Ibuprofen-induced multiorgan malformation during embryogenesis in Xenopus laevis (FETAX).

Ibuprofen, one of the most commonly prescribed nonsteroidal anti-inflammatory drugs, has not been fully assessed for embryonic toxicity in vertebrates. Here, we systematically assessed the embryotoxicity of ibuprofen in Xenopus laevis at various concentrations during embryogenesis. Embryos were treated with different concentrations of ibuprofen, ranging from 8 to 64 mg/L, at 23 °C for 96 h, and examined daily and evaluated at 72 hpf. Lethal or teratogenic effects were documented. For histological analysis, paraffin embedded embryos were transversely sectioned at a thickness of 10-µm and stained with hematoxylin and eosin. Total RNA was isolated from embryos at stages 6, 12, 22 and 36, and real-time quantitative PCR was performed. Ibuprofen-treated embryos showed delayed or failed dorsal lip formation and its closure at the beginning of gastrulation. This resulted in herniation of the endodermal mass after gastrulation under high concentrations of ibuprofen-treated embryos. Underdeveloped intestines with stage and/or intestinal malrotation, distorted microcephaly, and hypoplastic heart, lungs, and pronephric tubules were observed in ibuprofen-treated embryos. Cephalic, cardiac, and truncal edema were also observed in them. The severity of the deformities was observed in a concentration-dependent manner. The teratogenic index was 2.28. These gross and histological disruptions correlated well with the altered expression of each organ marker gene. In conclusion, ibuprofen induced delayed and disrupted gastrulation in the early developmental stage and multiorgan malformation later in the organogenesis stage of Xenopus laevis embryos.

Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Evaluation of the toxic effects of celecoxib on Xenopus embryo development.

Celecoxib is a non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 and is prescribed for severe pain and inflammation. The excellent therapeutic effects of celecoxib mean that it is frequently used clinically, including for women of child-bearing age. However, the prenatal effects of this compound have not been studied extensively in vertebrates. The present study examined the developmental toxicity of celecoxib using a frog embryo teratogenic assay-Xenopus (FETAX). In addition, we examined its effects on cell migration using co-cultures of human umbilical vein endothelial cells and 10T1/2 cells. These studies revealed that celecoxib induced concentration-dependent mortality and various malformations of the Xenopus internal organs, including gut miscoiling, haemorrhage, and oedema. Celecoxib also downregulated the expression of vascular wall markers (Msr and alpha smooth muscle actin) and other organ-specific markers (Nkx2.5, Cyl104 and IFABP). In vitro co-culture studies revealed that celecoxib inhibited pericyte migration and differentiation into vascular smooth muscle cells. In conclusion, celecoxib was both toxic and teratogenic in Xenopus embryos, where it produced serious heart and vessel malformation by inhibiting vascular wall maturation and vascular network formation.

Ablation of C/EBP homologous protein attenuates renal fibrosis after ureteral obstruction by reducing autophagy and microtubule disruption.

Fibrosis is an undesirable consequence of injury and a critical problem in many diseases. Recent studies have demonstrated an association of C/EBP homologous protein (CHOP) with fibrosis. We investigated the mechanism of CHOP in kidney fibrosis progression after unilateral ureteral obstruction (UUO) using Chop gene-deleted (Chop-/-) mice and their wild-type littermates (Chop+/+). UUO-induced kidney fibrosis was reduced in the Chop-/- than Chop+/+ mice. After UUO, CHOP expression was detected in the cytosol and nucleus of distal tubule cells and collecting duct cells of the kidney. UUO formed the autophagosome and increased the expression of autophagy proteins, Beclin-1, LC3-I and II, and p62 in the kidneys. These UUO-induced changes were significantly reduced in Chop-/- mice. Furthermore, Chop gene deletion attenuated mitochondrial fragmentation with lower expression of Fis-1, a mitochondrial fission protein, but higher expression of Opa-1, a mitochondrial fusion protein, than that seen in the wild-type mice. UUO disrupted the microtubule, which is involved in autophagosome formation, and this disruption was milder in the Chop-/- than Chop+/+ mouse kidney, with less reduction of histone deacetylase 6 and α­tubulin acetyl transferase, which acetylates tubulin, a component of the microtubule. After UUO, apoptosis, a consequence of autophagy and mitochondrial damage, was reduced in the Chop-/- mouse kidney cells than in Chop+/+ mice. Thus, the ablation of Chop attenuates renal fibrosis, accompanied by reduced autophagy, mitochondrial fragmentation, microtubule disruption, and apoptosis. Overall, these results suggest that CHOP plays a critical role in the progression of kidney fibrosis, likely through regulation of autophagy and apoptosis.

