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Hand 2在哺乳动物下颌发育过程中的功能。我们的假设是，Hand 2有助于大多数下颌骨结构的发展，通过建立一个远端形态发生域在下颌弓。在这个边界内，Hand 2限制基因参与下颌咽弓的发展，同时也抑制通常观察到的基因表达在这个域之外。此外，我们假设Hand 2的表达和功能至少部分地通过转录抑制和遗传相互作用由Twist 1调节。在小鼠和斑马鱼中，Hand 2在下颌咽弓内的颅神经嵴细胞（NCC）衍生的细胞中表达，下颌的骨和软骨由此产生。小鼠中Hand 2基因的靶向失活导致早期胚胎致死（E10.5）。然而，hand 2突变的斑马鱼可以存活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。我们已经表明，在长期的小鼠胚胎，手2的女儿细胞组成的大多数结构来自下颌arch. In我们的初步数据，我们表明，损失的手2在NCC的结果在颅面缺陷的NCC错误的指示。在本提案中，我们将在三个特定目标中解决Hand 2在面部形态发生中的作用。在目的1中，我们将定义的细胞和分子的变化，面部发育条件失活后的手2基因在颅神经嵴细胞。在目标2中，我们将使用手2斑马鱼突变体，以检查是否手2作为转录激活因子和阻遏物，并使用手2条件小鼠突变体，以确定目标的手2行动。在目标3中，我们将定义Twist 1在Hand 2下颌弓域中的作用，并确定Twist 1和Hand 2是否在该域中发生遗传相互作用。通过将我们对Hand 2条件性基因敲除小鼠的细胞和分子分析与我们对小鼠和斑马鱼中Hand 2作用的功能分析相结合，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个活产婴儿中的1个中，并且在我们的社会中代表了巨大的财政和社会负担。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。Hand 2突变斑马鱼的表型表明，Hand 2可能参与建立或维持下颌弓发育所需的发育域，尽管早期致死性阻止了对Hand 2在后来的弓图案形成事件和骨形成中的作用的分析。我们的研究将使用模型系统的组合直接解决下颌发育过程中Hand 2的功能和调节。总之，我们的研究将有助于确定面部发育所需的区域发展领域是如何建立的，以及不同的领域如何相互对抗以产生最终的面部计划。这些知识可以随后结合使用系统生物学方法来建立一个更全面的“基因图谱”参与面部形成，这可能会导致显着的进步，在未来的组织工程方法来治疗人类颅面异常。

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.