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Germ layers to organs: Using Xenopus to study "later" development
Abstract
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.
... The Xenopus liver has the same cell types as found in humans including hepatocytes, stellate cells, Kupffer cells and sinusoidal endothelial cells (Blitz et al., 2006). Similar to vertebrates, the liver is derived from the endoderm of the future gut tube, close to where the stomach and duodenum will meet (Nieuwkoop and Faber, 1994). ...
... By tadpole stage 37-39, the Xenopus liver is a sac-like structure, with thick walls that fold inwards and fills the liver cavity with hepatocytes. Also at this time, the liver and biliary ductal systems are developing and the gall bladder is a thin-walled sac structure (Blitz et al., 2006). At late stage development (stage 38-45) the Xenopus heart, liver and kidney Stage 38 embryos were exposed to paracetamol (0-5 mM) and harvested at stage 45. ...
Introduction:
Failure to predict drug-induced liver injury (DILI) remains a major contributing factor to lead compound drop-out during drug development. Xenopus embryos are amenable for early stage medium throughput small molecule screens and so have the potential to be used in pre-clinical screens. To begin to assess the usefulness and limitations of Xenopus embryos for safety assessment in the early phases of drug development, paracetamol was used as a model hepatotoxin. Paracetamol overdose is associated with acute liver injury. In mammals, the main mechanism of paracetamol-induced acute liver injury is an increased amount of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) combined with a reduction of free glutathione (GSH). Humans that have taken an overdose of paracetamol are often treated with N-acetyl cysteine (NAC).
Method:
Xenopus laevis embryos were treated with up to 5 mM paracetamol from stage 38 to stage 45 during development, when the liver is functional. The presence of paracetamol-induced liver injury was assessed by: (Dart et al., 2006) microRNA-122 (miR-122) expression (a hepatic marker), (Jaeschke, 2015) free GSH concentration (a marker of oxidative stress) and (Larson et al., 2005) NAC antioxidant intervention.
Results:
The amount of free GSH decreased significantly in embryos exposed to increasing paracetamol concentration. In embryos exposed to 5 mM paracetamol, 22.57 ± 4.25 nmol/mg GSH was detected compared to 47.11 ± 7.31 nmol/mg untreated embryos (mean ± SEM). In tail tissue, miRNA-122 expression increased 6.3-fold with 3 mM paracetamol concentration treatment compared to untreated embryos. NAC treatment altered the free GSH decline for embryos treated with up to 5 mM. Embryos exposed to 1 mM paracetamol and then exposed to 0.5 mM NAC 24 h prior to harvest, had an significantly higher amount GSH compared to embryos that were only exposed to 1 mM paracetamol (mean ± SEM; 97.1 ± 9.6 nmol/mg and 54.5 ± 6.6 nmol/mg respectively).
Conclusion:
Xenopus laevis embryos exhibit similar characteristics of paracetamol-induced liver injury observed in mammalian models. These data indicate that the Xenopus embryo could be a useful in vivo model to assess DILI and aid lead compound prioritisation during the early phase of drug development, in combination with pre-clinical in vitro studies. Consequently, the Xenopus embryo could contribute to the reduction principle as defined by the National Centre for the Replacement, Refinement and Reduction of Animals in Research.
... In the last decades, studies in amphibians such as the African clawed frog Xenopus laevis have substantially contributed to deciphering the mechanisms of early vertebrate heart development. Xenopus and humans share various anatomical, physiological as well as genetic similarities [1,2] suggesting that they have comparable underlying gene regulatory networks [1,3]. Several features make Xenopus an attractive model for studying cardiovascular development and disease. ...
... First, Xenopus husbandry is simple and breeding can be done year-round by inducing females to spawn after injection of human chorionic gonadotropin. The litter size can be as high as 2000 eggs per day per frog and embryos can be obtained by in vitro fertilization [3]. In addition, in vitro fertilization allows the synchronization of embryonic development between sibling embryos. ...
The African clawed frog, Xenopus, is a valuable non-mammalian model organism to investigate vertebrate heart development and to explore the underlying molecular mechanisms of human congenital heart defects (CHDs). In this review, we outline the similarities between Xenopus and mammalian cardiogenesis, and provide an overview of well-studied cardiac genes in Xenopus, which have been associated with congenital heart conditions. Additionally, we highlight advantages of modeling candidate genes derived from genome wide association studies (GWAS) in Xenopus and discuss commonly used techniques.
... The benefits of using these organisms are numerous: their pancreases develop faster, the number of animals is not limiting as large numbers of embryos can be injected and processed in a single day, early development is more accessible allowing easy isolation of endodermal or pancreatic tissue, overexpression studies are accomplished by simple microinjection of mRNA (targeting to specific regions of the endoderm is done by injection into single blastomeres at the 8-, 16-, or 32-cell stage), transgenic tadpoles can be produced quickly, gene knockdown studies can be performed quickly using antisense morpholinos, and earlier patterning and specification events can be addressed more easily. Indeed, recent results demonstrate that early pancreas development in Xenopus closely resembles that of mice and humans, and is applicable to mammalian cells as we previously demonstrated (Horb et al., 2003;Cao et al., 2004;Li et al., 2005;Afelik et al., 2006;Blitz et al., 2006;Jarikji et al., 2007). In fact, it is becoming clear that the same genes used in mammalian pancreas development are involved in Xenopus pancreas development ( Horb et al., 2003;Afelik et al., 2006;Jarikji et al., 2007). ...
... With the emergence of Xenopus as a model system for pancreas development, now is the appropriate time for a comprehensive review of Xenopus pancreas development to be written. Although there have been several recent reviews that have touched on some aspects of Xenopus pancreas development ( Blitz et al., 2006;Pieler and Chen, 2006;Spagnoli, 2007;Zorn and Wells, 2007;Pearl and Horb, 2008), none has comprehensively covered pancreas development in Xenopus from gastrulation through metamorphosis. This review aims to give detailed insight into pancreas development in Xenopus laevis from the end of gastrulation, when the endoderm becomes regionalized, through the emergence of pancreatic buds, to the cellular rearrangements that take place at metamorphosis resulting in a mature pancreas. ...
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.
... These pancreatic buds grow, fuse, and branch to eventually form the definitive pancreas. Like the mammalian pancreas, the pancreas of the frog Xenopus is composed of both endocrine and exocrine tissues and can be readily studied due to easy access to large numbers of externally developing embryos (Blitz et al., 2006). The exocrine tissue of both mammals and amphibians is organized into grape-like clusters of cells called acini and their associated ducts, which pro-duce and secrete enzymes into the duodenum that function in digestion of food. ...
... Xenopus has so far been under-used as a means of studying the processes of organogenesis (Blitz et al., 2006). However, among the advantages offered by Xenopus that could contribute significantly to a better understanding of the development of organs such as the pancreas is the provision of a fresh set of molecular markers. ...
The pancreas is both an exocrine and endocrine endodermal organ involved in digestion and glucose homeostasis. During embryogenesis, the anlagen of the pancreas arise from dorsal and ventral evaginations of the foregut that later fuse to form a single organ. To better understand the molecular genetics of early pancreas development, we sought to isolate markers that are uniquely expressed in this tissue. Microarray analysis was performed comparing dissected pancreatic buds, liver buds, and the stomach region of tadpole stage Xenopus embryos. A total of 912 genes were found to be differentially expressed between these organs during early stages of organogenesis. K-means clustering analysis predicted 120 of these genes to be specifically enriched in the pancreas. Of these, we report on the novel expression patterns of 24 genes. Our analyses implicate the involvement of previously unsuspected signaling pathways during early pancreas development. Developmental Dynamics 238:1455-1466, 2009. (c) 2009 Wiley-Liss, Inc.
... The Xenopus laevis (X.l.) embryo is an important vertebrate model used to assess the toxicity of compounds or environmental factors (including radiation and pollution) on embryonic development (FETAX test, Vismara et al., 1993) and to study biochemical, cellular, morphological, and physiological aspects of fertilization and development (Rizzo et al., 1994(Rizzo et al., , 1998Blitz et al., 2006;Hoke and Ankley, 2005). For these reasons, the biochemical assessment of the antioxidant potential of the embryos during the first days of development could be relevant (Ciccotelli et al., 1998;Dell'Orto et al., 1998). ...
... embryos increase after the first day of development and reach their maximum at stage 40. In this period, the hatching process occurs, and the embryo's organs, such as the liver, develop and start their metabolic functions (Blitz et al., 2006). The GSH concentration that was measured followed the pattern of liver development in embryos, suggesting that, in amphibians, the hepatic synthesis of this tripeptide is an important metabolic pathway for ROS detoxification. ...
Reactive oxygen species (ROS) are formed and degraded in all aerobic organisms, but their role during embryonic development has not yet been well established. In this paper, we report the activities of various enzymes involved in antioxidant metabolism during the first 7 days of embryonic development of Xenopus laevis embryos. During the first two days of development, embryo antioxidant metabolism is based on catalase and superoxide dismutase activities. Later, the glutathione system is activated, and the activity of all the enzymes involved increases. The results presented in this study, together with previously reported data, support the hypothesis that antioxidant defences may include enzymes that are genetically regulated, while the other systems that appear to be environmentally modulated become relevant later in development, probably to protect embryos from environmental and toxic factors.
... These studies indicate that the axolotl is potentially a useful model for studying the molecular mechanisms underlying lung regeneration in vertebrates. Other amphibians, such as Xenopus, could also be attractive models, because these species possess a high ability to regenerate other tissues, such as the tail (in tadpoles), limb and eye (Beck et al., 2006;Blitz et al., 2006). It is likely that the wellconserved FGF and BMP pathways also play a key role in lung regeneration in Xenopus, because they are involved in Xenopus lung development (Rankin et al., 2015;Shifley et al., 2012). ...
The ability to continuously grow and regenerate the gills throughout life is a remarkable property of fish and amphibians. Considering that gill regeneration was first described over one century ago, it is surprising that the underlying mechanisms of cell and tissue replacement in the gills remain poorly understood. By contrast, the mammalian lung is a largely quiescent organ in adults but is capable of facultative regeneration following injury. In the course of the past decade, it has been recognized that lungs contain a population of stem or progenitor cells with an extensive ability to restore tissue; however, despite recent advances in regenerative biology of the lung, the signaling pathways that underlie regeneration are poorly understood. In this Review, we discuss the common evolutionary and embryological origins shared by gills and mammalian lungs. These are evident in homologies in tissue structure, cell populations, cellular function and genetic pathways. An integration of the literature on gill and lung regeneration in vertebrates is presented using a comparative approach in order to outline the challenges that remain in these areas, and to highlight the importance of using aquatic vertebrates as model organisms. The study of gill regeneration in fish and amphibians, which have a high regenerative potential and for which genetic tools are widely available, represents a unique opportunity to uncover common signaling mechanisms that may be important for regeneration of respiratory organs in all vertebrates. This may lead to new advances in tissue repair following lung disease.
... Moreover, diploid X. tropicalis is a good alternative for genetic studies [254] and id convenient for Crisp/Cas [258], Zinc-finger nucleases (ZFNs) [259], and the TALENs method [260]. Overall, these features make it a good platform for toxicology [261], neurobiology [262], regenerative [263], developmental biology [264], embryology [265], immunology [266], and cancer studies [267]. ...