NSrp70 is significant for embryonic growth and development, being a crucial factor for gastrulation and mesoderm induction.

NSrp70 (nuclear speckle-related protein 70), a recently discovered protein and it belongs to the serine/arginine (SR) rich related protein family. NSrp70 is recognized as an important splicing factor comprising RNA recognition motif (RRM) and arginine/serine (RS)-like regions at the N- and C-terminus respectively, along with two coiled coil domains at each terminus. However, other functions of NSrp70 remain unelucidated. In this study, we investigated the role of NSrp70 in Xenopus embryogenesis and found that its maternal expression plays a critical role in embryonic development. Knockdown of NSrp70 resulted in dramatic reduction in the length of developing tadpoles and mild to severe malformation in Xenopus embryos. In addition, knockdown of NSrp70 resulted in an extremely short axis by blocking gastrulation and convergent extension. Further, animal cap assays along with activin A treatment revealed that NSrp70 is an essential factor for dorsal mesoderm induction as knockdown of NSrp70 caused a dramatic down-regulation of dorsal mesoderm specific genes and its loss significantly shortened the elongation region of animal caps. In conclusion, NSrp70 is crucial for early embryonic development, influencing gastrulation and mesoderm induction.

IFT46 plays crucial roles in craniofacial and cilia development.

The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development.

The splicing factor SRSF1 modulates pattern formation by inhibiting transcription of tissue specific genes during embryogenesis.

Alternative splicing is a major mechanism regulating pattern of gene expression through the production of multiple mRNAs from a single gene transcript. Any misregulation can cause various human diseases and also have severe effects on embryogenesis. SRSF1 is one of the critical factors regulating alternative splicing at many stages of vertebrate development and any disturbance in SRSF1 leads to serious consequences. In current study, we investigated the effects of loss of the SRSF1 gene using antisense morpholino oligonucleotides (MO) in Xenopus embryogenesis. It is evident from the results of RT-PCR and whole-mount in situ hybridization that SRSF1 is a maternal gene having strong expression in head, eyes and central nervous system. Moreover, SRSF1 morphants exhibited malformed phenotypes, including miscoiled guts, heart and cartilage formation, edema in the head and heart, and small eyes. Especially, in SRSF1 morphants, bone cartilage formation was reduced in the brain and Nkx-2.5 expression was dramatically reduced in the heart of SRSF1 morphants. In addition, a dramatic reduction in functional chordin RNA in SRSF1 morphants was observed suggesting that chordin is one of the targets of SRSF1. Thus, we concluded that SRSF1 is an essential factor for pattern formation including heart, cartilage and germ layers through the regulation of specific genes.

Pick1 modulates ephrinB1-induced junctional disassembly through an association with ephrinB1.

Members of the Eph family have been implicated in the formation of cell-cell boundaries, cell movement, and positioning during development in the context of cancer progression. De-regulation of this signaling system is linked to the promotion of more aggressive and metastatic tumor phenotypes in a large variety of human cancers, including breast, lung, and prostate cancer, melanoma, and leukemia. Thus, it is interesting to consider the case of cancer progression where de-regulation of the Eph/ephrin signaling system results in invasion and metastasis. Here, we present evidence that Pick1, one of the essential components of the adherens junction, recovers ephrinB1-induced cell-cell de-adhesion. Loss of Pick1 leads to dissociation of epithelial cells via disruption of the adherens junction, a phenotype similar to ephrinB1 overexpression. In addition, overexpressed ephrinB1-induced disruption of the adherens junction is rescued via binding to Pick1. These data indicate that Pick1 is involved in regulating the cell-cell junction in epithelial cells, and this may influence therapeutic strategy decisions with regards to cell adhesion molecules in metastatic disease.

The Role of Serum CD26 in the Diagnosis of Gastric Cancer.

