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Understanding how living tissues respond to changes in their mechanical environment is a key question in evolutionary biology. Invasive species provide an ideal model for this as they are often transplanted between environments that differ drastically in their ecological and environmental context. Spatial sorting, the name given to the phenomenon driving differences between individuals at the core and edge of an expanding range, has been demonstrated to impact the morphology and physiology of Xenopus laevis from the invasive French population. Here, we combined a structural analysis using micro-CT scanning and a functional analysis by testing the mechanical properties of the femur to test whether the increased dispersal at the range edge drives differences in bone morphology and function. Our results show significant differences in the inner structure of the femur as well as bone material properties, with frogs from the centre of the range having more robust and resistant bones. This is suggestive of an energy allocation trade-off between locomotion and investment in bone formation, or alternatively, may point to selection for fast locomotion at the range edge. Overall, our results provide insights on the growth of the long bones and the formation of trabecular bone in frogs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The microvascular anatomy of choriocapillaris, iris, ciliary body, and superficial vascular hyaloid system of eyes was studied in the permanent aquatic Xenopus laevis by scanning electron microscopy of vascular casts and was compared with that published in two semiaquatic ranid species (Rana esculenta and Rana temporaria), and the urodelian species Triturus criststus carnifex. Results showed that the choriocapillaris in Xenopus consisted of a dense meshwork of wide capillaries displaying polygonal arrays at the scleral side with venules leaving the centers and arterioles supplied from the periphery. The choriocapillaris lacked the multilayered capillary meshwork described in ranids. Iris and ciliary body were supplied by nasal and temporal branches of the iridial artery, which either originated with a common stem from the hyaloid artery or arose as individual vessels from the proximal portions of the semicircular nasal and temporal branches of the hyaloid artery. These branches ran in the pupillary margin and supplied the two-dimensional capillary network of the iris, as well as the three-dimensional network of the ciliary body. Iris and ciliary body drained via parallel running vasa recta into the choriocapillaris. The superficial vascular hyaloid bed (system) was supplied by the hyaloid artery. This artery coursed along the scleral surface of the ventrotemporal choriocapillaris toward the ora serrata, where it bifurcated into a temporal and a nasal semicircular branch. Seven to 10 arterial meridional twigs arose from these branches and supplied the superficial hyaloid capillary bed. Capillaries drained into branches of the hyaloid vein, which ascended toward the ora serrata, where the hyaloid vein joined the temporal branch of the ciliary vein., (© 2023 The Authors. Journal of Morphology published by Wiley Periodicals LLC.)

Robot swarms have, to date, been constructed from artificial materials. Motile biological constructs have been created from muscle cells grown on precisely shaped scaffolds. However, the exploitation of emergent self-organization and functional plasticity into a self-directed living machine has remained a major challenge. We report here a method for generation of in vitro biological robots from frog ( Xenopus laevis ) cells. These xenobots exhibit coordinated locomotion via cilia present on their surface. These cilia arise through normal tissue patterning and do not require complicated construction methods or genomic editing, making production amenable to high-throughput projects. The biological robots arise by cellular self-organization and do not require scaffolds or microprinting; the amphibian cells are highly amenable to surgical, genetic, chemical, and optical stimulation during the self-assembly process. We show that the xenobots can navigate aqueous environments in diverse ways, heal after damage, and show emergent group behaviors. We constructed a computational model to predict useful collective behaviors that can be elicited from a xenobot swarm. In addition, we provide proof of principle for a writable molecular memory using a photoconvertible protein that can record exposure to a specific wavelength of light. Together, these results introduce a platform that can be used to study many aspects of self-assembly, swarm behavior, and synthetic bioengineering, as well as provide versatile, soft-body living machines for numerous practical applications in biomedicine and the environment., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We studied urinary bladders of adult male and female Xenopus laevis using light microscopy of stained tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Results showed that bilaterally a vesical artery branched off the femoral artery. At the dorso-lateral serosal surface of the body of the bladder each artery splitted within a short distance into up to five smaller arteries that supplied body and neck regions. Arteries gave off short and long terminal arterioles, which fed the mucosal capillary meshwork. Long terminal arterioles followed dimensional changes of the bladder, while short ones anchored the capillary network to the arterial system. Capillary mesh sizes and shapes varied according to the filling state of the urinary bladder. In the highly to moderately distended (filled) bladder, capillaries were rather straight or undulated only slightly, in the contracted (emptied) bladder they undulated strongly and lay side by side. Postcapillary venules formed by two equally sized capillaries or from capillaries, which serially drained into a small postcapillary venule. Vesical venules formed a large dorsal vesical and a varying number of smaller lateral and ventral vesical veins. The dorsal vesical vein drained either directly or via the posterior hemorrhoidal vein into the common pelvic vein. Lateral and ventral vesical veins also drained into the latter. The vascular patterns found were discussed in respect to the bladder spatial movements during distention (filling) and relaxation (emptying). Furthermore, it was hypothesized that an extensively filled bladder could compress the overlaying abdominal vein forcing part of the blood otherwise drained towards the liver to be detoured via the renal portal veins to the kidneys., (© 2020 The Authors. Journal of Morphology published by Wiley Periodicals LLC.)

Ovaries and oviducts of the adult African Clawed Toad (Xenopus laevis DAUDIN, 1802) were studied by light microscopy (LM) of paraplast embedded tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Histomorphology revealed that ovarian vessels located in the thecal layers. Ovarian and interlobar arteries displayed a horse-shoe shaped longitudinally running bundle of vascular smooth muscle cells. Follicular blood vessels showed flattened profiles, which were confirmed by scanning electron microscopy in vascular corrosion casts. The flattened profiles obviously led to high intravasal pressures, which locally prevented filling of the follicular capillary bed. Oviduct arteries pierced the fibrous stroma surrounding the oviduct mucosa. In the pars convoluta, the mucosa consisted of a ciliated simple columnar epithelium and tubular oviduct glands that opened between ciliated epithelial cells into the oviduct lumen. Oviduct arteries branched at the basolateral surfaces of tubular glands. After a short tangential course, arterioles branched into capillaries which ran radially between oviduct glands towards the subepithelium. Anastomoses at different heights connected capillaries of neighbouring glands. Subepithelially, capillaries ran longitudinally and undulated. Postcapillary venules radiated centrifugally towards the stroma to finally drain into oviduct veins located in the stroma. Oviduct vascular densities clearly reflected non-ovulatory and ovulatory states., (© 2020 The Authors. Anatomia, Histologia, Embryologia published by Wiley-VCH GmbH.)

