Twist1 in Boundary Formation and Craniosynostosis

项目总结/摘要 这是一个建议，以调查组织边界的作用，在颅缝的发展和 颅缝早闭的病理生理学更广泛地说，这一建议的重点是边界如何控制模式， 一个复杂的多组分结构我们最近关于Saethre-Chotzen综合征机制的研究结果， 由Twist 1的杂合功能丧失引起，证明Twist 1突变小鼠在以下方面存在缺陷： 冠状缝处的神经嵴-中胚层边界。边界通常位于 中胚层来源的细胞和神经嵴来源的成骨细胞， 前额骨我们发现，由Twist 1控制的肝配蛋白-Eph信号传导在 该边界的维持：EphA 4在预期骨的颅外细胞层中表达， 成骨前体细胞通过其迁移。Twist 1和EphA 4的剂量减少导致 迁移性成骨前体（MOP）细胞向冠状缝的不适当靶向。这种寻路 我们提出，缺陷是Twist 1和EphA 4突变体中颅缝早闭的关键原因。在工作中， 提交，我们发现Notch配体Jagged 1在冠状缝中的一层细胞中表达， 划分成骨与非成骨的界限。在这些细胞中Jagged 1的表达显著降低 Twist 1突变体中。此外，这些细胞中Jagged 1的条件性失活导致骨结合，并导致骨形成。 Notch 2和Hes 1在缝合线中的上调。这些结果是我们三部分总体假设的基础 Twist 1位于调控层次的节点，控制ephrin-Eph和Jagged 1/Notch信号传导， Ephrin-Eph信号传导主要在颅外间充质中起作用以控制MOP细胞迁移，并且 Jagged 1在缝合中胚层中起作用，在缝合内指定边缘细胞。为了验证这一 假设，我们建议首先确定MOP细胞靶向缺陷是否是MOP细胞固有的，或者是一个 由于MOP细胞通过其迁移的外颅层的变化或 他们入侵。我们将使用条件靶向和Cre小鼠阵列来实现这一点。第二，我们会问 阻止缝细胞中Notch 2和β连环蛋白表达的扩增是否减轻了 颅缝早闭表型，以及缝细胞中Notch 2的强制表达是否导致缝早闭。 最后，我们将使用基因图谱来检验这一假设，即冠状细胞身份的变化 缝合是骨性结合的第一个事件，其次是靶向缺陷， 成骨前体细胞

Project Summary/Abstract 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.