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）：该项目的目标是确定哺乳动物下颌发育过程中基本螺旋-环-螺旋转录因子Hand2和Twist1的关系。我们的假设是 Hand2 和 Twist1 一起在下颌咽弓内建立一个驱动下颌发育所需的组织域。在此域内，Hand2 和 Twist1 通过效应器发挥作用，完成下颌形态发生计划。我们进一步假设这些效应器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 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) 衍生的间充质中表达，下颌的骨和软骨从中产生。我们已经证明，小鼠 NCC 内 Hand2 基因的条件性缺失会导致下颌弓衍生结构的模式变化。与 Hand2 相比，编码 bHLH 因子 Twist1 的基因在近端和中间下颌弓的 NCC 衍生间充质中表达。 NCC 中 Twist1 的靶向失活会导致迁移 NCC 中的细胞死亡以及随后的颌骨缺陷。虽然 Hand2 和 Twist1 被认为标记了两个不同的牙弓域，但我们的初步数据表明，这两个 bHLH 因子的表达域在下颌牙弓中显着重叠。此外，我们在初步数据中表明，在重叠域内删除 Hand2 和 Twist1 几乎完全废除了有组织的下颌发育。 在本提案中，我们将在三个具体目标中解决 Hand2 和 Twist1 在这个重叠域中的功能。在目标 1 中，我们将首次定义 Twist1 在整个下颌弓间充质迁移后 NCC 模式中的功能。有了这些知识，Aim 2 将分析在重叠表达域中 Hand2 和 Twist1 丢失所带来的细胞和分子变化。我们还将使用 RNA-seq 来识别这个组合信号网络的效应器。在目标 3 中，我们将使用斑马鱼中吗啉代敲低和过度表达来确定经过验证的介体的功能。通过将对 Hand2/Twist1 条件敲除小鼠的细胞和分子分析与对斑马鱼 Hand2/Twist1 效应器的功能分析相结合，我们将发现新的调节机制，在下颌弓中建立一个组织区域，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。我们期望这些发现能够改变研究人员如何看待下颌弓模式和有意义的再生疗法的前景。 公共健康相关性：每 800 名活产儿中就有 1 人患有颅面出生缺陷综合症，对我们的社会造成了巨大的经济和社会负担。虽然大量的小鼠发育遗传学研究已经阐明了其中一些综合征的基础，但还有更多综合征的原因仍然未知。编码 bHLH 因子 Hand2 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，其缺失会导致严重的面部缺陷。然而，我们的初步数据表明，Hand2 作为 Hand2/Twist1 通路的一部分发挥着更大的作用，该通路建立了整体下颌发育所需的信号网络。我们的研究将使用两个模型系统直接解决下颌形态发生过程中 Hand2 和 Twist1 的组合功能。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。随后可以使用系统生物学方法将这些知识结合起来，构建涉及面部形成的更全面的“基因图谱”，这可能会导致未来治疗人类颅面异常的组织工程方法取得重大进展。