This dissertation, "The Molecular Consequences of Indian Hedgehog Mutations in Distal Digit Patterning" by Kit-fong, Stephanie, Law, 羅潔芳, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled 'The Molecular Consequences of Indian Hedgehog mutations in Distal Digit Patterning.' Submitted by LAW KIT FONG For the degree of Master of Philosophy at the University of Hong Kong in September 2004 Indian hedgehog (IHH) is a member of the hedgehog protein family. It has an important role in endochondral ossification, regulating chondrocytes proliferation and differentiation. Recently, Ihh was shown to be associated with brachydactyly type A- 1 (BDA1) and acrocapitofemoral dysplasia (ACFD), suggesting a role in digit patterning. BDA1 (MIM 112500) is an autosomal dominant disorder, characterized by shortened or fused phalanges. Short stature is often associated with BDA1 patients, and mental retardation in women is sometimes observed. ACFD (MIM 607778) is a recessive congenital disorder, phenotypes of which include brachydactyly, short stature, and short limbs with cone-shaped epiphyses. Mutations in IHH causing BDA1 and ACFD lie on distinct regions of the protein. The BDA1 and ACFD mutations are likely to affect normal IHH functions in different ways and studies on the molecular consequences will provide insights into the different functional domains of the protein. To gain an understanding of the molecular consequences of these mutations, the first issue addressed was whether these mutations result in a loss of function for the mutant Ihh proteins, thus representing haploinsufficiency for Ihh. This mechanism was tested genetically using mice that are heterozygous for an Ihh-null allele (Ihh+/-), which contains only one functional allele and are therefore haploinsufficient for Ihh. Careful analysis of the distal digits showed no indication of shortened or missing middle phalanges and no indication of dwarfism. This is consistent with the absence of the BDA1 phenotype in these mice, suggesting that Ihh haploinsufficiency is not a mechanism for BDA1 in the mouse. The abilities of the mutant Ihh proteins to signal through the hedgehog signaling pathway was studied using an in vitro system. To establish this assay, wild-type cDNA Ihh constructs was created for expression in mammalian cells. In addition, mouse cDNA constructs containing the equivalence of human mutations identified to be associated with BDA1 and ACFD were generated. To test the function of the expressed wild-type and mutant Ihh proteins, a cell-based hedgehog induction assay using a newly established C3H 10T1/2-luc/lacZ reporter cell line was used, which gives quantitative luciferase signal upon hedgehog signaling. Preliminary data of induction assays using conditioned medium from transfected cells expressing wild- type Ihh and Ihh containing the E131K mutation showed both forms of the protein could signal with similar efficiency. While not all the mutant constructs have been fully tested, preliminary data indicate that using this in vitro assay, signaling at the cell level is not impaired. Base on the finding that loss-of-function is unlikely to be a factor, which is supported by the preliminary data that mutant Ihh proteins can transduce an appropriate intracellular signal, a mechanism involving the improper establishment of an Ihh gradient at the distal digital cartilage anlagen is proposed. This is a working model involving Gdf5, a gene important in joint formation. Further experiments are required to confirm the functional propert