Invasive species are a global scourge and often negatively impact native species. Understanding the expansion and dispersal limits of these species is essential. As previous studies have demonstrated increased locomotor performance for populations at the edge of the range of expanding populations, studies of locomotion including the anatomical and physiological traits underlying dispersal capacity are of interest. We focus here on an invasive population of Xenopus laevis introduced in France nearly forty years ago. Previous studies have demonstrated differences in mobility between populations from the centre and the edge of the invasive range, with individuals from the range edge possessing a higher endurance capacity. We test here whether range-edge frogs show anatomical differences in organs or muscles underlying these observed differences of performance. We dissected 10 males and 10 females from central and range-edge sites (40 animals in total) and measured the mass of their organs and the mass, the length, and the physiological cross-sectional area (PCSA) of 28 hind limb muscles. Our results show anatomical differences with individuals from the range edge possessing heavier, longer and more forceful muscles. Moreover, females from the range edge had a heavier heart but lighter stomach than those of the centre of the range. Future studies comparing the morphology between native and invasive populations in other regions or for other species will be especially insightful to better understand the possible adaptive changes in invasive populations and the limits on dispersal capacity., (© 2019 Anatomical Society.)

The cys-loop superfamily of ligand-gated ion channels are well recognized as important drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. The acetylcholine-gated chloride channel (ACC) family is a unique family of cholinergic receptors that have been shown, using Caenorhabditis elegans as a model, to have potential as anti-parasitic drug targets. However, there is little known about the function of these receptors in parasitic nematodes. Here, we have identified an acc gene (hco-acc-1) from the sheep parasitic nematode Haemonchus contortus. While similar in sequence to the previously characterized C. elegans ACC-1 receptor, Hco-ACC-1 does not form a functional homomeric channel in Xenopus oocytes. Instead, co-expression of Hco-ACC-1 with a previously characterized subunit Hco-ACC-2 produced a functional heteromeric channel which was 3x more sensitive to acetylcholine compared to the Hco-ACC-2 homomeric channel. We have also found that Hco-ACC-1 can be functionally expressed in C. elegans. Overexpression of both cel-acc-1 and hco-acc-1 in both C. elegans N2 and acc-1 null mutants decreased the time for worms to initiate reversal avoidance to octanol. Moreover, antibodies were generated against the Hco-ACC-1 protein for use in immunolocalization studies. Hco-ACC-1 consistently localized to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus. On the other hand, expression of Hco-ACC-1 in C. elegans was restricted to neuronal tissue. Overall, this research has provided new insight into the potential role of ACC receptors in parasitic nematodes., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Animals that are small compared to sound wavelengths face the challenge of localizing a sound source since the main cues to sound direction-interaural time differences (ITD) and interaural level differences (ILD)-both depend on size. Remarkably, the majority of terrestrial vertebrates possess internally coupled ears (ICE) with an air-filled cavity connecting the two eardrums and producing an inherently directional middle-ear system. Underwater, longer wavelengths and faster sound-speed reduce both ITD and ILD cues. Nonetheless, many animals communicate through and localize underwater sound. Here, a typical representative equipped with ICE is studied: the fully aquatic clawed frog Xenopus laevis . It is shown that two factors improve underwater sound-localization quality. First, inflated lungs function as Helmholtz resonator and generate directional amplitude differences between eardrum vibrations in the high-frequency (1.7-2.2 kHz) and low-frequency (0.8-1.2 kHz) range of the male advertisement calls. Though the externally arriving ILDs practically vanish, the perceived internal level differences are appreciable, more than 10 dB. As opposed to, e.g., lizards with thin and flexible eardrums, plate-like eardrums are shown to be Xenopus ' second key to successfully handling aquatic surroundings. Based on ICE, both plate-like eardrums and inflated lungs functioning as Helmholtz resonators explain the phonotaxis performance of Xenopus .

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities., (© 2018 Wiley Periodicals, Inc.)

To demonstrate the 3D microvascular anatomy of the brain of the model organism Xenopus laevis Daudin scanning electron microscopy of vascular corrosion casts was correlated with light microscopy of stained 7 µm thick serial tissues sections. Results showed that supplying arteries descended from the leptomeningeal surface without remarkable branchings straight to the subventricular zone where they branched and capillarized. Capillaries showed few H- and/or Y-shaped anastomoses during their centrifugal course toward the leptomeningeal surface where they drained into cerebral venules and veins. Apart from the accessory olfactory bulb and the vestibule-cochlear nucleus where capillaries were densely packed, capillaries formed a wide-meshed 3D network throughout the brain parenchyma and thus contrasted to urodelian brains where hairpin-shaped capillaries descend from the leptomeningeal vessels into varying depths of the brain parenchyma. In about two-third of specimens, a closed arterial circle of Willis was found at the base of the brain. If this circle in Xenopus might serve the same two functions as in men is briefly discussed. Choroid plexuses of third and fourth ventricle were found to have a high venous, but a low arterial inflow via one small choroidal artery only. Findings are compared with previous studies on the vascularization of the anuran brain and discrepancies in respect to presence or absence of particular arteries and/or veins in Ranids, Bufonids, and Pipids studied so far are discussed with particular emphasis on the techniques used in the various studies published so far., (© 2018 Wiley Periodicals, Inc.)

Here we propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. In this procedure, we used a newly developed fixative containing formalin, Triton X-100, and potassium hydroxide, which allows the fixation, decolorization, and transparentization of specimens at the same time. A bone staining solution containing alizarin red S with ethylene glycol and a clearing solution containing Tween 20 and potassium hydroxide also contributed the specificity and swiftness of this new system. As expected, although details of the skeletal system could be observed in specimens with high transparency, it was noteworthy that high-resolution fluorescence images acquired using zoom microscopes clearly delineated the shape of each bone. This new procedure would be expected to be widely used as a standard procedure for bone staining in the testing the developmental toxicity of chemicals and in the screening test of knockout or mutant animals.

