Search Results (194)

Craniofacial development is a complex, highly conserved process involving multiple tissue types and molecular pathways, with perturbations resulting in congenital defects that often require invasive surgical interventions to correct. Remarkably, some species, such as Xenopus laevis, can correct some craniofacial abnormalities during pre-metamorphic stages through thyroid hormone-independent mechanisms. However, the full scope of factors mediating remodeling initiation and coordination remain unclear. This study explores the differential remodeling responses of craniofacial defects by comparing the effects of two pharmacological agents, thioridazine-hydrochloride (thio) and ivermectin (IVM), on craniofacial morphology in X. laevis. Thio-exposure reliably induces a craniofacial defect that can remodel in pre-metamorphic animals, while IVM induces a permanent, non-correcting phenotype. We examined developmental changes from feeding stages to hindlimb bud stages and mapped the effects of each agent on the patterning of craniofacial tissue types including: cartilage, muscle, and nerves. Our findings reveal that thio-induced craniofacial defects exhibit significant consistent remodeling, particularly in muscle, with gene expression analysis revealing upregulation of key remodeling genes, matrix metalloproteinases 1 and 13, as well as their regulator, prolactin.2. In contrast, IVM-induced defects show no significant remodeling, highlighting the importance of specific molecular and cellular factors in pre-metamorphic craniofacial correction. Additionally, unique neuronal profiles suggest a previously underappreciated role for the nervous system in tissue remodeling. This study provides novel insights into the molecular and cellular mechanisms underlying craniofacial defect remodeling and lays the groundwork for future investigations into tissue repair in vertebrates. Full article

Plastic manufacturing involves using compounds that could be considered endocrine disruptors. Consequently, concern about the effect of these particles on the hormonal regulation of various systems, including the hypothalamic–pituitary–thyroid axis, has been increasing. By applying the Amphibian Metamorphosis Assay (AMA), the effects of irregular polystyrene microplastics (PS) MPs on the thyroid gland of Xenopus laevis were investigated. The histological effects on other organs of tadpoles were also studied. Tadpoles were exposed to 500 and 50 µg of virgin PS MP particles, (200 µm range)/L, administered by diet for 21 days. PS dietary exposure revealed statistically significant effects for the snout to vent length and the whole body length apical endpoints on day 21. The histological survey of both treatment groups revealed no noteworthy effects on the thyroid gland, digestive tract, or kidneys, but slight modifications to the liver. Mild ultrastructural modifications were detected in tadpoles’ enterocytes and hepatocytes in both treatment groups, but were likely to be reversible. Overall, our results contrast with previous research results in which effects were observed, but using different types, concentrations, and numbers of MPs. All this suggests the need for standardized methods for the environmental risk assessment of MPs/NPs (nanoplastics). Concern about the risk of NPs seems to be greater, and more studies with NP particles should be conducted. Full article

Hypoxic pulmonary vasoconstriction (HPV) optimizes gas exchange but, when impaired, can result in life-threatening hypoxemia. Moreover, under conditions of generalized alveolar hypoxia, HPV can result in pulmonary hypertension. Voltage-gated K⁺ channels (K_v channels) are key to HPV: a change in the intracellular hydrogen peroxide (H₂O₂) levels during acute hypoxia is assumed to modulate these channels’ activity to trigger HPV. However, there are longstanding conflicting findings on whether H₂O₂ inhibits or activates K_v channels. Therefore, we hypothesized that H₂O₂ affects K_v channels depending on the experimental conditions, i.e., the H₂O₂ concentration, the channel’s subunit configuration or the experimental clamping potential in electrophysiological recordings. Therefore, cRNAs encoding the K_v1.5 channel and the auxiliary K_vβ subunits (K_vβ1.1, K_vβ1.4) were generated via in vitro transcription before being injected into Xenopus laevis oocytes for heterologous expression. The K⁺ currents of homomeric (Kv1.5) or heteromeric (K_v1.5/K_vβ1.1 or K_v1.5/K_vβ1.4) channels were assessed by two-electrode voltage clamp. The response of the K_v channels to H₂O₂ was markedly dependent on (a) the clamping potential, (b) the H₂O₂ concentration, and (c) the K_v channel’s subunit composition. In conclusion, our data highlight the importance of the choice of experimental conditions when assessing the H₂O₂ sensitivity of K_v channels in the context of HPV, thus providing an explanation for the long-lasting controversial findings reported in the literature. Full article

