Xenopus Tropicalis Genome Project

Collaborators to date: Richard Harland (University of California at
Berkeley), Rob Grainger (University of Virginia)

Estimated Genome Size: 1.7 GB

a.  **Importance of *Xenopus tropicalis* to biomedical and biological
    research**

> Xenopus is a unique resource for two critical areas in vertebrate
> biology: early embryonic development and cell biology. In the former,
> *Xenopus laevis* has led the way in identifying the mechanisms of
> early fate decisions, patterning of the basic vertebrate body plan,
> and early organogenesis. Contributions in cell biology and
> biochemistry include seminal work on chromosome replication, chromatin
> and nuclear assembly, control of the cell cycle, in vitro
> reconstruction of cytoskeletal dynamics, and signaling pathways. In
> fact, Xenopus has become a major vertebrate model for the cellular and
> developmental biology research that is supported by most of the
> Institutes of the NIH.
>
> *Xenopus tropicalis*, a close relative of *X. laevis*, is well-placed
> to complement studies of *Xenopus laevis* and to extend research into
> new areas. Two aspects are obvious: First, *Xenopus tropicalis* offers
> one of the smallest genomes among the amphibians (which number more
> than 4,500 species). It is the only Xenopus species that is diploid,
> greatly simplifying genetic studies. When combined with the mass of
> research on *Xenopus laevis*, *Xenopus tropicalis* arguably has the
> most interesting genome yet to be sequenced. The amphibians occupy a
> key phylogenetic position between those genomes that are, or have been
> sequenced, namely the mammals and the fish. As comparative genomics
> develops, the Xenopus tropicalis genome will allow insights into the
> similarities in development and physiology of the vertebrates, as well
> as the important differences in the use of regulatory DNA sequences.
> For example, Amphibians have a limb skeleton that clearly resembles
> that of the mammals, but is radically different from the fin of the
> fish. Although similar to the mammalian limb, the Xenopus limb is
> different in the numbers of digits, and fusion of skeletal elements.
> We expect that these similarities and differences will ultimately be
> understood by a comparative analysis, and lead to a deep understanding
> of developmental mechanisms. When combined with the ease of analysis
> of regulatory information in transgenic animals (see below), we expect
> many new insights and principles to emerge from study of *Xenopus
> tropicalis*.

**The importance of the genome to DOE mission**

One of the most important uses for the genomic information will be to
investigate gene regulatory pathways, particularly those involved in
development and DNA repair. Comparisons between upstream regulatory
regions may lead to identification of transcription factors that
determine specific patterns of development. Having the genome sequence
will enable identification of conserved regulatory elements, which can
then be directly assayed in an experimental system. This work will build
off current functional genomics studies being carried out at the JGI on
*ciona intestinalis*.

Frogs and tadpoles absorb chemicals from air and water through their
thin porous skin. For this reason, they are good indicators of
environmental damage caused by pollution. Frogs and other amphibians
have been referred to as the canary in the coal mine, and may provide
and early warning to humans of environmental degradation or pending
disaster. Surveys of local frog populations have been used to monitor
water pollution around mining and industrial sites. The numbers of
amphibians and frogs have been declining worldwide for the past several
decades. Possible reasons for this decline include loss of habitat,
pesticide pollution, parasitic infestations, and increased uv light
exposure due to the decrease in the ozone layer. Having a better
understanding of the frog genome through this project will give greater
insight and allow for a more detailed analysis of this troubling
problem.

