Ali Nassif

Guilhem Lignon

Audrey Asselin

Charles C Zadikian

Stephane Petit

Hongwei W Sun

François C Klein

Côme Ferré

Maria Mi Morasso

Ariane Berdal

F C Ferré

M I Morasso

B P J Fournier

J Isaac

Transcriptional regulation of jaw osteoblasts: development to pathology

Keywords: Osteoblast(s), Transcription factor(s), Bioinformatics, Bone biology, Craniofacial anomalies, Gene expression, tables: 4 figures, 1 table Osteoblast, Transcriptome, RNA-seq, Jawbone, Tibia, Transcription Factor, Neural Crest, Bone disease, Development

"OB-core"; 54 TF genes are upregulated in Md-OB, referred to as "Mdset"; and 18 TF genes are upregulated in Tb-OB, referred to as "Tb-set". Notably, the expression of 29 additional TF genes depends on their RNA transcript variants. TF genes with no previously known role in OBs and bone were identified. Bioinformatics analysis combined with review of genetic disease databases and a comprehensive literature search showed a significant contribution of anatomical origin to the OB signatures. Mdset and Tb-set are enriched with site-specific TF genes associated with development and morphogenesis (neural crest versus mesoderm), and this developmental imprint persists during growth and homeostasis. Jaw and tibia site-specific OB signatures are associated with craniofacial and appendicular skeletal disorders as well as neurocristopathies, dental disorders and digit malformations. The present study demonstrates the feasibility of a new method to isolate pure OB populations and map their gene expression signature in the context of OB physiological environment, avoiding in vitro culture and its associated biases. Our results provide insights into the site-specific developmental pathways governing OBs and identify new major OB regulators of bone physiology. We also established the importance of the OB transcriptome as a prognostic tool for human rare bone diseases to explore the hidden pathophysiology of craniofacial malformations, among the most prevalent congenital defects in humans.

Introduction

Driven by a complex network of transcription factors (TFs), the osteoblast (OB) is traditionally considered to be a unique differentiated mesenchymal cell that synthesizes various peptides in charge of matrix deposition, biomineralization and remodeling during bone growth, homeostasis and healing (reviewed in [START_REF] Lian | Networks and hubs for the transcriptional control of osteoblastogenesis[END_REF]). However, craniofacial and jawbones exhibit a number of physiological specificities when compared to axial and appendicular bones (reviewed in [START_REF] Wang | Calvarial Versus Long Bone: Implications for Tailoring Skeletal Tissue Engineering[END_REF]). Craniofacial bones and long bones primarily develop through different ossification processes: intramembranous ossification for most craniofacial bones and endochondral ossification for long bones. Long bone growth and homeostasis, which ensure skeletal mobility and load bearing, are mainly regulated by mechanical strains and hormones.

Jawbone development additionally depends on teeth, as evidenced by its reduced volume in inherited tooth agenesis or after tooth extraction [START_REF] Bertl | Alveolar bone resorption after primary tooth loss has a negative impact on straightforward implant installation in patients with agenesis of the lower second premolar[END_REF][START_REF] Nowwarote | Decreased osteogenic activity and mineralization of alveolar bone cells from a patient with amelogenesis imperfecta and FAM83H 1261G>T mutation[END_REF].

Jawbones and long bones also respond differently to various agents. Estrogens and parathyroid hormone imbalances affect mineral density in long bones disproportionately [START_REF] Liu | Distinctive anabolic roles of 1,25-dihydroxyvitamin D(3) and parathyroid hormone in teeth and mandible versus long bones[END_REF][START_REF] Coutel | Mandibular bone is protected against microarchitectural alterations and bone marrow adipose conversion in ovariectomized rats[END_REF][START_REF] Watanabe | Regional variations of jaw bone characteristics in an ovariectomized rat model[END_REF]. Bisphosphonates and other antiresorptive drugs have few adverse effects on long bones but alter jawbone metabolism, sometimes culminating in jaw osteonecrosis (reviewed in [START_REF] Katsarelis | Infection and medication-related osteonecrosis of the jaw[END_REF]). Finally, autografts taken from craniofacial bones offer better outcomes regardless of the acceptor site when compared with grafts from long bones [START_REF] Akintoye | Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals[END_REF][START_REF] Leucht | Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration[END_REF].

Unsurprisingly then, jawbones and long bones show major differences at the cellular level.

Craniofacial bone progenitors show differential proliferation and osteogenic potential when compared to long bone progenitors [START_REF] Matsubara | Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells[END_REF][START_REF] Leucht | Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration[END_REF][START_REF] Aghaloo | Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells[END_REF][START_REF] Kelder | Cells Derived from Human Long Bone Appear More Differentiated and More Actively Stimulate Osteoclastogenesis Compared to Alveolar Bone-Derived Cells[END_REF]. Their cell lineages also differ: while the vast majority of craniofacial OBs derive from cranial neural crest, long bone OBs are of mesodermal origin [START_REF] Douarin | The ontogeny of the neural crest in avian embryo chimaeras[END_REF][START_REF] Gans | Neural crest and the origin of vertebrates: a new head[END_REF][START_REF] Chai | Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis[END_REF][START_REF] Yoshida | Cell lineage in mammalian craniofacial mesenchyme[END_REF]. Furthermore, in vivo and in vitro analyses suggest the existence of cell site-specific signatures between jawbones and long bones [START_REF] Kasperk | Human bone cell phenotypes differ depending on their skeletal site of origin[END_REF], notably for a number of TFs [START_REF] Rawlinson | Adult rat bones maintain distinct regionalized expression of markers associated with their development[END_REF][START_REF] Kingsmill | Gene expression profiles of mandible reveal features of both calvarial and ulnar bones in the adult rat[END_REF][START_REF] Reichert | Mesodermal and neural crest derived ovine tibial and mandibular osteoblasts display distinct molecular differences[END_REF][START_REF] Lee | Comparison of gene expression between mandibular and iliac bone-derived cells[END_REF]. However, to date, the transcriptome of the jaw OB in its physiological environment has remained elusive, as the studies focusing on jaws were performed using either cell culture or whole-tissue extracts. Moreover, jawbone cells, including OBs but also osteocytes and bone marrow cells, have been omitted from large-scale omics studies on the tissue-specific landscape [START_REF] Zhou | A mouse tissue transcription factor atlas[END_REF]Tabula Muris Consortium et al. 2018;[START_REF] Youlten | Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease[END_REF]. Here, we prove the feasibility of a new isolation method to obtain F o r P e e r R e v i e w pure populations of functional OBs within their physiological environment. This strategy allowed us to define the complete repertoires of TF genes expressed in jaw-and tibia-OBs in vivo. These data provide insights into the developmental and genetic pathways governing sitespecific OB differentiation and activity, and identify major OB regulators involved in jawbone physiology and disorders.

RNA Sequencing (RNA-Seq)

Mandible and tibia bones from P9 Col1a1*2,3-GFP mice were dissected (Appendix). Three independent dissections, each pooling bone tissues from approximately fifty P9 mice, were carried out. Immediately after dissection and collagenase I digestion, GFP-positive cells were FACS-sorted (Appendix). Protocols of RNA extraction, cDNA library construction and data processing pipeline are detailed in Appendix and are publicly available in the Gene Expression Omnibus (GEO), together with the RNA-Seq raw and processed data files (accession# GSE186535). Criteria for significant gene selection included q-values ≤ 0.1 (for multi-test adjustment), FC ≥ 1.4 or ≤ -1.4, and mean RPKM ≥ 1 for expressed genes.

Results

1-OB expression profiles include TFs conserved among bone sites as well as site-specific

TFs

The in vivo TF signatures expressed in functional OBs were defined by using a global gene profiling approach based on RNA-seq of flow-sorted GFP-positive cells from mandibular (Md) and tibia (Tb) bones of 9-day-old (P9) Col1a1*2,3-GFP mice (Fig. 1A). Our RNA-seq results were cross-referenced with a list of 1388 TFs (GEO accession# GSE186535). Md-OB or/and Tb-OB expressed 709 TF genes (977 transcripts), i.e. more than 50% of the known TF genes (Appendix Table 2). The largest group, termed "OB-core", comprises 608 TF genes (86%) similarly expressed in Md-OBs and Tb-OBs. The remaining TF genes were differentially expressed between the two skeletal sites: "Md-set" consists of 54 TF genes (8%) upregulated in Md-OBs, and "Tb-set" consists of 18 TF genes (2%) upregulated in Tb-OBs (Fig. 1B).

Notably, for 29 TF genes (4%), the bone site-specificity depended on their RNA transcript variants (TVs) (Appendix Fig. 1). To decipher how the anatomical origin contributed to the OB signatures, the three OB groups were further investigated with an enrichment analysis based on the Biological Processes (BPs) of the functional GO annotation using DAVID and a literature review (Fig. 1C-E and Appendix Fig. 2 and3).

OB and bone BPs:

Analysis of the OB transcriptome showed that OB-core and Md-set were enriched with TF genes associated with OB and bone-related BPs (Fig. 1C, Table 1). In OBcore, 21% of the TF genes (135/637) were previously established as regulators of OB (e.g. Atf3, Klf6, Runx2, Sp7, Tsc22d) (Appendix Fig. 2). The highest expressed TF genes of OB-core (with mean (RPKM) > 100) were all previously known for their involvement in OB (Atf3/4, Tsc22d1/3, Egr1, FosB, Hnrnpk, Klf6 and Nr4a). However, the percentage of TF genes with known roles in OB decreased along with the TF expression level, and between 75% and 80% of the TFs with mean (RPKM) < 25 have no previously known role in OB and bone (Appendix Fig. 2). To further investigate the expression of some of the TF genes of OB-core, qPCR and IF analyses were performed. In line with our RNAseq results, no significant difference in mRNA levels between Md-and Tb-bones isolated from P9 C57BL/6JR mice was detected for the eight investigated TF genes (Arid5b, Atf3, Klf6, Meox2, Runx2, Sp7, Tsc22d1, Ybx3) (Appendix Fig. 2 and4). Moreover, Runx2 and Sp7 IF analyses in E15.5 and P9 Col1a1*2,3-GFP mice showed similar protein expression pattern with almost all the functional OBs expressing both Sp7 and Runx2 independently of their localization (Appendix Fig. 4). In Md-

F o r P e e r R e v i e w
set, 62% of the TF genes (46/74) were previously known to regulate OB and bone (Fig. 1D).

Among them, 22 TF genes were expressed specifically in Md-OBs [including Alx genes (Alx1/3/4), Dlx genes (Dlx1/2), Pax genes (Pax3/9) and Hand2, Msx2, Six2, Sox5] and 24 TF genes showed lower expression in Tb-OBs [including Dlx genes (Dlx3/4/5), Sox genes (Sox9/11) and Msx1, Osr2, Pbx1, Sp6] (Fig. 1D).

Other connective/mesenchymal tissue BPs: OB-core was significantly enriched with TF genes associated with cartilage, skeletal muscle and fat BPs, whereas enrichment with TF genes associated with dental BPs was low (Fig. 1C and Appendix Fig. 2). Although Md-set displayed an enrichment profile similar to that of OB-core for bone and cartilage-related BPs, dental BPs were overrepresented in Md-set, whereas skeletal and fat-related BPs were low and mostly associated with negative regulation (Fig. 1C and Appendix Fig. 3). Gene-by-gene analysis of Md-set confirmed the high contribution of dental-associated genes, as 26% of the TF genes (19/74) have known roles in odontogenesis and odontoblast differentiation (e.g. Dlx4, Alx3, Lhx8, Dlx1) (Fig. 1D).

