Xenopus Laevis Embryos Research Articles

Novel Insights on 6:6 Perfluoroalkyl Phosphonic Acid-Induced Melanin Synthesis Disorders Leading to Pigmentation in Tadpoles.

As alternatives to traditional per- and polyfluoroalkyl substances, perfluoroalkyl phosphonic acids (PFPiAs) are widely present in aquatic environments and can potentially harm aquatic organisms. Pigmentation affects the probability of aquatic organisms being preyed on and serves as an important toxic endpoint of development, but little is known about the impacts of PFPiAs on the development of aquatic organisms. In this study, Xenopus laevis embryos were exposed to 6:6 PFPiA (1, 10, and 100 nM) for 14 days. The developed tadpoles exhibited evident pigmentation with increased melanin particle size and density on the skin. Pathological and behavioral experiments revealed that the retinal layers became thinner, reducing the photosensitivity and disturbing the circadian rhythm of the tadpoles. Compared to the control group, the exposed tadpoles showed higher levels but less changes of melanin throughout the light/dark cycle, as well as distinct oxidative damage. Consequently, the expression level of microphthalmia-associated transcription factor (MITF), a key factor inducing melanin synthesis, increased significantly. Molecular docking analysis suggested that 6:6 PFPiA forms strong interactions in the binding pocket of MITF, implying that it could activate MITF directly. The activation of MITF ultimately promotes melanin synthesis, resulting in pigmentation on tadpoles.

Water contamination by delorazepam induces epigenetic defects in the embryos of the clawed frog Xenopus laevis

Delorazepam, a derivative of diazepam, is a psychotropic drug belonging to the benzodiazepine class. Used as a nervous-system inhibitor, it treats anxiety, insomnia, and epilepsy, but is also associated with misuse and abuse. Nowadays benzodiazepines are considered emerging pollutants: conventional wastewater treatment plants indeed are unable to eliminate these compounds. Consequently, they persist in the environment and bioaccumulate in non-target aquatic organisms with consequences still not fully clear. To collect more information, we investigated the possible epigenetic activity of delorazepam, at three concentrations (1, 5 and 10 μg/L) using Xenopus laevis embryos as a model. Analyses demonstrated a significant increase in genomic DNA methylation and differential methylation of the promoters of some early developmental genes (otx2, sox3, sox9, pax6, rax1, foxf1, and myod1). Moreover, studies on gene expression highlighted an unbalancing in apoptosis/proliferation pathways and an aberrant expression of DNA-repair genes. Results are alarming considering the growing trend of benzodiazepine concentrations in superficial waters, especially after the peak occurred as a consequence of the pandemic COVID-19, and the fact that benzodiazepine GABA-A receptors are highly conserved and present in all aquatic organisms.

Identification of protein phosphatase 4 catalytic subunit as a Wnt promoting factor in pan-cancer and Xenopus early embryogenesis

Protein Phosphatase 4 Catalytic Subunit (PPP4C) is an evolutionarily conserved protein involved in multiple biological and pathological events, including embryogenesis, organogenesis, cellular homeostasis, and oncogenesis. However, the detailed mechanisms underlying these processes remain largely unknown. Thus, we investigated the potential correlation between PPP4C and biological processes (BPs) and canonical Wnt signaling using pan-cancer analysis and Xenopus laevis (X. laevis) embryo model. Our results indicate that PPP4C is a potential biomarker for specific cancer types due to its high diagnostic accuracy and significant prognostic correlation. Furthermore, in multiple cancer types, PPP4C-related differentially expressed genes (DEGs) were significantly enriched in pattern specification, morphogenesis, and canonical Wnt activation. Consistently, perturbation of Ppp4c in X. laevis embryos interfered with normal embryogenesis and canonical Wnt responses. Moreover, biochemical analysis of X. laevis embryos demonstrated that both endogenous and exogenous Ppp4c negatively regulated AXIN1 (Wnt inhibitor) abundance. This study provides novel insights into PPP4C roles in pattern specification and Wnt activation. The similarities in BPs and Wnt signaling regulation regarding PPP4C support the intrinsic link between tumorigenesis and early embryogenesis.

Cannabinoid receptor type 1 regulates sequential stages of migration and morphogenesis of neural crest cells and derivatives in chicken and frog embryos.

