Molecular and cellular consequences of Indian hedgehog mutations causing brachydactylies

Abstract

Hedgehogs are important morphogens essential for regulating a wide range of developmental processes. Mutations in IHH lead to digit abnormalities and skeletal defects such as Brachydactyly Type A1 (BDA1) and Acrocapitofemoral Dysplasia (ACFD). Our previous study focus on one mutation of IHH (p.E95K) causing BDA1 show altered signaling capacity and range, through impaired interaction with its receptor PTCH1 and modulator HIP1. E95K has been shown affecting on 〖Ca〗^(2+)binding sites critical for interaction with multiple signaling regulators, such as Ptch1, Hip1, Cdo, Boc and Gas1. As numerous mutations have been identified, we ask the question whether the previous pathogenesis model is common for all BDA1 mutations even ACFD mutations causing skeletal developmental defects. The structural analysis shows that all BDA1 mutations cluster within the 〖Ca〗^(2+) and 〖Zn〗^(2+)binding grooves, but ACFD mutations locate outside and away from this surface, implying different molecular mechanism underlie the similar skeletal defects. To further understand the molecular consequence, the current mutations are classified and selectively tested for their signaling activity in a fantastic chick neural tube in ovo electroporation system. The signaling activation is depicted by target genes expression including Class I and II transcription factors and quantitatively measured by Gli-luciferase reporter at signaling transcriptional level. These results show a common reduction in signaling capacity for all BDA1 and ACFD mutations. Furthermore, interaction study with Hip1, Cdo and Gas1, respectively and collectively, displays the different effects on each mutation in signaling activation, indicating that previous finding of impaired Hip1 interaction might not be the common consequence. Additional modulators, CDO and GAS1 also play important roles in regulating Ihh signaling pathway.

The regulation of Hh signaling pathway is complicated and characterized as spatial dynamic and tissue specific in developmental process. With the establishment of the regulation system in IHH signaling field in developing growth plate, I propose a mechanism whereby the impaired interaction with receptor PTCH1 in proliferative chondrocytes reduces signaling capacity with reduced cell proliferation. This signaling reduction assistant with the HIP1 defective restriction effect results in a further distribution of extracellular morphogen to periarticular and joint region. Subsequently, long-range signaling is enhanced, and through normal potentiation via CDO and GAS1 at the periarticular surface activating PTHrP, the consequence of which is an enhance feedback inhibiting chondrocyte hypertrophy. This together with reduced chondrocyte proliferation is a compounding effect, reducing bone growth in BDA1.

In developing digit, my findings further confirm and emphasize our previous hypothesis, which is the excessive IHH signal in the interzone has critical impact on distal digit outgrowth as a pathogenesis of short fingers and missing phalangeal joints. For ACFD, the mutations affected primarily interaction with PTCH1, and this could be a reason for the recessive nature of the disease.

My study of IHH mutations in neural tube and skeletal development provided new insights into the pathogenesis of BDA1 and ACFD thought the understanding of the molecular consequence of the mutations and the potential effect with the IHH signaling filed of the growth plate and synovial joint.