African Clawed Frog Xenopus Laevis Research Articles

Microplastics ingestion induces plasticity in digestive morphology in larvae of Xenopus laevis

Global changes in temperature, predator introductions, and pollution might challenge animals by altering food conditions. A fast-growing source of environmental pollution are microplastics. If ingested with the natural food source, microplastics act as artificial fibers that reduce food quality by decreasing nutrient and energy density with possible ramifications for growth and development. Animals might cope with altered food conditions with digestive plasticity. We examined experimentally whether larvae of the African clawed frog (Xenopus laevis) exhibit digestive morphology plasticity (i.e., gut length, mass, and diameter) in response to microplastics ingestion. As natural systems contain non-digestible particles similar in size and shape to microplastics, we included cellulose as a natural fiber control group. Gut length and mass increased in response to microplastics and cellulose ingestion indicating that both types of fibers induced digestive plasticity. Body mass and body condition were similar across experimental groups, indicating that larvae fully compensated for low nutrient and energy density by developing longer intestines. The ability of a species to respond plastically to environmental variation, as X. laevis responded, indicates that this species might have the potential to cope with new conditions during global change, although it is uncertain whether this potential may be reduced in a multi-stressor environment.

Rejeneratif tıpta model organizma; Aksolotl (Ambystoma Mexicanum)

The axolotl has an extraordinary capacity to regenerate damaged and lost structures, especially the nervous system, limbs, organs such as the eye and heart, without causing scarring. It has become a very important model organism by attracting the attention of scientists working in both developmental biology and regenerative medicine and stem cell biology. The axolotl, which is amphibian and tetrapod, is a more preferred model due to its ease of maintenance and reproduction compared to other organisms such as African clawed frog (Xenopus laevis) or zebrafish (Danio rerio), which are relatively difficult to study. The main purposes of this review are the definition and origin of the axolotl, its taxonomy, anatomy, reproduction, nutrition, habitat, to give a perspective to scientists who want to work on this model organism by giving examples to the scientific data and study fields of the last 20 years by addressing issues such as how it contributes to scientific studies as a model organism.

Impact of glyphosate-based herbicide on early embryonic development of the amphibian Xenopus laevis.

Worldwide, amphibian populations are declining drastically. One reason might be the use of pesticides including herbicides. The herbicide glyphosate is an inhibitor of the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase of the plant shikimate pathway, preventing the formation of aromatic amino acids and thus inducing plant death. Due to this specific action, GBH are considered nontoxic to non-target organisms. However, GBH impairs embryonic development of chickens, amphibians and fishes. So far, no detailed tissue- and organ-specific analysis of the effects of GBH during development in amphibians has been performed. We demonstrated that GBH Roundup® LB plus has a negative effect on embryonic development of the South African clawed frog Xenopus laevis. GBH treatment with sublethal concentrations resulted in a reduced body length and mobility of embryos. Furthermore, incubation with GBH led to smaller eyes, brains and cranial cartilages in comparison to untreated embryos. GBH incubation also resulted in shorter cranial nerves and had an effect on cardiac development including reduced heart rate and atrium size. On a molecular basis, GBH treatment led to reduced expression of marker genes in different tissues and developmental stages. GBH leads to disturbed embryonic development of Xenopus laevis.

Identification and establishment of type IV interferon and the characterization of interferon-\u03c5 including its class II cytokine receptors IFN-\u03c5R1 and IL-10R2

Interferons (IFNs) are critical soluble factors in the immune system and are composed of three types, (I, II and III) that utilize different receptor complexes IFN-αR1/IFN-αR2, IFN-γR1/IFN-γR2, and IFN-λR1/IL-10R2, respectively. Here we identify IFN-υ from the genomic sequences of vertebrates. The members of class II cytokine receptors, IFN-υR1 and IL-10R2, are identified as the receptor complex of IFN-υ, and are associated with IFN-υ stimulated gene expression and antiviral activity in zebrafish (Danio rerio) and African clawed frog (Xenopus laevis). IFN-υ and IFN-υR1 are separately located at unique and highly conserved loci, being distinct from all other three-type IFNs. IFN-υ and IFN-υR1 are phylogenetically clustered with class II cytokines and class II cytokine receptors, respectively. Therefore, the finding of this IFN ligand-receptor system may be considered as a type IV IFN, in addition to the currently recognized three types of IFNs in vertebrates.

