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• W3048925464 abstract "•MSCs are heterogeneous in the adult mouse incisor•Runx2+/Gli1+ cells are MSC niche cells, but not MSCs•Runx2+/Gli1+ cells coordinate the MSC-to-TAC transition and the incisor growth rate•Runx2-mediated IGF signaling controls cell fate of TACs Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche. Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche. Continuous cell replacement helps to maintain homeostasis in tissues such as the skin and gastrointestinal tract (Blanpain and Fuchs, 2014Blanpain C. Fuchs E. Stem cell plasticity. Plasticity of epithelial stem cells in tissue regeneration.Science. 2014; 344: 1242281Crossref PubMed Scopus (368) Google Scholar; Kaukua et al., 2014Kaukua N. Shahidi M.K. Konstantinidou C. Dyachuk V. Kaucka M. Furlan A. An Z. Wang L. Hultman I. Ahrlund-Richter L. et al.Glial origin of mesenchymal stem cells in a tooth model system.Nature. 2014; 513: 551-554Crossref PubMed Scopus (284) Google Scholar). Tissue homeostasis is supported by stem cells, which reside within specialized microenvironments, called niches, that in turn provide support and signals to regulate stem cell self-renewal and differentiation (Chacón-Martínez et al., 2018Chacón-Martínez C.A. Koester J. Wickström S.A. Signaling in the stem cell niche: regulating cell fate, function and plasticity.Development. 2018; 145: dev165399Crossref PubMed Google Scholar; Rezza et al., 2016Rezza A. Wang Z. Sennett R. Qiao W. Wang D. Heitman N. Mok K.W. Clavel C. Yi R. Zandstra P. et al.Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and their Niche in Developing Hair Follicles.Cell Rep. 2016; 14: 3001-3018Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Simons and Clevers, 2011Simons B.D. Clevers H. Strategies for homeostatic stem cell self-renewal in adult tissues.Cell. 2011; 145: 851-862Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). The complex dynamics of the stem cell niche are orchestrated by the supporting extracellular matrix (ECM), niche cells, and soluble signaling factors that act via autocrine or paracrine mechanisms (Morrison and Spradling, 2008Morrison S.J. Spradling A.C. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life.Cell. 2008; 132: 598-611Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar; Scadden, 2014Scadden D.T. Nice neighborhood: emerging concepts of the stem cell niche.Cell. 2014; 157: 41-50Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Several well-defined niches harbor stem cells necessary to maintain homeostasis and regenerate tissues after damage. The intestinal epithelium, for example, contains Paneth cells that secrete niche signals such as Wnt3, Egf, and Notch ligand Dll4 to intestinal stem cells (Ganz, 2000Ganz T. Paneth cells—guardians of the gut cell hatchery.Nat. Immunol. 2000; 1: 99-100Crossref PubMed Scopus (56) Google Scholar; Sato et al., 2011Sato T. van Es J.H. Snippert H.J. Stange D.E. Vries R.G. van den Born M. Barker N. Shroyer N.F. van de Wetering M. Clevers H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.Nature. 2011; 469: 415-418Crossref PubMed Scopus (1725) Google Scholar). In the hair follicle epidermis, transit-amplifying cells (TACs) crucially help regulate the stem cell niche by producing Sonic hedgehog (Shh) (Hsu et al., 2014Hsu Y.C. Li L. Fuchs E. Transit-amplifying cells orchestrate stem cell activity and tissue regeneration.Cell. 2014; 157: 935-949Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). In the mesenchyme, however, niche cells for mesenchymal stem cells (MSCs) have yet to be well defined. Mammalian teeth harbor MSCs in dental pulp that contribute to tooth homeostasis and repair. In particular, rodent incisors provide an excellent window into the activities of MSCs and their niches, because these teeth continue to grow throughout the animal’s life (Lapthanasupkul et al., 2012Lapthanasupkul P. Feng J. Mantesso A. Takada-Horisawa Y. Vidal M. Koseki H. Wang L. An Z. Miletich I. Sharpe P.T. Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors.Dev. Biol. 2012; 367: 140-153Crossref PubMed Scopus (42) Google Scholar; Wang et al., 2007Wang X.P. Suomalainen M. Felszeghy S. Zelarayan L.C. Alonso M.T. Plikus M.V. Maas R.L. Chuong C.M. Schimmang T. Thesleff I. An integrated gene regulatory network controls stem cell proliferation in teeth.PLoS Biol. 2007; 5: e159Crossref PubMed Scopus (180) Google Scholar). MSC and TAC populations can be clearly identified in the proximal region of the rodent incisor, residing between the labial and the lingual sides of the epithelial cervical loop (Sharpe, 2016Sharpe P.T. Dental mesenchymal stem cells.Development. 2016; 143: 2273-2280Crossref PubMed Scopus (202) Google Scholar; Shi et al., 2019Shi C. Yuan Y. Guo Y. Jing J. Ho T.V. Han X. Li J. Feng J. Chai Y. BMP Signaling in Regulating Mesenchymal Stem Cells in Incisor Homeostasis.J. Dent. Res. 2019; 98: 904-911Crossref PubMed Scopus (15) Google Scholar; Zhao et al., 2014Zhao H. Feng J. Seidel K. Shi S. Klein O. Sharpe P. Chai Y. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.Cell Stem Cell. 2014; 14: 160-173Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Recently, using genetic lineage tracing, several markers have been identified as labeling different MSC populations in vivo (An et al., 2018bAn Z. Sabalic M. Bloomquist R.F. Fowler T.E. Streelman T. Sharpe P.T. A quiescent cell population replenishes mesenchymal stem cells to drive accelerated growth in mouse incisors.Nat. Commun. 2018; 9: 378Crossref PubMed Scopus (53) Google Scholar; Feng et al., 2011Feng J. Mantesso A. De Bari C. Nishiyama A. Sharpe P.T. Dual origin of mesenchymal stem cells contributing to organ growth and repair.Proc. Natl. Acad. Sci. USA. 2011; 108: 6503-6508Crossref PubMed Scopus (309) Google Scholar; Kaukua et al., 2014Kaukua N. Shahidi M.K. Konstantinidou C. Dyachuk V. Kaucka M. Furlan A. An Z. Wang L. Hultman I. Ahrlund-Richter L. et al.Glial origin of mesenchymal stem cells in a tooth model system.Nature. 2014; 513: 551-554Crossref PubMed Scopus (284) Google Scholar; Zhao et al., 2014Zhao H. Feng J. Seidel K. Shi S. Klein O. Sharpe P. Chai Y. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.Cell Stem Cell. 2014; 14: 160-173Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), improving our understanding of the heterogeneity of stem cell populations. Specifically, our previous study has shown that quiescent Gli1+ cells are typical MSCs in the mouse incisor. These stem cells surround the neurovascular bundle in the proximal region of the incisor. This population of MSCs continuously gives rise to TACs, which actively divide and then differentiate into odontoblasts and dental pulp cells to support both homeostasis and injury repair (Zhao et al., 2014Zhao H. Feng J. Seidel K. Shi S. Klein O. Sharpe P. Chai Y. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.Cell Stem Cell. 2014; 14: 160-173Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Kaukua and colleagues showed that Plp1/Sox10+ glia-derived MSCs dwell in a niche in the proximal region of the mouse incisor (Kaukua et al., 2014Kaukua N. Shahidi M.K. Konstantinidou C. Dyachuk V. Kaucka M. Furlan A. An Z. Wang L. Hultman I. Ahrlund-Richter L. et al.Glial origin of mesenchymal stem cells in a tooth model system.Nature. 2014; 513: 551-554Crossref PubMed Scopus (284) Google Scholar). Although Gli1+ MSCs contribute to the entire dental pulp, these multipotent Plp1/Sox10+ Schwann cell precursors (SCPs) and Schwann cells contribute to approximately half of the pulp cells and odontoblasts during development, growth, and regeneration of the incisor (Kaukua et al., 2014Kaukua N. Shahidi M.K. Konstantinidou C. Dyachuk V. Kaucka M. Furlan A. An Z. Wang L. Hultman I. Ahrlund-Richter L. et al.Glial origin of mesenchymal stem cells in a tooth model system.Nature. 