The role of Twist family bHLH factors in limb morphogenesis

描述（由申请人提供）：Hand 1、Hand 2和Twist 1是基本螺旋-环-受阻（bHLH）蛋白的Twist类的成员，其在心脏、鳃弓、侧中胚层和发育中的肢芽中表达。从小鼠基因组中删除Hand 1或Hand 2会导致E9.5的胚胎死亡，原因是胚胎外缺陷或心脏缺陷。肢体内Hand 2的条件性消融表明Hand 2在肢体前后（AP）轴形成（桡骨和尺骨，拇指至小指）中发挥作用。肢体内Hand 1或Hand 2的异位表达导致轴前多指（趾）畸形，这是一种典型的AP图案缺陷。AP轴定义在极化活动区（ZPA），位于后肢间充质内，由形态发生素音刺猬（Shh）。Hand 2是负责使后肢间充质有能力表达Shh的转录因子之一。 Twist 1在发育肢体中与Hand 1和Hand 2部分共表达。TWIST 1中的突变是人类常染色体显性单倍不足疾病Saethre-Chotzen综合征（SCS）的病因，该疾病在许多表型特征中包括轴前多指（趾）。在SCS中发现的Twist 1杂合缺失小鼠模型表型和纯合Twist 1缺失小鼠表现出发育不良的肢芽，并在E11.5死亡。遗传学证据表明，Twist 1抑制Shh在前肢间充质中的表达，以帮助通过Hand 2和Twist 1之间的拮抗性遗传平衡来定义AP轴。Twist家族bHLH功能受时空表达（即表达水平及其位置）和转录复合物的形成（二聚化）控制。Twist和Hand蛋白可以与自身以及其他bHLH因子形成同源二聚体和异源二聚体。二聚体形成的控制由位于所有Twist家族蛋白的bHLH结构域的HHLH I内的进化上保守的苏氨酸和丝氨酸调节。这些保守残基通过蛋白激酶A（PKA）和含B564的蛋白磷酸酶2A（PP 2A）的作用进行磷酸化调节。Twist 1的点突变改变了HAT 1的磷酸化调节，导致Twist 1功能的改变。破坏HAND 1磷酸调节的TWIST 1中的人类突变与引起SCS相关，反映了这些SCS TWIST 1等位基因不能拮抗HAND 2功能。因此，我们假设Twist家族bHLH因子二聚化选择的控制支配它们在表达的组织内的特定生物学效应。该提案的目标是进一步确定控制Twist家族行为和调节已建立和新的肢体转录程序的遗传和分子机制。

DESCRIPTION (provided by applicant): 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.