In Tb-set, a gene-by-gene literature analysis revealed bone-related TFs known for their regulatory roles in cartilage (e.g. Tbx18, Hoxa5/d8, Nfia), skeletal muscle (e.g Heyl, Hoxa7, Crem, Nfya) and fat (e.g Creb3l3, Hoxa9/c5, Tbx15). Shox2 was the only TF known to have a role in odontogenesis but also in bone, cartilage, muscle and fat (Appendix Fig. 3). Notably, TFs known for their involvement in the regulation of mesenchymal cells and tissues other than OBs and bone and never described in differentiated OBs were identified in the RNA-seq panel.

These novel OB TFs include Arid5a/5b, Ybx3, Meox2 and Creb5 in OB-core; Zfp90/110, Sp6, Rxrb and Bhlhb9 in Md-set; and Sox7 and Irf7 in Tb-set (Fig. 1E and Appendix Fig. 2 and3). mRNA expression of some of these TF genes was validated by qPCR in raw bones from P9 C57BL/6JR mice (Appendix Fig. 2 and3). To further investigate one of the novel OB TFs identified in Md-set, the expression of Sp6, a known odontogenesis regulator, was analyzed by qPCR and IF from development (E10.5) to post-natal stages (P9). In line with our RNA-seq results, Sp6 was detected at mRNA and protein levels in craniofacial tissues, including in Mdbone and Md-OBs from E14.5 to P9 in C57BL/6JR mice, while no expression was detected in appendicular skeleton (Fig. 1F-I and Appendix Fig. 5).

2-Site-specific OB-omics: blueprints of embryonic signatures

Development and morphogenesis BPs:

To address the contribution of embryonic BPs to OB signatures at post-natal stage P9, we first performed enrichment analysis of the 3 OB sets (Fig. 2A). The 3 sets were enriched in TF genes associated with the following BPs: skeletal system, F o r P e e r R e v i e w limb and appendage development, and morphogenesis. Only OB-core and Md-set displayed enrichment in TF genes associated with head and craniofacial developmental BPs, while "limb bud formation" and "embryonic digit morphogenesis" were enriched exclusively in Md-set.

OB-core was enriched with TF genes associated with mesoderm development BPs, and Md-set with NC BPs, whereas Tb-set displayed no enrichment in any of these BPs. RNA-seq data were also analyzed in light of 278 TFs associated with NC, based on information generated from the literature (Fig. 2B-C, Appendix Table 3). 39% of Md-set TF genes (29/74) were associated with NC, and all these genes were directly associated either with cranial NC (CNC) (e.g. Alx1/3/4, Dlx1/2/3/5, Msx1/2, Sox5/9/11) or with organogenesis/patterning of craniofacial skeletal elements (Pax9) or NC differentiation (Nfatc4). Six Tb-set TF genes were associated with caudal NC and participated in positional identity in the anterior-posterior axis (Hoxa5/a7/c10/c5, Tbx18) and the epithelial-mesenchymal transition (Akna). The expression of two TFs associated with CNC, Dlx3 and Sox9, was further investigated from development (E10.5) to post-natal stages (P9). Both Dlx3 and Sox9 showed high mRNA expression levels in embryonic and peri-natal skeletal tissues as previously described (Appendix Table 4) and, in line with our RNA-seq data, were significantly higher from E10.5 to P9 in head and craniofacial tissues, including Md-bone, when compared to appendicular skeleton (Fig. 2D-E, Appendix Fig. 5). In situ IF imaging of Sox9 confirmed the known Sox9 localization in cartilaginous tissues and further demonstrated its expression in Md-bone and Md-OBs at protein level (Fig. 2F-H).

Long-term maintenance of in vivo and ex vivo OB embryonic positional identity:

To determine whether Md-OB origin remains neuroectodermal with age, we examined the distribution of NC-derived cells in the growing oral bones at embryonic (E15.5), postnatal (P9) and homeostasis stages [2 months (2M), 4 months (4M) and 8 months (8M)], taking advantage of the individual cell visibility of fluorescent markers in Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice (Fig. 3 and Appendix Fig. 5). Microscopic observations (Fig. 3A-C) and cell quantification using co-localization of tomato, GFP and DAPI signals on FACS-isolated cells (Fig. 3D) and tissue sections (Fig. 3E) revealed that OBs from both jaws (upper and lower) were of NC origin from development to adulthood. Dlx3, Msx1, Msx2 and Sox9 mRNA levels were significantly higher in mandible bone when compared to tibia bone at P9, 2M and 8M, whereas Hoxa10 and Hoxc10 mRNA was only detected in tibia bone (Fig. 3F). To investigate this embryonic signature ex vivo, we cultured primary osteoprogenitors from mandible (Mdcells) and tibia (Tb-cells) of P9 Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice (Fig. 3G-H). While the Md-cells that differentiated in vitro were double positive (GFP+, Tom+), Tb-cells displayed only GFP signal (Fig. 3G). RT-qPCR results showed differential expression of Dlx3, Msx1, Msx2 and Sox9 in Md-cells, and of Hoxa10 and Hoxc10 in Tb-cells persisted in vitro (Fig. 3H).

-Site-specific OB-omics: a prognostic tool for rare diseases

Using the OMIM database and a comprehensive literature review, we identified 29 TFs associated with human genetic disorders (out of 101 differentially expressed genes) in our omics data (Fig. 4A, Appendix Table 5). Twenty-six of these TF genes are directly associated with mineralized tissue pathologies, whereas 3 are linked to deficiencies in hematopoiesis and/or immune function (KLF1, IRF1 and IRF7) (Fig. 4B-C).

Twenty neurocristopathies are known to be caused by 9 TFs in Md-set (ALX1/3/4, DLX3, GLI3, GSC, MSX2, PAX3, SOX9). Except for IRF1, all the human disorders associated with Md-set genes display orofacial features. Thirteen TF genes are associated with jawbone phenotypes (11 in mandible, 5 in maxilla) as well as cranial bone pathologies (13/13), orofacial clefts (9/13), and dental abnormalities (6/13) (Fig. 4B). In humans, mutations of some Md-set TF genes are not associated with clinical jawbone features but instead with defects in cranial bones, palate and/or teeth (ALX3/4, DLX4, MSX1/2, PAX9, PBX1, SALL4, SOX11, SP6, TBX3) (Fig. 4C). Mutations of the Md-set genes DLX3, DLX5, GLI3, GSC, MSX2, SALL4, SOX9 and TBX3 cause not only craniofacial defects in humans, but also disorders in long bones and/or shoulder girdle/gluteal regions (Fig. 4C,Appendix Table 5).

The dysmorphological patterns of these human rare diseases are mirrored by the differential mRNA levels of TFs measured in mouse jaw and tibia OBs and bones by RNA-seq and qPCR (Fig. 1F, Fig. 2D-E 4). For example, mutations of TF genes highly expressed in Tb-OBs (Dlx3/5, Sox9) (Fig. 2D-E 5). This is also the case for NFIA and TBX15, which are more significantly expressed in Tb-set but at low levels and whose mutations in humans affect both long bones and craniofacial bones. Among the genes we found by RNA-Seq and which cause both oral and appendicular bone disorders in humans, are Dlx5, Pbx1 and Nfia; these genes are characterized by both bone site-specific TVs as well as TVs commonly expressed in OB-core (Appendix TF genes associated with limb bud formation and digit morphogenesis (Fig. 2A), mutations of 14 Md-set TF genes have been implicated in human digit malformations. Together, these data suggest that the OB transcriptome may be used as a diagnostic tool to predict the affected bone sites as well as other tissue disorders such as dental anomalies, neurocristopathies or digit malformations associated with TF mutations.

Discussion

This study defined the complete repertoires of TF genes expressed in jaw and tibia OBs in their native physiological environments. To do so, we established a new and easily reproducible approach by isolating functional OBs directly from tissues and thus avoiding cell culture, which may introduce biases in gene expression [START_REF] Ryu | Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation[END_REF]. Notably, our results evidenced differentially expressed transcript variants from a given gene between bone sites, including coding and non-coding RNA sequences, of key OB regulators such as Dlx5 and Runx2. These results are in line with osteocyte transcriptome mapping and strongly suggest additional levels of regulation of proteins synthesized by bone cells [START_REF] Youlten | Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease[END_REF].

In these OB populations free of contaminating cell-types and -stages, we identified a large TF set conserved between mandible and tibia OBs. This supports a model in which a central transcriptional network, OB-core, operates in all OBs of the skeleton independently of their localization. Other TF genes were site-specific, highlighting the importance of the experimental models chosen when studying bone disorders, notably those of the jaws. In line with previous studies [START_REF] Aïoub | Msx2 -/transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis[END_REF][START_REF] Duverger | In vivo impact of Dlx3 conditional inactivation in neural crest-derived craniofacial bones[END_REF][START_REF] Reichert | Mesodermal and neural crest derived ovine tibial and mandibular osteoblasts display distinct molecular differences[END_REF][START_REF] Nassif | Msx1 role in craniofacial bone morphogenesis[END_REF][START_REF] Lee | Comparison of gene expression between mandibular and iliac bone-derived cells[END_REF], the preferential expression of Msx/Dlx genes in mandible and Hox genes in tibia is supported here, and these genes are identified as being specifically related to functional OBs in vivo. Msx/Dlx/Hox homeoproteins operate under different contexts: (1) in early stages they drive rostro-caudal patterning and morphogenesis; and (2) thereafter, they trigger key OB functions, including the secretion of a number of non-collagenous peptides [START_REF] Molla | Enamel protein regulation and dental and periodontal physiopathology in MSX2 mutant mice[END_REF][START_REF] Duverger | Neural crest deletion of Dlx3 leads to major dentin defects through down-regulation of Dspp[END_REF]. Overall, the maintained spatial expression of several TFs supports the existence of developmental imprinting in differentiated OBs at postnatal and adult stages. Furthermore, in vivo NC cell tracking demonstrated the exclusivity of CNC and the absence of mesoderm participation in jaw OBs. In tibia, CNC participation was excluded. Thus, OBs maintain both a spatial and embryologic identity from growth to advanced post-natal stages. This implies that OBs differentiate from resident cell niches during growth and homeostasis, as fibroblasts do [START_REF] Rinkevich | Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential[END_REF][START_REF] Mah | Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts[END_REF].

In OB-core, although the cardinal bone TFs were found (e.g. Runx2, Sp7, Twist1), the vast majority of TF genes (almost 80%) had not been previously described as being expressed in differentiated OBs or associated with OB or bone. The same observation holds true for the Md and Tb sets. The present exhaustive inter-site comparative approach therefore completes the previously incomplete OB transcriptional profile and provides a better understanding of OBs within their physiological environments. The newly identified genes in the OB-core and sitespecific TF sets will allow reevaluation of the bone symptoms of the associated genetic diseases.

