The skull roof, or calvaria, is comprised of interlocking plates of bones that encase the brain. Separating these bones are fibrous sutures that permit growth. Currently, we do not understand the instructions for directional growth of the calvaria, a process which is error-prone and can lead to skeletal deficiencies or premature suture fusion (craniosynostosis, CS). Here, we identify graded expression of fibronectin (FN1) in the mouse embryonic cranial mesenchyme (CM) that precedes the apical expansion of calvaria. Conditional deletion of Fn1 or Wasl leads to diminished frontal bone expansion by altering cell shape and focal actin enrichment, respectively, suggesting defective migration of calvarial progenitors. Interestingly, Fn1 mutants have premature fusion of coronal sutures. Consistently, syndromic forms of CS in humans exhibit dysregulated FN1 expression, and we also find FN1 expression altered in a mouse CS model of Apert syndrome. 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.

The 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 of Fn1 in CM causes diminished frontal bone expansion by altering cell polarity and shape. To address how osteoprogenitors interact with the observed FN1 prepattern, we conditionally ablate Wasl/N-Wasp to disrupt F-actin junctions in migrating cells, impacting lamellipodia and cell-matrix interaction. Neural crest-targeted deletion of Wasl results in a diminished actin network and reduced expansion of frontal bone primordia similar to conditional Fn1 mutants. Interestingly, defective calvaria formation in both the Fn1 and Wasl mutants occurs without a significant change in proliferation, survival, or osteogenesis. Finally, we find that CM-restricted Fn1 deletion 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.

The coenocytic tip-growing alga Vaucheria exhibits positive and negative phototropism, apical expansion, polarotropism, and branch induction from the illuminated region of the cell, all of which are caused by blue light. The bending response of Vaucheria is a blue light-mediated growth response. Differently from diffuse-growing cells or organs, the apical hemispherical dome of the Vaucheria cell is the site of not only maximum growth activity but also the site of blue light perception. Thence the phototropic response is initiated by the bulging mechanism: that is, a quick shift of the growth center to the adjacent subapical flank region. Since tip growth is driven by localized exocytosis, both phototropic bending and branch induction are considered to be closely related blue light-responses. Here I describe first how to prepare a highly useful culture medium for most freshwater algae, to establish unialgal and axenic culture of Vaucheria, and then describe several simple illumination systems using ordinary and/or inverted microscopes for the measurements of tip growth and for analyses of phototropism, polarotropism, and blue light-induced branching. Brief information is also included concerning the nature and function of aureochrome, the newly discovered, ochrophyte-specific blue light receptor. Aureochrome mediates blue light-induced branching, but its role in the phototropic response is still not elucidated.

Most metazoans are able to grow beyond a few hundred cells and to support differentiated tissues because they elaborate multicellular, epithelial tubes that are indispensable for nutrient and gas exchange. To identify and characterize the cellular behaviors and molecular mechanisms required for the morphogenesis of epithelial tubes (i.e., tubulogenesis), we have turned to the D. melanogaster ovary. Here, epithelia surrounding the developing egg chambers first pattern, then form and extend a set of simple, paired, epithelial tubes, the dorsal appendage (DA) tubes, and they create these structures in the absence of cell division or cell death. This genetically tractable system lets us assess the relative contributions that coordinated changes in cell shape, adhesion, orientation, and migration make to basic epithelial tubulogenesis. We find that Dynamin, a conserved regulator of endocytosis and the cytoskeleton, serves a key role in DA tubulogenesis. We demonstrate that Dynamin is required for distinct aspects of DA tubulogenesis: DA-tube closure, DA-tube-cell intercalation, and biased apical-luminal cell expansion. We provide evidence that Dynamin promotes these processes by facilitating endocytosis of cell-cell and cell-matrix adhesion complexes, and we find that precise levels and sub-cellular distribution of E-Cadherin and specific Integrin subunits impact DA tubulogenesis. Thus, our studies identify novel morphogenetic roles (i.e., tube closure and biased apical expansion), and expand upon established roles (i.e., cell intercalation and adhesion remodeling), for Dynamin in tubulogenesis.