normally represented. This result indicates that the defect observed in Nid1 -/-mice is transient, and specific to the pro-B to pre-BI cell transition. In pre-BII cells, the pre-BCR induces a strong proliferation which may explain the recovery in Nid1 -/-mice. We thus analyzed de novo B cell differentiation in WT and Nid1 deficient mice 7 days after injection of the cytostatic agent hydroxyurea (HU) as described previously [START_REF] Espeli | Impaired B-cell development at the pre-BII-cell stage in galectin-1-deficient mice due to inefficient pre-BII/stromal cell interactions[END_REF]. Pre-pro-B cells were not affected but the frequency of B cells was significantly decreased from the pro-B/pre-BI cell transition to the following differentiation subsets, confirming the existence of homeostatic compensation at the pre-BII stage at steady state (Figure S5B).

To determine whether this bottleneck is due to reduced accessibility to IL7, we checked IL7 receptor signaling-mediated phosphorylation of STAT5 and early B cell proliferation. As expected, phospho-STAT5 (pSTAT5) levels and the proportion of proliferating cells were significantly higher in pro-B compared to pre-BI cells in wild-type (wt) animals (Figure 5C-D and Figure S5D-E). In Nid1 -/-mice, both pSTAT5 and pro-B cell proliferation were decreased to the levels observed in wt pre-BI cells, and this was not due to reduced IL7R expression levels (Figure S5F).

To exclude any contribution of Nidogen1 expressed by hematopoietic cells to the phenotype, wild type BM was transplanted into lethally irradiated Nid1 -/-mice (stroma KO) and B cell differentiation was compared to controls. While CLP frequency was similar between conditions, an accumulation of pre-pro-B cells was observed in stroma KO chimeras as compared to controls (Figure 5E). B cell differentiation was significantly decreased from the pro-B cell stage in the stroma KO chimeras only, confirming the importance of Nidogen-1 produced by the microenvironment for pro-B cell development.

Further analysis of Nidogen-1 immunostaining revealed that, in addition to sinusoidal expression (Figure 5A, lower panel), bright signals were associated to arterioles (Figure 6A), consistent with Nid1 expression in PSS cells and BMEC (Figure S6A). However, TdT + pro-B cells were found in close vicinity to Nid1 + LepR + PSS cells and mainly away from Nid1 hi arterioles (Figure 6A lower right panels). This localization was specific since randomly positioned cells were at a significantly greater distance from Nid1 + LepR + PSS cells but not from arterioles, suggesting that Nidogen-1 associated to PSS cells is necessary for pro-B cell localization in the peri-sinusoidal niche. This was confirmed by the finding that the distance between pro-B cells and LepR + PSS cells was significantly greater in Nid1 -/-mice as compared to control (Figure 6B, lower right panel).

In addition to pro-B cell localization with respect to LepR + cells, Nid1 deficiency affected the structure of sinusoids (Figure 6B, inserts). This was consistent with a change in H-type (Emcn hi CD31 hi ) vessel columns which were much shorter and lacked a straight columnar organization in Nid1 -/-mice (Figure S6B), as previously reported in vascular specific itgb1-and Lama5-deficient mice [START_REF] Langen | Cell-matrix signals specify bone endothelial cells during developmental osteogenesis[END_REF]. However, this modification in vascular organization had no effect on early hematopoiesis from LT-HSC to myeloid and lymphoid progenitors (Figure S5G). Of note, the LepR staining appeared more diffuse in Nid1 -/-mice. This may be attributed to a decreased capacity of PSS cells to spread on the Nidogen-1-depleted extra-cellular matrix (ECM) surrounding sinusoids instead of PSS cell defects. Indeed, the relative proportions of LepR + PSS cells and pericytes were similar between Nid1 -/-and WT mice, and PSS cells expressed normal LepR mRNA and protein levels (Figure S6C-D). In addition, Nid1 deficiency did not affect expression of HSC or B cell niche genes (Kitl, Cxcl12, Alpl, Angpt1, Il7, Vcam1), of Foxc1, master regulator of the HSC niche [START_REF] Omatsu | Foxc1 is a critical regulator of haematopoietic stem/progenitor cell niche formation[END_REF], or of ECM genes (Col1a1 and Lama4; Figure S6D). Finally, CXCR4 and CD49d (α4-integrin, VCAM1 ligand), shown to be crucial for B cell localization in the niche [START_REF] Fistonich | Cell circuits between B cell progenitors and IL-7(+) mesenchymal progenitor cells control B cell development[END_REF], were also normally expressed by pro-B cells in Nid1 -/-mice. Altogether these results suggest that Nidogen-1 regulates pro-B cell accessibility to IL7 secreting PSS cells.

Human pro-B cells and HSPC localize close to a human stromal cell subset related to the murine peri-sinusoidal stromal cell niche

Similarly to murine PSS cells, stromal cells expressing CD54, LepR and CD317, which represent 1 to 3% of low passage BM mesenchymal cells, have recently been identified in human BM samples [START_REF] James | Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes[END_REF]. In addition, and in opposition to CD317 -mesenchymal cells, human primary CD317 + cells express high levels of IL7 at the transcript and protein level. We thus compared gene expression profiles of murine PSS cells to the published datasets from CD317 + IL7 hi (Y102 and Y202 cell lines) and CD317 -IL7 lo (Y101 and Y201 cell lines) human stromal cells [START_REF] James | Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes[END_REF].

Unsupervised PCA analysis showed that mouse PSS cells clustered together with human IL7 hi stromal cells (Figure 7A, upper panel). Furthermore, when PCA analysis was restricted to the genes identified in murine PSS cells by the interactome approach, the distinction between IL7 lo and IL7 hi cells was even greater (Figure 7A lower panel).

