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.

项目描述（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和Twist1在哺乳动物下颌发育过程中的关系。我们的假设是Hand2和Twist1一起在下颌咽弓内建立了一个组织域，这是驱动下颌发育所必需的。在这个区域内，Hand2和Twist1通过效应器完成下颌形态发生计划。我们进一步假设这些效应物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地表型化在Hand2/Twist1双条件敲除小鼠胚胎中观察到的下颌缺陷。在小鼠和斑马鱼中，Hand2在下颌远端咽弓内的颅神经嵴细胞（NCC）衍生的间质中表达，下颌的骨和软骨就是从这里产生的。我们已经证明，小鼠NCCs中Hand2基因的条件缺失会导致下颌弓源性结构的模式变化。与Hand2相反，编码bHLH因子Twist1的基因在近端和中端下颌弓的nc来源的间质中表达。NCCs中Twist1的靶向失活导致迁移的NCCs细胞死亡和随后的颌骨缺陷。虽然Hand2和Twist1被认为标志着两个不同的弓域，但我们的初步数据表明，这两个bHLH因子的表达域在下颌弓中显着重叠。此外，我们在初步数据中表明，在重叠区域内删除Hand2和Twist1几乎完全取消了有组织的下颌发育。在本提案中，我们将在三个具体目标中解决Hand2和Twist1在这个重叠域的功能。在Aim 1中，我们将首次定义Twist1在整个下颌弓间质迁移后NCC模式中的功能。有了这些知识，Aim 2将分析伴随Hand2和Twist1重叠表达域缺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物。在Aim 3中，我们将在斑马鱼中使用morpholino敲除和过表达来确定经过验证的介质的功能。通过将Hand2/Twist1条件敲除小鼠的细胞和分子分析与斑马鱼中Hand2/Twist1效应物的功能分析结合起来，我们将发现在下颌弓中建立组织区域的新调控机制，该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者如何看待下颌弓的模式和有意义的再生治疗的前景。