Temporal and Spatial Expression Patterns of Sox1 Gene in Xenopus laevis Embryo

We describe the temporal and spatial expression pattern of Sox 1 gene during Xenopus laevis early development and compare the expression patterns of Sox 1-3 in the developing eye and brain. Alignment of Sox 1-3 amino acid sequences shows a high conservation within the HMG-box DNA binding domains. RT-PCR analysis indicates that Sox 1 is expressed throughout development from the unfertilized egg to at least the tadpole stage, although at different expression levels. The transcripts of XSox 1 are detected in the animal pole at cleavage and blastrula stages and mainly in the central nervous system （CNS） and the developing eye at neurula stages. The study of the developmental expression of XSox 1 will aid in the elucidation of the function of SoxB 1 subgroup genes in vertebrate neurogenesis.

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.

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.

Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.

A developmental biological study of aldolase gene expression in Xenopus laevis

We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.

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.

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.

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.

Harmful Effects on African Clawed Frog （Xenopus laevis） Reproduction as Expression of High Water Phosphates Levels

Amphibians are considered as reliable indicators of environmental quality. In Europe, a general decline of amphibians population parallels a worldwide decline, and some of the factors which are thought to be responsible to this decline are habitat loss, introduction of exotic species, disease and decreasing water quality. Recent investigations suggest that these eutrophic conditions may be associated with frog reproduction problems. Water quality criteria settled for the different species of amphibians do not currently exist, but in the present paper, the authors report their observation about water phosphate concentrations in association with hipofertility in African clawed frogs （Xenopus laevis）. The study considered two different groups of African clawed frog showing hypofertility （22 patients） hosted in captivity. The animals were visited, and the filtration systems functionality and the water quality were checked, i.e., temperature, pH, ammonia, nitrite, nitrate, phosphates, hardness and alkalinity. In the first group, it was decided to perform ultrasound scans, parasitological tests on fecal samples, and microscopical/macroscopical examination, as well as microbiological analysis on collected oocytes. In the second group, only fecal samples were collected in order to perform parasitologiacal exams. No pathological findings were showed by veterinary tests. Water changes were carried out in both facilities and after two months, the fertility in African clawed frogs improved, evidencing the involvement of phosphates values in the onset of the problem and even more in its resolution. More studies are needed to further define this correlation.

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.

Physical Blocking Neural Tube Closure Affects Radial Intercalation and Neural Crest Midline-directed Migration in Xenopus Dorsal Explants

Neural tube defects （NTDs） are severe congenital malformation diseases, which occur in 1 out of 1000 births in human. In Xenopus, several tissue movements are involved in the neural tube closure process. Immediately after the neural tube fusion, the neural crest cells get monopolar protrusion toward dorsal midline and migrate to form the roof of the neural tube. At the same time, radial intercalation takes place from the ventral neural tube and forces it to be single-layered. Here, we physically block the neural tube closure to test the cell movements and the following patterning in Xenopus laevis explants. The results show that the single-layered neural tube fails to form and the neural crest cells remain at the lateral regions in the explants with NTDs. However, the patterning of the neural tube is not affected as indicated by the normal expression of the preneural genes. These results indicate a requirement of the neural tube fusion for the radial intercalation and the dorsal midline directed neural crest migration, but not for the dorsal-ventral patterning of the neural tube.

Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts

We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.

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.

Characterization of the Xenopus homolog of animmediate early gene associated with cell activation: sequence analysis and regulation of its expression by thyroid hormone during amphibian metamorphosis

The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.

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.