Purpose: The value of serum cluster of differentiation 26 (CD26) in gastric cancer remains unknown. We investigated serum CD26 as a non-invasive serological marker for the diagnosis of gastric cancer and its relationship with serum human epidermal growth factor receptor-2 (HER2) levels. Patients and Methods: We enrolled 393 gastric cancer patients treated with endoscopic resection or surgery, and 90 healthy controls. HER2 positivity in tissue was evaluated by immunohistochemistry staining, and the serum CD26 and HER2 levels were measured using an enzyme-linked immunosorbent assay. Results: Serum CD26 levels were significantly lower in gastric cancer patients than in healthy controls (582.2 ± 254.3 vs 862.7 ± 410.6 ng/mL, P<0.001). Serum CD26 levels were significantly lower in advanced gastric cancer compared to early gastric cancer (642.2 ± 333.9 vs 503.4 ± 332.7 ng/mL, P<0.001), and tended to decrease with gastric cancer progression. To diagnose gastric cancer, the optimal cut-off value of serum CD26 was 762.7 ng/mL with 75.6% sensitivity and 64.4% specificity. Serum CD26 levels were weakly correlated with serum HER2 levels (rs=0.363, P<0.001). However, no difference in serum CD26 levels was observed between tissue HER2-negative and HER2-positive gastric cancer groups (586.2 ± 362.1 vs 579.6 ± 264.8 ng/mL, P=0.898). Conclusion: CD26 is a useful non-invasive serological marker for gastric cancer diagnosis; however, its levels do not correlate with HER2 status.

Omi/HtrA2 protease is associated with tubular cell apoptosis and fibrosis induced by unilateral ureteral obstruction.

Kidney fibrosis, a typical characteristic of chronic renal disease, is associated with tubular epithelial cell apoptosis. The results of our recent studies have shown that Omi/HtrA2 (Omi), a proapoptotic mitochondrial serine protease, performs a crucial function in renal tubular epithelial apoptotic cell death in animal models of acute kidney injury, including cisplatin toxicity and ischemia-reperfusion insult. However, the role of Omi in tubulointerstitial disease-associated fibrosis in the kidney remains to be clearly defined. We evaluated the potential function and molecular mechanism of Omi in ureteral obstruction-induced kidney epithelial cell apoptosis and fibrosis. The mice were subjected to unilateral ureteral obstruction (UUO) via the ligation of the left ureter near the renal pelvis. UUO increased the protein level of Omi in the cytosolic fraction of the kidney, with a concomitant reduction in the mitochondrial fraction. UUO reduced the X-linked inhibitor of apoptosis protein (XIAP), a substrate of Omi, and pro-caspase-3, whereas it increased cleaved poly(ADP-ribose) polymerase (cleaved PARP) and the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells. When mice were treated with ucf-101, an inhibitor of the proteolytic activity of Omi (6.19 microg/day ip), on a daily basis beginning 2 days before UUO and continuing until the end of the experiment, the Omi inhibitor protected XIAP cleavage after UUO and reduced the increment of PARP cleavage and the numbers of TUNEL-positive cells. Furthermore, the Omi inhibitor significantly attenuated UUO-induced increases in fibrotic characteristics in the kidney, including the atrophy and dilation of tubules, expansion of the interstitium, and increases in the expression of collagens, alpha-smooth muscle actin, and fibronectin. In conclusion, Omi/HtrA2 is associated with apoptotic signaling pathways in tubular epithelial cells activated by unilateral ureteral obstruction, thereby resulting in kidney fibrosis.

Xclaudin 1 is required for the proper gastrulation in Xenopus laevis.

Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.

Ultrastructural analysis of glutamate-, GABA-, and glycine-immunopositive boutons from supratrigeminal premotoneurons in the rat trigeminal motor nucleus.

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B-horseradish peroxidase (CTB-HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate-immunopositive boutons (69%) were more frequently observed than GABA- or glycine-immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate-immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA- or glycine-immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate-immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control.

Peroxiredoxin5 Controls Vertebrate Ciliogenesis by Modulating Mitochondrial Reactive Oxygen Species.

AIMS: Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development. RESULTS: We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells. INNOVATION: Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS. CONCLUSION: Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.

Spatiotemporal regulation of fibroblast growth factor signal blocking for endoderm formation in Xenopus laevis.

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.

Wen-pi-tang-Hab-Wu-ling-san attenuates kidney fibrosis induced by ischemia/reperfusion in mice.

Renal fibrosis is highly implicated as a cause of chronic renal failure, for which suitable therapeutics have not yet been developed. Recently, it was reported that Wen-pi-tang-Hab-Wu-ling-san (WHW) extract attenuates epithelial cells undergoing mesenchymal transition in cultured Madin-Darby canine kidney cells. This study investigated whether WHW extract prevents renal fibrosis induced by ischemia/reperfusion (I/R) in mice. Ischemia/reperfusion resulted in kidney fibrosis at 14 days after the procedure. When WHW was administered orally to mice beginning from 2 days after the onset of ischemia until killing, the fibrosis was significantly reduced. WHW administration significantly prevented a post-ischemic decrease of copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activities, leading to decreased lipid peroxidation and hydrogen peroxide production. In addition, WHW administration attenuated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) and attenuated the activation of nuclear factor-kappa B (NF-kappaB) in the kidneys subjected to ischemia. In conclusion, WHW extract attenuated the renal fibrosis and the attenuation was associated with a reduction of oxidative stress and an inhibition of ERK1/2, JNK1/2, p38 and NF-kappaB activation. WHW extract may be an attractive agent to attenuate the progression of fibrosis.

