Abstract
The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.
Highlights
In the formation and longitudinal growth of endochondral bones, committed mesenchymal cells condense and differentiate into chondrocytes to form a growth plate, within which chondrocytes undergo coordinated and sequential differentiation phases of proliferation, cell cycle exit and hypertrophy, resulting in longitudinal bone growth[1, 2]
In the development of the mammalian growth plate, while several transcription factors are individually well known for their key roles in regulating phases of chondrocyte differentiation, there is little information on how they interact and cooperate with each other
We developed a searchable library of differentially expressed genes, Growth Plate Differential Gene Expression Library (GP-DGEL), which has fine spatial resolution and global transcriptomic coverage for discovery of processes, pathways and disease candidates
Summary
In the formation and longitudinal growth of endochondral bones, committed mesenchymal cells condense and differentiate into chondrocytes to form a growth plate, within which chondrocytes undergo coordinated and sequential differentiation phases of proliferation, cell cycle exit and hypertrophy, resulting in longitudinal bone growth[1, 2]. As proliferating chondrocytes (PCs) mature, they exit the cell cycle and enter a prehypertrophic phase. This phase is an important transition, which produces signals for maintaining proliferation on the one hand and on the other, to regulate the progression from proliferation to cell cycle exit, entry into a prehypertrophic state, followed by the final stages of differentiation in which the cells enlarge to form hypertrophic chondrocytes (HCs) and become osteoblasts [1,2,3,4]. Disruption of the progression from one differentiation state to the and the relative proportions results in skeletal defects such as chondrodysplasia [5, 6]
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