We confirmed the relevance of the jaw and tibia OB signatures by comparing the detected TFs to genes implicated in monogenic skeletal diseases. Genes and rare diseases affecting other organs were also analyzed. Such was the case for the teeth, which share an embryological origin with orofacial bones. Jaw OBs display a unique mesenchymal profile highly enriched with dental-related TF genes (e.g Pax9, Msx2, Dlx2, Sp6), consistent with human tooth and bone genes/phenotypes (de La Dure- [START_REF] De | Elements of morphology: Standard terminology for the teeth and classifying genetic dental disorders[END_REF]. Additionally, our bioinformatics analyses showed enrichment of biological processes involved with limb bud formation and digit morphogenesis in Md-set. Analysis of human mutations confirmed that 14 of these genes are associated with digit malformations. Thus, in addition to a number of genes consistently associated with neurocristopathies and craniofacial development, the present study also suggests molecular similarities between craniofacial bones and digits.

Notably, mutations of some TF genes of Md-set have not yet been associated with jawbone disorders in humans. These synopses appear to contradict not only the RNA-Seq, qPCR data and IF analysis for OB and raw jaw/tibia bones reported here, but also the described mouse jawbone phenotypes for various mutations, such as Alx3 and Alx4 [START_REF] Beverdam | Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice[END_REF]), Dlx4 [START_REF] Wu | DLX4 is associated with orofacial clefting and abnormal jaw development[END_REF], Msx1 [START_REF] Nassif | Msx1 role in craniofacial bone morphogenesis[END_REF]) and Msx2 [START_REF] Aïoub | Msx2 -/transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis[END_REF]). These observations support that jawbone abnormalities might also be present but not diagnosed in human disorders and highlight the need for systematic exploration of the jawbones while investigating skeletal and dental diseases.

Apart from rare diseases, this molecular signature may underlie known site-specific bone behavior in pathophysiology. For instance, the exclusive CNC origin and Hox-negative status of jaw OBs are shared with other oral mesenchymal cells such as gingival fibroblasts (Isaac et al. 2018). This singular profile has been shown to provide these cells with increased stemness and regenerative potential [START_REF] Wang | Regeneration, repair and remembering identity: the three Rs of Hox gene expression[END_REF][START_REF] Jiang | Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring[END_REF]). These qualities are crucial for tissue regeneration and stem cell therapies, as consistently reflected in the superior efficiency of jaw-versus long-bone grafts [START_REF] Akintoye | Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals[END_REF][START_REF] Leucht | Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration[END_REF].

In conclusion, the OB TF transcriptome provides insights into the pathways governing sitespecific OB development and homeostasis, and identifies novel OB regulators involved in bone physiology and human rare diseases. Integrative analysis of the OB reveals that its TF signatures appear to be developmental imprints of distinct embryonic origin (NC/mesoderm) that may directly influence the behavior of bones and dictate their anatomical site specificities.

Finally, the present study also establishes the importance of the OB transcriptome as a prognostic tool to predict affected bone sites as well as other tissue disorders such as dental anomalies, neurocristopathies or digit malformations associated with TF mutations. Indeed, deciphering these Md-OB and Tb-OB signatures will facilitate our understanding of the pathophysiology of phenotypes associated to polymorphisms and/or identified by genome-wide association studies (GWAS). 2 (for 2M, 4M and 8M), embedded in OCT (VWR), sectioned with cryostat and scanned using a ZEISS Axio Scan.Z1 slide scanner (Zeiss) as previously described (Isaac et al. 2018). For IF analyses, frozen sections were warmed at room temperature for 20 minutes and blocked with 10 mg/mL bovine serum albumin (BSA) (Sigma-Aldrich) and 0.1% Triton (Thermo Fischer Scientific)-X100 in PBS (Thermo Fischer Scientific) for two hours. After two washes in 1X PBS, sections were incubated overnight at 4 °C with rabbit monoclonal Runx2 antibody at 1:500 (Abcam, ab192256), rabbit monoclonal Sox9 antibody at 1:500 (Abcam, ab185966), rabbit monoclonal Sp6 antibody at 1:500 (Abcam, ab246952) or rabbit monoclonal Sp7 antibody at 1:1000 (Abcam, ab209484). Sections were then washed and incubated for 2 hours at room temperature with Alexa Fluor® 647 dye (Cy5) conjugated to donkey anti-rabbit IgG (1∶500, Thermo Fischer Scientific). Sections were mounted using DAPI Fluoromount-G® (Clinisciences).

Pax3 M1 M2 M3 T1 T2 T3 Alx1 Alx3 Alx4 Barx1 Dlx1 Dlx2 Dlx3 Dlx5 (v54) Foxf1 Gli3 Gsc Hand2 Msx1 Msx2 Mycn Nfatc4 (v46) Pax3 Pax9 Pbx1 (v08) Prrx2 Rarb Rxrb (v16) Sall4 Smad5 (v42) Sox11 Sox5 Sox9 Tcf12 (v64) Tcf7l2 (v19) Tcf7l2 (
For cell counting, regions of interest (ROI) were manually selected on tissue sections and cell counts using Cyanine-5 (Cy5), Tomato (Tom), GFP and DAPI signals were determined by using Oncotopix software (Visiopharm) and QuPath v0.3.0 (Bankhead et al. 2017). The number of GFPpositive/Tom-positive and GFP-positive/Tom-negative cells or the number of GFP-positive/Cy5positive and GFP-positive/Cy5-negative cells was then divided by the total number of boneforming osteoblastic cells (GFP-positive) for each tissue section to obtain percentages. To limit artifacts, only DAPI-positive cells were counted.

Bone microdissection

For mouse E10.5 embryos, heads were meticulously dissociated from the body. At E14.5, the frontal part of the heads (referred as "Face") and the limbs were isolated. Of note, both forelimbs (FL) and hindlimbs (HL) were pooled and referred as "Limb". At E16.5, E18.5 and P1, the mandibles (with developing teeth) were meticulously isolated and referred as "Mandible (Md)".

At E16.5, whole hindlimbs (HL) were isolated. At E18.5 and P1, tibias (Tb) were individualized.

For mandibles at P9, 2M and 8M, the ascending branch, muscle tissue, molars and incisors were meticulously removed to preserve only basal and alveolar bones. For tibias, cartilage caps and epiphyses were removed, bone marrow cells were flushed, and the remaining cortical bone was harvested. All dissections were performed under a microscope (Zeiss).

The obtained tissue fragments were then rinsed in 1X PBS and processed for cell isolation (FACS and primary cell culture) and for RNA extraction and analyses (RNA-seq and RT-qPCR).

Cell isolation and primary cell culture

The aseptically dissected mandibles and tibias of Col1a1*2,3-GFP and Pax3-cre::Col1a1*2,3-GFP::Rosa tomato P9 mice were enzymatically digested with 0.25% collagenase type I (Sigma-Aldrich) in 1X PBS for 2 hours at 37 °C (two successive one-hour incubations). Dissociated cells were filtered using a 70-micron filter (Falcon, Thermo Fisher Scientific) and plated at a density of 8.4 × 10 4 cells/cm 2 in proliferative medium composed of DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS) (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). At confluency (D0), cells were grown in osteogenic medium composed of DMEM (Gibco) supplemented with 10% FBS, 50 µg/mL ascorbic acid (Sigma), 10 mM β-glycerophosphate (Sigma), and penicillin/streptomycin 1%. The cells were maintained at 37 °C in a fully humidified atmosphere in air with 5% CO2. Culture medium was changed at 48-h intervals. Cell RNA was isolated at Day 0, D9 and D18. extraction, reverse transcription and RT-qPCR analysis were performed as previously described [START_REF] Jacques | Tracking endogenous amelogenin and ameloblastin in vivo[END_REF]. Target genes were run in triplicate for each sample and normalized against the geometric mean of the mRNA levels of the housekeeping genes Hprt and Tbp. The specific primers used are listed in Appendix Table 1.

Flow cytometry cell sorting (FACS)

To analyze the cellular distribution in bone tissues and isolate cells for transcriptomics, FACS analysis was performed on the enzymatically dissociated and filtered cells from bone fragments of Col1a1*2,3-GFP and Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice using a BD LSRII instrument (BD) to collect cells according to their osteoblastic phenotype and embryonic origin.

RNA Sequencing (RNA-Seq)

Mandible and tibia bones from P9 Col1a1*2,3-GFP mice were dissected as described in the "Bone microdissection" section. Three independent dissections, each pooling bone tissues from approximately fifty P9 mice, were carried out. Immediately after dissection and collagenase I digestion, GFP-positive cells were FACS-sorted as described above, RNA was extracted using a single-cell RNA purification kit (Norgen) and RNA quantification and integrity were performed with a Bioanalyzer RNA (Agilent). Based on RNA quantification and RIN number, 3 experiments with RNA quantity ranging from 2 to 8 ng were selected and RNAseq analysis was performed by Oxford Gene Technology (OGT). Briefly, generation of cDNA was performed using SMART-Seq Raw sequencing data were processed as previously described (Isaac et al. 2014) with CASAVA 1.8.2 to generate FASTQ files. Reads of 100 bases were mapped to the mouse transcriptome and genome mm10 using TopHat version 2.1.0. Gene expression Reads Per Kilobase of transcript per Million mapped reads (RPKM) values and ANOVA scores were calculated using Partek GS 6.6.

Protocols of RNA extraction, cDNA library construction and data processing pipeline are publicly available in the Gene Expression Omnibus (GEO), together with the RNA-Seq raw and processed data files (accession# GSE186535)

Bioinformatics resources

The RNA-seq database was cross-referenced with a list of 1388 transcription factors (TF) generated using three databases [TcoF-DB v2 [START_REF] Schmeier | TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions[END_REF], TFcheckpoint [START_REF] Chawla | TFcheckpoint: a curated compendium of specific DNA-binding RNA polymerase II transcription factors[END_REF] and Universal Protein Resource (The UniProt Consortium 2021)]. This TF list has been deposited in the Gene Expression Omnibus (GEO) and is publicly available (accession# GSE186535). RNA-Seq-based transcriptome annotation and analysis and gene-ontology (GO) enrichment were performed using the DAVID Functional Annotation Clustering tool 6.8 (Huang et al. 2009a;Huang et al. 2009b), AmiGO2 [START_REF] Carbon | AmiGO: online access to ontology and annotation data[END_REF] and Metascape [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF].

Statistical analyses

All quantitative measurements were performed on at least three different animals per stage and per tissue. For cell counting, between 2 and 5 slides per animal were scanned and analyzed. Three independent primary cell cultures were performed, and one representative experiment is presented.

F

  GFP mice (JAX013134) and Pax3Cre KI mice (JAX-005549) were used to identify functional OBs and neural crest (NC) derivatives, respectively. These mouse models were crossed with Rosa tomato mice (JAX-008851) to obtain Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice. All experiments were presented to and approved by the Charles Darwin ethics committee (CEEACD/05) before the study began and conducted in accordance with the European legislation for the care and use of laboratory animals. This study adheres to the ARRIVE Guidelines for reporting animal. For ARRIVE guidelines checklist, mouse breeding, genotyping, histology, immunofluorescence (IF) analyses, bone microdissection, cell isolation, RNA extraction, RT-qPCR, bioinformatics resources and statistical analyses, see the Appendix.

