Microsurgical Manipulations to Isolate Collectively Migrating Mesendoderm

Mesendoderm mantle closure completes the gastrulation movements of the Xenopus laevis embryo and provides an unparalleled opportunity to study collective cell behaviors within a mesenchymal tissue. Free-edge sheet-like collective movements of these tissues contrast with movements of epithelial tissues in that mesendodermal cells are not constrained by tight junctions or adherens junctions, yet migrate in a coherent and persistent mode over several hours. Mesendoderm cells are the largest motile cells in the Xenopus embryo and complete a 500-µm migratory path. When mesendoderm is cultured on rigid glass substrates, these cells can exceed 100 µm in length and show a highly persistent leading lamellipodia that can exceed 20 µm from tip to base. These large collectively migrating cells provide a unique imaging opportunity to visualize polarized adhesive and cytoskeletal structures with high-numerical-aperture objectives. Mesendodermal cells in the early embryo originate from around the entirety of the marginal zone and may also be distinguished by their source along the animal-vegetal axis. Here we use the term mesendoderm but note alternative terms for these cells can include head mesoderm, endomesoderm, and prechordal mesoderm. This protocol summarizes microsurgical preparation of mesendoderm tissue explants and "windowed" embryos. Skills needed to dissect fragments of the mesendoderm mantle are marginally greater than those needed to isolate animal cap ectoderm and can be mastered within 2 weeks; skills needed to isolate the mesendoderm "donut" or "ring" or to prepare windowed embryos are significantly greater and may require several additional weeks of training.