This similarity between mouse and human CD317 + IL7 hi cells prompted us to analyze their distribution in human primary BM samples and their localization relative to pro-B cells and HSPC. We therefore analyzed expression of CD317, IL7 and CXCL12, and compared these patterns to CD34 expression which stains HSPC and endothelial cells. Cells expressing CD317, IL7 and CXCL12 were localized close to CD34 + endothelial cells (Figure 7B and Figure S7). As expected, strong interstitial staining was observed for CXCL12. Most remarkably, in some cases we observed highly vascularized regions with strong expression of CD317, IL7 and CXCL12 (Figure 7B, lower panel). In agreement with results obtained in mouse tissue, TdT + pro-B cells were localized close to vessels and IL7-expressing cells, and their frequency correlated with levels of IL7 expression. Interestingly, like mouse pro-B cells, human pro-B cells also expressed PLXDC1 (Figure 7C). Finally, CD34 + HSPC were also observed in contact with IL7 + cells, sometimes in close proximity to pro-B cells (Figure 7B and Figure S7B). Overall, our results demonstrate that PSS cells secreting IL7 and CXCL12 exist in humans and present a very similar gene expression profile to the mouse PSS subpopulation, suggesting that a number of biological questions related to leuko/stromal crosstalk could be addressed across multiple species.

Discussion

In the present study, we have developed antibody panels for flow cytometry to visualize and isolate different stromal cell subpopulations from wt mice. Such a phenotypic characterization is useful to precisely quantify the specificity of the different Cre recombinase reporter systems that have been used to analyze BM niches in the past. Our analysis indicates that BM mesenchymal cells are limited to four subsets: MSC, pericytes, PSS cells and OBs. Although the existence of minor subsets cannot be excluded, this result considerably reduces the number of mesenchymal subsets described using various Cre recombinase reporter systems and thus of potential hematopoietic niches. We have found that PSS cells are the main IL7-expressing cells [START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF], even though a small fraction of OBs and pericytes expressed IL7-Cre/YFP reporter but not Il7. Because OBs differentiate from PSS cells in adult life only [START_REF] Mizoguchi | Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development[END_REF] and do not express YFP in 2-3-week-old IL7 reporter mice, we can assume that YFP expression in adult OBs is due to IL7-driven Cre recombinase expression in PSS osteo-progenitors. Similar results were reported by Pereira and colleagues [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF]. In the case of pericytes, the partial recombination that we observed at the Rosa-YFP locus may be due to Il7 expression at levels below the qPCR detection threshold. Indeed, RNAseq studies previously indicated very low levels of IL7 in pericytes [START_REF] Kunisaki | Arteriolar niches maintain haematopoietic stem cell quiescence[END_REF].

Similarly, we did not detect IL7-Cre reporter expression in endothelial cells (Figure 2 and Figure S3), although one study reported detectable Il7 transcript expression in BMEC (Cordeiro [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF]. We believe that this is due to very low activity of Il7 promoter in endothelial cells since BMEC were shown to express 20-fold less IL7 than PSS cells. Approximately 60% of PSS cells were found to be YFP + using the IL7 reporter system (Figure 2C), suggesting that IL7 expressing cells represent a subset of PSS cells. However, YFP + and YFP -fractions were identical at the transcriptomic level, and both expressed Il7 (Figure 3A-B). This effect is most likely due to low Cre recombinase expression under the control of the IL7 promoter, as already reported by others [START_REF] Alves | Characterization of the thymic IL-7 niche in vivo[END_REF][START_REF] Mazzucchelli | Visualization and identification of IL-7 producing cells in reporter mice[END_REF]. Indeed, conditional deletion of Cxcl12 using the Il7-Cre or the LepR-Cre systems lead to a decrease in HSC in the BM with both Cre systems, but loss of retention was only observed in the compared to IL7 -PSS cells (Cordeiro [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF]. Based on our observations, it rather appears that CXCL12 deletion is more efficient in LepR-Cre than in IL7-Cre mice and thus that a slight variation in CXCL12 levels has a more dramatic effect on HSC maintenance than on their retention in the BM.

Having established the nature of IL7-expressing cells, we examined the localization of pro-B cells, which strongly rely on IL7 for their development. Pro-B cells were mainly distributed in the conduit close to LepR + PSS cells, and away from the endosteum, suggesting that OBs do not play a prominent role in B cell differentiation as previously thought [START_REF] Wu | Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}dependent signaling pathways[END_REF][START_REF] Zhu | Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells[END_REF]. This apparent discrepancy is most probably due to the fact that the systems used to specifically induce gene deletion in OB such as Osx-Cre or Bglap-Cre mice were also inducing deletion in PSS osteoprogenitors [START_REF] Mizoguchi | Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development[END_REF][START_REF] Zhang | Targeting of Mesenchymal Stromal Cells by Cre-Recombinase Transgenes Commonly Used to Target Osteoblast Lineage Cells[END_REF]. Alternatively, some systems may affect PSS cells indirectly. Acute ablation of OBs using thymidine kinase under the control of the Col1a1 promoter (Col2.3∆TK) leads to decreased numbers of early B cells [START_REF] Zhu | Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells[END_REF]. In this system, it is possible that a massive loss of OBs triggers PSS cell differentiation which affects the hematopoietic compartment as described in another study [START_REF] Visnjic | Hematopoiesis is severely altered in mice with an induced osteoblast deficiency[END_REF]. Finally, one could not exclude that PSS cells are directly ablated in this system since they express Col1a1 (Figure 3C).

Through our interactome analysis, we identified Nidogen-1, expressed by PSS cells, as a regulator of pro-B cell positioning with respect to the LepR-expressing peri-sinusoidal niche. Although the vascular structure is modified in Nid1 -/-mice and the abundance of LepR + structures seems affected, an indirect effect on pro-B cell differentiation is unlikely. Indeed, HSC, which strongly rely on secretion of CXCL12 and SCF by BMEC for their maintenance [START_REF] Asada | Differential cytokine contributions of perivascular haematopoietic stem cell niches[END_REF][START_REF] Xu | Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow[END_REF] are not affected, and the frequency of PSS cells as well as their expression of crucial HSC and B cell niche genes were not altered in Nid1 -/-mice. More specifically, PSS and pro-B cells from deficient animals express respectively normal levels of CXCL12/VCAM1 and of their respective ligands CXCR4/α4integrin, shown to be crucial for pro-B retention in the niche [START_REF] Arroyo | Differential requirements for alpha4 integrins during fetal and adult hematopoiesis[END_REF][START_REF] Park | Focal adhesion kinase regulates the localization and retention of pro-B cells in bone marrow microenvironments[END_REF]. In addition, adoptive transfer experiments show that the pro-B cell defect is not cell-autonomous, suggesting that CXCR4 and α4-integrin signaling may be necessary but not sufficient to explain pro-B cell retention in the niche. On the other hand, we cannot exclude a contribution of endothelial cells in the formation of the matrix surrounding the PSS cells since Nidogen-1 is expressed by BMEC. However, pro-B cells do not preferentially localize close to Nid1 hi arterioles indicating that Nidogen-1 expression is not sufficient to sustain B cell development. Instead, we believe that a complex network of factors and signaling, involving Nidogen-1, CXCL12 and VCAM1 is required for localization of pro-B cells near IL7-producing PSS cells.