2014; 513: 551-554Crossref PubMed Scopus (284) Google Scholar). Another study identified an MSC population derived from neuronal glia; it reported a subpopulation of MSCs that express CD90/Thy1 and contribute to 30% of differentiated cell progeny during incisor eruption and injury repair (An et al., 2018bAn Z. Sabalic M. Bloomquist R.F. Fowler T.E. Streelman T. Sharpe P.T. A quiescent cell population replenishes mesenchymal stem cells to drive accelerated growth in mouse incisors.Nat. Commun. 2018; 9: 378Crossref PubMed Scopus (53) Google Scholar). Collectively, these studies suggest there may be considerable heterogeneity among MSCs in the adult mouse incisor. Runx2 encodes a transcription factor that is known for its important role during bone and tooth development. In humans, RUNX2 mutations are responsible for an autosomal dominant disorder, cleidocranial dysplasia (CCD), which is associated with bone formation defects (Jaruga et al., 2016Jaruga A. Hordyjewska E. Kandzierski G. Tylzanowski P. Cleidocranial dysplasia and RUNX2-clinical phenotype-genotype correlation.Clin. Genet. 2016; 90: 393-402Crossref PubMed Scopus (51) Google Scholar; Wang et al., 2013Wang S. Zhang S. Wang Y. Chen Y. Zhou L. Cleidocranial dysplasia syndrome: clinical characteristics and mutation study of a Chinese family.Int. J. Clin. Exp. Med. 2013; 6: 900-907PubMed Google Scholar). Disruption of Runx2 in mice leads to maturational arrest of osteoblasts and therefore a complete lack of ossification during both endochondral and intramembranous bone formation, whereas tooth morphogenesis is arrested at the cap stage (D’Souza et al., 1999D’Souza R.N. Aberg T. Gaikwad J. Cavender A. Owen M. Karsenty G. Thesleff I. Cbfa1 is required for epithelial-mesenchymal interactions regulating tooth development in mice.Development. 1999; 126: 2911-2920PubMed Google Scholar; Komori et al., 1997Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. et al.Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts.Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3636) Google Scholar; Otto et al., 1997Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W.H. Beddington R.S.P. Mundlos S. Olsen B.R. et al.Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development.Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2405) Google Scholar). In the dental mesenchyme, Runx2 regulates mesenchymal odontogenic activity by modulating antagonistic interaction between transcription factors Msx1 and Osr2, and plays an essential role in upregulating mesenchymal Fgf signals during later stages of tooth morphogenesis (Kwon et al., 2015Kwon H.J. Park E.K. Jia S. Liu H. Lan Y. Jiang R. Deletion of Osr2 Partially Rescues Tooth Development in Runx2 Mutant Mice.J. Dent. Res. 2015; 94: 1113-1119Crossref PubMed Scopus (6) Google Scholar). In the dental epithelium, Runx2 and CCAAT/enhancer binding protein beta (Cebpb) act synergistically to prevent epithelial-mesenchymal transition (EMT) of odontogenic epithelial stem cells via snail family zinc finger2 (Snai2). Abrogating Cebpb and Runx2 results in EMT and promotes Sox2 expression and stemness of oral epithelial stem cells, leading to supernumerary teeth forming around the labial portion of the cervical loop in Cebpb−/−;Runx2+/− mice (Saito et al., 2018Saito K. Takahashi K. Huang B. Asahara M. Kiso H. Togo Y. Tsukamoto H. Mishima S. Nagata M. Iida M. et al.Loss of Stemness, EMT, and Supernumerary Tooth Formation in Cebpb−/−Runx2+/− Murine Incisors.Sci. Rep. 2018; 8: 5169Crossref PubMed Scopus (13) Google Scholar). Runx2 is clearly important for early tooth development, but its potential function in regulating MSCs in adult tooth homeostasis is unknown. In this study, we characterized the Gli1+ cell population in the mouse incisor using single-cell RNA sequencing (scRNA-seq). We revealed that Gli1+ cells constitute