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.

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.

Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos

AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One and half nanogram of IRE1α,1 ng of IRE1α-GR mRNA,1 ng of IRE1αΔC-GR mRNA,and 50 ng of IRE1α morpholino oligonucleotide(MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis.To rescue the effect of IRE1α MO,1 ng of IRE1α-GR mRNA was coinjected with 50 ng of MO.For the activation of the GR-fusion proteins,dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH.Embryos were kept in dexamethasone up to stage 41.Whole-mount in situ hybridization was used to determine specific gene expression,such as IRE1α,IRE1β,Xbra and Xsox17α.IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting.RESULTS:In the whole-mount in situ hybridization analysis,xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage.The relatively higher expression of IRE1α was observed in the pancreas,and significant transcription of IRE1β was found in the liver.IRE1α protein could be detected at all developmental stages analyzed,from stage 1 to stage 42.Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos.And at tadpole stage,the embryos injected with IRE1α-GR mRNA did not display overt phenotype,such as gut-coiling defect.Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages.We did not observe a significant change of mesodermal and endodermal marker gene expression,while after stage 40,about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil,but other structures outside the gastrointestinal tract were relatively normal.To test if the phenotypes were specifically caused by the knockdown of IRE1α,a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO.The data obtained demonstrated that the gut coiling defect was rescued.The deletion mutant of IRE1α was constructed,consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC,to investigate the functional domain of IRE1α.Injection of IRE1αΔCGR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α.In order to investigate if IRE1α/XBP1 pathway was involved in gut development,50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage.CONCLUSION:IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.

Developmental expression of three prmt genes in Xenopus

DEAR EDITOR Protein arginine methyltransferases (PRMTs)are involved in many cellular processes via the arginine methylation of histone or non-histone proteins.We examined the expression patterns of prmt4,prmt7,and prmt9 during embryogenesis in Xenopus using whole-mount in situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-PCR).Xenopus prmt4 and prmt7 were expressed in the neural crest,brain,and spinal cord,and also detected in the eye,branchial arches,and heart at the tailbud stage.Specific print9 signals were not detected in Xenopus embryos until the late tailbud stage when weak expression was observed in the branchial arches.Quantitative RT-PCR indicated that the expression of prmt4 and prmt7 was up-regulated during the neurula stage,whereas prmt9 maintained its low expression until the late tailbud stage,consistent with the whole-mount in situ hybridization results.Thus,the developmental expression patterns of these three print genes in Xenopus embryos provide a basis for further functional study of such genes.

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.

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.

Involvement of XZFP36L1,an RNA-binding protein,in Xenopus neural development

Xenopus ZFP36L1(zinc finger protein 36,C3H type-like 1)belongs to the ZFP36 family of RNA-binding proteins,which contains two characteristic tandem CCCH-type zinc-finger domains.The ZFP36 proteins can bind AU-rich elements in 3'untranslated regions of target mRNAs and promote their turnover.However,the expression and role of ZFP36 genes during neural development in Xenopus embryos remains largely unknown.The present study showed that Xenopus ZFP36L1 was expressed at the dorsal part of the forebrain,forebrain-midbrain boundary,and midbrain-hindbrain boundary from late neurula stages to tadpole stages of embryonic development.Overexpression of XZFP36L1 in Xenopus embryos inhibited neural induction and differentiation,leading to severe neural tube defects.The function of XZP36L1 requires both its zinc finger and C terminal domains,which also affect its subcellular localization.These results suggest that XZFP36L1 is likely involved in neural development in Xenopus and might play an important role in post-transcriptional regulation.

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.

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.

Expression and kinetic characteristics of muscle type acetylcholine receptors in Xenopus oocytes

Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa

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.

Expression Patterns of CAPN1 and CAPN8b Genes during Embryogenesis in Xenopus laevis

Calpains are a superfamily of Ca2+-dependent cysteine proteases, implicated in various cellular processes and thus probably necessary in all the stages of cell life. The first extended report of quantification of total RNAs within the developmental stages of Xenopus laevis was described in this study. Decreases of total RNAs were positively associated with waves of apoptotic cell death (onset of gastrulation, and morphogenesis). Using qPCR, the temporal expression pattern of CAPN1 and CPAN8b (XCL-2) were characterized during the Xenopus laevis embryogenesis. Transcripts of the CAPN1 and CAPN8 genes were detectable from gastrula stage and their levels oscillated throughout development. The expression of the CAPN1 (mu/I) gene was observed in earliest stage, indicating a maternal origin, while expression of the CAPN8b gene was detectable after midblastula transition. The levels of the two transcripts then started to rise again obviously as a result of zygotic expression (stage 11). The CAPN1 gene expression was particularly expressed at tailbud stage, while the CAPN8 transcripts were found at gastrula, neurula and tailbud stages. This is the first report of quantification of mRNAs CAPN8b and CAPN1 (mu/I) within the developmental stages of Xenopus laevis by qPCR.

