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The forkhead box transcription factor genesFoxc1andFoxc2are expressed in the condensing mesenchyme of the developing skeleton prior to the onset of chondrocyte differentiation. To determine the roles of these transcription factors in limb development we deleted bothFoxc1andFoxc2in lateral plate mesoderm using the Prx1-cre mouse line. Resulting compound homozygous mice died shortly after birth with exencephaly, and malformations to this sternum and limb skeleton. Notably distal limb structures were preferentially affected, with the autopods displaying reduced or absent mineralization. The radius and tibia bowed and the ulna and fibula were reduced to an unmineralized rudimentary structure. Molecular analysis revealed reduced expression of Ihh leading to reduced proliferation and delayed chondrocyte hypertrophy at E14.5. At later ages, Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δembryos exhibited restored Ihh expression and an expanded COLX-positive hypertrophic chondrocyte region, indicating a delayed exit and impaired remodeling of the hypertrophic chondrocytes. Osteoblast differentiation and mineralization were disrupted at the osteochondral junction and in the primary ossification center (POC). Levels of OSTEOPONTIN were elevated in the POC of compound homozygous mutants, while expression of Phex was reduced, indicating that impaired OPN processing by PHEX may underlie the mineralization defect we observe. Together our findings suggest that Foxc1 and Foxc2 act at different stages of endochondral ossification. Initially these genes act during the onset of chondrogenesis leading to the formation of hypertrophic chondrocytes. At later stages Foxc1 and Foxc2 are required for remodeling of HC and for Phex expression required for mineralization of the POC.

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by thePrg4gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to inducePrg4expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to inducePrg4expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to twoPrg4promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction ofPrg4by TGF-β. Our results indicate that Creb5 is a critical regulator ofPrg4/lubricin expression in the articular cartilage.