Horizontal gene transfer (HGT), the non-Mendelian transfer of genetic material between organisms, is relatively frequent in prokaryotes, whereas its extent among eukaryotes remains unclear. Here, we raise the hypothesis of a possible cross-phylum HGT event involving 5S ribosomal DNA (rDNA). A specific type of 5S rDNA sequence from the anuran Xenopus laevis was highly similar to a 5S rDNA sequence of the genome of its flatworm parasite Protopolystoma xenopodis. A maximum likelihood analysis revealed phylogenetic incongruence between the gene tree and the species trees, as the 5S rDNA sequence from Pr. xenopodis was grouped along with the sequences from the anurans. Sequence divergence analyses of the gene region and non-transcribed spacer also agree with an HGT event from Xenopus to Pr. xenopodis. Additionally, we examined whether contamination of the Pr. xenopodis genome assembly with frog DNA could explain our findings but found no evidence to support this hypothesis. These findings highlight the possible contribution of HGT to the high diversity observed in the 5S rDNA family. Full article

The significant decline in amphibians worldwide is demanding the development of reliable techniques to save species and their genetic diversity. Considerable efforts are currently in progress to develop assisted reproductive technologies (ARTs), focusing mainly on sperm cryopreservation and in vitro fertilization (IVF). In Xenopus, a simple and efficient transgenesis method based on the intracytoplasmic injection (ICSI) of cryoconserved sperm was developed several decades ago, allowing for quick generation of large numbers of transgenic animals, for biological research. Such a methodology could be critical for the recovery of species and their genetic diversity, contributing to amphibian conservation. However, this approach raised the question of whether the sperm preservation method used with ICSI is compatible with long-term storage. To address this question, animals were generated by ICSI using a twenty-year-old cryopreserved sperm preparation. Their development, behavior, and reproduction ability were compared with those of animals obtained using a recently frozen sperm preparation and those of animals obtained via IVF using fresh semen. Although lower than with IVF, we showed that fertilization rates using ICSI after 20 years of cryopreservation are similar to those of a recent preparation, with viable offspring leading to normal F2 generation. This pioneering achievement is proof of concept for long-term sperm cryopreservation using simple and readily available technologies for the conservation of endangered amphibians. Full article

Basic helix–loop–helix (bHLH) transcription factors, such as those in the atonal family, are important in cellular fate determination. The expression of the sponge ortholog of the atonal bHLH gene family, AmqbHLH1, in Xenopus laevis previously resulted in the formation of ectodermal ectopic neurons. However, the extent to which these neurons persist through development and the effects on the inner ear and lateral line, which require a critical level and timing of bHLH genes, remains unexplored. To test these long-term effects, we injected various concentrations of AmqbHLH1 mRNA into X. laevis embryos and assessed neurosensory development at developmental stages coinciding with fully developed neurosensory structures. The expression of AmqbHLH1 mRNA in X. laevis resulted in a dose-dependent reduction in or loss of ears and the lateral line system without eliminating ectopic neurons. At the lowest concentrations examined, we found that inner ear neurosensory development consisted sometimes of only a few scattered hair cells in a single-layer epithelium. Furthermore, low concentrations of AmqbHLH1 mRNA affected inner ear afferent guidance. Our data suggest that the AmqbHLH1 gene has some anti-neurosensory abilities in frogs and that the overexpression of a single gene may not be sufficient for stable long-term transdifferentiation in cells. Full article

The homotrimeric P2X7 receptor (P2X7R) contains three ATP⁴⁻ binding sites in its ectodomain. Here, we investigated the role of individual ATP⁴⁻ activation sites in rat P2X7R (rP2X7R) using trimeric concatemers consisting of either three wild-type subunits (7-7-7) or one to three subunits with ATP binding sites knocked out by the K64A mutation. Following expression in Xenopus laevis oocytes, ATP⁴⁻-elicited ion currents were recorded using the two-microelectrode voltage clamp technique. The 7-7-7 concatamer exhibited a biphasic ATP⁴⁻ concentration dependence, best fit by the sum of two Hill functions, confirming the existence of functionally distinct ATP⁴⁻ activation sites. The activation time course of the 7-7-7 was best approximated by the sum of a fast and a slow exponential saturating activation component. Similarly, deactivation exhibited both fast and slow exponential decay. Only one Hill function was required to best fit the ATP⁴⁻ concentration dependence of concatamers with only two or one ATP⁴⁻ binding sites, and their deactivation time courses largely lacked the slowly deactivating components. We conclude that the binding of one ATP⁴⁻ is sufficient for partial activation of the rP2X7R and that allosteric effects occur when all three ATP⁴⁻ binding sites are occupied, leading to distinct functional activation sites. Full article

