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Xenopus Genetics

Introduction:

The goal of this website is to collect and disseminate information for using the frog Xenopus tropicalis for genetic analysis. Xenopus tropicalis is an emerging model system that offers great promise for unraveling the mechanisms of embryonic development and many aspects of cell biology.

 

Why study the frog?

The embryonic development of the frog Xenopus laevis (X. laevis) has been studied for over 60 years and has been instrumental in improving our understanding of basic biological processes in cell and developmental biology. Robust methods for studying the development of Xenopus oocytes, eggs, and embryos have been established, thanks to the work of many Xenopus laevis researchers.

            There are numerous practical reasons why the Xenopus frog is such a good animal model for scientific study. For instance, Xenopus can be easily bred in the laboratory and produce hundreds to thousands of embryos in each mating. In addition, the animals can be induced to ovulate all year round so embryos can be produced at a time convenient for the experimenter. The embryos develop externally in relatively short periods of time. For that reason, every stage of cell division and differentiation, from a single cell, to swimming tadpole, to the adult frog can be easily examined. Frog oocytes and eggs are quite large. This simplifies experimental manipulations such as the ability to inject compounds like DNA or RNA into the egg. Manipulated eggs and embryos can be easily raised in simple salt solutions in Petri dishes for future analysis. The effects of any genetic alterations or experimental manipulations can be viewed directly in developing tadpoles. Experimental embryological manipulations are therefore much easier in frogs than in animals that develop in a hard egg or a womb. Since the early stages of frog and mammalian development proceed along remarkably similar paths, many events monitored in a developing frog can be extrapolated to human development. Therefore, we learn about human biology from studying frogs.

Frog genetics?

            The field of genetics, which studies the inheritance of genes and traits from one generation to the next, has been extremely powerful in unraveling biological mechanisms of vertebrate development. By mutating genes and studying the resulting inherited effects of these mutations, we can understand the roles these genes play in the development of the egg, embryo, or adult. In this manner, we can identify the function of each gene. For genetic analysis to work optimally, it is best to have a simple genome with few genes (less to mutate!). Also since genetics is always multigenerational, genetic analysis is easier when there is a short time from fertilization to sexual maturity. Unfortunately, Xenopus laevis is not well suited for genetic analysis. X. laevis frogs take several years to sexually mature and they are allotetraploid, meaning they have four copies of many genes. This greatly complicates the study of inheritance.

            Recently, another Xenopus frog species, Xenopus tropicalis, has gained a lot of attention from scientists wanting to develop frog genetics. X. tropicalis has all of the advantages described for X. laevis. In addition, it matures much faster (months) and is diploid (i.e. has two copies of every gene, just like humans). In recognition of the potential value of X. tropicalis genetics, several governments around the world have invested in developing this scientific resource. In the United States, the Department of Energy has committed to sequencing most of the genome of X. tropicalis. The availability of the genomic (DNA) sequence will be a powerful and exciting new resource for vertebrate genomic comparisons, gene identification, and genetic manipulation. The X. tropicalis genome sequencing project, projected to be done at the end of 2005, can be accessed at the website for the Joint Genome Institute. In addition, the National Institute of Health (NIH) has funded three projects to develop X. tropicalis genetics (http://www.nih.gov/science/models/xenopus/ ). Our lab received one of these grants, and along with an international effort of scientists, we are developing X. tropicalis as an animal model for studying genetics.

            In the laboratory of Richard Harland at the University of California, Berkeley, we have established a group of researchers consisting of two supervising Assistant Researchers (Mustafa Khokha and Timothy Grammer) and a team of talented undergraduates and research assistants. In order to rapidly disseminate our findings, protocols, and links to other important X. tropicalis information, we have created this website (http://tropicalis.berkeley.edu/home/).

Our goals:

We are establishing X. tropicalis genetics. In order for X. tropicalis to become a useful genetic model system, we need to develop three main areas: 1) We have to create molecular and genetic resources for the scientific community to give researchers the tools needed to study this frog. 2) We have to develop and optimize the conditions for raising X. tropicalis tadpoles and frogs. 3) We have to identify traits that can be studied genetically that will provide insight into understanding vertebrate development.

