Study On The Function Of Erk1/2 In Hypertrophic Chondrocyte Fate Transition And Its Progeny Cells

Bone develops through either intramembranous ossification or endochondral ossification.Most bones,including the long bones of the extremities,the base of the skull,vertebrae,and ribs,are formed by endochondral ossification.The hypertrophic chondrocytes（HCs）are located at the lowermost end of the growth plate of long bones and form the cartilage-bone junction with the trabecular bones adjacent to the growth plate.HC expresses abundant type X collagen（Col10a1）,and is considered to be the"engine"of endochondral ossification.In long bone growth plates,chondrocytes continue to undergo a continuous process from proliferation to terminal hypertrophic differentiation and eventually disappear at the cartilage-bone junction.Whether apoptosis is the final fate of HCs has long been a hot topic debated in the field.Using genetic lineage tracing technology,we and other international teams have demonstrated that HCs can convert into a considerable number of osteoblasts and osteocytes in bone marrow cavity,as well as some mesenchymal cells with multiple fate potentials.However,little is known about the regulatory mechanisms of the fate transition of HCs.Extracellular signal-regulated kinases 1 and 2（ERK1/2）directly or indirectly affect cell survival,metabolism,differentiation,and other cellular behaviors by responding to various extracellular signals and the mitogen-activated protein kinase（MAPK）signaling pathway.We analyzed our previous single-cell sequencing results of mouse HCs and their progeny cells,and found that ERK/MAPK signaling pathway was significantly enriched in a subpopulation of HCs.We confirmed that ERK1/2 proteins were accumulated continuously from the pre-hypertrophic zone to the bottom of the hypertrophic zone,and activated in HCs.We then employed chondrocyte-specific Cre recombinase（Col2a1-Cre）and HC-specific Cre recombinase（Col10a1-Cre）transgenic mice to study the function of ERK1/2 in HC fate transition and its progeny cells.Through gene recombination mediated by the Cre-Lox P system,ERK1/2 genes were specifically deleted in chondrocytes and HCs,respectively,and HCs and their progeny cells were traced by ROSA^LSL-EYFP/ROSA^{LSL-m Tm G} reporter gene.The two obtained mouse models,Col2a1-Cre;Erk1^-/-;Erk2^fl/fl;ROSA^LSL-EYFP and Col10a1-Cre;Erk1^-/-;Erk2^fl/fl;ROSA^{LSL-m Tm G} mice,were analyzed in terms of tissue morphology,in situ molecular expression and 3D structure by HE staining,multiple immunofluorescence staining and Micro-CT.In the mouse model with Erk1/2 gene knockout in Col2a1⁺chondrocytes,the results showed that loss of ERK1/2 in the hypertrophic zone inhibited the fate transition of HCs at the cartilage-osseous junction,resulting in the stagnation of HCs at the cartilage-osseous junction,which significantly lengthened the hypertrophic zone,and indirectly caused vascular invasion obstacle.Additional results demonstrate that deletion of Erk1/2 lead to apoptosis increment and the reduction of cells derived from HCs,especially the number of osteoblasts and osteocytes.In the mouse model with Erk1/2 gene knockout in HCs,the results showed that loss of ERK1/2 in the progeny of HCs led to decreased osteoblasts and osteocytes,thinning of femoral trabecular bones and reduction of bone mass,with a phenotype similar to osteoporosis.In addition,in order to distinguish progenies of HCs from those of periosteal cells in future studies,we generated the Indian hedgehog（Ihh）-m Kate2^tomm20-Dre knock-in mouse line.In this line,Dre recombinase is specifically expressed in Ihh⁺prehypertrophic and HCs.We bred the mouse with a reporter mouse strain Rosa26^confetti2,and demonstrated that the fluorescent protein m Kate2 can label Ihh⁺chondrocytes,and m CFP can label HCs and their progeny cells of Ihh-m Kate2^tomm20-Dre;Rosa26^confetti2 mice.We also found for the first time that Ihh expression is expressed in retinal ganglion cells,Purkinje cells and gallbladder epithelial cells.Taken together,we found that ERK1/2 signaling pathway is activated in HCs,and provided in vivo genetic evidence that ERK1/2 regulate the fate transition of HCs and the survival and osteogenic differentiation of their progeny cells,presenting new clues for understanding the regulatory mechanism of the fate transition of HCs.Furthermore,we generated a gene knock-in transgenic mouse line expressing Dre recombinase in Ihh⁺chondrocytes of growth plate,providing a new tool for in-depth study of the mechanism of HC fate transition.