It is known that cultured pro-B cells need high concentrations of IL7 to proliferate, while decreasing IL7 concentration induces differentiation toward the pre-BI stage, V to DJ recombination and intracellular expression of IgH chains [START_REF] Marshall | Modulation of the IL-7 dose-response threshold during pro-B cell differentiation is dependent on pre-B cell receptor expression[END_REF]. Early B cells lose their dependency on IL7 at the pre-BCR + pre-BII stage, as they mature toward the immature B cell stage. In line with these observations, we found that wt pro-B cells proliferate more than pre-BI cells (Figure 5). When pro-B cells are far from the PSS niche, as is the case in Nid1 -/-mice, their proliferation rate is decreased to the level of pre-BI cells. This suggests that pro-B cells need to move away from the source of IL7 to differentiate into pre-BI cells, as also supported by recent studies in which the amount of IL7 in the peri-sinusoidal niche or the integrin-mediated adhesion of pro-B cells were manipulated (Cordeiro [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF][START_REF] Fistonich | Cell circuits between B cell progenitors and IL-7(+) mesenchymal progenitor cells control B cell development[END_REF].

Our results and others suggest that distance of hematopoietic cells to their niche is important for their development [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF][START_REF] Itkin | Distinct bone marrow blood vessels differentially regulate haematopoiesis[END_REF][START_REF] Kunisaki | Arteriolar niches maintain haematopoietic stem cell quiescence[END_REF][START_REF] Mourcin | Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow[END_REF][START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF]. However, as LepR + stromal cells are widespread in the BM, we cannot exclude the presence of fine reticular extensions of PSS cells deeper in the parenchyma. These extensions may be implicated in the chemokine or growth factor gradients which are formed along cell surface in association with the ECM [START_REF] Laguri | Relationships between glycosaminoglycan and receptor binding sites in chemokines-the CXCL12 example[END_REF]. On the other hand, our interactome analysis confirms that accessibility of hematopoietic cells to a specific niche is not only related to distance per se, but also on the capacity to form specific interaction networks. As an example, pro-B cells expressed a higher number of genes involved in the interaction with PSS than pre-B cells, which are known to be associated with distinct Gal1 + stromal cells (Figure 4D). This would be consistent with recent results showing that a fraction of motile pre-B cells are located close to peri-sinusoidal niches but do not engage stable interactions with PSS cells [START_REF] Fistonich | Cell circuits between B cell progenitors and IL-7(+) mesenchymal progenitor cells control B cell development[END_REF]. We can then speculate that both the distance to the sinusoidal regions and adhesion to stromal cells or ECM are important for hematopoietic differentiation.

Our work not only shows the influence of PSS cells on early B cell development but also asks the question of BM compartmentalization and niche specificity. A PCA analysis performed previously showed the proximity of the different known PSS cell subsets using Nes-GFP and SCF-GFP reporter mice and their distance from other mesenchymal subsets [START_REF] Isern | The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function[END_REF]. We now confirm at the single cell level that PSS cells form a homogeneous cluster distinct from BMEC, pericytes, osteoblasts or MSC. However, because of technological limitations due to the detection of the most abundant polyA RNA only, we cannot rule out that a certain degree of intrinsic heterogeneity exists in the PSS cluster. For example, since it is known that PSS cells can differentiate in osteoblasts and adipocytes, we cannot exclude that PSS cells engaged in differentiation contribute to undetected intrinsic cluster heterogeneity. However, this should concern only a limited number of PSS cells and genes since the cluster is clearly homogeneous for the expression of a large number of genes (Table S1). Moreover, in addition to the maintenance of HSC through secretion of both SCF and CXCL12 [START_REF] Ding | Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches[END_REF][START_REF] Ding | Endothelial and perivascular cells maintain haematopoietic stem cells[END_REF][START_REF] Greenbaum | CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance[END_REF][START_REF] Sugiyama | Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches[END_REF] and of pre-pro-B cells through secretion of CXCL12 [START_REF] Ding | Haematopoietic stem cells and early lymphoid progenitors occupy distinct bone marrow niches[END_REF][START_REF] Greenbaum | CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance[END_REF][START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF], our results demonstrate that the same PSS cells sustain pro-B cells. Most importantly, HSC can be found together with pro-B cells in the same niche. Even if there is competition to access factors such as CXCL12 (by HSC, pre-pro-B and pro-B) or IL7 (by pre-pro-B and pro-B), HSC specifically interact with PSS cells through expression of "HSC niche genes", while early B cells engage specific interactions by upregulating "lymphoid niche genes". As a result, PSS cells appear to be able to accommodate different hematopoietic cells by supplying the appropriate crucial niche factors required for the development of distinct lineages.

