Hand2 Function and Regulation During Craniofacial Development

描述(申请人提供)：这个项目的目标是确定基本螺旋-环-螺旋转录因子Hand2和Twist1在哺乳动物下巴发育过程中的关系。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，这是驱动下颌骨发育所必需的。在这个区域内，Hand2和Twist1通过效应器完成下颌骨的形态发生计划。我们进一步假设这些效应器在斑马鱼中是保守的，这些信号的丢失或获得可以部分地修复在Hand2/Twist1双重条件基因敲除小鼠胚胎中观察到的下颌骨缺陷。在小鼠和斑马鱼中，Hand2表达于下颌下咽弓远端的脑神经脊细胞(NCC)来源的间充质中，下颌骨和软骨从NCC中产生。我们已经证明，在小鼠的NCC中有条件地缺失Hand2基因会导致下颌弓衍生结构的图案化变化。与Hand2不同，编码bHLH因子Twist1的基因在NCC来源的下颌近中牙弓间充质中表达。NCC中Twist1的靶向失活导致迁移中的NCC中的细胞死亡和随后的颌骨缺陷。虽然Hand2和Twist1被认为标记了两个不同的ARCH结构域，但我们的初步数据表明，这两个bHLH因子的表达结构域在下颌骨中显著重叠。此外，我们在初步数据中表明，重叠结构域中Hand2和Twist1的缺失几乎完全消除了有组织的下巴发育。在本提案中，我们将在三个具体目标中解决Hand2和Twist1在这个重叠领域中的功能。在目标1中，我们将首次确定Twist1在迁移后NCC模式贯穿整个下颌弓间充质中的功能。有了这些知识，AIM 2将分析伴随Hand2和Twist1在其重叠表达区域中丢失的细胞和分子变化。我们还将使用RNA-SEQ来识别这个组合信令网络的效应器。在目标3中，我们将利用吗啉基因敲除和斑马鱼中的过度表达来确定有效的介体的功能。通过结合我们对Hand2/Twist1条件基因敲除小鼠的细胞和分子分析，以及我们对斑马鱼Hand2/Twist1效应器的功能分析，我们将揭示新的调控机制，这些机制在下颌弓中建立一个组织区域，最终指导下颌骨的形态发生，其破坏可能导致人类面部出生缺陷。我们期望这些发现将改变研究者如何看待下颌弓模式和有意义的再生疗法的前景的范式。 与公共卫生相关：每800名活产儿中就有1人患有头面部出生缺陷综合征，在我们的社会中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症状的基础，但更多的原因仍不清楚。神经脊细胞特异性缺失编码bHLH因子Hand2的基因表明，该bHLH因子在下颌骨早期NCC模式的建立中发挥了作用，该模式的丧失会导致显著的面部缺陷。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，该通路建立了下颌骨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研究Hand2和Twist1在下颌骨形态发生中的组合功能。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在一起，以建立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畸形的组织工程方法取得重大进展。

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.