a heterogeneous population and identified a subpopulation characterized by expression of Runx2 in the proximal region of the adult incisor. These Runx2+ cells are located close to both MSCs and TACs and are thus strategically positioned to support the stem cell niche environment. Furthermore, insulin-like growth factor binding protein (Igfbp) 3 secreted by these Runx2+ cells activates insulin-like growth factor (IGF) 2 signaling to regulate TAC proliferation and differentiation. We demonstrated that this subpopulation of Runx2+/Gli1+ niche cells controls the rate of incisor growth in adult mice. This discovery improves our understanding of the stem cell niche microenvironment and has broad implications concerning the heterogeneity of Gli1+ cells and their roles in regulating tissue homeostasis. MSCs in the mouse incisor have been reported to be labeled by heterogeneous markers (An et al., 2018bAn Z. Sabalic M. Bloomquist R.F. Fowler T.E. Streelman T. Sharpe P.T. A quiescent cell population replenishes mesenchymal stem cells to drive accelerated growth in mouse incisors.Nat. Commun. 2018; 9: 378Crossref PubMed Scopus (53) Google Scholar; Feng et al., 2011Feng J. Mantesso A. De Bari C. Nishiyama A. Sharpe P.T. Dual origin of mesenchymal stem cells contributing to organ growth and repair.Proc. Natl. Acad. Sci. USA. 2011; 108: 6503-6508Crossref PubMed Scopus (309) Google Scholar; Kaukua et al., 2014Kaukua N. Shahidi M.K. Konstantinidou C. Dyachuk V. Kaucka M. Furlan A. An Z. Wang L. Hultman I. Ahrlund-Richter L. et al.Glial origin of mesenchymal stem cells in a tooth model system.Nature. 2014; 513: 551-554Crossref PubMed Scopus (284) Google Scholar; Zhao et al., 2014Zhao H. Feng J. Seidel K. Shi S. Klein O. Sharpe P. Chai Y. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.Cell Stem Cell. 2014; 14: 160-173Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). To elucidate the heterogeneity of the MSC population in the adult mouse incisor, we performed scRNA-seq analysis on incisors from 1-month-old wild-type mice (Figure 1A). Unbiased t-distributed stochastic neighbor embedding (t-SNE) showed Gli1 expression in the dental mesenchyme, as well as the epithelium (Figure 1B). Gli1+ cells were selected and re-clustered through t-SNE analysis. Results showed that Gli1+ cells consisted of nine distinct clusters (Figure 1C). We validated and mapped the clusters based on the selected marker gene expression (Figure S1A). We analyzed specific genes in different clusters and identified that Runx2 was highly expressed in a subpopulation located in the proximal region (Figure 1D). To validate the scRNA-seq analysis in vivo, we checked the expression pattern of Runx2 using immunofluorescence staining and confirmed that it is expressed in the proximal region of the incisor mesenchyme, mainly in the lateral portion close to the cervical loop (Figures 1E and 1F). Runx2 expression was also detected in the periodontal ligament and in some odontoblasts (Figure 1E). To visualize the relationship between Runx2+ cells and Gli1+ cells, we analyzed the colocalization of Runx2+ cells with Gli1+ cells using Gli1-LacZ mice. We found that Runx2+ cells overlapped with Gli1+ cells mainly in the region adjacent to the cervical loop (Figures 1G and 1H). To determine whether Runx2+ cells overlapped with TACs, we performed double staining of Runx2 and TAC marker Ki67, which revealed that Runx2+ cells and TACs were adjacent but mutually exclusive cell populations (Figures 1I and 1J). This expression of Runx2 in Gli1+ cells suggested that it may play a critical role in the mesenchymal tissue homeostasis of the mouse incisor. Mouse incisors self-renew throughout the animal’s life with the support of Gli1+ MSCs. Because Runx2 is expressed in a subpopulation of Gli1+ cells, we tested whether Runx2 is essential in regulating stem cell fate and therefore incisor homeostasis. We generated