V-ATPase Regulates Retinal Progenitor Cell Proliferation During Eye Regrowth in Xenopus.

Purpose: The induction of retinal progenitor cell (RPC) proliferation is a strategy that holds promise for alleviating retinal degeneration. However, the mechanisms that can stimulate RPC proliferation during repair remain unclear. Xenopus tailbud embryos successfully regrow functional eyes within 5 days after ablation, and this process requires increased RPC proliferation. This model facilitates identification of mechanisms that can drive in vivo reparative RPC proliferation. This study assesses the role of the essential H+ pump, V-ATPase, in promoting stem cell proliferation. Methods: Pharmacological and molecular loss of function studies were performed to determine the requirement for V-ATPase during embryonic eye regrowth. The resultant eye phenotypes were examined using histology and antibody markers. Misexpression of a yeast H+ pump was used to test whether the requirement for V-ATPase in regrowth is dependent on its H+ pump function. Results: V-ATPase inhibition blocked eye regrowth. Regrowth-incompetent eyes resulting from V-ATPase inhibition contained the normal complement of tissues but were much smaller. V-ATPase inhibition caused a significant reduction in reparative RPC proliferation but did not alter differentiation and patterning. Modulation of V-ATPase activity did not affect apoptosis, a process known to be required for eye regrowth. Finally, increasing H+ pump activity was sufficient to induce regrowth. Conclusions: V-ATPase is required for eye regrowth. These results reveal a key role for V-ATPase in activating regenerative RPC proliferation and expansion during successful eye regrowth.

Bimodal modulation of short-term motor memory via dynamic sodium pumps in a vertebrate spinal cord.

Dynamic neuronal Na+/K+ pumps normally only respond to intense action potential firing owing to their low affinity for intracellular Na+. Recruitment of these Na+ pumps produces a post-activity ultraslow afterhyperpolarization (usAHP) up to ∼10 mV in amplitude and ∼60 s in duration, which influences neuronal properties and future network output. In spinal motor networks, the usAHP underlies short-term motor memory (STMM), reducing the intensity and duration of locomotor network output in a manner dependent on the interval between locomotor bouts. In contrast to tonically active Na+ pumps that help set and maintain the resting membrane potential, dynamic Na+ pumps are selectively antagonized by low concentrations of ouabain, which, we show, blocks both the usAHP and STMM. We examined whether dynamic Na+ pumps and STMM can be influenced by neuromodulators, focusing on 5-HT and nitric oxide. Bath-applied 5-HT alone had no significant effect on the usAHP or STMM. However, this is due to the simultaneous activation of two distinct 5-HT receptor subtypes (5-HT7 and 5-HT2a) that have opposing facilitatory and suppressive influences, respectively, on these two features of the locomotor system. Nitric oxide modulation exerts a potent inhibitory effect that can completely block the usAHP and erase STMM. Using selective blockers of 5-HT7 and 5-HT2a receptors and a nitric oxide scavenger, PTIO, we further provide evidence that the two modulators constitute an endogenous control system that determines how the spinal network self-regulates the intensity of locomotor output in light of recent past experience.

Obtaining Xenopus tropicalis Embryos by In Vitro Fertilization.

Xenopus is a powerful model system for cell and developmental biology in part because frogs produce thousands of eggs and embryos year-round. In vitro fertilization (IVF) is ideal for obtaining developmentally synchronized embryos for microinjection or when natural mating has failed to produce a fertilization. In IVF, females are induced to ovulate, and then eggs are collected by manual expression. After testes are collected from a euthanized male frog, the eggs are fertilized in vitro. The embryos are then treated with cysteine to remove the sticky protective jelly coat. Dejellied embryos are much easier to manipulate during microinjection or when sorting in a Petri dish. The jelly coat is also very difficult to penetrate with an injection needle. After microinjection, embryos are maintained in Petri dishes until desired stages are reached. Although in vitro fertilization in X. laevis and X. tropicalis is similar, critical differences in solutions, handling of testis, response of fertilized eggs directly after introduction of sperm, and developmental timing are required for successful fertilization in X. tropicalis.

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease.

INTRODUCTION: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. METHODS: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. RESULTS: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. CONCLUSION: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.

Spinal Cord Transection In Xenopus laevis Tadpoles.

Spinal cord injury (SCI) is a permanent affliction, which affects the central nervous system (CNS) motor and sensory nerves, resulting in paralysis beneath the injury site. To date, there is no functional recovery therapy for SCI, and there is a lack of clarity regarding the many complexes and dynamic events occurring after SCI. Many non-mammalian organisms can regenerate after severe SCI, such as teleost fishes, urodele amphibians, and larval stages of anuran amphibians, including Xenopus laevis tadpoles. These are bona fide model organisms to study and understand the response to SCI and the mechanisms underlying successful regenerative processes. This type of research can lead to the identification of potential targets for SCI therapeutic intervention. This article describes how to perform Xenopus laevis tadpole spinal cord transection, including husbandry, surgery, postsurgery care, and functional test evaluation. This injury method can be applied for elucidating the different steps of spinal cord regeneration by studying the cellular, molecular, and genetic mechanisms, as well as histological and functional evolution after SCI and during spinal cord regeneration.

Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts.

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.

Thyroid hormone-induced expression of Foxl1 in subepithelial fibroblasts correlates with adult stem cell development during Xenopus intestinal remodeling.

In the Xenopus laevis intestine during metamorphosis, stem cells appear and generate the adult epithelium analogous to the mammalian one. We have previously shown that connective tissue cells surrounding the epithelium are essential for the stem cell development. To clarify whether such cells correspond to mammalian Foxl1-expressing mesenchymal cells, which have recently been shown to be a critical component of intestinal stem cell niche, we here examined the expression profile of Foxl1 in the X. laevis intestine by using RT-PCR and immunohistochemistry. Foxl1 expression was transiently upregulated only in connective tissue cells during the early period of metamorphic climax and was the highest just beneath the proliferating stem/progenitor cells. In addition, electron microscopic analysis showed that these subepithelial cells are ultrastructurally identified as telocytes like the mammalian Foxl1-expressing cells. Furthermore, we experimentally showed that Foxl1 expression is indirectly upregulated by thyroid hormone (TH) through Shh signaling and that TH organ-autonomously induces the Foxl1-expressing cells concomitantly with appearance of the stem cells in the tadpole intestine in vitro. The present results suggest that intestinal niche cells expressing Foxl1 are evolutionally conserved among terrestrial vertebrates and can be induced by TH/Shh signaling during amphibian metamorphosis for stem cell development.

Cross-Talk between P2X and NMDA Receptors.

Purinergic P2X receptors (P2X) are ATP-gated ion channels widely expressed in the CNS. While the direct contribution of P2X to synaptic transmission is uncertain, P2X reportedly affect N-methyl-D-aspartate receptor (NMDAR) activity, which has given rise to competing theories on the role of P2X in the modulation of synapses. However, P2X have also been shown to participate in receptor cross-talk: an interaction where one receptor (e.g., P2X2) directly influences the activity of another (e.g., nicotinic, 5-HT3 or GABA receptors). In this study, we tested for interactions between P2X2 or P2X4 and NMDARs. Using two-electrode voltage-clamp electrophysiology experiments in Xenopus laevis oocytes, we demonstrate that both P2X2 and P2X4 interact with NMDARs in an inhibited manner. When investigating the molecular domains responsible for this phenomenon, we found that the P2X2 c-terminus (CT) could interfere with both P2X2 and P2X4 interactions with NMDARs. We also report that 11 distal CT residues on the P2X4 facilitate the P2X4-NMDAR interaction, and that a peptide consisting of these P2X4 CT residues (11C) can disrupt the interaction between NMDARs and P2X2 or P2X4. Collectively, these results provide new evidence for the modulatory nature of P2X2 and P2X4, suggesting they might play a more nuanced role in the CNS.

Xela DS2 and Xela VS2: Two novel skin epithelial-like cell lines from adult African clawed frog (Xenopus laevis) and their response to an extracellular viral dsRNA analogue.

The skin epithelial layer acts as an important immunological barrier against pathogens and is capable of recognizing and responding to pathogen-associated molecular patterns (PAMPs) in human and mouse models. Although presumed, it is unknown whether amphibian skin epithelial cells exhibit the ability to respond to PAMPs such as viral double-stranded RNA (dsRNA). To address this, two cell lines from the dorsal skin (Xela DS2) and ventral skin (Xela VS2) of the African clawed frog (Xenopus laevis) were established. Xela DS2 and Xela VS2 cells have an epithelial-like morphology, express genes associated with epithelial cells, and lack senescence-associated beta-galactosidase activity. Cells grow optimally in 70% Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 26 °C. Upon treatment with poly(I:C), a synthetic analogue of viral dsRNA and known type I interferon inducer, Xela DS2 and Xela VS2 exhibit marked upregulation of key antiviral and pro-inflammatory transcripts suggesting frog epithelial cells participate in the recognition of extracellular viral dsRNA and production of local inflammatory signals; similar to human and mouse models. Currently, these are the only known Xenopus laevis skin epithelial-like cell lines and will be important for future research in amphibian epithelial cell biology, initial host-pathogen interactions, and rapid screening of the effects of environmental stressors, including contaminants, on frog skin epithelial cells.

Tissue mechanics drives regeneration of a mucociliated epidermis on the surface of Xenopus embryonic aggregates.