The proper design of experiments is a critical step for each study in order to obtain reproducible and reliable data. Taking into account constant competitiveness in the quickly developing biomedical sciences and the availability of sophisticated techniques, the choice and establishment of an experimental model system is essential for a successful research project. Currently, various sophisticated in vitro and in vivo models are being designed and developed in order to replace the use of mammalian models to investigate the mechanisms of action, activity and properties of novel compounds or treatment modalities. After the clinical success of photodynamic therapy (PDT) used against neovascular eye disorders, clinically approved PDT protocols for cancer are still being developed. Since several aspects of PDT should be examined, it is crucial to define the possible models that would guide the principle of the 3Rs (Reduction, Refinement, Replacement) practice, which is known as the fundamental strategy for designing more ethical animal studies. This review is focused on the usefulness of alternative in vivo as well as in vitro models to study important aspects of PDT, especially in the context of cancer research. 3D human-relevant cell culture models, followed by non-mammalian models such as the nematode (Caenorhabditis elegans), fly (Drosophila melanogaster), zebrafish (Danio rerio), frog (Xenopus laevis), or chicken chorioallantoic membrane (Gallus gallus), are discussed as an alternative to the widely used but ethically controversial mammalian models.
... These experimental advantages, together with their rapid external development, detailed temporal staging atlas, and relative transparency facilitate gene function assessment, making both Xenopus species versatile model systems for disease research and phenotypic drug screening (Harland and Grainger, 2011;Schmitt et al., 2014). Among aquatic vertebrate model animals, Xenopus excels by having comparable organ development and morphology to mammalian systems, but with the added benefit of being able to regenerate adult tissues, such as optic nerve, lens, spinal cord and limb tissue (Blitz et al., 2006;Muñoz et al., 2015;Slack et al., 2008). Xenopus animals and oocytes are used extensively to understand normal organ function and disease in humans (Labonne and Zorn, 2015), including cardiac congenital heart disorders and heterotaxy (Boskovski et al., 2013;Duncan and Khokha, 2016;Fakhro et al., 2011;Kaltenbrun et al., 2011;Langdon et al., 2012;, gastrointestinal and pancreatic diseases (Kofent and Spagnoli, 2016;Pearl et al., 2009;Salanga and Horb, 2015;Womble et al., 2016), endocrine functions and disorders (Buchholz, 2015), kidney disease (Lienkamp, 2016), lung development (Rankin et al., 2011;2015;Wallmeier et al., 2014), cancer (Chernet and Levin, 2013;Cross and Powers, 2009;Hardwick and Philpott, 2015;Haynes-Gilmore et al., 2014;Van Nieuwenhuysen et al., 2015;Wylie et al., 2015), ciliopathies (Kim et al., 2010;Klos Dehring et al., 2013;Ma et al., 2014), orofacial defects (Dickinson, 2016), and neurodevelopmental disorders (Erdogan et al., 2016;Pratt and Khakhalin, 2013). ...
The amphibian model Xenopus, has been used extensively over the past century to study multiple aspects of cell and developmental biology. Xenopus offers advantages of a non-mammalian system, including high fecundity, external development, and simple housing requirements, with additional advantages of large embryos, highly conserved developmental processes, and close evolutionary relationship to higher vertebrates. There are two main species of Xenopus used in biomedical research, Xenopus laevis and Xenopus tropicalis; the common perception is that both species are excellent models for embryological and cell biological studies, but only Xenopus tropicalis is useful as a genetic model. The recent completion of the Xenopus laevis genome sequence combined with implementation of genome editing tools, such as TALENs (transcription activator-like effector nucleases) and CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated nucleases), greatly facilitates the use of both Xenopus laevis and Xenopus tropicalis for understanding gene function in development and disease. In this paper, we review recent advances made in Xenopus laevis and Xenopus tropicalis with TALENs and CRISPR-Cas and discuss the various approaches that have been used to generate knockout and knock-in animals in both species. These advances show that both Xenopus species are useful for genetic approaches and in particular counters the notion that Xenopus laevis is not amenable to genetic manipulations.
... As highlighted throughout this chapter, the use of animal models is critical for identifying the roles of RBPs in vivo. Zebrafish and Xenopus are both popular choices for investigating RBPs in the developing heart due to their rapid development and ease of genetic manipulation (Blitz et al., 2006;Staudt and Stainier, 2012). Neither has a four-chambered heart, however, and both carry additional paralogs of some RBPs that are found in mammals. ...
The regulation of gene expression during development takes place both at the transcriptional and posttranscriptional levels. RNA-binding proteins (RBPs) regulate pre-mRNA processing, mRNA localization, stability, and translation. Many RBPs are expressed in the heart and have been implicated in heart development, function, or disease. This chapter will review the current knowledge about RBPs in the developing heart, focusing on those that regulate posttranscriptional gene expression. The involvement of RBPs at each stage of heart development will be considered in turn, including the establishment of specific cardiac cell types and formation of the primitive heart tube, cardiac morphogenesis, and postnatal maturation and aging. The contributions of RBPs to cardiac birth defects and heart disease will also be considered in these contexts. Finally, the interplay between RBPs and other regulatory factors in the developing heart, such as transcription factors and miRNAs, will be discussed.
... Endocrine cells are initially specified in the dorsal pancreas to make hormones such as Insulin, while the ventral pancreas produces mainly exocrine cells and digestive enzymes. As development proceeds, the dorsal and ventral buds fuse together to form one organ [4,[43][44][45], with endocrine and exocrine cells distributed throughout the fused structure. At somite stages, the development of the dorsal (endocrine) pancreas is regulated by signaling molecules secreted from neighboring tissues (e.g., the notochord), including TGF-β and FGF signals, which repress sonic hedgehog (shh) expression in the dorsal foregut endoderm, a prerequisite for pancreatic fate [46]. ...
The digestive system comprises numerous cells, tissues and organs that are essential for the proper assimilation of nutrients and energy. Many aspects of digestive organ function are highly conserved among vertebrates, yet the final anatomical configuration of the gut varies widely between species, especially those with different diets. Improved understanding of the complex molecular and cellular events that orchestrate digestive organ development is pertinent to many areas of biology and medicine, including the regeneration or replacement of diseased organs, the etiology of digestive organ birth defects, and the evolution of specialized features of digestive anatomy. In this review, we highlight specific examples of how investigations using Xenopus laevis frog embryos have revealed insight into the molecular and cellular dynamics of digestive organ patterning and morphogenesis that would have been difficult to obtain in other animal models. Additionally, we discuss recent studies of gut development in non-model frog species with unique feeding strategies, such as Lepidobatrachus laevis and Eleutherodactylous coqui, which are beginning to provide glimpses of the evolutionary mechanisms that may generate morphological variation in the digestive tract. The unparalleled experimental versatility of frog embryos make them excellent, integrative models for studying digestive organ development across multiple disciplines.
... A Tg system for urodele amphibians subsequently became available (Ueda et al. 2005;Sobkow et al. 2006;Casco-Robles et al. 2011;Hayashi et al. 2013). Several approaches have been tried for reproducible analyses of gene function with transgenesis (e.g, Cre-lox, FLP-FRT, heat shock promoter, tet regulatable promoter and GAL4-UAS; reviewed in Blitz et al. 2006). For investigation of a specific gene function in regeneration using transgenesis, we often have difficulty in obtaining larvae or adults because the gene of interest functionally interferes with normal development in transgenic animals. ...
Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single-cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR-LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration.
... Many of these gastrointestinal disorders are congenital defects that arise as a result of aberrant development. The precise molecular mechanisms underlying the onset or progression of gastrointestinal disorders is of great importance for the development of therapies and early diagnosis, and the amphibian Xenopus offers numerous advantages for elucidating the molecular signals controlling how the GI tract and associated organs are specified during embryogenesis [2]. ...
Diseases affecting endodermal organs like the pancreas, lung, and gastrointestinal tract have a substantial impact on human welfare. Since many of these are congenital defects that arise as a result of defects during development, broad efforts are focused on understanding the development of these organs so as to better identify risk factors, disease mechanisms, and therapeutic targets. Studies implementing model systems, like the amphibian Xenopus, have contributed immensely to our understanding of signaling pathways (e.g., Wnt, FGF, BMP, RA) and gene regulation (e.g., hhex, ptf1a, ngn3) that underlie normal development as well as disease progression. Recent advances in genome engineering further enhance the capabilities of the Xenopus model system for pursuing biomedical research, and will undoubtedly result in a boom of new information underlying disease mechanisms ultimately leading to advancements in diagnosis and therapy.
... Importantly, we used these animals at pre-metamorphic stages with most of the organs and neuronal structures being developed and functional. These animals are capable of performing complex behavior trials and show learning abilities and social interactions [21,22,26,27,42,43]. Such features are mostly not established in embryos thus, favoring the tadpole model for experiments estimating effects on human health. ...
Xenopus tadpoles are an emerging model for developmental, genetic and behavioral studies. A small size, optical accessibility of most of their organs, together with a close genetic and structural relationship to humans make them a convenient experimental model. However, there is only a limited toolset available to measure behavior and organ function of these animals at medium or high-throughput. Herein, we describe an imaging-based platform to quantify body and autonomic movements of Xenopus tropicalis tadpoles of advanced developmental stages. Animals alternate periods of quiescence and locomotor movements and display buccal pumping for oxygen uptake from water and rhythmic cardiac movements. We imaged up to 24 animals in parallel and automatically tracked and quantified their movements by using image analysis software. Animal trajectories, moved distances, activity time, buccal pumping rates and heart beat rates were calculated and used to characterize the effects of test compounds. We evaluated the effects of propranolol and atropine, observing a dose-dependent bradycardia and tachycardia, respectively. This imaging and analysis platform is a simple, cost-effective high-throughput in vivo assay system for genetic, toxicological or pharmacological characterizations.
... In addition to mammals, non-amniote vertebrates such as amphibians (Xenopus laevis and tropicalis) and teleosts (Danio rerio and Oryzas latipes) offer specific experimental advantages and have demonstrated their usefulness for biomedical research (Bedell et al. 2012;Blitz et al. 2006;Harland and Grainger 2011;Hellsten et al. 2010;Lieschke and Currie 2007;Wheeler and Brandli 2009). This is because gene expression is broadly conserved between homologous tissues of humans and other vertebrates (Chan et al. 2009). ...
Osteogenesis is the fundamental process by which bones are formed, maintained and regenerated. The osteoblasts deposit the bone mineralized matrix by secreting large amounts of extracellular proteins and by allowing the biochemical conditions for the nucleation of hydroxyapatite crystals. Normal bone formation requires a tight control of osteoblastic activity, and therefore, osteoblasts represent a major focus of interest in biomedical research. Several crucial features of osteogenesis can be readily recapitulated using murine, avian and fish primary and immortalized osteoblastic cultures. Here, we describe a novel and straightforward in vitro culture of primary osteoblasts from the amphibian Xenopus tropicalis, a major vertebrate model organism. X. tropicalis osteoblasts can readily be extracted from the frontoparietal bone of pre-metamorphosing tadpole skulls by series of gentle protease treatments. Such primary cultures efficiently proliferate and can conveniently be grown at room temperature, in the absence of CO2, on a variety of substrates. X. tropicalis primary osteoblasts express well-characterized genes known to be active during osteogenesis of teleost fish, chick, mouse and human. Upon differentiation, such cultures mineralize and activate DMP1, an osteocyte-specific gene. Importantly, X. tropicalis primary osteoblasts can be efficiently transfected and respond to the forced activation of the bone morphogenetic protein pathway by increasing their nuclear levels of phospho-Smad. Therefore, this novel primary culture is amenable to experimental manipulations and represents a valuable tool for improving our understanding of the complex network of molecular interactions that govern vertebrate bone formation.
... Recently the frog Xenopus has emerged as an alternative model to investigate early respiratory development Shifley et al., 2012;Wang et al., 2011;Yin et al., 2010). The experimental advantages of the abundant and large externally developing Xenopus embryos make them well suited to study complex signaling pathways (Blitz et al., 2006;Harland and Grainger, 2011) and are therefore also a promising model to examine the gene regulatory networks governing early respiratory system development. However the extent to which respiratory system development is conserved between Xenopus and mammals has not yet been systematically examined. ...
Background:
Respiratory system development is regulated by a complex series of endoderm-mesoderm interactions that are not fully understood. Recently Xenopus has emerged as an alternative model to investigate early respiratory system development, but the extent to which the morphogenesis and molecular pathways involved are conserved between Xenopus and mammals has not been systematically documented.
