BMP Signaling In Regulating Mesenchymal Stem Cells In Mouse Incisor Homeostasis

Objectives:Tissue regeneration could provide a better strategy as well as bring a bigger challenge to repair damaged tissues and organs of human body.To evaluate the success of tissue repair should be based on whether it can achieve the homeostasis balance as same as the normal tissue ultimately.Besides the scaffolds and the biological activity factors,the seed cells are the key part among the three elements of tissue engineering.Mesenchymal stem cells(MSCs)have become the research hotspot due to their excellent capacity of continuous self-renew,potential multi-directional differentiation,maintenance of tissue homeostasis and rehabilitation of the wounded tissue.Dental mesenchymal stem cells(DMSCs)are easy to obtain and cultivate,most importantly,they have incomparable advantages of tooth regeneration over other types of MSCs.Therefore,many other fields,including oral medical researchers are involved in the research of DSMCsMSCs regulate and maintain tissue homeostasis through a series of signaling pathways.BMP signaling pathway plays an important role in the tooth developmental stage of mammalian animals via regulating epithelial-mesenchymal interaction However,the function and mechanism of how BMP signaling pathway to maintain tissue homeostasis in adult stage which lasts for longer time and has more repair treatments is still unknown.The mouse incisor provides an excellent model for studying MSCs because the incisor grows continuously throughout the animal’s lifetime.Our research aims to explore the mechanism of BMP signaling in regulating MSCs to maintain tissue homeostasis using the mouse incisor as a model.This study would help to further elucidate the regulatory mechanism of BMP signaling pathway and the biological characteristics of MSCs,to provide more evidences for manipulating MSCs’ specific differentiation precisely,to enhance the therapeutic effect of the application of MSCs,to optimize4 the tooth regeneration strategies and to offer theoretical and basic experimental support to future clinical application and regenerative medicine trials.Methods:1.Immunofluorescence staining was used to detect the expression pattern of pSmad 1/5/9,the readout of activated BMP signal in adult mouse incisors,and lineage tracing was used to observe whether BMP signal was activated in Glil+MSCs(Glil positive Mesenchymal Stem Cells),TACs(Transit Amplifying Cells)and their derived cells.2.Gli1-CreERT2;Bmprlafl/fl transgenic mice,in which Bmprla was lost in Glil+MSCs derived cells,were generated by using Cre-Loxp system.In Glil-CreERT2;Bmpr1afl/fltransgenic mice,BMP signal was specifically knocked out in the Glil+MSCs derived cells of adult mice incisors.Micro-CT,HE staining,immunofluorescence staining and RNAscope in situ hybridization were used to observe the phenotype changes of incisors including the cervical loop structure,odontoblasts and ameloblasts differentiation and the growth of mice incisors.3.K14-rtTA;tetO-Cre;Bmprlafl/fl transgenic mice,in which BMP signaling was specifically ablated in Glil+derived dental epithelial cells,were generated by using rtTA-tetoCre system.Micro-CT,HE staining,immunofluorescence staining and RNAscope in situ hybridization were used to test the phenotype changes of incisors including the cervical loop structure,odontoblasts differentiation and deposition of dentin of proximal end.Then,the experiment outcomes of K14-rtTA;tetO-Cre.Bmprlafl/fl mice were used to compare to the control group Bmpr1afl/fl mice and another mutant group Glil-CreERT2;Bmpr1afl/fl mice to further clarify the role of BMP signaling pathway in maintaining adult mice incisors homeostasis by regulating MSCs.4.Immunofluorescence staining,TUNEL assays,RNAscope in situ hybridization,RNAscope multiple fluorescence staining,RT-PCR,Image J analysis and other methods were used to explore the cellular and molecular mechanisms of BMP signaling pathway regulating MSCs to maintain tissue homeostasis in mouse incisors.5.To observe and analyze the differentiation rate of Glil+MSCs progenitors and maintenance of Glil+MSCs population by immunofluorescence staining exerting on several transgenic mice including Gli1-LacZ,Gli1-CreER2";GlilLacZ,Gli1-CreERr2;Bmprlfl/fl,Glil-CreERT2;tdTomato,Glil-CreER-r2.Bmprlafl/fl;tdTomato models.Results:1.We found that BMP signaling was active(pSmadl/5/9 positive)in the preodontoblasts/odontoblasts,dental pulp,and a small number of TACs in one-month-old control mice,but was not detectable in the Glil+MSC region.Then,we performed double staining of Ki67 and pSmad1/5/9 in one-month-old control mice and found that BMP signaling activity was detected adjacent to,but not overlapping with the majority of TACs,except for a few TACs in the most distal region bordering pulp cells and preodontoblasts.In addition,we investigated co-localization of active BMP signaling and the progeny of Gli1+cells using lineage tracing.One day after tamoxifen induction of one-month-old Glil-CreERT2;tdTomato mice,Gli1+(tdTomato+)cells were located in the proximal region,where BMP activity was not detectable.One week after tamoxifen induction,as Gli1+MSCs begin to exit their niche and migrate to the TAC region,Gli1+progeny co-localized with BMP signaling activity in the transition zone between TACs and preodontoblasts.Four weeks after induction,as progeny of Glil1+cells differentiated into odontoblasts and dental pulp