Fgf8 Alters The Osteogenic Fate Of Mesenchymal Cells In Humerus

The ossification process of bones in vertebrates is mainly composed of intramembranous osteogenesis and endochondral ossification.The bones of the extremities are mainly ossified by cartilage osteogenesis.Interchondral osteogenesis is characterized by the formation of cartilage intermediates by bone mesenchymal cells,which are then mineralized into bone.The process of cell differentiation involves fine regulation of signaling pathways,and FGF signaling pathway plays an important role.Fgf8 is one of the important members of the FGF family.In vitro cell differentiation assays have shown that Fgf8 can promote the differentiation of mesenchymal cells into fat,cartilage,and neural tissue,but these studies are all in vitro cell culture studies,and there is no mouse genetics.The experiments elucidated the function of Fgf8 in osteogenic differentiation of mesenchymal cells.Shox2^Cre/+mice were constructed in our laboratory,and Shox2-specific expression in the tibia mesenchymal cells of limb buds were observed and involved in the process of osteogenic differentiation of limb bud mesenchymal cells.This study will use Shox2^Cre/+mice,R26R^Fgf8 mice(Fgf8 conditionally overexpressed mice)and R26R^{m Tm G} mice(Cre reporter mice)to specifically overexpress in tibial mesenchymal cells during limb development.Fgf8,to explore whether overexpression of Fgf8 alters the fate of the osteogenic differentiation of mesenchymal cells.First,we used mouse mating to collect Shox2^Cre/+;R26R^Fgf8;R26R^{m Tm G} mutant mice and littermate wild-type mouse embryos from various stages,and sorted Fgf8overexpressing mesenchymal cells by flow cytometry.Real-time PCR detection.The results showed that the expression level of Fgf8 in the quadruped tibia mesenchymal cells of the mutant mice was significantly higher than that of the control group.This indicates that Shox2^Cre/+;R26R^Fgf8;R26R^{m Tm G} mutant mice successfully achieved Fgf8 gene overexpression in limb tibia mesenchymal cells.Secondly,Shox2^Cre/+;R26R^Fgf8;R26R^{m Tm G} mice and normal littermate wild mice were subjected to whole bone staining and tissue section HE and Azan staining.The results showed that Shox2^Cre/+;R26R^Fgf8;R26R^{m Tm G} mice quadrilateral tibia The loss of hard bone tissue in the middle,replaced by cartilage tissue,indicates that overexpression of Fgf8 in the limb tibia mesenchymal cells lead to differentiation of mesenchymal cells into chondrocytes without differentiation into hard bone cells.Finally,in order to confirm Fgf8 overexpression from gene expression levels,the fate of osteogenic differentiation of the quadruple tibia mesenchymal cells was altered.We obtained immunofluorescence co-localization experiments and found that in Fgf8overexpressing mice,the short bone mesenchymal cells of the Shox2 ^Cre/+lineage do not express the osteoblasts marker genes SP7 and Runx2,but the marker genes SOX9 and chondrocytes are abundantly expressed.SOX9,demonstrated that overexpression of Fgf8in the limb tibia mesenchymal cells results in the differentiation of mesenchymal cells into chondrocytes without differentiation into hard bone cells.Further,through Western blot analysis,which FGF pathway was activated by overexpression of Fgf8,we detected multiple key signaling molecules of FGF pathway,p-AKT,Akt,and p-ERK,and finally found overexpression of Fgf8 in limb tibia mesenchymal cells.It activates the P-ERK signaling pathway in the FGF signaling pathway.In summary,in this experiment,the embryos of mice overexpressing the Fgf8 gene in the limb tibia mesenchymal cells were obtained by genetically modified mice.Further,by histomorphometric staining,real-time fluorescent quantitative PCR,Western blot,immunofluorescence co-localization and other techniques,it was proved that overexpression of Fgf8 in the tibia mesenchymal cells during activation of the tibia osteogenesis in the extremities would activate The P-ERK signaling pathway changes the fate of mesenchymal cells to differentiate into hard cells,causing them to differentiate into chondrocytes,and finally causing the disappearance of cartilage in the tibia.This study provides a theoretical basis for the study of the mechanism of cartilage dysplasia in the limbs.