Chris Showell

T‐box genes in early embryogenesis

The T‐box gene family, encoding related DNA‐binding transcriptional regulators, plays an essential role in controlling many aspects of embryogenesis in a wide variety of organisms. The T‐box genes exhibit diverse patterns of spatial and temporal expression in the developing embryo, and both genetic and molecular embryological studies have demonstrated their importance in regulating cell fate decisions that establish the early body plan, and in later processes underlying organogenesis. Despite these studies, little is known of either the regulation of the T‐box genes or the identities of their transcriptional targets. The aim of this review is to examine the diverse yet conserved roles of several T‐box genes in regulating early patterning in chordates and to discuss possible mechanisms through which this functional diversity might arise. Developmental Dynamics 229:201–218, 2004.

Xenopus: An emerging model for studying congenital heart disease

Congenital heart defects affect nearly 1% of all newborns and are a significant cause of infant death. Clinical studies have identified a number of congenital heart syndromes associated with mutations in genes that are involved in the complex process of cardiogenesis. The African clawed frog, Xenopus, has been instrumental in studies of vertebrate heart development and provides a valuable tool to investigate the molecular mechanisms underlying human congenital heart diseases. In this review, we discuss the methodologies that make Xenopus an ideal model system to investigate heart development and disease. We also outline congenital heart conditions linked to cardiac genes that have been well studied in Xenopus and describe some emerging technologies that will further aid in the study of these complex syndromes.

Developmental Expression Patterns of Tbx1, Tbx2, Tbx5, and Tbx20 in Xenopus tropicalis

T‐box genes have diverse functions during embryogenesis and are implicated in several human congenital disorders. Here, we report the identification, sequence analysis, and developmental expression patterns of four members of the T‐box gene family in the diploid frog Xenopus tropicalis. These four genes—Tbx1, Tbx2, Tbx5, and Tbx20—have been shown to influence cardiac development in a variety of organisms, in addition to their individual roles in regulating other aspects of embryonic development. Our results highlight the high degree of evolutionary conservation between orthologs of these genes in X. tropicalis and other vertebrates, both at the molecular level and in their developmental expression patterns, and also identify novel features of their expression. Thus, X. tropicalis represents a potentially valuable vertebrate model in which to further investigate the functions of these genes through genetic approaches. Developmental Dynamics 235:1623–1630, 2006.

Morpholino Injection in Xenopus

The study of gene function in developmental biology has been significantly furthered by advances in antisense technology made in the early 2000s. This was achieved, in particular, by the introduction of morpholino (MO) oligonucleotides. The introduction of antisense MO oligonucleotides into cells enables researchers to readily reduce the levels of their protein of interest without investing huge financial or temporal resources, in both in vivo and in vitro model systems. Historically, the African clawed frog Xenopus has been used to study vertebrate embryological development, due to its ability to produce vast numbers of offspring that develop rapidly, in synchrony, and can be cultured in buffers with ease. The developmental progress of Xenopus embryos has been extensively characterized and this model organism is very easy to maintain. It is these attributes that enable MO-based knockdown strategies to be so effective in Xenopus. In this chapter, we will detail the methods of microinjecting MO oligonucleotides into early embryos of X. laevis and X. tropicalis. We will discuss how MOs can be used to prevent either pre-mRNA splicing or translation of the specific gene of interest resulting in abrogation of that genes function and advise on what control experiments should be undertaken to verify their efficacy.

Natural mating and tadpole husbandry in the western clawed frog Xenopus tropicalis.

The routine generation and successful husbandry of tadpoles is essential for investigators using the Western clawed frog Xenopus tropicalis for developmental and genetic studies. We describe a method to induce natural mating by injecting a hormone into sexually mature X. tropicalis, and to raise the offspring through the embryonic, tadpole, and metamorphosis stages. X. tropicalis develop at a faster rate than Xenopus laevis, reaching metamorphosis in as little as 4 wk. Moreover, natural mating produces large numbers of offspring and is therefore particularly useful for mutation screening and genetic mapping. The method includes a technique for the anesthesia of frogs, which makes them easier to handle during injection of the hormone, reducing the likelihood of injury to the animals.

