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Genesis Special Issue - Xenopus: Advances and Emerging Technologies

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Genesis: January 2017, Volume 55, Issue 1-2

Issue Information

View Wiley Online Library Article - DOI: 10.1002/dvg.22967

PERSPECTIVES

Using Xenopus to understand human disease and developmental disorders

Amy K. Sater and Sally A. Moody

View Wiley Online Library Article - DOI: 10.1002/dvg.22997
Model animals are crucial to biomedical research. Among the commonly used model animals, the amphibian, Xenopus, has had tremendous impact because of its unique experimental advantages, cost effectiveness, and close evolutionary relationship with mammals as a tetrapod. Over the past 50&nbsp,years, the use of Xenopus has made possible many fundamental contributions to biomedicine, and it is a cornerstone of research in cell biology, developmental biology, evolutionary biology, immunology, molecular biology, neurobiology, and physiology. The prospects for Xenopus as an experimental system are excellent: Xenopus is uniquely well-suited for many contemporary approaches used to study fundamental biological and disease mechanisms. Moreover, recent advances in high throughput DNA sequencing, genome editing, proteomics, and pharmacological screening are easily applicable in Xenopus, enabling rapid functional genomics and human disease modeling at a systems level.

Maternal messages to live by: a personal historical perspective

Mary Lou King

View Wiley Online Library Article - DOI: 10.1002/dvg.23007
In the 1980s, the study of localized maternal mRNAs was just emerging as a new research area. Classic embryological studies had linked the inheritance of cytoplasmic domains with specific cell lineages, but the underlying molecular nature of these putative determinants remained a mystery. The model system Xenopus would play a pivotal role in the progress of this new field. In fact, the first localized maternal mRNA to be identified and cloned from any organism was Xenopus vg1, a TGF-beta family member. This seminal finding opened the door to many subsequent studies focused on how RNAs are localized and what functions they had in development. As the field moves into the future, Xenopus remains the system of choice for studies identifying RNA/protein transport particles and maternal RNAs through RNA-sequencing.

CELL BIOLOGY

Xenopus egg extract to study regulation of genome-wide and locus-specific DNA replication

Erica Raspelli, Lucia Falbo and Vincenzo Costanzo

View Wiley Online Library Article - DOI: 10.1002/dvg.22996

Faithful DNA replication, coupled with accurate repair of DNA damage, is essential to maintain genome stability and relies on different DNA metabolism genes. Many of these genes are involved in the assembly of replication origins, in the coordination of DNA repair to protect replication forks progression in the presence of DNA damage and in the replication of repetitive chromatin regions. Some DNA metabolism genes are essential in higher eukaryotes, suggesting the existence of specialized mechanisms of repair and replication in organisms with complex genomes. The impact on cell survival of many of these genes has so far precluded in depth molecular analysis of their function. The cell-free Xenopus laevis egg extract represents an ideal system to overcome survival issues and to facilitate the biochemical study of replication-associated functions of essential proteins in vertebrate organisms. Here, we will discuss how Xenopus egg extracts have been used to study cellular and molecular processes, such as DNA replication and DNA repair. In particular, we will focus on innovative imaging and proteomic-based experimental approaches to characterize the molecular function of a number of essential DNA metabolism factors involved in the duplication of complex vertebrate genomes.

Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts

Jessica G. Bermudez, Hui Chen, Lily C. Einstein and Matthew C. Good

View Wiley Online Library Article - DOI: 10.1002/dvg.23013

Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery.

Exosomal trafficking in Xenopus development

Michael Danilchik and Tess Tumarkin

View Wiley Online Library Article - DOI: 10.1002/dvg.23011

Exosomes are small extracellular vesicles (EVs) secreted by many cell types in both normal and pathogenic circumstances. Because EVs, particularly exosomes, are known to transfer biologically active proteins, RNAs and lipids between cells, they have recently become the focus of intense interest as potential mediators of cell-cell communication, particularly in long-range and juxtacrine signaling events associated with adaptive immune function and progression of cancer. Among the EVs, exosomes appear particularly adapted for long-range delivery of cargoes between cells. Because of their association with disease states, the exciting potential for exosomes to serve as diagnostic biomarkers and as target-specific biomolecule delivery vehicles has stimulated a broad range of biomedical investigations to learn how exosomes are generated, what their cargoes are, and how they might be tailored for uptake by remote targets. Addressing these questions requires experimental models in which biochemically useful amounts of material can be harvested, gene expression easily manipulated, and interpretable biological assays developed. The early Xenopus embryo fulfills these model-system ideals in an in vivo context: during morphogenesis the embryo develops several large, fluid-filled extracellular compartments across which numerous tissue-specifying signals must cross, and which are abundantly endowed with exosomes and other EVs. Importantly, certain surface-facing tissues avidly ingest EVs during gastrulation. Recent work has demonstrated that EVs can be isolated from these interstitial spaces in amounts suitable for proteomic and transcriptomic analysis. With its large numbers, great cell size, well-understood fate map, and tolerance of a variety of experimental approaches, the Xenopus embryo provides a unique opportunity to both understand and manipulate the basic cell biology of exosomal trafficking in the context of an intact organism.

