TWIST AND Msx2 IN BOUNDARY FORMATION AND CRANIOSYNOSTOSIS

描述（由申请人提供）：这是一项研究冠状缝形态发生和冠状缝连机制的建议。长期目标是了解细胞区室之间的边界如何形成，以及这些边界如何在生长控制和模式中发挥作用。颅缝早闭的细胞机制在很大程度上是未知的。流行的观点，主要是基于体外方法，试图解释颅缝早闭的分化或生存的成骨细胞种群的时间。我们的研究结果表明，理解颅缝早闭的根本原因需要在更高层次的生物组织分析。通过对Twist突变小鼠的检查，我们发现冠状缝的骨结合与形成额骨的神经嵴衍生的间充质和形成顶骨的中胚层衍生的间充质之间的边界缺陷相关。在努力确定其他基因的功能与扭曲冠状缝的发展，我们已经发现的证据表明，扰动Eph-ephrin和BMP信号可能有助于这些缺陷。我们的观察结果表明，肝配蛋白信号传导可能在Twist突变体表型中起作用：（i）肝配蛋白A2和A4以及它们的受体EphA 4在发育中的冠状缝中以高度定位的方式表达，（ii）它们的表达在Twist突变小鼠中改变，并在Msx 2-Twist双突变体中恢复到野生型模式。我们的观察结果表明，BMP信号转导参与，编码BMP拮抗剂的基因，头蛋白，在扭转突变小鼠的冠状缝中上调，并且头蛋白的转基因过表达导致类似于扭转突变体中所见的缝合缺陷。此外，神经嵴中Bmp 4的失活也会导致冠状缝处的额骨和顶骨融合。这些数据使我们的假设，颅缝早闭的扭曲突变体是由一个扰动的基因网络，控制边界的形成，这些网络很可能包括元素的Eph-肝配蛋白和BMP途径。在这里，我们建议（i）进行一系列的遗传实验，旨在测试的假设，扭转相互作用的Eph-ephrin和BMP途径的冠状缝的图案功能;（ii）测试的假设，Msx 2和扭转功能合作的遗传级联，调节边界形成和冠状缝的发展。这将需要分析Msx 2-Twist双突变体中冠状缝发育和ephrinA 2和EphA 4的表达。最后，我们将研究Msx 2和Twist之间的调节关系。我们将研究Twist功能降低导致Msx 2表达扩大的机制，并确定Msx 2的上调是否足以导致冠状缝中的边界缺陷和骨结合。这些研究的意义在于，首先，它们将为颅面疾病的一个重要类别-颅缝早闭的病理生理学提供信息。第二，他们将解决的生物学意义的边界形成模式和生长控制的一个基本问题，在发育生物学。

DESCRIPTION (provided by applicant): This is a proposal to investigate the morphogenesis of the coronal suture and mechanisms of coronal synostosis. The long-term aim is to understand how boundaries between cellular compartments form, and how such boundaries function in growth control and patterning. The cellular mechanisms underlying craniosynostosis are largely unknown. Prevalent views, based largely on ex vivo approaches, seek to explain craniosynostosis in terms of the timing of differentiation or survival of osteoblast populations. Our results suggest that understanding the fundamental causes of craniosynostosis requires an analysis at a higher level of biological organization. Through an examination of the Twist mutant mouse, we found that synostosis of the coronal suture is associated with a defect in the boundary between neural crest-derived mesenchyme that forms the frontal bone and mesodermal-derived mesenchyme that forms the parietal bone. In an effort to identify other genes that function together with Twist in coronal suture development, we have uncovered evidence that perturbations in Eph-ephrin and Bmp signaling may contribute to these defects. That ephrin signaling may have a part in the Twist mutant phenotype is suggested by our observations (i) that ephrins A2 and A4, as well as their receptor, EphA4, are expressed in a highly localized manner in the developing coronal suture, (ii) and that their expression is altered in Twist mutant mice and restored to their wild type pattern in Msx2-Twist double mutants. That BMP signaling is involved is suggested by our observation that the gene encoding the Bmp antagonist, noggin, is upregulated in the prospective coronal suture of Twist mutant mice, and that transgenic overexpression of noggin causes a sutural defect similar to that seen in Twist mutants. Moreover, inactivation of Bmp4 in neural crest also causes fusion of the frontal and parietal bones at the coronal suture. These data lead us to the hypothesis that craniosynostosis in the Twist mutant is caused in part by a perturbations in gene networks that control boundary formation, and that these networks are likely to include elements of the Eph-ephrin and BMP pathways. Here we propose (i) to carry out a series of genetic experiments aimed at testing the hypothesis that Twist interacts functionally with the Eph-ephrin and BMP pathways in the patterning of the coronal suture; (ii) to test the hypothesis that Msx2 and Twist function cooperatively in a genetic cascade that regulates boundary formation and coronal suture development. This will entail an analysis both of coronal suture development and the expression of ephrinA2 and EphA4 in Msx2-Twist double mutants. Finally, we will examine the regulatory relationship between Msx2 and Twist. We will investigate the mechanism by which reduced Twist function leads to expanded expression of Msx2, and we will determine whether upregulation of Msx2 is sufficient to cause a boundary defect and synostosis in the coronal suture. The significance of the proposed studies is, first, that they will contribute information on the pathophysiology of an important class of craniofacial disorders-the craniosynostoses. Second, they will address the biological significance of boundary formation in patterning and growth control-a fundamental problem in developmental biology.