MicroRNA, mRNA and protein responses to dehydration in skeletal muscle of the African-clawed frog, Xenopus laevis

During the dry season in southern Africa, the predominantly aquatic African-clawed frog (Xenopus laevis) burrows into the wet mud of ponds and marshes. As drying continues frogs undergo dehydration, often enduring the loss of over 30% of body water. The present study examines the role of miRNA action in mediating biochemical changes that support X. laevis endurance of dehydration. Three conditions were analyzed: control, medium dehydration (15% total body water lost) and high dehydration (30% total body water lost) and relative changes in skeletal muscle miRNA, mRNA and protein expression were measured. The results showed statistically significant increases of 1.8–2.4 fold in the expression, relative to controls, of two proteins involved in miRNA biogenesis, Dicer and Drosha, as well as reduced serine phosphorylation of a third protein, p-Ago2 (Ser 387). Levels of two specific miRNAs were also elevated during medium dehydration by 1.9 and 2.5 fold for miR-181a-3p and miR-210-5p, respectively. Gene transcript levels of selected muscle proteins were also altered: a 10–15 fold increase in mstn2 (myostatin 2), a 5–6 fold increase in mybpc3.S (myosin binding protein cardiac 3) and a 2.3-fold rise in tnnc2 (troponin C2) but oppositely a significant decrease in expression of myod1 (myoblast determination protein 1). These findings indicate an increase in miRNA biogenesis, muscle contractile proteins and hypoxia stress tolerance with an opposite decrease in muscle growth and differentiation under dehydrating conditions that could all contribute to X. laevis survival of extreme dehydration.

Blastomeres derived from the vegetal pole provide extra-embryonic nutrition to sturgeon (Acipenser) embryos: Transition from holoblastic to meroblastic cleavage

Generally, holoblastic cleavage in embryos (as in amphibians) in which all blastomeres contribute to one of the germ layers, are preserved as a stem lineage of vertebrates, and meroblastic cleavage has evolved independently in each vertebrate lineage. The increasing egg size: yolk volume is the key factor for transition from holoblastic to meroblastic cleavage patterns. Sturgeon (Acipenser) eggs are two times larger than those of the African clawed frog Xenopus laevis (amphibian); despite the varying size, sturgeon embryos retain nearly the same developmental characteristics as X. laevis. Comparatively, the fate of blastomeres derived from the vegetal pole (VP) of a sturgeon embryo is unspecified. Thus, the goal of this study was to determine whether the VP of the embryo contributes to embryonic development, or is simply extra-embryonic. This may also reveal whether the transition of the cleavage pattern (holoblastic to meroblastic) in the actinopterygian lineage is correlated with the egg size: yolk volume. Here, we found that sturgeon vegetal blastomeres formed only primordial germ cells, and the rest were made up of cellular yolk (yolk cells; YCs). Morphological and phenotypic characteristics revealed that after the 1 k-cell / mid-blastula transition, YCs became transcriptionally inactive and served only to provide nutrition to larvae as they developed. Furthermore, inhibition of vegetal blastomeres revealed that sturgeon can utilize their yolk in an acellular form, similar to teleosts, implying that meroblastic cleavage in the actinopterygians, like teleosts, might have evolved by the fusion of the vegetal blastomeres.

