Thyroid disruption by technical decabromodiphenyl ether (DE-83R) at low concentrations in Xenopus laevis

Decabromodiphenyl ether （decaBDE）,as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R （1-1000 ng/L） in water from stage 46/47 （free swimming larvae,system of Nieuwkoop and Faber） to stage 62.DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis （presented by forelimb emergence,FLE）.Histological examination showed that DE83R at all tested concentrations caused histological alterations-multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation,in thyroid glands.All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression.These results demonstrated that technical decaBDE disrupted the thyroid system in X.laevis tadpoles.Analysis of polybrominated diphenyl ethers （PBDEs） （sum of 39 congeners） in X.laevis indicated that mean concentrations of total PBDEs in X.laevis exposed to 1,10,100,1000 ng/L were 11.0,128.1,412.1,1400.2 ng/g wet weight,respectively.Considering that PBDEs burden of X.laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies,our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.

Bioconcentration kinetics of PCBs in various parts of the lifecycle of the tadpoles Xenopus laevis

Polychlorinated biphenyls （PCBs） in Xenopus laevis have been reported only for a few congeners. Additionally, there is very little information on the ability of Xenopus laevis to bioconcentrate PCBs. To address these issues, the tadpole Xenopus laevis was exposed to Aroclor 1254 mixtures in water at room temperature for 110 d followed by an additional 110 d of nonspiked PCBs in the water for the control group. During the whole process, bioconcentration factors （BCFs） of PCBs ranged from 1180 to 15670. For most PCB congeners, the highest and lowest bioconcentrations of the kinetic curves were found to be remarkably simultaneous, respectively. All 141 PCB congeners under the same experimental conditions had no linear correlation on the lgBCF versus lgKow relationship. The relationship between lgBCFs and lgKow followed a parabolic pattern indicative of selective bioconcentration, suggesting that the kinetic curves of the PCB congeners observed in the lifecycle of the tadpoles may be concentrated due to the amphibian special species and internal metabolism. In contrast, lgBCFs for PCBs were inversely related to lgKow, suggesting that a metabolism of the higher Kow PCB congeners occurred. These results support the author＇s conclusion that the tadpole Xenopus laevis plays major roles in the bioconcentration of PCB congeners, and demonstrated that the exposure kinetic curves of PCB congeners are complex. Besides the amphibian metamorphous development, the lifecycle of the tadpole Xenopus laevis also may be of importance in determining the bioconcentration of PCB congeners.

IRE1α knockdown rescues tunicamycin-induced developmental defects and apoptosis in Xenopus laevis

Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon endoplasmic reticulum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventional splicing of XBP1 mRNA, which activates unfolded protein response （UPR） to restore ER homeostasis. In mice, IRE1α plays an essential role in extraembryonic tissues. However, its precise action during the early stage of development is unknown. In this study, the gain and loss-of-function analyses were used to investigate the function of Xenopus IRE1α （xIRE1α）. The effects of xIRE1α during embryo development were detected with RT-PCR and whole mount in situ hybridization. ER stress was induced by tunicamycin. The apoptofic cells were measured by TUNNEL assays. Although both gain and loss of xlRE1α function had no significant effect on Xenopus embryogenesis, knockdown of xIRE1α could rescue tunicamycin-induced developmental defects and apoptosis. The finding indicates that xIRE1α is not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.

Developmental dependence for functional eye regrowth in Xenopus laevis

Introduction：A key challenge in designing tissue repair strategies is knowing whether and how developmental mechanisms are used for successful repair of mature/adult tissues.Although it is known that developmental components are used in repair,it remains mostly unclear which ones are required and whether they act similarly as during development.This issue is further complicated by the fact that it is difficult.

Low concentrations of atrazine,glyphosate,2,4-dichlorophenoxyacetic acid,and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis

Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides （atrazine, 2,4-dichloropheoxyacetic acid （2,4-D）, triadimefon, and glyphosate） in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.