Results:
In this study, we provide a histological and molecular atlas of Xenopus respiratory system development, focusing on Nkx2.1+ respiratory cell fate specification in the developing foregut. We document the expression patterns of Wnt/β-catenin, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling components in the foregut and show that the molecular mechanisms of respiratory lineage induction are remarkably conserved between Xenopus and mice. Finally, using several functional experiments we refine the epistatic relationships among FGF, Wnt, and BMP signaling in early Xenopus respiratory system development.
Conclusions:
We demonstrate that Xenopus trachea and lung development, before metamorphosis, is comparable at the cellular and molecular levels to embryonic stages of mouse respiratory system development between embryonic days 8.5 and 10.5. This molecular atlas provides a fundamental starting point for further studies using Xenopus as a model to define the conserved genetic programs controlling early respiratory system development.
... These two genes are initially expressed in a punctate pattern within the stomach and duodenum at NF41, and are only detected in the pancreas beginning at NF44/45. The rapidity of pancreas development in Xenopus, coupled with its embryological and molecular benefits, makes it a useful system for functional studies on pancreas development (Blitz et al., 2006;Harland and Grainger, 2011;Pearl et al., 2012). ...
Pancreas-specific transcription factor 1a (Ptf1a), a bHLH transcription factor, has two temporally distinct functions during pancreas development; initially it is required for early specification of the entire pancreas, while later it is required for proper differentiation and maintenance of only acinar cells. The importance of Ptf1a function was revealed by the fact that loss of Ptf1a leads to pancreas agenesis in humans. While Ptf1a is one of the most important pancreatic transcription factors, little is known about the differences between the regulatory networks it controls during initial specification of the pancreas as opposed to acinar cell development, and to date no comprehensive analysis of its downstream targets has been published. In this article, we use Xenopus embryos to identify putative downstream targets of Ptf1a. We isolated anterior endoderm tissue overexpressing Ptf1a at two early stages, NF32 and NF36, and compared their gene expression profiles using microarrays. Our results revealed that Ptf1a regulates genes with a wide variety of functions, providing insight into the complexity of the regulatory network required for pancreas specification. genesis 1-18, 2012. © 2012 Wiley Periodicals, Inc.
... The frog Xenopus has been a valuable model for the elucidation of gene function during early vertebrate development due to the combined use of the cell lineage fate map, cell microinjection, and embryo manipulation (Warkman and Krieg, 2007). Study of later embryonic stages is important for understanding organ development and metamorphic tissue remodeling (corresponding to events that occur during the fetal period in mammals), which may then help elucidate the developmental orgins of adult diseases (Blitz et al, 2006). However, inducible methods for altering gene expression in later development are limited in frogs compared to other model organisms. ...
We have characterized two new transgenic Xenopus lines enabling transgene expression using the Tet-On inducible system. An inducer line expresses the doxycycline- (Dox-) activated transcription factor rtTA under control of the ubiquitous promoter CMV. A responder line enables Dox-inducible expression of a dominant positive thyroid hormone receptor via a tetracycline responsive transgenic promoter (TRE). Dox-induced expression of transgenic GFP mRNA was detectable after 3 hr and increased up to 10- to 50-fold by 2 days depending on dose of Dox. Induced GFP mRNA expression returned to uninduced levels within 3 days upon Dox removal. Treatment of rtTA inducer and TRE responder double transgenic animals with Dox caused acceleration of metamorphic changes in thyroid hormone-response gene expression and morphology. These transgenic lines will be made available through the new Xenopus Stock Center and will serve as valuable tools for genetic analysis of development and metamorphosis.
... One classical approach adopted by experimental embryologists is to use excised tissue fragments, or explants, microsurgically removed from Xenopus embryos to study localized developmental processes [14]. These classical approaches have been complemented by more modern tools to visualize cells and analyze gene and protein expression [15][16][17]. ...
Embryonic development is guided by a complex and integrated set of stimuli that results in collective system-wide organization that is both time and space regulated. These regulatory interactions result in the emergence of highly functional units, which are correlated to frequency-modulated stimulation profiles. We have determined the dynamic response of vertebrate embryonic tissues to highly controlled, time-varying localized chemical stimulation using a microfluidic system with feedback control. Our approach has enabled localized spatiotemporal manipulation of the steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. Using this approach we investigated cell-scale responses to precisely controlled stimulation by tracking the redistribution of a GFP-tagged DEX-reporter constructed from the human glucocorticoid receptor (GR). We exposed defined regions of a single AC explant to different stimulation conditions--continuous stimulation, periodic stimulation, and no stimulation. We observed collective behavior of the GR transport into the nucleus was first-order. Furthermore, the dynamic response was well-modeled by a first-order differential equation with a single time derivative. The model predicted that responses to periodic stimulations closely matched the results of the frequency-based experiments. We find that stimulation with localized bursts versus continuous stimulation can result in highly distinct responses. This finding is critical as controlled space and time exposure to growth factors is a hallmark of complex processes in embryonic development. These complex responses to cellular signaling and transport machinery were similar to emergent behaviors in other complex systems, suggesting that even within a complex embryonic tissue, the overall system can converge toward a predictive first-order response.
... Moreover, while the term may not necessarily apply in the mammalian heart, it is certainly not applicable in the fishes. The term bulbus cordis has been used to describe the smooth muscle structure (the bulbus arteriosus) cranial to the teleost heart (e.g., Ekstro¨m and Korf 1986;Zhang et al. 2001) and the myocardial outflow of the lungfishes (e.g., Burggren and Johansen 1986) and of amphibians such as Xenopus (e.g., Horb and Thomsen 1999;Blitz et al. 2006). In fish embryogenesis, however, there is no myocardial structure that ever gives rise to a right ventricle, as the early fish heart tube gives rise to a single ventricle, and no other myocardial bulb-like structure is observed. ...
In chick and mouse embryogenesis, a population of cells described as the secondary heart field (SHF) adds both myocardium and smooth muscle to the developing cardiac outflow tract (OFT). Following this addition, at approximately HH stage 22 in chick embryos, for example, the SHF can be identified architecturally by an overlapping seam at the arterial pole, where beating myocardium forms a junction with the smooth muscle of the arterial system. Previously, using either immunohistochemistry or nitric oxide indicators such as diaminofluorescein 2-diacetate, we have shown that a similar overlapping architecture also exists in the arterial pole of zebrafish and some shark species. However, although recent work suggests that development of the zebrafish OFT may also proceed by addition of a SHF-like population of cells, the presence of a true SHF in zebrafish and in many other developmental biological models remains an open question. We performed a comprehensive morphological study of the OFT of a wide range of vertebrates. Our data suggest that all vertebrates possess three fundamental OFT components: a proximal myocardial component, a distal smooth muscle component, and a middle component that contains overlapping myocardium and smooth muscle surrounding and supporting the outflow valves. Because the middle OFT component of avians and mammals is derived from the SHF, our observations suggest that a SHF may be an evolutionarily conserved theme in vertebrate embryogenesis.
... In Xenopus, the endoderm is derived from the vegetal hemisphere, and much is now understood about how the endodermal germ layer is formed and how regional specification of the endoderm is regulated (Chen et al., 2003; Chen et al., 2004; Horb, 2000; Horb and Slack, 2001; Li et al., 2008; McLin et al., 2007; Pan et al., 2007; Pearl et al., 2009). Yet the relationship between specification, differentiation and proliferation of the various endodermal organs at later stages is unclear (Blitz et al., 2006). Although much is known about cell cycle regulation during early Xenopus development, how differentiation is coupled to the cell cycle at tail bud and tadpole stages is unknown (Philpott and Yew, 2008). ...
Developmental control of proliferation relies on tight regulation of protein expression. Although this has been well studied in early embryogenesis, how the cell cycle is regulated during organogenesis is not well understood. Bruno-Like RNA binding proteins bind to consensus sequences in the 3'UTR of specific mRNAs and repress protein translation, but much of this functional information is derived from studies on mainly two members, Drosophila Bruno and vertebrate BrunoL2 (CUGBP1). There are however, six vertebrate and three Drosophila Bruno family members, but less is known about these other family members, and none have been shown to function in the endoderm. We recently identified BrunoL1 as a dorsal pancreas enriched gene, and in this paper we define BrunoL1 function in Xenopus endoderm development. We find that, in contrast to other Bruno-Like proteins, BrunoL1 acts to enhance rather than repress translation. We demonstrate that BrunoL1 regulates proliferation of endoderm cells through translational control of cyclin A2 mRNA. Specifically BrunoL1 enhanced translation of cyclin A2 through binding consensus Bruno Response Elements (BREs) in its 3'UTR. We compared the ability of other Bruno-Like proteins, both vertebrate and invertebrate, to stimulate translation via the cyclin A2 3'UTR and found that only Drosophila Bru-3 had similar activity. In addition, we also found that both BrunoL1 and Bru-3 enhanced translation of mRNAs containing the 3'UTRs of Drosophila oskar or cyclin A, which have been well characterized to mediate repression. Lastly, we show that it is the Linker region of BrunoL1 that is both necessary and sufficient for this activity. These results are the first example of BRE-dependent translational enhancement and are the first demonstration in vertebrates of Bruno-Like proteins regulating translation through BREs.
... Therefore, to assess the functional conservation of hydra Noggin, ectopic expression of hydra noggin mRNA in a heterologous system was studied. X. laevis embryos have been used extensively to investigate such events in early embryogenesis due to the ease (Beck and Slack 2001;Blitz et al. 2006) with which such experiments can be carried out and the outcomes interpreted. Interestingly, when hydra noggin mRNA was injected in early Xenopus embryos, a secondary axis was induced in all of the injected embryos (Fig. 4B). ...
Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin-like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)-signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV-induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.
... Despite ongoing use of Xenopus as a model system of organogenesis (Thomsen, 2006) and the quickly growing field of skeletal developmental biology (Karsenty, 2003;Zelzer and Olsen, 2003;Hall, 2005;Goldring et al., 2006) few studies research skeletal development in Xenopus. This lack of attention is due largely to the limited array of techniques available for investigations of later development in Xenopus (Blitz et al., 2005). Functional experiments through the injection of morpholino oligonucleotides, antisense RNA, or RNA interference are limited to early stages (Nutt et al., 2001;Khokha et al., 2002). ...
This study characterizes regulatory elements of collagen 2 alpha 1 (col2a1) in Xenopus that enable transgene expression in cartilage-forming chondrocytes. The reporters described in this study drive strong cartilage-specific gene expression, which will be a valuable tool for further investigations of Xenopus skeletal development. While endogenous col2a1 mRNA is expressed in many embryonic tissues, its expression becomes restricted to tadpole and adult chondrocytes. This chondrocyte-specific expression is recapitulated by col2a1 reporter constructs, which were tested through I-SceI meganuclease-mediated transgenesis. These constructs contain a portion of the Xenopus tropicalis col2a1 intron, which aligns to a cartilage-specific intronic enhancer that has been well characterized in mammals. Two overlapping regions of the first intron that are 1.5-Kb and 665-bp long, both of which contain this enhancer sequence, drove EGFP expression in both larval and adult chondrocytes when connected to an upstream promoter. However, neither a truncated 155-bp region that also contains the enhancer, nor a separate 347-bp intronic region that lacks it, was able to drive cartilaginous transgene expression. The two cartilage-specific transgenes are heritable in F1 progeny, which exhibit none of the background expression observed in the injected founders. This study is the first to use the I-SceI technique to characterize an enhancer element in Xenopus, and the first to generate chondrocyte-specific gene expression in a non-mammalian vertebrate. The creation of novel cartilage-specific gene expression provides a new tool for further studies of anuran skeletal development.