  , Fig. 3F, Fig. 4D) and the site-specific in situ localization of TFs identified by IF (Fig. 1G-I and Fig. 2F-H) and previously reported (Appendix Table

Fig. 5 )

 5 Fig. 5) are associated with long-bone disorders in humans. Mutations of genes weakly expressed in both Md-OBs and Tb-OBs or with relatively low fold changes between the two OB populations affect both craniofacial bones and long bones (e.g. GSC, SALL4, TBX3) and/or are included in clinical synopses of short stature (GLI3, GSC, PBX1, SOX11, MYCN) (Appendix Table5). This is also the case for NFIA and TBX15, which are more significantly

Fig. 1 )

 1 Fig.1). Finally, in line with our bioinformatics analyses showing enrichment in Md-set with

  Figure and table legends

Figure 1 .

 1 Figure 1. Osteoblasts display a bone site-specific transcriptional profile and mesenchymal identity in their physiological environment (A) Workflow of the RNASeq transcriptional study of Md-OBs and Tb-OBs. (B) Total number of transcription factors (TFs) commonly expressed (OB-core), or overexpressed by Md-OBs (Md-Set) or by Tb-OBs (Tb-set). (C-E) Genes are classified according to their tissue affinities. (C) Gene ontology (GO) and enrichment analysis of TFs associated with mesenchymal tissues/cells in OB-core, Md-set and Tb-set (data are presented as -log10 (P value)). (D) RPKM expression heat map of TF genes in Md-set associated with mesenchymal tissues/cells (only RPKM values >1 are shown). (E) Genes known to be involved in different mesenchymal tissues but reported here for the first time as regulators of OBs and bone for the Md-set. (F) RT-qPCR analysis of in vivo gene expression of Sp6 in tissues isolated from C57BL/6JR mice at E10.5, E14.5, E16.5, E18.5, P1 and P9. mRNA levels are expressed in percentage of TF expression in P9 Md-bone. (G-I) Fluorescence imaging of functional OBs (GFP+) and Sp6expressing cells (pink+) in the first branchial arch (BA1) at E10.5 (G) and in jawbone (JB) at E15.5 (H) and P9 (I) in Col1a1*2,3-GFP mice (Scale bars: 200μm for the main images, 50μm for the inserts). BA1: first branchial arch, BP: biological process, GO: gene ontology, HL: Hindlimb, Inc: Incisor, JB: Jawbone, M: Meckel's cartilage, Md: Mandible, Mo: Molar, NT: neural tube, OB: osteoblast, OV: Otic vesicle, Tb: Tibia, TF: transcription factor, RPKM: Reads Per Kilobase of transcript per Million mapped reads, *site-specific genes (all the expressed transcripts of a given gene are increased in Md-OB), vXX: transcript variant identified by the last 2 numbers of the mRNA sequence NM number.

Figure 2 .

 2 Figure 2. The osteoblast transcriptome is characterized by site-specific developmental imprints (A) GO and enrichment analysis of TF genes associated with developmental processes and morphogenesis in OB-core, Md-set and Tb-set. (B) Venn diagram of TF genes associated with neural crest (NC) cells in OB-core, Md-set and Tb-set. (C) RPKM expression heat map of TF genes in Md-set and Tb-set associated with NC cells (only RPKM values >1 are shown). (D-E) RT-qPCR analysis of in vivo gene expression of Dlx3 (D) and Sox9 (E) in tissues isolated from C57BL/6JR mice at E10.5, E14.5, E16.5, E18.5, P1 and P9. mRNA levels are expressed in percentage of TF expression in P9 Mdbone. (F-H) Fluorescence imaging of functional OBs (GFP+) and Sox9-expressing cells (pink+) in the first branchial arch (BA1) at E10.5 (F) and jawbone (JB) at E15.5 (G) and P9 (H) in Col1a1*2,3-GFP mice (Scale bars: 200μm for the main images, 50μm for the inserts). BA1: first branchial arch, BP: biological process, CNC: Cranial Neural Crest, GO: gene ontology, HL: Hindlimb, Inc: Incisor, JB: Jawbone, M: Meckel's cartilage, Md: Mandible, Mo: Molar, NT: neural tube, OB: osteoblast, OV: Otic vesicle, Tb: Tibia, TF: transcription factor, RPKM: Reads Per Kilobase of transcript

Figure 3 .

 3 Figure 3. Osteoblast embryonic positional identity is maintained in vivo and ex vivo (A-D) Fluorescence imaging of functional OBs (GFP+) and NC-derived cells (Tomato+) in jawbone at E15.5 (A), P9 (B) and 2M (C) in Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice (Scale bars: 500μm for the main images, 50μm for the inserts). (D) Double labeling flow cytometry for GFP and Tomato of cells isolated from mandible and tibia of P9 Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice. (E) Quantification (cell counting) of NC-derived cells (tomato+) in mandible, maxillary and tibia from Pax3cre::Col1a1*2,3-GFP::Rosa tomato mice at 4M. (F) RT-qPCR analysis of in vivo gene expression of Dlx3, Msx1, Msx2, Sox9, Hoxa10 and Hoxc10 in mandible and tibia bone tissues isolated from C57BL/6JR mice at P9, 2M and 8M. mRNA levels are expressed in percentage of TF expression in P9 Md-bone (for Dlx3, Msx1, Msx2, Sox9) or in P9 Tb-Bone (for Hoxa10 and Hoxc10). (G) Primary cell culture of osteoprogenitors isolated from mandible and tibia of P9 Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice. At confluency, cells were grown in osteogenic medium and observed at day 0 (D0), day 9 (D9) and day 18 (D18) (Scale bar: 400μm). (H) RT-qPCR analysis of ex vivo gene expression of Dlx3, Msx1, Msx2, Sox9, Hoxa10 and Hoxc10 in mandible and tibia osteoprogenitors. mRNA levels are expressed in percentage of TF expression in D0 mandible cells (for Dlx3, Msx1, Msx2, Sox9) or in D0 tibia cells (for Hoxa10 and Hoxc10). Inc: Incisor, JB: Jawbone, M: Meckel's cartilage, Md: Mandible, Mo: Molar, NC: Neural Crest, OB: osteoblast, PH: phase, Tb: Tibia, TF: transcription factor, TG: Tooth Germ.

Figure 4 :

 4 Figure 4: Human mutations associated with TF genes of jaw and tibia site-specific osteoblast signatures and their consequences in pathophysiology. (A) RPKM expression heat map of TF genes in Md-set and Tb-set associated with human genetic disorders (only RPKM values >1 are shown). (B-C) OMIM and Orphanet based gene-by-gene analysis of genetic disorders and conditions caused by human mutations of Md-set and Tb-set according to tissue type and anatomical region. Pathological growth modification patterns are indicated with yellow arrows. (D) RTq-PCR analysis of in vivo gene expression of Alx3, Pax3 and Pax9 in mandible and tibia bone tissues isolated from C57BL/6JR mice at P9, 2M and 8M. mRNA levels are expressed in percentage of TF expression in P9 Md-bone. TF: transcription factor, OB: osteoblast, Md: Mandible, Tb: Tibia, RPKM: Reads Per Kilobase of transcript per Million mapped reads, *site-specific genes (all the expressed transcripts of a given gene are increased in Md-OBs), vXX: transcript variant identified by the last 2 numbers of the mRNA sequence NM number. Abbreviations of human genetic disorders: AI: Amelogenesis imperfecta, AML: Acute myelogenous leukemia, AMS: Ablepharon-macrostomia syndrome, BRMUTD: Brain malformations with or without

  Dissected bone samples were put in TRI-Reagent (Euromedex) and homogenized with a tissue homogenizer (Omni International) and cellular layers were scraped in TRI-reagent. RNA

  Input RNA Kit (Takara) and processed with an Illumina TruSeq RNA Sample Preparation kit (Illumina). Data were generated with an Illumina HiSeq 2000 sequencing system.

  -CCC-CAA-ACT-TCG-AC CTG-CGC-AAA-AGT-CCT-GTG-TG 175 mGfi1b NM_008114 CAA-GGT-GTT-CTC-CAC-CCC-TC GAA-GCT-TCG-CTC-CTG-TGA-GT 154 mHand2 NM_010402.4 AGA-AGA-GGA-AGA-AAG-AGC-TGA-ATG ACC-GGA-TGA-TCC-AAC-TGC-AA 228 mHoxa10 NM_001122950 ACT-AAG-AGC-AGC-ACG-GTA-CG GCT-GCA-TTT-TCG-CCT-TTG--CAA-GAA-GGC-ACA-GA TGG-AGT-GGA-TGG-ATG-GGG-AT 279 mSmad5 NM_001164042 GGA-ACC-CTA-AGC-TCT-GGG-AA CTT-AGT-GCA-AGT-CCT-CGA-CCA 121 mSox9 NM_011448 CAC-AAG-AAA-GAC-CACC-CCG-A GGA-CCC-TGA-GAT-TGC-CCA-GA 209 mSp6 NM_031183 CAC-TGC-TTC-TGC-CAG-CTC-T CCA-CAG-ACA-GCG-GTT-AGC-

  RTq-PCR analysis of in vivo gene expression of 3 TF genes from OB-core previously known for OB and bone regulation (Atf3, Klf6, Runx2 and Tsc22d1) and 3 TF genes from OB-core newly described in differentiated OB (Arib5b, Meox2 and Ybx3) in mandible and tibia bone tissues isolated from C57 BL/6JR mice at P9. mRNA levels are expressed in percentage of TF expression in P9 Md-bone. No difference in expression is observed for these 6 genes between bone and tibia. TF: transcription factor, OB:

	performed according to the number of parameters using either an unpaired t-test or two-way ANOVA followed by Tukey's multiple comparisons test. Data are expressed as mean ± standard error of mean (SEM) and significance values are shown in the figures. Non detected (ND), p>0.5 (ns), p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), p≤0.0001 (****). Area-proportional Venn diagrams transcription factor, OB: osteoblast, Md: Mandible, Tb: Tibia, TV: transcript variant, NM: mRNA RefSeq accession prefixes for validated protein-coding transcripts, NR: RNA RefSeq accession prefixes for non-protein-coding transcripts, FC: Fold Change. BC: Bone Collar, CB: Cortical Bone, Inc: Incisor, JB: JawBone, M: Meckel's cartilage, Md: Mandible, MC: for OB-core. (D-E) osteoblast, Md: Mandible, Tb: Tibia, Rpkm: Reads Per Kilobase of transcript per Million mapped reads, A. Medullary Cavity; MesC: Mesenchymal Condensation, Mo: Molar, OB: osteoblast, Tb: Tibia, TG: Tooth Germ.
	*site-specific genes (all the expressed transcripts of a given gene are commonly expressed in Md-OBs and were designed using BioVenn (Hulsen et al. 2008). Tb-OBs), vXX: transcript variant identified by the last 2 numbers of the mRNA sequence NM number.
	C.	o r F o r F o r F o r F o r F o r D. F o r F o r F o r	E.
		P e P e P e P e P e P e P e P e P e
		e r e r e r e r e r e r e r e r e r
		R e R e R e R e R e R e R e R e R e
		v v v v v v v v v
		i e i e i e i e i e i e i e i e i e
		w w w w w w w w w
			6 8 10 11 12 13 14

Normalized mRNA expression (Q-PCR)

Table 2 : RNA-seq data summary of the bone site-specific TF profile of functional OB isolated from mouse mandible and tibia using RNA-seq.