Using metabolomic profiling to inform use of surrogate species in ecological risk assessment practices

The U.S. EPA frequently uses avian or fish toxicity data to set protective standards for amphibians in ecological risk assessments. However, this approach does not always adequately represent aquatic-dwelling and terrestrial-phase amphibian exposure data. For instance, it is accepted that early life stage tests for fish are typically sensitive enough to protect larval amphibians, however, metamorphosis from tadpole to a terrestrial-phase adult relies on endocrine cues that are less prevalent in fish but essential for amphibian life stage transitions. These differences suggest that more robust approaches are needed to adequately elucidate the impacts of pesticide exposure in amphibians across critical life stages. Therefore, in the current study, methodology is presented that can be applied to link the perturbations in the metabolomic response of larval zebrafish (Danio rerio), a surrogate species frequently used in ecotoxicological studies, to those of African clawed frog (Xenopus laevis) tadpoles following exposure to three high-use pesticides, bifenthrin, chlorothalonil, or trifluralin. Generally, D. rerio exhibited greater metabolic perturbations in both number and magnitude across the pesticide exposures as opposed to X. laevis. This suggests that screening ecological risk assessment surrogate toxicity data would sufficiently protect amphibians at the single life stage studied but care needs to be taken to understand the suite of metabolic requirements of each developing species. Ultimately, methodology presented, and data gathered herein will help inform the applicability of metabolomic profiling in establishing the risk pesticide exposure poses to amphibians and potentially other non-target species.

Embryotoxicity of Selective Serotonin Reuptake Inhibitors—Comparative Sensitivity of Zebrafish (Danio rerio) and African Clawed Frog (Xenopus laevis) Embryos

Over the past twenty years, the prescription of antidepressant drugs has increased all over the world. After their application, antidepressants, like other pharmaceuticals, are excreted and enter the aquatic environment. They are dispersed among surface waters mainly through waste water sources, typically at very low concentrations— from a tenth up to hundreds of ng/L. Frequently detected antidepressants include fluoxetine and citalopram—both selective serotonin reuptake inhibitors. The aim of our study was to assess the embryotoxicity of fluoxetine hydrochloride and citalopram hydrochloride on the early life stages of zebrafish (Danio rerio) and the African clawed frog (Xenopus laevis). The embryos were exposed to various concentrations of the individual antidepressants and of their mixtures for 96 h. The tested levels included both environmentally relevant and higher concentrations for the evaluation of dose-dependent effects. Our study demonstrated that even environmentally relevant concentrations of these psychiatric drugs influenced zebrafish embryos, which was proven by a significant increase (p < 0.01) in the embryos’ heart rates after fluoxetine hydrochloride exposure and in their hatching rate after exposure to a combination of both antidepressants, and thus revealed a potential risk to aquatic life. Despite these results, we can conclude that the African clawed frog is more sensitive, since exposure to the highest concentrations of fluoxetine hydrochloride (10,000 μg/L) and citalopram hydrochloride (100,000 μg/L) resulted in total mortality of the frog embryos.

Cardiac and Cerebellar Histomorphology and Inositol 1,4,5-Trisphosphate (IP3R) Perturbations in Adult Xenopus laevis Following Atrazine Exposure

Despite several reports on the endocrine-disrupting ability of atrazine in amphibian models, few studies have investigated atrazine toxicity in the heart and cerebellum. This study investigated the effect of atrazine on the unique Ca2+ channel-dependent receptor (Inositol 1,4,5-trisphosphate; IP3R) in the heart and the cerebellum of adult male Xenopus laevis and documented the associated histomorphology changes implicated in cardiac and cerebellar function. Sixty adult male African clawed frogs (Xenopus laevis) were exposed to atrazine (0 µg/L (control), 0.01 µg/L, 200 µg/L, and 500 µg/L) for 90 days. Thereafter, heart and cerebellar sections were processed with routine histological stains (heart) or Cresyl violet (brain), and IP3R histochemical localization was carried out on both organs. The histomorphology measurements revealed a significant decrease in the mean percentage area fraction of atrial (0.01 µg/L and 200 µg/L) and ventricular myocytes (200 µg/L) with an increased area fraction of interstitial space, while a significant decrease in Purkinje cells was observed in all atrazine groups (p < 0.008, 0.001, and 0.0001). Cardiac IP3R was successfully localized, and its mean expression was significantly increased (atrium) or decreased (cerebellum) in all atrazine-exposed groups, suggesting that atrazine may adversely impair cerebellar plasticity and optimal functioning of the heart due to possible disturbances of calcium release, and may also induce several associated cardiac and neural pathophysiologies in all atrazine concentrations, especially at 500 µg/L.

Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Xenopus Oocytes and Eggs by Selective Antioxidants.

Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog Xenopus laevis. The fluorescent cell-permeable dye DCFDA, which is widely employed for ROS detection in cultured mammalian cells, was used to monitor ROS levels in the fresh and bench-aged oocytes and eggs by an optimized protocol. It was found that intracellular ROS contents were increased in frog oocytes and eggs aged for 48 h. It was further demonstrated using selective cell-permeable AOXs targeting different ROS-generating mechanisms, that the major source of ROS in Xenopus oocytes and eggs is the plasma membrane NADPH oxidase, and that mitochondrial generation contributes to the intracellular ROS content to a lesser extent. Targeted inhibition of NADPH oxidase with a natural organic compound apocynin reduced ROS levels significantly in Xenopus oocytes and eggs, maintained their normal phenotype and supported their functional competence. To our knowledge this is the first report concerning beneficial effects of apocynin on the isolated gamete cells, such as oocytes and eggs.

Copepod consumption by amphibians and fish with implications for transmission of Dracunculus species.

Parasitic nematodes in the genus Dracunculus have a complex life cycle that requires more than one host species in both aquatic and terrestrial habitats. The most well-studied species, Dracunculus medinensis, is the causative agent of human Guinea worm disease (dracunculiasis). There are several other Dracunculus species that infect non-human animals, primarily wildlife (reptiles and mammals). The classic route of D. medinensis transmission to humans is through the ingestion of water containing the intermediate host, a cyclopoid copepod, infected with third-stage larvae (L3s). However, many animal hosts (e.g., terrestrial snakes, dogs) of other Dracunculus sp. appear unlikely to ingest a large number of copepods while drinking. Therefore, alternative routes of infection (e.g., paratenic or transport hosts) may facilitate Dracunculus transmission to these species. To better understand the role of paratenic and transport hosts in Dracunculus transmission to animal definitive hosts, we compared copepod ingestion rates for aquatic species (fish, frogs [tadpoles and adults], and newts) which may serve as paratenic or transport hosts. We hypothesized that fish would consume more copepods than amphibians. Our findings confirm that African clawed frogs (Xenopus laevis) and fish consume copepods, but that fish ingest, on average, significantly higher numbers (68% [34/50]) than adult African clawed frogs (36% [18/50]) during a 24-h time period. Our results suggest that amphibians and fish may play a role in the transmission of Dracunculus to definitive hosts. Still, additional research is required to determine whether, in the wild, fish or frogs are serving as paratenic or transport hosts. If so, they may facilitate Dracunculus transmission. However, if these animals simply act as dead-end hosts or as means of copepod population control, they may decrease Dracunculus transmission.

Susceptibility of anurans, lizards, and fish to infection with Dracunculus species larvae and implications for their roles as paratenic hosts

Dracunculus spp. are parasitic nematodes that infect numerous species of mammals and reptiles. The life cycles of Dracunculus species are complex, and unknowns remain regarding the role of paratenic and transport hosts in transmission to definitive hosts. We had two primary objectives: to assess the susceptibility of several species of anurans, lizards, and fish as paratenic hosts for Dracunculus species, and to determine the long-term persistence of Dracunculus infections in African clawed frogs (Xenopus laevis). Animals were orally exposed to copepods infected with infectious third-stage larvae (L3s) of either Dracunculus insignis or D. medinensis. Dracunculus L3s were recovered from four anuran species, two lizard species, and one fish species, demonstrating that Dracunculus can infect tissues of a diversity of species. In long-term persistence trials, D. medinensis L3s were recovered from African clawed frogs tissues up to 58 days post-infection, and D. insignis L3s were recovered up to 244 days post-infection. Our findings regarding the susceptibility of novel species of frogs, lizards, and fish to infection with Dracunculus nematodes, and long-term persistence of L3s in paratenic hosts, address pressing knowledge gaps regarding Dracunculus infection in paratenic hosts and may guide future research regarding the transmission of Dracunculus to definitive mammalian hosts.