In addition to HSC, pre-pro-B and pro-B cells, other hematopoietic subsets are also associated with sinusoids. Indeed, plasma cells have been shown to be in contact with peri-sinusoidal CAR cells and to rely on CXCL12, CD54 and CD106 for homing and maintenance in the BM [START_REF] Dilillo | Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice[END_REF][START_REF] Tokoyoda | Cellular niches controlling B lymphocyte behavior within bone marrow during development[END_REF]. More recently, multi potent progenitors (MPP) and CLP were also found associated with PSS cells (Cordeiro [START_REF] Gomes | Hematopoietic Stem Cell Niches Produce Lineage-Instructive Signals to Control Multipotent Progenitor Differentiation[END_REF]. Results from this study demonstrated that CXCL12 favors positioning of CLP close to PSS to gain access to IL7, suggesting that PSS cells organize hematopoietic cell positioning in the BM. This capacity of PSS cells to accommodate hematopoietic cells at distinct stages of development fits with the hemosphere model proposed earlier [START_REF] Wang | Identification of a clonally expanding haematopoietic compartment in bone marrow[END_REF]. In their study, Adams and colleagues described HSC forming units with both mesenchymal and endothelial cells. Interestingly, this specific niche allowed local clonal expansion of HSC into more mature hematopoietic lineages. In line with this organization, we found that half of the HSC were located at less than 41.7 µm from pro-B cells, suggesting that factors involved in the development of distinct hematopoietic subsets are supplied locally and that migration of developing cells relative to mother cell is relatively limited.

Although increasing evidences demonstrate the localization of numerous hematopoietic subsets close to sinusoids, this does not preclude the existence of other cellular niches. Indeed, arteriolar Nestin + stromal cells have been shown to maintain HSC into quiescence [START_REF] Kunisaki | Arteriolar niches maintain haematopoietic stem cell quiescence[END_REF]. In addition, the low permeability of arteries as compared to sinusoids would be a favorable niche for HSC quiescence through the maintenance of low reactive oxygen species levels [START_REF] Itkin | Distinct bone marrow blood vessels differentially regulate haematopoiesis[END_REF].

Finally, CXCL12 and SCF expression by aBMEC is crucial for HSC maintenance [START_REF] Xu | Stem cell factor is selectively secreted by arterial endothelial cells in bone marrow[END_REF].

According to these results, it can be proposed that in order to differentiate, quiescent HSC enter the G1 phase of the cycle, but retain their capacity to self-renew, while moving from the arteriolar to the sinusoidal niche. Once in the sinusoidal region, the HSC will find growth factors allowing both their maintenance and their commitment into distinct lneages.

Although considerable knowledge has been acquired with respect to murine stromal cell niches in situ over recent years, less is known in humans. Comparison of the transcriptomic profiles for murine 20 PSS cells with IL7 hi stromal cells isolated from human BM revealed a close relationship between them. Furthermore, human IL7 hi cells expressed genes involved in murine PSS cell interactions with HSC and early B cells, indicating that they probably share the capacity to accommodate distinct hematopoietic subsets, as already suggested by the preferential localization of CD34 + progenitors in the vicinity of PSS cells [START_REF] Tormin | CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization[END_REF]. We confirmed CD317, IL7 and CXCL12 expression by human PSS cells in situ, and identified TdT + pro-B cells and CD34 + HSPC in close vicinity. In addition, we observed that pro-B cells were more frequently associated with IL7-rich regions, suggesting that human pro-B cells are also dependent on IL7. This finding appears to contradict older studies which suggested that IL7 was not required for human B cell development since B cells were present in SCID patients where no IL7R signaling occurs [START_REF] Russell | Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development[END_REF]. However, more recently, it was shown that IL7 induces human pro-B cell proliferation, suggesting that the B cells found in SCID patients correspond to a wave of IL7-independent fetal B cells [START_REF] Bendall | Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development[END_REF][START_REF] Johnson | Murine and human IL-7 activate STAT5 and induce proliferation of normal human pro-B cells[END_REF][START_REF] Milford | TSLP or IL-7 provide an IL-7Ralpha signal that is critical for human B lymphopoiesis[END_REF].

In conclusion, our results demonstrate that murine and human PSS cells specifically supply niche factors required for HSC and early B cell developmental programs. Although mesenchymal cells are certainly heterogeneous, our results demonstrate that hematopoietic niches in the BM can be multifunctional and that their number may be lower than previously thought. Finally, the existence of a similar niche in humans will have considerable impact on the development of regenerative medicine aiming to manipulate hematopoietic differentiation in pathological situations. A. Immunostaining of pro-B cells (TdT + ) and HSC (Lin -CD48 -CD150 + ; Arrowhead) on bone sections of WT mice. The images were obtained from maximum intensity projection of tile Z scans. The region in the dotted square was magnified for better visualization of the contact between a LepR + PSS cell, a HSC and a pro-B cell (right panels). See also video S1. B. Quantification in 3D of the distance between each HSC and the nearest pro-B or the nearest randomly positioned cell (n=158) as indicated. C. Gene datasets for YFP + PSS cells from IL7 reporter mice were compared to those of LT-HSC, ST-HSC, pre-pro-B, pro-B and pre-B cells to generate the respective interactomes. D. Genes of the interactome expressed by LT-HSC and by pre-pro-B or pro-B cells were compared to ST-HSC and pre-B cells, respectively. The Venn diagrams display the number of upregulated genes for each of the indicated hematopoietic subsets. E. The genes overexpressed by each subset compared to their nearest related subset as defined in (D) were used to perform a functional profiling by defining the enrichment for particular GO annotations (arrows point to the corresponding histogram). GO terms are represented on the y-axis and the significance on the x-axis (±Log(p-value); See also Table S3). F.

Main figure titles and legends

Heat map showing genes expressed by LT-HSC, pre-pro-B or pro-B cells and involved in the interactome with YFP + PSS cells. The fold-change for expression between extremes ranged from 1.7 to 31.8. Unsupervised clustering was performed on rows and columns. G. GSEA assessing enrichment of the stromal cell interactome as determined with LT-HSC, pre-pro-B and pro-B, on the pairwise comparisons of PSS cells (red) with OBs (Alcam -or Alcam + ) or sBMEC (blue). NES: Normalized enrichment score; FDR: False Discovery Rate. See also Figure S4 A. PCA of murine PSS cells with human CD317+IL7hi and CD317-IL7lo stromal cells with all expressed genes in mouse and human (upper panel) or with genes identified from PSS cell interactome with LT-HSC, pre-pro-B and pro-B (lower panel). B. IHC was performed on human bone marrow sections using anti-CD34, anti-CD317, anti-IL7, anti-CXCL12 and anti-TdT antibodies (see controls in Figure S7A).

