Twist1 in Boundary Formation and Craniosynostosis

项目摘要/摘要 这是一项研究组织边界在颅缝发育和颅缝发育中的作用的建议 颅缝早闭的病理生理学研究。更广泛地说，该建议关注的是边界控制模式在 一种复杂的、多组件的结构。我们关于Saethre-Chotzen综合征发病机制的最新结果， 由Twist1杂合性功能丧失引起的，表明Twist1突变小鼠在 冠状缝合处的神经脊-中胚层交界处。边界通常位于 预期缝合的中胚层来源的细胞和神经嵴来源的成骨细胞 未来的额骨。我们证明了由Twist1控制的ePhin-Eph信号在 维持这一边界：EphA4在预期的骨骼外颅外的一层细胞中表达， 成骨前体细胞通过其迁移。减少Twist1和EphA4的剂量导致 不适当地将移行性成骨前体(MOP)细胞定位于冠状缝合。这次探路 我们认为，缺陷是Twist1和EphA4突变体中颅缝融合的关键原因。目前正在工作中 提交时，我们发现Notch配体Jagged1在冠状缝合中的一层细胞中表达 划定成骨与非成骨边界。Jagged1在该细胞中的表达显著降低 在Twist1突变体中。此外，这些细胞中Jagged1的条件性失活会导致联会，并导致 Notch2和Hes1在缝线中表达上调。这些结果是我们三部分总体假设的基础 Twist1在一个节点上是一个管理层级，控制着ePhin-Eph和Jagged1/Notch信令， EPhin-Eph信号主要在外颅间质中起作用，控制MOP细胞的迁移，并且 Jagged1在缝合中胚层中起作用，说明缝合中的边缘细胞。为了测试这一点 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 MOP细胞迁移通过的外颅层的改变或缝合间充质的改变 他们会入侵。我们将使用条件靶向和一组CRE小鼠来实现这一点。第二，我们会问 阻止缝合细胞中Notch2和β连环蛋白表达的扩张是否缓解 颅骨融合的表型，以及强制在缝合细胞中表达Notch2是否会导致融合。 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 缝合是融合的第一个事件，它之后是靶向缺陷，并因此而加重。 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.