Interspecific sensitivity of European amphibians towards two pesticides and comparison to standard test species

BackgroundAlthough debates about the assessment of potential effects of pesticides on amphibians are ongoing, amphibians are not yet considered in the current EU environmental risk assessment of pesticides. Instead, the risk assessment of potential effects on aquatic amphibian life stages relies on use of data of surrogate species like the standard temperate fish species rainbow trout (Oncorhynchus mykiss). This assumption is mainly based on the comparison to amphibian species not native to Europe such as the aquatic African clawed frog (Xenopus laevis). It remains unclear whether these surrogate species cover semi-aquatic Central European amphibian sensitivities. Therefore, we assessed the acute sensitivity of aquatic stages of eight European amphibian species native in Germany (Bufo bufo, Bufotes viridis, Epidalea calamita, Hyla arborea, Pelobates fuscus, Pelophylax sp., Rana dalmatina, R. temporaria) towards commercial formulations of the fungicide folpet (Folpan® 500 SC, Adama) and the insecticide indoxacarb (Avaunt® EC, Cheminova). The determined acute sensitivities (median lethal concentration, LC50) were included in species sensitivity distributions and compared to experimentally determined LC50 values of X. laevis and literature values of O. mykiss.ResultsThe results showed that native amphibian sensitivities differed between the tested pesticides with a factor of 5 and 11. Depending on the pesticide, X. laevis was five and nine times more tolerant than the most sensitive native amphibian species. Comparing literature values of O. mykiss to the experimentally determined sensitivities of the native amphibian species showed that the O. mykiss sensitivity was in the same range as for the tested amphibians for the formulation Folpan® 500 SC. The comparison of sensitivities towards the formulation Avaunt® EC showed an eight times lower sensitivity of O. mykiss than the most sensitive amphibian species.ConclusionsA risk assessment using the 96-h LC50 values for fish covers the risk for the assessed aquatic stages of European amphibians after the application of the recommended uncertainty factor of 100 and thus may be adequate for lower tier risk assessment of the studied pesticides. If aquatic amphibian testing will be required for pesticide risk assessment nevertheless, acute tests with the model organism X. laevis and the application of an appropriate uncertainty factor might be a promising approach.

Progeny of Xenopus laevis from altitudinal extremes display adaptive physiological performance.

Environmental temperature variation generates adaptive phenotypic differentiation in widespread populations. We used a common garden experiment to determine whether offspring with varying parental origins display adaptive phenotypic variation related to different thermal conditions experienced in parental environments. We compared burst swimming performance and critical thermal limits of African clawed frog (Xenopus laevis) tadpoles bred from adults captured at high (∼2000 m above sea level) and low (∼ 5 m above sea level) altitudes. Maternal origin significantly affected swimming performance. Optimal swimming performance temperature (Topt) had a >9°C difference between tadpoles with low altitude maternal origins (pure- and cross-bred, 35.0°C) and high-altitude maternal origins (pure-bred, 25.5°C; cross-bred, 25.9°C). Parental origin significantly affected critical thermal (CT) limits. Pure-bred tadpoles with low-altitude parental origins had higher CTmax (37.8±0.8°C) than pure-bred tadpoles with high-altitude parental origins and all cross-bred tadpoles (37.0±0.8 and 37.1±0.8°C). Pure-bred tadpoles with low-altitude parental origins and all cross-bred tadpoles had higher CTmin (4.2±0.7 and 4.2±0.7°C) than pure-bred tadpoles with high-altitude parental origins (2.5±0.6°C). Our study shows that the varying thermal physiological traits of Xenopus laevis tadpoles are the result of adaptive responses to their parental thermal environments. This study is one of few demonstrating potential intraspecific evolution of critical thermal limits in a vertebrate species. Multi-generation common garden experiments and genetic analyses would be required to further tease apart the relative contribution of plastic and genetic effects to the adaptive phenotypic variation observed in these tadpoles.