Muscarinic acetylcholine receptors (mAChRs) mediate effects of acetylcholine (ACh) in many systems, including those involved in spinal functions like locomotion. In Xenopus laevis tadpoles at two days old, a model vertebrate for motor control research, we investigated the role of mAChRs in the skin mechanosensory pathway. We found that mAChR activation by carbachol did not affect the sensory Rohon-Beard neuron properties. However, carbachol could hyperpolarise sensory interneurons and decrease their voltage responses to outward currents. Carbachol could increase the threshold for the mechanosensory pathway to start swimming, preventing the initiation of swimming at higher concentrations altogether. Recording from the sensory interneurons in carbachol showed that their spiking after skin stimulation was depressed. However, the general muscarinic antagonist atropine did not have a clear effect on the swimming threshold or the modulation of sensory interneuron membrane conductance. Our results suggest the skin mechanosensory pathway may be subject to muscarinic modulation in this simple vertebrate system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Xenopus laevis is one of the most widely used model organism in neurobiology. It is therefore surprising, that no detailed and complete description of the cranial nerves exists for this species. Using classical histological sectioning in combination with fluorescent whole mount antibody staining and micro-computed tomography we prepared a detailed innervation map and a freely-rotatable three-dimensional (3D) model of the cranial nerves and anterior-most spinal nerves of early X. laevis tadpoles. Our results confirm earlier descriptions of the pre-otic cranial nerves and present the first detailed description of the post-otic cranial nerves. Tracing the innervation, we found two previously undescribed head muscles (the processo-articularis and diaphragmatico-branchialis muscles) in X. laevis. Data on the cranial nerve morphology of tadpoles are scarce, and only one other species (Discoglossus pictus) has been described in great detail. A comparison of Xenopus and Discoglossus reveals a relatively conserved pattern of the post-otic and a more variable morphology of the pre-otic cranial nerves. Furthermore, the innervation map and the 3D models presented here can serve as an easily accessible basis to identify alterations of the innervation produced by experimental studies such as genetic gain- and loss of function experiments., (© 2017 Wiley Periodicals, Inc.)

The tendency of animals to follow boundaries within their environment can serve as a strategy for spatial learning or defensive behaviour. We examined whether Xenopus laevis tadpoles and froglets employ such a strategy by characterizing their swimming pattern in a square tank with shallow water. Trajectories obtained from video recordings were analysed for proximity to the nearest wall. With the exception of young larvae, the vast majority of animals (both tadpoles and froglets) spent a disproportionately large amount of time near the wall. The total distance covered was not a confounding factor, but animals were stronger wall followers in smaller tanks. Wall following was also not influenced by whether the surrounding walls of the tank were black or white, illuminated by infrared light, or by the presence or absence of tentacles. When given a choice in a convex tank to swim straight and leave the wall or turn to follow the wall, the animals consistently left the wall, indicating that wall following in X. laevis is barrier-driven. This implies that wall following behaviour in Xenopus derives from constraints imposed by the environment (or the experimenter) and is unlikely a strategy for spatial learning or safety seeking.

Methods for using exogenous fluorophore and general anesthetic 1-aminoanthracene (1-AMA) and its photoactive derivative 1-azidoanthracene (1-AZA) are provided. 1-AMA potentiates GABA A chloride currents and immobilizes Xenopus laevis tadpoles. Cellular and tissue anesthetic distribution can be imaged for quantifying "on-pathway" and "off-pathway" targets. 1-AZA shares targets with 1-AMA and offers further optoanesthetic spatial and temporal control upon near-UV laser irradiation. Furthermore, 1-AZA adduction provides screening of possible relevant anesthetic protein targets and binding site characterization. We highlight several useful imaging and binding assays to demonstrate utility of 1-AMA and its derivative 1-AZA., (© 2018 Elsevier Inc. All rights reserved.)

Xenopus laevis is widely used as a model organism in biological research. Morphological descriptions of the larval cartilaginous skeleton are more than half a century old and comprehensive studies of early cartilage differentiation and development are missing. A proper understanding of early cranial skeletal development in X. laevis requires a detailed description that can function as a baseline for experimental studies. This basis makes it possible to evaluate skeletal defects produced by experiments on gene interactions, such as gain- or loss-of function experiments. In this study, we provide a detailed description of the pattern and timing of early cartilage differentiation and development in the larval head of X. laevis. Methods used include antibody staining, confocal laser scanning microscopy and 3D-reconstruction. Results were than compared to earlier studies based on classical histological approaches and clearing-and-staining. The first cartilage to chondrify is, in contrast to other vertebrates investigated so far, the ceratohyal. The components of the branchial basket chondrify in anterior-to-posterior direction as reported for other amphibians. Chondrification of different cartilages begins at different stages and the majority of cartilages are fully developed at Ziermann and Olsson stage 17. Our baseline data on the pattern and timing of early cartilaginous development in X. laevis is useful for evaluation of experiments which alter head skeletal development as well as for identifying heterochronic shifts in head development in other amphibians., (© 2017 Wiley Periodicals, Inc.)

During nervous system development growing axons can interact with each other, for example by adhering together in order to produce bundles (fasciculation). How does such axon-axon interaction affect the resulting axonal trajectories, and what are the possible benefits of this process in terms of network function? In this paper we study these questions by adapting an existing computational model of the development of neurons in the Xenopus tadpole spinal cord to include interactions between axons. We demonstrate that even relatively weak attraction causes bundles to appear, while if axons weakly repulse each other their trajectories diverge such that they fill the available space. We show how fasciculation can help to ensure axons grow in the correct location for proper network formation when normal growth barriers contain gaps, and use a functional spiking model to show that fasciculation allows the network to generate reliable swimming behaviour even when overall synapse counts are artificially lowered. Although we study fasciculation in one particular organism, our approach to modelling axon growth is general and can be widely applied to study other nervous systems.

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007-1020, 2017., (© 2017 Wiley Periodicals, Inc.)

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10-PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL-1β and TNF-α, were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086-1100, 2017., (© 2017 Wiley Periodicals, Inc.)

The African clawed frog, Xenopus laevis, is one of the most widely used model organisms in biological research. However, the most recent anatomical description of X. laevis was produced nearly a century ago. Compared with other anurans, pipid frogs - including X. laevis - exhibit numerous unusual morphological features; thus, anatomical descriptions of more 'typical' frogs do not detail many aspects of X. laevis skeletal and soft-tissue morphology. The relatively new method of using iodine-based agents to stain soft tissues prior to high-resolution X-ray imaging has several advantages over gross dissection, such as enabling dissection of very small and fragile specimens, and preserving the three-dimensional topology of anatomical structures. Here, we use contrast-enhanced computed tomography to produce a high-resolution three-dimensional digital dissection of a post-metamorphic X. laevis to successfully visualize: skeletal and muscular anatomy; the nervous, respiratory, digestive, excretory and reproductive systems; and the major sense organs. Our digital dissection updates and supplements previous anatomical descriptions of this key model organism, and we present the three-dimensional data as interactive portable document format (PDF) files that are easily accessible and freely available for research and educational purposes. The data presented here hold enormous potential for applications beyond descriptive purposes, particularly for biological researchers using this taxon as a model organism, comparative anatomy and biomechanical modelling., (© 2017 Anatomical Society.)