Immunostainings were performed on serial sections. Upper and lower panels correspond to sections from two different samples. IL7 + cells are shown with arrow heads. Hematopoietic stem and progenitor cells (HSPC) are indicated by an arrow in the CD34/TdT co-staining to show their proximity to both CD34 + vessels and TdT + pro-B cells. Large fields from which the lower panels were extracted are presented in Figure S7B. C. IHC was performed on human bone sections using anti-TdT and anti-PLXDC1 antibodies. Hematoxylin was used as counter-stain. The regions in the dotted squares were magnified to better visualize PLXDC1/TdT co-staining (lower panels).

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Stéphane J.C. Mancini (stephane.mancini@inserm.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Mice

C57BL/6J and C57NL/6N mice were purchased from Janvier Laboratories, France. IL7cre Rosa26-eYFP [START_REF] Repass | IL7-hCD25 and IL7-Cre BAC transgenic mouse lines: new tools for analysis of IL-7 expressing cells[END_REF] were backcrossed for more than eight generations onto a C57BL/6J background.

Nid1 -/-mice [START_REF] Murshed | The absence of nidogen 1 does not affect murine basement membrane formation[END_REF] backcrossed on a C57BL/6N background were a kind gift from Pr.

Krieg (University of Cologne). Both male and female mice were used between 6 and 15 weeks of age and were housed under specific pathogen-free conditions. Animals were randomly included For the generation of bone marrow chimeras, recipient mice were lethally irradiated with 7 Gy of total body irradiation (X-ray source, RS-2000 irradiator) one day before transplantation. Amoxicilline was added to the drinking water at 0.25 mg/ml and left during 2 weeks. Transplantation of 5x10 6 donor bone marrow cells was performed by intra venous injection via the retro-orbital sinus. BM B cell development was analyzed 15 week post transplantation.

Hydroxyurea treatments were performed by injecting intraperitoneally 1 mg/g of body weight at a rate of 2 injections separated by 7 hours per day for 2 days as previously described [START_REF] Espeli | Impaired B-cell development at the pre-BII-cell stage in galectin-1-deficient mice due to inefficient pre-BII/stromal cell interactions[END_REF]. BM B cell development was analyzed 7 days post injection.

Human tissues

Osteo-medullary biopsies from four normal male and female adult subjects from 18 to 56 years old were used. Patients provided informed consent in line with the Declaration of Helsinki before sample collection. The study protocol was approved by the institutional review board at Institut Paoli-Calmettes (agreement #IPC 2018-002).

METHOD DETAILS

Flow cytometry and cell sorting

For flow cytometry experiments, single-cell suspensions were stained using standard protocols and the antibodies listed in Supplemental Experimental Procedures. Dead cells were excluded using Sytox Blue, LIVE/DEAD® Fixable Aqua Dead Cell Stain (ThermoFisher) or DRAQ7 (Beckman Coulter).

To analyze hematopoietic cell populations, BM cells were extracted, lysed (1X RBC lysis buffer, eBiosciences) and stained. Cell-cycle analysis was performed by staining double-stranded nucleic acids with TO-PRO-3 (ThermoFisher Scientific) following fixation and permeabilization of the cells using the Cytofix/Cytoperm kit (BD Biosciences). Phosphorylated STAT5 was detected using the antiphosphoprotein pSTAT5 antibody following fixation and permeabilization of the cells with the PerFix EXPOSE kit (Beckman Coulter). The lineage cocktail (Lin) used to identify the hematopoietic progenitors contains the following markers: CD3, CD4, CD8α, DX5, B220, CD19, CD11b, CD11c, Gr1 and Ter119. Hematopoietic subsets were defined as follow: LT-HSC: Lin -Sca1 + CD11 + (LSK)CD150 + CD48 - CD135 -; ST-HSC: LSK CD150 -CD48 -CD135 -; MPP2: LSK CD150 + CD48 + CD135 -; MPP3: LSK CD150 - CD48 + CD135 -; MPP4: LSK CD150 -CD135 + ; Myeloid progenitors: Lin -CD117 + Sca1 -; CLP: Lin - CD117loCD127 + CD135 + ; pre-pro-B: B220 + CD19 -CD43 + CD24 -CD117 + CD127 + ; pro-B: CD19 + B220 + IgM - CD23 -BP1 -CD2 -CD117 + ; pre-BI: CD19 + B220 + IgM -CD23 -BP1 + CD2 -CD117 + ; pre-BII: CD19 + B220 + IgM -CD23 - CD2 + ; Immature B cells: CD19 + B220 + IgM + CD23 -; Recirculating B cells: CD19 + B220 + IgM + CD23 + .

For analysis of BM stromal cells, femur and tibia from mice were crushed, deposited on a 100µm cell strainer and rinsed three times with PBS to recover cells from the conduit fraction in the eluate.

Mesenchymal and endothelial cells associated to the sub-endosteal region were recovered from the crushed bones. Conduit fractions were incubated with 0.1mg/mL of Liberase TL Research Grade (Roche) and 10 U/mL DNaseI (Invitrogen) at 37 °C for 20 minutes. The bone fraction was treated with 2 mg/ml collagenase I and 10 U/mL DNaseI at 37 °C for 40 minutes. BM stromal cells were enriched by negative selection using immunomagnetic anti-rat IgG Dynabeads (Invitrogen) and antibodies recognizing CD45 (H129-326 supernatant), Ter-119 (BD Biosciences) and B220 (eBioscience). The stroma lineage cocktail is composed of the following markers: B220, CD19, CD11b, CD11c, Gr1, Ter119 and CD117. For transcriptomic analysis, CD54 + BP1 + stromal cells were sorted from WT mice, and CD54 + BP1 + YFP + and CD54 + BP1 + YFP -cells were sorted from IL7-Cre/Rosa-eYFP mice. CD31 + Sca1 low sinusoidal BMEC were sorted from WT mice. FACS analysis was performed on a FACS LSRII and cell sorting on a FACSAria III (BD Biosciences). Data were analyzed using DiVa Version 8.01 (BD Biosciences) or FlowJo Version 10 software (TreeStar).