... To follow the fate of dorsal and ventral pancreatic bud-derived cells, we took advantage of the embryological benefits of Xenopus (Blitz et al., 2006;Pearl and Horb, 2008) and created chimeric transgenic Elas-GFP/wild-type embryos. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion of the two buds during normal development, whereas the dorsal pancreas-derived cells do not. ...
During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each region to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild-type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas-derived cells. To identify ventral pancreas-specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from NF38/39 Xenopus laevis tadpoles and compared their gene expression profiles (NF refers to the specific stage of Xenopus development). As a result of this screen, we have identified several new ventral pancreas-specific genes, all of which are expressed in the same location within the ventral pancreas at the junction where the two ventral pancreatic buds fuse. Morpholino-mediated knockdown of one of these ventral-specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion, as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.
... Several model organisms have been informative in pancreatic developmental studies. Mouse and chick are the traditional models, while recently Xenopus and zebrafish have also been utilized (7)(8)(9)(10)(11)(12)(13). The two aquatic species, Xenopus and zebrafish, are cheaper to work with, grow faster than mice, and have a shorter generation time allowing experiments with higher throughput. ...
Diabetes is a disease that could be treated more effectively with a better understanding of pancreas development. This review examines the role of master regulator genes driving crucial steps in pancreas development, from foregut specification to differentiation of the five endocrine cell types. The roles of Pdx1, Ptf1a, and Ngn3 are particularly examined as they are both necessary and sufficient for promoting pancreatic cell fates (Pdx1, Ptf1a) and endocrine cell development (Ngn3). The roles of Arx and Pax4 are studied as they compose part of the regulatory mechanism balancing development of different types of endocrine cells within the iselts and promote the development of alpha/PP and beta/delta cell progenitors, respectively. The roles of the aforementioned genes, and the consequences of misexpression of them for functionality of the pancreas, are examined through recent studies in model organisms, particularly Xenopus and zebrafish. Recent developments in cell replacement therapy research are also covered, concentrating on stem cell research (coaxing both adult and embryonic stem cells toward a beta cell fate) and transdifferentiation (generating beta cells from other differentiated cell types).
... They used the elastase promoter-GFP construct, which was also used in our study, as a real time marker of the transdifferentiation process. Various transcription factors, eg, Ptf1a/p48 or ESR10, can now be expressed ectopically under the control of a tissue-specific promoter in an effort to examine the potential of these transcription factors to induce ectopic pancreas (Blitz et al., 2005). ...
Several experimental approaches have been described to generate transgenic frogs. Here, we report on the application of a novel method in Xenopus, making use of I-SceI meganuclease. The characteristic feature of this endonuclease is that it has an extended recognition site of 18 bp, which is expected to exist only once in 7 x 10(10) bp of random DNA sequences. Various reporter constructs flanked by two I-SceI recognition sites were injected together with the I-SceI meganuclease into one-cell stage Xenopus embryos. We observed an overall transgenesis frequency of 10% or more under optimized condition. The injected genes were integrated into the genome and transmitted to F1 offspring. Southern blot analysis showed that between one and eight copies of the transgene were integrated. Meganuclease-aided transgenesis, thus, provides a simple and highly efficient tool for transgenesis in Xenopus.
... Much of the studies in Xenopus have focused on the early inductive events of cardiogenesis. 35 Our work clearly indicates that Xenopus provides an efficient model for in vivo analysis of the consequences of loss-of-function of cardiac regulators. In this respect, it is interesting to note the consistency of loss-of-function phenotypes observed in Xenopus, rodents and human (Table 1). ...
Unlike other organs, the adult heart has limited regenerative potential owing to the inability of postnatal cardiomyocytes to undergo proliferative growth. As a result, ischemic heart disease continues to be a major cause of morbidity and mortality worldwide. Elucidating the molecular pathways of cardiomyocyte differentiation and proliferation holds great promise for human health. In a recent paper we employed a multidisciplinary approach to identify a novel pathway required for cardiomyocyte growth and differentiation. Starting with the dissection of a new regulatory sequence required for cardiac specific expression, we identified the cognate DNA binding protein as KLF13, a tissue-restricted member of the newly identified KLF family of zinc-finger proteins. We took advantage of the ease in manipulating Xenopus embryos to genetically alter KLF13 levels thus demonstrating a requirement for KLF13 in cardiac progenitor cell proliferation and heart morphogenesis. Furthermore, we combined biochemical approaches with genetic manipulations in Xenopus to show that KLF13 is a GATA4 interacting protein and a genetic modifier of GATA4 function. Cyclin D1 was identified as a direct transcriptional target for KLF13 that may account for the proliferation defects observed in embryos with downregulated KLF13 levels. Thus, tissue-specific regulators of the cell cycle may be potential congenital heart disease causing genes in humans.
... Technology is now firmly in place to explore the developmental and genetic mechanisms central to the divergence of anurans and the subsequent maintenance of their Bauplan (for detailed discussion of recent advances in Xenopus technology see Carruthers & Stemple, 2006;Blitz, Andelfinger & Horb, 2006). Xenopus species are readily amenable to genetic manipulation by mRNA and morpholino injection as well as transgenesis (for elegant examples of each technique in use see : Blitz, Cho & Chang, 2003;Horb et al., 2003). ...
Anurans (frogs, toads, and their larvae) are among the most morphologically derived of vertebrates. While tightly conserved across the order, the anuran Bauplan (body plan) diverges widely from that of other vertebrates, particularly with respect to the skeleton. Here we address the adaptive, ontogenetic, and genetic bases of three such hallmark anuran features: (1) the absence of discrete caudal vertebrae, (2) a truncated axial skeleton, and (3) elongate hind limbs. We review the functional significance of each as it relates to the anuran lifestyle, which includes locomotor adaptations to both aquatic and terrestrial environments. We then shift our focus to the proximal origins of each feature, namely, ontogeny and its molecular regulation. Drawing on relatively limited data, we detail the development of each character and then, by extrapolating from comparative vertebrate data, propose molecular bases for these processes. Cast in this light, the divergent morphology of anurans emerges as a product of evolutionary modulation of the generalised vertebrate developmental machinery. Specifically, we hypothesise that: (1) the formation of caudal vertebrae is precluded due to a failure of sclerotomes to form cartilaginous condensations, perhaps resulting from altered expression of a suite of genes, including Pax1, Pax9, Msx1, Uncx-4.1, Sonic hedgehog, and noggin; (2) anteriorised Hox gene expression in the paraxial mesoderm has led to a rostral shift of morphological boundaries of the vertebral column; and, (3) spatial and temporal shifts in Hox expression may underlie the expanded tarsal elements of the anuran hind limb. Technology is currently in place to investigate each of these scenarios in the African clawed frog Xenopus. Experimental corroboration will further our understanding of the molecular regulation of the anuran Bauplan and provide insight into the origin of vertebrate morphological diversity as well as the role of development in evolution.
... Thus, with the current explosion of interest in evolutionary developmental physiology (Warburton et al. 2006), the breadth and depth of developmental information and techniques available for amphibians makes them the obvious choice with which to study the physiology of developing organs and organ systems. Xenopus is currently the focus of intense sequencing efforts () and in situ expression studies in organogenesis (Blitz et al. 2006 ). Unfortunately, physiological studies have not similarly focused on Xenopus. ...
The concept of animal models is well honored, and amphibians have played a prominent part in the success of using key species to discover new information about all animals. As animal models, amphibians offer several advantages that include a well-understood basic physiology, a taxonomic diversity well suited to comparative studies, tolerance to temperature and oxygen variation, and a greater similarity to humans than many other currently popular animal models. Amphibians now account for approximately 1/4 to 1/3 of lower vertebrate and invertebrate research, and this proportion is especially true in physiological research, as evident from the high profile of amphibians as animal models in Nobel Prize research. Currently, amphibians play prominent roles in research in the physiology of musculoskeletal, cardiovascular, renal, respiratory, reproductive, and sensory systems. Amphibians are also used extensively in physiological studies aimed at generating new insights in evolutionary biology, especially in the investigation of the evolution of air breathing and terrestriality. Environmental physiology also utilizes amphibians, ranging from studies of cryoprotectants for tissue preservation to physiological reactions to hypergravity and space exploration. Amphibians are also playing a key role in studies of environmental endocrine disruptors that are having disproportionately large effects on amphibian populations and where specific species can serve as sentinel species for environmental pollution. Finally, amphibian genera such as Xenopus, a genus relatively well understood metabolically and physiologically, will continue to contribute increasingly in this new era of systems biology and "X-omics."
... Since it has been shown that the ontogenic decline of regenerative capacity is due to intrinsic change in the Xenopus limb bud itself (Sessions & Bryant 1988), the Xenopus provides an excellent model to investigate essential differences between regenerative limbs and non-regenerative ones within the same animal. Furthermore, there are several technical advantages in Xenopus, such as highly efficient generation of transgenic individuals and availability of information on the entire genome due to the completion of a genome project in Xenopus tropicalis (Blitz et al. 2006). ...
While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.
X-box-binding protein 1 (XBP1) is a protein containing the basic leucine zipper structure. It belongs to the cAMP-response element binding protein (CREB)/activating transcription factor transcription factor family. As the main transcription factor, spliced XBP1 (XBP1s) participates in many physiological and pathological processes and plays an important role in embryonic development. Previous studies showed that XBP1-knockout mice died because of pancreatic exocrine function deficiency, indicating that XBP1 plays an important role in pancreatic development. However, the exact role of XBP1 in pancreatic development remains unclear. This study aimed to investigate the role of XBP1 in the pancreatic development of Xenopus laevis embryos. Whole-mount in situ hybridization and quantitative real-time PCR (qRT-PCR) results revealed that the expression levels of pancreatic progenitor marker genes pdx1, p48, ngn3, and sox9 were downregulated in XBP1s morpholino oligonucleotide (MO)-injected embryos. The expression levels of pancreatic exocrine and endocrine marker genes insulin and amylase were also downregulated. Through the overexpression of XBP1s, the phenotype and gene expressions were opposite to those in XBP1s MO-injected embryos. Luciferase and chromatin immunoprecipitation assays showed that XBP1s could bind to the XBP1-binding site in the foxa2 promoter. These results revealed that XBP1 is required in the pancreatic development of Xenopus laevis and might function by regulating foxa2.
Purpose of review:
Given the enormous impact congenital heart disease has on child health, it is imperative that we improve our understanding of the disease mechanisms that underlie patient phenotypes and clinical outcomes. This review will outline the merits of using the frog model, Xenopus, as a tool to study human cardiac development and left-right patterning mechanisms associated with congenital heart disease.
Recent findings:
Patient-driven gene discovery continues to provide new insight into the mechanisms of congenital heart disease, and by extension, patient phenotypes and outcomes. By identifying gene variants in CHD patients, studies in Xenopus have elucidated the molecular mechanisms of how these candidate genes affect cardiac development, both cardiogenesis as well as left-right patterning, which can have a major impact on cardiac morphogenesis. Xenopus has also proved to be a useful screening tool for the biological relevance of identified patient-mutations, and ongoing investigations continue to illuminate disease mechanisms.
Summary:
Analyses in model organisms can help to elucidate the disease mechanisms underlying CHD patient phenotypes. Using Xenopus to disentangle the genotype-phenotype relationships of well-known and novel disease genes could enhance the ability of physicians to efficaciously treat patients and predict clinical outcomes, ultimately improving quality of life and survival rates of patients born with congenital heart disease.
