Investigating morphogenesis in Xenopus embryos: imaging strategies, processing, and analysis

Abstract

Methods have been developed for visualizing cell movement and protein dynamics during morphogenesis within live multicellular tissues isolated from Xenopus laevis embryos. These include the preparation and use of reporter constructs in Xenopus embryos, microsurgical techniques for isolating embryonic tissues, and methods for culturing live tissues for extended periods. In this article, we present strategies for successful imaging of large thick embryonic tissues by improving the signal and minimizing damage to cells and tissues from overexposure. We also describe strategies for image analysis, including construction of kymographs, use of time- and z-projected confocal stacks, and approaches to segment images using regions of interest. With these imaging tools, the "cut-and-paste" embryology of the Xenopus model system allows remarkable access to both the mechanics of cells and tissues as well as the complex cell biology of adhesion and the cytoskeleton during morphogenesis.

Figure 1

Live-cell confocal microscopy during morphogenesis. (A) Mediolaterally intercalating mesoderm cells labeled with both rhodamine-dextran to highlight the cytoplasm and nucleus and negatively mark yolk granules and intracellular vesicles and mem-GFP to highlight the plasma membrane at cell boundaries. Mem-GFP appears twice as intense when two membranes fall within the confocal depth of focus or in a single set of pixels. (B) GFP expressing live mesoderm assembles fibronectin fibrils along the interface where mesoderm abuts agarose. Synthesized fibronectin fibrils are labeled with a Cy3-conjugated fibronectin mAb (4H2) in real time. (C) Paxillin-GFP expressing animal cap edge cells cultured on a fibronectin coated glass substrate. (D) Frames from a confocal time lapse sequences of utrophin-mCherry expressing mesendoderm cells cultured on a fibronectin coated substrate.

Figure 2

Processing and Analyzing Images. (A) Two confocal z-sections of mem-GFP are collected from a marginal zone explant, color encoded, and combined into a single merged image. Membrane protrusions are visible at z =0 and cell boundary is clear at the 5 μm level. Merged z-levels encoded by different colors can show how mesoderm cell protrusions on the substrate are coordinated with shape changes in the cell body. (B) A single frame from a confocal time lapse of tau-GFP collected for 10 minutes at a 10 second interval. Average or maximum time projections of tau-GFP present the dynamics of microtubule within elongated mesoderm cells over the time course of the movie. (C) Frames from a time-lapse of mem-GFP expressing cells can be used to extract cell shapes (C′), designate ROIs, and follow the shape changes over time for individual cells (marked in red and blue in C′).