Multiciliated cells generate fluid flow along epithelial surfaces, and defects in their development or function cause primary ciliary dyskinesia. The fluid flow is generated by the coordinated beating of motile cilia, which are microtubule-based organelles. The base of each cilium, the basal body, is anchored to the apical cell membrane and surrounded by a dense apical cytoskeleton of actin, microtubules, and intermediate filaments. Several cell adhesion proteins play a role in the connection of the basal body to the apical cytoskeleton. Here, we show that the desmosomal protein plakophilin3, a member of the armadillo family of proteins, localizes to the striated rootlet in Xenopus laevis multiciliated cells. Knockdown of plakophilin 3 leads to significant defects in cilia-generated fluid flow and basal body docking. These defects are cell-autonomous and independent of cell intercalation and gross changes in the actin cytoskeleton. These findings suggest a crucial role for PKP3 in basal body apical migration and docking in multiciliated cells, highlighting a novel connection between desmosomal proteins and ciliary function. Full article

The conopeptide αD-FrXXA was previously isolated by our team from the venom of the vermivorous snail Conus fergusoni. This toxin is composed of two chains of 47 amino acids and inhibits neuronal and muscular subtypes of nAChR. In this study, we explored its effects on voltage-gated potassium channels heterologously expressed in Xenopus laevis oocytes using the two-electrode voltage-clamp technique (TEVC). At a concentration of 15 μM, αD-FrXXA was able to inhibit by 50% or more the currents of four subtypes of the Kv1 subfamily and slightly inhibit (<20%) two subtypes of the EAG subfamily. The conopeptide αD-FrXXA inhibits in a concentration-dependent manner the subtypes Kv1.3 (IC₅₀ 0.38 ± 0.06 μM) and Kv1.6 (IC₅₀ 0.52 ± 0.14 μM). The results reported here are noteworthy because this α-conopeptide behaves similarly to the α/κJ-PlXIVA conopeptide that inhibits nAChR and Kv channels. Full article

Motile cilia play essential roles in various physiological processes including fluid flow generation and sperm motility. In this study, we identified 1,3-diphenyl-6-(4-phenylpiperazin-1-yl)benzo[e][1,2,4]triazin-7(1H)-one as a potent and reversible modulator of ciliary function using the Xenopus laevis model. This benzotriazinone derivative inhibits ciliary-driven fluid flow by inducing cilia detachment without causing toxicity in developing embryos. Unlike traditional deciliation agents that rely on calcium signaling, this compound induces cilia loss through a shear stress-driven mechanism at the transition zone, without disrupting tissue morphology or the apical actin network. Importantly, it also induces flagellar loss and impairs sperm motility at picomolar concentrations. Our findings highlight the potential of this 6-(4-phenylpiperazin-1-yl)-substituted benzotriazinone as a non-hormonal male contraceptive and underscore a novel mechanism of cilia modulation that may have broader implications for the treatment of cilia-related disorders. Full article

The control of cyclin-dependent kinase 1 (CDK1) kinase activity is crucial for cell cycle progression. Cell division cycle 6 (CDC6) inhibits this activity in embryonic mitoses, and thus regulates the timing of cell division progression. The meiotic cell cycle differs greatly from the mitotic one. Metaphase II (MII)-arrested oocytes remain in prolonged M-phase state due to the high activity of CDK1 in the presence of CytoStatic Factor (CSF). The role of CDC6 in the control of CDK1 during MII and oocyte activation remains unknown. Here, we studied the role of CDC6/CDK1 interactions in Xenopus laevis cell-free extracts arrested in MII (CSF extract) and upon calcium activation leading to meiotic-to-mitotic transition. The CSF extract allows analysis of biochemical processes based on immunodepletion of selected proteins and facilitates manipulations using addition of recombinant proteins. We show by glutathione S-transferase (GST)-CDC6 pull-down that CDC6 associates with CDK1 in CSF extract and by histone H1 kinase assay that it downregulates CDK1 activity. Thus, CDC6-dependent inhibition of CDK1 is involved in the homeostasis of the MII-arrest. Upon CSF extract activation with calcium exogenous GST-CDC6 provokes accelerated transition from MII to interphase, while the depletion of endogenous CDC6 results in a slower transition to interphase. We demonstrate this by following both the phosphorylation state of CDK1 substrate cell division cycle 27 (CDC27) and histone H1 kinase assay. Importantly, increasing doses of GST-CDC6 proportionally accelerate CDK1 inactivation showing that CDC6 controls the dynamics of MII to interphase transition in a dose-dependent manner. Thus, CDC6 is a CDK1 silencer acting upon both the MII arrest and CSF extract activation by assuring the physiological activity of CDK1 during this meiotic arrest and correct timely inactivation of this kinase during the second process. Thus, we show that CDC6 controls CDK1 not only during mitotic divisions, but also in MII-arrest and the meiotic-to-mitotic transition in Xenopus laevis cell-free extracts. This study aims to bridge that gap by investigating CDC6 function using a biochemically controlled system. Full article

