Aflatoxin M1 effects on Xenopus laevis development

BACKGROUND: The principal Aflatoxin B(1) (AFB(1)) hydroxylated metabolite excreted in milk is Aflatoxin M(1) (AFM(1)) classified in group 2B by the International Agency for Research on Cancer (IARC). Human exposure to AFM(1) is due to the consumption of contaminated dairy products and partly to endogenous production through AFB(1) liver metabolism. METHODS: Since no data are available on AFM(1) embryotoxicity, its lethal and teratogenic potential was investigated using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Stage-8 blastulae were exposed to AFM(1) at 1, 4, 16, 64, and 256 microg/L concentrations until stage 47, free-swimming larva. RESULTS: A slight increase of mortality and malformed larva percents was found in AFM(1)-exposed groups but these differences were not statistically significant in comparison with the controls. CONCLUSIONS: Therefore, AFM(1) is a non-embryotoxic compound when evaluated with a FETAX model at concentrations under the conditions tested. However, AFM(1) merits further studies using mammals as experimental models to identify a possible risk during human pregnancy.     

Cell cycle transition in early embryonic development of Xenopus laevis

This article reviews cell cycle changes that occur during midblastula transition (MBT) in Xenopus laevis based on research carried out in the authors' laboratory. Blastomeres dissociated from the animal cap of blastulae, as well as those in an intact embryo, divide synchronously with a constant cell cycle duration in vitro, up to the 12th cell cycle regardless of their cell sizes. During this synchronous cleavage, cell sizes of blastomeres become variable because of repeated unequal cleavage. After the 12th cell cycle blastomeres require contact with an appropriate protein substrate to continue cell division. When nucleocytoplasmic (N/C) ratios of blastomeres reach a critical value during the 13th cycle, their cell cycle durations lengthen in proportion to the reciprocal of cell surface areas, and cell divisions become asynchronous due to variations in cell sizes. The same changes occur in haploid blastomeres with a delay of one cell cycle. Thus, post-MBT cell cycle control becomes dependent not only on the N/C relation but also on cell surface activities of blastomeres. Unlike cell cycle durations of pre-MBT blastomeres, which show monomodal frequency distributions with a peak at about 30 min, those of post-MBT blastomeres show polymodal frequency distributions with peaks at multiples of about 30 min, suggesting 'quantisement' of the cell cycle. Thus, we hypothesised that MPF is produced periodically during its unit cycle with 30 min period, but it titrates, and is neutralized by, an inhibitor contained in the nucleus in a quantity proportional to the genome size; however, when all of the inhibitor has been titrated, excess MPF during the last cycle triggers mitosis. At MBT, cell cycle checkpoint mechanisms begin to operate. While the operation of S phase checkpoint to monitor DNA replication is initiated by N/C relation, the initiation of M phase checkpoint operation to monitor chromosome segregation at mitosis is regulated by an age-dependent mechanism.     

Recovery of the Xenopus laevis heart from ROS-induced stress utilizes conserved pathways of cardiac regeneration

Urodele amphibians and some fish are capable of regenerating up to a quarter of their heart tissue after cardiac injury. While many anuran amphibians like Xenopus laevis are not capable of such feats, they are able to repair lesser levels of cardiac damage, such as that caused by oxidative stress, to a far greater degree than mammals. Using an optogenetic stress induction model that utilizes the protein KillerRed, we have investigated the extent to which mechanisms of cardiac regeneration are conserved during the restoration of normal heart morphology post oxidative stress in X. laevis tadpoles. We focused particularly on the processes of cardiomyocyte proliferation and dedifferentiation, as well as the pathways that facilitate the regulation of these processes. The cardiac response to KillerRed-induced injury in X. laevis tadpole hearts consists of a phase dominated by indicators of cardiac stress, followed by a repair-like phase with characteristics similar to mechanisms of cardiac regeneration in urodeles and fish. In the latter phase, we found markers associated with partial dedifferentiation and cardiomyocyte proliferation in the injured tadpole heart, which, unlike in regenerating hearts, are not dependent on Notch or retinoic acid signaling. Ultimately, the X. laevis cardiac response to KillerRed-induced oxidative stress shares characteristics with both mammalian and urodele/fish repair mechanisms, but is nonetheless a unique form of recovery, occupying an intermediate place on the spectrum of cardiac regenerative ability. An understanding of how Xenopus repairs cardiac damage can help bridge the gap between mammals and urodeles and contribute to new methods of treating heart disease.     

