Project I: From Skull Shape to Cell Activity in Coronal Craniosynostosis

项目I，从颅骨形状到冠状面颅缝早闭症的细胞活性 颅缝早闭是一种常见的出生缺陷，可以作为综合征的一部分或作为孤立的异常发生。 对与颅缝早闭疾病相关的颅骨畸形的分析通常集中在颅缝早闭上。 穹窿缝和颅穹窿形状的改变。我们有来自人类和老鼠的新数据， 颅缝早闭症颅骨表型涉及所有颅骨，除了颅穹窿的缝， 和颅骨软组织。解剖颅骨和软组织发育的整体变化如何驱动 颅缝早闭颅骨表型，我们将量化破坏骨形成的影响，在小鼠模型 在细胞水平上使用双光子激光显微镜，结合多尺度计算模型， 头骨生长我们将首先确定成骨细胞系细胞（OLC）活性在产生特定颅骨细胞中的作用。 通过表征增殖和增殖的时间和空间分布， 在小鼠头骨发育中区分OLC。这将通过开发一种新的转基因 系，Runx 2-RFP，将用于产生Osx-GFP; Runx 2-RFP小鼠和双光子激光显微镜， 可视化颅胚胎发生期间OLC分化的阶段。我们将开发一个分期系统， 定量比较包括小鼠在内的各种转基因系中OLC的增殖和分化模式 冠状面颅缝早闭和未受影响的同窝仔（特定目标1）。这将阐明细胞水平 颅骨发育中发生的变化为将细胞行为与3D形状变化结合起来提供了基础 发生在个体发育过程中。为了严格理解OLC分化的变化如何引起 全球颅骨畸形，我们将创建一个多尺度的计算模型的颅骨形态发生 （具体目标2）。计算建模方法将增强假设驱动的调查 颅缝早闭表型的产生受实际测量参数的限制。细胞数以 初始“骨化中心”、OLC分化和增殖率、颅内压梯度（从 生长诱导的颅骨-软组织相互作用和缝合闭合率可以参数化和修改， 该模型结果可以持续定量地与我们广泛的骨骼图像存档进行比较 特征和颅器官的形状。协同作用：本项目与 项目III将基于我们检测到的OLC增殖和分化模式的差异， 发育中和颅缝早闭小鼠，因为这可以直接有助于了解信号通路 参与OLC分化的时空调节，将纳入网络分析中 项目三.在已知致病突变的小鼠中， 为项目II完成的人类颅缝早闭病例的形态测量分析提供信息， 计算模型可用于排除或识别特定参数对严重性的贡献。 在小鼠中的颅面表型，并通过扩展在人类中。

PROJECT I, From Skull Shape to Cell Activity in Coronal Craniosynostosis Craniosynostosis is a common birth defect that can occur as part of a syndrome or as an isolated anomaly. Analysis of skull malformations associated with craniosynostosis disorders often focus on premature closure of vault sutures and change in cranial vault shape. We have novel data from humans and mice that demonstrate that craniosynostosis cranial phenotypes involve all skull bones, sutures other than those of the cranial vault, and cranial soft tissues. To dissect how global alteration of cranial bone and soft tissue development drive craniosynostosis cranial phenotypes, we will quantify the effects of disrupted bone formation in a mouse model at the cellular level using two-photon laser microscopy, combined with a multiscale computational model of skull growth. We will first establish the role of osteoblast lineage cell (OLC) activity in producing specific cranial dysmorphologies through characterization of the temporal and spatial distribution of proliferating and differentiating OLCs in developing mouse skulls. This will be accomplished by developing a new transgenic line, Runx2-RFP, that will be used to generate Osx-GFP;Runx2-RFP mice and two-photon laser microscopy to visualize stages in OLC differentiation during cranial embryogenesis. We will develop a staging system to quantitatively compare OLC proliferation and differentiation patterns in various transgenic lines including mice with coronal craniosynostosis and unaffected littermates (Specific Aim1). This will elucidate the cellular-level changes that occur in cranial development providing the basis for joining cell behavior with 3D shape changes that occur during ontogeny. To rigorously understand how changes in OLC differentiation can give rise to global skull dysmorphology, we will create a multiscale computational model of cranial morphogenesis (Specific Aim 2). The computational modeling approach will enhance a hypothesis driven investigation of the production of craniosynostosis phenotypes constrained by actual, measured parameters. Numbers of cells in initial 'ossification centers', rate of OLC differentiation and proliferation, intracranial pressure gradients from growth induced skull-soft tissue interaction, and rate of suture closure can be parameterized and modified in the model. The results can be continually quantitatively compared to our extensive image archive of bone characteristics and cranial organ shapes in developing mice. Synergy: Interaction between this project and Project III will be based on the differences we detect in OLC proliferation and differentiation patterns in typically developing and craniosynostosis mice as this can contribute directly to knowledge of signaling pathways involved in the spatiotemporal regulation of OLC differentiation to be incorporated in the network analysis of Project III. Precise phenotyping of cranial shapes in mice in which the disease causing mutation is known will inform the morphometric analyses of human craniosynostosis cases accomplished for Project II while the computational model can be used to rule out, or identify the contribution of specific parameters to severity of craniofacial phenotypes in mice, and by extension in humans.