Hand2 Function and Regulation During Craniofacial Development

描述（由申请人提供）：本项目的目标是确定基本螺旋-环-螺旋转录因子Hand2在哺乳动物下颌发育过程中的功能。我们的假设是Hand2通过在下颌弓中建立远端形态发生域来促进大多数下颌结构的发育。在这个边界内，Hand2限制了参与下颌咽弓发育的基因，同时也抑制了通常在该区域外观察到的基因的表达。此外，我们假设Hand2的表达和功能至少部分受到Twist1通过转录抑制和遗传相互作用的调节。在小鼠和斑马鱼中，Hand2在下颌咽弓内的颅神经嵴细胞（NCC）来源的细胞中表达，下颌的骨和软骨就是由这些细胞产生的。在小鼠中，Hand2基因的靶向失活导致早期胚胎致死（E10.5）。然而，hand2突变斑马鱼可以存活5天；在这些突变体中，颅面软骨的广泛畸形伴随着基因表达的阳性和阴性变化。我们已经在足月小鼠胚胎中表明，hand2 -子细胞构成了大部分来源于下颌弓的结构。在我们的初步数据中，我们发现NCC中Hand2的缺失导致颅面缺陷，表明NCC模式错误。在本提案中，我们将在三个具体目标中讨论Hand2在面部形态发生中的作用。在Aim 1中，我们将定义颅神经嵴细胞中Hand2基因条件失活后面部发育的细胞和分子变化。在目标2中，我们将使用斑马鱼hand2突变体来检查hand2是否同时作为转录激活因子和抑制因子，并使用hand2条件小鼠突变体来确定hand2作用的靶标。在Aim 3中，我们将定义Twist1在Hand2下颌弓结构域中的作用，并确定Twist1和Hand2是否在该结构域中遗传相互作用。通过将Hand2条件敲除小鼠的细胞和分子分析与Hand2在小鼠和斑马鱼中的作用的功能分析结合起来，我们希望发现控制ncc命运和身份的新调控机制，这些ncc最终导致面部发育，其破坏可能导致人类面部出生缺陷，包括小颌畸形。公共卫生相关性：颅面出生缺陷综合征发生在每250例活产中1例，在我们的社会中是一个巨大的经济和社会负担。虽然许多小鼠发育遗传学研究已经阐明了其中一些综合征的基础，但更多的原因仍然未知。hand2突变斑马鱼的表型表明，hand2可能参与建立或维持下颌弓发育所需的发育结构域，尽管早期的致病性阻碍了对hand2在后期弓模式事件和骨形成中的作用的分析。我们的研究将通过组合模型系统直接解决Hand2在下颌发育过程中的功能和调控问题。总之，我们的研究将有助于确定面部发育所必需的区域发育域是如何建立的，以及不同的区域如何相互对抗以产生最终的面部计划。这些知识随后可以使用系统生物学方法结合起来，建立一个更全面的涉及面部形成的“基因图谱”，这可能会在未来的组织工程方法中取得重大进展，以治疗人类颅面异常。

DESCRIPTION (provided by applicant): The goal of this project is to define the function of the basic helix-loop-helix transcription factor Hand2 during mammalian lower jaw development. Our hypothesis is that Hand2 contributes to the development of most lower jaw structures by establishing a distal morphogenetic domain in the mandibular arch. Within this boundary, Hand2 confines genes involved in mandibular pharyngeal arch development while also repressing the expression of genes normally observed outside this domain. Further, we hypothesize that Hand2 expression and function is at least partially regulated by Twist1 through both transcriptional repression and genetic interactions. In both mouse and zebrafish, Hand2 is expressed in cranial neural crest cell (NCC)-derived cells within the mandibular pharyngeal arch, from which bone and cartilage of the lower jaw arise. Targeted inactivation of the Hand2 gene in mice results in early embryonic lethality (by E10.5). However, hand2-mutant zebrafish are viable for five days; in these mutants, extensive malformations in craniofacial cartilages are accompanied by positive and negative changes in gene expression. We have shown in term mouse embryos that Hand2-daughter cells compose most structures derived from the mandibular arch. In our Preliminary Data, we show that loss of Hand2 in NCCs resultst in craniofacial defects indicative of NCC mispatterning. In this proposal, we will address the role of Hand2 in facial morphogenesis in three Specific Aims. In Aim 1, we will define the cellular and molecular changes in facial development following conditional inactivation of the Hand2 gene in cranial neural crest cells. In Aim 2, we will use hand2 zebrafish mutants to examine whether Hand2 acts as both a transcriptional activator and repressor and use Hand2 conditional mouse mutants to identify targets of Hand2 action. In Aim 3, we will define the role of Twist1 in the Hand2 mandibular arch domain and determine whether Twist1 and Hand2 interact genetically within this domain. By coupling our cellular and molecular analysis of Hand2 conditional knockout mice with our functional analysis of Hand2 action in mouse and zebrafish, we expect to uncover novel regulatory mechanisms governing fate and identity of NCCs that ultimately lead to facial development and whose disruption can lead to human facial birth defects, including micrognathia. PUBLIC HEALTH RELEVANCE: Craniofacial birth defect syndromes occur in 1 out of every 250 live births and represent a large financial and social burden within our society. While numerous mouse developmental genetics studies have elucidated the basis of some of these syndromes, the cause of many more remains unknown. The phenotype of the hand2 mutant zebrafish indicates that Hand2 may be involved in establishing or maintaining a developmental domain necessary for mandibular arch development, though early lethality prevents analysis of the role of Hand2 in later arch patterning events and bone formation. Our study will directly address the function and regulation of Hand2 during lower jaw development using a combination of model systems. Together, our studies will help define how regional developmental domains necessary for facial development are established and how different domains may antagonize each other to produce a final facial plan. Such knowledge can be subsequently combined using a systems biology approach to build a more comprehensive "gene map" involved in facial formation, which could lead to significant advances in future tissue engineering approaches to treat human craniofacial anomalies.