While an injury to the central nervous system (CNS) in humans and mammals is irreversible, amphibians and teleost fish have the capacity to fully regenerate after severe injury to the CNS. Xenopus laevis has a high potential to regenerate the brain and spinal cord during larval stages (47-54), and loses this capacity during metamorphosis. The optic nerve has the capacity to regenerate throughout the frog's lifespan. Here, we review CNS regeneration in frogs, with a focus in X. laevis, but also provide some information about X. tropicalis and other frogs. We start with an overview of the anatomy of the Xenopus CNS, including the main supraspinal tracts that emerge from the brain stem, which play a key role in motor control and are highly conserved across vertebrates. We follow with the advantages of using Xenopus, a classical laboratory model organism, with increasing availability of genetic tools like transgenesis and genome editing, and genomic sequences for both X. laevis and X. tropicalis. Most importantly, Xenopus provides the possibility to perform intra-species comparative experiments between regenerative and non-regenerative stages that allow the identification of which factors are permissive for neural regeneration, and/or which are inhibitory. We aim to provide sufficient evidence supporting how useful Xenopus can be to obtain insights into our understanding of CNS regeneration, which, complemented with studies in mammalian vertebrate model systems, can provide a collaborative road towards finding novel therapeutic approaches for injuries to the CNS., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Theoretical models predict that spatial sorting at the range edge of expanding populations should favor individuals with increased mobility relative to individuals at the center of the range. Despite the fact that empirical evidence for the evolution of locomotor performance at the range edge is rare, data on cane toads support this model. However, whether this can be generalized to other species remains largely unknown. Here, we provide data on locomotor stamina and limb morphology in individuals from two sites: one from the center and one from the periphery of an expanding population of the clawed frog Xenopus laevis in France where it was introduced about 30 years ago. Additionally, we provide data on the morphology of frogs from two additional sites to test whether the observed differences can be generalized across the range of this species in France. Given the known sexual size dimorphism in this species, we also test for differences between the sexes in locomotor performance and morphology. Our results show significant sexual dimorphism in stamina and morphology, with males having longer legs and greater stamina than females. Moreover, in accordance with the predictions from theoretical models, individuals from the range edge had a greater stamina. This difference in locomotor performance is likely to be driven by the significantly longer limb segments observed in animals in both sites sampled in different areas along the range edge. Our data have implications for conservation because spatial sorting on the range edge may lead to an accelerated increase in the spread of this invasive species in France., (© 2017. Published by The Company of Biologists Ltd.)

Microvascular anatomy and histomorphology of larval and adult spleens of the Clawed Toad, Xenopus laevis were studied by light microscopy of paraplast embedded serial tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Histology showed i) that white and red pulp are present at the onset of metamorphic climax (stage 57) and ii) that splenic vessels penetrated deeply into the splenic parenchyma at the height of metamorphic climax (stage 64). Scanning electron microscopy of VCCs demonstrated gross arterial supply and venous drainage, splenic microvascular patterns as well as the structure of the interstitial (extravasal) spaces representing the "open circulation routes." These spaces identified themselves as interconnected resin masses of two distinct forms, namely "broccoli-shaped" forms and highly interconnected small resin structures. Arterial and venous trees were clearly identified, as were transitions from capillaries to interstitial spaces and from interstitial spaces to pulp venules. Venous sinuses were not diagnosed (nonsinusal spleen). The splenic circulation in Xenopus laevis is "open." It is hypothesized that red blood cells circulate via splenic artery, central arteries, penicillar arteries, and red pulp capillaries primarily via "broccoli-shaped" interstitial spaces, pulp venules and veins into subcapsular veins to splenic veins while lymphocytes circulate also via the interstitial spaces represented by the highly interconnected small resin structures in vascular corrosion casts. In physiological terms, the former most likely represent the fast route for blood circulation, while the latter represent the slow route. J. Morphol. 277:1559-1569, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Nkx5 family members are homeobox transcription factors important for sensory organ development. Several members of the Nkx5 family are expressed in the eye, brain, developing ear, and lateral line. Members of this family have been previously identified in medaka, chick, and mouse. Here, we characterize two members of the Nkx5 family, Nkx5.3 and SOHo, in Xenopus laevis. We verify the identity of X. laevis Nkx5.3 and SOHo by phylogenetic comparison to chicken, medaka, and zebrafish orthologs. Both Nkx5.3 and SOHo are expressed in the developing eye, ear, lateral line system, and cranial neurons as determined by in situ hybridization.

The embryonic kidney of Xenopus laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

The amphibian olfactory system undergoes massive remodeling during metamorphosis. The transition from aquatic olfaction in larvae to semiaquatic or airborne olfaction in adults requires anatomical, cellular, and molecular modifications. These changes are particularly pronounced in Pipidae, whose adults have secondarily adapted to an aquatic life style. In the fully aquatic larvae of Xenopus laevis, the main olfactory epithelium specialized for sensing water-borne odorous substances lines the principal olfactory cavity (PC), whereas a separate olfactory epithelium lies in the vomeronasal organ (VNO). During metamorphosis, the epithelium of the PC is rearranged into the adult "air nose," whereas a new olfactory epithelium, the adult "water nose," forms in the emerging middle cavity (MC). Here we performed a stage-by-stage investigation of the anatomical changes of the Xenopus olfactory organ during metamorphosis. We quantified cell death in all olfactory epithelia and found massive cell death in the PC and the VNO, suggesting that the majority of larval sensory neurons is replaced during metamorphosis in both sensory epithelia. The moderate cell death in the MC shows that during the formation of this epithelium some cells are sorted out. Our results show that during MC formation some supporting cells, but not sensory neurons, are relocated from the PC to the MC and that they are eventually eliminated during metamorphosis. Together our findings illustrate the structural and cellular changes of the Xenopus olfactory organ during metamorphosis., (© 2015 Wiley Periodicals, Inc.)

