The hedgehog target Vlk genetically interacts with Gli3 to regulate chondrocyte differentiation during mouse long bone development

• Inactivation of both Gli3 and Vlk causes severe shortening of specifically the long limb bones.
• The temporally coordinated establishment of the different types of chondrocytes is disrupted in Vlk∆/∆Gli3∆/∆ limbs.
• VLK is an essential modulator of IHH/GLI3 controlled long bone development.

Endochondral bone development is orchestrated by the spatially and temporally coordinated differentiation of chondrocytes along the longitudinal axis of the cartilage anlage. Initially, the slowly proliferating, periarticular chondrocytes give rise to the pool of rapidly dividing columnar chondrocytes, whose expansion determines the length of the long bones. The Indian hedgehog (IHH) ligand regulates both the proliferation of columnar chondrocytes and their differentiation into post-mitotic hypertrophic chondrocytes in concert with GLI3, one of the main transcriptional effectors of HH signal transduction. In the absence of Hh signalling, the expression of Vlk (vertebrate lonesome kinase, also called Pkdcc) is increased. We now show that the shortening of limb long bones in Vlk-deficient mouse embryos is aggravated by additional inactivation of Gli3. Our analysis establishes that Vlk and Gli3 synergize to control the temporal kinetics of chondrocyte differentiation during long bone development. Whereas differentiation of limb mesenchymal progenitors into chondrocytes and the initial formation of the cartilage anlagen of the limb skeleton are not altered, Vlk and Gli3 are required for the temporally coordinated differentiation of periarticular into columnar and ultimately hypertrophic chondrocytes in long bones. In limbs lacking both Vlk and Gli3, the appearance of columnar and hypertrophic chondrocytes is severely delayed and zones of morphologically distinct chondrocytes are not established until E16.5. At the molecular level, these morphological alterations are reflected by delayed activation and lowered expression of Ihh, Pth1r and Col10a1 in long bone rudiments of double mutant limbs. In summary, our genetic analysis establishes that VLK plays a role in the IHH/GLI3 interactions and that Vlk and Gli3 cooperate to regulate long bone development by modulating the temporal kinetics of establishing columnar and hypertrophic chondrocyte domains.