Effect Of Tooth Development In Mice With Constitutive Activation Of YAP In Neural Crest Derived Mesenchymal Cells

Background and ObjectiveIn the process of development of vertebrates,the fate of multifunctional neural crest cells is driven by environmental related external signal and cell differentiation of the integrated control of the internal signals..Synergistic and antagonistic signal networks can modulate the generation of neural crest derivatives and produce accurate numbers of cells at appropriate time and position.Neural crest derived odontogenic mesenchymal cells include pluripotent stem cells which can differentiate into odontoblasts,chondrocytes and osteoblasts cells.However,the key indicators of the signal network regulating the fate of dental mesenchymal cells remain unclear.In the process of dentin formation,dentin cells differentiated from cranial neural crest play an important role in dentin secretion after dentin precursor and terminal differentiation.The terminal differentiation of odontoblasts is controlled by enamel epithelium and depends on matrix mediated interaction.Wnt1-CRE transgenic mice have been widely used to detect the relationship between gene function and cell line in a variety of developmental environments,including the migration of early neural crest to the second palate,skull,cardiac output tract and the development of midbrain.Its role is based on the high expression of Wnt1 gene in dorsal neural canal before neural crest migration and middle posterior margin(MHB)in early midbrain development.Wnt1-CRE combined with a variety of Cre has been widely used in the tracking of neural crest and midbrain cells.The Hippo/YAP cascade growth factor is a very conserved signal pathway.The mutation of somatic pathway components in Drosophila melanogaster leads to high tissue proliferation.In addition,the discovery of some biochemical links in these genes has led to the elucidation of a series of biochemical phosphorylation transcripts.Until recently,all major members of Hippo signaling pathway were found in adult mice.Although we have described many main elements of Hippo trajectory,our understanding of signal trajectory is far from comprehensive.Materials and MethodsMouse maintenance and genotypingThe Wnt1-Cre transgenic line,YAP Tg and R26R conditional knockout alleles have been described previously.Wnt1-Cre;YAP Tg;R26R embryos were generated by mating Wntl-Cre male mice with YAP Tg;R26R female mice.The tail DNA was genotyped by PCR.Identification of CRE,YAP Tg,R26R,gene in mutant embryos by PCR genotyping.At noon of the day,the vaginal obstruction was found e0.5.The embryos were accurately classified by ganglion counting and compared with the control embryos.All mouse embryos used in this study were preserved in C57BL6/J.Histology and skeletal analysisThe samples were collected for histological analysis.Embryos were collected from female micss on a specific pregnancy date.The samples were fixed in 4%paraformaldehyde(PFA)at an ambient temperature of 4? for 24 hours.The samples were dehydrated by gradient ethanol series.The tissue samples were embedded in paraffin and treated with 8?m thickness for continuous tissue sections.The sections were stained with Hematoxylin and Eosin for histological analysis.Immunofluorescence staining and in situ hybridizationThe samples were collected for immunofluorescence staining and in situ hybridization.The embryos were treated as described above and embedded in paraffin.Indirect immunofluorescence staining was performed according to the technical procedure in this paper.The in-situ hybridization of sections was carried out according to the technical protocal in this paper.Finally,the RNA probes were labeled with digoxigenin.Analysis of cell proliferation and apoptosisThe samples were collected for Edu incorporation test to detect cell proliferation.Pregnant mice were intraperitoneal injection with 10 mM/ml Edu solution every 100g body weight,and embryos were collected 2 hours later.The samples were fixed in 4%PFA overnight and embedded in paraffin.Tissue sections were prepared according to standard techniques.Use the Edu staining kit to detect Edu according to the protocols provided by the manufacturer.Cell apoptosis was detected by terminal deoxynucleotide trans esterase mediated dUTP biotin nick end labeling(TUNEL)using an in-situ cell death detection kit(fluorescein)according to the manufacturer's protocols.Paraffin sections were rehydrated and treated with protease K according to the manufacturer's protocols.After substrate reaction,the slides were fixed with vectashield fixed medium and observed under a fluorescence microscope.Obtaining the mouse embryo head,washing the mouse head embryo with 4?PBS solution,the mouse jaw was extracted under the microscope,and the mouse molar embryo were separated.Then total RNA was extracted and purified by DNase digestion of Rnaeasy Mini Kit(Qiagen),and the