A Comparative Survey of the Frequency and Distribution of Polymorphism in the Genome of Xenopus tropicalis

Naturally occurring DNA sequence variation within a species underlies evolutionary adaptation and can give rise to phenotypic changes that provide novel insight into biological questions. This variation exists in laboratory populations just as in wild populations and, in addition to being a source of useful alleles for genetic studies, can impact efforts to identify induced mutations in sequence-based genetic screens. The Western clawed frog Xenopus tropicalis (X. tropicalis) has been adopted as a model system for studying the genetic control of embryonic development and a variety of other areas of research. Its diploid genome has been extensively sequenced and efforts are underway to isolate mutants by phenotype- and genotype-based approaches. Here, we describe a study of genetic polymorphism in laboratory strains of X. tropicalis. Polymorphism was detected in the coding and non-coding regions of developmental genes distributed widely across the genome. Laboratory strains exhibit unexpectedly high frequencies of genetic polymorphism, with alleles carrying a variety of synonymous and non-synonymous codon substitutions and nucleotide insertions/deletions. Inter-strain comparisons of polymorphism uncover a high proportion of shared alleles between Nigerian and Ivory Coast strains, in spite of their distinct geographical origins. These observations will likely influence the design of future sequence-based mutation screens, particularly those using DNA mismatch-based detection methods which can be disrupted by the presence of naturally occurring sequence variants. The existence of a significant reservoir of alleles also suggests that existing laboratory stocks may be a useful source of novel alleles for mapping and functional studies.

The western clawed frog (Xenopus tropicalis): an emerging vertebrate model for developmental genetics and environmental toxicology.

Xenopus tropicalis, also known as Silurana tropicalis or the Western clawed frog, is a small, wholly aquatic frog that is found in the countries that lie along the west coast of equatorial Africa from Gabon to Sierra Leone. It is a diploid relative of Xenopus laevis, a frog that has found many uses in laboratories throughout the world. X. tropicalis closely resembles its relative in anatomy throughout its life cycle. It shares its advantages as a model organism for studying many aspects of vertebrate biology, particularly the genetic, biochemical, and environmental factors that influence vertebrate development from embryonic stages through adulthood. It also shares much of its biology with more distantly related species, including mammals, but unlike mammalian model organisms, routine manipulation of X. tropicalis in the laboratory can produce thousands of embryos for experimental analysis. X. tropicalis is also finding uses as an important test species for assessing the impact of environmental toxins and disease on amphibians, which are in decline in many areas of the world due to waterborne pollutants and infectious agents such as the chytrid fungus.

Egg Collection and In Vitro Fertilization of the Western Clawed Frog Xenopus tropicalis

The eggs of Xenopus laevis have been widely used in studies investigating a variety of aspects of biology, such as control of the cell cycle, RNA processing, and the cytoskeleton. The Western clawed frog Xenopus tropicalis is likely to prove useful for such studies in the future, because of the potential to combine traditional experimental approaches with genetic analysis and the available genome sequence. The eggs of X. tropicalis are also a key starting material for transgenesis. Here, we describe a method for the routine collection of eggs from X. tropicalis, together with a method for in vitro fertilization. Very large numbers of eggs, often more than 2000, can be obtained from a single X. tropicalis female. In vitro fertilization is a valuable alternative to natural mating for generating embryos. It is particularly useful for microinjection experiments and when collecting embryos at a series of defined developmental stages (e.g., for studies of gene expression), because it produces embryos that develop synchronously during early embryonic development. The typical yield of embryos ranges from several hundred to more than 1000 per fertilization.

Tissue Sampling and Genomic DNA Purification from the Western Clawed Frog Xenopus tropicalis

The extraction of genomic DNA from Western clawed frog (Xenopus tropicalis) tissue samples is an essential step for genotyping known mutations, identifying live animals carrying nonfluorescent transgenes, and reverse genetic screening techniques. We describe here a method for sampling tail tissue from X. tropicalis tadpoles at stage 48 and later. This method of tissue sampling allows a significant amount of genomic DNA to be obtained from tadpoles without killing them. In both Xenopus laevis and X. tropicalis, the tip of the tail is able to regenerate following surgery and its removal does not have a significant effect on the survival of the tadpole when performed carefully. Genomic DNA purification can be carried out in “deep-well” 96-well plates, making it amenable to high-throughput applications. Typically, the DNA yield is sufficient for more than 100 standard 50-μL polymerase chain reactions (PCRs) (using 1–5 μL of DNA per reaction), so it can be used for genetic screening and mapping studies.