Function Study Of IHH And FGF8 In The Development Of The Distal Skeletal Elements In Vertebrate Limbs

In 1903, Farabee reported the first recorded disorder of human autosomal dominant Mendelian trait, a kind of inherited digit malformation. In 1951, Bell classified this disorder as brachydactyly type A1 (BDA1), which is characterized by shortening or missing of the middle phalanges. In 2001, Gao et al reported that heterozygous missense mutations in the Ihh gene caused BDA1. In this project, we analyzed three mutants and explored the molecular mechanism of digit formation.We studied the activity of Ihh signal by in vitro induction and in vivo induction. In in vitro study, we compared the induction effect of wildtype Ihh with three mutants. When C3H10T1/2 cells were induced by the plasmids which expressed the full length wildtype and mutant Ihh proteins, the mutants showed decreased effect, but didn't completely lose it. The induction of purified N-Ihh proteins showed similar results. Ihh expression pattern in chicks implicates that Ihh expressed in the tip of the digit plays a role in digit patterning. In in vivo study, Application of wildtype N-Ihh to the interdigital spaces in chick embryos resulted in up-regulated activity of Ihh signal pathway in mesenchyme and elongation of penultimate phalange, even an appearance of extra phalange. These results demonstrated that the reduced Ihh signal was the fundamental cause of BDA1.To discover the role of Ihh in digit formation, we further studied the changes of digit development induced by N-Ihh. This protein prolonged FGF8 expression in AER and expanded Sox9 expression in mesenchyme. FGF8 protein also expanded Sox9 expression, but its induction effect on digit formation was different from N-Ihh. Application of FGF8 at early stage induced extra digit and at later stage inhibited distal phalange formation. Application of both N-Ihh and FGF8 induced a large distal phalange. These results suggest Ihh and FGF8 play different roles and act synergistically to promote chondrogenesis during digit primordial elongation. Moreover, N-Ihh delayed AER regression and FGF8 accelerated AER regression. Both of them restrained mesenchymal cell apoptosis.Sox9 expression was not adjacent to the source of FGF signal. After analysis of Sef expression, we found that FGF signal induced Sef expression in the short range and Sox9 expression in the long range. Sef is an antagonist of FGF signal and possibly inhibites the intracellular pathway which activates Sox9 expression. We proposed a new model for negative feedback to explain this phenomenon. Further study on this model will provide important evidence for revealing pivotal mechanisms of bone development in which survival and differentiation of mesenchymal cells are coordinated.