Xenopus laevis

SIGNIFICANCE:Xenopus laevis, the African clawed frog, has been used widely in the field of developmental biology. By following the development of this unique organism, scientists have identified and now understand the role of many genes in frog development, providing insight into vertebrate development.

The Organism

The African clawed frog, Xenopus laevis, is in the class Amphibia, order Anura, suborder Opisthocoela, family Pipidae, and genus Xenopus. This genus includes five other species that inhabit silt-filled ponds throughout much of Southern Africa. Members of this species share a distinctive habitat and morphology. The organism’s name alone provides insight into its structure and habitats: The root xeno stems from Greek for “strange,” while pus is from the Greek for “foot” and laevis is Latin for “slippery.” Xenopus laevis is entirely aquatic, a feature that makes it unique among the members of the genus, feeding and breeding underwater. It is believed that they evolved from terrestrial anurans, organisms that are aquatic as tadpoles but are terrestrial as adults. Migration across land from pond to pond has been observed but is limited by distance and time of year (occurring during the rainy season) because out of water, the frogs will dry out and die within a day. In instances of extreme drought, adult frogs will bury themselves in the mud and wait until the next rainfall.

Xenopus laevis is mottled greenish-brown on its dorsal surface and yellowish-white on its ventral surface. In appearance, these frogs are flattened dorsoventrally, with dorsally oriented eyes as adults. The members of the genus are collectively known as platannas, from the word “plathander,” meaning flat-handed. Three toes of the hind limbs are clawed, and a line of specialized sensory organs (the lateral line organs) is found on both the dorsal and ventral surfaces and encircles the eyes. The breeding season for X. laevis depends on temperature and rainfall. The tadpoles are herbivorous, feeding on algae, whereas the adults are carnivorous, feeding on worms, crustaceans, and other creatures living in the mud.

A Model Organism

A is defined as one that breeds quickly, is easily managed in the laboratory, and has large numbers of offspring or broods. Xenopus laevis meets these requirements nicely. An interesting feature of this organism is its responsiveness to human chorionic gonadotropin, a hormone secreted by the placenta and present in the urine of pregnant women. When exposed to the hormone, female frogs will spawn (lay eggs). As a result of this phenomenon, X. laevis was once used as an indicator in human pregnancy tests, whereby the female frogs were injected with human female urine. At present, researchers take advantage of this phenomenon to produce large numbers of offspring by injecting frogs with the hormone. Another characteristic that makes X. laevis a good model organism is that it is hardy and can survive in captivity for long periods of time with relatively low mortality rates.

A final requirement for an to be useful is that research on the animal should add to the understanding of biological principles in other organisms. Xenopus laevis is widely used in the field of developmental biology. For many decades, amphibian embryologists used salamander embryos, such as Triturus, and embryos of the frog Rana species. As mentioned above, amphibian embryos have several advantages over other organisms: amphibian embryos are large, can be obtained in large numbers, and can be maintained easily and inexpensively in the laboratory. However, one disadvantage of traditional amphibian species is that they are seasonal breeders. As a result, investigators cannot conduct experiments throughout the year on most amphibians. Xenopus laevis is a notable exception, because it can be induced to breed year-round.

As the fertilized X. laeviszygote develops, the yolk-laden cytoplasm, known as the vegetal pole, is oriented downward by gravity. The rest of the cytoplasm, termed the animal pole, orients itself upward. The animal pole is the main portion of the cell, giving rise to the embryo proper. Cell division, or cleavage of cells, in the animal pole increases the number of cells greatly. Movement and migration of these cells, under the influences of interactions with neighboring cells, give rise to a multilaminar embryo that includes the ectoderm (which gives rise to skin and nervous system), the mesoderm (which gives rise to muscle), and the endoderm (which gives rise to many of the “tubes” of the organism, such as the intestines and the respiratory tract).

By following embryos from the very earliest stages, researchers have been able to create “fate maps” of fertilized eggs, which can be used to predict adult derivatives of specific regions in a developing embryo. Early researchers introduced many different techniques to create these kinds of maps. One technique involves destroying single cells during early development and following the development of the embryo to see what tissue is altered. Other methods include transplantation of individual cells or small groups of cells into a host organism and following the fate of the transplanted tissue.

Genetic Manipulation in Xenopus

Much of what is now known about the interactions between cells in developing vertebrate embryos has come from X. laevis. The early work of embryologists Hans Spemann and Pieter Nieuwkoop has been supported with molecular techniques, and many genes have been identified that control nearly every aspect of Xenopus development. A few examples include the Xenopus Brachury gene (Xbra), which is involved in the establishment of the dorsal-ventral axis; Xenopus ventral (vent1), which aids in the differentiation of ventral mesoderm and epidermal structures; and Xenopus nodal-related 1 (Xnr1), a gene that is responsible for the specification of the left-right axis.

Xenopus embryos possess a number of advantages that have allowed investigators to study many aspects of developmental biology. One of the struggles that early researchers faced was the lack of dependable techniques for creating transgenic embryos to study the functions and role of individual genes. One can isolate and clone the genes of Xenopus and inject RNA into zygotes. RNA, however, is an unstable molecule and relatively short-lived. Therefore, the study of molecular events in the embryo after the period when the embryonic genes are turned on remained problematic. Attempts to inject cloned DNA to be expressed in the embryo were complicated by the fact that it does not integrate into the frog genomic chromosomes during cleavage. Exogenous DNA is then unequally distributed in embryonic cells and, therefore, is always expressed in random patterns. In 1996, Kristen L. Kroll and Enrique Amaya developed a technique to make stable transgenic Xenopus embryos. This technique has the potential to boost the utility of Xenopus tremendously. One significant advantage of using transgenic frogs over transgenic mice is that one can produce first-generation transgenics, making it unnecessary to wait until the second generation to examine the effects of the on development.

The transgenic technique has several steps, and each step is full of problems. Because exogenous DNA is not incorporated into the zygotic genome, Kroll and Amaya decided to attempt to introduce it into sperm nuclei. Sperm nuclei are treated with the enzyme lysolecithin to remove the plasma membrane prior to incubation with the linearized DNA containing the exogenous gene. The sperm nuclei are then incubated with restriction enzyme to introduce nicks in the nuclear DNA. The nicks facilitate incorporation of the plasmid DNA. The nuclei are then placed in an egg extract, which causes the nuclei to swell as if they were male pronuclei. This technique has been used in many laboratories to introduce into the frog genes that are not normally expressed, allowing the researcher to study the function of these genes.

The National Institutes of Health is supporting the Trans-NIH Xenopus Initiative, specifically developed to support research in the areas of genomics and genetics in Xenopus research. While there is still much to be learned from these unique organisms, it is clear that the advantages of this animal model far outweigh the disadvantages. With continued work in laboratories around the world, scientists may soon fully understand the genetics involved in vertebrate development. Xenopus laevis is ideally suited to provide critical breakthroughs in embryonic body patterning and cell fate determination, later development and the formation of organs, and cell biological and biochemical processes.

Key Terms

• embryologythe study of developing embryos
• fate mapa map created by following the adult fate of embryonic cells
• model organisman organism well suited for genetic research because it has a well-known genetic history, a short life cycle, and genetic variation between individuals in the population
• transgenic organisman organism that contains a gene not normally expressed in its genome

Bibliography

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