Shoko Ishibashi

A method for generating transgenic frog embryos.

The amphibian embryo has classically been one of the best systems for elucidating the molecular mechanisms of early development, in particular for studies of mesodermal and neural induction. Amphibian embryos develop externally and are large and robust. Therefore, tissues can be dissected, isolated, or transplanted with high precision and ease in these embryos. In addition, it is relatively easy to manipulate the expression of gene products by injecting in-vitro transcribed RNAs into developing embryos. However, since RNAs are translated soon after injection, this method has been used mainly for studying early stages of development. Manipulating genes specifically during later stages of development requires fine control over the time and place of expression, which can be achieved only through transgenic technology. In this chapter, we describe a very efficient method of transgenesis developed for Xenopus laevis and Xenopus tropicalis. 1.2. Background Understanding the molecular basis of pattern formation and differentiation in frog embryos was previously hindered by the lack of a system for temporal and tissue-specific expression of wild-type and mutant forms of developmentally important genes. RNA injection, the most common transient expression method in Xenopus, has been effectively used to study maternally expressed genes. However, since RNAs are translated immediately after injection, this method is unfavorable for the study of zygotic gene products that are expressed only after the midblastula transition. Direct injection of DNA can be used to express genes behind temporal and tissue-specific promoters after the midblastula transition.

Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis

Summary In the past decade, Xenopus tropicalis has emerged as a powerful new amphibian genetic model system, which offers all of the experimental advantages of its larger cousin, Xenopus laevis. Here we investigated the efficiency of transcription activator-like effector nucleases (TALENs) for generating targeted mutations in endogenous genes in X. tropicalis. For our analysis we targeted the tyrosinase (oculocutaneous albinism IA) (tyr) gene, which is required for the production of skin pigments, such as melanin. We injected mRNA encoding TALENs targeting the first exon of the tyr gene into two-cell-stage embryos. Surprisingly, we found that over 90% of the founder animals developed either partial or full albinism, suggesting that the TALENs induced bi-allelic mutations in the tyr gene at very high frequency in the F0 animals. Furthermore, mutations tyr gene were efficiently transmitted into the F1 progeny, as evidenced by the generation of albino offspring. These findings have far reaching implications in our quest to develop efficient reverse genetic approaches in this emerging amphibian model.

pTransgenesis:a cross-species, modular transgenesis resource

As studies aim increasingly to understand key, evolutionarily conserved properties of biological systems, the ability to move transgenesis experiments efficiently between organisms becomes essential. DNA constructions used in transgenesis usually contain four elements, including sequences that facilitate transgene genome integration, a selectable marker and promoter elements driving a coding gene. Linking these four elements in a DNA construction, however, can be a rate-limiting step in the design and creation of transgenic organisms. In order to expedite the construction process and to facilitate cross-species collaborations, we have incorporated the four common elements of transgenesis into a modular, recombination-based cloning system called pTransgenesis. Within this framework, we created a library of useful coding sequences, such as various fluorescent protein, Gal4, Cre-recombinase and dominant-negative receptor constructs, which are designed to be coupled to modular, species-compatible selectable markers, promoters and transgenesis facilitation sequences. Using pTransgenesis in Xenopus, we demonstrate Gal4-UAS binary expression, Cre-loxP-mediated fate-mapping and the establishment of novel, tissue-specific transgenic lines. Importantly, we show that the pTransgenesis resource is also compatible with transgenesis in Drosophila, zebrafish and mammalian cell models. Thus, the pTransgenesis resource fosters a cross-model standardization of commonly used transgenesis elements, streamlines DNA construct creation and facilitates collaboration between researchers working on different model organisms.

BDNF promotes target innervation of Xenopus mandibular trigeminal axons in vivo

BackgroundTrigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland.ResultsWe found that the cement gland is enriched in BDNF mRNA transcripts compared to the other neurotrophins NT3 and NT4 during mandibular trigeminal nerve innervation. BDNF knockdown in Xenopus embryos or specifically in cement glands resulted in the failure of mandibular trigeminal axons to arborise or grow into the cement gland. BDNF expressed ectodermal grafts, when positioned in place of the cement gland, promoted local trigeminal axon arborisation in vivo.ConclusionBDNF is necessary locally to promote end stage target innervation of trigeminal axons in vivo, suggesting that BDNF functions as a short-range signal that stimulates mandibular trigeminal axon arborisation and growth into the cement gland.

cis-Regulatory Remodeling of the SCL Locus during Vertebrate Evolution

ABSTRACT Development progresses through a sequence of cellular identities which are determined by the activities of networks of transcription factor genes. Alterations in cis-regulatory elements of these genes play a major role in evolutionary change, but little is known about the mechanisms responsible for maintaining conserved patterns of gene expression. We have studied the evolution of cis-regulatory mechanisms controlling the SCL gene, which encodes a key transcriptional regulator of blood, vasculature, and brain development and exhibits conserved function and pattern of expression throughout vertebrate evolution. SCL cis-regulatory elements are conserved between frog and chicken but accrued alterations at an accelerated rate between 310 and 200 million years ago, with subsequent fixation of a new cis-regulatory pattern at the beginning of the mammalian radiation. As a consequence, orthologous elements shared by mammals and lower vertebrates exhibit functional differences and binding site turnover between widely separated cis-regulatory modules. However, the net effect of these alterations is constancy of overall regulatory inputs and of expression pattern. Our data demonstrate remarkable cis-regulatory remodelling across the SCL locus and indicate that stable patterns of expression can mask extensive regulatory change. These insights illuminate our understanding of vertebrate evolution.

