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Nearly a century ago, studies by Lancelot Hogben and others demonstrated that ovulation in female Xenopus laevis can be induced via injection of mammalian gonadotropins into the dorsal lymph sac, allowing for egg production throughout the year independent of the normal reproductive cycles. Hormonally induced females are capable of producing thousands of eggs in a single spawning, which can then be fertilized to generate embryos or used as a substrate for generation of egg extracts. The protocol for induction of ovulation and subsequent egg collection is straightforward and robust, yet some of its details may vary among laboratories based on prior training, availability of necessary reagents, or the experimental objectives. As the goal of this protocol is not to describe every single variation possible for acquiring eggs but to provide a simple and clear description that can be easily applied by researchers with no prior working experience with X. laevis, we focus on describing the method we use at the National Xenopus Resource-that is, inducing ovulation in X. laevis via dorsal lymph sac injection of gonadotropic hormones and the stimulation of egg laying through application of gentle pressure to the females.
The embryos of the African clawed frog, Xenopus laevis, are a powerful substrate for the study of complex fundamental biological and disease mechanisms in neurobiology, physiology, molecular biology, cell biology, and developmental biology. A simple and straightforward technique for generating a large number of developmentally synchronized embryos is in vitro fertilization (IVF). IVF permits simultaneous fertilization of thousands of eggs but requires the death of the parental male, which may not be feasible if the male comes from a stock of precious animals. An alternative to euthanizing a precious male is to use a natural mating, which allows for the collection of many embryos with minimal preparation but with the potential loss of the experimental advantage of developmental synchronization. Here we present both strategies for obtaining X. laevis embryos.
Modular recirculating animal aquaculture systems incorporate UV sterilization and biological, mechanical, and activated carbon filtration, creating a nearly self-contained stable housing environment for Xenopus laevis Nonetheless, minimal water exchange is necessary to mitigate accumulation of metabolic waste, and regular weekly, monthly, and yearly maintenance is needed to ensure accurate and efficient operation. This protocol describes the methods for establishing a new recirculating system and the necessary maintenance, as well as water quality parameters, required for keeping Xenopus laevis.
Robust and efficient protocols for fertilization and early embryo care of Xenopus laevis and Xenopus tropicalis are essential for experimental success, as well as maintenance and propagation of precious animal stocks. The rapid growth of the National Xenopus Resource has required effective implementation and optimization of these protocols. Here, we discuss the procedures used at the National Xenopus Resource, which we found helpful for generation and early upkeep of Xenopus embryos and tadpoles.
Maintenance of optimal conditions such as water parameters, diet, and feeding is essential to a healthy Xenopus laevis and Xenopus tropicalis colony and thus to the productivity of the lab. Our prior husbandry experience as well as the rapid growth of the National Xenopus Resource has given us a unique insight into identifying and implementing these optimal parameters into our husbandry operations. Here, we discuss our standard operating procedures that will be of use to both new and established Xenopus facilities.
During the climax of amphibian metamorphosis many tadpole organs remodel. The different remodeling strategies are controlled by thyroid hormone (TH). The liver, skin, and tail fibroblasts shut off tadpole genes and activate frog genes in the same cell without DNA replication. We refer to this as "gene switching". In contrast, the exocrine pancreas and the intestinal epithelium dedifferentiate to a progenitor state and then redifferentiate to the adult cell type. Tadpole and adult globin are not present in the same cell. Switching from red cells containing tadpole-specific globin to those with frog globin in the liver occurs at a progenitor cell stage of development and is preceded by DNA replication. Red cell switching is the only one of these remodeling strategies that resembles a stem cell mechanism.
Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.
Chromatin immunoprecipitation and deep sequencing (ChIP-SEQ) represents a powerful tool for identifying the genomic targets of transcription factors, chromatin remodeling factors, and histone modifications. The frogs Xenopus laevis and Xenopus tropicalis have historically been outstanding model systems for embryology and cell biology, with emerging utility as highly accessible embryos for genome-wide studies. Here we focus on the particular strengths and limitations of Xenopus cell biology and genomics as they apply to ChIP-SEQ, and outline a methodology for ChIP-SEQ in both species, providing detailed strategies for sample preparation, antibody selection, quality control, sequencing library preparation, and basic analysis.
Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages. This protocol can be used to understand the molecular and cellular mechanisms involved in nervous system regeneration, including neural stem and progenitor cell (NSPC) proliferation and neurogenesis, extrinsic and intrinsic mechanisms involved in axon regeneration, glial response and scar formation, and trophic factors. For experienced personnel, husbandry takes 1-2 months; SCI can be achieved in 5-15 min; and swimming recovery takes 20-30 d.