Injury, surgery, and disease often disrupt tissues and it is the process of regeneration that aids the restoration of architecture and function. Regeneration can occur through multiple strategies including stem cell expansion, transdifferentiation, or proliferation of differentiated cells. We have identified a case of regeneration in Xenopus embryonic aggregates that restores a mucociliated epithelium from mesenchymal cells. Following disruption of embryonic tissue architecture and assembly of a compact mesenchymal aggregate, regeneration first restores an epithelium, transitioning from mesenchymal cells at the surface of the aggregate. Cells establish apico-basal polarity within 5 hours and a mucociliated epithelium within 24 hours. Regeneration coincides with nuclear translocation of the putative mechanotransducer YAP1 and a sharp increase in aggregate stiffness, and regeneration can be controlled by altering stiffness. We propose that regeneration of a mucociliated epithelium occurs in response to biophysical cues sensed by newly exposed cells on the surface of a disrupted mesenchymal tissue.

Relationship between the Induced Iron Overload Model and Hepatic Erythropoiesis in Xenopus laevis.

Iron is an essential element for hemoglobin synthesis during erythropoiesis. Iron overload, in contrast, adversely affects erythropoiesis and causes organ dysfunction. Research using various animal models may help to elucidate pathophysiological mechanisms induced by excess iron. In the present study, we evaluated the relationship between iron metabolism and erythropoietic activity in the African clawed frog, Xenopus laevis. In X. laevis, both erythropoiesis and iron metabolism occur in the liver. First, we developed a method to quantify iron levels in the liver and plasma using 2-nitroso-5-[N-n-propyl-N-(3-sulfopropyl) amino] phenol (Nitroso-PSAP). We then measured iron levels and analyzed hematopoietic parameters in frogs that were orally administered sodium ferrous citrate (SFC). The hepatic iron level increased in the SFC group, but the number of erythrocytes, hematocrit, and hemoglobin concentration did not change, suggesting that the regulation of the production and release of mature erythrocytes in the liver was not directly affected by dietary iron. At four days after administration of 2 mg/kg SFC, the number of immature erythrocytes decreased in the liver. Interestingly, atypical blood cells with hyper-segmented nuclei were observed, identified by acridine orange cell staining; these atypical blood cells were hardly detectable under the steady state. Compared with previously reported results in mice, the increase in the hepatic iron levels was small, but our results indicate that SFC affects hematopoietic activity. These results establish a novel model for iron metabolism and provide new insights into the relationship between iron metabolism and erythropoiesis in vertebrates.

P2X Electrophysiology and Surface Trafficking in Xenopus Oocytes.

Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.

The TFF Peptides xP1 and xP4 Appear in Distinctive Forms in the Xenopus laevis Gastric Mucosa: Indications for Different Protective Functions.

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

Xenopus laevis as a Bioindicator of Endocrine Disruptors in the Region of Central Chile.

One of the direct causes of biodiversity loss is environmental pollution resulting from the use of chemicals. Different kinds of chemicals, such as persistent organic pollutants and some heavy metals, can be endocrine disruptors, which act at low doses over a long period of time and have a negative effect on the reproductive and thyroid system in vertebrates worldwide. Research on the effects of endocrine disruptors and the use of bioindicators in neotropical ecosystems where pressure on biodiversity is high is scarce. In Chile, although endocrine disruptors have been detected at different concentrations in the environments of some ecosystems, few studies have been performed on their biological effects in the field. In this work, Xenopus laevis (African clawed frog), an introduced species, is used as a bioindicator for the presence of endocrine disruptors in aquatic systems with different degrees of contamination in a Mediterranean zone in central Chile. For the first time for Chile, alterations are described that can be linked to exposure to endocrine disruptors, such as vitellogenin induction, decreased testosterone in male frogs, and histological changes in gonads. Dioxin-like and oestrogenic activity was detected in sediments at locations where it seem to be related to alterations found in the frogs. In addition, an analysis of land use/cover use revealed that urban soil was the best model to explain the variations in frog health indicators. This study points to the usefulness of an invasive species as a bioindicator for the presence of endocrine-disruptive chemicals.

The plus maze and scototaxis test are not valid behavioral assays for anxiety assessment in the South African clawed frog.

There are no behavioral models for testing anxiety in amphibians, a group of animals widely used for developmental, ecotoxicological, and genetic research. We aimed to validate two common rodent paradigms, the plus maze and the scototaxis test, for use in the aquatic African clawed frog (Xenopus laevis). We predicted: (a) that frogs would prefer the dark, vs. light, portions of the testing arenas (face validity), (b) that this behavior could be altered with acute administration of anxio-selective drugs (construct validity), and (c) that time spent in the dark portions of the arenas would be positively correlated (predictive validity). Prior to testing, frogs were treated with fluoxetine (selective serotonin reuptake inhibitor [SSRI]), desipramine (serotonin- and norepinephrine-reuptake inhibitor), caffeine (methylxanthine, adenosine receptor antagonist, phosphodiesterase inhibitor), saline, or were left unmanipulated. Each drug was administered acutely (1 h prior to testing; caffeine) or subacutely (24, 3, and 1 h prior to testing; fluoxetine, desipramine) at one of three doses. Plus maze and scototaxis testing were separated by 1 week; each frog completed both behavioral tasks and was treated with the same drug regimen prior to testing. Overall, both tests showed face validity, however, data suggest these paradigms lack both construct and predictive validity.