Tools for live imaging of active Rho GTPases in Xenopus

Rachel E. Stephenson and Ann L. Miller

View Wiley Online Library Article - DOI: 10.1002/dvg.22998

Rho family GTPases are signaling molecules that orchestrate cytoskeletal dynamics in a variety of cellular processes. Because they effect localized changes to the cytoskeleton only in their active (GTP-bound) conformation, the ability to monitor the active state of Rho GTPases in space and time is critical for understanding their function. Here, we summarize popular tools used for live imaging of active Rho GTPases, outlining advantages and drawbacks of these approaches. Additionally, we highlight key features of the Xenopus laevis embryo that make it well-suited for epithelial cell biology and discuss how application of Rho activity reporters in the Xenopus laevis embryo led to the discovery of a novel phenomenon, junctional Rho flares.

SIGNALING AND MORPHOGENESIS

Genome-wide analysis of canonical Wnt target gene regulation in Xenopus tropicalis challenges &beta,-catenin paradigm

Yukio Nakamura and Stefan Hoppler

View Wiley Online Library Article - DOI: 10.1002/dvg.22991

Wnt/&beta,-catenin signaling is an important cell-to-cell signaling mechanism that controls gene expression during embryonic development and is critically implicated in human diseases. Developmental, cellular, and transcriptional responses to Wnt signaling are remarkably context-specific in different biological processes. While nuclear localization of &beta,-catenin is the key to activation of the Wnt/&beta,-catenin pathway and target gene expression, the molecular mechanisms of how the same Wnt/&beta,-catenin signaling pathway induces specific responses remain undetermined. Recent advances in high-throughput sequencing technologies and the availability of genome information for Xenopus tropicalis have enabled us to uncover a genome-wide view of Wnt/&beta,-catenin signaling in early vertebrate embryos, which challenges previous concepts about molecular mechanisms of Wnt target gene regulation. In this review, we summarize our experimental approaches, introduce the technologies we employed and focus on recent findings about Wnt target gene regulation from Xenopus research. We will also discuss potential functions of widespread &beta,-catenin binding in the genome that we discovered in this species.

A frog's view of EphrinB signaling

Yoo-Seok Hwang and Ira O. Daar

View Wiley Online Library Article - DOI: 10.1002/dvg.23002

Cell&ndash,cell and cell&ndash,substrate adhesion are essential to the proper formation and maintenance of tissue patterns during development, and deregulation of these processes can lead to invasion and metastasis of cancer cells. Cell surface adhesion and signaling molecules are key players in both normal development and cancer progression. One set of cell surface proteins, the Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, are significant regulators of these processes. During embryonic development, the Eph/ephrin signaling system is involved in cell&ndash,cell contact events that result in cell sorting and boundary formation between receptor and ligand bearing cells. When migrating cells that display the membrane bound ligands or receptors come in contact with cells bearing the cognate partner, the response may be adhesion or repulsion, ultimately leading to the proper positioning of these cells. During cancer progression, the signaling between these receptor/ligand pairs is often deregulated, leading to increased invasion and metastasis. To gain mechanistic insight into the pathways that mediate Eph receptor and ephrin signaling we have relied upon a very tractable system, the frog Xenopus. This model system has proven to be extremely versatile, and represents a relatively quick and manipulable system to explore signaling events and the in vivo processes affected by these signals.

Xenopus as a model for studies in mechanical stress and cell division

Georgina A. Stooke-Vaughan, Lance A. Davidson and Sarah Woolner

View Wiley Online Library Article - DOI: 10.1002/dvg.23004

We exist in a physical world, and cells within biological tissues must respond appropriately to both environmental forces and forces generated within the tissue to ensure normal development and homeostasis. Cell division is required for normal tissue growth and maintenance, but both the direction and rate of cell division must be tightly controlled to avoid diseases of over-proliferation such as cancer. Recent studies have shown that mechanical cues can cause mitotic entry and orient the mitotic spindle, suggesting that physical force could play a role in patterning tissue growth. However, to fully understand how mechanics guides cells in vivo, it is necessary to assess the interaction of mechanical strain and cell division in a whole tissue context. In this mini-review we first summarise the body of work linking mechanics and cell division, before looking at the advantages that the Xenopus embryo can offer as a model organism for understanding: (1) the mechanical environment during embryogenesis, and (2) factors important for cell division. Finally, we introduce a novel method for applying a reproducible strain to Xenopus embryonic tissue and assessing subsequent cell divisions.

