Mechanisms of apical expansion in calvarial bone morphogenesis

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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.