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融合的病理生理学中的作用的建议。更广泛地说，这项建议侧重于边界如何控制复杂、多组件结构中的模式。我们最近对由Twist1杂合性功能丧失引起的Saethre-Chotzen综合征的机制的研究结果表明，Twist1突变小鼠在冠状缝合处的神经脊-中胚层边界存在缺陷。边界通常位于预期缝合的中胚层来源的细胞和预期额骨的神经脊来源的成骨细胞之间。我们发现，由Twist1控制的Ephin-Eph信号在这一边界的维持中发挥了作用：EphA4在预期的骨外颅外细胞层中表达，成骨前体细胞通过该细胞层迁移。减少Twist1和EphA4的剂量会导致迁移成骨前体细胞(MOP)不适当地靶向冠状缝合。我们认为，这种寻路缺陷是Twist1和EphA4突变体中颅性融合的关键原因。在目前正在提交的工作中，我们发现Notch配体Jagged1在冠状缝合中的一层细胞中表达，该细胞层划分了成骨-非成骨边界。在Twist1突变体中，Jagged1的表达在这样的细胞中显著减少。此外，这些细胞中Jagged1的条件性失活会导致融合，并导致缝合中Notch2和Hes1的上调。这些结果是我们三部分总体假设的基础，即Twist1是在一个节点上的一个调控层级，控制ePhin-Eph和Jagged1/Notch信号，EPhin-Eph信号主要在外颅间充质中发挥功能以控制MOP细胞迁移，Jagged1在缝合中胚层中起作用，规范缝合内的边缘细胞。为了验证这一假设，我们建议首先确定MOP细胞靶向缺陷是MOP细胞固有的，还是MOP细胞迁移通过的外颅层的变化或它们侵袭的缝合间充质的结果。我们将使用条件靶向和一组CRE小鼠来实现这一点。其次，我们会问，阻止Notch2和βcatenin在缝合细胞中的表达是否可以减轻颅缝融合的表型，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，即冠状缝合细胞身份的改变是融合的第一个事件，然后是成骨前体细胞靶向的缺陷，并因此而加剧。 与公共卫生相关：这是一项研究颅缝是如何形成的，以及三个基因--Twist1，EphA4和Jagged1--如何控制缝合内特定细胞的发育。这些基因特别令人感兴趣，因为携带这些基因突变的人有先天缺陷，会影响他们的头骨。通过研究这些基因的工作原理，我们将更多地了解颅骨发育的基本过程，以及这些基因的突变如何导致出生缺陷。从长远来看，这项工作可能有助于设计出治疗此类出生缺陷的新方法。

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.