A mutation in the second transmembrane domain of the GABA receptor subunit, Rdl, is associated with resistance to insecticides such as dieldrin and fipronil. Molecular cloning of Rdl cDNA from a strain of the malaria mosquito, Anopheles gambiae, which is highly resistant to dieldrin revealed this mutation (A296G) as well as another mutation in the third transmembrane domain (T345M). Wild-type, A296G, T345M and A296G + T345M homomultimeric Rdl were expressed in Xenopus laevis oocytes and their sensitivities to fipronil, deltamethrin, 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT), imidacloprid and spinosad were measured using two-electrode voltage-clamp electrophysiology. Spinosad and DDT had no agonist or antagonist actions on Rdl. However, fipronil, deltamethrin and imidacloprid decreased GABA-evoked currents. These antagonistic actions were either reduced or abolished with the A296G and the A296G + T345M mutations while T345M alone appeared to have no significant effect. In conclusion, this study identifies another mutation in the mosquito Rdl that is associated with insecticide resistance. While T345M itself does not affect insecticide sensitivity, it may serve to offset the structural impact of A296G. The present study also highlights Rdl as a potential secondary target for neonicotinoids and pyrethroids. We show for the first time that deltamethrin (a pyrethroid insecticide) and imidacloprid (a neonicotinoid insecticide) act directly on the insect GABA receptor, Rdl. Our findings highlight Rdl as a potential secondary target of pyrethroids and neonicotinoids mutations in which may contribute to resistance to these widely used insecticides., (© 2015 International Society for Neurochemistry.)

Second harmonic generation (SHG) microscopy is a powerful tool for studying submicron architecture of muscles tissues. Using this technique, we show that the canonical single frequency sarcomeric SHG intensity pattern (SHG-IP) of premetamorphic xenopus tadpole tail muscles is converted to double frequency (2f) sarcomeric SHG-IP in metamorphic climax stages due to massive physiological muscle proteolysis. This conversion was found to rise from 7% in premetamorphic muscles to about 97% in fragmented muscular apoptotic bodies. Moreover a 66% conversion was also found in non-fragmented metamorphic tail muscles. Also, a strong correlation between predominant 2f sarcomeric SHG-IPs and myofibrillar misalignment is established with electron microscopy. Experimental and theoretical results demonstrate the higher sensitivity and the supra resolution power of SHG microscopy over TPEF to reveal 3D myofibrillar misalignment. From this study, we suggest that 2f sarcomeric SHG-IP could be used as signature of triad defect and disruption of excitation-contraction coupling. As the mechanism of muscle proteolysis is similar to that found in mdx mouse muscles, we further suggest that xenopus tadpole tail resorption at climax stages could be used as an alternative or complementary model of Duchene muscular dystrophy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Many recent accounts of the frog peripheral auditory system have reproduced Wever's (1973) schematic cross-section of the ear of a leopard frog. We sought to investigate to what extent this diagram is an accurate and representative depiction of the anuran inner ear, using three-dimensional reconstructions made from serial sections of Rana pipiens, Eleutherodactylus limbatus and Xenopus laevis. In Rana, three discrete contact membranes were found to separate the posterior otic (=endolymphatic) labyrinth from the periotic (=perilymphatic) system: those of the amphibian and basilar recesses and the contact membrane of the saccule. The amphibian 'tegmentum vasculosum' was distinguishable as a thickened epithelial lining within a posterior recess of the superior saccular chamber. These features were also identified in Eleutherodactylus, but in this tiny frog the relative proportions of the semicircular canals and saccule resemble those of ranid tadpoles. There appeared to be a complete fluid pathway between the right and left periotic labyrinths in this species, crossing the cranial cavity. Xenopus lacks a tegmentum vasculosum and a contact membrane of the saccule; the Xenopus ear is further distinguished by a lateral passage separating stapes from periotic cistern and a more direct connection between periotic cistern and basilar recess. The basilar and lagenar recesses are conjoined in this species. Wever's diagram of the inner ear of Rana retains its value for diagrammatic purposes, but it is not anatomically accurate or representative of all frogs. Although Wever identified the contact membrane of the saccule, most recent studies of frog inner ear anatomy have overlooked both this and the amphibian tegmentum vasculosum. These structures deserve further attention.

The Xenopus tadpole optic tectum is a multisensory processing center that receives direct visual input as well as nonvisual mechanosensory input. The tectal neurons that comprise the optic tectum are organized into layers. These neurons project their dendrites laterally into the neuropil where visual inputs target the distal region of the dendrite and nonvisual inputs target the proximal region of the same dendrite. The Xenopus tadpole tectum is a popular model to study the development of sensory circuits. However, whole cell patch-clamp electrophysiological studies of the tadpole tectum (using the whole brain or in vivo preparations) have focused solely on the deep-layer tectal neurons because only neurons of the deep layer are visible and accessible for whole cell electrophysiological recordings. As a result, whereas the development and plasticity of these deep-layer neurons has been well-studied, essentially nothing has been reported about the electrophysiology of neurons residing beyond this layer. Hence, there exists a large gap in our understanding about the functional development of the amphibian tectum as a whole. To remedy this, we developed a novel isolated brain preparation that allows visualizing and recording from all layers of the tectum. We refer to this preparation as the "horizontal brain slice preparation." Here, we describe the preparation method and illustrate how it can be used to characterize the electrophysiology of neurons across all of the layers of the tectum as well as the spatial pattern of synaptic input from the different sensory modalities., (Copyright © 2015 the American Physiological Society.)

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Background and Aims: Amphibian intestinal remodeling, where thyroid hormone (T3) induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis., Methods/findings: Our quantitative RT-PCR (qRT-PCR) and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2) and receptor tyrosine kinase-like orphan receptor 2 (Ror2) are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation., Significance: Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.

Anuran larvae have been classified into four morphological types which reflect intraordinal macroevolution. At present, complete characterizations of the lateral line system are only available for Xenopus laevis (Type I) and Discoglossus pictus (Type III). We analyzed the morphology, arrangement, and innervation of neuromasts related to the anterodorsal and anteroventral lateral line nerves in 10 anuran species representing Types I, II, and IV with the aim of interpreting the existing variation and discussing the evolution of the lateral line in anuran larvae. We found: (1) the presence of two orbital and three mandibular neuromast lines in all anuran larvae studied, (2) the ventral arrangement of mandibular neuromast lines appears to have evolved convergently in Larval Types I and II, and the lateroventral arrangement of mandibular lines of neuromasts appears to have evolved in Larval Types III and IV; (3) interspecific variation in the organization, size, and number of sensory cells per neuromast within the lines; and (4) the supralabial extension of the Angular line in Lepidobatrachus spp. and the tentacular location of the Oral neuromasts in X. laevis are concomitant with their particular morphologies. Based on the variation described we find that the lateral line system in anuran larvae seems to have been maintained without significant changes, with the exception of Lepidobatrachus spp. and Xenopus. These unique features added to other of Lepidobatrachus tadpoles are sufficient to propose a new Larval Type (V)., (Copyright © 2014 Wiley Periodicals, Inc.)