quality of RNA samples was evaluated.The number of RNA integrity of the six samples was 10,indicating that the quality of RNA samples was high.In order to reduce the difference between individuals and embryos,each RNA SEQ sample was extracted from tooth embryos extracted from three different biological embryos to ensure the same amount of total RNA.Gene sequencing sequences were extracted and compared with Wnt1-cre;Transcriptome profiles of YAP Tg transgenic mice and control groups.Results1.Constitutive Activation of YAP in the CNC Cell,Resulting in Identical Defects of Meckel's and the mandibular bones.Since the expression of Wnt1 was limited to the migration of neural crest cells,we produced conditional YAP gene activated mice(Wntl CRE;YAP Tg)in all neural crest cells.All Wntl CRE;YAP Tg mutant mice have develop the same mandibular defects,including mandibular defects,condylar and coronal atrophy and angular reduction.At E18.5,the Meckel cartilage was formed,but smaller at Wnt1-CRE;YAP Tg mutant mice.There was significant difference between Wnt1-CRE;YAP Tg mutant mice and control group.In addition,we also observed the abnormal shape,curved shape and uneven thickness of Merkel cartilage at this stage.2.In situ hybridization of mouse embryonic molar embryos showed that Sprouty2 was weakly expressed in the control odontogenic epithelium and enhanced in the odontogenic epithelium and mesenchymal cells of transgenic mice.FGF3 was expressed in the cusp of control mice,but decreased significantly in transgenic mice.FGF9 was normally expressed in odontogenic epithelial basal cells and only slightly expressed in the same part of transgenic mice.FGF10 was expressed in the cusp and dental papilla,inhibited in transgenic mice,and only slightly expressed in the local dental papilla.Sprouty gene encodes the inhibitor of FGF,and the expression of signal molecules such as FGF3,FGF9 and FGF10 was significantly inhibited in transgenic mice.TGF?1 was mainly expressed in the cervical ring and inner enamel epithelium of control mice.In YAP Tg transgenic mice,TGF?1 expressed in epithelium was significantly inhibited.The expression of BMP4 was located in the apical region of dental papilla in control mice.In YAP Tg transgenic mice,the expression range was extended to the dental papilla region,and the signal was slightly enhanced.Shh gene was mainly expressed in bell shaped inner enamel epithelial cells in control mice,and there was no obvious abnormality in expression intensity and pattern in mouse tooth germ.The folding of inner enamel epithelium was reduced in most mice,accompanied by abnormal shape of the second enamel node.YAP expression was enhanced in cartilage and bone of YAP Tg transgenic mice,and the expression of b-catenin was significantly inhibited in Meckel's cartilage.Edu staining showed that there was no statistically significant change in the ratio of proliferating cells in Wnt1-Cre;YAP Tg transgenic mice compared with control mice.TUNEL was used to detect apoptosis,whether in control mice or Wnt1-CRE;YAP Tg transgenic mice did not detect apoptotic cells in the enamel organ.3.RNA-seq data showed that the expression of FGF gene family changed significantly with the expression of YAP gene.The changes of FGF3,FGF9 and FGF10 transcription levels coincided with the changes of YAP overexpression.The expression of Sprouty gene family also changed significantly.The transcriptional level of Sprouty was up-regulated and the overexpression of YAP gene occurred simultaneously.qPCR data and in situ hybridization also proved the up-regulated expression of Sprouty2 gene.Conclusion1.As an important part of Hippo signaling pathway,YAP is continuously expressed in the development of normal mouse teeth.By using WNT1-CRE and R26R gene,YAP gene can be constructed to be continuously overexpressed in cranial-face and tooth embryo at a specific stage of embryonic development.2.Constitutive activation of YAP can seriously affect the development of craniomaxillofacial.Neonatal mice show short skull development and facial fissure.Embryonic mice showed abnormal calcification of craniomaxillofacial bone,abnormal cartilage development and skull deformity.3.Constitutive activation of YAP can affect odontoblast differentiation.TGF-?/BMP signaling has been shown to function during odontoblast differentiation and dentin formation.We also observed abnormal cell polarization in odontoblasts of E18.5 Wntl-Cre;YAP Tg;mice,in which TGF? signaling is blocked in the dental epithelium.Resulting in abnormal development of mouse molar tooth germ and reduction of tooth germ volume,but the tissue differentiation of odontogenic epithelium and stem cells has not been significantly affected.Multiple signal factors related to tooth development are inhibited or enhanced.YAP can inhibit the expression of FGF signal family by enhancing the expression of Sprouty signal family,thus affecting the development of tooth germ.