Maturation experiments reveal bias in the chemistry of fossil melanosomes

Abstract Fossil melanosomes are a major focus of paleobiological research because they can inform on the original coloration, phylogenetic affinities, and internal anatomy of ancient animals. Recent studies of vertebrate melanosomes revealed tissue-specific trends in melanosome-metal associations that can persist in fossils. In some fossil vertebrates, however, melanosomes from all body regions are enriched only in Cu, suggesting diagenetic overprinting of original chemistry. We tested this hypothesis using laboratory experiments on melanosomes from skin and liver of the African clawed frog Xenopus laevis. After maturation in Cu-rich media, the metal chemistry of melanosomes from these tissues converged toward a common composition, and original differences in Cu oxidation state were lost. Elevated Cu concentrations and a pervasive Cu(II) signal are likely indicators of diagenetically altered melanosomes. These results provide a robust experimental basis for interpretating the chemistry of fossil melanosomes.

EDNA is a useful tool to evaluate the success of the eradication program of Xenopus laevis in Portugal

Biological invasions are widely recognized as a major driver of global biodiversity loss. The most cost-effective answer is often population eradication, while the number of individuals is still limited. The detection of the invasive species at low densities is essential for eradication success, which can be difficult using traditional methods. Environmental DNA (eDNA) can facilitate the detection and monitoring of invasive aquatic species at low densities and can be more sensitive than traditional sampling. The African clawed frog (Xenopus laevis) is a highly invasive species and has been recorded in several European countries. In Portugal, the species was discovered in 2006 and since 2010, streams of the Lisbon area, suspected or at risk of invasion, are being monitored every year and removal is ongoing. The main method of detection is electrofishing but methods like draining artificial ponds have also been used. This program so far succeeded in containing the spread and reduced frog abundance, although total eradication has not yet been accomplished. To evaluate the success of this program, we collected water samples from fifteen sites. In each site we sampled both pools and riifles to test if lotic microhabitat influences X. laevis eDNA detection and concentration. Surface water velocity in each sampling point was also measured. We filtered the samples, extracted DNA from filters, and assayed the extracted DNA for X. laevis DNA using quantitative polymerase chain reaction (qPCR). From the fifteen sites sampled, five were positive for X. laevis eDNA: four lotic sites and one lentic site. Local eradication success was evident mainly in lentic habitats, and three potential failures were also identified. Finding X. laevis at one of these sites only after X. laevis eDNA detection was an important contribution of this technique for the success of the control and eradication program. No significative differences were found between lotic microhabitat (riffles and pools) regarding X. laevis eDNA concentration and detection but velocity affected the concentration of X. laevis eDNA captured. Our results corroborate other studies that recommend eDNA as a complementary sampling approach to other traditional methods and suggests its suitability as a tool for the detection of invasive amphibians. To be presented as a flash oral presentation at the DNAQUA International Conference - First international conference on the use of DNA for water biomonitoring.

Land cover, individual's age and spatial sorting shape landscape resistance in the invasive frog Xenopus laevis.

