Robert M. Grainger

The Genome of the Western Clawed Frog Xenopus tropicalis

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633, see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes. Assembly, annotation, and analysis of the frog genome compares gene content and synteny with the human and chicken genomes. The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Embryonic lens induction: shedding light on vertebrate tissue determination

The principle of embryonic induction was defined by early studies of lens determination, and because of the relative simplicity of the developing lens and its interaction with presumptive retinal tissue it has been a favored system for examining mechanisms of induction. Recent studies have led to substantial alterations of the classic model for this process, introducing several elements that significantly refine our view of vertebrate tissue determination.

Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.

We have assessed the efficacy of the recently developed CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system for genome modification in the amphibian Xenopus tropicalis. As a model experiment, targeted mutations of the tyrosinase gene were verified, showing the expected albinism phenotype in injected embryos. We further tested this technology by interrupting the six3 gene, which is required for proper eye and brain formation. Expected eye and brain phenotypes were observed when inducing mutations in the six3 coding regions, as well as when deleting the gene promoter by dual targeting. We describe here a standardized protocol for genome editing using this system. This simple and fast method to edit the genome provides a powerful new reverse genetics tool for Xenopus researchers. genesis 51:835–843.

Frog genetics: Xenopus tropicalis jumps into the future

. The two mainreasons for this are that the embryosdevelop externally and their relativelylarge size allows embryologists toperform microsurgery and manipu-late the embryos experimentally inways that are not as easy in other ver-tebrate embryos (e.g. mouse or zebra-fish). Most of the early experimentswere done on embryos from

Xenopus, the next generation: X. tropicalis genetics and genomics.

A small, fast‐breeding, diploid relative of the frog Xenopus laevis, Xenopus tropicalis, has recently been adopted for research in developmental genetics and functional genomics. X. tropicalis shares advantages of X. laevis as a classic embryologic system, but its simpler genome and shorter generation time make it more convenient for multigenerational genetic, genomic, and transgenic approaches. Its embryos closely resemble those of X. laevis, except for their smaller size, and assays and molecular probes developed in X. laevis can be readily adapted for use in X. tropicalis. Genomic manipulation techniques such as gynogenesis facilitate genetic screens, because they permit the identification of recessive phenotypes after only one generation. Stable transgenic lines can be used both as in vivo reporters to streamline a variety of embryologic and molecular assays, or to experimentally manipulate gene expression through the use of binary constructs such as the GAL4/UAS system. Several mutations have been identified in wild‐caught animals and during the course of generating inbred lines. A variety of strategies are discussed for conducting and managing genetic screens, obtaining mutations in specific sequences, achieving homologous recombination, and in developing and taking advantage of the genomic resources for Xenopus tropicalis.

Vertebrate eye development

Vertebrate eye determination is mediated by a series of inductive interactions that have now been more precisely defined with the use of regional markers. Analyses of the genes responsible for eye mutations and the cloning of genes delimiting spatial domains within the developing eye have begun to elucidate the molecular basis of this process.

Conservative assembly and segregation of nucleosomal histories

The assembly of new histones into nucleosomes and the segregation of old histones during replication were investigated using a density gradient, sedimentation equilibrium analysis of histones labeled in vivo with dense amino acids. After a 1 hr pulse of dense amino acids and 3H-lysine, nucleosomes were isolated from chick myoblast organ cultures, and the histones were cross-linked to octamers. The octamers were purified from DNA and then banded to equilibrium in cesium-formate guanidinium-HCI density gradients. The cross-linked dense octamers have the same density as the noncross-linked dense histones, and both were significantly heavier than histones synthesized in the presence of light amino acids. This experiment shows that new histone does not mix with old histone in the new nucleosomes, since the labeling protocol allows density labeling of only one histone for every seven preexisting unlabeled histones. Thus the assembly of new histone octamers is conservative. Using essentially the same experimental design, but varying the details of the labeling procedures, we also show that the dense histone octamer is stable over 3-4 generations, that neighboring octamers tend to be synthesized at the same time, and that old and new histone octamers segregate conservatively over 2-3 generations.

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.

Xenopus research: metamorphosed by genetics and genomics

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.

Inductive interactions in the spatial and temporal restriction of lens-forming potential in embryonic ectoderm of Xenopus laevis

The process of lens cell determination in amphibians is currently viewed as one involving a series of inductive interactions. On the basis of previous investigations, these interactions are thought to begin during gastrulation when the presumptive foregut endoderm and then the heart mesoderm come into contact with the presumptive lens ectoderm. This earlier period of induction is followed by the later interaction of the optic vesicle with the lens-forming ectoderm. Transplantation experiments were performed to determine the relative significance of the early and later periods of induction in the process of lens cell determination in the anuran Xenopus laevis. Various ectodermal tissues were transplanted either into the lens-forming region of open neural plate stage host embryos or over the newly formed optic vesicle of later neurula stage embryos. All transplanted tissues were labeled with the intracellular marker horseradish peroxidase to assess the exact origins of any induced lens structures. The results indicate that all nonneural ectodermal tissues have some lens-forming potential early during gastrulation; however, this potential is restricted to the lens-forming region, and perhaps nearby regions, later in development during the time of neurulation. Furthermore, the results show that the optic vesicle is not a substantial inductor of the lens in tissues that have not been previously exposed to the earlier series of inductive interactions that take place during gastrulation and neurulation. Since the optic vesicle does not appear to be a sufficient inductor of the lens, these earlier inductive interactions are, therefore, essential in the process of lens cell determination in Xenopus. These earlier inductive interactions lead to a steady increase in what may be called a lens-forming bias in the presumptive lens ectoderm during this period of development. The eventual loss in the ability of nonlens ventral ectoderm to respond to these lens inductors is presumably the result of other determinative processes that occur in this tissue.