 2 Filtering steps included: q-values ≤ 0.1 (for multi-test adjustment), FC ≥ 1.4 or ≤ -1.4, and mean RPKM ≥ 1 for expressed genes. 20 highlighted in red are related to cranial neural crest (CNC). Corresponding human gene symbols and genes ID were established using ToppGene Suite[START_REF] Chen | ToppGene Suite for gene list enrichment analysis and candidate gene prioritization[END_REF]).Zebrafish 24 hpf embryos: NC cells of frontonasal bourgeon and anterior neurocranium skeletal progenitors

	r
	P e
	e r
	R e
	v
	i e
	w
	18

TF: transcription factor, OB: osteoblast, Md: Mandible, Tb: Tibia, RPKM: Reads Per Kilobase of transcript per Million mapped reads, NM: mRNA RefSeq accession prefixes for validated protein-coding transcripts, NR: RNA RefSeq accession prefixes for non-protein-coding transcripts .

Table 4 : Non exhaustive compilation of in situ localization in bone tissues of selected TFs previously reported in the literature using immunohistochemistry, immufluorescence, in situ hybridization and transgenic animals with reporter genes.

 4 : Apical Ectodermal Ridge, E: Embryonic Day, EH: Embryonic Hour, HPF: Hours Post Fertilization, ISH: in situ hybridization, M: Month, NC: neural crest, P: Post Natal Day, PW: Post Natal Weeks, OB: osteoblast,

	TG: Transgenic,

AER

Table 5 : Compilation of human clinical synopses associated with Md-set and TB-set TF gene mutations with Mendelian association (OMIM and Orphanet)

 5 AER: apical ectodermal ridge, IDM: interdigital mesenchyme, NCP: neurocristopathies[START_REF] Vega-Lopez | Neurocristopathies: New insights 150 years after the neural crest discovery[END_REF], ZPA: zone of polarizing activity.Abbreviations of human genetic disorders: CAKUTHED: Congenital anomalies of kidney and urinary tract syndrome with or without hearing loss, abnormal ears, or developmental delay, ICF2: Immunodeficiencycentromeric instability-facial anomalies syndrome 2, , IVIC: Instituto Venezolano de Investigaciones Cientificas, PFM: Parietal foramina 1, , RMS: Rhabdomyosarcoma 2, alveolar, SAMS: Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities, SHFM: split hand/foot malformation), TDO:

	trichodontoosseous syndrome

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Acknowledgements

This work was supported by the Université de Paris and the French National Research Agency (ANR) [IdEx Université de Paris (grant ANR-18-IDEX-0001) and the Osteodiversity-ANR consortium (ANR-12-BSV1-0018)], grants from "Fondation des Gueules Cassées" and the Federation Hospitalo-Universitaire FHU DDS-ParisNet, a grant from Assistance Publique-Hôpitaux de Paris (AP-HP), Inserm and Université de Paris. We thank Dr. Benoit Robert and Dr. Yvan Lallemand of the Institut Pasteur, the INSERM UMRS 1138 Histology, Cell Imaging and Flow Cytometry Platform (CICH) and the animal core facilities of INSERM UMRS 1138 for their expert technical support. 1 Transcriptional regulation of jaw osteoblasts: development to pathology A. Nassif, G. Lignon, A. Asselin, C.C. Zadikian, S. Petit, H.W. Sun, C. Klein, F.C. Ferré, M.I. Morasso, A. Berdal, B.P.J. Fournier, J. Isaac

Data presentation and statistical analyses were performed using Prism 9 software (GraphPad Software). After the normality was tested using the Shapiro-Wilk test, statistical analyses were http://mc.manuscriptcentral.com/jdr Journal of Dental Research

Author contributions

A.N: Contributed to acquisition, and interpretation, drafted the manuscript critically and revised the manuscript G. L: Contributed to acquisition, and interpretation, drafted the manuscript critically and revised the manuscript A. A: Contributed to conception and design, to acquisition and interpretation, and critically revised the manuscript S. P: Contributed to acquisition and critically revised the manuscript C.C. Z: Contributed to acquisition and critically revised the manuscript H.W.S: Contributed to analysis and critically revised the manuscript C. K: Contributed to acquisition and critically revised the manuscript A. B: Contributed to analysis and interpretation and critically revised the manuscript B.J.P.F: Contributed to analysis and interpretation and critically revised the manuscript J. I: Contributed to conception and design, acquisition, analysis, and interpretation, performed all statistical analyses, drafted the manuscript and critically revised the manuscript All authors gave their final approval and agree to be accountable for all aspects of the work.

Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

GO:0045667 14 Cebpb, Cebpd, Dlx5, Gli1, Hey1, Jund, Mef2c, Nfatc1, Runx2, Smad1, Smad3, Smad5, Snai2, Twist1

Appendix Appendix Materials and Methods

Animal models and study design

To identify functional OBs, Col1a1*2,3-GFP mice that express GFP under the control of a 2.3-kb rat procollagen, type 1, alpha 1 promoter (JAX013134) were used [START_REF] Kalajzic | Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage[END_REF]. Neural crests (NC) cells and their derivatives were investigated using a Pax3-cre mouse strain expressing Cre recombinase from the endogenous Pax3 locus (JAX-005549) [START_REF] Lang | Pax3 functions at a nodal point in melanocyte stem cell differentiation[END_REF]. These mouse models were crossed with transgenic mice carrying a tomato flox/flox reporter, Rosa tomato mice (JAX-008851) to obtain Pax3-cre::Col1a1*2,3-GFP::Rosa tomato mice. C57BL/6JR mice were used as an inbred strain and for a control. Mice were genotyped as previously described (Isaac et al. 2018).

Histology, immunofluorescence (IF) imaging and cell counting analyses

Heads and hindlimbs (HL) from transgenic mice at embryonic day 10.5 (E10.5), 14.5 (E14.5), 15.5 (E15.5), 16.5 (E16.5), 18.5 (E18.5), post-natal day 1 (P1), post-natal day 9 (P9), 2 months (2M), 4 months (4M) and 8 months (8M) were harvested, fixed, decalcified with EDTA (Sigma-Aldrich) Bone-related TF genes TF OB-core

Appendix figures and

Appendix Tables

Appendix Table 1: List of specific primers used for RT-qPCR.

Each primer pair was designed with primer-BLAST to span an exon-exon junction.


C Angotti

M C Venier-Julienne

C Penna

S Femminò

L Sindji

C Paniagua

C N Montero-Menei

Prof Pasquale Pagliaro
email: pasquale.pagliaro@unito.it

Venier-Julienne Mc

Montero-Menei Cn

O.M.P S Associate

Nanoprecipitated catestatin released from

Keywords: microcarriers, drug delivery, catestatin, mesenchymal stem cells, hypoxia

published or not. The documents may come

Introduction

Prolonged hypoxia an oxidative stress conditions are responsible of the damage observed in the cardiovascular system in several pathological situations; consequently prolonged effects of pro-survival factors are necessary tools to limit such a damage. Among emerging peptides in the cardiovascular system Chromogranin-A (CgA) derived peptides are occupying a role of paramount importance. CgA is a key player in the neuroendocrine regulation of cardiac function. In fact, CgA is ubiquitously distributed in the nervous system, immune system and diffuse neuroendocrine system of both vertebrates and invertebrates [START_REF] Helle | The granin family of uniquely acidic proteins of the diffuse neuroendocrine system: comparative and functional aspects[END_REF][START_REF] Penna | Cardioprotection against ischemia/reperfusion injury and chromogranin A-derived peptides[END_REF][START_REF] Pieroni | Myocardial production of chromogranin A in human heart: a new regulatory peptide of cardiac function[END_REF]. CgA is a pro-hormone which is stored in vesicles [START_REF] Taupenot | The chromogranin-secretogranin family[END_REF] and its processing gives rise to several peptides of biological importance, including serpinin, pancreastatin, vasostatin, and Catestatin (CST) [START_REF] Penna | Cardioprotection against ischemia/reperfusion injury and chromogranin A-derived peptides[END_REF].

CST is a 21-amino acid derivate of CgA and initially it was described as a potent inhibitor of catecholamine release from adrenal medulla [START_REF] Ratti | Structure-activity relationships of chromogranin A in cell adhesion. Identification of an adhesion site for fibroblasts and smooth muscle cells[END_REF][START_REF] Tota | Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart[END_REF], but in the last years several new biological activities have been described, including a cardioprotective role. Moreover a myocardial production of CST was demonstrated recently [START_REF] Bassino | A novel catestatin-induced antiadrenergic mechanism triggered by the endothelial PI3K-eNOS pathway in the myocardium[END_REF][START_REF] Bassino | Catestatin exerts direct protective effects on rat cardiomyocytes undergoing ischemia/reperfusion by stimulating PI3K-Akt-GSK3β pathway and preserving mitochondrial membrane potential[END_REF][START_REF] Biswas | Chromogranin/secretogranin proteins in murine heart: myocardial production of chromogranin A fragment catestatin (Chga(364-384))[END_REF][START_REF] Penna | Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart[END_REF][START_REF] Penna | Cardioprotection against ischemia/reperfusion injury and chromogranin A-derived peptides[END_REF][START_REF] Perrelli | Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling[END_REF]. The anti-adrenergic effects of CST in the heart muscle seem to be mediated by the PI3K-Akt-eNO-Synthase signaling axis [START_REF] Bassino | A novel catestatin-induced antiadrenergic mechanism triggered by the endothelial PI3K-eNOS pathway in the myocardium[END_REF][START_REF] Bassino | Catestatin exerts direct protective effects on rat cardiomyocytes undergoing ischemia/reperfusion by stimulating PI3K-Akt-GSK3β pathway and preserving mitochondrial membrane potential[END_REF].

Moreover a positive effect of CST on monocyte migration in-vitro and the induction of angiogenesis, arteriogenesis and vasculogenesis in the unilateral mouse hind limb ischemia model by a basic fibroblast growth factor (bFGF)-dependent mechanism has been demonstrated [START_REF] Egger | Monocyte migration: a novel effect and signaling pathways of catestatin[END_REF][START_REF] Theurl | The neuropeptide catestatin acts as a novel angiogenic cytokine via a basic fibroblast growth factor-dependent mechanism[END_REF]. Based on the above-mentioned findings, it is clear that CST exerts a protective and proangiogenetic role via the activation of survival kinases.