The description of functional connectivity is based on the quantification of landscape resistance, which represents species-specific movement costs across landscape features. Connectivity models use these costs to identify movement corridors at both individual and population levels and provide management recommendations for populations of conservation interest. Typically, resistance costs assigned to specific land cover types are assumed to be valid for all individuals of the population. Little attention has been paid to intraspecific variation in resistance costs due to age or dispersal syndrome, which may significantly affect model predictions. We quantified resistance costs in an expanding invasive population of the African clawed frog Xenopus laevis in Western France. In this principally aquatic amphibian, juveniles, sub-adults and adults disperse overland. The enhancement of dispersal traits via spatial sorting has been also observed at the range periphery of the population. Resistance costs, and thus connectivity, might vary as a function of life stage and position within the invaded range. We assessed multiple dimensions of functional connectivity. On various land cover types, we measured locomotion, as crossing speed, in different post-metamorphic age classes, and dehydration, sensitivity of locomotion to dehydration and substrate preference in juveniles. We also tested the effect of the position in the invaded range (core vs. periphery) on individual performances. In juveniles, general trends towards higher resistance costs on grass and lower resistance costs on bare soil and asphalt were observed, although not all experiments provided the same cost configurations. Resistance to locomotion varied between age classes, with adults and sub-adults facing lower costs than juveniles, particularly when crossing structurally complex land cover types such as grass and leaf litter. The position in the range had a minor effect on landscape resistance, and only in the dehydration experiment, where water loss in juveniles was lower at the range periphery. Depicting functional connectivity requires (a) assessing multiple dimensions of behavioural and physiological challenges faced by animals during movement; (b) considering factors, such as age and dispersal syndrome, that may affect movement at both individual and population levels. Ignoring this complexity might generate unreliable connectivity models and provide unsupported management recommendations for conservation.

Retinol binding protein 1 affects Xenopus anterior neural development via all-trans retinoic acid signaling.

Retinol binding protein 1 (Rbp1) acts as an intracellular regulator of vitamin A metabolism and retinoid transport. In mice, Rbp1 deficiency decreases the capacity of hepatic stellate cells to take up all-trans retinol and sustain retinyl ester stores. Furthermore, Rbp1 is crucial for visual capacity. Although the function of Rbp1 has been studied in the mature eye, its role during early anterior neural development has not yet been investigated in detail. We showed that rbp1 is expressed in the eye, anterior neural crest cells (NCCs) and prosencephalon of the South African clawed frog Xenopus laevis. Rbp1 knockdown led to defects in eye formation, including microphthalmia and disorganized retinal lamination, and to disturbed induction and differentiation of the eye field, as shown by decreased rax and pax6 expression. Furthermore, it resulted in reduced rax expression in the prosencephalon and affected cranial cartilage. Rbp1 inhibition also interfered with neural crest induction and migration, as shown by twist and slug. Moreover, it led to a significant reduction of the all-trans retinoic acid target gene pitx2 in NCC-derived periocular mesenchyme. The Rbp1 knockdown phenotypes were rescued by pitx2 RNA co-injection. Rbp1 is crucial for the development of the anterior neural tissue.

Insights from a vertebrate model organism on the molecular mechanisms of whole-body dehydration tolerance.

Studies on the molecular mechanisms of dehydration tolerance have been largely limited to plants and invertebrates. Currently, research in whole body dehydration of complex animals is limited to cognitive and behavioral effects in humans, leaving the molecular mechanisms of vertebrate dehydration relatively unexplored. The present review summarizes studies to date on the African clawed frog (Xenopus laevis) and examines whole-body dehydration on physiological, cellular and molecular levels. This aquatic frog is exposed to seasonal droughts in its native habitat and can endure a loss of over 30% of its total body water. When coping with dehydration, osmoregulatory processes prioritize water retention in skeletal tissues and vital organs over plasma volume. Although systemic blood circulation is maintained in the vital organs and even elevated in the brain during dehydration, it is done so at the expense of reduced circulation to the skeletal muscles. Increased hemoglobin affinity for oxygen helps to counteract impaired blood circulation and metabolic enzymes show altered kinetic and regulatory parameters that support the use of anaerobic glycolysis. Recent studies with X. laevis also show that pro-survival pathways such as antioxidant defenses and heat shock proteins are activated in an organ-specific manner during dehydration. These pathways are tightly coordinated at the post-transcriptional level by non-coding RNAs, and at the post-translational level by reversible protein phosphorylation. Paired with ongoing research on the X. laevis genome, the African clawed frog is poised to be an ideal animal model with which to investigate the molecular adaptations for dehydration tolerance much more deeply.

Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 μL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.