The main cannabinoid receptor CB1R first shows expression during early neurula stage in chicken (Gallus gallus) embryos, and at early tailbud stage in the frog (Xenopus laevis) embryos. This raises the question of whether CB1R regulates similar or distinct processes during the embryonic development of these two species. Here, we examined whether CB1R influences the migration and morphogenesis of neural crest cells and derivatives in both chicken and frog embryos. Early neurula stage chicken embryos were exposed to arachidonyl-2'-chloroethylamide (ACEA; a CB1R agonist), N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; a CB1R inverse agonist) or Blebbistatin (nonmuscle Myosin II inhibitor) in ovo and examined during migration of neural crest cells and at condensing cranial ganglia stage. Early tailbud stage frog embryos were bathed in ACEA, AM251 or Blebbistatin, and analyzed at late tailbud stage for changes in craniofacial and eye morphogenesis, and in patterning and morphology of melanophores (neural crest-derived pigment cells). In chicken embryos exposed to ACEA and Myosin II inhibitor, cranial neural crest cells migrated erratically from the neural tube, and the right, but not the left, ophthalmic nerve of the trigeminal ganglia was affected in ACEA- and AM251-treated embryos. In frog embryos with inactivation or activation of CB1R, or inhibition of Myosin II, the craniofacial and eye regions were smaller and/or less developed, and the melanophores overlying the posterior midbrain were more dense, and stellate in morphology, than the same tissues and cells in control embryos. This data suggests that despite differences in the time of onset of expression, normal activity of CB1R is required for sequential steps in migration and morphogenesis of neural crest cells and derivatives in both chicken and frog embryos. In addition, CB1R may signal through Myosin II to regulate migration and morphogenesis of neural crest cells and derivatives in chicken and frog embryos.

Calcium dynamics at the neural cell primary cilium regulate Hedgehog signaling–dependent neurogenesis in the embryonic neural tube

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.

The complete dorsal structure is formed from only the blastocoel roof of Xenopus blastula: insight into the gastrulation movement evolutionarily conserved among chordates

Gastrulation is a critical event whose molecular mechanisms are thought to be conserved among vertebrates. However, the morphological movement during gastrulation appears to be divergent across species, making it difficult to discuss the evolution of the process. Previously, we proposed a novel amphibian gastrulation model, the “subduction and zippering (S&Z) model”. In this model, the organizer and the prospective neuroectoderm are originally localized in the blastula’s blastocoel roof, and these embryonic regions move downward to make physical contact of their inner surfaces with each other at the dorsal marginal zone. The developmental stage when contact between the head organizer and the anterior-most neuroectoderm is established is called “anterior contact establishment (ACE).” After ACE, the A-P body axis elongates posteriorly. According to this model, the body axis is derived from limited regions of the dorsal marginal zone at ACE. To investigate this possibility, we conducted stepwise tissue deletions using Xenopus laevis embryos and revealed that the dorsal one-third of the marginal zone had the ability to form the complete dorsal structure by itself. Furthermore, a blastocoel roof explant of the blastula, which should contain the organizer and the prospective neuroectoderm in the S&Z model, autonomously underwent gastrulation and formed the complete dorsal structure. Collectively, these results are consistent with the S&Z gastrulation model and identify the embryonic region sufficient for construction of the complete dorsal structure. Finally, by comparing amphibian gastrulation to gastrulation of protochordates and amniotes, we discuss the gastrulation movement evolutionarily conserved among chordates.

Mortality and malformation effects of acute vanadium (V) exposure on the African clawed frog (Xenopus laevis) embryos

Vanadium (V) is a transition metal that is found in low concentrations in aquatic ecosystems. These levels increase due to anthropogenic activities. The mortality and teratogenicity effects of V remain unexplored in amphibian species. To address this gap in the knowledge base, a standard Frog Embryo Teratogenic Index – Xenopus (FETAX) assessment was conducted. Vanadium pentoxide (V2O5) was chosen for its known toxicity in other aquatic biota and its solubility in water. A range-finding test was conducted in two different mediums, V2O5 in distilled water (VDH2O) and V2O5 in FETAX medium (VMED), to determine concentration ranges where effects occurred. Thereafter, definitive tests were conducted using two separate breeding pairs, with two replicate dishes per concentration containing 15 embryos each. Multiple endpoints were assessed including mortality, malformations, minimum concentration to inhibit growth (MCIG), and the teratogenic index (TI). Mortality and malformation effects occurred at different ranges, and therefore, the exposures were conducted in low dose and high dose ranges. The high dose range for mortality effects was conducted at 0, 10, 20, 40, 80, and 160 mg/L of V. The low dose exposures to assess malformation effects were conducted at 0.0001, 0.00025, 0.0005, 0.00075, and 0.001 mg/L. Binary logistic regression was used to determine the LC50 and EC50 for the two sets of definitive tests. The LC50s were determined to be 46.10 mg/L and 26.91 mg/L for VDH2O and 34.50 and 25.25 for VMED for the two breeding pairs respectively. The EC50 was calculated as 0.00053 mg/L and 0.00037 mg/L for VDH2O and 0.00036 mg/L and 0.00017 mg/L for VMED for the two definitive tests respectively. The TI was calculated as 86,981 and 72,729 for VDH2O and 95,833 and 148,526 for VMED. Ultimately, there were severe malformation effects in embryos exposed to low doses of V and V was determined to be a very strong teratogen.