Extracellular adenosine belongs to the purinergic signalling pathway and regulatesvarious physiological processes through activation of specific receptors named adora. Theextracellular concentration of adenosine is regulated by several ecto-enzymes involved eitherin its generation or in its degradation but also by nucleoside transporters enabling its exitoutside or entry inside the cell. In adults, the functions of adenosine are quite well known,however, the its involvement during embryogenesis remains poorly studied. An excess ofadenosine in early phases of development is lethal in mouse and sea urchins, demonstratingthe importance of the extracellular adenosine level regulation during embryogenesis. The aimof my phD is to understand the role of adenosine during embryogenesis using Xenopus as avertebrate model. Indeed, the first in vivo evidence of the implication of the purinergic signallingpathway during vertebrate development, and in particular of ADP during eye formation hasbeen demonstrated using this model. The first part of this project was to characterize all theadenosine signalling pathway actors in Xenopus in order to generate the first comprehensiveand comparative embryonic expression map of these genes. This work allowed me to selectthe alkaline phosphatase alpl for functional studies based on its specific expression profile, inthe retina and kidney. These functional studies, mostly carried out by knockdown experiments,constituted the second part of this phD and showed the implication of this enzyme during theeye and kidney development.
The Xenopus embryonic kidney, the pronephros, serves as a useful model for the study of nephron development as well as tubulogenesis in general. The history of embryological studies on pronephric development through transplantation, explant, and animal cap experiments provides a valuable body of knowledge on which to base current studies. Continued investigation of the molecular underpinnings of nephric formation and the sustained development of innovative techniques complement the fundamental embryological advantages of this system. Because the Xenopus pronephros is composed of a single functional nephron that lies just beneath the ectoderm through all stages of development, it can be easily manipulated via microdissection techniques and visualized both in living embryos and in fixed tissue. The ability to target molecular alterations specifically to the pronephric anlagen through the application of kidney-specific transgenic promoters along with cell fate-based microinjections of mRNAs or morpholinos provides the ability to manipulate gene expression in a spatially restricted manner. Recently, light-inducible morpholinos have provided a means to temporally regulate gene expression in Xenopus, as have transgenic promoters. Application of targeted nuclease technologies should also soon provide the ability to manipulate amphibian models at the level of genomic DNA. Because the Xenopus pronephros progresses into a functional nephron in 3–4 days within an embryo that develops outside the mother, it provides unique advantages for observations of nephrogenesis. Additionally, recent work suggests that the Xenopus kidney will facilitate studies on nephron regeneration. In this chapter, we will review key studies in Xenopus pronephric development and highlight current innovations that will accelerate our understanding of kidney formation and tubulogenesis in the near future.
The E3 ubiquitin ligase CRL4Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell
nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits
CRL4Cdt2 while binding to PCNA. In vertebrates, CRL4Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that
CRL4Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains
a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and
PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4Cdt2. Our results expand the group of vertebrate CRL4Cdt2 substrates to include a bona fide DNA repair enzyme.
The genome of Xenopus laevis codes for two genes of peroxiredoxin 6, i.e., xen1 (Acc. no. EMBL Data Bank-BCO54278) and xen2 (Acc. no. EMBL Data Bank-BCO54309). Both the genes were cloned and expressed in Escherichia coli. The amino acid sequences of Xen1 and Xen2 enzymes are identical by 95%, and they possess the same peroxidase activity as well as similar optimums of temperature, pH, and thermostability. The genes of peroxiredoxin 6 of Xenopus laevis considerably differ in the period of expression during ontogenesis; i.e., xen2 is expressed during every stage of development, somewhat more intensively after stages 0–5; the expression of xen1 is initiated later, i.e., during the developmental stages of 47–48 h. Expression of xen2 increases after the incubation of embryos in a medium with hydrogen peroxide. Comparison of the amino acid sequences of Xen1 and Xen2 proteins shows that only Xen2 can possess phospholipase activity because its amino acid sequence contain residues of the phospholipase A2 active center: Ser31, His25, and Asp139.
There are diseases and injuries in which a patient's cells or tissues are destroyed that can only be adequately corrected by tissue or organ transplants. Stem cells may be able to generate new tissue and even cure diseases for which there is no adequate therapy. Type 1 diabetes (T1DM), an insulin-dependent diabetes, is a chronic disease affecting genetically predisposed individuals, in which insulin-secreting beta (β)-cells within pancreatic islets of Langerhans are selectively and irreversibly destroyed by autoimmune assault. Type 2 diabetes (T2DM) is characterized by a gradual decrease in insulin sensitivity in peripheral tissues and the liver (insulin resistance), followed by a gradual decline in β-cell function and insulin secretion. Successful replacing of damaged β-cells has shown considerable potential in treating T1DM, but lack of adequate donors is a barrier. The literature suggests that embryonic and adult stem cells are promising alternatives in long-term treatment of diabetes. However, any successful strategy should address both the need for β-cell replacement and controlling the autoimmune response to cells that express insulin. This review summarizes the current knowledge of options and the potential of stem cell transplantation in diabetes treatment.
How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.
Liver function is crucial for maintaining metabolic homeostasis in mammals. Numerous genes must be properly regulated for the liver to develop and perform a variety of activities. Several recent gene-knockout studies in mice have clarified the roles of GATA6, HNF4alpha, and Foxa1/Foxa2 in early stages of liver formation. After the liver forms, transcriptional changes continue to occur; during the perinatal period, certain genes such as alpha-fetoprotein and H19 are silenced, others are activated, and position-dependent (or zonal) regulation is established. Zhx2 was recently identified as one factor involved in postnatal repression of alpha-fetoprotein and other genes. Furthermore, several studies indicate that negative regulation is involved in the zonal control of glutamine synthetase. Finally, exciting new evidence indicates that signaling through the Wnt/beta-catenin pathway is also involved in zonal regulation in the adult liver.
Morphogenesis integrates a wide range of cellular processes into a self-organizing, self-deforming tissue. No single molecular "magic bullet" controls morphogenesis. Wide ranging cellular processes, often without parallels in conventional cell culture systems, work together to generate the architecture and modulate forces that produce and guide shape changes in the embryo. In this review we summarize the early development of the frog Xenopus laevis from a biomechanical perspective. We describe processes operating in the embryo from whole embryo scale, the tissue-scale, to the cellular and extracellular matrix scale. We focus on describing cells, their behaviors and the unique microenvironments they traverse during gastrulation and discuss the role of tissue mechanics in these processes.
The transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1beta gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-binding properties and acts as a gain-of-function mutation with increased transactivation potential in transfection experiments. Expression of this mutated factor in the Xenopus embryo leads to defective development and agenesis of the pronephros, the first kidney form of amphibians. Very similar defects are generated by overexpressing in Xenopus the wild-type HNF1beta, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1beta mutant R137-K161del, which is associated with a reduced number of nephrons with hypertrophy of the remaining ones and which has an impaired DNA binding, shows only a minor effect on pronephros development in Xenopus. Thus, the overexpression of both human mutants has a different effect on renal development in Xenopus, reflecting the variation in renal phenotype seen with these mutations. We conclude that mutations in human HNF1beta can be functionally characterized in Xenopus. Our findings imply that HNF1beta not only is an early marker of kidney development but also is functionally involved in morphogenetic events, and these processes can be investigated in lower vertebrates.
We have isolated and sequenced a cDNA encoding Xenopus laevis pancreatic trypsin, which has approximately 70% amino acid sequence identity to mammalian trypsinogen. Northern blotting analysis shows that the trypsin gene is activated just before the tadpole starts to feed, reaches peak activity in the swimming tadpole (premetamorphosis), and is then repressed during prometamorphosis, attaining its lowest activity at the climax of metamorphosis. The same gene is then activated again in frogs but to a much lower level. The pattern of the changes in trypsin gene expression is followed by at least two other pancreas-specific genes and marks the remodeling of the pancreas of the animal at metamorphosis. Thyroid hormone, which is the causative agent of metamorphosis, can down-regulate trypsin gene expression prematurely.
The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.
The pronephros serves as the embryonic kidney of the lower vertebrates. In this report we describe the development of the pronephric system of Xenopus laevis utilizing scanning electron microscopy and novel monoclonal antibodies that specifically recognize different parts of the pronephros. Antibody 3G8 recognizes the tubules and nephrostomes of the pronephroi only and does not react with the duct. Antibody 4A6 stains only the duct and the nephrostomes. These antibodies thus allow the positive identification of these two intermediate mesoderm derivatives. Both reagents detect antigens expressed some time after the pronephric structures first form and probably represent markers of terminal differentiation. When the tubules and duct first form they are separate structures that can easily be distinguished; the connective tubules have a distinctive organization, the collecting (or common) tubule is broader than other tubules, and the narrow pronephric duct has a specific shape and position. In later stages the collecting tubule and the rostral portion of the duct undergo a considerable amount of convolution, and both contribute to the final coiled tubular body of the pronephros. The ability of 3G8 and 4A6 to distinguish these two elements of the nephric system was used to reexplore classical experiments on the interaction between these two structures during development of the pronephric system. The use of whole-mount analysis has allowed us to examine large numbers of embryos from different stages and dissected in a variety of planes. These experiments demonstrate the dynamic nature of the intermediate mesoderm and indicate that although the pronephros may be specified by mid-neurula stages, patterning is not complete until tailbud stages.
Heart induction in Xenopus has been thought to be dependent primarily on the interaction of the heart primordia with the Spemann organizer. We demonstrate, however, that signals derived from the deep dorsoanterior endoderm during early gastrulation are also essential for heart formation. The presence of deep endoderm dramatically enhances heart formation in explants of heart primordia, both in the presence and absence of organizer. Likewise, extirpation of the entire endoderm can decrease the frequency of heart formation in embryos that retain organizer activity. Finally, we show that the combined presence of both endoderm and organizer is necessary and sufficient to induce heart in ventral mesoderm explants that would not otherwise form heart tissue. Xenopus heart induction, therefore, may be a multistep process requiring separate dorsalization and cardiogenic signalling events. This is the first demonstration of a heart-inducing role for the endoderm in Xenopus, indicating that the mechanism of heart formation may be similar in most vertebrates.
LFB1 (HNF1) is a tissue-specific transcription factor found in the livers, stomachs, intestines, and kidneys of vertebrates.
By analyzing the promoter of the Xenopus LFB1 gene, we identified potential autoregulation by LFB1 and regulation by HNF4,
a transcription factor with a tissue distribution similar to that of LFB1. Injection of LFB1 promoter-chloramphenicol acetyltransferase
constructs into Xenopus eggs revealed embryonic activation that is restricted to the region of the developing larvae expressing
endogeneous LFB1. Proper embryonic activation was also observed with a rat LFB1 promoter. Deletion analysis of the Xenopus
and rat promoters revealed that in both promoters embryonic activation is absolutely dependnet on the presence of an element
that contains CCNCTCTC as the core consensus sequence. Since this element is recognized by the maternal factor OZ-1 previously
described by N. Ovsenek, A. M. Zorn, and P. A. Krieg (Development 115:649-655, 1992), we might have identified the main constituents
of a hierarchy that leads via LFB1 to the activation of tissue-specific genes during embryogenesis.
We have developed a simple approach for large-scale transgenesis in Xenopus laevis embryos and have used this method to identify in vivo requirements for FGF signaling during gastrulation. Plasmids are introduced into decondensed sperm nuclei in vitro using restriction enzyme-mediated integration (REMI). Transplantation of these nuclei into unfertilized eggs yields hundreds of normal, diploid embryos per day which develop to advanced stages and express integrated plasmids nonmosaically. Transgenic expression of a dominant negative mutant of the FGF receptor (XFD) after the mid-blastula stage uncouples mesoderm induction, which is normal, from maintenance of mesodermal markers, which is lost during gastrulation. By contrast, embryos expressing XFD contain well-patterned nervous systems despite a putative role for FGF in neural induction.
Drosophila tinman is an NK-class homeobox gene required for formation of the dorsal vessel, the insect equivalent of the vertebrate heart. Vertebrate sequences related to tinman, such as mouse Nkx-2.5, chicken cNkx-2.5, Xenopus XNkx-2.5 and XNkx-2.3 are expressed in cardiac precursors and in tissues involved in induction of cardiac mesoderm. Mice which lack a functional Nkx-2.5 gene die due to cardiac defects. To determine the role of tinman-related sequences in heart development, we have overexpressed both XNkx-2.3 and XNkx-2.5 in Xenopus laevis embryos. The resulting embryos are morphologically normal except that they have enlarged hearts. The enlarged heart phenotype is due to a thickening of the myocardium caused by an increase in the overall number of myocardial cells (hyperplasia). Neither ectopic nor precocious expression of cardiac differentiation markers is detectable in overexpressing embryos. These results suggest that both XNkx-2.3 and XNkx-2.5 are functional homologues of tinman, responsible for maintenance of the heart field.
