PROJECT SUMMARY/ABSTRACT Although the critical roles Hox genes play in establishing skeletal morphology has been known for decades, virtually nothing is understood regarding the molecular mechanisms by which Hox genes function in the skeleton. Utilizing a unique collection of genetic tools that permit live visualization of Hox expression (Hoxa11eGFP), Cre-mediated lineage labeling and/or conditional deletion (HoxCreERT2, Hoxd11LoxP/LoxP) and assessment of Hox11 chromatin binding sites (unpublished, validated Hoxa113XFLAG and Hoxd113XFLAG alleles), the overall objective of this application is to dissect the pathways and targets regulated by Hox transcription factors in skeletal stem/progenitor cells to regulate osteogenic and chondrogenesis differentiation. Previous work has demonstrated that Hox-expressing stem/progenitors are maintained in the skeleton in the absence of Hox function, and osteo- and chondrogenic lineages continue to emerge (Sox9-, Osx-/Runx2-expressing), but differentiation is incomplete. Osteoblasts do not progress to mature stages, and chondrocytes fail to undergo normal apoptosis and replacement by bony matrix in Hox mutants. This differentiation defect can be recapitulated in vitro. Based on previously published work and preliminary data, the central hypothesis is that Hox transcription factors regulate critical downstream events at the top of the hierarchy during osteochondrogenic differentiation from skeletal stem/progenitor cells in parallel with canonical differentiation factors. This project will utilize the Hoxa11eGFP reporter and Hoxa11CreERT2-mediated lineage labeling in the presence and absence of adult conditional deletion of Hoxd11 to probe the single cell trajectories of Hox11- expressing progenitors as they expand and differentiate into cartilage and bone in response to injury (Aim 1). The recapitulation of osteo- and chondrogenic differentiation defects in Hox11 mutants in vitro permits a comparative assessment of differential gene expression during temporally controlled differentiation (Aim 2). Newly generated and validated Hoxa113XFLAG; Hoxd113XFLAG epitope-tagged alleles will be utilized to interrogate the sites of chromatin binding in Hox-expressing progenitors and early differentiating cells (Aim 3). The research proposal is innovative in its use of sophisticated genetic tools generated by the research team, the combined in vivo and in vitro approaches, and critical inclusion of a co-investigator and her team with biostatistics expertise. The proposed research is significant as it addresses the longstanding and highly significant question of the molecular mechanism of Hox function in the skeleton. As Hox expression is only observed in skeletal stem/progenitors and early differentiation markers initiate as cells exit the Hox lineage, dissecting the downstream targets and pathways regulated by Hox that are critical to complete successful osteogenic and chondrogenic differentiation will provide impactful new ...