The Role Of Primary Cilia And Ciliary Transport Protein IFT140 In Mouse Molar Development

Background:Primary cilia are"antenna-like"organelles extending outward from the cell surface that detect and transduce chemical and mechanical signals in the extracellular environment,regulate cell behavior,and in turn regulate tissue development and homeostasis.state.Primary cilia have been shown to be present in almost all mammalian cells except lymphocytes.Gene mutations of ciliary constituent proteins can cause abnormal ciliary structure and dysfunction by interfering with ciliary assembly,depolymerization or signal transport process,resulting in serious diseases such as abnormal bone and tooth development,polycystic kidney disease,and developmental delay.These diseases are collectively referred to as Ciliopathies.Intraflagellar transport（IFT）is responsible for the complex signal transport in cilia,and IFT is very important for the formation and function of cilia.IFT consists of multiprotein complexes that can be biochemically broken down into IFT complexes B（IFT-B）and A（IFT-A）.Among them,the IFT-A complex binds to microtubules through dynein and is responsible for retrograde transport.The IFT140 protein is one of the core components of IFT-A and is responsible for the retrograde transmission of extracellular signals into cells.Tooth development goes through a strict regulatory process,including the critical stages of tooth embryo development,tooth crown and root formation,and depends on the coordination and participation of many signaling pathways.Primary cilia,as the signal conduction center protruding from the cell surface,act as the cell antenna and sense the environment and morphogenesis during tooth development.In recent years,many studies have shown that they play an important role in the regulation of tooth development.Meanwhile,ciliary gene mutations lead to numerous defects in tooth development of both humans and mice,including differences in tooth number,shape and structure.Human teeth have complex shapes for cutting,grinding and other functions,including incisors,cusps and molars.The key scientific questions are:how can tooth embryo be regulated and developed into different teeth,and what are its key functional cells and regulatory genes?In addition,how does primary cilia,as a bridge of signal transmission,regulate and influence tooth development through the IFT140?Therefore,this study will use the primary cilia-related gene IFT140 conditional knockout mouse as a model to deeply understand how the primary cilia,a specialized organelle for sensing functions,regulates the key signaling pathways of tooth development,and affects the differentiation of odontoblasts and odontoblast.Objective:The aim of this study was to understand the role of primary cilia on tooth development in mice by adjusting the expression of IFT140,to explore the mechanism of primary cilia/IFT140 in regulating tooth development of mice,and to preliminatively attempt to regulate tooth development by primary cilia in vitro.Methods:Part 1:Expression characteristics of primary cilia and IFT140 during tooth development1.Immunofluorescence staining was used to observe the distribution of primary cilia in tooth embryo development,crown and root formation,so as to analyze the possible role of primary cilia in signal transduction and odontoblast differentiation during tooth development.2.R26R^tdtomato/IFT140cre ER mice were used to trace the expression location and time point of IFT140 in molars and incisors of mice,and to predict the possible stages of IFT140 in cells,providing clues for phenotypic analysis of gene knockout animals.3.Immunofluorescence staining was used to confirm the relationship between primary cilia and IFT140.Part 2:Phenotype of conditional knockout of IFT140 in dental epithelial and mesenchymal cells of molars1.Since IFT140 global knockout mice are embryonically lethal,occurring at embryonic day 13.5（E13.5）,the conditional knockout mouse model of IFT140 gene was adopted.K14-cre,Prx1-cre,Osx-cre and Dmp1-cre mice were hybridized with Ift140^f/f mice,respectively,to obtain IFT140 gene knockout mice in epithelial stem cells,mesenchymal stem cells,preodontoblast cells and mature odontoblast cells.2.Phenotypic analysis of teeth in epithelial stem cell knockout IFT140 mice:Micro CT scanning,H&E staining and immunofluorescence staining were used to observe the differences of tooth morphology,key structure of tooth and distribution of primary cilia between Ift140^f/f/K14-cre mice and Ift140^f/f mice from the beginning of tooth embryo development.3.Phenotypic analysis of mouse teeth with a series of