Xenopus laevis as a model system to study cytoskeletal dynamics during axon pathfinding

Paula G Slater, Laurie Hayrapetian and Laura Anne Lowery

View Wiley Online Library Article - DOI: 10.1002/dvg.22994

The model system, Xenopus laevis, has been used in innumerable research studies and has contributed to the understanding of multiple cytoskeletal components, including actin, microtubules, and neurofilaments, during axon pathfinding. Xenopus developmental stages have been widely characterized, and the Xenopus genome has been sequenced, allowing gene expression modifications through exogenous molecules. Xenopus cell cultures are ideal for long periods of live imaging because they are easily obtained and maintained, and they do not require special culture conditions. In addition, Xenopus have relatively large growth cones, compared to other vertebrates, thus providing a suitable system for imaging cytoskeletal components. Therefore, X. laevis is an ideal model organism in which to study cytoskeletal dynamics during axon pathfinding.

Xenopus as a model organism to study heterotrimeric G-protein pathway during collective cell migration of neural crest

G. Toro-Tapia, S. Villaseca, J. I. Leal, A. Beyer, J. Fuentealba and M. Torrej&oacute,n

View Wiley Online Library Article - DOI: 10.1002/dvg.23008

Collective cell migration is essential in many fundamental aspects of normal development, like morphogenesis, organ formation, wound healing, and immune responses, as well as in the etiology of severe pathologies, like cancer metastasis. In spite of the huge amount of data accumulated on cell migration, such a complex process involves many molecular actors, some of which still remain to be functionally characterized. One of these signals is the heterotrimeric G-protein pathway that has been studied mainly in gastrulation movements. Recently we have reported that Ric-8A, a GEF for G&alpha, proteins, plays an important role in neural crest migration in Xenopus development. Xenopus neural crest cells, a highly migratory embryonic cell population induced at the border of the neural plate that migrates extensively in order to differentiate in other tissues during development, have become a good model to understand the dynamics that regulate cell migration. In this review, we aim to provide sufficient evidence supporting how useful Xenopus model with its different tools, such as explants and transplants, paired with improved in vivo imaging techniques, will allow us to tackle the multiple signaling mechanisms involved in neural crest cell migration.

HUMAN DISEASE AND DEVELOPMENTAL DEFECTS

Frogs model man: In vivo thyroid hormone signaling during development

Laurent M. Sachs and Daniel R. Buchholz

View Wiley Online Library Article - DOI: 10.1002/dvg.23000

Thyroid hormone (TH) signaling comprises TH transport across cell membranes, metabolism by deiodinases, and molecular mechanisms of gene regulation. Proper TH signaling is essential for normal perinatal development, most notably for neurogenesis and fetal growth. Knowledge of perinatal TH endocrinology needs improvement to provide better treatments for premature infants and endocrine diseases during gestation and to counteract effects of endocrine disrupting chemicals. Studies in amphibians have provided major insights to understand in vivo mechanisms of TH signaling. The frog model boasts dramatic TH-dependent changes directly observable in free-living tadpoles with precise and easy experimental control of the TH response at developmental stages comparable to fetal stages in mammals. The hormones, their receptors, molecular mechanisms, and developmental roles of TH signaling are conserved to a high degree in humans and amphibians, such that with respect to developmental TH signaling &ldquo,frogs are just little people that hop.&rdquo, The frog model is exceptionally illustrative of fundamental molecular mechanisms of in vivo TH action involving TH receptors, transcriptional cofactors, and chromatin remodeling. This review highlights the current need, recent successes, and future prospects using amphibians as a model to elucidate molecular mechanisms and functional roles of TH signaling during post-embryonic development.

