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Abstract

Bones of vertebrate limbs are formed by endochondral ossification. During this process a cartilage model of the future skeletal elements initially forms. Chondrocytes in the cartilage model undergo various steps of differentiation from proliferating into hypertrophic chondrocytes, which are subsequently replaced by bone. One of the critical steps during this process is the regulation of chondrocyte proliferation and differentiation in the developing cartilage anlagen. Previous studies have demonstrated that Indian hedgehog (Ihh) and Parathyroid hormone-like peptide (Pthlh) interact in a negative feedback loop to regulate chondrocyte differentiation. Ihh, which is expressed in prehypertrophic chondrocytes, activates the expression of Pthlh in the periarticular region of the developing skeletal elements. Pthlh in turn signals back to its receptor (Pthr) in proliferating and hypertrophic chondrocytes, and prevents the onset of hypertrophic differentiation of chondrocytes, thereby inhibiting Ihh expression. In addition, Ihh was shown to positively regulate chondrocyte proliferation. Several lines of evidence suggest an interaction of the Ihh/Pthlh pathway with other signaling factors, including Bone morphogenetic proteins (BMPs) and Fibroblast growth factors (FGFs). Previous studies on chicken limbs have shown that ectopically expressed activated BMP receptor 1A upregulates the expression of Pthlh and blocks chondrocyte differentiation. Therefore, it was hypothesized that BMPs act downstream of Ihh and mediate the Ihh signals to induce Pthlh expression. In this study, a potential interaction of the Ihh/Pthlh and BMP signaling pathways was analyzed using a culture system for embryonic mouse and chick limbs, which was supplemented with activators and inhibitors of both signaling pathways. The results have shown that BMPs are not acting as secondary signals of Ihh in mediating the induction of Pthlh expression. Instead BMP signaling acts at various steps of chondrocyte differentiation and interacts with Ihh signaling in different ways. First, both BMP and Ihh signals are essential for chondrocyte proliferation and act in parallel. Second, BMP signaling regulates the expression level of Ihh thereby coordinating the rate of chondrocyte proliferation and the onset of hypertrophic differentiation. Third, a negative role for BMP signaling was identified in regulating the differentiation of terminal hypertrophic cells. A third signaling pathway critical for proliferation and differentiation of chondrocytes is the FGF signaling pathway. Constitutive activation of the FGF receptor 3 (Fgfr3) gene results in specific dwarfism syndromes in human and mice. On the molecular level activated FGF signaling results in reduced proliferation and differentiation of chondrocytes. In this study the epistatic relationship of FGF signaling with the Ihh/Pthlh and BMP signaling pathways was analyzed. Activation of FGF signaling in the limb culture system showed decreased chondrocyte proliferation and reduced Ihh expression similar to that in a mouse model for human dwarfism (FGFach mouse). Moreover FGF signaling acts upstream of Ihh/Pthlh signaling and regulates the onset of hypertrophic differentiation indirectly by regulating Ihh expression. Furthermore, FGF signaling advances the hypertrophic differentiation process instead of inhibiting it, as it was proposed in previous studies. Additionally, as activation of FGF signaling resembles a loss of BMP signals the interaction of FGF and BMP signaling was analyzed. Both pathways were found to have antagonistic functions during chondrocyte development. Moreover, activation of BMP signaling in FGFach mice in the limb culture system can increase the reduced rate of chondrocyte proliferation and Ihh expression thus rescuing the dwarfism phenotype. Summarizing, the results of this study allowed to integrate three signaling pathways, Ihh/Pthlh, BMP and FGF, into a common control network regulating chondrocyte development.