Mechanisms of apical expansion in calvarial bone morphogenesis

先天性颅骨畸形，如颅骨融合和发育不良，在1/2500活人中发生 对大脑和感官发育有不利影响的新生儿。我们缺乏基本的 了解颅骨如何以及为什么朝向顶端生长，这会影响方向 膜内矿化和缝线融合。信号因子与细胞 颅骨祖细胞顶端扩张的机制尚不清楚。 我们最近发现小鼠的颅骨祖细胞从眶上弓区域迁移 到顶端，这种运动在条件间充质缺失突变体中被破坏，即 缺乏所有的配体分泌。我们还鉴定了纤维连接蛋白细胞外基质的分级表达。 在依赖于间充质Wnts的颅骨间充质中，提示细胞运动 通过细胞沿纤维连接蛋白的刚性梯度迁移的趋性过程。这 一项提案打算在活体内定义颅骨间充质非经典性WNT在 引导细胞的极性和趋化性。在此多PI计划中，我们将利用我们的综合 在条件小鼠遗传学、活细胞成像和新兴生物物理方法方面的专业知识 在活体内解决我们的中心假设，即间充质Wnts依赖的纤维连接蛋白 通过以下方式定向颅骨祖细胞朝向顶端的集体移动 多药趋向性。对于假设，在Aim1中，我们将确定间充质Wnts的作用 通过实时光片成像调节细胞运动行为的信号。在AIM2中，我们将测试 如果纤维连接蛋白通过以下方式引导组织刚性梯度和SOM细胞的集体运动 多药趋向性。R21提案的主要成果包括：1.开发蜂窝网络的框架 颅骨形态发生过程中的行为，2.SOM-WNT作为全局空间线索的作用， 3.在颅骨发育过程中，趋杜性作用驱动细胞在分级的纤维连接蛋白基质上运动。 骨骼生长。 影响：目前，我们缺乏一个概念性的框架来理解颅骨中的细胞运动 骨扩张，尽管它在高度流行的颅缝融合和颅骨中发挥作用 发育不良。这些概念验证实验的结果将作为一种新的范式 在我们对间充质细胞三维运动的理解中，为我们提供了新的见解 颅骨形态发生和先天缺陷。

Congenital skull defects such as craniosynostosis and dysplasias occur in 1/2500 live births with detrimental consequences for brain and sensory organ development. We lack basic understanding of how and why the skull bones grow towards the apex, which impacts the direction of intramembranous mineralization and fusion of sutures. The signaling factors and cellular mechanisms underlying the apical expansion of skull bone progenitors remain unidentified. We recently found mouse skull bone progenitors migrate from the supraorbital arch region to the apex and this movement is disrupted in conditional mesenchyme Wntless mutants, that lack all ligand secretion. We also identified a graded expression of fibronectin extracellular matrix in the cranial mesenchyme that is dependent on mesenchyme Wnts, suggesting cell movement by a process of durotaxis in which cells migrate along a stiffness gradient of fibronectin. This proposal intends to define, in vivo, the role of the cranial mesenchyme non-canonical Wnts in directing cellular polarity and durotaxis. In this multi-PI proposal, we will leverage our integrative expertise in conditional mouse genetics, live cell imaging, and emerging biophysical approaches in vivo to address our central hypothesis that mesenchyme Wnts-dependent fibronectin orients the collective movement of calvarial bone progenitors toward the apex by durotaxis. Towards the hypothesis, in Aim1 we will identify the role of mesenchyme Wnts signaling in regulating cell movement behaviors with live light-sheet imaging. In Aim2, we will test if fibronectin directs a tissue-stiffness gradient and collective movement of SOM cells apically by durotaxis. Key deliverables of this R21 proposal include: 1. Developing a framework of cellular behaviors during calvarial bone morphogenesis, 2. the role of SOM-Wnt as a global spatial cue, and 3. the role of durotaxis driven cell movements on a graded fibronectin matrix during calvarial bone growth. Impact: Currently, we lack a conceptual framework for understanding cell movement in calvarial bone expansion, despite its role in highly prevalent craniosynostosis and cranial skeletal dysplasias. The results from these proof-of-concept experiments will serve as a new paradigm in our understanding of cell movement in 3D in mesenchymal cells and provide us fresh insights into skull bone morphogenesis and congenital defects.