What we can learn from a tadpole about ciliopathies and airway diseases: Using systems biology in Xenopus to study cilia and mucociliary epithelia

Peter Walentek and Ian K. Quigley

View Wiley Online Library Article - DOI: 10.1002/dvg.23001

Over the past years, the Xenopus embryo has emerged as an incredibly useful model organism for studying the formation and function of cilia and ciliated epithelia in vivo. This has led to a variety of findings elucidating the molecular mechanisms of ciliated cell specification, basal body biogenesis, cilia assembly, and ciliary motility. These findings also revealed the deep functional conservation of signaling, transcriptional, post-transcriptional, and protein networks employed in the formation and function of vertebrate ciliated cells. Therefore, Xenopus research can contribute crucial insights not only into developmental and cell biology, but also into the molecular mechanisms underlying cilia related diseases (ciliopathies) as well as diseases affecting the ciliated epithelium of the respiratory tract in humans (e.g., chronic lung diseases). Additionally, systems biology approaches including transcriptomics, genomics, and proteomics have been rapidly adapted for use in Xenopus, and broaden the applications for current and future translational biomedical research. This review aims to present the advantages of using Xenopus for cilia research, highlight some of the evolutionarily conserved key concepts and mechanisms of ciliated cell biology that were elucidated using the Xenopus model, and describe the potential for Xenopus research to address unresolved questions regarding the molecular mechanisms of ciliopathies and airway diseases.

Xenopus, an ideal model organism to study laterality in conjoined twins

Matthias Tisler, Axel Schweickert and Martin Blum

View Wiley Online Library Article - DOI: 10.1002/dvg.22993

Conjoined twins occur at low frequency in all vertebrates including humans. Many twins fused at the chest or abdomen display a very peculiar laterality defect: while the left twin is normal with respect to asymmetric organ morphogenesis and placement (situs solitus), the organ situs is randomized in right twins. Although this phenomenon has fascinated already some of the founders of experimental embryology in the 19th and early 20th century, such as Dareste, Fol, Warynsky and Spemann, its embryological basis has remained enigmatic. Here we summarize historical experiments and interpretations as well as current models, argue that the frog Xenopus is the only vertebrate model organism to tackle the issue, and outline suitable experiments to address the question of twin laterality in the context of cilia-based symmetry breakage.

Seeing the future: using Xenopus to understand eye regeneration

Ai-Sun Tseng

View Wiley Online Library Article - DOI: 10.1002/dvg.23003

Studies of Xenopus eye development have contributed considerably to the understanding of vertebrate neurogenesis, including eye field specification, cell fate determination and identification of genes critical for eye formation. This knowledge has served as a solid foundation for cellular and molecular examinations of the robust regenerative capacity of the Xenopus eye. The retina, lens, and the optic nerve are capable of regeneration after injury in both larval and adult stages. Here, we discuss the current models for studying eye regeneration in Xenopus and their potential applications for providing insights into human eye diseases. As Xenopus has many of the same tools that are available for other regeneration models, we thus highlight the distinct strengths and versatility of this organism that make it especially suited for extrapolating and testing strategies aimed at promoting regeneration and repair in eye tissues. Furthermore, we outline a promising future for the use of new techniques and approaches to address outstanding questions in understanding eye regeneration.

Transcriptional dynamics of tail regeneration in Xenopus tropicalis

Jessica Chang, Julie Baker and Andrea Wills

View Wiley Online Library Article - DOI: 10.1002/dvg.23015

In contrast to humans, many amphibians are able to rapidly and completely regenerate complex tissues, including entire appendages. Following tail amputation, Xenopus tropicalis tadpoles quickly regenerate muscle, spinal cord, cartilage, vasculature and skin, all properly patterned in three dimensions. To better understand the molecular basis of this regenerative competence, we performed a transcriptional analysis of the first 72 h of tail regeneration using RNA-Seq. Our analysis refines the windows during which many key biological signaling processes act in regeneration, including embryonic patterning signals, immune responses, bioelectrical signaling and apoptosis. Our work provides a deep database for researchers interested in appendage regeneration, and points to new avenues for further study.

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

Thomas Naert, Tom Van Nieuwenhuysen and Kris Vleminckx

View Wiley Online Library Article - DOI: 10.1002/dvg.23005

The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models. We believe that due to particular advantageous characteristics, outlined within this review, GEXM will become a valuable alternative animal model for modeling human cancer. Furthermore, we provide perspectives of how GEXM will be used as a platform for elucidation of novel therapeutic targets and for preclinical drug validation. Finally, we also discuss some future prospects on how the recent expansions and adaptations of the CRISPR/Cas9 toolbox might influence and push forward X. tropicalis cancer research.

