Hand1, Hand2, and Twist1 are members of the Twist class of Basic Helix-Loop-Helix (bHLH) proteins, which are expressed within the heart, branchial arches, lateral mesoderm and developing limb buds. Deletion of either Hand1 or Hand2 from the mouse genome results in embryonic lethality at E9.5 due to either extraembryonic or cardiac defects. Conditional ablation of Hand2 within the limb indicates that Hand2 plays a role in limb antero-posterior (AP) axis formation (radius and ulna, thumb to little finger). Ectopic expression of either Hand1 or Hand2 within the limb results in preaxial polydactyly, a typical AP patterning defect. The AP axis is defined in the zone of polarizing activity (ZPA), located within the posterior limb mesenchyme, by the morphogen sonic hedgehog (Shh). Hand2 is one of the transcription factors responsible for making the posterior limb mesenchyme competent to express Shh. Twist1 is partially coexpressed with Hand1 and Hand2 within the developing limb. Mutations in TWIST1 are causative of the human autosomal dominant haploinsufficient disease Saethre-Chotzen Syndrome (SCS), which, among a number of phenotypic characteristics, include preaxial polydactyly. Twist1 heterozygous null mice model phenotypes found in SCS and homozygous Twist1 null mice exhibit hypoplastic limb buds and die at E11.5. Genetic evidence indicates that Twist1 represses Shh expression in the anterior limb mesenchyme to help define the AP axis via an antagonistic genetic balance between Hand2 and Twist1. Twist family bHLH function is controlled by spatial-temporal expression (i.e. the level of expression and its location) and the formation of transcriptional complexes (dimerization). Twist and Hand proteins can form both homo- and heterodimers with themselves, as well as other bHLH factors. Control of dimer formation is regulated by an evolutionarily conserved threonine and serine located within Helix I of the bHLH domain of all Twist family proteins. These conserved residues are phosphoregulated by the actions of Protein Kinase A (PKA) and the B564-containing protein phosphatase 2A (PP2A). Point mutations in Twist1 that alter Helix I phosphoregulation result in changes in Twist1 function. Human mutations in TWIST1 that disrupt Helix I phosphoregulation are associated with causing SCS, reflecting an inability of these SCS TWIST1 alleles to antagonize HAND2 function. Thus, we hypothesize that the control of Twist family bHLH factor dimerization choice governs their specific biological effects within the tissues in which they are expressed. The goal of this proposal is to further define the genetic and molecular mechanisms that control Twist-family behavior and regulate both established and novel limb transcriptional programs.
This proposal seeks to define the role of Twist family bHLH factors in shoulder, pelvic girdle and limb formation. By using a series of gain-of-function and loss-of-function Hand and Twist mouse mutant alleles, we can activate/delete these factors in combination using several limb expressing Cre Recombinase mouse lines. Morphology/histology and gene expression analysis will then be obtained and compared across experiments.
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