Molecular and Cellular Basis of Craniosynostosis

这是一个建议，以调查干细胞调节在颅缝发育和 颅缝融合症的维护及其病理生理学。更广泛地说，这项建议的重点是如何阻止 细胞在空间和时间上受到控制，以促进脊椎动物器官的发育和维护。我们的 最近的研究结果表明，两个相关转录因子Twist1和TCF12中的任何一个的杂合性丢失， 解释了大多数Saethre-Chotzen患者的冠状缝合缺陷。在体内使用复杂的 在小鼠和斑马鱼的成像和遗传学中，我们将测试Tcf12修饰Twist1的功能以维持 在缝合规范和维护过程中的骨骼祖细胞。Twist1和Twist1的共同角色 TCF12在发育中和出生后冠状缝合中有可能解释最初的 联会和术后联会的高复发率。我们的一个特殊优势是 研究计划是三位在颅面遗传学方面有成就的研究人员的互补专业知识。罗布 Maxson在小鼠联会模型方面拥有长期的专业知识，为鉴定 Twist1和TCF12是Saethre-Chotzen综合征中受影响最大的两个基因。杨柴最近 确定了缝合线中长期缝合通畅所需的Gli1+干细胞群体 颅骨生长。盖奇·克伦普开创了斑马鱼体内成像技术，以检查 头面部缺陷的细胞学基础。首先，这个团队将测试Twist1和Tcf12在相同组织中的功能 抑制IHH诱导的缝合祖细胞向成骨细胞的分化，如果Tcf12作为一种 Twist1的缝合线特定的二聚伙伴。其次，我们将检查Twist1的持续需求 和Tcf12通过有条件地删除出生后Gli1+缝合干细胞中的这些基因来维持缝合。 第三，我们将使用Twist1和Tcf12的新的敲入标记等位基因来确定Twist1和Tcf12的直接基因组靶 Twist1-Tcf12二聚体在出生后缝合干细胞中的表达，以及Tcf12如何改变Twist1对 参与缝合维护所需的监管区域。第四，我们将使用强大的成像技术 揭示Twist1-Tcf12二聚体在体内的空间分布。第五，我们将利用 首次建立Saethre-Chotzen综合征的斑马鱼模型，直观地显示随着时间的推移斑马鱼模式是如何变化的 成骨细胞分化的时机导致较晚的冠状缝合缺陷。这些目标的结果将检验 我们的模型认为Tcf12作为Twist1的缝合线特定合作伙伴，部分是通过引导Twist1到特定的 缝合间充质中抑制早期成骨细胞分化所需的基因组区域。这些新的 对缝合维护中融合基因的长期需求的洞察将有可能 为预防术后联会带来新的方法，从而减少高风险手术的数量 目前正在对患有Saethre-Chotzen综合征的幼儿进行治疗。

This is a proposal to investigate the role of stem cell regulation in cranial suture development and maintenance, and its pathophysiology in craniosynostosis. More broadly, this proposal focuses on how stem cells are controlled in space and time to promote the development and maintenance of vertebrate organs. Our recent results show that heterozygous loss of either of two related transcription factors, TWIST1 and TCF12, account for coronal suture defects in the majority of Saethre-Chotzen patients. Using sophisticated in vivo imaging and genetics in mice and zebrafish, we will test that Tcf12 modifies the function of Twist1 to maintain skeletal progenitors during both the specification and maintenance of sutures. A common role for Twist1 and Tcf12 in the developing and postnatal coronal suture would have the potential to explain both the initial synostosis and the high recurrence rate of postoperative synostosis in patients. A particular strength of our research plan is the complementary expertise of three accomplished investigators in craniofacial genetics. Rob Maxson has long-standing expertise in mouse models of synostosis, having contributed to the identification of TWIST1 and TCF12 as the two most affected genes in Saethre-Chotzen syndrome. Yang Chai recently identified a population of Gli1+ stem cells in the suture that are required for long-term suture patency and calvarial bone growth. Gage Crump has pioneered in vivo imaging techniques in zebrafish to examine the cellular basis of craniofacial defects. First, this team will test that Twist1 and Tcf12 function in the same tissues to repress the Ihh-driven differentiation of sutural progenitors into osteoblasts, as predicted if Tcf12 serves as a suture-specific dimerization partner for Twist1. Second, we will examine continuous requirements for Twist1 and Tcf12 in suture maintenance by conditionally deleting these genes in postnatal Gli1+ sutural stem cells. Third, we will use new knock-in tagged alleles of Twist1 and Tcf12 to identify the direct genomic targets of Twist1-Tcf12 dimers in postnatal sutural stem cells, as well as how Tcf12 modifies the ability of Twist1 to engage regulatory regions necessary for suture maintenance. Fourth, we will use powerful imaging techniques to reveal the in vivo spatial patterns of Twist1-Tcf12 dimers within sutures. Fifth, we will take advantage of the first zebrafish model of Saethre-Chotzen syndrome to directly visualize over time how changes in the pattern and timing of osteoblast differentiation result in later coronal suture defects. The results of these aims will test our model that Tcf12 functions as a suture-specific partner for Twist1, in part by guiding Twist1 to particular genomic regions necessary to inhibit premature osteoblast differentiation in suture mesenchyme. These new insights into the long-term requirements of synostosis genes in suture maintenance will have the potential to lead to new ways of preventing post-operative synostosis, thus reducing the number of risky operations currently performed on young children with Saethre-Chotzen syndrome.