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Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions (e.g. amphibian dorsal morula, mammalian anterior visceral endoderm) require stabilised nuclear β-catenin, but the role of localised Wnt ligand signalling activity in their establishment remains unclear. In Xenopus, dorsal β-catenin is initiated by vegetal microtubule-mediated symmetry breaking in the fertilised egg, known as 'cortical rotation'. Localised wnt11b mRNA and ligand-independent activators of β-catenin have been implicated in dorsal β-catenin activation, but the extent to which each contributes to axis formation in this paradigm remains unclear. Here, we describe a CRISPR-mediated maternal-effect mutation in Xenopus laevis wnt11b.L. We find that wnt11b is maternally required for robust dorsal axis formation and for timely gastrulation, and zygotically for left-right asymmetry. Importantly, we show that vegetal microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs. In addition, we show that activated Wnt coreceptor Lrp6 and Dishevelled lack behaviour consistent with roles in early β-catenin stabilisation, and that neither is regulated by Wnt11b. This work thus implicates Wnt11b in the distribution of putative dorsal determinants rather than in comprising the determinants themselves. This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interests D.W.H. is director of the Developmental Studies Hybridoma Bank and serves on the Xenbase advisory board; M.E.H. is director of the National Xenopus Resource., (© 2022. Published by The Company of Biologists Ltd.)

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.

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., (© 2021 Cold Spring Harbor Laboratory Press.)

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., (© 2021 Cold Spring Harbor Laboratory Press.)

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 ., (© 2020 Cold Spring Harbor Laboratory Press.)

CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F 0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F 0 generation.

Studying genes involved in organogenesis is often difficult because many of these genes are also essential for early development. The allotetraploid frog, Xenopus laevis , is commonly used to study developmental processes, but because of the presence of two homeologs for many genes, it has been difficult to use as a genetic model. Few studies have successfully used CRISPR in amphibians, and currently there is no tissue-targeted knockout strategy described in Xenopus The goal of this study is to determine whether CRISPR/Cas9-mediated gene knockout can be targeted to the Xenopus kidney without perturbing essential early gene function. We demonstrate that targeting CRISPR gene editing to the kidney and the eye of F0 embryos is feasible. Our study shows that knockout of both homeologs of lhx1 results in the disruption of kidney development and function but does not lead to early developmental defects. Therefore, targeting of CRISPR to the kidney may not be necessary to bypass the early developmental defects reported upon disruption of Lhx1 protein expression or function by morpholinos, antisense RNA, or dominant negative constructs. We also establish a control for CRISPR in Xenopus by editing a gene ( slc45a2 ) that when knocked out results in albinism without altering kidney development. This study establishes the feasibility of tissue-specific gene knockout in Xenopus , providing a cost-effective and efficient method for assessing the roles of genes implicated in developmental abnormalities that is amenable to high-throughput gene or drug screening techniques., (Copyright © 2018 by the Genetics Society of America.)

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.

Completion of the Xenopus laevis genome sequence from inbred J strain animals has facilitated the generation of germline mutant X. laevis using targeted genome editing. In the last few years, numerous reports have demonstrated that TALENs are able to induce mutations in F0 Xenopus embryos, but none has demonstrated germline transmission of such mutations in X. laevis. In this report we used the oocyte host-transfer method to generate mutations in both tyrosinase homeologs and found highly-penetrant germline mutations; in contrast, embryonic injections yielded few germline mutations. We also compared the distribution of mutations in several F0 somatic tissues and germ cells and found that the majority of mutations in each tissue were different. These results establish that X. laevis J strain animals are very useful for generating germline mutations and that the oocyte host-transfer method is an efficient technique for generating mutations in both homeologs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Injection of human Chorionic Gonadotropin (hCG) directly into the dorsal lymph sac of Xenopus is a commonly used protocol for induction of ovulation, but recent shortages in the stocks of commercially available hCG as well as lack of a well tested alternative have resulted in frustrating experimental delays in laboratories that predominantly use Xenopus in their research. Mammalian Luteinizing Hormones (LH) share structural similarity, functional equivalency, and bind the same receptor as hCG; this suggests that LH may serve as a good alternative to hCG for promoting ovulation in Xenopus. LH has been found to induce maturation of Xenopus oocytes in vitro, but whether it can be used to induce ovulation in vivo has not been examined. Here we compared the ability of four mammalian LH proteins, bovine (bLH), human (hLH), ovine (oLH), porcine (pLH), to induce ovulation in Xenopus when injected into the dorsal lymph sac of sexually mature females. We find that both ovine and human LH, but not bovine or porcine, are good substitutes for hCG for induction of ovulation in WT and J strain Xenopus laevis and Xenopus tropicalis., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