It has been shown that lens regeneration from outer cornea of larval Xenopus laevis is dependent on neural retina both in vivo and in tissue culture. The isolated outer cornea cultured in the presence of bovine brain-derived acidic Fibroblast Growth Factor (aFGF) is able to reprogram the differentiation into lens fibers, although this transdifferentiative process is not coupled with the formation of a normally organized lens. The capacity of aFGF to promote lens differentiation from cornea is not linked to its mitogenic activity. The cultured corneal cells can transdifferentiate into lens fibers in the presence of aFGF when DNA replication and cell proliferation are prevented by addition of aphidicolin, a specific inhibitor of DNA polymerase in eukaryotes, to the culture medium.
The proglucagon gene encodes several hormones that have key roles in the regulation of metabolism. In particular, glucagon-like peptide (GLP-1), a potent stimulus of insulin secretion, is being developed as a therapy for the treatment of non-insulin-dependent diabetes mellitus. To define structural moieties of the molecule that convey its insulinotropic activity, we have cloned and characterized the proglucagon gene from the amphibian, Xenopus laevis. Unexpectedly, these cDNAs were found to encode three unique glucagon-like-1 peptides, termed xenGLP-1A, xenGLP-1B, and xenGLP-1C in addition to the typical proglucagon-derived hormones glucagon and GLP-2. xenGLP-1A, -1B, and -1C were synthesized and tested for their ability to bind and activate the human GLP-1 receptor (hGLP-1R), and to stimulate insulin release from rat pancreas. All three Xenopus GLP-1-like peptides bind effectively to the hGLP-1R and stimulate cAMP production. Surprisingly, xenGLP-1B(1-30) demonstrated higher affinity for the hGLP-1R than hGLP-1 (IC50 of 1.1 +/- 0.4 nM vs. 4.4 +/- 1.0 nM, respectively, P < 0.02) and was equipotent to hGLP-1 in stimulating cAMP production (EC50 of 0.17 +/- 0.02 nM vs. 0.6 +/- 0. 2 nM, respectively, P > 0.05). Further studies demonstrated that hGLP-1, xenGLP-1A, -1B, and -1C stimulate comparable insulin release from the pancreas. These results demonstrate that despite an average of nine amino acid differences between the predicted Xenopus GLPs and hGLP-1, all act as hGLP-1R agonists.
The role of the notochord in inducing and patterning adjacent neural and mesodermal tissues is well established. We provide evidence that the notochord is also required for one of the earliest known steps in the development of the pancreas, an endodermally derived organ. At a developmental stage in chick embryos when the notochord touches the endoderm, removal of notochord eliminates subsequent expression of several markers of dorsal pancreas bud development, including insulin, glucagon and carboxypeptidase A. Pancreatic gene expression can be initiated and maintained in prepancreatic chick endoderm grown in vitro with notochord. Non-pancreatic endoderm, however, does not express pancreatic genes when recombined with the same notochord. The results suggest that the notochord provides a permissive signal to endoderm to specify pancreatic fate in a stepwise manner.
The ontogeny of the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM), and pancreatic polypeptide (PP) as well as insulin-like growth factor I (IGF-I) in the gastro-entero-pancreatic (GEP) system of Xenopus laevis (stages 41-66) was studied using double immunofluorescence and morphometric analysis. As early as stage 41, clustered INS-immunoreactive (-IR) and isolated GLUC-IR cells occurred in the pancreas. The first SOM-IR cells appeared at stage 43, followed by PP-IR cells at stage 46. About 79% of the PP immunoreactivity was confined to a subpopulation of the GLUC-IR cells. Both the GLUC/PP-IR cells and the PP-IR cells were located in a distinct area of the pancreas. The first islets occurred in premetamorphosis (around stage 50) and comprised mainly INS-IR and GLUC-IR cells. The majority of SOM-IR, PP-IR, and GLUC/PP-IR cells was dispersed. The numbers of hormone cells remained quite constant until the end of prometamorphosis (stage 58). Around stages 60-62, the islets were partly disintegrated and the numbers of islet cells slightly decreased. At stage 63, the cell number began to increase and reached the levels typical for the adult around stage 66. After metamorphic climax, the islets were reformed. In the gastrointestinal tract, transient INS-IR cells occurred prior to the adaptation of the gastrointestinal tract to feeding (stages 41-44) and during metamorphosis when there is remodeling of the gastrointestinal tract (stages 60-63). Therefore, INS released from the transient mucosal INS-IR cells may be involved in the temporary proliferation of mucosal epithelial cells. The first GLUC-IR and SOM-IR cells were seen at stage 41. PP-IR cells followed at stage 46. In contrast to the islets, GLUC-IR and PP-IR cells constituted different cell populations. Around stage 46, the first IGF-I immunoreactions appeared in the GEP-system. In pancreas, IGF-I immunoreactivity was found in the GLUC/PP-IR, cells (85-99%) but was absent from INS-IR, GLUC-IR, and SOM-IR cells. The IGF-I-IR gastro-entero-endocrine cells, however, seemed to contain none of the classical islet hormones.
Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-betaB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.
Mutations in the Tbx5 transcription factor cause heart septal defects found in human Holt-Oram Syndrome. The complete extent to which Tbx5 functions in heart development, however, has not been established. Here we show that, in Xenopus embryos, Tbx5 is expressed in the early heart field, posterior to the cardiac homeobox transcription factor, Nkx2.5. During morphogenesis, Tbx5 is expressed throughout the heart tube except the anterior portion, the bulbus cordis. When Tbx5 activity is antagonized with a hormone-inducible, dominant negative version of the protein, the heart fails to develop. These results suggest that, in addition to its function in heart septation, Tbx5 has a more global role in cardiac specification and heart development in vertebrate embryos.
In most mammals the pancreas develops from the foregut endoderm as ventral and dorsal buds. These buds fuse and develop into a complex organ composed of endocrine, exocrine and ductal components. This developmental process depends upon an integrated network of transcription factors. Gene targeting experiments have revealed critical roles for Pdx1, Isl1, Pax4, Pax6 and Nkx2-2 (refs 3,4,5,6,7, 8,9,10). The homeobox gene HLXB9 (encoding HB9) is prominently expressed in adult human pancreas, although its role in pancreas development and function is unknown. To facilitate its study, we isolated the mouse HLXB9 orthologue, Hlxb9. During mouse development, the dorsal and ventral pancreatic buds and mature beta-cells in the islets of Langerhans express Hlxb9. In mice homologous for a null mutation of Hlxb9, the dorsal lobe of the pancreas fails to develop. The remnant Hlxb9-/- pancreas has small islets of Langerhans with reduced numbers of insulin-producing beta-cells. Hlxb9-/- beta-cells express low levels of the glucose transporter Glut2 and homeodomain factor Nkx 6-1. Thus, Hlxb9 is key to normal pancreas development and function.
Xenopus embryos have several experimental advantages for studying development. Although these advantages have traditionally been used to elucidate mechanisms of early development, they can also be exploited to investigate issues later in development such as organogenesis. We have begun to study pancreatic organogenesis in Xenopus. Using histological and molecular marker analysis, we characterized the anatomy of the developing pancreas in Xenopus embryos from the time of initial pancreatic rudiment formation to the time when the tadpole starts to feed. We examined the expression of various endocrine hormones, exocrine gene products, and pancreatic transcription factors. Interestingly, the endocrine hormone insulin has restricted expression in the dorsal pancreas. Investigation of pancreatic specification during gastrulation demonstrates that insulin expression is regionalized along the dorsoventral axis early in development. © 2000 Wiley-Liss, Inc.
Not since the early 1970s has there been an attempt to describe and illustrate the anatomy of the developing mouse embryo. More than ever such material is needed by biologists as they begin to unravel the molecular mechanisms underlying development and differentiation. After more than ten years of painstaking work, Matt Kaufman has completed The Atlas of Mouse Development--the definitive account of mouse embryology and development. For all those researching or studying mammalian development, The Atlas of Mouse Development will be the standard reference work for many years to come. Key Features * Provides a comprehensive sequential account of the development of the mouse from pre-implantation to term * Contains clear and concise descriptions of the anatomical features relevant to each stage of development * Large format for easy use * Contains explanatory notes and legends, and more than 180 meticulously labeled plates, 1,300 photographs of individual histological sections, and 200 electron micrographs, illustrating: * Intermittent serial histological sections through embryos throughout embryogenesis and organogenesis * Differentiation of specific organs and organ systems, including the spinal cord, eyes, gonads, kidneys, lungs and skeletal system * External appearance of intact embryos throughout development
Correlation between activin and retinoic acid (RA), both of which affect early amphibian development, was studied using Xenopus laevis embryos. In the first set of experiments, two isomers of RA, all-trans RA and 13-cis RA, were compared in terms of stability of biological activity against light. Xenopus blastulae were dipped in RA solutions which had either been kept away from light, or had been exposed to light for a few hours. At doses ranging from 10–4to 10–6M, RA elicited head deformity. All-trans RA, under both dark and light conditions, had similarly potent effects. On the other hand, 13-cis RA under dark conditions had much weaker effects than it did under light conditions.
In the second set of experiments, activin was mixed with all-trans RA, and the inducing effects on the animal cap explants were investigated. Activin at a concentration of 10 ng/ml induced notochord. In combination with 10–6M RA, muscle was well induced instead of notochord. In combination with 10–5M RA, pronephric tubules were markedly induced. Pronephric tubules were never induced by activin alone, at any of the various concentrations employed. This is the first report on the very high frequency of induction of pronephric tubules by the combination of activin A and all-trans RA in the Xenopus ectoderm.
In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas-like structures accompanied by an intestinal epithelium-like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus-like structure, including secretory granules. The pancreas-specific molecular markers insulin and XlHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well-differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3–5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15 h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.
The lining of the gut, together with the pancreas, liver, gall bladder, and respiratory system, is formed from the endoderm. The gut also contains smooth muscle and connective tissue of mesodermal origin. The amphibian Xenopuslaevis is potentially an excellent model organism for studying how the cells of the endoderm and mesoderm become programmed to produce these internal organs. However, the anatomical complexity of the coiled gut presents a problem in studying its development. In order to overcome this problem we here present a comprehensive guide to the anatomy and histology of the developing Xenopus gut. We use a simple dissection to display its anatomy and the expression of four endodermal markers (alkaline phosphatase, IFABP, XlHbox8, and endodermin). We present schematic diagrams that show how the gut is arranged in three dimensions and how this organisation changes during development. We also present drawings of histological sections of the gut which allow any region to be identified and so represent an atlas for working with sections. Finally, we describe the histology of the cells of the various organs of the gut. This histological identification may be necessary for the identification of parts following experiments in which the normal pattern is disturbed. Dev. Dyn. 1998;212:509–521. © 1998 Wiley-Liss, Inc.
Xenopus and zebrafish serve as outstanding models in which to study vertebrate heart development. The embryos are transparent, allowing observation during organogenesis; they can be obtained in large numbers; and they are readily accessible to embryologic manipulation and microinjection of RNA, DNA, or protein. These embryos can live by diffusion for several days, allowing analysis of mutants or experimental treatments that perturb normal heart development. Xenopus embryos have been used to understand the induction of the cardiac field, the role of Nkx genes in cardiac development, and the role transforming growth factor β molecules in the establishment and signaling of left-right axis information. Large-scale mutant screens in zebrafish and the development of transgenics in both Xenopus and zebrafish have accelerated the molecular identification of genes that regulate conserved steps in cardiovascular development. Am. J. Med. Genet. (Semin. Med. Genet.) 97:248–257, 2000. © 2001 Wiley-Liss, Inc.