Gli1-CreERT2;Runx2fl/fl mice in which Runx2 was inactivated in the Gli1+ lineage after induction with tamoxifen at one month of age. Immunofluorescence staining confirmed that Runx2 was efficiently deleted from the incisor mesenchyme (Figure S1B). We observed that the incisors of these mice had decreased in length 1 month after deletion of Runx2 (Figures 2A and 2B ), which was confirmed by micro-computed tomography (micro-CT) (Figures 2C and 2D). Three months later, we noticed more significant shortening of incisors in Runx2 mutant mice (Figures 2E–2I). To observe the incisor growth rate dynamically, we made a notch in the incisor enamel close to the junction with the gingiva one day after induction and measured the growth of the incisor as revealed by the movement of the notch 3 and 6 days later. The notch movement was significantly slower in Runx2 mutant mice after 6 days (Figure S2A). This is consistent with our finding that the ability to repair the incisor after clipping was compromised in Runx2 mutant mice (Figure S2B). These data indicate that loss of Runx2 affected tissue homeostasis and eventually retarded the growth rate of incisors in Gli1-CreERT2;Runx2fl/fl mice. Histological analysis revealed abnormal dentin formation and disorganized epithelium near the cervical loop (Figures 2J–2M) one month after induction. The expression of odontoblast differentiation marker dentin sialophosphoprotein (Dspp) was closer to the proximal end of the incisor in the Runx2 mutants compared with control mice (Figures 2N–2Q), suggesting that Runx2 plays an essential role in regulating odontoblast differentiation. Because Gli1+ cells contribute to the periodontal ligament in the adult mouse incisor (Figures 4Ba and 4Bc), Runx2 was also deleted from the periodontal ligament in Gli1-CreERT2;Runx2fl/fl mice. We found defects in the periodontal ligament and alveolar bone in Gli1-CreERT2;Runx2fl/fl mice (Figure S2C). These data indicated that Runx2+ cells also play an important role in the homeostasis of the periodontium. It has been reported that Gli1+ cells contribute to both mesenchymal and epithelial cell lineages (Seidel et al., 2010Seidel K. Ahn C.P. Lyons D. Nee A. Ting K. Brownell I. Cao T. Carano R.A. Curran T. Schober M. et al.Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor.Development. 2010; 137: 3753-3761Crossref PubMed Scopus (143) Google Scholar; Zhao et al., 2014Zhao H. Feng J. Seidel K. Shi S. Klein O. Sharpe P. Chai Y. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.Cell Stem Cell. 2014; 14: 160-173Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar) and that Runx2 is expressed in mature ameloblasts (Figure S3B). To rule out the possibility that the changes we observed in the mesenchyme after deletion of Runx2 were secondary to changes in the epithelium, we generated Sox2-CreERT2;Runx2fl/fl mice with inducible deletion of Runx2 in the Sox2+ cells, which exclusively contribute to the epithelium (Figure S3A) (Juuri et al., 2012Juuri E. Saito K. Ahtiainen L. Seidel K. Tummers M. Hochedlinger K. Klein O.D. Thesleff I. Michon F. Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ progenitors.Dev. Cell. 2012; 23: 317-328Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). We induced these mice with tamoxifen at one month of age. Four weeks after induction, Runx2 was lost only in the epithelium in Sox2-CreERT2;Runx2fl/fl mice (Figure S3D). Dentin formation was unaffected in the incisors, based on micro-CT analysis and H&E staining (Figures S3F–S3J). These data suggested that loss of Runx2 in the ameloblasts had no effect on the proximal dental mesenchyme. Because Runx2+ cells colocalized with Gli1+ cells, we sought to determine whether Runx2+/Gli1+ cells are a subpopulation of MSCs. We generated Runx2-rtTA;tetO-Cre;tdTomato mice to perform lineage tracing of Runx2+ cells in the adult mouse incisor. One week after induction at one month