Background

Pseudostratified epithelia (PSE) are epithelia in which the interphase nuclei of the epithelial cells dynamically position at different depths within the epithelial monolayer, while mitotic nuclei localize at the most apical side of the epithelium. PSE are usually highly proliferative tissues acting as organ precursors in many organisms, including the building blocks of the epiblast in the gastrulating mouse embryo, the pancreatic buds and different areas of the central nervous system in vertebrates, as well as the imaginal discs and the optic lobe anlage in Drosophila (Meyer et al., 2011; Ichikawa et al., 2013; Strzyz et al., 2016).

Almost a century ago, Sauer observed that in PSE nuclei moved to the apical surface to divide (Sauer, 1935). The apical migration of nuclei is linked to the cell cycle progression, occurring at G2 phase, and it is known as Interkinetic Nuclear Migration (INM) or Pre-mitotic Rapid Apical Migration (PRAM). Thus, INM is responsible for the spatial organization of proliferating cells in PSE and the localization of mitotic nuclei at the PSE basal side is frequently linked to failures in INM (Tsuda et al., 2009). The apical “mitotic zone” serves as a niche for signaling and spatial control of mitotic entry, and it has been recently shown that apical daughter cells reintegrate better into the PSE (reviewed in Norden, 2017).

Better known is the control of the mitotic spindle orientation, which will determine tissue architecture or cell fate specification in the context of symmetric or asymmetric cell divisions, respectively. As failures in both processes are implicated in tumorigenesis, spindle misorientation might promote or, at least, facilitate tumor development (Pease and Tirnauer 2011; Noatynska et al., 2012, Bergstralh and St Johnston 2014). Epithelial cells tend to divide with their mitotic spindle parallel to the plane of the epithelial sheet. The significance of tightly regulating this process during early development, when epithelia are in a high proliferative phase, is manifest by the existence of several neurological diseases associated with mitotic spindle orientation failures, such as microcephaly, lissencephaly and Huntington disease (Noatynska et al., 2012).

Several protocols for 3D automated measurements of mitotic spindles have been recently published (Juschkeet al., 2014; Lázaro-Diéguez et al., 2014). Although these protocols are very reliable as they eliminate any bias due to hand processing of the images, they are established to work in vitro, in cell culture. In Franco and Carmena (2019), we have developed a simple and reliable method to determine the angle of the mitotic spindle and the positioning of the mitotic nucleus of neuroepithelial cells in fixed whole brains of Drosophila third instar larvae, making possible to visualize in vivo, the morphogenetic consequences of failures in any of these processes. Since planar divisions and INM are considered hallmarks of some neural progenitors, these measurements can be an effective way of studying the architecture and organization of a developing neuroepithelium.