Immunohistofluorescence

Femurs and tibias were incubated for four hours in PFA 4% prepared in PBS, decalcified and incubated in cryoprotective buffer as previously described [START_REF] Kusumbe | Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue[END_REF]. Bones embedded in OCT were cryosectioned on Superfrost+ slides and immediately fixed in acetone for 5 min at -20 °C. After drying, sections were rehydrated in PBS for 5 min, saturated in blocking solution (Purified Goat anti-mouse, Jackson ImmunoResearch, BSA 5%, Saponin 0.05% in PBS) for 30 min and finally incubated with primary antibodies in a humidified chamber overnight at 4 °C or for 1 h at room along the orthogonal, y axis, from each position of each real pro-B and randomly positioned cells to the border of the mask created from the convex DAPI staining.

Immunohistochemistry

Osteo-medullary biopsies were formalin-fixed and decalcified in 0.27 M Ethylene diamine tetra acetic acid (EDTA) before embedding in paraffin. 1.5 to 3 µm sections were used. Immunohistochemistry was performed on a Ventana Discovery XT biomarker platform. Staining with the anti-CD34 (QBEnd-10, Beckman Coulter) and anti-TdT (Sen28, Novocastra) antibodies was performed after antigen retrieval at pH 8. Staining with the anti-CD317 (ab134061, Abcam), anti-IL7 (HPA019590, Sigma Aldrich), anti-CXCL12 (D8G6H, Cell Signaling Technology) and anti-PLXDC1 (197C193, Novus Biologicals) antibodies was performed after antigen retrieval at pH 6. Multiplex staining was performed using the Discovery purple kit (Ventana), together with DAB substrate or the Discovery yellow kit (Ventana). Slides were counterstained with hematoxylin.

Gene expression profiling qPCR analysis

Single cells were sorted in two separate experiments using the autoclone module on an AriaIII sorter (Becton Dickinson) directly into C1 chips (Fluidigm). Single cell loading, cell lysis and the preamplification steps using Taqman primers (see Supplemental Experimental Procedures) were performed using the C1 auto-prep system according to the manufacturer's instructions (Fluidigm).

Pre-amplified products were diluted in 25 µL of C1 DNA dilution reagents before further processing using Biomark (Fluidigm). Bulk stromal cells were directly sorted into 96-well plates containing Cells Direct Reaction Mix (Life Technologies) using the autoclone module on an AriaIII sorter (Becton Dickinson). Each subpopulation analyzed was obtained from two independent sorting processes. Cell lysis and pre-amplification with Taqman primers (22 Cycles) were performed according to the Fluidigm Advanced Development Protocol. Transcript quantization in single or bulk sorted cells was performed using microfluidic real-time PCR on Dynamic Array IFCs (Biomark,96.96 Dynamic Arrays,Fluidigm). Ct values were calculated using the system's software (BioMark Real-time PCR Analysis; Fluidigm) and filtered based on expression levels for GAPDH and expression of the surface cell markers used for the cell sorting. Gene expression analysis was performed with heat maps generated using Morpheus (Broad institute). Unsupervised hierarchical clustering was performed using one minus Pearson's correlation distance metrics and the average linkage method.

Single cell RNAseq analysis

CD54+BP1+ PSS cells, CD51+BP1-mesenchymal cells and CD31+ BMEC were sorted by flow cytometry and mixed together. They were then subjected to the 10X Genomics Chromium Single-Cell 3′ v2 protocol according to the manufacturer's instructions by HalioDX (Marseille, France), and the resulting libraries were sequenced on an Illumina NextSeq500. A second replicate was performed in the same conditions. Both datasets are available in the GEO database (GSE121568).

Cell Ranger software v2.1.1 was used to processes FASTQ raw files: reads alignment to the mm10 genomes, filtering, barcode counting and unique molecular identifier (UMI) counting. Cells outside 2 medians absolute deviation (MADs) from the median for UMI log-counts and 3 MADs from the median for log-transformed number of genes detected were excluded, as well as cells above 5 MADs from the median for the percentage of mitochondrial genes. Genes not expressed in any cells of the remaining cells were removed. Unsupervised clustering of single-cell RNA-seq 10X Chromium data and t-distributed stochastic neighbour embedding (tSNE) for 875 cells with expression detected for 15,872 genes were performed with the Seurat package (v2.3.4) in R as described previously [START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF]. Gene expression measurements were normalized for each cell by the total expression, multiplied by a scale factor of 10,000 and log transformed. Variable genes were identified with the FindVariableGenes function of Seurat. Cell to cell variation driven by the number of detected molecules as well as the percentage of mitochondrial genes were regressed with the ScaleData function. A principal component analysis (PCA) was then performed on variable genes and revealed that the first 11 principal components (PCs) were significant with the jackstraw procedure [START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF] and by identifying in the graph the elbow of the standard deviation of PCs. These 11 PCs were then used to cluster the cells using the Louvain algorithm with the function FindClusters with the resolution parameters set to 0.6 (k parameter set to 30 for the construction of the K-nearest neighbour graph). 7 clusters were obtained, among which 2 clusters of contaminant hematopoietic cells identified with specific markers (Ptprc,Cd69,Fcer1a,Ccl3,Ccl4 and Cxcr4) expressed by at least one these clusters but not by the others. The cells of these clusters were removed and the remaining cells were re-analysed using the same procedure from normalization to clustering. This time the first 7 PCs of PCA were found to be significant. Unsupervised clustering of the cells on the 7 PCs with the same parameters as before resulted in 6 clusters. Positive and negative markers for each cluster were determined with the function FindAllMarkers of the Seurat package using the default Wilcoxon rank sum test and Bonferroni p-values correction for multiple testing. Finally a tSNE were run on the 7 PCs using the RunTSNE function of Seurat with default parameters except the do.fast option set to true. Number of cells analyzed: PSS cells, n=271; L-type BMEC, n=163; H-type BMEC, n=137; OB, n=86; Pericytes, n=54; MSC, n=18.