Hence, CST is a very promising agent for cardiovascular protective purposes; however, usually, proteins have a very short half-life after administration and must be conveniently protected. The combination of cardioprotective agents and the use of biomaterials to improve cell behavior within the infarcted area is an interesting approach in cardiovascular regenerative medicine. Pharmacologically active microcarriers (PAMs), biodegradable and biocompatible polymeric microspheres containing a bioactive protein, have the ideal characteristics for this purpose. Besides being able to deliver physiological doses of a relevant bioactive protein released in a controlled manner, they may also convey cells on their 3-dimensional (3D) biomimetic surface of extracellular matrix molecules. In this way the combined action of the bioactive protein and the biomimetic surface can favor the survival and engraftment of the transported cells after their transplantation in different pathological conditions [START_REF] Delcroix | The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats[END_REF][START_REF] Karam | Combining adult stem cells and polymeric devices for tissue engineering in infarcted myocardium[END_REF][START_REF] Musilli | Pharmacologically active microcarriers for endothelial progenitor cell support and survival[END_REF][START_REF] Quittet | Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat[END_REF][START_REF] Savi | Enhanced engraftment and repairing ability of human adipose-derived stem cells, conveyed by pharmacologically active microcarriers continuously releasing HGF and IGF-1, in healing myocardial infarction in rats[END_REF][START_REF] Tatard | Pharmacologically active microcarriers releasing glial cell line -derived neurotrophic factor: Survival and differentiation of embryonic dopaminergic neurons after grafting in hemiparkinsonian rats[END_REF]. Recently, it has been shown that PAMs releasing vascular-endothelial-growth-factor (VEGF) and conveying MSCs (derived from the adipose tissue) may induce their proliferation, angiogenic differentiation, and a decrease in cell death in vitro [START_REF] Penna | Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival[END_REF][START_REF] Madonna | Transplantation of adipose tissue mesenchymal cells conjugated with VEGF-releasing microcarriers promotes repair in murine myocardial infarction[END_REF]. Moreover, in an in vivo mice model of post-ischemic myocardium these functionalized PAMs implanted in the infarcted myocardium enhanced MSC engraftment and differentiation into vascular structures resulting in myocardial fractional shortening improvement [START_REF] Madonna | Transplantation of adipose tissue mesenchymal cells conjugated with VEGF-releasing microcarriers promotes repair in murine myocardial infarction[END_REF]. Within this line, obtaining a prolonged release of physiological doses of bioactive CST, a small peptide that can diffuse within the tissue parenchyma and exert cardioprotective properties, is an interesting strategy. The delivered peptide may not only repair the damaged tissue, but also in combination with the 3D biomimetic surface of the PAMs act synergistically to enhance the engraftment of the transported MSCs. Fibronectin-covered PAMs that stimulate MSC survival (Garbayo el at 2011) and CST, a catecholamine release inhibitor with antihypertensive, proangiogenetic and cardioprotective properties [START_REF] Penna | Cardioprotection against ischemia/reperfusion injury and chromogranin A-derived peptides[END_REF], may be a novel ideal combination to repair the organ damage caused by an hypoxic insult.

However, the prolonged and complete delivery of proteins at physiological doses still remains a technological challenge for effective clinical applications, mainly due to proteinpolymer interaction during the process and protein degradation related to acidic environment following degradation of PLGA (D,L-lactide-co-glycolide acid) [START_REF] Aubert-Pouessel | A novel in vitro delivery system for assessing the biological integrity of protein upon release from PLGA microspheres[END_REF][START_REF] Park | A new preparation method for protein loaded poly(D, L-lactic-co-glycolic acid) microspheres and protein release mechanism study[END_REF]. With the aim to enhance protection of the protein from degradation and to obtain a more efficient release, we developed a strategy based on the use of a reversible nano-precipitated protein associated to a triblock copolymer P188 (poly (ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-poly(ethylene oxide)PEO) leading to protein protection, especially during the release step [START_REF] Giteau | Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres[END_REF]. Protein precipitates were formed from aqueous solution at the pH corresponding to the isoeletric point (pI) of the protein by the addition of sodium chloride to a water-miscible organic solvent, Glycofurol [START_REF] Karam | Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac tissue-engineering[END_REF]).

Our purpose in this study is to determine an appropriate method of nanoprecipitation of this small peptide, CST, for the encapsulation in PLGA-based microspheres preserving CST bioactivity. P188 will be used as a component of the microcarrier covalently linked to PLGA (PLGA-P188-PLGA) to render it more hydrophilic, thus avoiding denaturation by adsorption to hydrophobic surfaces, but also to retain the protective polymer inside the microcarriers during their degradation [START_REF] Morille | New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells[END_REF]. We thereafter evaluated the release kinetics of CST and the ability of the bioactive CST released from functionalized PAMs to protect bone marrow MSCs in standard in vitro conditions.

Materials and methods

Chemicals and drugs

Chemical reagents in general were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Polyvinyl alcohol (Mowiol ® 4-88) was obtained from Kuraray Specialities Europe (Frankfurt, Germany). P188 poloxamer or Lutrol® was kindly supplied by BASF (Levallois-Perret, France). PLGA-P188-PLGA were synthesized by IBMM-CRBA CNRS UMR 5247 (Montpellier, France) as previoulsy described [START_REF] Morille | New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells[END_REF].

Polytetrafluoroethylene (PTFE) filters Millex®-FH (pore size 0.45 μm) were obtained from Millipore (Millipore SA, Guyancourt, France). Culture mediums, penicillin, streptomycin and trypsin were obtained from M-Medical (Milan, Italy).

Protein precipitation and PAM formulation

Protein, glycofurol, and sodium chloride were used without further purification. Experiments were carried out at 4° C and the overall mixture was prepared directly inside a teflon tube.

The protein powder was first dissolved in a non-buffered aqueous solution. In previous experiments [START_REF] Karam | Combining adult stem cells and polymeric devices for tissue engineering in infarcted myocardium[END_REF][START_REF] Karam | Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac tissue-engineering[END_REF], we showed that the best protein precipitation efficiency corresponds to the isoeletric point (pI) of the protein; for CST nanoprecipitation (pI CST 11.72). Then, we studied the possibility to adjust the pH of the solution by dissolving the salt into Glycine-NaOH buffer (pH 8.6). Sodium chloride was added at different concentrations, and at a later stage the poloxamer P188 was added in Glycine-Na-OH buffer to a 20:1 poloxamer-protein ratio; finally this solution was introduced into glycofurol. Thirtyminutes later at +4°C, the protein particles were recovered by centrifugation (10,000g, 30 min, +4°C). Mixing and centrifugation times were selected in order to optimize the quantity of precipitated protein with an experimental design.

After this, microspheres where made by a non-denaturing solid-in-oil-in-water (s/o/w) emulsion evaporation/extraction technique [START_REF] Delcroix | The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats[END_REF]. Protein precipitates were dispersed in the organic phase (670 μL of 50 mg PLGA or PLGA-P188-PLGA dissolved in a 3:1 methylene chloride:acetone solution). The organic suspension was then emulsified in a poly (vinyl alcohol) aqueous solution (30 mL, 4% w/v at 1 °C) and mechanically stirred at 550 rpm for 1 min. After addition of 33 mL of deionized water and stirring for 10 min, the emulsion was added to 167 mL deionized water and stirred for 20 min to extract the organic solvent. Finally, the microspheres were filtered on a 5 μm Small Volume Low Pressure (SVLP) type filter, washed and freeze-dried (Paillard- [START_REF] Paillard-Giteau | Effect of various additives and polymers on lysozyme release from PLGA microspheres prepared by an s/o/w emulsion technique[END_REF]. PAM without protein were prepared following the same process, and called blank-microspheres (PAM) or FN-PAMs when covered with fibronectin (FN). To obtain FN-PAMs, PLGA-P188-PLGA microspheres were coated with FN and poly-D-Lysine (PDL). Coating solutions were prepared in Dulbecco's Phosphate-Buffered Saline DPBS.Each tube was covered with sigmacote® to prevent product loss on the tube walls. The concentration of the coating molecules was 9 μg/mL of FN and 6 μg/mL of PDL (corresponding to a 60:40 ratio of FN:PDL). 5 mg of PAM was added to DPBS and sonicated until full dispersion of the microspheres. The solution containing PDL and FN molecules was mixed to the microspheres suspension (final volume: 10 mL) and placed under rotation at 15 rpm at 37 °C during 1.5h. After coating, FN-PAMs were washed 3 times in distilled water, lyophilized and kept at -20 °C.

PAM characterization

The surface morphology of the microspheres was investigated by scanning electron microscopy (SEM, JSM 6310F, JEOL, Paris, France). Freeze-dried microspheres were mounted onto metal stubs using double-sided adhesive tape, vacuum-coated with a film of carbon using a MED 020 (Bal-Tec, Balzers, Lichtenstein). The average particle size and size distribution were determined using a Coulter Multisizer (Beckman Coulter, Roissy, France).

The microspheres were suspended in isotonic saline solution and sonicated for a few minutes prior to analysis. The mean particle sizes are expressed as volume distributions (N=3).

Zeta potential measurement was performed using a Zetasizer 2000 (Malvern Instruments, Orsay, France) operating at 150 V at RT was used to assess the variations of electrical surface charges of PAMs. Briefly, a suspension of PAMs at 0.3 mg/ml in 1 mM NaCl was measured after sonication (42 kHz, 10 s). Results are presented after conversion of electrophoretic mobility values to zeta-potential using Smoluchowski's equation. Experiments were performed in triplicate (N=3).

The fibronectin surface was characterized by confocal microscopy after FN immunostaining.

Lyophilized PAMs (1 mg) were suspended in DPBS containing 4% bovine serum albumin (BSA), 0.2% Tween 20 (DPBSBT) and incubated for 30 min at room temperature (RT) under 15 rpm stirring. Samples were then washed three times with DPBS and centrifuged (9000 g, 5 min, R.T.). Anti-FN mouse monoclonal antibody (1:100 in DPBSBT) was incubated at 37 °C for 1.5 h under rotation or overnight at +4°C. Samples were then washed 3 times before incubation with biotinylated anti-mouse IgG antibody (2.5 μg/mL in DPBS) for 1 h, at RT, under rotation. After three washes, samples were incubated with streptavidin-fluoroprobe 547 H (1:500 in DPBS) at RT, for 40 min, under rotation. Samples were observed under confocal microscopy (Olympus Fluoview TM FV2 300, Rungis, France). Three independent experiments were performed and every condition was observed in triplicate.

Experimental design

To define the optimum conditions of protein precipitation, an experimental design was used.

We performed a preliminary experimental design (2 4-1 fractional factorial design) with the aim to study the effect of 4 parameters and 3 interactions between these factors, supposing to influence the CST precipitation yield: the centrifugation speed, the contact time of mixture and the ionic strength of the protein aqueous solution (NaCl or ZnCl 2 concentration). The studied interactions are: NaCl concentration-ZnCl 2 concentration, NaCl concentrationcontact time, Time-ZnCl 2 concentration . The effect of the tested experimental factors was calculated using NEMROD software [START_REF] Mathieu | NEMROD®-W (New Efficient Methodology for Research using Optimal Design[END_REF]. Subsequently, the parameters identified as influent on protein precipitation yield, were optimised with a Doehlert matrix. This experimental design represents an optimal design for the spherical domain defined by the two factors and it allows a sequential approach [START_REF] Giteau | Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres[END_REF] .

Experiments required for this design are described in Fig. 1. It consists in a set of 7 distinct experiments uniformly distributed in a hexagonal plan (step 1). To approach the optimal conditions, three complementary experiments were added for the matrice expansion (step 2), and two supplementary experiments were performed (step 3). The measured response was the precipitation efficiencies of CST (%). The same experiments were performed for each condition: this allowed a direct comparison of responses.

Protein encapsulation efficiency

Protein encapsulation yield was determined considering the CST total protein entrapped.

Protein PLGA-P188-PLGA microspheres (5 mg) were dissolved in 500 l Dimethylformamide (DMF) in Teflon™ tube and vortexed. After 10 min 500 l of DMF was added and the solution was vortexed for 10 min more. The encapsulation efficiency was measured using NanoDrop 2000 Spectrophotometer (Thermo Scientific,France) immediately to avoid artifacts (N=3).

