Molecular and cellular consequences of Indian hedgehog mutations causing brachydactylies

Abstract

Hedgehogs are important morphogens essential for 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). Previous study in a BDA1 mouse model with a p.E95K mutation in IHH result in an impaired signaling capacity and but increased range of signaling, through impaired interaction with its receptor PTCH1 and modulator HIP1, respectively. As numerous mutations have been identified for BDA1, we ask that whether this is a common molecular consequence for BDA1 mutations, and what is the relationship with recessive disorder, ACFD. We aim to understanding the molecular pathogenesis of BDA1 and ACFD in relation to the hedgehog-signaling field within the cartilage ends of a developing long bone and synovial joints. 3D structural analysis showed that mutations are clustered within Ca2+ and Zn2+ binding grooves that mediate interactions with PTCH, HIP1, CDO, BOC and GAS1. Additionally, ACFD mutations are away from these binding centers. To understand the molecular consequence, IHH mutations are grouped and generated in expression constructs to evaluate the signaling capacity in an in ovo electroporation system, using SHH downstream responsive genes, as well as a Gli luciferase reporting system for quantitative analyses. Results showed a common reduced signaling capacity for all selected mutations, but differential effect on the binding with signaling modulators. With the establishment of IHH signaling field within growth plate, a mechanism is proposed whereby the impaired interaction with the receptor PTCH1 reduces signaling capacity with reduced cell proliferation. The mutations also affected interaction of HIP1, a negative regulator that restricts IHH distribution through the zone of proliferative chondrocytes. As a consequence, signaling range is enhanced, and through normal potentiation via CDO and GAS1 at the periarticular surface activating PTHrP, the consequence of which is an enhance negative feedback inhibiting chondrocyte hypertrophy. This together with reduced chondrocyte proliferation is a compounding effect, reducing bone growth as part of the pathogenesis in the BDA1 mouse. Within the joint, this finding further confirms previous hypothesis where IHH signals further into the interzone, because of the long-range effect of the mutations. In BDA1, this excessive IHH signal in interzone has an impact on distal digit outgrowth as a pathogenesis of BDA1 with short fingers and missing phalangeal joints. For ACFD, the mutations affected primarily PTCH1 interaction, and this could be a reason for the recessive nature of the disease.