The microvasculariaztion of the lateral line organs (LLOs) of the adult pipid frog, Xenopus laevis was studied by scanning electron microscopy of vascular corrosion casts (VCCs) and correlative light microscopy of paraplast embedded tissues sections. Scanning electron micrographs of VCCs revealed that each neuromast within the LLO rests on a distinct bowl-like capillary network (vascular bowl). One to three vascular bowls were supplied by an ascending arteriole and drained by a descending venule towards the skin deep dermal vascular network. Blood flow regulation mechanisms in form of intimal cushions were present at the origin of ascending arterioles supplying LLOs, microvenous valves were present at the confluence of deep dermal venules and veins. This together with sprouting and nonsprouting angiogenesis (intussusceptive microvascular growth) found in vascular bowls demonstrate that in adult Xenopus the capillary bed of LLO's still can be adjusted to changing energetic needs., (Copyright © 2013 Wiley Periodicals, Inc.)

The expression patterns of conserved developmental regulatory transcription factors and neuronal markers were analyzed in the basal hypothalamus of Xenopus laevis throughout development by means of combined immunohistochemical and in situ hybridization techniques. The connectivity of the main subdivisions was investigated by in vitro tracing techniques with dextran amines. The basal hypothalamic region is topologically rostral to the basal diencephalon and is composed of the tuberal (rostral) and mammillary (caudal) subdivisions, according to the prosomeric model. It is dorsally bounded by the optic chiasm and the alar hypothalamus, and caudally by the diencephalic prosomere p3. The tuberal hypothalamus is defined by the expression of Nkx2.1, xShh, and Isl1, and rostral and caudal portions can be distinguished by the distinct expression of Otp rostrally and Nkx2.2 caudally. In the mammillary region the xShh/Nkx2.1 combination defined the rostral mammillary area, expressing Nkx2.1, and the caudal retromammillary area, expressing xShh. The expression of xLhx1, xDll4, and Otp in the mammillary area and Isl1 in the tuberal region highlights the boundary between the two basal hypothalamic territories. Both regions are strongly connected with subpallial regions, especially those conveying olfactory/vomeronasal information, and also possess abundant intrahypothalamic connections. They show reciprocal connections with the diencephalon (mainly the thalamus), project to the midbrain tectum, and are bidirectionally related to the rhombencephalon. These results illustrate that the basal hypothalamus of anurans shares many features of specification, regionalization, and hodology with amniotes, reinforcing the idea of a basic bauplan in the organization of this prosencephalic region in all tetrapods., (Copyright © 2013 Wiley Periodicals, Inc.)

Normal development in anurans includes a free swimming larva that goes through metamorphosis to develop into the adult frog. We have investigated cranial muscle development and adult cranial muscle morphology in three different anuran species. Xenopus laevis is obligate aquatic throughout lifetime, Rana(Lithobates) pipiens has an aquatic larvae and a terrestrial adult form, and Eleutherodactylus coqui has direct developing juveniles that hatch from eggs deposited on leaves (terrestrial). The adult morphology shows hardly any differences between the investigated species. Cranial muscle development of E. coqui shows many similarities and only few differences to the development of Rana (Lithobates) and Xenopus. The differences are missing muscles of the branchial arches (which disappear during metamorphosis of biphasic anurans) and a few heterochronic changes. The development of the mandibular arch (adductor mandibulae) and hyoid arch (depressor mandibulae) muscles is similar to that observed in Xenopus and Rana (Lithobates), although the first appearance of these muscles displays a midmetamorphic pattern in E. coqui. We show that the mix of characters observed in E. coqui indicates that the larval stage is not completely lost even without a free swimming larval stage. Cryptic metamorphosis is the process in which morphological changes in the larva/embryo take place that are not as obvious as in normal metamorphosing anurans with a clear biphasic lifestyle. During cryptic metamorphosis, a normal adult frog develops, indicating that the majority of developmental mechanisms towards the functional adult cranial muscles are preserved.

Formation of the follicular envelopes surrounding oocytes in the developing ovary and their subsequent morphological differentiation go hand-in-hand with succession of the steroidogenesis stages, arrest of meiosis and its maintenance, establishment of the conditions necessary for vitellogenesis, oocyte growth, and maturation. Metabolites are exchanged via gap junctions and receptor-mediated transport through the perioocytic space. The ion transport in follicular cells (FCs) regulates the plasma membrane potential, creating the conditions for efficient directed transport through gap junctions. Manifold biologically active substances accepted by follicular cells are an additional adjusting lever for regulating the state of follicle system. In this review, we have attempted to emphasize the amphibian FCs as key players in the follicle system; the more so as we have failed to find any review that would bring together the data on the origin of amphibian FCs, their morphology, as well as regulation of oocyte growth and development. As a rule, recent works in this field focus on the molecular mechanisms providing for regulation of individual stages in oocyte development. This review describes the origin and changes in the morphology of follicular cells during the development of Xenopus laevis oocyte as well as the data on their regulatory functions in vitellogenesis and their involvement in steroidogenesis, maintenance of meiotic arrest, and subsequent maturation.

An ambitious goal in biology is to understand the behaviour of cells during development by imaging-in vivo and with subcellular resolution-changes of the embryonic structure. Important morphogenetic movements occur throughout embryogenesis, but in particular during gastrulation when a series of dramatic, coordinated cell movements drives the reorganization of a simple ball or sheet of cells into a complex multi-layered organism. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants, in fixed embryos, in vivo using fluorescence microscopy or microscopic magnetic resonance imaging. None of these methods allows cell behaviours to be observed with micrometre-scale resolution throughout the optically opaque, living embryo over developmental time. Here we use non-invasive in vivo, time-lapse X-ray microtomography, based on single-distance phase contrast and combined with motion analysis, to examine the course of embryonic development. We demonstrate that this powerful four-dimensional imaging technique provides high-resolution views of gastrulation processes in wild-type X. laevis embryos, including vegetal endoderm rotation, archenteron formation, changes in the volumes of cavities within the porous interstitial tissue between archenteron and blastocoel, migration/confrontation of mesendoderm and closure of the blastopore. Differential flow analysis separates collective from relative cell motion to assign propulsion mechanisms. Moreover, digitally determined volume balances confirm that early archenteron inflation occurs through the uptake of external water. A transient ectodermal ridge, formed in association with the confrontation of ventral and head mesendoderm on the blastocoel roof, is identified. When combined with perturbation experiments to investigate molecular and biomechanical underpinnings of morphogenesis, our technique should help to advance our understanding of the fundamentals of development.