8 Å structure of the outer rings of the Xenopus laevis nuclear pore complex obtained by cryo-EM and AI

The nuclear pore complex(NPC),one of the largest protein complexes in eukaryotes,serves as a physical gate to regulate nucleocytoplasmic transport.Here,we determined the 8Åresolution cryo-electron microscopic(cryo-EM)structure of the outer rings containing nuclear ring(NR)and cytoplasmic ring(CR)from the Xenopus laevis NPC,with local resolutions reaching 4.9Å.With the aid of AlphaFold2,we managed to build a pseudoatomic model of the outer rings,including the Y complexes and flanking components.In this most comprehensive and accurate model of outer rings to date,the almost complete Y complex structure exhibits much tighter interaction in the hub region.In addition to two copies of Y complexes,each asymmetric subunit in CR contains five copies of Nup358,two copies of the Nup214 complex,two copies of Nup205 and one copy of newly identified Nup93,while that in NR contains one copy of Nup205,one copy of ELYS and one copy of Nup93.These in-depth structural features represent a great advance in understanding the assembly of NPCs.

Molecular analyses of Xenopus laevis Mesp-related genes

During vertebrate somitogenesis,somites bud off from the anterior end of the presomitic mesoderm(PSM).Meso-dermal posterior(Mesp)-related genes play essential roles in somitogenesis,particularly in the definition of the somite boundary position.Among vertebrates,two types of Mesp-related genes have been identified:Mesp1 and Mesp2 in the mouse;Meso-1 and Meso-2 in the chicken;Xl-mespa and Xl-mespb(also known as Thylacine1)in the African clawed frog(Xenopus laevis);and mesp-a and mesp-b in the zebrafish.However,the functional differences between two Mesp-related genes remain unknown.In the present study,we carried out comparative analyses of the Xl-mespa and Xl-mespb genes.The amino acid sequences of the Xl-mespa and Xl-mespb proteins showed a high level of similarity.The expression of Xl-mespa started broadly in the ventrolateral mesoderm and gradually shifted to a striped pattern of expression.In contrast,Xl-mespb showed a striped pattern of expression from the start.These expression profiles completely overlapped at the PSM during somitogenesis.To investigate the functional differ-ences between Xl-mespa and Xl-mespb in terms of target gene regulation,we carried out a luciferase assay using the murine Lunatic fringe(L-fng)promoter.Transcription of the L-fng promoter was activated more strongly by Xl-mespb than by Xl-mespa.This same pattern was observed for the murine Mesp-related proteins.These results suggest that the functional differences between the two types of Mesp-related genes are evolutionally conserved in vertebrates.

IRE1α is essential for Xenopus pancreas development

Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon the endoplasmic reticu- lum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventionally splicing XBP1 mRNA, which activates the unfolded protein response （UPR） to restore ER homeostasis. In mice, IREla inactivity leads to embryonic death and IREla plays an essential role in extraembryonic tissues and the placenta. However, its precise action in the embryo proper is still unknown. In this study, the loss of function ana/ysis was performed to investigate the function of Xenopus IREla （xlREla） during pancreas development. Firstly, the complete open reading frame of xIRE1α was amplified and the expression pattern was detected. The effects of Xenopus IRE1α and XBP1 during embryo development were detected with whole-mount in situ hybridization. The results demonstrated that xIRE1α was much closer to human IREla when compared with their sequence alignment, xlREla was expressed strongly in developing pancreas and the knockdown of xIREla inhibited the differentiation and specification of the pancreas, xlREltt, which was required for cytoplasmic splicing of XBP1 pre-mRNA and XB- P1MO, also showed inhibitory effects on pancreas development. These results suggest that xlREla is essential for pancreas development during embryogenesis and functions via the XBP1 dependent pathway.

Tcf7l1 promotes transcription of Kruppel-like factor 4 during Xenopus embryogenesis

Kruppel-like factor 4（Klf4） is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711（dnTcf711）, which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.

