The role of Twist family bHLH factors in limb morphogenesis

描述(申请人提供)：Hand1、Hand2和Twist1是基本螺旋-环-螺旋(BHLH)蛋白Twist类的成员，在心脏、鳃弓、外侧中胚层和发育中的肢芽中表达。小鼠基因组中Hand1或Hand2的缺失会导致胚胎死亡，原因是胚胎外缺陷或心脏缺陷。有条件地消融肢体内的Hand2表明Hand2在肢体前后(AP)轴的形成(桡骨和尺骨、拇指到小指)中发挥作用。肢体内Hand1或Hand2的异位表达导致轴前多指，这是一种典型的AP模式缺陷。AP轴位于后肢间充质内的极化活动区(ZPA)，由形态发生的声波刺猬(Shh)确定。Hand2是使后肢间充质细胞能够表达Shh的转录因子之一。Twist1与Hand1和Hand2在发育肢体中部分共表达。Twist1基因突变是人类常染色体显性遗传性单倍体缺乏症(SCS)的原因，在许多表型特征中，包括轴前多指。在SCS中发现的Twist1杂合缺失小鼠模型表型和纯合子Twist1缺失小鼠表现出发育不全的肢芽，并在E11.5死亡。遗传证据表明，Twist1抑制了前肢间充质中Shh的表达，通过Hand2和Twist1之间的拮抗遗传平衡来帮助确定AP轴。Twist家族的功能受时空表达(即表达水平和位置)和转录复合体的形成(二聚化)的控制。Twist和Hand蛋白可以与自身形成同源和异源二聚体，也可以与其他bHLH因子一起形成。二聚体形成的控制是由进化上保守的苏氨酸和丝氨酸调控的，它们位于所有Twist家族蛋白的bHLH域的螺旋I内。这些保守残基受蛋白激酶A(PKA)和含有B564的蛋白磷酸酶2A(PP2A)的作用而受到磷酸化调节。Twist1上的点突变改变了Helix I的磷酸调节，导致了Twist1功能的变化。人类Twist1基因突变破坏了Helix I的磷酸调节，与引起SCS有关，反映了这些SCS Twist1等位基因无法对抗HAND2功能。因此，我们假设，Twist家族bHLH因子二聚化选择的控制控制了它们在表达它们的组织中的特定生物学效应。这项建议的目标是进一步确定控制扭转家族行为的遗传和分子机制，并调节已建立的和新的肢体转录程序。 公共卫生相关性：这项建议试图确定Twist家族bHLH因子在肩部、骨盆带和四肢形成中的作用。通过使用一系列功能获得和功能丧失的Hand和Twist小鼠突变等位基因，我们可以在几个表达Cre重组酶的肢体小鼠系中联合激活/删除这些因子。然后将获得形态/组织学和基因表达分析，并在实验中进行比较。

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. PUBLIC HEALTH RELEVANCE: 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.