The tinman homeobox gene of Drosophila is absolutely required for development of the insect heart. This observation prompted the isolation of tinman-related genes from vertebrates, in the hope that the developmental function of the gene would be conserved between evolutionarily distinct species. The first vertebrate tinman gene, Nkx2-5, was isolated from mouse and subsequently, orthologues of Nkx2-5 have been isolated from a number of different species. In all cases, a conserved pattern of Nkx2-5 expression is observed in the developing heart, commencing prior to differentiation. Genetic ablation of Nkx2-5 in the mouse results in embryonic lethality due to heart defects, but most myocardial genes are expressed normally and a beating heart tube forms. This observation raises the possibility that additional genes related to Nkx2-5 are partially rescuing Nkx2-5 function in the null mouse. Recently, additional members of the tinman-related gene family have been discovered and characterized in a number of different species. Somewhat surprisingly, orthologous genes in different organisms can be rather divergent in sequence and may show completely different expression patterns. In at least some organisms, expression of the tinman-related genes is not observed in the heart. Due to the increasing number of family members and the somewhat divergent expression patterns, the precise role of the tinman-related genes in cardiac development remains an open question. In a search for additional tinman-related genes in the frog, Xenopus laevis, we have identified Nkx2-9, a novel member of the tinman-related gene family. Preliminary characterization reveals that Nkx2-9 is expressed in the cardiogenic region of the embryo prior to differentiation, but transcript levels decrease rapidly, in the heart, at about the time that differentiation commences. Dev. Genet. 22:230–238, 1998. © 1998 Wiley-Liss, Inc.
The mechanisms that regulate cell fate within the pronephros are poorly understood but are important for the subsequent development of the urogenital system and show many similarities to nephrogenesis in the definitive kidney. Dynamic expression of Notch-1, Serrate-1, and Delta-1 in the developing Xenopus pronephros suggests a role for this pathway in cell fate segregation. Misactivation of Notch signaling using conditionally active forms of either Notch-1 or RBP-J/Su(H) proteins prevented normal duct formation and the proper expression of genetic markers of duct cell differentiation. Inhibition of endogenous Notch signaling elicited the opposite effect. Taken together with the mRNA expression patterns, these data suggest that endogenous Notch signaling functions to inhibit duct differentiation in the dorsoanterior region of the anlage where cells are normally fated to form tubules. In addition, elevated Notch signaling in the pronephric anlage both perturbed the characteristic pattern of the differentiated tubule network and increased the expression of early markers of pronephric precursor cells, Pax-2 and Wilms' tumor suppressor gene (Wt-1). We propose that Notch signaling plays a previously unrecognized role in the early selection of duct and tubule cell fates as well as functioning subsequently to control tubule cell patterning and development.
FGF-8has attracted attention particularly because of its importance for limb development in the chick and mouse, although it also has a number of earlier expression domains in these species. We have now cloned anFGF-8homologue fromXenopusin which it is easier to do functional studies on early development. There is no maternal expression, while zygotic expression is highest in the gastrula and neurula stages.XFGF-8is expressed as a ring around the blastopore and subsequently in the tail bud. There are several domains in the head including the hatching gland, the branchial clefts, and the midbrain–hindbrain border. At later stages there is a prominent band of expression in the limb bud epidermis. Although there is no morphological apical ridge, this band of expression suggests that theXenopuslimb bud contains a cryptic region with a similar ability to stimulate mesenchymal outgrowth. The mesoderm-inducing activity of XFGF-8 is somewhat lower than that of other FGFs, while the posteriorizing activity is similar. These differences are probably due to the different receptor specificity. The posterior expression and high posteriorizing activity suggest that XFGF-8 contributes to the patterning of the anterior–posterior axis by FGF family members during gastrulation. In contrast to the amniotes,Xenopuslimb buds can regenerate following damage. We show that regeneration is correlated with the reexpression ofXFGF-8in the distal epidermis, suggesting that this ability is critical for successful limb regeneration.
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.
Zebrafish and Xenopus, genetically accessible vertebrates with an externally developing, optically clear embryo, are ideally suited for in vivo functional dissection of the embryonic development of the circulatory system. Physiological characterizations of the cardiovascular system are still imperative for a more complete understanding of the connections between genetic/epigenetic factors and cardiovascular development. Here, we review experimental tools and methods that have been developed to measure numerous cardiovascular parameters in these millimetre-sized animals
Duisburg, Essen, Univ., Diss., 2004. Computerdatei im Fernzugriff.
The disposition of prospective areas and the course of morphogenetic movements during gastrulation and neurulation were investigated by vital staining. The prospective lining of the archenteron, the prospective neural area, and the prospective epidermal area are represented on the surface of the early gastrula. The prospective lining of the archenteron occupies the area within 65–70° of the vegetal pole and is divided into prospective archenteron roof and prospective archenteron floor by the blastopore pigment line which functions as the locus of invagination. A crescent-shaped neural area lies immediately above the prospective archenteron roof, rising from it at 125° lateral to the dorsal midline to a point 130° above the vegetal pole in the dorsal midline. In the early gastrula, most, if not all, mesoderm is deep to the surface layer and is mapped by the insertion of dyed agar spikes. Results thus far indicate that the prospective notochord lies in the dorsal deep marginal zone, followed laterally by the medial region of the somites, the lateral region of the somites, and the lateral plate.The morphogenetic significance of the comparative disposition of the anlagen in Xenopus is discussed.
The prospective areas in the deep layer of the early gastrula were mapped and their morphogenetic movements during gastrulation and neurulation were followed by vital dye marking of cells. It was found that the deep layer of the early gastrula consists of prospective mesoderm and prospective neural and epidermal ectoderm. At Stage 10+ the prospective mesoderm lies deep to the suprablastoporal endoderm in the form of a thick collar which has already begun involution in the dorsal sector. The prospective notochord is located middorsally and is followed laterally, in turn, by prospective somite and lateral mesoderm. Prospective anterior mesoderm involutes first, followed in succession by prospective mesoderm of more posterior regions. The relative movements of the neural and epidermal ectoderm and the underlying mesodermal mantle during neurulation were mapped by simultaneous marking of both layers. These movements show the highly coordinated nature of the thickening of somite mesoderm and folding of the neural plate. Strong dorsal convergence and ventral divergence occur in both the prospective mesoderm and ectoderm. In the ectoderm, dorsal convergence of cells results in anteroposterior extension; however, such dorsal movement in the mesoderm is absorbed in the thickening of the somite region. The extension of the dorsal mesoderm is brought about during neurulation by addition of cells posteriorly from the uninvoluted circumblastoporal mesoderm. The significance of the location and movements of the mesoderm in Xenopus laevis is discussed.
Morphological studies using both light and electron microscope were carried out with the aim of characterizing cells present in the larval and adult pancreas of Xenopus laevis. The following cell types have been seen: (1) exocrine cells, with a very well developed r.e.r. (rough endoplasmic reticulum), well defined Golgi complexes and numerous large secretory granules (A cells);(2) cells without either r.e.r. or secretory granules but with a large number of well developed mitochondria (B cells); (3) endocrine cells often clustered in the typical islets and with small membrane-coated granules showing a very dense central core surrounded by a light halo (C cells).
pDuring development, the aspect is seen to change from an unorganized tissue in which the acinar structures are still not clearly visible (stage 42), to a more organized form in which the exocrine cells (A cells) are seen to be arranged around the lumen of the acinus together with some B cells.
At the stages 54–56, an increasing number of acini surrounded both by A and B cells was observed. At about stage 61, large quantities of necrotic cells were seen and it became more difficult to individualize the acinar organization found in the preceding stages. Finally, there are no necrotic cells in the adult but only A, B cells which are organized in well developed acinar structures and C cells.
The investigation also included a study of some pancreatic enzymes (lipase and amylase) synthesized during larval life. Lipase activity shows a peak at stage 54–56 in which the most well organized tissue of the entire larval life was observed. The activity then decreases, reaching a minimum at stage 66, after which it rapidly rises. Maximum amylase activity occurs at stage 51 after which there is a decrease, to a minimum at stage 66. The activity then remains at constant level.
FORTY-FIVE FIGURES The preparation of a series of normal stages of the chick embryo does not need justification at a time when chick ernbryos are not only widely used in descriptive and experimental embryology but are proving to be increasingly valuable in medical research, as in work on viruses and cancer. The present series was planned in connection with the preparation of a new edition of Lillie’s DeueZopmerzt of the Chick by the junior author. It is being published separately to make it accessible immediately to a large group of workers. Ever since Aristotle “discovered” the chick embryo as the ideal, object for embryological studies, the embryos have been described in terms of the length of time of incubation, and this arbitrary method is still in general use, except for the first three days of incubation during which more detailed characteristics such as the numbers of somites are applied. The shortcomings of a classification based on chronological age are obvious to every worker in this field, for enormous variations may occur in embryos even though all eggs in a setting are plmaced in the incubator at the same time. Many factors are responsible for the lack of correlation between chronological and structural age. Among these are : genetic differences in the rate of development of different breccls (eg., the embryo of the White Leghorn breed develops more 49
The current study was designed to determine if insulin, glucagon and somatostatin-containing cells are present in the pancreas of adult Xenopus laevis. Localization methods utilized included cytochemical aldehyde fuchsin (AF) staining as well as the immunochemical peroxidase antiperoxidase (PAP) procedure for light microscopy. The results show numerous large clusters of AF-positive cells within a network of highly vascularized acinar tissue. PAP immunochemical localization with insulin antibody on adjacent sections demonstrates positive immunoreactivity to AF-positive cell groups and also the presence of immunoreactive insulin (IRI). Cells exhibiting this immunoreactivity are located in the central region of the islet-like structures. Serial sections not only show PAP immunoreactivity for IRI, but also for immunoreactive glucagon (IRG) and immunoreactive somatostatin (IRS) in the same islet-like structure. IRG and IRS-containing cells are situated around the periphery of the islet-like structures, surrounding the central core of IRI-containing cells. Antibody specificity was confirmed by homologous and heterologous antigen immuno-absorbance assays, as well as incubation of adjacent sections in preimmune sera. Based on this data we conclude that: the distribution of cells of the endocrine pancreas of metamorphosed Xenopus laevis is similar to that of many mammals and certain urodeles. Given the apparent specificity of the antigen-antibody reactions, it appears that Xenopus insulin, glucagon and somatostatin are structurally conserved.
We report the isolation of a new homeobox gene from Xenopus laevis genomic DNA. The homeodomain sequence is highly diverged from the prototype Antennapedia sequence, and contains a unique histidine residue in the helix that binds to DNA. The homeodomain is followed by a 65 amino acid carboxyterminal domain, the longest found to date in any vertebrate homeobox gene. We have raised specific antibodies against an XlHbox 8-beta-gal fusion protein to determine the spatial and temporal expression of this gene. The nuclear protein first appears in a narrow band of the endoderm at stage 33 and develops into expression within the epithelial cells of the pancreatic anlagen and duodenum. Expression within the pancreatic epithelium persists into the adult frog. This unprecedented restriction to an anteroposterior band of the endoderm suggests that vertebrate homeobox genes might be involved in specifying positional information not only in the neuroectoderm and mesoderm, but also in the endoderm. Our data suggest that XlHbox 8 may therefore represent the first member of a new class of position-dependent transcription factors affecting endodermal differentiation.