Release Kinetics

The release profile of protein from PLGA-P188-PLGA microspheres was determined by adding 500 l of PBS buffer, pH 7.4 to 5 mg of PAM-CST into eppendorf tubes. The tubes were closed and incubated in a shaking water bath (37°C, 125 r.p.m.). The tubes were centrifuged for 5-min. at 2800 g and 500 l of the supernatant were collected for analysis and replaced by fresh buffer. This procedure was repeated at different time-points (1, 2, 3, 4, 7, 10, 14 and 21 days) and the released CST present in the collected aliquots was measured by NanoOrange ® protein quantification kit (Invitrogen, Cergy Pontoise, France) following the manufacturer's guidelines. The theoretical amount of CST present in the 5 mg PAM-CST was corrected using the results of the encapsulation yield, thus allowing the actual amount of CST contained in the PAM-CST to be established. The cumulative release of CST-A over time was then calculated. Three independent experiments were performed.

MSC isolation and cell culture

Mesenchymal stem cells were extracted from bone marrow of femurs of Wistar rats 6-12 months of age (weight 450-550 g; Janvier, Le Genest St Isle, France). MSCs were extracted by inserting a 21-gauge needle into the shaft of the bone and flushing with a solution of minimum essential medium eagle  (α-MEM) and 20% fetal bovine serum (FBS) (Sigma-Aldrich, Milan, Italy) implemented with 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin (Lonza); the cell suspension was filtered and cultured at 37°C. After 24hrs the medium was replaced with α -MEM containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. We allowed MSCs to grow up to passage 3 (P3), replacing the medium every 2-3 days as reported in the literature [START_REF] Gallo | Limited plasticity of mesenchymal stem cells cocultured with adult cardiomyocytes[END_REF][START_REF] Muscari | Polyamine depletion reduces TNFalpha/MG132-induced apoptosis in bone marrow stromal cells[END_REF][START_REF] Penna | Early homing of adult mesenchymal stem cells in normal and infarcted isolated beating hearts[END_REF][START_REF] Raimondo | Morphological characterization of GFP stably transfected adult mesenchymal bone marrow stem cells[END_REF][START_REF] Wang | Cell fusion is the principal source of bonemarrow-derived hepatocytes[END_REF].

All animal experiments comply with the ARRIVE guidelines and are carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, as well as with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The animal protocols followed in this study were approved by the local "Animal Use and Care" committees (RR # 145674720268, dated 08/04/2013).

In vitro study: survival after hypoxia/reoxygenation with and without pretreatment with free-CST, FN-PAMs or FN-PAM-CST

Experiments were carried out with MSCs alone or after pre-treatment of MSCs with either FN-PAMs, FN-PAM-CST or CST alone (free CST) in normoxic and hypoxic experimental condition, as previously described [START_REF] Penna | Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival[END_REF]. Briefly, the bone marrow-MSCs were pre-treated with free CST (5 nM), FN-PAMs (0.5 mg) or FN-PAM-CST (0.5 mg) for 24-hrs.

Before exposure to hypoxic condition, MSCs were incubated with trypsin-EDTA 0.25% solution and subjected to mild centrifugations (250-300 g for 5-min.) in order to separate cells from FN-PAMs or FN-PAM-CST. Then the medium was changed and cells were cultured in 2% FBS without treatment factors. Thereafter, subgroups of cells were subjected to a protocol of hypoxia/reoxygenation (H/R) (72/3 hrs) in a hypoxic chamber (INVIVO 2 200,Belsar,Varese,Italy). Accordingly the groups considered in the study of cell survival were the following (Fig. 2 In other words and in brief, in each of the above four groups, we considered normoxic (-N) and hypoxic (-H) protocols for comparative purpose.

At the end of experiments, in triplicate in a blinded fashion, cell survival was assessed, using the cell viability test 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, Italy) [START_REF] Wang | Cell fusion is the principal source of bonemarrow-derived hepatocytes[END_REF].

Statistical analysis

Data are presented as mean +/-SEM. Statistical significance was performed with t test or one-way ANOVA (post-hoc test Newman-Keuls Multiple Comparison Test) using GraphPad Prisma. The results were considered significant when P< 0.05.

Results

Characterization of PAM

Morphological features of PAM and release profile of CST from PAM are reported in Fig 3-I and 3-II, respectively. The average diameter and size distribution of the resulting microspheres were evaluated using a Multisizer® Coulter Counter. We obtained microspheres with a diameter of 60 μm ± 22 (Fig. 3-Ia). The morphological aspect of MS, determined by scanning electron microscopy (SEM), showed a spherical shape (Fig. 3-Ib,c).

To obtain FN-PAMs, PLGA-P188-PLGA microspheres were coated with FN and Poly-D-Lysine (PDL) (FN:PDL is 60:40). The microspheres had a zeta potential of 42,9±1,75 indicating the coating of PDL on their surface allowing cell attachment. The fibronectin surface characterized by confocal microscopy after FN immunostaining (Fig. 3-Id,e) showed that they were covered by this extracellular matrix protein. The effects of the parameters studied and of the interactions between these parameters are shown in Table 1 and graphically represented in Fig. 4. Preliminary experiments, testing multiple parameters supposed influencing CST precipitation, reveal how centrifugation speed and use of ZnCl 2 have a negligible effect on nanoprecipitation yield of CST (Fig. 4). As

Experimental design

N°Exp

shown in Table 1, we obtain low percentage of nanoprecipitation yield using ZnCl 2 0.008M in combination with different centrifugation speeds. However, the difference in the ionic strength of the protein aqueous solution, using NaCl instead of ZnCl 2 , and the increased contact time of mixture have a positive effect (Fig. 4) and lead to a higher nanoprecipitation yield (63.4%), as shown in Table 1. Subsequently, these two parameters identified as influent on protein precipitation yield, were optimised with a Doehlert matrix (Fig. 5). We found that the optimal conditions to obtain a good percentage of CST nanoprecipitation of 76% of the initial protein (Step 3), was obtained by increasing the contact time of the mixture until 90 min. at a 2.175 M NaCl. Furthermore, in order to obtain a good precipitation yield the protein needs to be in a pH which is similar to its isoeletric point [START_REF] Karam | Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac tissue-engineering[END_REF], so we dissolved the salt in Glycine-NaOH buffer (pH 8.6). This condition allowed to obtain the best protein precipitation efficiency.

Release of CST from FN-PAM-CST

The encapsulation yield of total protein into FN-PAM-CST was 62%. After the release kinetics study we found a continuous release of CST from these PAMs. The release profile showed a burst effect during the first 24 h of almost 50% of CST corresponding to 15 µg for 5mg of PAM (Fig. 3-II). This initial burst is followed by a quite homogeneous and continuous release until the end of the release period. The total amount released in three weeks for 5 mg PAMs corresponded to around 45 µg, which is within the range of the physiological doses of CST that may be used with around 1 mg PAMs in vitro. The release profile observed at the end of 3 weeks shows a cumulative release representing 100% of the entrapped protein. experiments [START_REF] Penna | Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart[END_REF][START_REF] Bassino | Catestatin exerts direct protective effects on rat cardiomyocytes undergoing ischemia/reperfusion by stimulating PI3K-Akt-GSK3β pathway and preserving mitochondrial membrane potential[END_REF].

Survival

As can be seen in Fig 6a, in the absence of pre-treatment (CTRL), the H/R protocol induced a 20% reduction in MSC number (p< 0.01 vs control). While this H/R protocol was unable to induce cell death in the presence of FN-PAM, in the presence of either free CST or FN-PAM-CST, the MSC continued to even proliferate despite the H/R protocol (p< 0.01 vs H/R only, for both). Therefore, pre-treatment with either CST and/or FN-PAM are able to counteract hypoxia effects.

We also analyzed MSC proliferative behavior in normoxia (Fig 6b) and found out that CST was able to induce a slight proliferation, which was ameliorated by FN-PAM-CST. In fact, in normoxia MSC proliferation increased significantly with FN-PAM-CST only.

These results suggest that there is no loss of bio-activity of CST after nanopreciptation procedure, which actually slightly improved the activity of free CST, when slowly released from FN-PAM. Moreover, in H/R experiments, pre-treatment with FN-PAM-CST not only limited cell mortality, but also improved cell growth.

Discussion

Catestatin is a promising agent for cardiovascular protective purposes and use of PAMs to convey MSCs, and/or to deliver CST could be a good approach to repair the damage observed in the cardiovascular system in several pathological situations. In this study, we have hypothesized that a continuous release of physiological low doses of CST by FN-PAM may act on cells and counteract in a better way prolonged oxidative-and hypoxic-induced stress. Within this context, the major new findings of this study are: 1) CST can be nanoprecipitated in order to preserve its structure for encapsulation studies; 2) Nanoprecipitated CST can be encapsulated in FN-PAMs; 3) functionalized FN-PAMs can slowly release a bioactive CST; 4) FN-PAMs induce MSC proliferation in normoxia potentiated by the functionalization with CST and 5) FN-PAM-CST protect bone marrow MSC from hypoxia/reoxygenation stress.

The encapsulation and the sustained release of a protein from microspheres is a difficult task and depends on the physicochemical characteristics of each protein such as molecular weight, hydrophilicity and stability [START_REF] Bilati | Strategic approaches for overcoming peptide and protein instability within biodegradable nano-and microparticles[END_REF]. In particular, the low molecular weight of CST represented a challenge for the prolonged release of this small peptide in an active state from the microspheres. We have previously shown that proteins encapsulated under a nanosolid state within a PLGA polymer preserve their structure and integrity, allowing a better and prolonged release profile and the maintenance of their biological activity (Penna et al., 2013). In order to optimize the preparation of small peptides, various methods like spraydrying or spray-freeze drying have been reported [START_REF] Lam | Sustained release of recombinant human insulin-like growth factor-I for treatment of diabetes[END_REF][START_REF] Wang | Stabilization and encapsulation of human immunoglobulin G into biodegradable microspheres[END_REF]. Even if these methods can generate protein particles, they have several disadvantages, such as the complexity of the technique, leading to low protein recovery and, above all, they may denature proteins. From an aqueous solution, protein precipitation can be induced for example with the addition of polymers or salts, with the reduction of the protein charge by changing the pH, or with the reduction of the dielectric constant by the addition of watermiscible organic solvents [START_REF] Cleland | Stable formulations of recombinant human growth hormone and interferon-gamma for microencapsulation in biodegradable microspheres[END_REF] increasing ionic strength. The saltinduced precipitation is an extensively used method in biotechnology for separating target proteins from multi-components protein solutions as the first purification [START_REF] Arakawa | Protein--solvent interactions in pharmaceutical formulations[END_REF][START_REF] Foster | Salting-out of enzymes with ammonium sulphate[END_REF][START_REF] Rothstein | Differential precipitation of proteins[END_REF]. In our experimental setup, a high amount of salt is necessary for CST nanoprecipitation and encapsulation, while preserving its bioactivity.