In many vertebrates, mature ovulated eggs are arrested at metaphase II prior to fertilization. The eggs exit meiotic arrest after fertilization-induced or parthenogenetic activation, followed by embryo development or egg degradation, respectively. Calcium-dependent activation of meiotically-arrested eggs has been thoroughly investigated in various species. In addition, several recent studies have detailed the excessive activation of ovulated frog eggs, so-called overactivation. This overview highlights the major events of overactivation observed in mature ovulated eggs of the African clawed frog Xenopus laevis with a focus on similarities and differences between spontaneous, oxidative stress-induced, and mechanical stress-induced overactivation. The dramatically different cell death scenarios that unfold in activated and overactivated eggs are also exposed in the article. Full article

The chytrid fungus, Batrachochytrium dendrobatidis (Bd), infects amphibian skin, causing chytridiomycosis, which is a contributing cause of worldwide declines and extinctions of amphibians. Relatively little is known about the roles of amphibian skin-resident immune cells, such as macrophages, in these antifungal defenses. Across vertebrates, macrophage differentiation is controlled through the activation of colony-stimulating factor-1 (CSF1) receptor by CSF1 and interleukin-34 (IL34) cytokines. While the precise roles of these respective cytokines in macrophage development remain to be fully explored, our ongoing studies indicate that frog (Xenopus laevis) macrophages differentiated by recombinant forms of CSF1 and IL34 are functionally distinct. Accordingly, we explored the roles of X. laevis CSF1- and IL34-macrophages in anti-Bd defenses. Enriching cutaneous IL34-macrophages, but not CSF1-macrophages, resulted in significant anti-Bd protection. In vitro analysis of frog macrophage-Bd interactions indicated that both macrophage subsets phagocytosed Bd. However, IL34-macrophages cocultured with Bd exhibited greater pro-inflammatory gene expression, whereas CSF1-macrophages cocultured with Bd showed greater immunosuppressive gene expression profiles. Concurrently, Bd-cocultured with CSF1-macrophages, but not IL34-macrophages, possessed elevated expression of genes associated with immune evasion. This work marks a step forward in our understanding of the roles of frog macrophage subsets in antifungal defenses. Full article

The ability of an organism to regrow tissues is regulated by various signaling pathways. One such pathway that has been studied widely both in the context of regeneration and development is the Notch signaling pathway. Notch is required for the development of the eye and regeneration of tissues in multiple organisms, but it is unknown if Notch plays a role in the regulation of Xenopus laevis embryonic eye regrowth. We found that Notch1 is required for eye regrowth and regulates retinal progenitor cell proliferation. Chemical and molecular inhibition of Notch1 significantly decreased eye regrowth by reducing retinal progenitor cell proliferation without affecting retinal differentiation. Temporal inhibition studies showed that Notch function is required during the first day of regrowth. Interestingly, Notch1 loss-of-function phenocopied the effects of the inhibition of the proton pump, vacuolar-type ATPase (V-ATPase), where retinal proliferation but not differentiation was blocked during eye regrowth. Overexpression of a form of activated Notch1, the Notch intracellular domain (NICD) rescued the loss of eye regrowth due to V-ATPase inhibition. These findings highlight the importance of the Notch signaling pathway in eye regeneration and its role in inducing retinal progenitor cell proliferation in response to injury. Full article

Recent advancements in tissue engineering and stem cell science have positioned bone disease treatment as a promising frontier in regenerative medicine. This review explores the hormonal and signaling pathways critical to bone regeneration, with a focus on their clinical relevance. Key endocrine factors, including thyroid hormones (T3 and T4), insulin-like growth factor 1 (IGF-1), bone morphogenetic proteins (BMPs), parathyroid hormone (PTH), calcitonin, and fibroblast growth factor 23 (FGF23), play pivotal roles in bone remodeling by regulating osteoblast activity, bone resorption, and mineralization. These factors primarily act through the Wnt/β-catenin, BMP, and FGF signaling pathways, which govern bone repair and regeneration. While animal models, such as axolotls, zebrafish, and Xenopus laevis, provide valuable findings about these mechanisms, translating these findings into human applications presents challenges. This review underscores the therapeutic potential of modulating these hormonal networks to enhance bone regeneration while cautioning against possible adverse effects, such as uncontrolled tissue proliferation or metabolic imbalances. By integrating knowledge from regenerative models, this work provides a foundation for optimizing hormone-based therapies for clinical applications in bone repair and disease treatment. Full article