conditional IFT140knockout in mesenchymal stem cells:Teeth from IFT140 knockout mice were collected with different stages.Micro CT scanning and H&E staining were performed to observe the different effects of conditional IFT140 gene in mesenchymal stem cells,preodontoblast cells and mature odontoblast cells during mouse tooth development.Part 3:Mesenchymal stem cell（PRX1 labeled）deletion of IFT140 regulates the mechanism of tooth development1.R26R^tdtomato/Prx1-cre mouse was used to trace the locations and time points of PRX1⁺cells during the development of molars and incisors in mice,providing clues to explain the tooth morphological changes of Ift140^f/f/Prx1-cre mice.2.HE staining,immunofluorescence staining and q RT-PCR were used to investigate the morphogenesis mechanism of the molars in Ift140^f/f/Prx1-cre mice.HE staining was used to observe the morphogenesis pattern of the molars at continuous time points in the embryonic stage.P21 staining was used to observe the development of enamel knot.ARL13B?Arl13b staining and related indicators（Ptch1,Smo,Gli1,Gli2,Gli3）by q RT-PCR were used to detect the occurrence rate of primary cilia and changes in Shh signaling of stem cells.Ed U injection and staining were used to evaluate the effects on tooth development and proliferation.RUNX2 and SP7 staining were used to evaluate osteogenic/odontogenic differentiation.Root differentiation was assessed by NFIC staining.3.The changes of tooth development in Ift140^f/f/Prx1-cre mice were analyzed by transcriptome sequencing.Part 4:The regulation of tooth morphology by IFT140 in dental mesenchymal stem cells1.In vitro 3D culture of tooth embryo,Micro CT scanning and H&E staining were used to observe the changes of tooth crown morphological development after in vitro knockout of IFT140 in E13.5 tooth germ.2.Under the renal capsule culture technology,Micro CT scanning and H&E staining were used to observe the changes of crown morphology and root number of molars cultured under the renal capsule after in vitro removal of IFT140.3.In this study,Micro CT scanning and H&E staining were used to observe the morphological and developmental changes of the tooth embryo after IFT140 was knocked out in vitro and replanted into the mandibular bone of mice with missing teeth.Part 5:The role of PRX1 labeled dental mesenchymal stem cells in tooth development and periodontal membrane reconstruction1.In the previous studies,it was found that knockout of IFT140 only in PRX1labeled mesenchymal stem cells changed the morphological development pattern of mouse teeth,and different molars were affected differently.Therefore,we used R26R^tdtomato/Prx1-cre mice to observe the distribution of PRX1 labeled cells during tooth development in mice.The timepoint when PRX1⁺cells appeared and their distribution in different molars were observed.2.After determining the distribution range of PRX1⁺cells in tooth development,immunofluorescence staining（RUNX2,SP7,COL1A1,AMELX）was used to observe the differentiation of PRX1⁺cells’progeny.3.To characterize PRX1⁺cells in healthy human molars based on published single-cell sequencing data.4.The function of PRX1⁺cells in the process of allograft replantation was observed.PRX1 was knocked out in vitro cell culture model to observe the change of angiogenesis ability.Results:Part 1:Primary cilia/IFT140 are involved in the development of mouse tooth embryo,molars and incisors1.Primary cilia are highly expressed at the initial stage of mouse tooth embryo development,including bud stage,cap stage,bell stage and tooth tissue formation stage.2.Primary cilia showed different patterns in the differentiation process of epithelial stem cells and mesenchymal stem cells:from the absence of primary cilia in epithelial basal layer cells at the beginning to the dense distribution in inner enamel epithelial cells located near the endothelial side,while ameloblasts almost all had primary cilia located near the odontoblast side.Mesenchymal stem cells have primary cilia from the beginning of tooth development,and the primary cilia are most dense when they differentiate into preodontoblasts,while the primary cilia are rarely distributed when they have differentiated into odontoblasts.3.Comparison of IFT140 expression levels in various organs showed that the expression level of IFT140 was relatively highest in teeth and bone.IFT140 positive cells at root initiation after birth were marked,and the subsequent progeny cells were mainly concentrated in the active region of root end of molars.Part 2:Conditional knockout of IFT140 in dental epithelium and mesenchymal cells have different effects on molar development1.Knockout