Cell cycle oscillations driven by two interlinked bistable switches.

Regular transitions between interphase and mitosis during the cell cycle are driven by changes in the activity of the enzymatic protein complex cyclin B with cyclin-dependent kinase 1 (Cdk1). At the most basic level, this cell cycle oscillator is driven by negative feedback: active cyclin B-Cdk1 activates the anaphase-promoting complex/cyclosome, which triggers the degradation of cyclin B. Such cell cycle oscillations occur fast and periodically in the early embryos of the frog Xenopus laevis, where several positive-feedback loops leading to bistable switches in parts of the regulatory network have been experimentally identified. Here, we build cell cycle oscillator models to show how single and multiple bistable switches in parts of the underlying regulatory network change the properties of the oscillations and how they can confer robustness to the oscillator. We present a detailed bifurcation analysis of these models.

A molecular network of conserved factors keeps ribosomes dormant in the egg.

Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.

Somitic mesoderm morphogenesis is necessary for neural tube closure during Xenopus development.

Neural tube closure is a fundamental process during vertebrate embryogenesis, which leads to the formation of the central nervous system. Defective neural tube closure leads to neural tube defects which are some of the most common human birth defects. While the intrinsic morphogenetic events shaping the neuroepithelium have been studied extensively, how tissues mechanically coupled with the neural plate influence neural tube closure remains poorly understood. Here, using Xenopus laevis embryos, live imaging in combination with loss of function experiments and morphometric analysis of fixed samples we explore the reciprocal mechanical communication between the neural plate and the somitic mesoderm and its impact on tissue morphogenesis. We show that although somitic mesoderm convergent extension occurs independently from neural plate morphogenesis neural tube closure depends on somitic mesoderm morphogenesis. Specifically, impaired somitic mesoderm remodelling results in defective apical constriction within the neuroepithelium and failure of neural tube closure. Last, our data reveal that mild abnormalities in somitic mesoderm and neural plate morphogenesis have a synergistic effect during neurulation, leading to severe neural tube closure defects. Overall, our data reveal that defective morphogenesis of tissues mechanically coupled with the neural plate can not only drastically exacerbate mild neural tube defects that may arise from abnormalities within the neural tissue but can also elicit neural tube defects even when the neural plate is itself free of inherent defects.

Developmental exposure to the pesticide malathion enhances expression of Prdm12, a regulator of nociceptor development, in Xenopus laevis.

The transcription factor Prdm12 exerts important influences on the development of nociceptors, peripheral touch and pain-sensing neurons, and has been implicated in human pain sensation disorders. We examined the consequences of exposing developing Xenopus laevis embryos to the commonly used pesticide malathion on Prdm12 expression. Using qPCR and western blot analysis we observed that malathion treatment for the first six days of tadpole development significantly increased both prdm12 mRNA levels and Prdm12 protein levels compared to controls. Consequently, early exposure to this pesticide has potential to alter nociceptor development.

Active inference, morphogenesis, and computational psychiatry.

