IADR Abstract Archives
Cxcl12-Hedgehog Signaling Regulates Calvaria Homeostasis and Injury Repair
Objectives: Craniofacial bones support the face and protect the brain with a rigid cavity. Unlike long bones, cranial bones possess unique properties in terms of developmental origin, osteogenesis progress, and anatomical structure. The major stem cell population for cranial bone homeostasis has been identified to reside in the suture mesenchyme, in which Gli1 was found to mark suture stem cells (SuSCs) in vivo. However, how SuSCs respond to cranial bone injury distant from the suture and migrate there for regeneration remains elusive.
Methods: We used genetic lineage tracing, conditional gene expression/deletion, and single-cell RNA sequencing (scRNA seq) analysis to identify the behaviors and regulations of the Gli1+ SuSCs during cranial injury repair.
Results: CXCL12 and Hedgehog signaling was activated shortly after cranial bone injury. Activated Hedgehog signaling is concomitantly amplified by injury-induced Sonic hedgehog (Shh) expression. Blocking CXCL12-CXCR4 axis by AMD3100, a CXCR4 antagonist, drastically hampered injury repair and resulted in non-unions, which was further confirmed via genetic knockout (cKO) of Cxcr4 in Gli1-lineage cells. CXCR4 is a GPCR coupled to Gαi, which inhibits Gαs in activating protein kinase A (PKA). Intriguingly, we observed that Gli1+ SuSCs expressing gain-of-function Gαs mutant ‘phenocopied’ Cxcr4 cKO mice with fewer Gli1+ SuSCs recruited to the injury site and impaired healing. Conversely, Gli1+ cells with Gαs loss-of-function exhibited accelerated bone regeneration with increased Gli1+ SuSCs activation, activated Shh expression, enhanced osteogenesis and craniosynostosis. These phenotypes were partially rescues by an anti-Shh monoclonal antibody (mAb) treatment.
Conclusions: We found that upon calvaria injury, Gli1+ SuSCs were expanded, and participated in tissue regeneration by long-range migration and osteogenic differentiation. Molecularly, we found the CXCL12-CXCR4 axis played a pivotal role to guide Gli1+ SuSCs migration. The CXCL12-Hedgehog signaling axis is essential in calvaria injury repair by regulating stem cell migration, proliferation, and osteogenic differentiation, which sheds light on the SuSC-mediated regeneration. Our data also provide mechanistic insights into the cranial bone defects in human genetic diseases caused by mutations in the Gαs protein.
Methods: We used genetic lineage tracing, conditional gene expression/deletion, and single-cell RNA sequencing (scRNA seq) analysis to identify the behaviors and regulations of the Gli1+ SuSCs during cranial injury repair.
Results: CXCL12 and Hedgehog signaling was activated shortly after cranial bone injury. Activated Hedgehog signaling is concomitantly amplified by injury-induced Sonic hedgehog (Shh) expression. Blocking CXCL12-CXCR4 axis by AMD3100, a CXCR4 antagonist, drastically hampered injury repair and resulted in non-unions, which was further confirmed via genetic knockout (cKO) of Cxcr4 in Gli1-lineage cells. CXCR4 is a GPCR coupled to Gαi, which inhibits Gαs in activating protein kinase A (PKA). Intriguingly, we observed that Gli1+ SuSCs expressing gain-of-function Gαs mutant ‘phenocopied’ Cxcr4 cKO mice with fewer Gli1+ SuSCs recruited to the injury site and impaired healing. Conversely, Gli1+ cells with Gαs loss-of-function exhibited accelerated bone regeneration with increased Gli1+ SuSCs activation, activated Shh expression, enhanced osteogenesis and craniosynostosis. These phenotypes were partially rescues by an anti-Shh monoclonal antibody (mAb) treatment.
Conclusions: We found that upon calvaria injury, Gli1+ SuSCs were expanded, and participated in tissue regeneration by long-range migration and osteogenic differentiation. Molecularly, we found the CXCL12-CXCR4 axis played a pivotal role to guide Gli1+ SuSCs migration. The CXCL12-Hedgehog signaling axis is essential in calvaria injury repair by regulating stem cell migration, proliferation, and osteogenic differentiation, which sheds light on the SuSC-mediated regeneration. Our data also provide mechanistic insights into the cranial bone defects in human genetic diseases caused by mutations in the Gαs protein.