Microarray analysis

pre-B and pro-B vs. pre-B. For the comparisons, Venn diagrams were generated, assuming that genes were specific for a population when the expression level was at least 2-fold higher.

GO enrichment

GO enrichment was analyzed for each population by hypergeometric testing associated with the custom multiple testing correction procedure g:SCS implemented in gProfiler R version [START_REF] Reimand | g:Profiler--a web server for functional interpretation of gene lists (2011 update)[END_REF]. The threshold for significance was set to 5%, and the reference annotation set was the same size as the annotated domain.

Gene set enrichment analysis

The GSEA method from the Broad Institute was used to statistically test whether the stromal cell interactome was enriched within CD54 + BP1 + stromal cells, sBMEC and osteoblasts (Alcam -Sca1 -or Alcam + Sca1 -) compared to each other. The stromal cell interactome GeneSet was added to the c2v5.1 public GeneSet collection (4726 GeneSets) from the Molecular Signatures Database (MSigDB) [Subramanian, Tamayo, et al. (2005, PNAS 102, 15545-15550)] to provide the adequate statistical power. Statistical analysis was performed by determining the false discovery rate (q-value) based on 1,000 random permutations between all the GeneSets. Results were considered significant when the FDR q-value was below 0.25, as recommended by the software developers.

QUANTIFICATION AND STATISTICAL ANALYSIS

The number of biological replicates is indicated in the relevant figure legends. Error bars for pooled replicates represent standard error of the mean (SEM). The distribution of samples was determined by applying the Kolmogorov-Smirnov test. Samples were compared using a two-tailed unpaired t test or a Mann-Whitney test when normality was not reached. All statistical analyses were performed with GraphPad Prism 6. *, p<0.05; **, p<0.01; ***, p<0.001.

DATA AND SOFTWARE AVAILABILITY

Microarray and single cell RNAseq datasets generated in this study have been deposited in the GEO database under accession number GSE90588 and GSE121568 respectively.

KEY RESOURCES TABLE

Figure 1 .Figure 2 .

 12 Figure 1. Phenotypic characterization of the BM microenvironment and pro-B cell localization

Figure 3 .

 3 Figure 3. IL7, CXCL12 and SCF are co-expressed by the PSS cell subset.

Figure 4 .

 4 Figure 4. HSC and pro-B cells are frequently found in the same niche and form specific interaction

Figure 5 .Figure 6 . 0019 Figure 7 .

 5600197 Figure 5. Nidogen-1 allows pro-B cell access to IL7.

  in the experiments according to genotyping results. The numbers of mice used per experiment are stated in the Figure Legends. Mice were handled in accordance with the French Guidelines for animal handling (Agreement #02294.01) and the EU Directive 2010/63/EU.

Acknowledgments

We are grateful to the core flow cytometry, animal and microscopy facilities of the CRCM and CIML for providing supportive help, to Pr. Thomas Krieg for the kind gift of important material and to Emilie Agavnian from the ICEP platform for technical help. This work was partly supported by grants from the SIRIC (Inca-Inserm-DGOS 6038), the Cancéropôle PACA funded by Inca, the ARC Foundation (PJA#20131200298 and PJA#20161204555 to SJCM, PJA#20141201990 to MAL), the ANR (OSTEOVALYMPH, ANR-17-CE14-0019-03) and from the Excellence Initiative of Aix-Marseille University -A*MIDEX, a French "Investissements d'Avenir" program. MB was the recipient of a PhD grant from FRM (#FDT20150532380), MDG's post-doctoral fellowship was supported by the Fondation de France (#2013-00042638).

Declaration of interests:

The authors declare no competing interests. temperature. After washing three times, slides were incubated with the appropriate secondary antibodies for 1 h at room temperature and mounted with Prolong (Pierce). Images were acquired in z on 10 to 15µm for 3D reconstruction using LSM780 or LSM880 confocal microscopes (Carl Zeiss) and processed in Fiji (https://fiji.sc/).

Quantification of confocal images

Custom Matlab scripts were developed for calculation of the 3D distance between pro-B and nearest LepR + cells, LepR + Nid + cells, Nid1 hi arteries and bone surface. LepR and Nidogen images were segmented by successively median filtering over 3 by 3 pixels, optimized thresholding and erosion, dilatation over 1 pixel. Finally, objects smaller than 70 voxels (128.7 µm3) were discarded from the final segmented result (binary images). Double positive structures, LepR + Nid + , were defined as pixels segmented both for LepR and for Nidogen. Arteries were detected using a higher threshold coefficient, and by discarding objects smaller than 1 000 voxels (1 838 µm3). To get the envelope of the entire tissue, DAPI filtering was performed over 5 by 5 pixels and erosion, dilatation over 12 pixels, to ultimately keep the convex shape surrounding the largest segmented object. Matlab codes used to calculate threshold values, freely obtained from the Matlab file-exchange website, were written by Martín D., based on the work of Tsai W. for LepR staining (optimal threshold), and by Marti, F.T. for Nidogen and DAPI staining (moment threshold).

For each 3D image stack, pro-B cells were manually identified by TdT staining on the maximum projection of each stack using the ImageJ cell count plugin. Their axial position was then defined as corresponding to the maximum fluorescence intensity along z, for each x, y position. Since the TdT staining can be more or less diffuse from cell to cell, the fluorescence intensity was averaged in each plane over 3 by 3 pixels around the detected position. For control, random positions were generated, with a number equal to the number of pro-B cells detected in each stack. A mask was created in 3D to allow selecting these random positions. This mask was defined by delineating the entire tissue, using the convex envelope surrounding all DAPI-stained cells. The LepR segmented image stack was removed from this mask to allow cells to be positioned only in the parenchyma. This mask was dilated by 4 µm, corresponding to the average cell radius, to avoid random cells overlapping the sinusoids. A random set of pixel positions was selected within the mask to allow comparison between real and randomized cells.

3D Euclidian distances from each segmented staining were computed over the whole image stack using bwdistsc1. This Matlab function provides the distance from any point to the segmented structure and allowed to accommodate for anisotropic calibrations: 1.1 µm in x, y and 1.5 µm in z.

