Bimonte, Sabrina (2010) Study of the role of Ofd1 in limb and endochondral bone development. [Tesi di dottorato] (Unpublished)

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Oral-facial-digital type I (OFDI) syndrome is a X-linked male lethal developmental disorder and belongs to the heterogeneous group of developmental disorders known as Oral-facial-digital syndromes (OFDs). This syndrome is ascribed to ciliary dysfunction and is characterized by malformation of the face, oral cavity and digits. Conditional inactivation using different Cre lines allowed us to study the role of the Ofd1 transcript in limb and skeletal development at embryonic and post-natal stages. We generated three conditional mutants. The first mutants were generated by crossing the Ofd1fl line with the transgenic line (Msx2Cre) that express the Cre recombinase under the control of the Msx2 promoter in the AER and the ventral ectoderm starting from E9.5-10. Limbs of Ofd1fl|Msx2Cre mice, display no skeletal phenotype indicating that Ofd1 does not play a role in limb patterning and outgrowth, although we cannot exclude a role for Ofd1 at earlier stages or in the dorsal ectoderm. The second mutants were obtained by crossing Ofd1fl female mice with the Prx1Cre transgenic male mice that express the Cre recombinase in the limb bud mesenchyme from E9.5, when the expression of Prx1Cre is predominant in the forelimb mesenchyme. By E10.5 the expression is evident in both limbs. Ofd1fl|Prx1Cre mice display a more severe skeletal phenotype in the forelimbs than in the hindilmbs. Skeletal defects include polydactyly with unpatterned digits, partial fusion of carpal joint elements and shortened long bones. We demonstrated that polydactyly in Ofd1fl|Prx1Cre mice was associated with progressive loss of Shh signaling and an impaired processing of Gli3. Shortened long bones were due to defective Ihh signaling, decreased proliferation and to premature differentiation of hypertrophic chondrocytes. In addition Ofd1fl|Prx1Cre mice display defective formation of the bone collar. Immunofluorescence and ultrastructural studies allowed us to demonstrate that Ofd1 is necessary for correct ciliogenesis in the limb bud mesenchyme and chondrocytes of long bones. These results indicate that, contrary to what previously shown for the embryonic node, in the limb bud mesenchyme and in the chondrocytes, Ofd1 is necessary for normal ciliogenesis but not for cilia outgrowth. Overall, these results suggest that Ofd1 is required in the mesenchyme at early stages of limb morphogenesis for Shh signaling to determine anteroposterior patterning of the digits and at later stages for proper endochondral bone formation. Finally we generated a thirth mouse model with inactivation of Ofd1 in limb chondrocytes via Col2a-Cre-mediated recombination. Ofd1fl|Col2aCre display dwarfism by P30 days after birth that was accompanied by complete loss of growth plate and depletion of chondrocyte cilia. The results demonstrate a role for Ofd1 in the process of post-natal skeletal development.

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