A major roadblock in the biomedical treatment of human sensory disorders, including blindness, has been an incomplete understanding of the nervous system and its ability to adapt to changes in sensory modality. Likewise, fundamental insight into the evolvability of complex functional anatomies requires understanding brain plasticity and the interaction between the nervous system and body architecture. While advances have been made in the generation of artificial and biological replacement components, the brain's ability to interpret sensory information arising from ectopic locations is not well understood. We report the use of eye primordia grafts to create ectopic eyes along the body axis of Xenopus tadpoles. These eyes are morphologically identical to native eyes and can be induced at caudal locations. Cell labeling studies reveal that eyes created in the tail send projections to the stomach and trunk. To assess function we performed light-mediated learning assays using an automated machine vision and environmental control system. The results demonstrate that ectopic eyes in the tail of Xenopus tadpoles could confer vision to the host. Thus ectopic visual organs were functional even when present at posterior locations. These data and protocols demonstrate the ability of vertebrate brains to interpret sensory input from ectopic structures and incorporate them into adaptive behavioral programs. This tractable new model for understanding the robust plasticity of the central nervous system has significant implications for regenerative medicine and sensory augmentation technology.

Development of the amphibian vestibular organ is regulated by molecular and neuronal mechanisms and by environmental input. The molecular component includes inductive signals derived from neural tissue of the hindbrain and from the surrounding mesoderm. The integrity of hindbrain patterning, on the other hand, depends on instructive signals from the isthmus organizer of the midbrain, including the transcription factor XTcf-4. If the development of the vestibular system depends on the integrity of the isthmus as the organizing centre, suppression of isthmus maintenance should modify vestibular morphology and function. We tested this hypothesis by downregulation of the transcription factor XTcf-4. 10 pmol l(-1) XTcf-4-specific antisense morpholino oligonucleotide was injected in one blastomere of two-cell-stage embryos of Xenopus laevis. For reconstitution experiments, 500 pg mRNA of the repressing XTcf-4A isoform or the activating XTcf-4C isoform were co-injected. Overexpression experiments were included using the same isoforms. Otoconia formation and vestibular controlled behaviour such as the roll-induced vestibuloocular reflex (rVOR) and swimming were recorded two weeks later. In 50% of tadpoles, downregulation of XTcf-4 induced (1) a depression of otoconia formation accompanied by a reduction of the rVOR, (2) abnormal tail development and (3) loop swimming behaviour. (4) All effects were rescued by co-injection of XTcf-4C but not, or only partially, by XTcf-4A. (5) Overexpression of XTcf-4A caused similar morphological and rVOR modifications as XTcf-4 depletion, while overexpression of XTcf-4C had no effect. Because XTcf-4C has been described as an essential factor for isthmus development, we postulate that the isthmus is strongly involved in vestibular development.

The present study represents a detailed spatiotemporal analysis of the localization of calbindin-D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions., (Copyright © 2012 Wiley Periodicals, Inc.)

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay. In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Live imaging studies of the processes of demyelination and remyelination have so far been technically limited in mammals. We have thus generated a Xenopus laevis transgenic line allowing live imaging and conditional ablation of myelinating oligodendrocytes throughout the CNS. In these transgenic pMBP-eGFP-NTR tadpoles the myelin basic protein (MBP) regulatory sequences, specific to mature oligodendrocytes, are used to drive expression of an eGFP (enhanced green fluorescent protein) reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. This enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. Using two-photon imaging in vivo, we show that pMBP-eGFP-NTR tadpoles display a graded oligodendrocyte ablation in response to MTZ, which depends on the exposure time to MTZ. MTZ-induced cell death was restricted to oligodendrocytes, without detectable axonal damage. After cessation of MTZ treatment, remyelination proceeded spontaneously, but was strongly accelerated by retinoic acid. Altogether, these features establish the Xenopus pMBP-eGFP-NTR line as a novel in vivo model for the study of demyelination/remyelination processes and for large-scale screens of therapeutic agents promoting myelin repair.

A morphofunctional analysis of the thymus from differently aged Xenopus laevis tadpoles during regeneration of the tail is reported. In stage 50 larvae, competent to regenerate, the appendage cut provoked thymic structural modifications that affected the medullary microenvironment cells and changes in TNF-α immunoreactivity. Mucocyte-like cells, multicellular epithelial cysts, myoid cells and cells immunoreactive to TNF-α increased in number. Increased numbers of lymphocytes were also found in regenerating areas and, at the end of regeneration, thymic structural and immunocytochemical patterns were restored to control levels. The observed cellular responses and the induction of molecules critical for thymus constitutive processes suggest a stimulation of thymic function after tail amputation. In older larvae, whose capacity to form a new complete and correctly patterned tail was reduced, thymic morphological changes were more severe and may persist throughout the regeneration process with a significant reduction in organ size. In these larvae the histological patterns and the marked thymic decrease may be related to the events occurring during regeneration, i.e. the higher inflammatory response and the reduced tail regenerative potential., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Studies on the amphibian respiratory tract microvascular anatomy are few and contradictory. Using scanning electron microscopy of vascular corrosion casts, correlative light microscopy of paraplast-embedded Goldner-stained serial tissue sections, and three-dimensional morphometry, we studied the topographic microvascular anatomy in the trachea of the adult South African Clawed Toad, Xenopus laevis Daudin. Histomorphology showed that the cartilaginous portion of the trachea contained irregularly shaped hyaline cartilage plates in its cranial and caudal portions and C-shaped hyaline cartilage rings in the middle portion. Tracheal cartilages formed large continuous plates on the ventral circumference, numerous small discontinuous plates on the dorsal circumference, and large vertical plates on the caudolateral circumference. The muscular portion of the trachea consisted of bands of smooth muscle that joined the free ends of cartilage plates. The supply of the trachea was via pulmonal artery-tracheobronchial trunk artery-tracheobronchial artery-tracheal artery. The subepithelial capillary network consisted of rectangular meshes which are in the area of the tracheal cartilages located between the cartilages and the respiratory epithelium. Small tracheal veins merged into a single tracheal vein that emptied into the pulmonary vein. Because of its dense subepithelial capillary network and its drainage into the pulmonal vein, the trachea could actively take part in respiration., (Copyright © 2012 Wiley Periodicals, Inc.)