The South African clawed toad, Xenopus laevis, is a versatile laboratory model of vertebrate development. To study the role of insulin during embryogenesis, we have recently cloned preproinsulin cDNAs from this species. Unexpectedly, we identified two preproinsulin cDNAs corresponding to two different nonallelic genes that code for similar but distinctly different insulins. We now report the isolation, amino acid sequence, and characterization of both of these insulins from pancreatic extracts of adult toads, confirming that both Xenopus preproinsulin genes are expressed. Xenopus insulins represent the first amphibian insulins to be characterized. Xenopus insulin I and Xenopus insulin II are more similar to each other than they are to insulins of other species. In addition, Xenopus insulins are more similar to mammalian and bird insulins, than they are to fish insulins, implying a closer evolutionary link to terrestrial vertebrates than to most aquatic vertebrates. A homogeneous preparation of Xenopus insulin I showed high reactivity in a pork insulin RIA. Xenopus insulin I was approximately 2-fold more potent than pork insulin in binding to insulin receptors on human IM-9 lymphocytes and 1.5-fold more potent than pork insulin in stimulating glucose oxidation in rat adipocytes. We were unable to purify Xenopus insulin II sufficiently for immunological and biological characterization.
By transfecting various Xenopus albumin-CAT fusion genes into the mouse hepatoma cell line BW1J a 13 base-pair hepatocyte-specific promoter element (HP1) could be identified. A similar sequence element is also present in the promoter of the albumin and alpha-fetoprotein genes of other vertebrates. Introduction of single point mutations into HP1 destroys its function. Binding studies with nuclear proteins identify a factor interacting with HP1 which is specific for hepatic cells. In-vitro transcription in a rat liver nuclear extract demonstrates that HP1 leads to an increased transcriptional activity. This increased transcription is specifically inhibited by the addition of an HP1-containing oligonucleotide, establishing that the interaction of factors with HP1 is essential for increased transcription. Since HP1 derived from a Xenopus gene functions in mammalian hepatocytes, we conclude that a regulatory system involved in liver-specific gene expression has been conserved during evolution.
Primary cultures of male Xenopus liver parenchymal cells that retained their competence to respond to estrogen were used to study the hormone-induced activation of the vitellogenin gene in vitro. The accumulation of vitellogenin mRNA in these cells was monitored by a quantitative diazotized paper disc hybridization procedure with a sensitivity of at least 6 pg of sequences complementary to the probe in total RNA samples of 10 micrograms. A short-term time-course analysis showed that vitellogenin mRNA was detectable within 3 hr of exposure to estrogen during primary stimulation, and that the maximum rate of accumulation was reached at 5--6 hr. A long-term time-course analysis of the accumulation of vitellogenin mRNA showed that it is possible to obtain a primary response, a hormone withdrawal effect and an enhanced secondary response in the same batch of cells in a manner analogous to that observed in vivo. Measurement of hormone concentration dependence showed a response at 10(-9) M estradiol, which continued to increase up to at least 10(-6) M estradiol. This requirement for large doses of estradiol for maximal response can be explained by the rapid metabolism of estradiol by the cultured cells.
We have isolated a Xenopus homeodomain sequence, XNkx-2.5, which shows significant similarity to mouse Nkx-2.5 and to the Drosophila tinman gene product. In Drosophila, tinman is required for formation of the heart and visceral mesoderm structures. In situ hybridization studies show that XNkx-2.5 is expressed in the heart region during early Xenopus development and later is also expressed in gut tissue. The observed similarity of sequences and expression patterns suggests that the regulatory mechanisms underlying heart formation may be conserved between distant species.
The pancreas is an organ containing two distinct populations of cells, the exocrine cells that secrete enzymes into the digestive tract, and the endocrine cells that secrete hormones into the bloodstream. It arises from the endoderm as a dorsal and a ventral bud which fuse together to form the single organ. Mammals, birds, reptiles and amphibians have a pancreas with similar histology and mode of development, while in some fish, the islet cells are segregated as Brockmann bodies. Invertebrates do not have a pancreas, but comparable endocrine cells may be found in the gut or the brain.
The early pancreatic bud shows uniform expression of the homeobox gene IPF-1 (also known as IDX-1, STF-1 or PDX), which when mutated to inactivity leads to total absence of the organ. The occurrence of heterotopic pancreas in the embryo, and also the metaplasias that can be displayed by a regenerating pancreas in the adult, both suggest that only a few gene products distinguish the pancreatic cell state from that of the surrounding tissues of duodenum, gall bladder and liver.
In the developing pancreatic buds, the endocrine cells start to differentiate before the exocrine cells, and coexpression of different hormones by the same cell is often observed at early stages. Although pancreatic endocrine cells produce many gene products also characteristic of neurons, evidence from in vitro cultures and from quail-chick grafts shows that they are of endogenous and not of neural crest origin. Observational studies suggest strongly that both endocrine and exocrine cells arise from the same endodermal rudiment.
Development of the pancreas in embryonic life requires a trophic stimulus from the associated mesenchyme. In postnatal life, all cell types in the pancreas continue to grow. Destruction of acinar tissue by duct ligation or ethionine treatment is followed by rapid regeneration. Surgical removal of parts of the pancreas is followed by moderate but incomplete regeneration of both acini and islets. Poisoning with alloxan or streptozotocin can lead to permanent depletion of β cells. Although the cell kinetics of the pancreas are not understood, it seems likely that there is a continuous slow turnover of cells, fed from a stem cells population in the ducts, and that the controls on the production rate of each cell type are local rather than systemic.
The Wilms' tumor suppressor gene (WT1) is required for the formation of the mammalian metanephros, or adult kidney, and for the normal development of the mesonephros, the major mammalian embryonic kidney. In this report the isolation of a Xenopus gene closely related to the mammalian WT1 gene in both sequence and splicing pattern is described. Expression of this gene, xWT1, is restricted to the developing nephric system until late tadpole stages, which expression also begins to be observed in the heart. Within the nephric system, expression is observed in the dorsal portion of the splanchnic lateral plate in tailbud embryos, and in the glomus of early tadpoles. No expression is observed in the pronephric tubules or pronephric duct. The WT1 gene is therefore expressed in a similar temporal and spatial pattern in the vascularized portion of the amphibian pronephroi and in the mammalian metanephroi, arguing that it probably plays a similar crucial role in the morphogenesis of these very different kidney forms. The absence of expression in the developing pronephric tubules indicates that xWT1 is not required for the epithelialization of the tubular portion of the pronephros.
Most vertebrate organs, once formed, continue to perform the function for which they were generated until the death of the organism. The kidney is a notable exception to this rule. Vertebrates, even those that do not undergo metamorphosis, utilize a progression of more complex kidneys as they grow and develop. This is presumably due to the changing conditions to which the organism must respond to retain what Homer Smith referred to as our physiological freedom. To quote, "Recognizing that we have the kind of blood we have because we have the kind of kidneys we have, we must acknowledge that our kidneys constitute the major foundation of our physiological freedom. Only because they work the way they do has it become possible for us to have bones, muscles, glands, and brains. Superficially, it might be said that the function of the kidneys is to make urine; but in a more considered view one can say that the kidneys make the stuff of philosophy itself" ("From Fish to Philosopher," Little, Brown and Co., Boston, 1953). Different kidneys are used to make the stuff of philosophy at different stages of development depending on the age and needs of the organism, rather than the usual approach of simply making embryonic organs larger as the animal grows. Although evolution has provided the higher vertebrates with complex adult kidneys, they continue to utilize simple kidneys in embryogenesis. In lower vertebrates with simple adult kidneys, even more simple versions are used during early developmental stages. In this review the anatomy, development, and gene expression patterns of the embryonic kidney, the pronephros, will be described and compared to the more complex kidney forms. Despite some differences in anatomy, similar developmental pathways seem to be responsible for the induction and the response to induction in both evanescent and permanent kidney forms. Gene expression patterns can, therefore, be added to the morphological and functional data indicating that all forms of the kidney are closely related structures. Given the similarities between the development of simple and complex kidneys, the embryonic kidneys may be an ideal model system in which to investigate the genesis of multicomponent organ systems.
FGF-8 has attracted attention particularly because of its importance for limb development in the chick and mouse, although it also has a number of earlier expression domains in these species. We have now cloned an FGF-8 homologue from Xenopus in which it is easier to do functional studies on early development. There is no maternal expression, while zygotic expression is highest in the gastrula and neurula stages. XFGF-8 is expressed as a ring around the blastopore and subsequently in the tail bud. There are several domains in the head including the hatching gland, the branchial clefts, and the midbrain-hindbrain border. At later stages there is a prominent band of expression in the limb bud epidermis. Although there is no morphological apical ridge, this band of expression suggests that the Xenopus limb bud contains a cryptic region with a similar ability to stimulate mesenchymal outgrowth. The mesoderm-inducing activity of XFGF-8 is somewhat lower than that of other FGFs, while the posteriorizing activity is similar. These differences are probably due to the different receptor specificity. The posterior expression and high posteriorizing activity suggest that XFGF-8 contributes to the patterning of the anterior-posterior axis by FGF family members during gastrulation. In contrast to the amniotes, Xenopus limb buds can regenerate following damage. We show that regeneration is correlated with the reexpression of XFGF-8 in the distal epidermis, suggesting that this ability is critical for successful limb regeneration.
We have examined the timing of specification of the pronephric tubules and duct in Xenopus laevis by explanting the presumptive pronephric rudiments into blastula ectodermal wraps. We have established the time point of specification using the monoclonal antibody markers 3G8 and 4A6 which recognize antigens in pronephric tubule and duct, respectively. We show that, by experimental analysis in explants, kidney tubules are specified by stage 12.5 in the pronephric anlagen whereas pronephric duct is specified later between stages 13 and 14. Furthermore we show that signals involved in tubulogenesis of the pronephric tubules are normally received between stage 12.5 and 13. These experiments unambiguously pinpoint the timing of pronephros specification analyzed by explant experimentation to a developmental stage prior to that demonstrated for urodele amphibia, and provide an essential biological backdrop to a search for the molecular nature of pronephric inducers.
The tumor suppressor WT1 has been demonstrated to have a wide variety of activities in vitro and is required for metanephric development in vivo. In the experiments presented here, the Xenopus pronephros was used as a simple model system to examine the activity of Xenopus WT1 (xWT1) during kidney development. xWT1 was ectopically expressed in Xenopus embryos by mRNA injection and found to inhibit pronephric tubule development. Confocal microscopy confirmed this observation and revealed that the inhibition was the result of a failure to form a pronephric anlage of appropriate size rather than a defect in epithelialization. Examination of Xlim-1 expression, an early molecular marker of pronephric specification, in tailbud embryos indicated that injected xWT1 mRNA inhibited pronephric specification prior to any overt sign of morphogenesis (Xenopus stage 21). These results suggest that xWT1 may act to repress tubule-specific gene expression in the portion of the pronephros fated to form its vascular structure, the glomus.
Data from gene ablation studies in mice have indicated critical roles for Lim-1, Wnt4, WT-1, and Pax-2 in the coordination and execution of kidney patterning and differentiation. However, the precise roles of these molecules, their ordering within a genetic hierarchy, and the manner in which they contribute to establishing the fates of cells of each of the components of the nephron have yet to be elucidated in any system. In this report, the temporal and spatial expression patterns of these genes within the Xenopus pronephric system were examined in detail by single- and double-probe in situ hybridization. We describe restrictions of these gene expression patterns within the pronephros which indicate a model for the partitioning of the common pronephric anlage into its three component parts--the tubules, the glomus, and the duct.
Beating hearts can be induced under in vitro conditions when the dorsal blastopore lip (including the zone of Spemann organizer) is treated with Suramin. In contrast, untreated organizer forms dorsal mesodermal derivatives as notochord and somites. When those in vitro produced heart precursor tissues are transplanted ectopically in the posterior trunk area of early larvae, secondary beating heart structures will be formed. Furthermore, the replacement of the heart primordium of the host embryo by heart tissue induced under in vitro conditions will result in the rescue of the heart anlage. This model could be a valuable tool for the study of the multi-step molecular mechanisms of heart structure induction under in vitro conditions and vasculogenesis after transplantation into the host embryo.