With the aim to determine an appropriate method of precipitation of CST for the encapsulation in PLGA-P188-PLGA microspheres preserving CST bioactivity we performed an experimental design testing different parameters. Indeed the ionic strength can be used to stimulate the salt induced precipitation: sodium chloride reduces the electrostatic repulsive interaction between the charged proteins, promoting in this way attractive hydrophobic interaction [START_REF] Tobler | Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry[END_REF][START_REF] Vonhippel | Neutral Salts: The Generality of Their Effects on the Stability of Macromolecular Conformations[END_REF]. However the increase of ionic strength was limited by the solubility of the salt used (NaCl 2.175 M) and by the formation of artefacts during the mixing. Moreover, excess salt-induced precipitation may induce proteins to incorrectly fold or to form insoluble aggregates upon re-dissolution [START_REF] Tobler | Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry[END_REF].

We have furthermore employed an organic solvent to induce the formation of fine protein particles. Nanosized protein particles were formed by the addition of excess glycofurol, an organic solvent miscible in water, to the aqueous solution [START_REF] Giteau | Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres[END_REF]. Glycofurol has been previously used to induce the formation of nanosized solid protein particles [START_REF] Giteau | Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres[END_REF][START_REF] Karam | Pharmacologically active microcarriers associated with thermosensitive hydrogel as a growth factor releasing biomimetic 3D scaffold for cardiac tissue-engineering[END_REF], but to our knowledge, this is the first time that such a small peptide (21-amino acids) is efficiently precipitated with this method. Another parameter tested in the experimental design was the time of incubation of the mix of CST with sodium chloride and organic solvents. We observed an increased percentage of nanoprecipitation yield correlated to the increase in time of incubation. We obtained in fact a very good CST precipitation at 90 minutes of incubation.

Furthermore, P188 poloxamer was added to the mix to co-precipitate with the protein. This non-ionic surfactant, comprised of hydrophobic and hydrophilic segments will surround and protect the protein by limiting its adsorption onto the hydrophobic surface of the PLGA polymer [START_REF] Morille | New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells[END_REF][START_REF] Mustafi | Multi-block poloxamer surfactants suppress aggregation of denatured proteins[END_REF]. It was co-precipitated with CST at the ratio of 1:20 of protein:additive polymer, and allowed a complete release of CST. This formulation allows a complete CST release from the PAMs, probably due to a better diffusion of the protein and an enhanced protection within the core. In this study CST precipitation with glycofurol, sodium chloride and P188 in optimum conditions allowed obtaining stabilization during the encapsulation process without loss of bioactivity. In particular, the hydrophilic P188, limiting the protein interaction with the PLGA hydrophobic polymer surface, protects the protein, increases their biological activity and may favor protein release into the media. Actually, PAMs containing higher proportions of free P188 with the same protein content enhance active protein release [START_REF] Morille | New PLGA-P188-PLGA matrix enhances TGF-beta3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells[END_REF]. Therefore, P188 may certainly influence active protein release from triblock PLGA-P188-PLGA PAMs. In fact, 50% of CST was released during the first 24h in vitro. Such an abundant release in the initial phase of a post-ischemic therapeutic approach may be a positive feature. Indeed the first phase of reperfusion is the more critical for tissue salvage [START_REF] Penna | Cardioprotection against ischemia/reperfusion injury and chromogranin A-derived peptides[END_REF]. The later phases may be critical for tissue repair and regeneration and a continuous slow release of a therapeutic peptide (e.g.

VEGF and CST) released by microspheres transporting cells on their 3D functionalized surface may be beneficial for tissue engineering strategies [START_REF] Elçin | Extensive in vivo angiogenesis following controlled release of human vascular endothelial cell growth factor: implications for tissue engineering and wound healing[END_REF][START_REF] Madonna | Transplantation of adipose tissue mesenchymal cells conjugated with VEGF-releasing microcarriers promotes repair in murine myocardial infarction[END_REF]. However, this needs to be ascertained in vivo for CST, and it should be ascertained whether other conditions, such as fibronectin coating, may affect the release of the active peptide.

As demonstrated by us and other authors, CST is a very promising agent for a cardiovascular protective purpose [START_REF] Bassino | A novel catestatin-induced antiadrenergic mechanism triggered by the endothelial PI3K-eNOS pathway in the myocardium[END_REF][START_REF] Bassino | Catestatin exerts direct protective effects on rat cardiomyocytes undergoing ischemia/reperfusion by stimulating PI3K-Akt-GSK3β pathway and preserving mitochondrial membrane potential[END_REF][START_REF] Egger | Monocyte migration: a novel effect and signaling pathways of catestatin[END_REF][START_REF] Penna | Catestatin improves post-ischemic left ventricular function and decreases ischemia/reperfusion injury in heart[END_REF][START_REF] Perrelli | Catestatin reduces myocardial ischaemia/reperfusion injury: involvement of PI3K/Akt, PKCs, mitochondrial KATP channels and ROS signalling[END_REF]. In order to reduce damage observed in cardiovascular system following oxidative stress and prolonged hypoxic conditions, therapies with cell transplantation could represent a good strategy to follow; however approaches adapted to the varying conditions are needed. We have recently shown that VEGF-A as such or slowly released by FN-PAM can differently affect survival kinases and anti-apoptotic mediators, in stem cells [START_REF] Penna | Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival[END_REF].

On the basis of the above reported data, we have hypothesized that the prolonged release of CST by FN-PAM may act on cells and counteract in a better way prolonged hypoxic-induced stress. The in vitro study with CST-PAM suggested that it is possible to yield a good and effective complex to be used against hypoxia/reoxygenation damage. PAMs represents a multifunction vehicle that can be used in many contexts and with a variety of cells and proteins , and could be considered a new approach to improve MSC survival in the pathological hostile environment.

A way to improve the efficiency of cell graft, for instance, is to design a scaffold able to deliver a bioactive therapeutic protein that is released in a controlled manner but also a scaffold that provides a biomimetic surface. PAMs represent an efficient and adaptable device for tissue engineering; they are biodegradable and non-cytotoxic microcarriers that provide an adequate 3D support for cell culture and/or for their administration [START_REF] Delcroix | Adult cell therapy for brain neuronal damages and the role of tissue engineering[END_REF]. Depending on the choice of the encapsulated protein and also of the molecule used for the biomimetic coating, PAM represents a multifunction vehicle that can be used in many contexts and with a variety of cells.

Conclusions

Our data are in line with the viewpoint that the use of FN-PAMs complexed with therapeutic proteins could be considered a new approach to improve cell survival in hostile environments such as inflammatory and post-ischemic tissue. Within this context, it has been previously

shown that the 3D biomimetic support provided by FN-PAMs on their own, without delivering a therapeutic factor, already increases the survival of MSCs transported by these PAMs and implanted in the brain after global ischemia [START_REF] Garbayo | Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers[END_REF]. Moreover, they enhanced the neuroprotective effect of the implanted MSCs.

In this in vitro study, we have shown that CST can be encapsulated into PAMs and that this functionalized PAMs can protect bone marrow MSC from hypoxia/reoxygenation injury. If confirmed in an appropriate pre-clinical model, this approach may be envisaged for future clinical studies on tissue regeneration. Mesenchymal stem cells (MSCs) were kept at 21% O2 conditions for 1 day during the pretreatments, subsequently they were subjected to 3 days hypoxia and 3 hrs reoxygenation. Fibronectin-coated pharmacologically-active-microcarriers (FN-PAM), FN-PAM incorporating CST (FN-PAM-CST) or free CST were added at time 0. For comparative purpose we considered normoxic (-N) and hypoxic (-H) protocols.

FIGURE CAPTATIONS

  ):1) Untreated MSCs kept under standard conditions for 3-days (MSC-3-N), and untreated MSCs exposed to H/R (MSC-3-H);2) MSCs pre-treated with FN-PAM and kept under standard conditions (FN-PAM-3-N), and MSCs pre-treated with FN-PAMs and exposed to H/R (FN-PAM-3-H);3) MSCs pre-treated with FN-PAM-CST and kept under standard conditions (FN-PAM-CST-3-N), and MSCs pre-treated with FN-PAM-CST and exposed to H/R (FN-PAM-CST-3-H); 4) MSCs pre-treated with free CST and kept under standard conditions (CST-3-N), and MSCs pre-treated with free CST and exposed to H/R (CST-3-H).

  after H/R with and without pre-treatment with free CST, FN-PAMs or FN-PAM-CSTData of survival analysis after H/R in pre-treated cells are presented as percent variation with respect to mean value of the cell count in control condition (MSC-3-N). To choose the optimal dose of CST, we performed experiments testing at least three different concentrations of free CST (5 nM, 10 nM and 2.5 μM). While the higher concentration resulted toxic (induced cell death), the two lower concentrations similarly increased cell survival (data no shown), thus we decided to continue to test the lower concentration and to use 0.5 mg PAMs/15x10 4 cells in order to obtain a similar concentration of CST in dishes in the following experiments. This proportion corresponds to what has been used in different

Fig. 1 :

 1 Fig.1: Spatial representation of experiments varying in the defined experimental domain for CST nanoprecipitation. The table represents the values of each studied factor (NaCl and Time) for the different experiments.

Fig. 2 :

 2 Fig.2: Protocols and time-lines for experimental groups in hypoxia/reoxygenation. Timing of various interventions is shown in relation to the onset of cell culture.Mesenchymal stem cells (MSCs) were kept at 21% O2 conditions for 1 day during the pretreatments, subsequently they were subjected to 3 days hypoxia and 3 hrs reoxygenation. Fibronectin-coated pharmacologically-active-microcarriers (FN-PAM), FN-PAM incorporating CST (FN-PAM-CST) or free CST were added at time 0. For comparative purpose we considered normoxic (-N) and hypoxic (-H) protocols.

Fig. 3 :

 3 Fig.3: Panel I, PAM characterization. PAM size distribution had an average diameter of 60 μm (a). SEM images show a smooth surface and confirm the size distribution (b,c). The surrounding biomimetic coating was distributed homogenously at their surface as shown by the confocal image of fibronectin immunofluorescence (c, d). Panel II, Cumulative release of CST from microspheres up to 21 days. Each point represents the mean of triplicate experiments.

Fig. 4 :

 4 Fig.4: Parameters and interaction studied. Graphical representation of the effects of the parameters and interaction studied (Nemrod Software).

Fig. 5 :Fig. 6 :

 56 Fig.5: Graphical representation of the measured responses as precipitation efficiencies. In the 3D view of the experimental domain, the directions of variation of the factor values are represented by arrows and the results by dots. *mean of three values : (a) : 57±2% (b) : 45 ±3.5% (c) : 43±2% (d) : 76±3.5%

Table 1 :

 1 Fractional factorial design and nanoprecipitation yields for each experiment.

		NaCl	Contact	Centrifugation	ZnCl 2	Nanoprecipitation
			time	speed		yield
	(nemrod) M	Min	G	M	%
	1	0	30	10 000	0	16.2
	2	1	30	10 000	0.008	24.3
	3	0	60	10 000	0.008	32.3

Acknowledgements

We would like to thank Dr Fabien Violet and Dr Francesca Tullio for their invaluable support in performing some of the experiments. Also we thank the SCIAM "Service commun d'imagerie et d'analyse microscopique" for help with SEM and confocal images. We thank the Italo-French University (Galileo project) and RILO (MeccaSaric) of the University of Torino for financial support.