Keller, Raymond E.
Biophysics, Development, Molecular Biology, Neuroscience, Stem Cells & Regeneration
Cellular and molecular mechanisms of morphogenesis
The goal of our research is to learn the cellular, molecular and biomechanical mechanisms underlying morphogenetic movements during embryogenesis. One of the fundamental problems in developmental biology is determining what local cellular activities underlie the morphogenesis of cell populations. Analysis of this problem requires an integrated, multilevel approach. We examine the cell motility or shape changes that constitute the "motor" of morphogenetic movement with time lapse recording of low light videomicroscopy, or with confocal microscopy of labeled cells both in the whole embryo and explants. We learn how these types of cell motility generate forces, and determine the mechanical properties of the tissues transmitting these forces, by correlating videomicroscopy of cell behavior with mechanical measurements, using a computer-controlled biomechanical measuring device. To understand the molecular mechanisms of cell motility, and the biomechanics of how it is harnessed, we use pharmacologcal and molecular biological manipulations to alter the function of molecular components and analyze the resulting changes in cell behavior, force production, and mechanical properties of the tissue. Since the mechanical function of a region-specific cell motility is dependent on the spatial and temporal pattern of its expression, we test what tissue interactions induce and pattern specific cell motilities by making microsurgical rearrangements of inducing and responding tissues. Since movements feedback on inductions, we also physically block movements and monitor changes in patterning of motility.
Our analysis of the convergent extension movements in the embryo of the frog, Xenopus laevis, serves as an example of this approach. The dorsal tissues of the vertebrate embryo narrow (converge) and elongate (extend) greatly during gastrulation and neurulation in movements collectively called "convergent extension." We used videomicroscopy of fluorescently labeled cells to show that cells bias their protrusive activity in the mediolateral direction, exert traction on adjacent cells in this direction, and pull themselves between one another along the mediolateral axis, to form a longer, narrower array. Mechanical measurements showed that the tissue becomes stiffer as it extends, enabling it to push strongly enough to stretch the remaining passive tissues of the embryo without buckling. Our current work seeks to learn the molecular and mechanical basis of this directed protrusive activity and stiffening, and also how these properties are induced. We inject RNAs coding for proteins that act as dominant inhibitors of molecules thought to be important in organizing the directed protrusive activity, along with RNA coding for green fluorescent protein (GFP). This enables us to visualize the resulting changes in behavior of the affected cells, with low light fluorescence videomicroscopy. Correlated changes in the forces produced and in tissue mechanics are measured, and changes in the terminal cell phenotype are monitored with molecular marker expression. With this multilevel, integrated approach, we can perturb a molecular function and directly analyze the effect on cell motility, tissue mechanics, patterning, and cell differentiation, enabling us to learn what components function in a particular morphogenetic event, and the mechanism of that function.