> **The size and interest of the research community who will use the
> proposed BAC library**
>
> NIH currently funds 402 research grants utilizing Xenopus with a total
> award amount of \$86,289,711. In addition, NSF funds an additional 13
> awards in the amount of \$2,000,000. The current mailing list for
> researchers interested in Xenopus research numbers 328 researchers
> worldwide. A search of Xenopus in the pubMed database revealed
> citations for over 25,000 papers. There is a Xenopus database
> ([http://www.xenbase.org](http://www.xenbase.org/)) available that
> among other useful data contains contact information for over 200
> researchers in the field.
>
> **Resources available to complement the sequence.**
>
> NIH has funded several areas of research related to Xenopus genomics.
> Xenopus laevis and tropicalis EST projects are currently underway with
> 118,996 ESTs available for Xenopus laevis and 6,632 ESTs from Xenopus
> tropicalis. The limiting step for tropicalis has been the availability
> of high quality cDNA libraries. Bruce Blumberg at the University of
> California, Irvine has recently received a grant from NCRR to generate
> eighteen normalized, full-length enriched cDNA libraries from *Xenopus
> tropicalis* over the next three years. These will be made available to
> the community at large without restriction and could serve as an
> important genomic resource for functional studies and working out the
> details of transcription units from the BACs and genomic sequence when
> it becomes available.
>
> Ali Hemmati-Brivanlou at Rockefeller University has been funded to
> make cDNA microarray chips available to the community. Peter Vise
> (University of Texas, Austin) is funded to maintain and expand the
> Xenopus database. Funding opportunities also exist to conduct pilot
> genetic studies, prepare radiation hybrid maps and develop training
> and stock centers.
>
> Prof. Rob Grainger is leading an effort to produce a large insert BAC
> library that will be for mapping and end sequencing. A second smaller
> insert BAC library is currently being constructed at the Institute for
> Systems Biology in Seattle. These two libraries will be valuable
> resources that will be freely available to the research community.
>
> **Other funding support possible.**
>
> NIH has conducted discussions about a Xenopus sequencing project.
> Discussions will take place to determine if an agreement can be
> reached for NIH contribution to the sequencing effort to be undertaken
> at the JGI. This funding will likely be for fiscal year 2003.
>
> **Next Steps.**
>
> The JGI is organizing a Xenopus workshop that will take place in March
> 2003 in Walnut Creek to discuss aspects of the genome project with
> leading experts in the field. Steve Klein and representatives of NIH
> will be present to determine how best to utilize available resources
> to complete the project. Teams will be set up to coordinate activities
> and ensure that communication between groups is kept open.
>
> The embryological techniques and molecular assays which have been
> described for *X. laevis* are readily applied to *X. tropicalis*, but
> may be supported by multigeneration genetic analyses. The diploid
> genome of *X. tropicalis* will facilitate uncovering recessive
> phenotypes, and pseudogenes are far less likely to complicate promoter
> analysis than in the tetraploid X. laevis. The development of genetic
> analysis will allow a quantum leap forward in the potential of
> *Xenopus*. As a potential genetic system X. tropicalis presents very
> important advantages. When compared to *Xenopus laevis* it develops to
> sexual maturity in 1/3 the time, has 1/2 the genome size (and is
> believed to be diploid), and requires 1/5 the housing space. It
> nonetheless shares the embryological advantages of Xenopus laevis and
> gene sequences are sufficiently similar that probes cross-react
> between the two species. Haploid and gynogenetic diploid embryos are
> easy to generate; this greatly reduces the time (and space) needed to
> make inbred lines and to perform genetic screens when compared, for
> example, to the resources needed for mouse genetics. As a vertebrate
> genetic model, this system offers significant advantages over the
> mouse system, which costs far more to house and maintain, and has
> raised more controversy as a model because it is a mammal. The
> evolutionary conservation of developmental processes revealed in
> recent years among all vertebrates gives us confidence that
> information revealed from studies of lower vertebrates will, however,
> at least in large part, apply to mammalian systems.

The utility of a genetic approach in frogs over mice is highlighted in
other ways as well. Simply having the large numbers of eggs in a typical
Xenopus spawning is also of great value. Of course, ability to
manipulate the frog embryo is significantly better; an advantage that is
underscored when one considers the possibility of making genetic
chimeras. In frogs it should be possible to make chimeras of mutant and
wildtype tissue to be able to very readily study the effects of gene
manipulation on particular developmental events by obviating early, more
general lethal effects that have diminished the ability to interpret
many mouse knockouts. The urgent need for developing a genetic component
to Xenopus research now emerges when one considers the implications of
recent breakthroughs arising from this system. Very novel findings, for
example, regarding signal transduction (e.g. concerning the Wnt and BMP
pathways) have resulted from analysis in X. laevis. However, as we try
to integrate the many components of these pathways the ability of
reagents traditionally used in this system (e.g. antisense and dominant
negatives) are very limited when compared to the prospect of having
mutations in these genes for epistasis studies. The same principle
applies to tissue and organ formation, where, for example, in the eye,
brain and heart, many regulatory genes have been identified but the
subtleties of gene interaction remain poorly understood because there
are no developed genetic tools. To take full advantage of the Xenopus
system the genetic dimension must be developed.