The distance was then assessed for the 3D position of each real pro-B and randomly positioned cells.

Since bone sections were cut along the x axis, the distance from the bone border was computed Stromal cells were directly sorted in RLT Buffer for mRNA purification using an RNeasy plus micro-kit (QIAGEN). mRNA quality was evaluated using an Agilent 2100 (Pico Chip) before sending samples to the GenomEast Platform (http://genomeast.igbmc.fr/, Strasbourg, France). Affymetrix MoGene 1.0 ST and 430 2.0 raw CEL files, either obtained from in-house experiments or downloaded from the Gene Expression Omnibus (GEO) public database (see Supplemental Experimental Pocedures) were processed and normalized by applying the Robust Multichip Average (RMA) method, using the Oligo package through Bioconductor in the R statistical environment (version 3.2.0).

Quality-control for array hybridization and compatibility between the different sources was also assessed using Oligo. To do so, boxplots and density plots of raw and RMA-normalized expression data and subsequent classification analyses were examined. Probe sets for which the normalized expression value was less than 6 (log2 scale) across all arrays were considered as not expressed, and were therefore removed from the analysis. PCA was performed on the samples using the MultiExperiment Viewer (MeV version 4.8.1) on Probesets that showed a minimal 2-fold differential expression (in linear scale) between at least two cell populations. Heatmaps were generated using Morpheus software. Unsupervised hierarchical clustering was performed using one minus Pearson's correlation distance metrics and the average linkage method.

The human and mouse stromal cell datasets were aggregated as previously described [START_REF] Charbord | A systems biology approach for defining the molecular framework of the hematopoietic stem cell niche[END_REF]. Probes with multiple matching sequences were removed using version 17 of the custom ChIP definition file [START_REF] Dai | Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data[END_REF]. PCA analysis was performed either on the 14437 genes found in common between the datasets or on the genes found in the interactome between PSS cells and LT-HSC, pre-pro-B or pro-B cells.Affymetrix datasets generated in the course of this study were uploaded to the GEO database under accession number GSE90588.

Interactome analysis

The bioinformatics pipeline can be split into four main steps: collection of interaction data, building interactome reference map, normalization of microarray data with conversion of gene identifiers conversions, and comparison of genes present in the different cellular populations.

Interactome reference map

Interactome integration and analysis was done with tools initially developed for Interactome-Transcriptome analysis [START_REF] Garcia | Detection of driver protein complexes in breast cancer metastasis by large-scale transcriptome-interactome integration[END_REF]. For the interactome analysis, several protein-protein interaction (PPI) databases were downloaded, parsed and integrated by superimposing all the interactions found. PPIs from the Database of Interacting Proteins, Human Protein Reference Database, IntAct, the Molecular INTeraction database were integrated to build the interactome. A total of 13202 genes and 70530 interactions were mapped in the interactome. Protein identifiers were mapped to Entrez GeneID accession numbers using correspondence tables downloaded from the NCBI FTP site (date: November 5th, 2014; ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene_info.gz). In addition to the cells profiled in-house, publicly available datasets were downloaded from the GEO as raw CEL files. All datasets studied were analyzed on the Affymetrix Mouse Gene 1.0 ST platform data.

Microarray data were parsed, underwent quality-control and renormalized using the R/Bioconductor (Oligo package) and the RMA algorithm. Probes were then mapped to Entrez Gene identifiers by maximum median value [START_REF] Reyal | Visualizing chromosomes as transcriptome correlation maps: evidence of chromosomal domains containing co-expressed genes--a study of 130 invasive ductal breast carcinomas[END_REF]. To compare gene expression from multiple samples, genes were classed as expressed/not expressed for each sample using the postprocessing approach described for the Immgen dataset [START_REF] Heng | The Immunological Genome Project: networks of gene expression in immune cells[END_REF]. Briefly, gene expression data was modeled by applying a Gaussian mixture distribution (the number of chosen Gaussians was k=4) (Package R Nor1Mix). The first Gaussians modeled were considered non-expressed genes. The threshold was set to 95% for the second Gaussian.

Gene accession number conversion

To compare gene expression profiles for mouse cell populations using a human interactome reference map, gene IDs had to be homogenized. Mus musculus Entrez GeneID accession numbers were automatically converted to Homo sapiens accession numbers using the NCBI Homologene database downloaded as a flat file from the NCBI FTP site (ftp://ftp.ncbi.nih.gov/pub/HomoloGene/).

The final conversion to gene symbols was performed using the gene_info.gz file from the NCBI FTP site (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene_info.gz).

GO filtering

To focus on genes coding for surface or extracellular proteins, GO annotation-based filtering was performed on the genes from the mouse genome. The GO terms selected for this filtering were: GO:0009897, GO:0005887, GO:0030246, GO:0031012, GO:0005581, GO:0005923, GO:0048535, GO:0030595 and GO:0008083.

Population comparison

Interactions between YFP + CD54 + BP1 + stromal cells from IL7-Cre/Rosa-eYFP mice and LT-HSC, ST-HSC, pre-pro-B, pro-B or pre-B cells were identified using the human interactome described above. Cis interactions were considered to be false positives, and thus only trans interactions were considered.

Trans-homotypic interactions were included in the analysis. The relevance of the genes identified in the interactome for LT and ST-HSC was further improved by crossing data from two different datasets and considering only genes identified in both (Table S2). The gene expression signatures obtained for each hematopoietic subset were compared as follow: LT-HSC vs. ST-HSC, pre-pro-B vs.

Supplemental video and Excel table titles

Video S1. 3D reconstruction of z-stacked confocal images showing the contact of a LepR + PSS cell with a HSC and a pro-B cell. Related to Figure 4A. Table S1. Genes significantly up-and down-regulated in each cluster shown in Figure 3D Table S2. Interactome of hematopoietic cell subsets with IL7-YFP + peri-sinusoidal stromal cells. Related to Figure 4. Table S3. Gene Ontology enrichment for HSC and early B cell specific genes involved in the interaction network with peri-sinusoidal stromal cells. Related to Figure 4.