The role of Twist family bHLH factors in limb morphogenesis

描述（由申请人提供）：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体芽中表达。从小鼠基因组中删除Hand1或Hand2中的任何一个都会导致E9.5胚胎死亡，原因可能是胚胎外缺陷或心脏缺陷。肢体内Hand2的条件消融表明Hand2在肢体前后轴形成（桡骨、尺骨、拇指到小指）中起作用。Hand1或Hand2在肢体内的异位表达导致轴前多指畸形，这是一种典型的AP型缺陷。AP轴由形态因子sonic hedgehog （Shh）定义在位于后肢间质内的极化活动区（ZPA）。Hand2是使后肢间质能够表达Shh的转录因子之一。Twist1在发育中的肢体中与Hand1和Hand2部分共表达。TWIST1的突变是人类常染色体显性单倍体不足疾病sae3 - chotzen综合征（SCS）的病因，该疾病在许多表型特征中包括前轴多指畸形。在SCS中发现的Twist1杂合缺失小鼠和纯合缺失小鼠的模型表型表现为肢芽发育不全，并在E11.5死亡。遗传证据表明，Twist1通过Hand2和Twist1之间的拮抗遗传平衡，抑制前肢间质Shh的表达，从而帮助确定AP轴。Twist家族bHLH功能受时空表达（即表达水平及其位置）和转录复合物形成（二聚化）的控制。Twist和Hand蛋白可以与自身以及其他bHLH因子形成同源和异源二聚体。控制二聚体的形成是由一个进化上保守的苏氨酸和丝氨酸位于所有Twist家族蛋白的bHLH结构域的螺旋I。这些保守残基受蛋白激酶A （PKA）和含b564的蛋白磷酸酶2A （PP2A）的磷酸化调控。Twist1的点突变改变了Helix I的磷酸化调控，导致Twist1功能的改变。人类TWIST1基因突变破坏Helix I磷酸化调控与引起SCS有关，反映了这些SCS TWIST1等位基因无法拮抗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其表达的组织内的特定生物学效应。本提案的目标是进一步定义控制twist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。

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.