We utilize novel biophysical strategies in concert with modern molecular genetic and reverse genetic approaches in Drosophila to explore the forces that are responsible for cell shape change and movements during morphogenesis. This work is a collaboration with Glenn Edwards' group in Physics and with Stephanos Venakide's and John Harer's groups in Mathematics.
What is dorsal closure?
Dorsal closure is a key step in Drosophila morphogenesis in which two embryonic tissues, the amnioserosa and lateral epidermis, undergo cell shape changes and movements to enclose the amnioserosa and form a seamless dorsal epithelium. A canthus forms where two opposing sheets of lateral epidermis first
meet and begin to produce a seam. Through studies of the
three-dimensional aspect of each canthus and studies of closure when
both canthi have been removed, we discover the importance of this
structure for native closure behavior.
We show that both the amnioserosa and a supracellular purse string in the leading edge of the lateral epidermis contribute to the movements of dorsal closure. We have developed techniques to extract data from confocal fluorescent 4D data sets of dorsal closure in order to analyze the cell shape changes and movements associated with wild type and genetically or biophysically disrupted closure.
The Forces in Dorsal Closure
Dorsal closure proceeds even if we laser ablate one (but NOT both!) of the tissues responsible for closure. This indicates that this model epithelial cell sheet movement depends on redundant forces that, in concert, drive morphogenesis (Kiehart et al, 2000; Hutson et al, 2003). We show that the magnitude of each force is significantly larger than their vector sum, indicating that there is both potential for generating larger forces and the successful morphogenesis requires that the forces applied be precisely balanced (Hutson et al, 2003).
Motors generate forces in closure
We have also explored the molecules responsible for generating those movements. We showed that conventional nonmuscle myosin (myosin II), encoded by the Drosophila gene zipper, provides key contractile forces in different tissues where the supramolecular complexes that incorporate this motor protein are distinct (Young et al, 1993; Franke et al, 2005).
Regulation of forces and mechanosensing
Cells are capable of sensing and responding to forces, from cells in culture on stretchable substrates to cells in vivo dealing with the impact of body movement, such as walking. As embryos develop into complex multicellular organisms, they generate cell and tissue movements and shape changes. In several model systems, including dorsal closure, these behaviors are associated with forces.
How molecular events are regulated such that large, opposing forces efficiently drive morphogenesis remains a mystery, but we are pursuing leads that point to two distinct pathways: the bidirectionally signaling integrin cell surface receptors and mechanically gated ion channels.
To investigate cellular junctions in tissue movements and mechanics, we follow junctional proteins fused to fluorescent proteins in Drosophila embryos (left). Junctional molecules required for force coordination and tissue morphogenesis are explored following genetic, pharmacological and laser perturbations. These experiments altogether reveal different roles of junctional complexes during development.
Mechanically gated ion channels are transmembrane proteins whose function is regulated by mechanical force. Taking a pharmacological approach (right) in combination with forward and reverse genetics, we have identified genes encoding channels associated with mechanosensing that are required for dorsal closure.