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community's knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

Developmental exposure to chemicals associated with unconventional oil and gas extraction alters immune homeostasis and viral immunity of the amphibian Xenopus.

Although aquatic vertebrates and humans are increasingly exposed to water pollutants associated with unconventional oil and gas extraction (UOG), the long-term effects of these pollutants on immunity remains unclear. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable and sensitive model for evaluating the effects of waterborne pollutants. X. laevis tadpoles were exposed to a mixture of equimass amount of UOG chemicals with endocrine disrupting activity (0.1 and 1.0 µg/L) for 3 weeks, and then long-term effects on immune function at steady state and following viral (FV3) infection was assessed after metamorphosis. Notably, developmental exposure to the mixture of UOG chemicals at the tadpole stage affected metamorphic development and fitness by significantly decreasing body mass after metamorphosis completion. Furthermore, developmental exposure to UOGs resulted in perturbation of immune homeostasis in adult frogs, as indicated by significantly decreased number of splenic innate leukocytes, B and T lymphocytes; and a weakened antiviral immune response leading to increased viral load during infection by the ranavirus FV3. These findings suggest that mixture of UOG-associated waterborne endocrine disruptors at low but environmentally-relevant levels have the potential to induce long-lasting alterations of immune function and antiviral immunity in aquatic vertebrates and ultimately human populations.

Development of a Photoswitchable Lithium-Sensitive Probe to Analyze Nonselective Cation Channel Activity in Migrating Cancer Cells.

This is the first work to use a newly designed Li+-selective photoswitchable probe Sabrina Heng Lithium (SHL) in living colon cancer cells to noninvasively monitor cation channel activity in real time by the appearance of lithium hot spots detected by confocal microscopy. Punctate Li+ hot spots are clustered in the lamellipodial leading edges of HT29 human colon cancer cells and are colocalized with aquaporin-1 (AQP1) channels. AQP1 is a dual water and cyclic-nucleotide-gated cation channel located in lamellipodia and is essential for rapid cell migration in a subset of aggressive cancers. Both the Li+ hot spots and cell migration are blocked in HT29 cells by the AQP1 ion channel antagonist AqB011. In contrast, Li+ hot spots are not evident in a poorly migrating colon cancer cell line, SW620, which lacks comparable membrane expression of AQP1. Knockdown of AQP1 by RNA interference in HT29 cells significantly impairs Li+ hot spot activity. The SHL probe loaded in living cells shows signature chemical properties of ionic selectivity and reversibility. Dynamic properties of the Li+ hot spots, turning on and off, are confirmed by time-lapse imaging. SHL is a powerful tool for evaluating cation channel function in living cells in real time, with particular promise for studies of motile cells or interlinked networks not easily analyzed by electrophysiological methods. The ability to reset SHL by photoswitching allows monitoring of dynamic signals over time. Future applications of the Li+ probe could include high-throughput optical screening for discovering new classes of channels, or finding new pharmacological modulators for nonselective cation channels.

Etv6 activates vegfa expression through positive and negative transcriptional regulatory networks in Xenopus embryos.

VEGFA signaling controls physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VEGFA have been uncovered, vegfa transcriptional regulation in vivo remains unclear. Here, we show that the ETS transcription factor, Etv6, positively regulates vegfa expression during Xenopus blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits expression of the repressor foxo3, to prevent Foxo3 from binding to and repressing the vegfa promoter. Etv6 also directly activates expression of the activator klf4; reflecting a genome-wide paucity in ETS-binding motifs in Etv6 genomic targets, Klf4 then recruits Etv6 to the vegfa promoter to activate its expression. These two mechanisms (double negative gate and feed-forward loop) are classic features of gene regulatory networks specifying cell fates. Thus, Etv6's dual function, as a transcriptional repressor and activator, controls a major signaling pathway involved in endothelial and blood development in vivo.

Studies of Limb Regeneration in Larval Xenopus.

A basic protocol is given for animal maintenance and surgery in studies of hindlimb regeneration in larval Xenopus laevis Unlike urodele limbs, those of larval frogs typically show much more variation in the extent of regeneration after amputation. Such variation can be reduced by optimizing the conditions of larval maintenance to regulate the rates of growth and development, by selecting only larvae with normal rates of growth and morphological development for experimental use, and by attention to precision and consistency in the proximo-distal level of surgical amputation.