Active inference is a leading theory in neuroscience that provides a simple and neuro-biologically plausible account of how action and perception are coupled in producing (Bayes) optimal behavior; and has been recently used to explain a variety of psychopathological conditions. In parallel, morphogenesis has been described as the behavior of a (non-neural) cellular collective intelligence solving problems in anatomical morphospace. In this article, we establish a link between the domains of cell biology and neuroscience, by analyzing disorders of morphogenesis as disorders of (active) inference. The aim of this article is three-fold. We want to: (i) reveal a connection between disorders of morphogenesis and disorders of active inference as apparent in psychopathological conditions; (ii) show how disorders of morphogenesis can be simulated using active inference; (iii) suggest that active inference can shed light on developmental defects or aberrant morphogenetic processes, seen as disorders of information processing, and perhaps suggesting novel intervention and repair strategies. We present four simulations illustrating application of these ideas to cellular behavior during morphogenesis. Three of the simulations show that the same forms of aberrant active inference (e.g., deficits of sensory attenuation and low sensory precision) that have been used to explain psychopathological conditions (e.g., schizophrenia and autism) also produce familiar disorders of development and morphogenesis when implemented at the level of the collective behavior of a group of cells. The fourth simulation involves two cells with too high precision, in which we show that the reduction of concentration signaling and sensitivity to the signals of other cells treats the development defect. Finally, we present the results of an experimental test of one of the model's predictions in early Xenopus laevis embryos: thioridazine (a dopamine antagonist that may reduce sensory precision in biological systems) induced developmental (anatomical) defects as predicted. The use of conceptual and empirical tools from neuroscience to understand the morphogenetic behavior of pre-neural agents offers the possibility of new approaches in regenerative medicine and evolutionary developmental biology.

Facile methods for reusing laboratory plastic in developmental biology experiments

Plastic pollution negatively affects ecosystems and human health globally, with single-use plastic representing the majority of marine litter in some areas. Life science laboratories prefer pristine conditions for experimental reliability and therefore make use of factory standardized single-use plastic products. This contributes to overall plastic waste in the United States and globally. Here, we investigate the potential of reusing plastic culture dishes and subsequently propose methods to mitigate single-use plastic waste in developmental biology research laboratories. We tested the efficacy of bleach and ethyl alcohol in sterilizing used dishes. We then tested the feasibility of washing and reusing plastic to culture Xenopus laevis embryos subjected to various manipulations. Cleaning and reusing laboratory plastic did not affect the development or survival of X. laevis, indicating that these cleaning methods do not adversely affect experimental outcome and can be used to sterilize plastic before reuse or recycling. Lastly, we performed a survey of various life science laboratories to estimate both waste reduction and savings associated with recycling single-use plastics. Standardization of these procedures would allow research laboratories to benefit economically while practicing environmentally conscious consumption.

Abnormal left-right organizer and laterality defects in Xenopus embryos after formin inhibitor SMIFH2 treatment.

Left-right symmetry breaking in most studied vertebrates makes use of so-called leftward flow, a mechanism which was studied in detail especially in mouse and Xenopus laevis embryos and is based on rotation of monocilia on specialized epithelial surface designated as left-right organizer or laterality coordinator. However, it has been argued that prior to emergence of leftward flow an additional mechanism operates during early cleavage stages in Xenopus embryo which is based on cytoskeletal processes. Evidence in favour of this early mechanism was supported by left-right abnormalities after chemical inhibition of cytoskeletal protein formin. Here we analyzed temporal dimension of this effect in detail and found that reported abnormalities arise only after treatment at gastrula-neurula stages, i.e. just prior to and during the operation of left-right organizer. Moreover, molecular and morphological analysis of the left-right organizer reveals its abnormal development. Our results strongly indicate that left-right abnormalities reported after formin inhibition cannot serve as support of models based on early symmetry breaking event in Xenopus embryo.

Microsurgical Manipulations to Isolate Collectively Migrating Mesendoderm.

Mesendoderm mantle closure completes the gastrulation movements of the <i>Xenopus laevis</i> embryo and provides an unparalleled opportunity to study collective cell behaviors within a mesenchymal tissue. Free-edge sheet-like collective movements of these tissues contrast with movements of epithelial tissues in that mesendodermal cells are not constrained by tight junctions or adherens junctions, yet migrate in a coherent and persistent mode over several hours. Mesendoderm cells are the largest motile cells in the <i>Xenopus</i> embryo and complete a 500-µm migratory path. When mesendoderm is cultured on rigid glass substrates, these cells can exceed 100 µm in length and show a highly persistent leading lamellipodia that can exceed 20 µm from tip to base. These large collectively migrating cells provide a unique imaging opportunity to visualize polarized adhesive and cytoskeletal structures with high-numerical-aperture objectives. Mesendodermal cells in the early embryo originate from around the entirety of the marginal zone and may also be distinguished by their source along the animal-vegetal axis. Here we use the term mesendoderm but note alternative terms for these cells can include head mesoderm, endomesoderm, and prechordal mesoderm. This protocol summarizes microsurgical preparation of mesendoderm tissue explants and "windowed" embryos. Skills needed to dissect fragments of the mesendoderm mantle are marginally greater than those needed to isolate animal cap ectoderm and can be mastered within 2 weeks; skills needed to isolate the mesendoderm "donut" or "ring" or to prepare windowed embryos are significantly greater and may require several additional weeks of training.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Nodal asymmetry and hedgehog signaling during vertebrate left-right symmetry breaking.