Specification of the dorsoventral (DV) axis is critical for the subsequent differentiation of regional fate in the primary germ layers of the vertebrate embryo. We have identified a novel factor that is essential for dorsal development in embryos of the frog Xenopus laevis. Misexpression of Xenopus mab21-like 3 (Xmab21l3) dorsalizes gastrula-stage mesoderm and neurula-stage ectoderm, while morpholino-mediated knockdown of Xmab21l3 inhibits dorsal differentiation of these embryonic germ layers. Xmab21l3 is a member of a chordate-specific subclass of a recently characterized gene family, all members of which contain a conserved, but as yet ill-defined, Mab21 domain. Our studies suggest that Xmab21l3 functions to repress ventralizing activity in the early vertebrate embryo, via regulation of BMP/Smad and Ras/ERK signaling.
Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.
Fertilization of eggs from the African clawed frog Xenopus laevis is characterized by an increase in cytosolic calcium, a phenomenon that is also observed in other vertebrates such as mammals and birds. During fertilization in mammals and birds, the transfer of the soluble PLCζ from sperm into the egg is thought to trigger the release of calcium from the endoplasmic reticulum (ER). Injecting sperm extracts into eggs reproduces this effect, reinforcing the hypothesis that a sperm factor is responsible for calcium release and egg activation. Remarkably, this occurs even when sperm extracts from X. laevis are injected into mouse eggs, suggesting that mammals and X. laevis share a sperm factor. However, X. laevis lacks an annotated PLCZ1 gene, which encodes the PLCζ enzyme. In this study, we attempted to determine whether sperm from X. laevis express an unannotated PLCZ1 ortholog. We identified PLCZ1 orthologs in 11 amphibian species, including 5 that had not been previously characterized, but did not find any in either X. laevis or the closely related Xenopus tropicalis . Additionally, we performed RNA sequencing on testes obtained from adult X. laevis males and did not identify potential PLCZ1 orthologs in our dataset or in previously collected ones. These findings suggest that PLCZ1 may have been lost in the Xenopus lineage and raise the question of how fertilization triggers calcium release and egg activation in these species.
Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.
Neuronal nuclear antigen (NeuN), discovered in mice brain cell nuclei by Mullen et al. (1992), is used as an excellent marker of post-mitotic neurons in vertebrates. In this study, the expression pattern of NeuN was examined in the Xenopus brain to explore phylogenetic differences in NeuN expression. Anti-NeuN antibody showed selective staining in mouse and Xenopus brain extracts, but the number and molecular weight of the bands differed in Western blotting analysis. In immunostaining, anti-NeuN antibody showed selective staining of neurons, but not glial cells, in the Xenopus brain. Most neurons, including olfactory bulb mitral cells and cerebellar Purkinjie cells, which show no immunoreactivity in birds/mammals, showed NeuN immunoreactivity in Xenopus. This study revealed that anti-NeuN antibody is a useful marker of post-mitotic neurons in amphibians, but it also stains neurons that show no reactivity in more derived animals.
Remarkable progress has recently been made in molecular biology of double axis formation in Xenopus laevis. Leaving aside, for the time being, the problem of the gene expressions regulating Xenopus laevis development, here I show that pulse treatment could induce formation of a secondary axis in a fertilized Xenopus laevis egg. At 3 min after insemination, metal oxides were added to Xenopus fertilized eggs, and then twin embryos appeared. Zirconium oxide (ZrO2) was the most effective metal oxide for producing twin embryos. ZrO2 was added to the fertilized eggs, and 30 sec later, the eggs were dejellied with cysteine solution and washed within 7 min after insemination. The fertilized eggs began flattening at around 15 min after insemination. When the degree of flattening (the vertical length of the egg divided by the horizontal length) of the eggs at the 16- and 32-cell stages became less than 0.4 degrees, production of twin embryos occurred. Many flattened eggs at less than 0.4 degrees formed twin embryos. The third cleavage of eggs treated with metal oxides was meridional, while the normal third cleavage was horizontal.