of IFT140 in dental epithelial stem cells(Ift140^f/f/K14-cre)did not significantly affect tooth morphology and mineralization.K14-cre began to label dental epithelial cells at the bud stage,but primary cilia still existed in the bud stage of Ift140^f/f/K14-cre mice,and the primary cilia disappeared at the late cap stage.2.Knockout of IFT140 at different stages of tooth mesenchymal cells had different effects on tooth development.There was no significant change in tooth development when IFT140 was knocked out in mature odontoblasts(Ift140^f/f/Dmp1-cre),and the root became shorter and mineralization weakened after IFT140 was knocked out in preodontoblast cells(Ift140^f/f/Osx-cre).Notably,after knockout of IFT140 in mesenchymal stem cells(Ift140^f/f/Prx1-cre),the morphogenesis of molar crown and root was significantly changed.Part 3:The absence of IFT140 in mesenchymal stem cells results in the loss of primary cilium structure and blocked Shh signaling1.The developmental changes of tooth morphological pattern in Ift140^f/f/Prx1-cre mice began at the early cap stage,and only primary enamel knot was formed.2.In Ift140^f/f/Prx1-cre mice,the occurrence rate of primary cilia in dental mesenchymal cells was significantly reduced,Shh signaling was impaired,and the expression levels of downstream molecules（Ptch1,Smo,Gli1）were decreased.3.In Ift140^f/f/Prx1-cre mice,dental mesenchymal cell proliferation did not change significantly in the early stage,but decreased in the late stage.4.The differentiation of mesenchymal cells in Ift140^f/f/Prx1-cre mice was firstly enhanced and then weakened.The trend was the same in root formation period.5.Transcriptomic sequencing of Ift140^f/f/Prx1-cre mice showed decreased expression of odontogenic genes.Part 4:In vitro regulation of IFT140 expression can alter the morphogenesis of mouse first molars1.At the bud stage,IFT140 was knocked out in vitro and 3D culture of the tooth germ was performed.Compared with the control group,the number of tooth cups and the shape of tooth crowns were reduced.2.After IFT140 was knocked out in vitro and transplanted to subrenal capsule for culture,the crown shape and the number of teeth were changed.3.After IFT140 was knocked out in vitro and the tooth germ was reimplanted into the jaw,the resulting tooth morphology was similar to that of IFT140 knocked out in mesenchymal stem cells in vivo,i.e.,double cusp single tooth.Part 5:PRX1 labeled dental mesenchymal stem cells play an important role in tooth development and can function as vascular pericytes in adult periodontal membrane reconstruction1.PRX1⁺cells were abundant in the early development of the first molar embryo,and almost all types of mesenchymal cells can be differentiated in the subsequent tooth formation,but the distribution of PRX1⁺cells in the early development of the third molar was limited.2.Sc RNA-seq data analysis identified PRX1 expressing cells with high expression of perivascular markers;In vivo staining also found PRX1⁺cells surrounding vascular endothelial cells,that is,PRX1⁺cells showed perivascular characteristics.3.In the process of periodontal membrane reconstruction after allograft,PRX1+cells from recipient mice migrated to the periodontal membrane of donor molars,and participate in the formation of new blood vessels as vascular pericytes.4.In vitro 3D co-culture of human periodontal membrane stem cells and umbilical vein endothelial cells showed decreased ability of angiogenesis after PRX1deletion.Conclusions:1.Primary cilia/IFT140 are involved in tooth development,especially during odontoblast differentiation.2.The absence of primary cilia/IFT140 in dental epithelial cells and mesenchymal cells has different effects on tooth development.The absence of IFT140in the epithelium has no significant effect on dental morphogenesis and mineralization.3.Knockout of IF140 in mesenchymal stem cells results in significant changes in molar morphology.4.The loss of IFT140 in mesenchymal stem cells results in the change of the morphogenesis of the first molars.The odontogenic differentiation ability of stem cells is first enhanced and then weakened.The proliferation ability has no obvious change in the early stage and is weakened in the late stage.5.The absence of IFT140 in mesenchymal stem cells significantly reduces the occurrence rate of primary cilia.Shh signal transport is blocked.6.The mesenchymal stem cell marker PRX1 marks most of the mesenchymal cells,but the distribution of mesenchymal stem cells is less in the early stage of the third molar.PRX1⁺cells can participate in the angiogenesis of periodontal regeneration as perivascular cells.