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Abstract

Chondrogenesis is a developmental process that is central to the formation of cartilage and most bone, during which an initial cartilaginous template gradually ossifies in a process termed endochondral ossification. Bone morphogenetic protein (BMP) signaling is a highly conserved signaling pathway that induces a gene regulatory network (GRN) which drives chondrogenesis and endochondral ossification, with disruption of BMP signaling leading to severe skeletal defects. Despite its importance, it remains unclear the specific molecular mechanisms by which BMP signaling exerts its actions during chondrogenesis. This thesis seeks to define the BMP-activated GRN during chondrogenesis, identify targets of BMP signaling during this process, and determine how BMP signaling interacts with other critical regulatory inputs during chondrogenesis. To address these questions, we used murine primary limb mesenchymal (PLM) cells as a model of BMP-activated chondrogenesis. We assessed the transcriptome of BMP4-treated PLMs using RNA-seq, and determined the binding landscape of phospho-SMADs transcription factors (TFs) and the chondrocyte lineage determinant TF SOX9 using the novel sequencing technique CUT&Tag. We found that BMP signaling is required to activate a complete and robust chondrogenic program. SMAD target genes are predominantly comprised of genes with pleiotropic and indirect functions in chondrogenesis, such as cell adhesion. We also identified candidate sets of BMP-activated TFs which, despite lacking known chondrogenic roles, have multiple lines of evidence supporting probable novel chondrogenic function. SMADs in this context also function almost exclusively in collaboration with SOX9, whereas SOX9 can operate independently from SMADs. We proposed a model where SOX9 is the primary regulatory signal in specifying chondrocyte cell identity during BMP-activated chondrogenesis, whereas BMP signaling cooperates with SOX9 to establish a permissive environment for chondrogenesis. Finally, we examined how the target genes of SMADs, SOX9, and the osteogenic TF RUNX2 changes during late endochondral ossification. We found that all three regulatory inputs cooperate to mediate a switch from a chondrogenic to osteogenic program, and that BMP signaling is now capable of establishing osteoblast identity independently from SOX9 and RUNX2. Many new target genes for all three TFs were identified, providing a novel resource to interrogate the changing GRN during endochondral ossification.