Scx-cre Mice Were Constructed To Investigate The Differences In The Development Of Maxillofacial And Forelimb Tendons

Backgroud:Tendon is a dense connective tissue mainly composed of type I collagen and fibroblasts for force transmission.During tenogenesis,tenogeni cells interact with adjacent myogenic and chondrogenic primordium to form musculoskeletal system.It is known that maxillofacial tendons are different from those of limbs and trunk in terms of cell origin:maxillofacial tendons are derived from Cranial Neural Crest ecdermal Cells（CNCCs）,while the tendons of extremities are derived from lateral plate mesoderm,and trunk tendons from syndetome in the paraxial mesoderm derived somite.Scleraxis（Scx）is a highly specific marker of tendon pedigree,which is continuously expressed in tendon precursor and mature tendon cells to play an important role in the differentiation,recruitment,chemotaxis and agglutination of tendon precursor cells,as well as tendon extracellular matrix（ECM）generation.Fibroblast growth factors（FGF）pathway plays a role in the tendon progenitor specification,tendon differentiation and mechanical force transduction.Fgf8 and Fgf4 are expressed in the tendon and muscle cells at the myotendinous junction（MTJ）of chicken limbs,respectively.Fgf4 could positively regulate the expression of Scx and Tenasin C in chicken limbs,but Fgf8 could not induce the expression of Scx and Tenascin C.However,overexpression of Fgf8 in mouse maxillofacial tenogenic cells can inhibit the expression of Scx and impairs the development of tendons.There is no Fgf8 in the developing tendons of mice.Therefore,the specific role of Fgf8 in the tendon development needs to be further clarified.In this study,Scx-cre mouse was constructed to eliminate Scx and overexpression of Fgf8 in tendon,through which its influence on tendon development in maxillofacial and forelimb was explored.Objective:In this thesis,Scx^cre/cregene mice were constructed to knock out Scx gene in tendon precursor cells and eliminate Scx function in tendon development.Scx-cre;Rosa26R-Fgf8 mice were generated specifically overexpressed Fgf8 in differentiated tendon cells to obtain Fgf8 gene function.So that the role of Scx and Fgf8 in tendon differentiation and their differential effects on the development of maxillofacial tendon and forelimb tendon could be proved.Methods:Scx-cre mouse was constructed by CRISPR/Cas9,by which the first exon in Scx gene was replaced by cre.Scx-cre mice were crossed with Scx-cre mice or Rosa26R-Fgf8 mice to get Scx^cre/cremice and Scx-cre;Rosa26R-Fgf8 mice.Scx^cre/creembryos and wild-type embryos（WT）in the same litter were fixed,dehydrated,embedded and sectioned,respectively.Similarly,Scx-cre;Rosa26R-Fgf8 mice and control mice（Control）in the same litter were fixed,decalcified,immobilized,dehydrated,embedded and sectioned.Gross views and Masson staining were performed to determine the phenotype.The changes of maxillofacial and forelimb secondary structure were observed by Alcian blue/Alizarin red bone staining.The differences in cell proliferation and apoptosis were compared by Brd U labeled and TUNEL assay.TTRUST transcription factor database was used to search for transcription factor relationship network in tendon development.The subpopulation clustering of tendon cells was performed by single cell sequencing combined with R packet.Results:（1）Scx-cre mice were constructed by replacing the first exon of Scx with cre.After birth,the Scxcre/cremice showed the same phenotype as Scx-/-mice,indicating that the Scx-cre mice were successfully constructed.（2）Alcian blue/Alizarin red bone staining showed that the coracoid process of the mandible in E16.5 Scxcre/cremice was significantly shorter,the humerus triangular nodule of the forelimb was not formed,and the forelimb column was significantly thinner.（3）Masson staining showed that Scxcre/cremice,both in maxillofacial and forelimb,whose long tendon（force transmitting tendon）was severely damaged without blue-stained dense fibrous tissue structure,and with scattered cells.In contrast,muscle anchoring tendons can be formed.（4）Masson results showed that the coracoid process of the mandible in Scx^cre/cremice was short,the diameter of theprocessus angularis was reduced,the temporomandibular joint disc was not agglutinated,and the condylar cartilage was disordered.Humeral nodules were not formed,and the mineralized components of the bone cortex were reduced.（5）Brd U/TUNEL assay showed that compared with WT mice,there was no significant difference in the proliferation/apoptosis of maxillofacial tendon precursor cells in Scxcre/cre mice at E13.5 days,indicating that the damage in Scxcre/cremaxillofacial tendons was not achieved by affecting the cell proliferation and apoptosis.（6）Bone staining showed that the humeral nodule of Scx-cre;Rosa26R-Fgf8 mice showed obvious chondrogenesis after birth,and its shape became round and dull with increased blueness.（7）Masson staining showed that E18.5 days of Scx-cre;Rosa26R-Fgf8 mouse forelimb deltoid tuberosity development was inhibited.While only part of the maxillofacial masseter fibers were loose,and almost nothing obvious abnormality was observed in secondary bone.（8）Masson staining showed that compared with control mice,P3 weeks of Scx-cre;Rosa26R-Fgf8 mice had a smooth boundary instead of specific finger-crossing structure was formed in the masseter MTJ.In these mice,the anchor tendon was sparse,the coracoid process and the angular process enthese fibrous layers were reduced,and the temporomandibular joint disc was thickened and the condyle expansion was reduced.Whereas the nonmineralized fibrochondral layer of the humeral tubercle of the forelimb was thickened and the cells were disordered.It is suggested that overexpression of Fgf8in differentiated tendon cells has different effects on maxillofacial and forelimb tendons.（9）The forelimb tendon development gene regulatory network showed that Scx indirectly regulated Fgf8 through Six2.Single cell sequencing UMAP showed that the Scx and Osr2 lineages were mutually exclusive in distribution.Conclusion:1.The effect of Scx on the secondary structure of the forelimb bone was more serious than that of the mandible,suggesting that the development of maxillofacial secondary bone is less dependent on Scx lineage cells.2.The loss of Scx function has the same effect on the tendons of forelimb and maxillofacial region.The teansfer tendons are severely damaged,but muscle anchor tendons can be formed.There was no significant change in proliferation/apoptosis of damaged maxillofacial tendon cells.The results indicated that Scx also recruited and agglutinated tendon precursor cells for maxillofacial tendon elongation.3.Overexpression of Fgf8 in differentiated tendon cells inhibited the differentiation and maturation of Scx lineage cells in maxillofacial region,however promoted the increase of chondrogenic transdifferentiation of Scx lineage cells in the humeral tubercle of forelimb,suggesting that Fgf8 plays different roles in the development of maxillofacial tendon and forelimb tendon.