High-Magnification In Vivo Imaging of Xenopus Embryos for Cell and Developmental Biology

Abstract

INTRODUCTIONEmbryos of the frog Xenopus laevis are an ideal model system for in vivo imaging of dynamic biological processes, from the inner workings of individual cells to the reshaping of tissues during embryogenesis. Their externally developing embryos are more amenable to in vivo analysis than internally developing mammalian embryos, and the large size of the embryos make them particularly suitable for time-lapse analysis of tissue-level morphogenetic events. In addition, individual cells in Xenopus embryos are larger than those in other vertebrate models, making them ideal for imaging cell behavior and subcellular processes (e.g., following the dynamics of fluorescent fusion proteins in living or fixed cells and tissues). Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large number of embryos available allows even an inexperienced researcher to perform hundreds of such manipulations per day. Transgenesis is quite effective as well. Finally, because the fate map of Xenopus embryos is stereotypical, simple targeted microinjections can reliably deliver reagents into specific tissues and cell types for gene manipulation or for imaging. Although yolk opacity can hinder deep imaging in intact embryos, almost any cell in the early embryo can be placed into organotypic culture, such that the cells of interest are directly apposed to the cover glass. Furthermore, live imaging techniques can be complemented with immunostaining and in situ hybridization approaches in fixed tissues. This protocol describes methods for labeling and high-magnification time-lapse imaging of cell biological and developmental processes in Xenopus embryos by confocal microscopy.

Zeta-Tubulin Is a Member of a Conserved Tubulin Module and Is a Component of the Centriolar Basal Foot in Multiciliated Cells

“…Morphant cells had fewer sub-apical foci, and the annular aspect of the apical actin network was less defined (Figures 4B and S4G). Phalloidin labeling intensity was also reduced in zeta-tubulin morphant cells compared to neighboring uninjected cells in mosaic embryos [24], but not in those injected with a mismatch morpholino based on MO2 (MM-MO2) (Figures S4H and S4I). Thus, depletion of zeta-tubulin disrupts the actin cytoskeleton associated with basal bodies.…”

Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure andCaenorhabditis elegansGastrulation

“…To test whether genes we found by screening in C. elegans have conserved cellular functions, we turned to Xenopus embryos, which allow for imaging neural tube closure at the level of individual cells. Xenopus is a valuable vertebrate model in NTD research (see, e.g., Wallingford and Harland 2002;Haigo et al 2003;Gray et al 2009;Liu et al 2011;Ossipova et al 2014) owing in part to its external development, which facilitates in vivo imaging of neural tube closure (Kieserman et al 2010). To determine whether Xenopus might serve as a model for probing the WAVE complex's roles in neural tube formation, we tested whether a homolog of gex-3, nckap1, affects neural tube closure in Xenopus.…”

Isolation and live imaging of enteric progenitors based on Sox10‐Histone2BVenus transgene expression

“…Time lapse imaging of developmental processes is most readily achieved in embryos that develop externally like zebrafish (Andersen et al ., 2010), chick (Kulesa and Fraser, 1998, 2000), and frog (Kieserman et al ., 2010). Indeed induction and early stages of NC migration have been particularly amenable to live imaging at the dorsal aspect of these embryos (Ahlstrom and Erickson, 2009; Krull et al ., 1995; McLennan and Kulesa, 2007).…”