Neurolemma-Injected Xenopus Oocytes: An Innovative Ex Vivo Approach to Study the Effects of Pyrethroids on Ion Channels in Their Native State

Microtransplantation of rat brain neurolemma into the plasma membrane of Xenopus laevis oocytes is an ex vivo method used to study channels and receptors in their native state using standard electrophysiological approaches.In this review,we show that oocytes injected with adult rat brain neurolemma elicited tetrodotoxin-sensitive inward ion currents upon membrane depolarization,which were increased in a concentration-dependent manner by treatment with the pyrethroid insecticides permethrin and deltamethrin.Under our initial protocols,oocyte health was reduced over time and neurolemma incorporation varied between batches of oocytes from different frogs,limiting the usefulness of the assay for regulatory issues.A collection of changes to the assay procedure,data acceptance criteria,and analysis method yield substantially improved precision and,hence,assay performance.These changes established this ex vivo approach as a toxicologically relevant assay to study the toxicodynamic action of pyrethroids on ion channels in their native state using neurolemma fragments prepared from juvenile and adult rat brains.

Purinergic P2Y Receptors Are Involved in Xenopus Head Formation

P2Y receptors belong to the family of G protein-coupled receptors and are activated by nucleotides in the extracellular space. We showed that Xenopus P2Y1 and P2Y11 were expressed in the dorsal marginal zone from early gastrula stage and enriched in the central nervous system from neurula stages. They were expressed in the prospective head region during early development. Knockdown of P2Y1 and P2Y11 caused head malformation, such as small eyes, brain atrophy, and defect in cartilage tissues, as well as reduced expression of neural, placode, and neural crest markers. Furthermore, the expression of neural plate and epidermal markers was affected by P2Y1 or P2Y11 depletion at early neurula stage, suggesting that P2Y1 or P2Y11 might be required for the neural induction. Our findings suggested that P2Y receptors might be involved in distinguishing between neural and non-neural fates. The results also suggested that P2Y1 or P2Y11 could play a role in neural induction and/or maintenance of neural tissues in the head formation processes.

非洲爪蟾与牛蛙骨骼标本制作及结构比较

为了解非洲爪蟾(Xenopus laevis)与牛蛙(Rana catesbiana Shaw)骨骼构造及其生理功能的差异,制作了非洲爪蟾和牛蛙骨骼标本,研究其各部骨骼结构,探讨骨骼结构与生理功能的相关性。结果表明,牛蛙有锄骨和锄骨齿,非洲爪蟾没有;牛蛙与非洲爪蟾四肢骨骼组成相同,但牛蛙前肢中各指指骨数目与非洲爪蟾相反;牛蛙与非洲爪蟾脊柱均由8块椎骨构成,但椎弓方向和脊柱形状不同;牛蛙肩带和胸部有肩胛骨、中胸骨和剑胸软骨,但非洲爪蟾没有;牛蛙与非洲爪蟾腰带组成相同但髋臼不同。同一种族的不同物种形态结构与生理功能都与其长期生长坏境密切相关,揭示了蛙类骨骼形态的环境适应性特征。

Effects of Dietary Protein Variations at Different Life-stages on Vocal Dominance of the African Clawed Frogs

How nutritional conditions during early development affect an organism’s phenotype at adulthood is still poorly understood despite a plethora of research on developmental plasticity.The"environmental matching"hypothesis predicts that individuals will have high fitness providing that their adult environment"matches"what they experienced during development.In contrast,the"silver spoon"hypothesis predicts that individuals who obtain better developmental resources will be generally superior.Here we tested these two hypotheses and examined the underlying hormonal mechanisms by manipulating the early dietary protein content of African clawed frogs(Xenopus laevis)for a year with a 2×2 factorial experimental design.We found that only a lowprotein food during development enhanced the vocal competition ability of male X.laevis,and that vocal dominance was associated with higher cortisol levels but not related with testosterone content.These results were not congruent with the"environmental matching"hypothesis or with the"silver spoon"hypothesis,suggesting the behavioral plasticity during development is more complex than our expectation in amphibians.

