Stage-dependent sequential organization of nascent smooth muscle cells and its implications for the gut coiling morphogenesis in Xenopus larva.

In vertebrates, gut coiling proceeds left-right asymmetrically during expansion of the gastrointestinal tract with highly organized muscular structures facilitating peristalsis. In this report, we explored the mechanisms of larval gut coiling morphogenesis relevant to its nascent smooth muscle cells using highly transparent Xenopus early larvae. First, to visualize the dynamics of intestinal smooth muscle cells, whole-mount specimens were immunostained with anti-smooth muscle-specific actin (SM-actin) antibody. We found that the nascent gut of Xenopus early larvae gradually expands the SM-actin-positive region in a stage-dependent manner. Transverse orientation of smooth muscle cells was first established, and next, the cellular longitudinal orientation along the gut axis was followed to make a meshwork of the contractile cells. Finally, anisotropic torsion by the smooth muscle cells was generated in the center of gut coiling, suggesting that twisting force might be involved in the late phase of coiling morphogenesis of the gut. Administration of S-(-)-Blebbistatin to attenuate the actomyosin contraction in vivo resulted in cancellation of coiling of the gut. Development of decapitation embryos, trunk 'torso' explants, and gut-only explants revealed that initial coiling of the gut proceeds without interactions with the other parts of the body including the central nervous system. We newly developed an in vitro model to assess the gut coiling morphogenesis, indicating that coiling pattern of the nascent Xenopus gut is partially gut-autonomous. Using this gut explant culture technique, inhibition of actomyosin contraction was performed by administrating either actin polymerization inhibitor, myosin light chain kinase inhibitor, or calmodulin antagonist. All of these reagents decreased the extent of gut coiling morphogenesis in vitro. Taken together, these results suggest that the contraction force generated by actomyosin-rich intestinal smooth muscle cells during larval stages is essential for the normal coiling morphogenesis of this muscular tubular organ.

In vitro bioassay of allelopathy of Arabidopsis thaliana by sandwich method and protoplast co-culture method with digital image analysis.

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

Homologous chromosome pairing is completed in crossover defective atzip4 mutant.

In transposon-tagged lines of Arabidopsis, we found a mutant that was defective in meiotic chromosome segregation. This mutant, named atzip4-4, was due to a novel mutant allele of AtZIP4, which has sequence similarity to yeast ZIP4/SPO22, which codes a ZMM protein that is a proposed unit of the synapsis initiation complex. The chiasma distribution in atzip4-4 differed from that in the wild-type, involved in a deficiency of interfering crossovers in the mutant genome. On the other hand, FISH staining of loci on two independent chromosomes in mutant meiocytes indicated that homologous chromosome pairing to synapse progresses normally until the pachytene stage, yet homologous chromosomes often separated abruptly at diplotene and diakinesis. These results suggest that AtZIP4 plays an important role in normal crossover formation and meiotic chromosome segregation, but not in homolog search. The relationship of AtZIP4 and other related proteins in meiotic events is discussed and compared with that in yeast.

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen.

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.

BIN4, a novel component of the plant DNA topoisomerase VI complex, is required for endoreduplication in Arabidopsis.

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication. Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM- and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA damage response and endocycle control.

Glycosylation of bisphenol A by freshwater microalgae.

The endocrine disruptor bisphenol A (BPA, 4,4'-isopropylidenediphenol) is used to manufacture polycarbonate plastic and epoxy resin linings of food and beverage cans, and the residues from these products are then sometimes discharged into rivers and lakes in waste leachates. However, the fate of BPA in the environment has not yet been thoroughly elucidated. Considering the effect of BPA on aquatic organisms, it is important that we estimate the concentration of BPA and its metabolites in the aquatic environment, but there are few data on the metabolites of BPA. Here, we focused on freshwater microalgae as organisms that contribute to the biodegradation or biotransformation of BPA in aquatic environments. When we added BPA to cultures of eight species of freshwater microalgae, a reduction in the concentration of BPA in the culture medium was observed in all cultures. BPA was metabolized to BPA glycosides by Pseudokirchneriella subcapitata, Scenedesmus acutus, Scenedesmus quadricauda, and Coelastrum reticulatum, and these metabolites were then released into the culture medium. The metabolite from P. subcapitata, S. acutus, and C. reticulatum was identified by FAB-MS and (1)H-NMR as bisphenol A-mono-O-beta-d-glucopyranoside (BPAGlc), and another metabolite, from S. quadricauda, was identified as bisphenol A-mono-O-beta-d-galactopyranoside (BPAGal). These results demonstrate that freshwater microalgae that inhabit universal environments can metabolize BPA to its glycosides. Because BPA glycosides accumulate in plants and algae, and may be digested to BPA by beta-glycosidase in animal intestines, more attention should be given to levels of BPA glycosides in the environment to estimate the ecological impact of discharged BPA.

Arabidopsis SPO11-2 functions with SPO11-1 in meiotic recombination.

The Spo11 protein is a eukaryotic homologue of the archaeal DNA topoisomerase VIA subunit (topo VIA). In archaea it is involved, together with its B subunit (topo VIB), in DNA replication. However, most eukaryotes, including yeasts, insects and vertebrates, instead have a single gene for Spo11/topo VIA and no homologues for topo VIB. In these organisms, Spo11 mediates DNA double-strand breaks that initiate meiotic recombination. Many plant species, in contrast to other eukaryotes, have three homologues for Spo11/topo VIA and one for topo VIB. The homologues in Arabidopsis, AtSPO11-1, AtSPO11-2 and AtSPO11-3, all share 20-30% sequence similarity with other Spo11/topo VIA proteins, but their functional relationship during meiosis or other processes is not well understood. Previous genetic evidence suggests that AtSPO11-1 is a true orthologue of Spo11 in other eukaryotes and is required for meiotic recombination, whereas AtSPO11-3 is involved in DNA endo-reduplication as a part of the topo VI complex. In this study, we show that plants homozygous for atspo11-2 exhibit a severe sterility phenotype. Both male and female meiosis are severely disrupted in the atspo11-2 mutant, and this is associated with severe defects in synapsis during the first meiotic division and reduced meiotic recombination. Further genetic analysis revealed that AtSPO11-1 and AtSPO11-2 genetically interact, i.e. plants heterozygous for both atspo11-1 and atspo11-2 are also sterile, suggesting that AtSPO11-1 and AtSPO11-2 have largely overlapping functions. Thus, the three Arabidopsis Spo11 homologues appear to function in two discrete processes, i.e. AtSPO11-1 and AtSPO11-2 in meiotic recombination and AtSPO11-3 in DNA replication.

Xenopus neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein.

A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.

Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein.

Interactions between homologs in meiotic prophase I, such as recombination and synapsis, are critical for proper homolog segregation and involve the coordination of several parallel events. However, few regulatory genes have been identified; in particular, it is not clear what roles the proteins similar to the mitotic cell cycle regulators might play during meiotic prophase I. We describe here the isolation and characterization of a new Arabidopsis mutant called solo dancers that exhibits a severe defect in homolog synapsis, recombination and bivalent formation in meiotic prophase I, subsequently resulting in seemingly random chromosome distribution and formation of abnormal meiotic products. We further demonstrate that the mutation affects a meiosis-specific gene encoding a novel protein of 578 amino acid residues with up to 31% amino acid sequence identity to known cyclins in the C-terminal portion. These results argue strongly that homolog interactions during meiotic prophase I require a novel meiosis-specific cyclin in Arabidopsis.