of age, Runx2+ cells were present in the proximal region of the mesenchyme (Figures 3A and 3B ). Analysis at one and three months after induction showed that Runx2+ cells remained in the proximal region of the incisor and did not differentiate into odontoblasts or dental pulp cells, suggesting that Runx2+ cells do not contribute to the dental mesenchyme during incisor growth (Figures 3C–3F). To assess whether Runx2+ cells contribute to injury repair, we clipped one incisor to approximately half of its length (Figure 3H). Three days after clipping, the injured side was almost repaired such that its length was comparable to that of the uninjured contralateral incisor (Figure 3K). However, Runx2+ cells still did not move away from the proximal region of the incisor or differentiate into odontoblasts during injury repair (Figures 3I and 3L). We also quantified the percentage of Runx2+ cells at different stages. The statistical analysis showed that there was no significant difference in the percentage of Runx2+ cells during growth or injury repair (Figures 3G and 3N). To evaluate the relationship between Runx2+ cells and label-retaining cells (LRCs), which are considered a population of quiescent stem cells, we injected EdU daily into pups for 4 weeks beginning at postnatal day 5 and traced 5-Ethynyl-2'-deoxyuridine (EdU) incorporation for another 8 weeks. The EdU staining showed that LRCs did not overlap with Runx2+ cells (Figure S4). Collectively, based on these data, we concluded that Runx2+/Gli1+ cells are not MSCs. Nevertheless, they reside in the MSC niche and play an important role in regulating growth and tissue homeostasis of adult mouse incisors. Although Runx2+/Gli1+ cells are not MSCs, Gli1-CreERT2;Runx2fl/fl mice showed a reduced incisor growth rate. To elucidate how Runx2+ cells control growth and tissue homeostasis via regulating the incisor MSC niche, we first quantified the Gli1+ MSC population in Gli1-CreERT2;Runx2fl/fl;Gli1-LacZ mice. The number of Gli1+ cells in Gli1-CreERT2;Runx2fl/fl;Gli1-LacZ mice remained the same as in Gli1-LacZ mice one week after induction (Figures 4Aa and 4Ac ) but significantly decreased two weeks after induction (Figures 4Ae and 4Ag). To investigate the effect of Runx2 loss on the differentiation of Gli1+ MSCs, we assessed the contribution of MSCs to their progeny, comparing Gli1-CreERT2;tdTomato and Gli1-CreERT2;Runx2fl/fl;tdTomato mice. We measured the length of dental pulp that was positive for the tdTomato signal, representing the Gli1+ cells’ progeny in the incisor; we then computed this length as a percentage of the length of the whole dental pulp (Figure 4Be). One week after induction, the percentage of Gli1+ cells’ progeny measured in this manner was unchanged (Figures 4Ba, 4Bb, and 4Bf), but it significantly decreased 2 weeks after induction in Runx2 mutant mice (Figures 4Bc, 4Bd, and 4Bg), suggesting that the differentiation rate had slowed. We also checked the number and differentiation of TACs. The number of TACs decreased significantly one week after induction in Gli1-CreERT2;Runx2fl/fl mice, as detected by Ki67 immunofluorescence staining (Figures 4Ca, 4Cc, and 4Ci); there was no concomitant increase in apoptosis detected by TUNEL assay (Figure S5A). Next, we assessed whether there was a change in the TAC differentiation rate in Runx2 mutant incisors. We first identified that the number of TACs was comparable in Runx2 mutant and control mice 5 days after induction (Figure S5B). Then, we injected EdU 5 days after induction and assessed the TAC differentiation rate upon euthanizing the mice 48 h later. Because TACs retain EdU during differentiation in this short time frame, the length of overlap between Dspp+ odontoblasts and EdU+ cells reflected the number of TACs that underwent odontogenic differentiation in this period. Strikingly, there were fewer double-positive cells in Runx2 mutant incisors