Optimization of the T3-induced Xenopus metamorphosis assay for detecting thyroid hormone signaling disruption of chemicals

T3-induced Xenopus metamorphosis is an ideal model for detecting thyroid hormone（TH）signaling disruption of chemicals. To optimize the T3-induced Xenopus assay and improve its sensitivity and reproducibility, we intend to develop quantitatively morphological endpoints and choose appropriate concentrations and exposure durations for T3 induction.Xenopus laevis at stage 52 were exposed to series of concentrations of T3（0.31–2.5 nmol/L）for 6 days. By comparing morphological changes induced by T3, we propose head area,mouth width, unilateral brain width/brain length, and hindlimb length/snout-vent length as quantitative parameters for characterizing T3-induced morphological changes, with body weight as a parameter for indicating integrated changes. By analyzing time-response curves, we found that following 4-day exposure, T3-induced grossly morphological changes displayed linear concentration–response curves, with moderate morphological changes resulting from 1.25 nmol/L T3 exposure. When using grossly morphological endpoints to detect TH signaling disruption, we propose 4 days as exposure duration of T3, with concentrations close to 1.25 nmol/L as induction concentrations. However, it is appropriate to examine morphological and molecular changes of the intestine on day 2 due to their early response to T3. The quantitative endpoints and T3 induction concentrations and durations we determined would improve the sensitivity and the reproducibility of the T3-induced Xenopus metamorphosis assay.

An ex vivo assay for screening glucocorticoid signaling disruption based on glucocorticoid-response gene transcription in Xenopus tails

There is a pressing need for developing in vivo or ex vivo assays to screen the glucocorticoid（GC） signaling disruption of chemicals. Thus, we aimed to establish an ex vivo assay for screening GC signaling disruption based on the GC-response gene transcription in Xenopus laevis tails cultured ex vivo. Firstly, we investigated effects of corticosterone（CORT, a main GC in frogs） on GC-response gene expression, and determined the six genes as molecular endpoints for assaying the GC signaling disruption. CORT in the range of 1.56–400 nmol/L was found to up-regulate transcription of the six GC-response genes, exhibiting comparable or higher sensitivity than previously reported assays. To validate this ex vivo assay, then, we examined effects of dexamethasone（a known GC signaling agonist） on GC-response gene expression. Dexamethasone displayed an agonistic action in a concentration-dependent manner, further demonstrating the efficiency of the established assay. Finally, we applied the ex vivo assay to evaluate the GC signaling disruption of bisphenol A（BPA）. In accordance with previous reports, we found a concentration-dependent agonistic activity of BPA,showing that the established assay is effective for detecting the GC signaling disrupting activity of environmental chemicals. Correspondingly, the GC signaling agonistic actions of CORT and BPA in ex vivo tails accorded with the observations in vivo, indicating that the ex vivo assay is able to detect the actions of chemicals in vivo. Overall, we established an ex vivo assay that can effectively screen GC signaling disruption of environmental chemicals.

Gut microbiota dysbiosis involves in host non-alcoholic fatty liver disease upon pyrethroid pesticide exposure

A growing body of evidence has demonstrated the significance of the gut microbiota in host health,while the association between gut microbiota dysbiosis and multiple diseases is yet elusive in the scenario of exposure to widely used pesticides.Here,we show that gut microbiota dysbiosis involves in host's abnormal lipid metabolism and consequently the non-alcoholic fatty liver disease in Xenopus laevis upon exposure to cis-bifenthrin,one of the most prevalent pyrethroid insecticides in the world.With the guidance of gut microbiota analysis,we found that cis-bifenthrin exposure significantly perturbed the gut microbial community,and the specific taxa that served as biomarkers were identified.Metabolomics profiling and association analysis further showed that a significant change of intestinal metabolites involved in lipid metabolic pathways were induced along with the microbiota dysbiosis upon exposure to cis-bifenthrin.Detailed investigation showed an altered functional regulation of lipids in the liver after cis-bifenthrin exposure and the accumulation of lipid droplets in hepatocytes.Specifically,a change in deoxycholic acid alters bile acid hepatoenteral circulation,which affects lipid metabolism in the liver and ultimately causes the development of fatty liver disease.Collectively,these findings provide novel insight into the gut microbiota dysbiosis upon pesticide exposure and their potential implication in the development of chronic host diseases related to liver metabolic syndrome.