Extrasynaptic homomeric glycine receptors in neurons of the rat trigeminal mesencephalic nucleus.

The neurons in the trigeminal mesencephalic nucleus (Vmes) innervate jaw-closing muscle spindles and periodontal ligaments, and play a crucial role in the regulation of jaw movements. Recently, it was shown that many boutons that form synapses on them are immunopositive for glycine (Gly+), suggesting that these neurons receive glycinergic input. Information about the glycine receptors that mediate this input is needed to help understand the role of glycine in controlling Vmes neuron excitability. For this, we investigated the expression of glycine receptor subunit alpha 3 (GlyRα3) and gephyrin in neurons in Vmes and the trigeminal motor nucleus (Vmo), and the Gly+ boutons that contact them by light- and electron-microscopic immunocytochemistry and quantitative ultrastructural analysis. The somata of the Vmes neurons were immunostained for GlyRα3, but not gephyrin, indicating expression of homomeric GlyR. The immunostaining for GlyRα3 was localized away from the synapses in the Vmes neuron somata, in contrast to the Vmo neurons, where the staining for GlyRα3 and gephyrin were localized at the subsynaptic zones in somata and dendrites. Additionally, the ultrastructural determinants of synaptic strength, bouton volume, mitochondrial volume, and active zone area, were significantly smaller in Gly+ boutons on the Vmes neurons than in those on the Vmo neurons. These findings support the notion that the Vmes neurons receive glycinergic input via putative extrasynaptic homomeric glycine receptors, likely mediating a slow, tonic modulation of the Vmes neuron excitability.

Peroxiredoxin1, a novel regulator of pronephros development, influences retinoic acid and Wnt signaling by controlling ROS levels.

Peroxiredoxin1 (Prdx1) is an antioxidant enzyme belonging to the peroxiredoxin family of proteins. Prdx1 catalyzes the reduction of H2O2 and alkyl hydroperoxide and plays an important role in different biological processes. Prdx1 also participates in various age-related diseases and cancers. In this study, we investigated the role of Prdx1 in pronephros development during embryogenesis. Prdx1 knockdown markedly inhibited proximal tubule formation in the pronephros and significantly increased the cellular levels of reactive oxygen species (ROS), which impaired primary cilia formation. Additionally, treatment with ROS (H2O2) severely disrupted proximal tubule formation, whereas Prdx1 overexpression reversed the ROS-mediated inhibition in proximal tubule formation. Epistatic analysis revealed that Prdx1 has a crucial role in retinoic acid and Wnt signaling pathways during pronephrogenesis. In conclusion, Prdx1 facilitates proximal tubule formation during pronephrogenesis by regulating ROS levels.

Nerve growth factor activates brain-derived neurotrophic factor promoter IV via extracellular signal-regulated protein kinase 1/2 in PC12 cells.

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.

Evaluation of developmental toxicity and teratogenicity of diclofenac using Xenopus embryos.

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) with analgesic and anti-pyretic properties. This compound is therefore used to treat pain, inflammatory disorders, and dysmenorrhea. Due to its multimodal mechanism of action and ability to penetrate placenta, diclofenac is known to have undesirable side effects including teratogenicity. However, limited data exist on its teratogenicity, and a detailed investigation regarding harmful effects of this drug during embryogenesis is warranted. Here, we analyzed the developmental toxic effects of diclofenac using Xenopus embryos according to the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) protocol. Diclofenac treatment exerted a teratogenic effect on Xenopus embryos with a teratogenic index (TI) value of 2.64 TI; if this value is higher than 1.2, the cut-off value indicative of toxicity. In particular, mortality of embryos treated with diclofenac increased in a concentration-dependent manner and a broad spectrum of malformations such as shortening and kinking of the axis, abdominal bulging, and prominent blister formation, was observed. The shape and length of internal organs also differed compared to the control group embryos and show developmental retardation on histological label. However, the expression of major tissue-specific markers did not change when analyzed by reverse transcription-polymerase chain reaction (RT-PCR). In conclusion, diclofenac treatment can promote teratogenicity that results in morphological anomalies, but not disrupt the developmental tissue arrangement during Xenopus embryogenesis.