We characterize the genetic diversity of Xenopus laevis strains using RNA-seq data and allele-specific analysis. This data provides a catalogue of coding variation, which can be used for improving the genomic sequence, as well as for better sequence alignment, probe design, and proteomic analysis. In addition, we paint a broad picture of the genetic landscape of the species by functionally annotating different classes of mutations with a well-established prediction tool (PolyPhen-2). Further, we specifically compare the variation in the progeny of four crosses: inbred genomic (J)-strain, outbred albino (B)-strain, and two hybrid crosses of J and B strains. We identify a subset of mutations specific to the B strain, which allows us to investigate the selection pressures affecting duplicated genes in this allotetraploid. From these crosses we find the ratio of non-synonymous to synonymous mutations is lower in duplicated genes, which suggests that they are under greater purifying selection. Surprisingly, we also find that function-altering ("damaging") mutations constitute a greater fraction of the non-synonymous variants in this group, which suggests a role for subfunctionalization in coding variation affecting duplicated genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Mass spectrometry-based proteomics enables the global identification and quantification of proteins and their posttranslational modifications in complex biological samples. However, proteomic analysis requires a complete and accurate reference set of proteins and is therefore largely restricted to model organisms with sequenced genomes., Results: Here, we demonstrate the feasibility of deep genome-free proteomics by using a reference proteome derived from heterogeneous mRNA data. We identify more than 11,000 proteins with 99% confidence from the unfertilized Xenopus laevis egg and estimate protein abundance with approximately 2-fold precision. Our reference database outperforms the provisional gene models based on genomic DNA sequencing and references generated by other methods. Surprisingly, we find that many proteins in the egg lack mRNA support and that many of these proteins are found in blood or liver, suggesting that they are taken up from the blood plasma, together with yolk, during oocyte growth and maturation, potentially contributing to early embryogenesis., Conclusion: To facilitate proteomics in nonmodel organisms, we make our platform available as an online resource that converts heterogeneous mRNA data into a protein reference set. Thus, we demonstrate the feasibility and power of genome-free proteomics while shedding new light on embryogenesis in vertebrates., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In mammals, it has been well established that gastrointestinal and pancreatic endocrine cells are specified by a cascade of different transcription factors, but whether these same pathways (or linear relationships) operate in Xenopus is currently unknown. We recently identified the endocrine-specific zinc finger transcription factor insulinoma associated protein 1 (insm1) as a dorsal-enriched gene. We found that insm1 is expressed in the dorsal pancreas as early as NF28, making it one of the earliest markers to be localized to the dorsal pancreas. Through morpholino-mediated knockdown, we demonstrate that insm1 is essential for proper specification of both gastrointestinal and pancreatic endocrine cells. In addition, we place insm1 downstream of ngn3 and upstream of pax6 and neuroD in the endocrine cell transcription factor cascade. These are the first results showing that the endodermal endocrine cell development in Xenopus uses the same transcriptional cascade as in mammals.

Understanding how the pancreas develops is vital to finding new treatments for a range of pancreatic diseases, including diabetes and pancreatic cancer. Xenopus is a relatively new model organism for the elucidation of pancreas development, and has already made contributions to the field. Recent studies have shown benefits of using Xenopus for understanding both early patterning and lineage specification aspects of pancreas organogenesis. This review focuses specifically on Xenopus pancreas development, and covers events from the end of gastrulation, when regional specification of the endoderm is occurring, right through metamorphosis, when the mature pancreas is fully formed. We have attempted to cover pancreas development in Xenopus comprehensively enough to assist newcomers to the field and also to enable those studying pancreas development in other model organisms to better place the results from Xenopus research into the context of the field in general and their studies specifically. Developmental Dynamics 238:1271-1286, 2009. (c) 2009 Wiley-Liss, Inc.