The Cell Sorting Process of Xenopus Gastrula Cells Progresses in a Stepwise Fashion Involving Concentrification and Polarization

Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.

Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in <i>Xenopus laevis</i>embryogenesis

To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.

Xenopus transgenic to screen candidate molecules favoring myelin repair

To facilitate live imaging of demyelination and remyelination, we have generated a Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelinating oligodendrocytes. In this line, the proximal portion of mouse myelin basic protein (MBP) regulatory sequence, specific to mature myelin-forming oligodendrocytes, drives the expression of a transgene protein formed by the GFP reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. The NTR enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. The demyelination response of MBP-GFP-NTR tadpoles to MTZ is followed by spontaneous remyelination after cessation of MTZ treatment. Thanks to the transparency of tadpoles, these events can be monitored in vivo by two-photon imaging and easily quantified on a simple fluorescence macroscope. We have used the MBP-GFP-NTR model to screen in vivo molecules favoring remyelination. At the end of MTZ-induced demyelination, tadpoles were switched to water containing the compounds to be tested. After 3 days of treatment remyelination was assayed by counting the number of GFP+ oligodendrocytes per optic nerve. Using Crispr/Cas9 strategy, the target of the candidate molecule can be easily deleted in the MBPGFP-NTR line to examine the mechanism of action of the candidate molecule. Therefore the Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelinating oligodendrocytes, constitutes a new medium-throughput screening platform for myelin repair therapeutics in demyelinating diseases.

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.

Toxic effects of long-term dual or single exposure to oxytetracycline and arsenic on Xenopus tropicalis living in duck wastewater

Direct discharge of aquaculture wastewater may have toxic effects,due to the presence of heavy metals,antibiotics,and even resistant pathogens,but little attention has been given.Here,tanks simulating a wild ecosystem were built to study the effects of long-term exposure to duckwastewater containing oxytetracycline(OTC)and/or arsenic(As)on the growth,physiological function,and gut microbiota evolution of Xenopus tropicalis.The results showed that duck wastewater had no apparent impact on X.tropicalis,but the impact increased significantly(P<0.05)with exposure to OTC and/or As,especially the impact on body weight and growth rate.Biochemical indicators revealed varying degrees of oxidative stress damage,hepatotoxicity(inflammation,necrosis,and sinusoids),and collagen fibrosis of X.tropicalis in all treated groups after 72 days of exposure,which indirectly inhibited X.tropicalis growth.Moreover,16S rDNA amplicon sequencing results showed that the gut microbiota structure and metabolic function were perturbed after chronic exposure,which might be the leading cause of growth inhibition.Interestingly,the abundance of intestinal resistance genes(RGs)increased with exposure time owing to the combined direct and indirect effects of stress factors in duck wastewater.Moreover,once the RGs were expressed,the resistance persisted for at least 24 days,especially that conferred by tetA.These results provide evidence of the toxic effects of DW containing OTC(0.1–4.0 mg/L)and/or As(0.3–3.5μg/L)on amphibians and indicate that it is vital to limit the usage of heavy metals and antibiotics on farms to control the biotoxicity of wastewater.

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.

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in Xenopus egg extracts

Nuclear envelope is a dynamic structure in the cell cycle. At the beginning of mitosis, nuclear envelope breaks down and its components disperse into the cytoplasm. At the end of mitosis, nuclear envelope reassembles using the dispersed components. Searching for the mechanisms of the nuclear disassembly and reassembly has for a long time been one of the key projects for cell biologists. In this report we show that microtubules take a role in the nuclear envelope assembly around the sperm chromatin in Xenopus egg ex-tracts. Microtubule cytoskeleton has been demonstrated to take roles in the transport of intracellular membranes such as Golgi and ER vesicles. We found that the nuclear envelope assembly needs functional microtubules. At the beginning of the nuclear assembly, microtubules nucleated to form a microtubule aster around the centrosome at the base of the sperm head. Using the microtubule drug colchicine to dis-rupt the microtubule nucleation, nuclear envelope reassem-bly was seriously inhibited. If the microtubules were stabi-lized by taxol, another microtubule drug, the nuclear enve-lope reassembly was also interfered, although a significantly large aster formed around the chromatin. Based on these observations, we propose that microtubules play an impor-tant role in the nuclear envelope reassembly maybe by transporting the nuclear envelope precursors to the chroma-tin surfaces.

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.