Imaging and quantitative analysis of cytokinesis in developing brains of Kinesin-6 mutant mice.

Cytokinesis in neural progenitors occurs with specialized constraints due to their highly polarized structure and the need for both symmetric and asymmetric divisions. They must produce proper numbers of progenitors, neurons, and glia in a precise order. Yet very few functional studies of cytokinesis have been done in the developing brain. To elucidate mechanisms of cytokinesis during brain development, we designed a novel method to study cytokinesis in whole mount "slabs" of embryonic mouse cerebral cortex. It takes advantage of cytokinesis occurring on the ventricular surface of the cortex and allows examination of cytokinesis across many cells in the context of an intact brain tissue. The cortical slabs can be fixed for immunohistochemistry or used in live imaging experiments. In particular, we investigated mutants of the Kinesin-6, Kif20b, which show defects in cytokinetic abscission and have small brains. Here, we describe how to dissect neocortex from embryonic cerebral hemispheres, immunostain the cortical slabs for cytokinetic midbodies and other structures, and image the apical surface. We show how to quantitatively analyze apical structures including midbody numbers, organization, and morphology. New images and analyses of Kif20b(magoo) loss of function mutants are shown. Applying and adapting these types of analyses to other cytoskeletal proteins and mouse mutants will help advance our understanding on how the embryonic neuroepithelium generates neurons and builds the brain.