Hand2 Function and Regulation During Craniofacial Development

Hand2 颅面发育过程中的功能和调节

• 批准号: 8401463
• 负责人: David E. Clouthier
• 金额: $ 35.22万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-20 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401463
• 关键词: Address; Amaze; BHLH Protein; Biological Models; Branchial arch structure; Cartilage; Cell Death; Cephalic; Chromosome Mapping; Complement; Congenital Abnormality; Connective Tissue; Coupling; Data; Defect; Development; Developmental Process; Distal; Embryo; Embryonic Development; Endothelin-1; Event; Face; Future; Gap Junctions; Gene Deletion; Genes; Genetic; Goals; Helix-Turn-Helix Motifs; Human; Human Development; Jaw; Knock-out; Knockout Mice; Knowledge; Lead; Live Birth; Mandible; Maxilla; Mediator of activation protein; Mesenchyme; Molecular; Molecular Analysis; Morphogenesis; Mus; Neck; Neural Crest Cell; Osteogenesis; Pathway interactions; Pattern; Phenocopy; Phenotype; RNA; Regulation; Research; Research Personnel; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Societies; Structure; Syndrome; System; Systems Biology; Techniques; Time; Tissue Engineering; Tissues; Zebrafish; arm; base; bone; combinatorial; craniofacial; developmental genetics; middle ear; mutant; novel; overexpression; regenerative therapy; social; transcription factor

DESCRIPTION (provided by applicant): The goal of this project is to define the relationship of the basic helix-loop-helix transcription factors Hand2 and Twist1 during mammalian lower jaw development. Our hypothesis is that Hand2 and Twist1 together establish an organizing domain within the mandibular pharyngeal arch that is required to drive lower jaw development. Within this domain, Hand2 and Twist1 act through effectors to accomplish a lower jaw morphogenetic plan. We further hypothesize that these effectors are conserved in zebrafish and that loss or gain of these signals can partially phenocopy the lower jaw defects observed in Hand2/Twist1 double conditional knockout mouse embryos. In both mouse and zebrafish, Hand2 is expressed in cranial neural crest cell (NCC)-derived mesenchyme within the distal mandibular pharyngeal arch, from which bone and cartilage of the lower jaw arise. We have shown that conditional deletion of the Hand2 gene within NCCs of mice results in patterning changes in mandibular arch-derived structures. In contrast to Hand2, the gene encoding the bHLH factor Twist1 is expressed in the NCC-derived mesenchyme of the proximal and intermediate mandibular arch. Targeted inactivation of Twist1 in NCCs leads to cell death in migrating NCCs and subsequent jaw defects. While Hand2 and Twist1 are thought to mark two distinct arch domains, our Preliminary Data illustrates that the expression domains of these two bHLH factors significantly overlap in the mandibular arch. Further, we show in Preliminary Data that deletion of both Hand2 and Twist1 within the overlapping domain almost completely abolishes organized lower jaw development. In this proposal, we will address the function of Hand2 and Twist1 in this overlapping domain in three Specific Aims. In Aim 1, we will, for the first time, define the function of Twist1 in post-migratory NCC patterning throughout the mandibular arch mesenchyme. Armed with this knowledge, Aim 2 will analyze the cellular and molecular changes that accompany loss of both Hand2 and Twist1 in their overlapping expression domain. We will also use RNA-seq to identify effectors of this combined signaling network. In Aim 3, we will determine the function of validated mediators using morpholino knockdown and overexpression in zebrafish. By coupling our cellular and molecular analysis of Hand2/Twist1 conditional knockout mice with our functional analysis of Hand2/Twist1 effectors in zebrafish, we will uncover novel regulatory mechanisms that establish an organizing region in the mandibular arch that ultimately directs lower jaw morphogenesis and whose disruption can lead to human facial birth defects. We expect these findings to shift the paradigm of how investigators view both mandibular arch patterning and the prospects of meaningful regenerative therapies. PUBLIC HEALTH RELEVANCE: Craniofacial birth defect syndromes occur in 1 out of every 800 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. Neural crest cell-specific deletion of the gene encoding the bHLH factor Hand2 illustrates a role for this bHLH factor in establishing early NCC patterning in the lower jaw, the loss of which results in significant facial defects. However, our preliminary data suggests that Hand2 has a far bigger role as part of a Hand2/Twist1 pathway that establishes the signaling network needed for overall lower jaw development. Our study will directly address the combinatorial function of Hand2 and Twist1 during lower jaw morphogenesis using two model systems. Together, our studies will help define how regional signaling pathways complement each other to establish facial plans. 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.

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和Twist 1在哺乳动物下颌发育过程中的关系。我们的假设是，Hand 2和Twist 1共同在下颌咽弓内建立了一个组织域，这是驱动下颌发育所需的。在这个领域内，手2和扭曲1通过效应器来完成下颌形态发生计划。我们进一步假设这些效应子在斑马鱼中是保守的，并且这些信号的丢失或获得可以部分表型复制在Hand 2/Twist 1双条件性敲除小鼠胚胎中观察到的下颌缺陷。 在小鼠和斑马鱼中，Hand 2在下颌咽弓远端的颅神经嵴细胞（NCC）来源的间充质中表达，下颌骨和软骨由此产生。我们已经表明，有条件地删除小鼠NCC内的Hand 2基因导致下颌弓衍生结构的图案变化。与Hand 2相反，编码bHLH因子Twist 1的基因在近端和中间下颌弓的NCC来源的间充质中表达。NCC中Twist 1的靶向失活导致迁移性NCC中的细胞死亡和随后的颌骨缺损。虽然Hand 2和Twist 1被认为标志着两个不同的弓域，我们的初步数据表明，这两个bHLH因子的表达域显着重叠在下颌arch. Further，我们在初步数据中显示，删除Hand 2和Twist 1内的重叠域几乎完全废除了有组织的下颌发育。 在本提案中，我们将在三个特定目标中解决Hand 2和Twist 1在这个重叠域中的功能。在目标1中，我们将首次定义Twist 1在整个下颌弓间充质中迁移后NCC模式中的功能。有了这些知识，Aim 2将分析伴随Hand 2和Twist 1在其重叠表达结构域中丢失的细胞和分子变化。我们还将使用RNA-seq来识别这种组合信号网络的效应子。在目标3中，我们将使用斑马鱼中的吗啉代敲低和过表达来确定经验证的介质的功能。通过将我们对Hand 2/Twist 1条件性基因敲除小鼠的细胞和分子分析与我们对斑马鱼中Hand 2/Twist 1效应子的功能分析相结合，我们将发现新的调控机制，这些机制在下颌弓中建立了一个组织区域，最终指导下颌形态发生，其破坏可能导致人类面部出生缺陷。我们希望这些发现能够改变研究人员如何看待下颌牙弓模式和有意义的再生治疗前景的范式。 公共卫生相关性：颅面出生缺陷综合征发生在每800个活产婴儿中的1个，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。编码bHLH因子Hand 2的基因的神经嵴细胞特异性缺失说明了该bHLH因子在建立下颌早期NCC模式中的作用，其损失导致显著的面部缺陷。然而，我们的初步数据表明，Hand 2作为Hand 2/Twist 1通路的一部分，具有更大的作用，该通路建立了整个下颌发育所需的信号网络。我们的研究将直接解决的组合功能的手2和扭转1在下颌形态发生使用两个模型系统。总之，我们的研究将有助于确定区域信号通路如何相互补充，以建立面部计划。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。