A lot of progress has been made in all three areas:

1) Molecular and Genetic Resources

            With regard to molecular and genetic resources, progress has been tremendous. Molecular reagents for X. tropicalis are becoming available at a staggering pace. By the end of 2005, a high-quality draft of the X. tropicalis genome will be available. At the same time, over 1 million ESTs will also be deposited into the public databases. In addition, physical (BAC fingerprinting and BAC end sequencing) and genetic (microsatellite) maps are being generated. Therefore, the amount of molecular data that is available and will soon become available has grown enormously (before 2000 there were less than 20 entries in Genbank for X. tropicalis and less than 10,000 in 2001). Our lab has accumulated a lot of valuable genetic probes that are available to the community upon request. In addition, many protocols developed for X. laevis work for X. tropicalis with minor modifications. Our lab published a recent paper describing some of these protocols and modifications. Therefore, the molecular reagents for X. tropicalis have become substantial.

            Genetic resources are also developing quickly. With the efforts of the Grainger lab at the University of Virginia and our work at the University of California (Berkeley), multiple frog strains are available in the United States and are further being inbred. These strains appear to be adequately polymorphic from one another for generating a linkage map, and we are developing additional lines that may be even more polymorphic.

2) Raising X. tropicalis tadpoles and frogs

            Of course for genetics to be optimal, animals must be raised from fertilization to sexual maturity in as short a time as possible. Our lab and the Grainger lab (U. Virginia) have invested a significant effort in optimizing these husbandry conditions. Our lab can consistently raise males to sexual maturity within four months and have made these protocols available on our website. We have found that X. tropicalis is a hardy animal and requires only minimal attention as an adult when compared to mammalian model systems. In addition, X. tropicalis has a long fertile lifespan of at least 10 years, which will aid in the maintenance of particular lines. However, early tadpoles are considerably more fragile and need careful attention for the first few weeks of life. We have developed and published protocols that can reliably raise hundreds of tadpoles at a time. We continue to test feeding regimens and other conditions that optimize the time from fertilization to sexual maturity and will continue to update our website. In addition, we have characterized and described disease and health issues of the frogs both on our website and in the published literature.

3) Identify developmentally interesting traits that can be studied genetically

            Finally, to develop X. tropicalis genetics, we have to identify genetic mutants and characterize them. Therefore, we and others have developed forward and reverse genetic screens to identify mutations in the genome.

            Multiple groups are testing forward genetic screens, which use random mutagenesis followed by screens to find an interesting inheritable phenotype. Once a phenotype is found, work then proceeds to try to identify what gene is mutated that causes that phenotype. Our lab is developing the use of gamma ray mutagenesis and while other (Zimmerman and Grainger) labs are using chemical mutagenesis.

In reverse genetics, the scienitst identifies the mutated gene first and then determines what phenotype results from the mutation of this gene. Two labs (Stemple and Conlon) are trying to develop this approach.

            Another lab is working on creating transgenic frogs to be used as both important research tools as well as for the possibility of future insertional mutagenesis screens (Meade).

Our mutagenesis progress so far:

            We have already identified mutants from our initial pilot gamma ray mutagenesis screen and are in the process of characterizing them. We showed in our recent paper that X. tropicalis multigenerational analysis is very feasible and demonstrated that mutations can be propagated and easily identified using monohybrid, dihybrid, and even trihybrid crosses (meaning we can mate parents that have not just one, but three separate mutations!).

What’s next?

            Due to the success of our pilot study, we are now initiating a much larger gamma ray mutagenesis screen. We will update the website as this progresses. Also, we are continuing to study the mutants we already have.

            Now that we have identified various mutants, we must do two things: 1) characterize each mutation to identify the tissues and structures that are malformed and 2) identify the gene that is mutated.

            Our mutant characterization is ongoing and will help us in identifying the precise process of development that is affected. As for identifying the genes that have been mutated, at this point we are limited to a candidate-based approach. We are using known zebrafish mutations that have similar phenotypes to guess what frog genes might be mutated. We will also develop and use genome-wide microarrays to identify changes in gene expression in mutant versus normal animals. Using a combination of these approaches, we will identify candidate genes that may be mutated. Once candidate genes are identified, we will use morpholino oligos to specifically “knock out” the gene in nonmutated embryos and see if we can phenocopy the mutant (ie. get the same defect). Morpholino oligos are a powerful loss of function approach that we have used very effectively in X. tropicalis. Currently, a genetic linkage map is being assembled which will allow us in the future to identify genes by mapping the mutation to nearby linkage markers. By this point, Xenopus tropicalis genetics should be well underway!