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 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.

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.

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.

Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity.

Acinetobacter baumannii is an emerging opportunistic pathogen responsible for healthcare-associated infections. The outer membrane protein A of A. baumannii (AbOmpA) is the most abundant surface protein that has been associated with the apoptosis of epithelial cells through mitochondrial targeting. The nuclear translocation of AbOmpA and the subsequent pathology on host cells were further investigated. AbOmpA directly binds to eukaryotic cells. AbOmpA translocates to the nucleus by a novel monopartite nuclear localization signal (NLS). The introduction of rAbOmpA into the cells or a transient expression of AbOmpA-EGFP causes the nuclear localization of these proteins, while the fusion proteins of AbOmpADeltaNLS-EGFP and AbOmpA with substitutions in residues lysine to alanine in the NLS sequences represent an exclusively cytoplasmic distribution. The nuclear translocation of AbOmpA induces cell death in vitro. Furthermore, the microinjection of rAbOmpA into the nucleus of Xenopus laevis embryos fails to develop normal embryogenesis, thus leading to embryonic death. We propose a novel pathogenic mechanism of A. baumannii regarding the nuclear targeting of the bacterial structural protein AbOmpA.