Transient receptor potential (TRP) cation channel genes code for an extensive family of conserved proteins responsible for a variety of physiological processes, including sensory perception, ion homeostasis, and chemical signal transduction. The TRP superfamily consists of seven subgroups, one of which is the transient receptor potential vanilloid (trpv) channel family. While trpv channels are relatively well studied in adult vertebrate organisms given their role in functions such as nociception, thermoregulation, and osmotic regulation in mature tissues and organ systems, relatively little is known regarding their function during embryonic development. Although there are some reports of the expression of specific trpv channels at particular stages in various organisms, there is currently no comprehensive analysis of trpv channels during embryogenesis. Here, performing in situ hybridization, we examined the spatiotemporal expression of trpv channel mRNA during early Xenopus laevis embryogenesis. Trpv channels exhibited unique patterns of embryonic expression at distinct locations including the trigeminal ganglia, spinal cord, cement gland, otic vesicle, optic vesicle, nasal placode, notochord, tailbud, proctodeum, branchial arches, epithelium, somite and the animal pole during early development. We have also observed the colocalization of trpv channels at the animal pole (trpv 2/4), trigeminal ganglia (trpv 1/2), and epithelium (trpv 5/6). These localization patterns suggest that trpv channels may play diverse roles during early embryonic development.
Vitronectin (vn) is a cell-adhesive glycoprotein present in blood and extracellular matrix of all vertebrates. In the present study we reported the cDNA cloning of Xenopus laevisvitronectin and its spatial and temporal expression pattern during the embryonic development of this important model organism. The deduced amino acid sequence of Xenopus laevis vn showed 49%, 47% and 43% identity with human, chicken and zebrafish orthologs, respectively, whereas the comparison with Xenopus tropicalis vn presented 85% identity. The structural organization consisting of a somatomedin B domain and two hemopexin-like domains was similar to higher vertebrate vitronectins. The vn transcripts were detected from stage 28 onward. At tadpole stages, vn is expressed in heart, gut derivatives and in the notochord. The protein was detected in heart, liver, foregut, pronephros and notochord at stages 43 and 47 of Xenopus embryos. Our results suggest that vitronectin is developmentally regulated and could participate in embryo organogenesis.
Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. Developmental Dynamics 238:1433-1443, 2009. (c) 2009 Wiley-Liss, Inc.
The initial opening between the gut and the outside of the deuterostome embryo breaks through at the extreme anterior. This region is unique in that ectoderm and endoderm are directly juxtaposed, without intervening mesoderm. This opening has been called the stomodeum, buccopharyngeal membrane or oral cavity at various stages of its formation, however, in order to clarify its function, we have termed this the "primary mouth". In vertebrates, the neural crest grows around the primary mouth to form the face and a "secondary mouth" forms. The primary mouth then becomes the pharyngeal opening. In order to establish a molecular understanding of primary mouth formation, we have begun to examine this process during Xenopus laevis development. An early step during this process occurs at tailbud and involves dissolution of the basement membrane between the ectoderm and endoderm. This is followed by ectodermal invagination to create the stomodeum. A subsequent step involves localized cell death in the ectoderm, which may lead to ectodermal thinning. Subsequently, ectoderm and endoderm apparently intercalate to generate one to two cell layers. The final step is perforation, where (after hatching) the primary mouth opens. Fate mapping has defined the ectodermal and endodermal regions that will form the primary mouth. Extirpations and transplants of these and adjacent regions indicate that, at tailbud, the oral ectoderm is not specifically required for primary mouth formation. In contrast, underlying endoderm and surrounding regions are crucial, presumably sources of necessary signals. This study indicates the complexity of primary mouth formation, and lays the groundwork for future molecular analyses of this important structure.
We have analyzed the developing expression pattern of x-Shh in the Xenopus forebrain, interpreting the results within the framework of the neuromeric model to assess evolutionary trends and clues. To achieve this goal, we have characterized phenotypically the developing x-Shh expressing forebrain subdivisions and neurons by means of the combination of in situ hybridization for x-Shh and immunohistochemistry for the detection of forebrain essential regulators and markers, such as the homeodomain transcription factors Islet 1, Orthopedia, NKX2.1 and NKX2.2 and tyrosine hydroxylase. Substantial evidence was found for x-Shh expression in the telencephalic commissural preoptic area and this is strongly correlated with the presence of a pallidum and/or a basal telencephalic cholinergic system. In the diencephalon, x-Shh was demonstrated in the zona limitans intrathalamica and the x-Shh expressing cells were extended into the prethalamus. Throughout development and in the adult hypothalamic x-Shh expression was strong in basal regions but, in addition, in the alar suprachiasmatic region. The findings obtained in the forebrain of Xenopus revealed a largely conserved pattern of Shh expression among tetrapods. However, interesting differences were also noted that could be related to evolutive changes in forebrain organization.
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