Abstract
Purpose
Despite enormous progress regarding the genetic regulation of limb development, little attention has been paid to the cellular and tissue mechanisms that govern outgrowth. How does the limb bud acquire its peculiar shape? Previous models have focused on isotropic growth resulting from distally based proliferation. However, recent models and proliferation data and models suggest that differential proliferation cannot explaing the morphogenesis of the limb bud. We tested the possibility that oriented cell behaviours underlie early outgrowth.
Method
We visualised early limb buds in living mouse and zebrafish embryos at cellular resolution by using transgenic subcellular fluorescent labels that mark either the cell nucleus or the cell membrane. We acquired time lapse and static images using a confocal microscope and generated velocity vector fields to track cell movements, and also tracked cell division planes through the entire tissue. To complement the live imaging, we also undertook lineage tracing experiments in chick and zebrafish embryos. The molecular determinants of these cell movements were tested by crossing the reporter transgenes onto mutant backgrounds.
Results
Lateral plate contributes mesoderm to the early limb bud through directional cell movement. The direction of cell motion, longitudinal cell axes and bias in cell division planes lie largely parallel to one another along the rostrocaudal (head-tail) axis in lateral plate mesoderm. Transition of these parameters from a rostrocaudal to a mediolateral (outward from the body wall) orientation accompanies early limb bud outgrowth. Furthermore, we provide evidence that Wnt5a established cell polarity that likely underlies the oriented cell behaviours.
Conclusion
Anisotroic, oriented cell behaviours likely underlie early limb bud outgrowth. The driving forces of cell movements have yet to be determined. This morphogenetic perspective will also help to place information regarding genetic regulation of limb development and human mutations into a dynamic context. Of great interest to us is how dynamic cell movements might contribute to the establishment of early limb prepattern, prior to overt differentiation of the skeletal template.