cells,we found that they co-localized extensively with activated BMP signaling in the preodontoblast region and dental pulp cells in close proximity to this region.2.After loss of Bmpr1a in Gli1+derived cells,we observed slightly shorter incisor dentin two weeks after tamoxifen induction in adult Gli1-CreERT;Bmpr1afl/fl mice,but significantly shorter incisor dentin and a severe defect of the proximal region of the incisor were recorded four weeks after tamoxifen induction in adult Gli1-CreERT2;Bmpr1afl/fl mice using microCT analysis.Eight and twelve weeks after induction,we observed more significant shortening of the distal region of the incisor dentin compared to control mice,suggesting that loss of BMP signaling affects turnover and tissue homeostasis,eventually disrupting incisor growth in Gli1-CreERT2;Bmprlafl/flmice.HE staining revealed that the cervical loop and cells were disorganized,normal anatomical layers was hard to distinguished,enamel and dentin in the proximal region was not detectable four weeks after tamoxifen induction in Glil-CreERT2;Bmprlafl/fl/fl incisors.Moreover,expression of Amelogenin(Amelx)and dentin sialophosphoprotein(Dspp),was decreased from the proximal preodontoblast region to distal region with induction extended.Eight weeks after tamoxifen induction,Glil-CreERT2;Bmpr1afl/flmice exhibited severely disorganized dental pulp tissue.Additionally,some ectopic cartilage-like structures which were positive for both Collagen Ⅱ and tdTomato were present in the dental pulp cavity in Gli1-CreERT2;Bmpr1afl/fl;tdTomato mouse incisors,suggesting that the Glil+progeny switched to a chondrogenic fate following loss of BMP signaling.3.We generated K14-rtTA;tetO-Cre;Bmprlafl/fl mice,in which BMP signaling was specifically ablated in Glil+derived dental epithelial cells.Four weeks after doxycycline induction at one month of age,dentin formation was unaffected in the proximal end of incisors of K14-rtTA;tetO-Cre;Bmprlafl/fl mice based on Micro-CT analysis.HE staining showed that the cervical loop had changed slightly,we still could distinguish the outer and inner enamel epithelium.More importantly,expression of odontoblast differentiation marker Dspp in K14-rtTA;tetO-Cre;Bmprlafl/fl mice was indistinguishable compared to Bmpr1afl/fl control mice.The results demonstrate that BMP signaling in the dental mesenchyme,rather than in the dental epithelium,is specifically required to regulate odontoblast differentiation and dentin formation in adult mouse incisors.4.We detected ectopic proliferating cells in the preodontoblast/odontoblast region in Gli1-CreERT2;Bmpr1afl/fl mice one week after TMX induction at one month of age,indicated by Ki67 immunostaining.In contrast,apoptosis appeared unaffected as assessed by TUNEL assay.During the proliferation and differentiation process,distinct signaling pathways are required to regulate the MSC differentiation hierarchy.We analyzed the activities of WNT and FGF signaling in mice incisors using Axin2 and Etv4 as readouts,respectively.In control incisors,WNT and FGF signaling were highly active in the TAC region,proximal to the region where BMP signaling was activated.In Glil-CreERT2;Bmprlafl/fl mice one week after tamoxifen induction,WNT and FGF signaling were increased in dental pulp cells close to the preodontoblast region compared to controls.Similarly,we found that Wnt10a and Fgf3 expression was upregulated in the same region.We confirmed the upregulation of WNT and FGF signaling by qPCR analysis.In addition,we found that ectopic WNT and FGF signaling activity overlapped with the region in which proliferation was upregulated,suggesting that increased WNT and FGF signaling may be responsible for ectopic proliferation and odontoblast differentiation defects in Glil-CreERT2;Bmpr1afl/al mice.5.We examined Glil expression in Glil-CreERT2;Bmpr1afl/fl;Gli1-LacZ mice and found that Gli1+MSCs were greatly reduced in number compared to the controls after BMP signaling was specifically ablated in Gli1+MSCs derived cells.Meanwhile,we observed a reduced number of Gli1+progeny in Gli1-CreERT2;Bmprlafl/fl;tdTomato mutant incisors after two weeks TMX induction,particularly in the more distal region,suggesting that the differentiation rate of Gli1+ MSCs was slower,rather than faster Considering these findings,BMP signaling in the preodontoblast/odontoblast region may provide feedback to MSCs in the mouse incisor to sustain the MSC population and maintain tissue homeostasisConclusions:This study investigated the important role and regulatory mechanism of BMP signaling in maintaining tissue homeostasis in adult mouse incisor through regulating MSCs.Our results show that activated BMP signaling is associated with preodontoblasts/odontoblasts and dental pulp cells.Loss of Bmpr1a in the lineage derived from Gli1+cells led to compromised odontoblast differentiation and incisor growth defects.Furthermore,our study demonstrates that BMP signaling serves as a key regulator that antagonizes WNT and FGF signaling to regulate the fate of MSCs Importantly,we found that compromised BMP signaling in the Gli1+lineage also led to a diminished Gli1+ MSC population,suggesting that BMP is not only important for odontoblast differentiation,but also plays a crucial role in providing feedback to maintain the MSC population.This study highlights the essential and dual roles of BMP signaling in the molecular network that regulates adult mouse incisor tissue homeostasis.