Noonan syndrome is one of the most common causes of human congenital heart disease and is frequently associated with missense mutations in the protein phosphatase SHP-2. Interestingly, patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), juvenile myelomonocytic leukemia (JMML) and LEOPARD syndrome frequently carry a second, somatically introduced subset of missense mutations in SHP-2. To determine the cellular and molecular mechanisms by which SHP-2 regulates heart development and, thus, understand how Noonan-associated mutations affect cardiogenesis, we introduced SHP-2 encoding the most prevalent Noonan syndrome and JMML mutations into Xenopus embryos. Resulting embryos show a direct relationship between a Noonan SHP-2 mutation and its ability to cause cardiac defects in Xenopus; embryos expressing Noonan SHP-2 mutations exhibit morphologically abnormal hearts, whereas those expressing an SHP-2 JMML-associated mutation do not. Our studies indicate that the cardiac defects associated with the introduction of the Noonan-associated SHP-2 mutations are coupled with a delay or arrest of the cardiac cell cycle in M-phase and a failure of cardiomyocyte progenitors to incorporate into the developing heart. We show that these defects are a result of an underlying malformation in the formation and polarity of cardiac actin fibers and F-actin deposition. We show that these defects can be rescued in culture and in embryos through the inhibition of the Rho-associated, coiled-coil-containing protein kinase 1 (ROCK), thus demonstrating a direct relationship between SHP-2(N308D) and ROCK activation in the developing heart.

Major common features have been reported for the organization of the basal telencephalon in amniotes, and most characteristics were thought to be acquired in the transition from anamniotes to amniotes. However, gene expression, neurochemical, and hodological data obtained for the basal ganglia and septal and amygdaloid complexes in amphibians (anamniotic tetrapods) have strengthened the idea of a conserved organization in tetrapods. A poorly characterized region in the forebrain of amniotes has been the bed nucleus of the stria terminalis (BST), but numerous recent investigations have characterized it as a member of the extended amygdala. Our study analyzes the main features of the BST in anuran amphibians to establish putative homologies with amniotes. Gene expression patterns during development identified the anuran BST as a subpallial, nonstriatal territory. The BST shows Nkx2.1 and Lhx7 expression and contains an Islet1-positive cell subpopulation derived from the lateral ganglionic eminence. Immunohistochemistry for diverse peptides and neurotransmitters revealed that the distinct chemoarchitecture of the BST is strongly conserved among tetrapods. In vitro tracing techniques with dextran amines revealed important connections between the BST and the central and medial amygdala, septal territories, medial pallium, preoptic area, lateral hypothalamus, thalamus, and prethalamus. The BST receives dopaminergic projections from the ventral tegmental area and is connected with the laterodorsal tegmental nucleus and the rostral raphe in the brainstem. All these data suggest that the anuran BST shares many features with its counterpart in amniotes and belongs to a basal continuum, likely controlling similar reflexes, reponses, and behaviors in tetrapods., (Copyright © 2011 Wiley-Liss, Inc.)

We analyzed the function of neural progenitors in the developing central nervous system of Xenopus laevis tadpoles by using in vivo time-lapse confocal microscopy to collect images through the tectum at intervals of 2-24 hours over 3 days. Neural progenitor cells were labeled with fluorescent protein reporters based on expression of endogenous Sox2 transcription factor. With this construct, we identified Sox2-expressing cells as radial glia and as a component of the progenitor pool of cells in the developing tectum that gives rise to neurons and other radial glia. Lineage analysis of individual radial glia and their progeny demonstrated that less than 10% of radial glia undergo symmetric divisions resulting in two radial glia, whereas the majority of radial glia divide asymmetrically to generate neurons and radial glia. Time-lapse imaging revealed the direct differentiation of radial glia into neurons. Although radial glia may guide axons as they navigate to the superficial tectum, we find no evidence that radial glia function as a scaffold for neuronal migration at early stages of tectal development. Over 3 days, the number of labeled cells increased 20%, as the fraction of radial glia dropped and the proportion of neuronal progeny increased to approximately 60% of the labeled cells. Tadpoles provided with short-term visual enhancement generated significantly more neurons, with a corresponding decrease in cell proliferation. Together these results demonstrate that radial glial cells are neural progenitors in the developing optic tectum and reveal that visual experience increases the proportion of neurons generated in an intact animal., (Copyright © 2011 Wiley-Liss, Inc.)

The diffusion coefficient of fluorescein in detached cilia of Xenopus laevis olfactory receptor neurons was measured using spatially-resolved FRAP, where the dye along half of the ciliary length was photobleached and its spatiotemporal fluorescence redistribution recorded. Fitting a one-dimensional numerical simulation of diffusion and photobleaching for 35 cilia resulted in a mean value of the diffusion coefficient (1.20 ± 0.23) · 10(-10)m(2)/s and thus a reduction by a factor of 3.4 compared to free diffusion in aqueous solution.

Small multicellular organisms such as nematodes, fruit flies, clawed frogs, and zebrafish are emerging models for an increasing number of biomedical and environmental studies. They offer substantial advantages over cell lines and isolated tissues, providing analysis under normal physiological milieu of the whole organism. Many bioassays performed on these alternative animal models mirror with a high level of accuracy those performed on inherently low-throughput, costly, and ethically controversial mammalian models of human disease. Analysis of small model organisms in a high-throughput and high-content manner is, however, still a challenging task not easily susceptible to laboratory automation. In this context, recent advances in photonics, electronics, as well as material sciences have facilitated the emergence of miniaturized bioanalytical systems collectively known as Lab-on-a-Chip (LOC). These technologies combine micro- and nanoscale sciences, allowing the application of laminar fluid flow at ultralow volumes in spatially confined chip-based circuitry. LOC technologies are particularly advantageous for the development of a wide array of automated functionalities. The present work outlines the development of innovative miniaturized chip-based devices for the in situ analysis of small model organisms. We also introduce a new term "wormometry" to collectively distinguish these up-and-coming chip-based technologies that go far beyond the conventional meaning of the term "cytometry.", (Copyright © 2011 International Society for Advancement of Cytometry.)