13-P121 Investigating the dynamics of Notch signalling in the embryonic chick inner ear using a reporter-based approach

The vertebrate body is supported by a bony skeleton, which is largely formed by endochondral ossification. To ensure normal bone growth, differentiation of chondrocytes has to be tightly regulated. One of the major regulators of this process is the growth factor Indian hedgehog (Ihh). Endochondral ossification is impaired in Ihh mutant mice due to disturbed chondrocyte proliferation and differentiation. In vertebrates hedgehog signalling is transduced by the transcription factors Gli2 and Gli3, which can form both a transcriptional activator and a transcriptional repressor in various tissues. Analyses of Ihh/Gli3 double mutants have demonstrated role for Gli3 as a transcriptional repressor in this context. By using a floxed Gli2 allele we created Ihh/Gli2 and Gli3/Gli2 double mutant mice. In these double mutants endochondral ossification is severely disturbed. Ihh/Gli2 mutant limbs display a reduced size and chondrocytes remain undifferentiated until E16.5. Like in Ihh single mutant limbs at E16.5 almost all chondrocytes of the anlagen turn hypertrophic. Hybridiziation of chondrocyte markers like ColX, Gli1 and UCMA on Ihh/Gli2 double mutant and Ihh single mutant limbs show identical expression patterns thus making a repressor function of Gli2 unlikely. In Gli2/Gli3 double mutants exhibit severe defects in bone formation and delayed chondrocyte differentiation, closely resembling the Ihh/Gli3 double mutant phenotype. As Ihh/Gli3 double mutants Gli2/Gli3 mutant bones do not express Gli1. Nevertheless either single mutant expresses Gli1 strongly indicating that Gli3 as well as Gli2 can activate gene expression during endochondral ossification.