Xenopus research: metamorphosed by genetics and genomics

Abstract

Research using Xenopus takes advantage of large, abundant eggs and readily manipulated embryos in addition to conserved cellular, developmental and genomic organization with mammals. Research on Xenopus has defined key principles of gene regulation and signal transduction, embryonic induction, morphogenesis and patterning as well as cell cycle regulation. Genomic and genetic advances in this system, including the development of Xenopus tropicalis as a genetically tractable complement to the widely used Xenopus laevis, capitalize on the classical strengths and wealth of achievements. These attributes provide the tools to tackle the complex biological problems of the new century, including cellular reprogramming, organogenesis, regeneration, gene regulatory networks and protein interactions controlling growth and development, all of which provide insights into a multitude of human diseases and their potential treatments.

Figure 1

Photograph of adult X. laevis (left) together with X. tropicalis (right) (from ref. [61]). Development of both species is quite similar, though egg and embryo size is somewhat smaller for X. tropicalis and development can proceed more rapidly since the embryos are adapted to a higher temperature. Detailed features of the two systems have been described elsewhere [61].

Figure 2

Summary of induced mutations found predominantly during the first ENU screen of X. tropicalis [30]. Mutations in a multitude of organ systems have been uncovered. Eight classes are presented here with images from one mutant in each class illustrated in this figure, and other members of the class denoted below or adjacent to the images. The images are paired: left is wildtype and right is mutant. Clockwise from upper left: eye phenotypes [brightfield of variegated retinal pigment epithelium in kaleidoscope (kal)]; inner ear [dysmorphic otoconia in komimi (kom)]; axial [the dwarf issunboushi (iss)]; neural crest [melanocytes in lumen of neural tube in cyd vicious (cyd)]; myofibrillogenesis [skeletal muscle stained with anti-alpha actinin (green) and phalloidin (red) showing disorganized sarcomeres in dicky ticker (dit)]; limb development [skeletal preparation showing complete absence of forelimb formation in xenopus de milo (xdm)]; cardiovascular [confocal image of muzak (muz) stained with anti-myosin heavy chain (green) and phalloidin (red)]; and blood [white hart (wha) showing reduced globin staining]. Figure courtesy of L. Zimmerman (National Institute for Medical Research, Mill Hill, London).

Figure 3

Analysis of the Six3 locus in X. tropicalis (from ref. [33]). This analysis demonstrates that the X. tropicalis genome is well placed for comparative genomic studies to identify enhancers in conserved non-coding sequences (CNS) and that the fast transgenic methodology in Xenopus is particularly useful in testing their relevance as gene regulatory elements. In the middle of the figure one can see that in the 100 Kb surrounding the Six3 gene the mouse genome is so highly conserved compared to the base genome used here (human) that potentially functional conserved non-coding sequences (CNS) cannot be readily identified. However, when human is compared to Xenopus, seven highly conserved non-coding regions are found. Each was tested by co-transgenesis where a PCR product for the CNS is mixed with a basal promoter-green fluorescent protein (GFP) fusion construct in a suspension of sperm nuclei and injected into embryos, as summarized in the top part of the figure. Mixed enhancer and promoter fragments concatamerize (co-transgenesis) and are integrated rapidly, allowing efficient testing for enhancer activity in the conserved non-coding regions. At the top left, the endogenous expression of Six3 in eye and brain is shown by in situ hybridization. At the bottom of the figure, CNS3 and CNS5 were identified as enhancers of Six3 by the transgenic assay. The two enhancers together account for all embryonic expression of Six3. Note that the evolutionary distance of the Fugu genome is such that one of the key enhancers (location demarcated by red arrow) would not have been identified by this method.