Mutation of interfaces in domain-swapped human betaB2-crystallin

The superfamily of eye lens betagamma-crystallins is highly modularized, with Greek key motifs being used to form symmetric domains. Sequences of monomeric gamma-crystallins and oligomeric beta-crystallins fold into two domains that pair about a further conserved symmetric interface. Conservation of this assembly interface by domain swapping is the device adopted by family member betaB2-crystallin to form a solution dimer. However, the betaB1-crystallin solution dimer is formed from an interface used by the domain-swapped dimer to form a tetramer in the crystal lattice. Comparison of these two structures indicated an intriguing relationship between linker conformation, interface ion pair networks, and higher assembly. Here the X-ray structure of recombinant human betaB2-crystallin showed that domain swapping was determined by the sequence and not assembly conditions. The solution characteristics of mutants that were designed to alter an ion pair network at a higher assembly interface and a mutant that changed a proline showed they remained dimeric. X-ray crystallography showed that the dimeric mutants did not reverse domain swapping. Thus, the sequence of betaB2-crystallin appears well optimized for domain swapping. However, a charge-reversal mutation to the conserved domain-pairing interface showed drastic changes to solution behavior. It appears that the higher assembly of the betagamma-crystallin domains has exploited symmetry to create diversity while avoiding aggregation. These are desirable attributes for proteins that have to exist at very high concentration for a very long time.     

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 35.67% 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 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental 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 uninjected 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.     

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.     

Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development

Background information. pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. Results. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)‐based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross‐rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. Conclusion. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.     

Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the “agarose-embedded heat shock (AeHS) method” involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.     

Crystal structure of truncated human betaB1-crystallin

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.     

Resources and transgenesis techniques for functional genomics in Xenopus

Recent developments in genomic resources and high‐throughput transgenesis techniques have allowed Xenopus to ‘metamorphose’ from a classic model for embryology to a leading‐edge experimental system for functional genomics. This process has incorporated the fast‐breeding diploid frog, Xenopus tropicalis, as a new model‐system for vertebrate genomics and genetics. Sequencing of the X. tropicalis genome is nearly complete, and its comparison with mammalian sequences offers a reliable guide for the genome‐wide prediction of cis‐regulatory elements. Unique cDNA sets have been generated for both X. tropicalis and X. laevis, which have facilitated non‐redundant, systematic gene expression screening and comprehensive gene expression analysis. A variety of transgenesis techniques are available for both X. laevis and X. tropicalis, and the appropriate procedure may be chosen depending on the purpose for which it is required. Effective use of these resources and techniques will help to reveal the overall picture of the complex wiring of gene regulatory networks that control vertebrate development.     

Successful reconstitution of the non-regenerating adult telencephalon by cell transplantation in Xenopus laevis

The South African clawed frog (Xenopus laevis) can regenerate the anterior half of the telencephalon only during larval life, but such regeneration is no longer possible after metamorphosis. In order to gain a better understanding of differences between larvae and adults that are potentially related to regeneration, several experiments were conducted on larvae and froglets after the partial removal of the telencephalon. As a result, it was found that the cells in the brain proliferated actively, even in non-regenerating froglets, just as was observed in regenerating larvae after the partial removal of the telencephalon. Moreover, it was shown that although the structure was usually imperfect, even isolated single cells derived from the frog brain were able to reconstitute the lost portion when the cells were transplanted to the partially truncated telencephalon. It is therefore likely to be critical for massive organ regeneration that ependymal layer cells promptly cover the cerebral lateral ventricles at an initial stage of wound healing, as is the case observed in larvae. However, in froglets, these cells strongly adhere to one another, and they are therefore unable to move to seal off the exposed ventricle, which in turn is likely to render the froglet brain non-regenerative.     

