Molecular and Cellular Basis of Craniosynostosis

在胎儿发育和儿童早期，骨性颅骨的生长适应了骨的快速扩张。 底层大脑这首先是由生长个体头骨的祖细胞完成的，然后是干细胞。 细胞驻留在称为缝合线的柔韧骨关节中。在一个常见的出生缺陷称为颅缝早闭症（1 2000例活产），颅缝的缺失导致骨骼融合，阻碍大脑生长，从而导致 认知障碍如果不治疗的话。手术矫正涉及对婴儿进行侵入性和风险性手术， 分离融合的骨头不幸的是，头骨经常会重新融合，需要反复手术。有 因此，迫切需要更好地了解颅缝早闭的原因，这样我们就可以开发出 尽量减少重复手术干预。在上一个融资周期中，我们生成并描述了第一个 Saethre-Chotzen综合征的斑马鱼模型，其优先影响冠状缝。我们这样做 明确指出胚胎头骨生长速度的早期变化是骨缝融合的主要原因。在 这次更新我们解决了颅缝早闭领域中三个悬而未决的问题。在目标1中，我们研究 胚胎期的缝干细胞生长并维持头骨。虽然缝合干细胞具有 在出生后的阶段进行了研究，无论它们是来自生长骨骼尖端的祖细胞，还是 从迁移细胞，仍然存在争议。通过产生发育中小鼠的第一个单细胞转录组 和斑马鱼冠状缝，我们已经发现了保守的胚胎细胞类型和分子标记， 缝合祖细胞。在小鼠和鱼类中使用新的谱系追踪工具，我们将测试骨前祖细胞 表达ETS家族转录因子是缝驻留干细胞的来源。在目标2中，我们研究 Saethre-Chotzen基因Twist 1和Tcf 12如何调节骨前缘祖细胞向骨缝的转变 干细胞初步数据显示，Twist 1和Tcf 12上调BMP拮抗剂Grem 1和Noggin 在骨缝形成过程中，这表明Bmp信号的更严格调节对于减缓骨生长至关重要， 防止融合。利用小鼠条件遗传学和新的斑马鱼突变体，我们将测试直接调节 Twist 1和Tcf 12表达Grem 1和Noggin对于调节骨生长是必要和充分的， 正常的缝线形成。在目标3中，我们解决了颅缝早闭症领域的一个核心谜团-为什么特别是 突变往往只影响特定的缝合线通过生成和对比新的斑马鱼模型， 冠状和7中线颅缝早闭基因，我们将测试冠状缝的形成是否特别是 对扰乱骨骼生长速度的突变敏感。为此，我们将利用新的斑马鱼 转基因报告基因，允许成骨细胞添加和缝线形成的定量体内测量。一 该提案的优势在于斑马鱼、小鼠和人类颅面遗传学方面的独特专家团队。通过 利用模式生物来了解不同类型的颅缝早闭的发育基础，我们努力 为具有特定基因突变的颅缝早闭患者开发更有针对性的治疗方法。

During fetal development and early childhood, growth of the bony skull accommodates a rapid expansion of the underlying brain. This is accomplished first by progenitors that grow the individual skull bones, and then by stem cells residing in the flexible bony joints called sutures. In a common birth defect called craniosynostosis (1 in 2000 live births), loss of the cranial sutures results in bony fusions that impede brain growth, thus leading to cognitive impairment if left untreated. Surgical correction involves invasive and risky surgeries on infants to break apart the fused bones. Unfortunately, the skull bones often re-fuse, necessitating repeated surgeries. There is thus a critical need to better understand the causes of craniosynostosis, such that we can develop therapies that minimize repeated surgical interventions. In the previous funding cycle, we generated and characterized the first zebrafish model of Saethre-Chotzen Syndrome, which preferentially affects the coronal suture. In so doing, we pinpointed early changes in the growth rates of the embryonic skull bones as a major cause of suture fusions. In this renewal we address three outstanding questions in the field of craniosynostosis. In Aim 1, we investigate the embryonic origins of the suture stem cells that grow and maintain the skull. While suture stem cells have been studied at postnatal stages, whether they arise from progenitors at the tips of growing bones, or alternatively from migrating cells, remains debated. By generating the first single-cell transcriptomes of the developing mouse and zebrafish coronal sutures, we have uncovered conserved embryonic cell types and molecular markers for suture progenitors. Using new lineage tracing tools in mouse and fish, we will test that bone front progenitors expressing ETS-family transcription factors are the origin of suture-resident stem cells. In Aim 2, we investigate how the Saethre-Chotzen genes Twist1 and Tcf12 regulate the transition from bone front progenitors to suture stem cells. Preliminary data reveal that Twist1 and Tcf12 upregulate the Bmp antagonists Grem1 and Noggin during suture formation, suggesting that tighter regulation of Bmp signaling is essential to slow bone growth and prevent fusions. Using mouse conditional genetics and new zebrafish mutants, we will test that direct regulation of Grem1 and Noggin expression by Twist1 and Tcf12 is necessary and sufficient for regulated bone growth and normal suture formation. In Aim 3, we address a central mystery of the craniosynostosis field – why do particular mutations tend to affect only particular sutures? By generating and contrasting new zebrafish models for 11 coronal and 7 midline craniosynostosis genes, we will test whether coronal suture formation is particularly sensitive to mutations that perturb the rate of bone growth. To do so, we will make use of new zebrafish transgenic reporters that allow quantitative in vivo measurements of osteoblast addition and suture formation. A strength of the proposal is the unique team of experts in zebrafish, mouse, and human craniofacial genetics. By using model organisms to understand the developmental bases for diverse types of craniosynostosis, we strive toward developing more targeted treatments for craniosynostosis patients with particular genetic mutations.