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的形态发生和冠状缝合机制的提案。长期目标是了解细胞区室之间的边界是如何形成的，以及这些边界如何在生长控制和模式中起作用。颅缝闭闭的细胞机制在很大程度上是未知的。流行的观点主要基于离体方法，试图从成骨细胞群体分化或存活的时间来解释颅缝闭合。我们的结果表明，了解颅缝闭锁的根本原因需要在更高的生物组织水平上进行分析。通过对Twist突变小鼠的检查，我们发现冠状缝合线的结合与形成额骨的神经嵴源性间充质和形成顶骨的中胚层源性间充质之间的边界缺陷有关。在努力鉴定与Twist一起在冠状缝线发育中起作用的其他基因时，我们已经发现了证据，表明ephrin和Bmp信号的扰动可能导致这些缺陷。我们的观察结果表明，ephrin信号可能在Twist突变表型中起一定作用(i) ephrin A2和A4及其受体EphA4在发育中的冠状缝线中以高度局部的方式表达，（ii）它们的表达在Twist突变小鼠中发生改变，在Msx2-Twist双突变小鼠中恢复到野生型模式。我们观察到编码BMP拮抗剂noggin的基因在Twist突变小鼠的预期冠状缝线中上调，并且转基因noggin的过表达导致与Twist突变小鼠相似的缝线缺陷，这表明BMP信号通路参与其中。此外，神经嵴Bmp4失活也会导致冠状缝处额顶骨融合。这些数据使我们提出这样的假设：Twist突变体的颅缝闭锁部分是由控制边界形成的基因网络的扰动引起的，这些网络可能包括ephrin和BMP通路的元件。在这里，我们建议(i)进行一系列的基因实验，旨在测试Twist在冠状动脉缝合的模式中与ephrin和BMP途径在功能上相互作用的假设；（ii）验证Msx2和Twist在调控边界形成和冠状缝线发育的遗传级联中协同作用的假设。这将需要分析冠状缝线发育和ephrinA2和EphA4在Msx2-Twist双突变体中的表达。最后，我们将研究Msx2和Twist之间的调节关系。我们将研究Twist功能降低导致Msx2表达增加的机制，并确定Msx2的上调是否足以导致冠状缝合线的边界缺陷和滑膜紧闭。所提出的研究的意义在于，首先，它们将为颅面疾病的一个重要类别——颅缝闭锁的病理生理学提供信息。其次，他们将讨论边界形成在图案和生长控制中的生物学意义，这是发育生物学的一个基本问题。

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.