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. 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.Purpose
Method
Limb regeneration as it occurs in amphibians has two basic requirements: a source of multipotent cells capable of generating various tissues, and reorganization of those cells to form the one and only pattern of tissue appropriate to restore the missing parts. In the current biomedical world, there is much work dedicated to tissue engineering and to the differentiation of stem cells into various mature cell types. Neither of these approaches however, will by themselves succeed in regenerating a complex structure such as a limb. In our lab, we decided to focus on the pattern organization side of the equation by testing the potential of mammalian limb bud tissue to change its positional identity, and to manipulate that potential. We used mouse embryos for our mammalian model. Small groups of cells were transplanted from one region of the limb bud into another, and the resulting effect on the positional identity of those cells was assessed using molecular markers of the upper arm, forearm and hand. We knocked out a genetic regulator of cell fate named Ezh2 specifically in the limb bud to test its role in committing cells to a given positional identity along the proximodistal limb axis.Purpose
Method
