SummaryThe skull roof, or calvaria, is comprised of interlocking plates of bone. Premature suture fusion (craniosynostosis, CS) or persistent fontanelles are common defects in calvarial development. Although some of the genetic causes of these disorders are known, we lack an understanding of the instructions directing the growth and migration of progenitors of these bones, which may affect the suture patency. Here, we identify graded expression of Fibronectin (FN1) protein in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvarial osteoblasts. Syndromic forms of CS exhibit dysregulated FN1 expression, and we find FN1 expression is altered in a mouse CS model as well. Conditional deletion ofFn1in CM causes diminished frontal bone expansion by altering cell polarity and shape. To address how osteoprogenitors interact with the observed FN1 prepattern, we conditionally ablateWasl/N-Waspto disrupt F-actin junctions in migrating cells, impacting lamellipodia and cell-matrix interaction. Neural crest-targeted deletion ofWaslresults in a diminished actin network and reduced expansion of frontal bone primordia similar to conditionalFn1mutants. Interestingly, defective calvaria formation in both theFn1andWaslmutants occurs without a significant change in proliferation, survival, or osteogenesis. Finally, we find that CM-restrictedFn1deletion leads to premature fusion of coronal sutures. These data support a model of FN1 as a directional substrate for calvarial osteoblast migration that may be a common mechanism underlying many cranial disorders of disparate genetic etiologies.

Congenital defects affecting the formation of the skull roof, such as craniosynostosis (CS) or persistent fontanelles, can occur as a result of abnormal calvarial bone differentiation and growth. Our genetic lineage tracing experiments showed mouse calvarial bone progenitors originate from supraorbital arch mesenchyme cells and migrate to the apex. We lack a basic understanding of how calvarial bones grow towards the skull apex, which impacts the position, patterning, and fusion of sutures. Here, we identified a baso‐apical graded expression of Fibronectin (FN1) protein preceding calvarial bone expansion. Conditional deletion of fibronectin in cranial mesenchyme causes diminished frontal bone apical expansion, patent fontanelles and partial CS of the coronal suture. Differential expression of FN1 protein is associated with human CS syndromes and present in the Apert syndrome mouse and zebrafish models. We hypothesize that calvarial bone primordia extend apically by Fn matrix‐mediated cell migration and with dysregulation of this cell‐substrate interaction can contribute to calvarial bone defects. To this end, we identified a specific role for Wiskott‐Aldrich Syndrome Like (Wasl), a gene required for lamellipodia formation, in regulating apical expansion of calvarial bone progenitors without perturbing their differentiation or proliferation. Together, these data allow us to propose a new model where graded FN1 protein expression creates a directional substrate on which calvarial bone progenitors use Wasl‐dependent lamellipodia to migrate. FN dysregulation can be a point of convergence for the diverse etiologies of CS and serve as a therapeutic target for calvarial bone defects and diseases.