Molecular and Cellular Basis of Craniosynostosis

在胎儿发育和儿童早期，骨头骨的生长适应快速扩张的 潜在的大脑。这首先是由生长个体头骨的祖细胞完成的，然后是通过茎 位于弹性骨关节中的细胞称为缝合线。在一种常见的出生缺陷中称为颅缝早闭(1/2 2,000名活产儿)，颅缝的丢失会导致骨融合，阻碍大脑发育，从而导致 如果不治疗，就会造成认知障碍。手术矫正包括对婴儿进行侵入性和高风险的手术以使其破裂 把融合的骨头分开。不幸的是，头骨经常会重新融合，需要反复手术。的确有 因此，迫切需要更好地了解颅缝早闭的原因，以便我们能够开发出 尽量减少重复的手术干预。在上一个资金周期中，我们生成并描述了第一个 斑马鱼Saethre-Chotzen综合征模型，优先影响冠状缝合。在此过程中，我们 准确地指出胚胎颅骨生长速度的早期变化是缝合融合的主要原因。在……里面 这次更新，我们解决了颅缝融合领域的三个悬而未决的问题。在目标1中，我们调查 生长和维持头骨的缝合干细胞的胚胎起源。而缝合干细胞已经 在出生后阶段进行了研究，无论它们是来自生长骨尖的祖先，还是另一种情况 从细胞迁移的角度来看，仍然存在争议。通过产生发育中的小鼠的第一个单细胞转录本 和斑马鱼冠状缝合，我们发现了保守的胚胎细胞类型和分子标记 缝合线的祖先。在老鼠和鱼身上使用新的血统追踪工具，我们将测试骨骼的前祖细胞 表达Ets家族转录因子是缝线驻留干细胞的起源。在目标2中，我们调查 Saethre-Chotzen基因Twist1和Tcf12如何调控骨前祖细胞向骨缝的转变 干细胞。初步数据显示，Twist1和Tcf12上调BMP拮抗剂Grem1和Noggin的表达 在缝合形成过程中，提示对BMP信号的更严格的调控对于减缓骨生长和 防止融合。利用小鼠条件遗传学和新的斑马鱼突变体，我们将测试这种直接调控 Twist1和Tcf12对Grem1和Noggin的表达是调节骨生长和 正常的缝线形成。在目标3中，我们解决了颅缝早闭领域的一个中心谜团--为什么特别 突变倾向于只影响特定的缝合线？通过生成和对比11个新的斑马鱼模型 冠状和7个中线颅缝融合基因，我们将测试冠状缝的形成是否特别 对扰乱骨骼生长速度的突变敏感。为了做到这一点，我们将利用新的斑马鱼 转基因记者，允许在体内定量测量成骨细胞的添加和缝合形成。一个 该提案的优势在于斑马鱼、老鼠和人类颅面遗传学方面的独特专家团队。通过 使用模式生物来了解不同类型的颅缝融合症的发育基础，我们努力 为具有特殊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.