MTORC1 Signaling Promotes Limb Bud Cell Growth And Chondrogenesis

Objective:mTORC1 signaling has been shown to promote limb skeletal growth through stimulation of protein synthesis in chondrocytes.However,potential roles of mTORC1 in prechondrogenic mesenchyme have not been explored.In this study,we observed the effects of Raptor^f/f on mouse limb bud development though comparing the Raptor^f/f mouse embryos and normal mouse embryos.Meanwhile,we investigated the effects of Both pharmacological and genetic disruption of mTORC1 on cell proliferation,cartilage nodule formation and correlative gene expression in vitro.Methods:1.With mTORC1 pathway inhibitor rapamycin treating ADTC5,we used Western blot to test whether the mTORC1 was activated in ADTC5 and the inhibitory effect of rapamycin on mTORC1.We counted the number of ADTC5 in one or two day after the treatment of rapamycin and compared it with the control group.2.We compared limb buds isolated from E11.5 RapCKO with the control group of normal mouse embryos using the technique of flow cytometry analysis,microscopic imaging,whole-mount in situ hybridization,primary cell culturing into cartilage,Alcian blue staining to explore the difference between the two groups.3.We got ADTC5 for cell culture and induced ADTC5into cartilage.so we could use the methods of Alcian blue staining and gene expression to compare the experimental group mixed with 20nM rapamycin and the control groupResults:In this study,First we used rapamycin which was the inhibitor of of the mTORC1 to treat ADTC5.Western blot showed that the expression of phosphorylated S6 protein was inhibited,which indicated that the mTOR pathway was activated and rapamycin can inhibit the mTORC1 pathway effectively in ADTC5.Next we deleted Raptor,a unique and essential component of mTORC1,in prechondrogenic limb mesenchymal cells.Deletion of Raptor reduced the size of limb bud cells,resulting in overall diminution of the limb bud without affecting skeletal patterning.Then we examined the potential role of mTORC1 in chondrogenic differentiation in vitro.Both pharmacological and genetic disruption of mTORC1 significantly suppressed the number and size of cartilage nodules in micromass cultures of limb bud mesenchymal cells.Similarly,inhibition of mTORC1 signaling in ATDC5 cells greatly impaired cell proliferation,cartilage nodule formation.Inhibition of mTORC1 signaling decreased the expression of the master transcriptional factor Sox9,along with the cartilage matrix genes Acan and Col2a1.Conclusion:we have identified an important role for mTORC1 signaling in promoting limb mesenchymal cell growth and chondrogenesis during embryonic development.