Insulin-producing beta-cells are present as single cells or in small clusters distributed throughout the pancreas of the Xenopus laevis tadpole. During metamorphic climax when the exocrine pancreas dedifferentiates to progenitor cells, the beta-cells undergo two changes. Insulin mRNA is down regulated at the beginning of metamorphic climax (NF62) and reexpressed again near the end of climax. Secondly, the beta-cells aggregate to form islets. During climax the increase in insulin cluster size is not caused by cell proliferation or by acinar-to-beta-cell transdifferentiation, but rather is due to the aggregation of pre-existing beta-cells. The total number of beta-cells does not change during the 8 days of climax. Thyroid hormone (TH) induction of premetamorphic tadpoles causes an increase in islet size while prolonged treatment of tadpoles with the goitrogen methimazole inhibits this increase. Expression of a dominant negative form of the thyroid hormone receptor (TRDN) driven by the elastase promoter not only protects the exocrine pancreas of a transgenic tadpole from TH-induced dedifferentiation but also prevents aggregation of beta-cells at climax. These transgenic tadpoles do however undergo normal loss and resynthesis of insulin mRNA at the same stage as controls. In contrast transgenic tadpoles with the same TRDN transgene driven by an insulin promoter do not undergo down regulation of insulin mRNA, but do aggregate beta-cells to form islets like controls. These results demonstrate that TH controls the remodeling of beta-cells through cell-cell interaction with dedifferentiating acinar cells and a cell autonomous program that temporarily shuts off the insulin gene.

The amphibian embryo is a highly successful model system with great promise for organogenesis research. Since the late 1800s, amphibians have been employed to understand vertebrate development and since the 1950s, the African clawed frog Xenopus laevis has been the amphibian of choice. In the past two decades, Xenopus has led the way forward in, among other things, identifying transcription factors, gene regulatory networks and inter- and intracellular signaling pathways that control early development (from fertilization through gastrulation and neurulation). Perhaps the best measure of how successful Xenopus has been as a model for early mammalian development is the observation that much of the knowledge gleaned from Xenopus studies has subsequently directly translated to discoveries of similar mechanisms operating in mouse development. Despite this great success in early development, research on organogenesis in Xenopus has lagged behind the mouse. However, recent technical advances now make Xenopus amenable for studies on later development, including organogenesis. Here, we discuss why Xenopus is well suited for such research and, we believe, permits addressing questions that have been difficult to approach using other model systems. We also highlight how Xenopus researchers have already begun studying a number of major organs, pancreas, liver, kidney and heart, and suggest how Xenopus might contribute more to these areas in the near future.

It is known from work with amniote embryos that regional specification of the gut requires cell-cell signalling between the mesoderm and the endoderm. In recent years, much of the interest in Xenopus endoderm development has focused on events that occur before gastrulation and this work has led to a different model whereby regional specification of the endoderm is autonomous. In this paper, we examine the specification and differentiation of the endoderm in Xenopus using neurula and tail-bud-stage embryos and we show that the current hypothesis of stable autonomous regional specification is not correct. When the endoderm is isolated alone from neurula and tail bud stages, it remains fully viable but will not express markers of regional specification or differentiation. If mesoderm is present, regional markers are expressed. If recombinations are made between mesoderm and endoderm, then the endodermal markers expressed have the regional character of the mesoderm. Previous results with vegetal explants had shown that endodermal differentiation occurs cell-autonomously, in the absence of mesoderm. We have repeated these experiments and have found that the explants do in fact show some expression of mesoderm markers associated with lateral plate derivatives. We believe that the formation of mesoderm cells by the vegetal explants accounts for the apparent autonomous development of the endoderm. Since the fate map of the Xenopus gut shows that the mesoderm and endoderm of each level do not come together until tail bud stages, we conclude that stable regional specification of the endoderm must occur quite late, and as a result of inductive signals from the mesoderm., (Copyright 2001 Academic Press.)