Development of visceral left–right asymmetry in bilateria is based on initial symmetry breaking followed by subsequent asymmetric molecular patterning. An important step is the left-sided expression of transcription factor pitx2 which is mediated by asymmetric expression of the nodal morphogen in the left lateral plate mesoderm of vertebrates. Processes leading to emergence of the asymmetric nodal domain differ depending on the mode of symmetry breaking. In Xenopus laevis and mouse embryos, the leftward fluid flow on the ventral surface of the left–right organizer leads through intermediate steps to enhanced activity of the nodal protein on the left side of the organizer and subsequent asymmetric nodal induction in the lateral plate mesoderm. In the chick embryo, asymmetric morphogenesis of axial organs leads to paraxial nodal asymmetry during the late gastrulation stage. Although it was shown that hedgehog signaling is required for initiation of the nodal expression, the mechanism of its asymmetry remains to be clarified. In this study, we established the activation of hedgehog signaling in early chick embryos to further study its role in the initiation of asymmetric nodal expression. Our data reveal that hedgehog signaling is sufficient to induce the nodal expression in competent domains of the chick embryo, while treatment of Xenopus embryos led to moderate nodal inhibition. We discuss the role of symmetry breaking and competence in the initiation of asymmetric gene expression.

Enhancers of Host Immune Tolerance to Bacterial Infection Discovered Using Linked Computational and Experimental Approaches (Adv. Sci. 26/2022)

Drug Repurposing Linked computational and experimental approaches identified that Xenopus laevis embryos can naturally tolerate certain bacteria by modulating their metal ion binding and hypoxia gene networks to maintain health despite high pathogen load. In article number 2200222, using natural tolerance mechanisms as inspiration, Megan Sperry, Richard Novak, Donald E. Ingber, and co-workers induce an artificial tolerant state in susceptible Xenopus using drug repurposing.

ANT1 overexpression models: Some similarities with facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by progressive muscle weakness. Adenine nucleotide translocator 1 (ANT1), the only 4q35 gene involved in mitochondrial function, is strongly expressed in FSHD skeletal muscle biopsies. However, its role in FSHD is unclear. In this study, we evaluated ANT1 overexpression effects in primary myoblasts from healthy controls and during Xenopus laevis organogenesis. We also compared ANT1 overexpression effects with the phenotype of FSHD muscle cells and biopsies.Here, we report that the ANT1 overexpression-induced phenotype presents some similarities with FSHD muscle cells and biopsies. ANT1-overexpressing muscle cells showed disorganized morphology, altered cytoskeletal arrangement, enhanced mitochondrial respiration/glycolysis, ROS production, oxidative stress, mitochondrial fragmentation and ultrastructure alteration, as observed in FSHD muscle cells. ANT1 overexpression in Xenopus laevis embryos affected skeletal muscle development, impaired skeletal muscle, altered mitochondrial ultrastructure and led to oxidative stress as observed in FSHD muscle biopsies. Moreover, ANT1 overexpression in X. laevis embryos affected heart structure and mitochondrial ultrastructure leading to cardiac arrhythmia, as described in some patients with FSHD.Overall our data suggest that ANT1 could contribute to mitochondria dysfunction and oxidative stress in FSHD muscle cells by modifying their bioenergetic profile associated with ROS production. Such interplay between energy metabolism and ROS production in FSHD will be of significant interest for future prospects.

Distinct spatiotemporal contribution of morphogenetic events and mechanical tissue coupling during Xenopus neural tube closure.

ABSTRACTNeural tube closure (NTC) is a fundamental process during vertebrate development and is indispensable for the formation of the central nervous system. Here, using Xenopus laevis embryos, live imaging, single-cell tracking, optogenetics and loss-of-function experiments, we examine the roles of convergent extension and apical constriction, and define the role of the surface ectoderm during NTC. We show that NTC is a two-stage process with distinct spatiotemporal contributions of convergent extension and apical constriction at each stage. Convergent extension takes place during the first stage and is spatially restricted at the posterior tissue, whereas apical constriction occurs during the second stage throughout the neural plate. We also show that the surface ectoderm is mechanically coupled with the neural plate and its movement during NTC is driven by neural plate morphogenesis. Finally, we show that an increase in surface ectoderm resistive forces is detrimental for neural plate morphogenesis.