Molecular and Cellular Basis of Craniosynostosis

这是一个研究干细胞调节在颅缝发育中的作用的建议， 维持，及其在颅缝早闭的病理生理学。更广泛地说，这一建议的重点是如何干 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持。我们 最近的结果表明，两种相关转录因子TWIST 1和TCF 12中的任何一种的杂合缺失， 是大多数Saethre-Chotzen患者冠状缝缺损的原因。使用复杂的体内 在小鼠和斑马鱼的成像和遗传学研究中，我们将测试Tcf 12修饰Twist 1的功能，以维持 在缝线的规格和维护过程中的骨骼祖细胞。Twist 1和 在发育和出生后冠状缝中的tcf 12有可能解释最初的 骨性关节连接和术后骨性关节连接的高复发率。我们的一个特殊优势 研究计划是三位在颅面遗传学方面有成就的研究者的互补专长。罗布 Maxson在小鼠骨结合模型方面具有长期的专业知识， TWIST 1和TCF 12是Saethre-Chotzen综合征中最受影响的两个基因。杨柴近日在 确定了缝线中的Gli 1+干细胞群，这是长期缝线通畅所需的， 颅骨生长Gage Crump是斑马鱼体内成像技术的先驱， 颅面缺损的细胞基础首先，该团队将测试Twist 1和Tcf 12在相同组织中的功能。 抑制Ihh驱动的缝祖细胞分化成成骨细胞，如预测的那样，如果Tcf 12作为一种抑制剂， Twist 1的缝合特异性二聚化配偶体。其次，我们将研究Twist 1的连续需求 和Tcf 12在缝合维持中的作用，通过有条件地删除出生后Gli 1+缝合干细胞中的这些基因。 第三，我们将使用新的敲入标记的Twist 1和Tcf 12等位基因来鉴定Tcf 12的直接基因组靶标。 出生后缝干细胞中的Twist 1-Tcf 12二聚体，以及Tcf 12如何改变Twist 1的能力， 参与缝线维护所需的监管区域。第四，我们将使用强大的成像技术 揭示缝线内Twist 1-Tcf 12二聚体的体内空间模式。第五，我们将利用 第一个Saethre-Chotzen综合征的斑马鱼模型，可以直接可视化模式随时间的变化 成骨细胞分化的时间导致较晚的冠状缝缺损。这些目标的结果将检验 我们的模型表明，Tcf 12作为Twist 1的缝线特异性伙伴发挥作用，部分原因是通过引导Twist 1特定于 抑制缝间充质中过早成骨细胞分化所必需的基因组区域。这些新 深入了解缝骨形成基因在缝合维持中的长期需求将有可能 导致新的方法来预防术后骨连接，从而减少风险手术的数量 目前在患有Saethre-Chotzen综合征的幼儿中进行。

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.