A simple method of transgenesis using I-sce I meganuclease in Xenopus.

Here we present a protocol for generating transgenic embryos in Xenopus using I-SceI meganuclease. This method relies on integration of DNA constructs, containing one or two I-SceI meganuclease sites. It is a simpler method than the REMI method of transgenesis, and it is ideally suited for generating transgenic lines in Xenopus laevis and Xenopus tropicalis. In addition to it being simpler than the REMI method, this protocol also results in single copy integration events rather than tandem concatemers. Although the protocol we describe is for X. tropicalis, the method can also be used to generate transgenic lines in X. laevis. We also describe a convenient method for designing and generating complex constructs for transgenesis, named pTransgenesis, based on the Multisite Gateway(®) cloning, which include I-SceI sites and Tol2 elements to facilitate genome integration.

Xenopus: An in vivo model for imaging the inflammatory response following injury and bacterial infection.

A major goal in regenerative medicine is to identify therapies to facilitate our body׳s innate abilities to repair and regenerate following injury, disease or aging. In the past decade it has become apparent that the innate immune system is able to affect the speed and quality of the regenerative response through mechanisms that are not entirely clear. For this reason there has been a resurgent interest in investigating the role of inflammation during tissue repair and regeneration. Remarkably, there have only been a handful of such studies using organisms with high regenerative capacity. Here we perform a study of the inflammatory response following injury in Xenopus larvae, which are able to achieve scarless wound healing and to regenerate appendages, as a preamble into understanding the role that inflammation plays during tissue repair and regeneration in this organism. We characterized the morphology and migratory behavior of granulocytes and macrophages following sterile and infected wounding regimes, using various transgenic lines that labeled different types of myeloid lineages, including granulocytes and macrophages. Using this approach we found that the inflammatory response following injury and infection in Xenopus larvae is very similar to that seen in humans, suggesting that this model provides an easily tractable and medically relevant system to investigate inflammation following injury and infection in vivo.

Reverse Genetic Studies Using Antisense Morpholino Oligonucleotides

Here we present a protocol, which allows loss-of-function studies in Xenopus embryos using antisense morpholino oligonucleotides (MOs). Gene knockdown studies provide a critical method for assessing gene function in vitro and in vivo. Such studies are currently performed in Xenopus using primarily one of the two main methods: (1) overexpression of dominant negative constructs or (2) inhibition of gene function by using MOs targeting either the initiation of translation or mRNA splicing. While a dominant negative approach is very effective, it often suffers from specificity. Given that MOs target very specific nucleotide sequences in the target RNA, it suffers considerably less from issues of specificity. The most convenient method for introducing MOs into embryos is through microinjection, which is a simple procedure. Therefore, a reverse genetics approach in Xenopus using MOs is an extremely powerful tool to study gene function, particularly when taking advantage of available sequence data in the post-genomic era. Furthermore, given the well-established fate map in Xenopus, it is also very easy to generate mosaic knockdown embryos, where the gene of interest is affected in defined regions of the embryo. Finally it should be noted that MOs can also be used to block miRNA function and processing, so that it provides a convenient method to not only perform gene knockdown studies on protein coding genes, but also noncoding genes. The protocol we describe here is for both Xenopus laevis and Xenopus tropicalis.

Generating Transgenic Frog Embryos by Restriction Enzyme Mediated Integration (REMI).

Here we present a protocol for generating transgenic embryos in Xenopus laevis and Xenopus tropicalis. The method includes three steps: (1) The preparation of high-speed egg extracts, which facilitates the replacement of protamines in sperm nuclei with nucleosomes and decondenses the chromatin of sperm nuclei; (2) The isolation of sperm nuclei; and (3) The mixing of sperm nuclei, restriction enzyme, and high-speed extract in vitro, following by nuclear transplantation into unfertilized eggs to generate the transgenic embryos. This procedure generates non-mosaic transgenic embryos at high frequency and efficiency.

Ca2+-Induced Mitochondrial ROS Regulate the Early Embryonic Cell Cycle

Summary While it is appreciated that reactive oxygen species (ROS) can act as second messengers in both homeostastic and stress response signaling pathways, potential roles for ROS during early vertebrate development have remained largely unexplored. Here, we show that fertilization in Xenopus embryos triggers a rapid increase in ROS levels, which oscillate with each cell division. Furthermore, we show that the fertilization-induced Ca2+ wave is necessary and sufficient to induce ROS production in activated or fertilized eggs. Using chemical inhibitors, we identified mitochondria as the major source of fertilization-induced ROS production. Inhibition of mitochondrial ROS production in early embryos results in cell-cycle arrest, in part, via ROS-dependent regulation of Cdc25C activity. This study reveals a role for oscillating ROS levels in early cell cycle regulation in Xenopus embryos.