Chapter 17 - Electron Microscopy of the Amphibian Model Systems Xenopus laevis and Ambystoma mexicanum
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
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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.
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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.
References (120)
- et al.
Frog genetics: Xenopus tropicalis jumps into the future
Trends Genet.
(1998) - et al.
Progressive patterning precedes somite segmentation in the Mexican axolotl (Ambystoma mexicanum)
Dev. Biol.
(1988) - et al.
Combined intrinsic and extrinsic influences pattern cranial neural crest migration and pharyngeal arch morphogenesis in axolotl
Dev. Biol.
(2004) - et al.
Molecular nature of Spemann’s organizer: The role of the Xenopus homeobox gene goosecoid
Cell
(1991) Spemann’s organizer and the self-regulation of embryonic fields
Mech. Dev
(2009)- et al.
Immunohistochemical demonstration of hyaluronan and its possible involvement in axolotl neural crest cell migration
J. Struct. Biol.
(2000) - et al.
Improved preservation of the subepidermal extracellular matrix in axolotl embryos using electron microscopical techniques based on cryoimmobilization
J. Struct. Biol.
(1997) - et al.
Tight juntion biogenesis in the early Xenopus embryo
Mech. Dev.
(2000) - et al.
Appearance and distribution of laminin during development of Xenopus laevis
Differentiation
(1990) - et al.
Experimental evidence for a proteinaceous presegmental wave required for morphogenesis of axolotl mesoderm
Dev. Biol.
(1985)
On the preparation of cryosections for immunocytochemistry
J Ultrastruct. Res.
Beta-catenin signaling activity dissected in the early Xenopus embryo: A novel antisense approach
Dev. Biol.
Bone morphogenetic protein-4 and noggin signaling regulates pigment cell distribution in the axolotl trunk
Differentiation
A study of fixation of early amphibian embryos for electron microscopy
J. Ultrastruct. Res.
How we are shaped: The biomechanics of gastrulation
Differentiation
Bottle cell formation in relation to mesodermal patterning in the Xenopus embryo
Mech. Dev.
Actomyosin contractility and microtubules drive apical constriction in Xenopus bottle cells
Dev. Biol.
Stimulation of initial neural crest cell migration in the axolotl embryo by tissue grafts and extracellular matrix transplanted on microcarriers
Dev. Biol.
Timing in the regulation of neural crest cell migration: Retarded “maturation” of regional extracellular matrix inhibits pigment cell migration in embryos of the white axolotl mutant
Dev. Biol.
Cryopreparation methods for electron microscopy of selected model systems
Methods Cell Biol.
Formation of functional tight junctions in Xenopus embryos
Dev. Biol.
Fibronectin is visualized by scanning electron microscopy immnuocytochemistry on the substratum for cell migration in Xenopus laevis gastrulae
Dev. Biol.
Dorsoventral differences in cell–cell interactions modulate the motile behaviour of cells from the Xenopus gastrula
Dev. Biol.
Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy
Dev. Biol.
Beta-Catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos
Mech. Dev.
Microwave-assisted tissue processing for same-day-EM-diagnosis of potential bioterrorism and clinical samples
Micron
Subcellular distribution of distinct nucleolin subfractions recognized by two monoclonal antibodies
Exp. Cell Res.
Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis
Dev. Biol.
A germline GFP transgenic axolotl and its use to track cell fate: Dual origin of the fin mesenchyme during development and the fate of blood cells during regeneration
Dev. Biol.
A low viscosity epoxy resin embedding medium for electron microscopy
J. Ultrastr. Res.
Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy
J. Cell Biol.
A protocol for isolating Xenopus oocyte nuclear envelope for visualization and characterization by scanning electron microscopy (SEM) or transmission electron microscopy (TEM
Nat. Protoc.
Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for western blotting and scanning electron microscopy (SEM)
Nat. Protoc.
Developmental-stage series of axolotl embryos
Tight junction formation in early Xenopus laevis embryos: Identification and ultrastructural characterization of junctional crests and junctional vesicles
Cell Tissue Res.
Functions of hyaluronan in wound repair
Wound Repair Regen.
Cell-free extract systems and the cytoskeleton: Preparation of biochemical experiments for transmission electron microscopy
Methods Mol. Biol.
Using Xenopus oocyte extracts to study signal transduction
Methods Mol. Biol.
Learning about cancer from frogs: Analysis of mitotic spindles in Xenopus egg extracts
Dis. Model Mech.
Furrow microtubules and localized exocytosis in cleaving Xenopus laevis embryos
J. Cell. Sci.
Membrane dynamics of cleavage furrow closure in Xenopus laevis
Dev. Dyn
Localization of gold in biological tissue
Histochemistry
Essential role of non-canonical wnt signalling in neural crest migration
Development
The development of the larval pigment patterns in Triturus alpestris and Ambystoma mexicanum
Adv. Anat. Embryol. Cell. Biol.
Analysis of cranial neural crest migratory pathways in axolotl using cell markers and transplantation
Development
Migratory patterns and developmental potential of trunk neural crest cells in the axolotl embryo
Dev. Dyn.
Cranial neural crest emergence and migration in the Mexican axolotl (Ambystoma mexicanum)
Zoology
Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs
J. Cell. Sci.
Type I cadherins are required for differentiation and coordinated rotation in Xenopus laevis somitogenesis
Int. J. Dev. Biol.
Filaments made from A- and B- type lamins differ in structure and organization
J. Cell. Sci.
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