EMERGING APPROACHES

Targeted integration of genes in Xenopus tropicalis

Zhaoying Shi, Dandan Tian, Huhu Xin, Jingru Lian, Xiaogang Guo and Yonglong Chen

View Wiley Online Library Article - DOI: 10.1002/dvg.23006
With the successful establishment of both targeted gene disruption and integration methods in the true diploid frog Xenopus tropicalis, this excellent vertebrate genetic model now is making a unique contribution to modelling human diseases. Here, we summarize our efforts on establishing homologous recombination-mediated targeted integration in Xenopus tropicalis, the usefulness, and limitation of targeted integration via the homology-independent strategy, and future directions on how to further improve targeted gene integration in Xenopus tropicalis.

AmphiBase: A new genomic resource for non-model amphibian species

Taejoon Kwon

View Wiley Online Library Article - DOI: 10.1002/dvg.23010

More than five thousand genes annotated in the recently published Xenopus laevis and Xenopus tropicalis genomes do not have a candidate orthologous counterpart in other vertebrate species. To determine whether these sequences represent genuine amphibian-specific genes or annotation errors, it is necessary to analyze them alongside sequences from other amphibian species. However, due to large genome sizes and an abundance of repeat sequences, there are limited numbers of gene sequences available from amphibian species other than Xenopus. AmphiBase is a new genomic resource covering non-model amphibian species, based on public domain transcriptome data and computational methods developed during the X. laevis genome project. Here, I review the current status of AmphiBase, including amphibian species with available transcriptome data or biological samples, and describe the challenges of building a comprehensive amphibian genomic resource in the absence of genomes. This mini-review will be informative for researchers interested in functional genomic experiments using amphibian model organisms, such as Xenopus and axolotl, and will assist in interpretation of results implicating &ldquo,orphan genes.&rdquo, Additionally, this study highlights an opportunity for researchers working on non-model amphibian species to collaborate in their future efforts and develop amphibian genomic resources as a community.

Probing forebrain to hindbrain circuit functions in Xenopus

Darcy B. Kelley, Taffeta M. Elliott, Ben J. Evans, Ian C. Hall, Elizabeth C. Leininger, Heather J. Rhodes, Ayako Yamaguchi and Erik Zornik

View Wiley Online Library Article - DOI: 10.1002/dvg.22999

The vertebrate hindbrain includes neural circuits that govern essential functions including breathing, blood pressure and heart rate. Hindbrain circuits also participate in generating rhythmic motor patterns for vocalization. In most tetrapods, sound production is powered by expiration and the circuitry underlying vocalization and respiration must be linked. Perception and arousal are also linked, acoustic features of social communication sounds&mdash,for example, a baby's cry&mdash,can drive autonomic responses. The close links between autonomic functions that are essential for life and vocal expression have been a major in vivo experimental challenge. Xenopus provides an opportunity to address this challenge using an ex vivo preparation: an isolated brain that generates vocal and breathing patterns. The isolated brain allows identification and manipulation of hindbrain vocal circuits as well as their activation by forebrain circuits that receive sensory input, initiate motor patterns and control arousal. Advances in imaging technologies, coupled to the production of Xenopus lines expressing genetically encoded calcium sensors, provide powerful tools for imaging neuronal patterns in the entire fictively behaving brain, a goal of the BRAIN Initiative. Comparisons of neural circuit activity across species (comparative neuromics) with distinctive vocal patterns can identify conserved features, and thereby reveal essential functional components.

New-generation mass spectrometry expands the toolbox of cell and developmental biology

Camille Lombard-Banek, Erika P. Portero, Rosemary M. Onjiko and Peter Nemes

View Wiley Online Library Article - DOI: 10.1002/dvg.23012

Systems cell biology understanding of development requires characterization of all the molecules produced in the biological system. Decades of research and new-generation sequencing provided functional information on key genes and transcripts. However, there is less information available on how differential gene expression translates into the domains of functionally important proteins, peptides, and metabolites, and how changes in these molecules impact development. Mass spectrometry (MS) is the current technology of choice for the detection and quantification of large numbers of proteins and metabolites, because it requires no use of antibodies, functional probes, or a priori knowledge of molecules produced in the system. This review focuses on recent technologies that have improved MS sensitivity for proteins and metabolites and enabled new functionalities to assess their temporal and spatial changes during vertebrate embryonic development. This review highlights case studies, in which new-generation MS tools have enabled the study of hundreds-to-thousands of proteins and metabolites in tissues, cell populations, and single cells in model systems of vertebrate development, particularly the frog (Xenopus), zebrafish, and mouse. New-generation MS expands the toolbox of cell and developmental studies, raising exciting potentials to advance basic and translational research in the life sciences.

Reproduced with permission from John Wiley &amp, Sons, copyright 2017.

Genesis: January 2017 Volume 55, Issue 1-2

Last Updated: 2017-02-07

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