compared with the controls (Figures 4Cf, 4Ch, and 4Cj), showing that differentiation of TACs was perturbed in Runx2 mutant mice one week after induction. Clearly, loss of Runx2 in the Gli1+ lineage first led to compromised TAC proliferation and differentiation and then affected the MSC population, which suggests that Runx2+ cells maintain the incisor MSC niche by regulating TAC proliferation and differentiation. To identify downstream components of the molecular mechanism by which Runx2 regulates the MSC niche, we analyzed gene expression in the proximal region of the incisor by performing RNA-seq on adult Runx2fl/fl and Gli1-CreERT2;Runx2fl/fl mice one week after induction. Hierarchical clustering showed that gene expression profiles of Runx2 mutant and control mice were well separated (Figure 5A). Five hundred and eleven differentially expressed genes were identified (>2-fold, p < 0.05), of which 299 were upregulated and 212 were downregulated (Figure 5B). Analysis of these genes using Ingenuity Pathways Analysis (IPA, QIAGEN) revealed that several signaling pathways related to cell-cycle regulation were highly involved, such as p53 signaling, cyclins and cell-cycle regulation, IGF signaling, and Wnt/β-catenin signaling (Figure 5C). We focused on IGF signaling, which is known to play an important role in the MSC niche (Youssef et al., 2017Youssef A. Aboalola D. Han V.K. The Roles of Insulin-Like Growth Factors in Mesenchymal Stem Cell Niche.Stem Cells Int. 2017; 2017: 9453108PubMed Google Scholar; Ziegler et al., 2019Ziegler A.N. Feng Q. Chidambaram S. Testai J.M. Kumari E. Rothbard D.E. Constancia M. Sandovici I. Cominski T. Pang K. et al.Insulin-like Growth Factor II: An Essential Adult Stem Cell Niche Constituent in Brain and Intestine.Stem Cell Reports. 2019; 12: 816-830Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Specifically, we confirmed that Igf2 protein was downregulated in the Runx2+ and TAC regions of Runx2 mutant incisors (Figure 5Dc) compared with controls (Figure 5Da). The IGF1 receptor (Igf1r) was detected in the TAC region by RNAscope in both control and Runx2 mutant mice (Figure S6A). Igf1r could be phosphorylated in control mice (Figure 5De) but failed to be phosphorylated in mesenchymal TACs in Runx2 mutant mice (Figure 5Dg). The expression of phosphorylated insulin receptor substrate 1 (p-Irs1) was also decreased in Runx2 mutant mice (Figures 5Di and 5Dk). Phosphorylated Akt (p-Akt), a downstream target of IGF signaling, was downregulated (Figures 5Dm and 5Do). These results were confirmed by western blot (Figure 5E). To explore how Runx2+ cells regulate Igf2, we first checked the mRNA expression pattern of Igf2. Igf2 was highly expressed in the MSC region in both control and Runx2 mutant incisors (Figures S6Ba and S6Bc). Analysis following real-time PCR showed that there was no significant difference in Igf2 mRNA expression levels between control and Runx2 mutant groups (Figure S6Be). The activity of the IGF ligands is regulated by a family of six IGFBPs in vertebrates. Therefore, we performed RNAscope to visualize the mRNA expression of each of the IGFBPs (Figure S6C). We then narrowed our focus to Igfbp3, which was specifically downregulated in Runx2+ cell region following the loss of Runx2. We detected the expression pattern of Igfbp3 at the mRNA level using RNAscope. In control mice, Runx2 proteins and Igfbp3 mRNA were colocalized in the same cells (Figure 5Fa). After deleting Runx2, the expression of Igfbp3 was undetectable specifically in the apical region, where Runx2+ cells w" @default.
• W3048925464 created "2020-08-18" @default.
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• W3048925464 date "2020-08-01" @default.
• W3048925464 modified "2023-10-17" @default.
• W3048925464 title "Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling" @default.
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