Sox1基因在爪蟾早期发育中的时空表达图式

克隆了非洲爪蟾的Sox1基因并研究了它在非洲爪蟾早期发育过程中的时空表达图式,比较了Sox1—3基因在发育的脑和眼中的表达图式。序列比对分析显示Sox1—3蛋白在其HMG框结构域具有高度的保守性。通过RT-PCR方法分析了Sox1基因在爪蟾早期不同发育时段的表达情况,结果显示Sox1基因从未受精卵到尾芽期均有表达,但表达强度有所差异。原位杂交结果显示,在早期卵裂阶段和囊胚期,Sox1基因主要在动物极表达;从神经板期开始,Sox1基因主要在中枢神经系统和眼原基中表达。在蝌蚪期,Sox1与Sox2、Sox3在脑部和眼睛的表达区域有所不同。对于爪蟾Sox1基因时空表达图式的研究将有助于阐明SoxB1基因家族在脊椎动物神经系统发生过程中的作用。     

Novel double promoter approach for identification of transgenic animals: A tool for in vivo analysis of gene function and development of gene-based therapies

Advances in vertebrate genetics have allowed studies of gene function in developing animals through gene knockout and transgenic analyses. These advances have encouraged the development of gene-based therapies through introduction of exogenous genes to enhance and/or replace dysfunctional or missing genes. However, in vertebrates, such analyses often involve tedious screening for transgenic animals, such as PCR-based genotype determinations. Here, we report the use of double-promoter plasmids carrying the transgene of interest and the crystallin-promotor-driven Green fluorescent protein (GFP) in transgenic Xenopus laevis tadpoles. This strategy allows a simple examination for the presence of GFP in the eyes to identify transgenic animals. PCR-based genotyping and functional characterization confirms that all animals expressing GFP in the eyes indeed carry the desired promoter/transgene units. Thus, the use of this and other similar vectors should dramatically improve current transgenesis protocols and reduce the time and cost for identifying transgenic animals.     

Upregulation of matrix metalloproteinase triggers transdifferentiation of retinal pigmented epithelial cells in Xenopus laevis: A Link between inflammatory response and regeneration

In adult Xenopus eyes, when the whole retina is removed, retinal pigmented epithelial (RPE) cells become activated to be retinal stem cells and regenerate the whole retina. In the present study, using a tissue culture model, it was examined whether upregulation of matrix metalloproteinases (Mmps) triggers retinal regeneration. Soon after retinal removal, Xmmp9 and Xmmp18 were strongly upregulated in the tissues of the RPE and the choroid. In the culture, Mmp expression in the RPE cells corresponded with their migration from the choroid. A potent MMP inhibitor, 1,10‐PNTL, suppressed RPE cell migration, proliferation, and formation of an epithelial structure in vitro. The mechanism involved in upregulation of Mmps was further investigated. After retinal removal, inflammatory cytokine genes, IL‐1β and TNF‐α , were upregulated both in vivo and in vitro. When the inflammation inhibitors dexamethasone or Withaferin A were applied in vitro, RPE cell migration was severely affected, suppressing transdifferentiation. These results demonstrate that Mmps play a pivotal role in retinal regeneration, and suggest that inflammatory cytokines trigger Mmp upregulation, indicating a direct link between the inflammatory reaction and retinal regeneration. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1086–1100, 2017     

Functional regeneration of the olfactory bulb requires reconnection to the olfactory nerve in Xenopus larvae

Larvae of the South African clawed frog (Xenopus laevis) can regenerate the telencephalon, which consists of the olfactory bulb and the cerebrum, after it has been partially removed. Some authors have argued that the telencephalon, once removed, must be reconnected to the olfactory nerve in order to regenerate. However, considerable regeneration has been observed before reconnection. Therefore, we have conducted several experiments to learn whether or not reconnection is a prerequisite for regeneration. We found that the olfactory bulb did not regenerate without reconnection, while the cerebrum regenerated by itself. On the other hand, when the brain was reconnected by the olfactory nerve, both the cerebrum and the olfactory bulb regenerated. Morphological and histological investigation showed that the regenerated telencephalon was identical to the intact one in morphology, types and distributions of cells, and connections between neurons. Froglets with a regenerated telencephalon also recovered olfaction, the primary function of the frog telencephalon. These results suggest that the Xenopus larva requires reconnection of the regenerating brain to the olfactory nerve in order to regenerate the olfactory bulb, and thus the regenerated brain functions, in order to process olfactory information.