Microsurgical Methods to Make the Keller Sandwich Explant and the Dorsal Isolate.

Abstract

This protocol summarizes preparation of the dorsal marginal zone sandwich explant (a.k.a. the "Keller sandwich") and the dorsal isolate from Xenopus embryos. The Keller sandwich is assembled from two early gastrula stage dorsal marginal zone (DMZ) explants. DMZ explants isolated before involution maintain planar patterning processes and block radial signals that might be exchanged between pre- and postinvolution tissues. DMZ explants isolated later in gastrulation, but subsequently opened and flattened may have both planar and radial patterning. The epithelial margins of DMZ explants in Keller sandwiches heal and basal contacts form between the deep layers of the two DMZ explants. The dorsal isolate is dissected from mid- to late-gastrula stage embryos after involution and archenteron formation. Germ-layer contacts between dorsal endoderm, mesoderm, and ectoderm generated by gastrulation movements are maintained in the dorsal isolate. These two explants can be used to study tissue, cell, and subcellular processes relevant to convergent extension, from patterning to cell behaviors, and their collective biomechanics. Skills needed to dissect the Keller sandwich are greater than those needed to dissect animal cap ectoderm and can be mastered in a few weeks; skills needed to dissect the dorsal isolate are similar to those needed to dissect animal caps and can be learned in a week.