Twist1 in Boundary Formation and Craniosynostosis

描述（由申请人提供）：这是一项研究组织边界在颅缝发育和颅缝闭合病理生理中的作用的提案。更广泛地说，这个建议关注的是边界如何在一个复杂的、多组件的结构中控制模式。我们最近对由Twist1杂合性功能丧失引起的saethree - chotzen综合征机制的研究结果表明，Twist1突变小鼠在冠状缝处的神经嵴-中胚层边界处存在缺陷。边界通常位于未来缝线的中胚层来源细胞和未来额骨的神经嵴来源成骨细胞之间。我们发现，由Twist1控制的ephrin-Eph信号在维持这一边界中起作用：EphA4在外颅至未来骨的细胞层中表达，成骨前细胞通过该细胞迁移。Twist1和EphA4的剂量减少导致迁移性成骨前体（MOP）细胞不恰当地靶向冠状缝合线。我们提出，这种寻路缺陷是Twist1和EphA4突变体颅缝闭合的关键原因。在目前提交的工作中，我们发现Notch配体Jagged1在冠状缝线的一层细胞中表达，该细胞划分成骨-非成骨边界。在Twist1突变体中，Jagged1在这些细胞中的表达明显降低。此外，这些细胞中Jagged1的条件性失活会导致关节闭锁，并导致缝合线中Notch2和Hes1的上调。这些结果是我们的三部分假设的基础，即Twist1处于调节层次的节点，控制ephrin-Eph和Jagged1/Notch信号，ephrin-Eph信号主要在外颅间质中起作用，控制MOP细胞的迁移，Jagged1在缝合的中胚层中起作用，规范缝合线内的边界细胞。为了验证这一假设，我们建议首先确定MOP细胞靶向缺陷是MOP细胞固有的，还是由于MOP细胞迁移所经过的外颅层或它们侵入的缝合间质发生了变化。我们将使用条件靶向和一组Cre小鼠来解决这个问题。其次，我们将探讨阻止缝合细胞中Notch2和β - catenin表达的增加是否会减轻颅缝闭锁表型，以及强迫缝合细胞中Notch2表达是否会导致颅缝闭锁。最后，我们将使用基因谱来验证这样的假设，即冠状缝合线细胞身份的改变是骨膜融合的第一个事件，随后是成骨前体细胞靶向缺陷，并加剧了这种变化。

DESCRIPTION (provided by applicant): This is a proposal to investigate the role of tissue boundaries in cranial suture development and the pathophysiology of craniosynostosis. More broadly, this proposal focuses on how boundaries control pattern in a complex, multicomponent structure. Our recent results on the mechanism of Saethre-Chotzen syndrome, caused by heterozygous loss of function of Twist1, demonstrated that Twist1 mutant mice have a deficiency in the neural crest-mesoderm boundary at the coronal suture. The boundary normally lies between the mesoderm-derived cells of the prospective suture and the neural crest derived osteogenic cells of the prospective frontal bone. We showed that ephrin-Eph signaling, controlled by Twist1, has a role in the maintenance of this boundary: EphA4 is expressed in a layer of cells ectocranial to the prospective bone, through which osteogenic precursor cells migrate. Reduced dosage of Twist1 and EphA4 results in inappropriate targeting of migratory osteogenic precursor (MOP) cells to the coronal suture. This pathfinding defect, we proposed, is a key cause of craniosynostosis in Twist1 and EphA4 mutants. In work now under submission, we found that the Notch ligand, Jagged1, is expressed in a layer of cells in the coronal suture that demarcate the osteogenic-non-osteogenic boundary. Expression of Jagged1 is markedly reduced in such cells in Twist1 mutants. Moreover, conditional inactivation of Jagged1 in these cells results in synostosis, and to an upregulation of Notch2 and Hes1 in the suture. These results are the basis of our three-part overall hypothesis that Twist1 is at a node a regulatory hierarchy, controlling ephrin-Eph and Jagged1/Notch signaling, that Ephrin-Eph signaling functions primarily in the ectocranial mesenchyme to control MOP cell migration, and that Jagged1 functions in sutural mesoderm in the specification of border cells within the suture. To test this hypothesis, we propose first to determine whether the MOP cell targeting defect is inherent in MOP cells or is a result of a change in the ectocranial layer through which MOP cells migrate or of the sutural mesenchyme that they invade. We will approach this using conditional targeting and an array of Cre mice. Second, we will ask whether preventing the expansion of Notch2 and beta catenin expression in sutural cells mitigates the craniosynostosis phenotype, and whether forcing expression of Notch2 in sutural cells causes synostosis. Finally, we will use gene profiling to test the hypothesis that a change in the identity of cells of the coronal suture is the first event in synostosis, and that it is followed by-and exacerbated by-a defect in the targeting of osteogenic precursor cells. PUBLIC HEALTH RELEVANCE: This is a proposal to study how cranial sutures form and how three genes-Twist1, EphA4 and Jagged1- control the development of specific cells within the sutures. These genes are of special interest because humans with mutations in them have birth defects that affect their skulls. By studying how these genes work, we will learn more about basic processes of skull development as well as how mutations in these genes lead to birth defects. In the long term, this work may help to devise new treatments of such birth defects.