Objective We generated knock-in mice that express a tamoxifen-inducible Cre recombinase from the Prg4 locus (Prg4GFPCreERt2), and used these animals to fate-map the progeny of Prg4-positive articular cartilage cells at various ages. Methods We crossed Prg4GFPCreERt2 mice to Rosa26floxlacZ or Rosa26mTmG reporter strains, administered tamoxifen to the double heterozygous offspring at different ages, and assayed Cre-mediated recombination by histochemistry and/or fluorescence microscopy. Results In 1-month-old mice, the expression of the Prg4GFPCreERt2 allele mirrors expression of endogenous Prg4 and, when tamoxifen is given for 10 days, causes Cre-mediated recombination in ~70% of the superficial-most chondrocytes. Prg4GFPCreERt2 expressing cells are mostly confined to the top three cell layers of the articular cartilage in 1-month-old mice, but descendants of these cells are located in deeper regions of the articular cartilage in aged mice. At embryonic day 17.5, Prg4GFPCreERt2 expressing cells are largely restricted to the superficial-most cell layer of the forming joint, yet at approximately 1 year, progeny of these cells span the depth of the articular cartilage. Conclusions Our results indicate that Prg4-expressing cells located at the joint surface in the embryo serve as a progenitor population for all deeper layers of the mature articular cartilage. Also, our data reveal that Prg4GFPCreERt2 is expressed by superficial chondrocytes in young mice, but expands into deeper regions of the articular cartilage as the animals age. The Prg4GFPCreERt2 allele should be a useful tool for inducing efficient Cre-mediated recombination of floxed alleles at sites of Prg4 expression.

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor ␤. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.

No abstract

During endochondral ossification small immature chondrocytes enlarge to form hypertrophic chondrocytes, which express collagen X. In this work, we demonstrate that FoxA factors are induced during chondrogenesis, bind to conserved binding sites in the collagen X enhancer, and can promote the expression of a collagen X-luciferase reporter in both chondrocytes and fibroblasts. In addition, we demonstrate by both gain and loss of function analyses that FoxA factors play a crucial role driving the expression of both endogenous collagen X and other hypertrophic chondrocyte-specific genes. Mice engineered to lack expression of both FoxA2 and FoxA3 in their chondrocytes display defects in chondrocyte hypertrophy, alkaline phosphatase expression, and mineralization in their sternebrae and in addition exhibit postnatal dwarfism that is coupled to significantly decreased expression of both collagen X and MMP13 in their growth plates. Together, our findings indicate that FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program.

No abstract