Chapter 17 - Electron Microscopy of the Amphibian Model Systems Xenopus laevis and Ambystoma mexicanum

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Abstract

In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr’s resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.

Introduction

Amphibians are classic model organisms for vertebrates in cell and developmental biology. Urodeles (newts and salamanders, such as Triturus and Ambystoma) and anurans (frogs and toads, such as Rana, Bufo, Xenopus) are the model species studied most intensively since the late 19th century. In the early days of embryology, many studies on developmental processes were performed especially in amphibians. The main advantages of amphibian models are the generation of vast amounts of embryos, the external development, as well as the robustness of the embryos tolerating experimental manipulations, such as tissue grafting (explantation, implantation, or transplantation), even across species. Many important concepts, such as cell fate determination or embryonic induction, were formulated in these times (Hamburger, 1988), mainly based on studies with urodele embryos. Later, the South African clawed frog, Xenopus laevis, was introduced as the most important amphibian model, due to practical considerations such as the reliable induction of ovulation by injection of hormones or the easy chemical removal of the jelly coat around the eggs (Sive et al., 2000). Over the past 100 years, some of the hallmark experiments in developmental biology were performed with amphibians as model organisms, for example, the “organizer experiment.” In this experiment, an area in the future dorsal region of the embryo was defined that, upon transplantation into a host embryo, was able to instruct the formation of a complete secondary body axis from graft and host tissues (Spemann and Mangold, 1924). Further important experiments were the first cloning of a vertebrate by microinjection of nuclei from differentiated cells into enucleated eggs (Gurdon et al., 1975), the identification of the first organizer gene, the homeobox gene goosecoid (after more than 60 years of frustrating efforts; Cho et al., 1991), followed by the subsequent molecular characterization of the organizer (reviewed in De Robertis, 2009), and recently, the introduction of antisense-Morpholinos for specific gene knock-down in embryos (Heasman et al., 2000).

The major disadvantage of Xenopus laevis as a model for genetic studies is its pseudotetraploid genome and the long generation time (1–2 years). Therefore, the interest had shifted to other model organisms, such as Drosophila melanogaster, Caenorhabditis elegans, or the zebrafish Danio rerio. But Xenopus laevis and the axolotl, Ambystoma mexicanum, persisted as important models in developmental and regeneration biology. In fact, Xenopus laevis still is the best-understood model for mesoderm induction and patterning, or the regulation of cell movements in the early gastrulating or neurulating embryo (Heasman, 2006, Keller et al., 2003, Kimelman, 2006). Recently, a close relative, Xenopus tropicalis, emerged as a new amphibian model organism with a diploid genome and a shorter generation time of 3–6 months (Amaya et al., 1998).

In addition, the Xenopus egg is a powerful system for biochemical studies using egg extracts (Crane and Ruderman, 2006, Cross and Powers, 2009, Mandato et al., 2001, Maresca and Heald, 2006) and for patch-clamp studies of ion-channels that are ectopically expressed in the oocyte membrane (Tammaro et al., 2008). The axolotl also continues to be an important model in a variety of research areas, from physiology to development and regeneration (Voss et al., 2009).

In the past few years, the regenerative potential of both Xenopus and the axolotl has gained increased interest (Slack et al., 2008, Voss et al., 2009). In this respect, the neoteny of the axolotl is especially remarkable as it allows the animal to regenerate complex body structures, such as limbs, tail, spinal cord, heart, or jaws (e.g., Kragl et al., 2009, Mchedlishvili et al., 2007, Sobkow et al., 2006). Recently, it has become possible to study the genomic background for this regenerative potential (Habermann et al., 2004, Putta et al., 2004), which opens the door for deeper insights into the underlying processes, especially with respect to possible medical implications, and a potential to stimulate tissue regeneration in humans.

Many of the questions that arise during developmental or regeneration studies finally focus on basic cell biological questions concerning subcellular protein localization, macromolecular dynamics, vesicle trafficking, signaling regulation, etc. Often these cell biological questions can only be answered by using fine structural imaging methods, including electron microscopy.

Section snippets

Rationale

Our goal is to provide a set of different methods for the ultrastructural analysis of amphibian tissues, mostly of embryonic origin. The protocols include ultrastructural and immunolabeling approaches for TEM and SEM. In general, embryos are considered as “problematic samples” due to their yolk content, the concentration of very different tissues within a small specimen and, in many cases, the impossibility of dissecting them before fixation. Here, most of the presented methods are modified

Xenopus

Although Xenopus is widely used as a laboratory animal, information on the histology of embryonic stages is rather limited. There is a detailed light microscopical description of tissue sections through early embryos (Hausen and Riebesell, 1991), but later stages, from tailbud stage 27 onward, and the histogenesis of tissues and organs, are poorly documented. The current knowledge about these later events is deduced mainly from low-resolution gene expression patterns (Sive et al., 2000).

Materials

  • Embryo culture media: For Xenopus: MBSH (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 10 mM HEPES pH 7.4; 10 mg/ml streptomycinsulfate and penicillin. For Ambystoma: Steinberg solution, 1000 ml (Steinberg, 1957): Solution A: 3.4 g NaCl, 0.05 g KCl, 0.205 g MgSO4 × 7 H2O, 4 ml 1 N HCl (about 9 ml 1N HCl in 100 ml aqua bidest), 0.56 g TRIS (Sigma) and 900 ml aqua bidest; plus Solution B: 0.08 g Ca(NO3)2× 4H2O and 100 ml aqua bidest.

  • Fixatives: For ultrastructure/SEM: Modified

Discussion and Outlook

For more than a century, amphibian embryos have been classic models in cell and developmental biology. They were at the forefront of biological sciences in the early 20th century, culminating in the most influential organizer experiment (Spemann and Mangold, 1924). However, for more than 50 years the molecular nature of many discovered processes remained mysterious. Many researchers switched to models, such as Drosophila or the zebrafish that seemed to be more suitable for genetic analysis.

Acknowledgments

The authors would like to thank Inge Zimmermann, and Metta Riebesell for excellent technical assistance, and E.M. Tanaka for the kind donation of reagents.

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