Notch (N) is a transmembrane receptor that mediates cell–cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O -fucose glycan modification that ...regulates N-ligand binding. This modification requires GDP- l -fucose as a donor of fucose. The GDP- l -fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy- d -mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP- l -fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila . We found that these mutants functioned cell-nonautonomously, and that GDP- l -fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP- l -fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP- l -fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP- l -fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.
Many animals exhibit stereotypical left–right (LR) asymmetry in their internal organs. The mechanisms of LR axis formation required for the subsequent LR asymmetric development are well understood, ...especially in some vertebrates. However, the molecular mechanisms underlying LR asymmetric morphogenesis, particularly how mechanical force is integrated into the LR asymmetric morphogenesis of organs, are poorly understood. Here, we identified
zipper (
zip), encoding a
Drosophila non-muscle myosin II (myosin II) heavy chain, as a gene required for LR asymmetric development of the embryonic anterior midgut (AMG). Myosin II is known to directly generate mechanical force in various types of cells during morphogenesis and cell migration. We found that myosin II was involved in two events in the LR asymmetric development of the AMG. First, it introduced an LR bias to the directional position of circular visceral muscle (CVMU) cells, which externally cover the midgut epithelium. Second, it was required for the LR-biased rotation of the AMG. Our results suggest that myosin II in CVMU cells plays a crucial role in generating the force leading to LR asymmetric morphogenesis. Taken together with previous studies in vertebrates, the involvement of myosin II in LR asymmetric morphogenesis might be conserved evolutionarily.
The pearl oyster Pinctada fucata has great potential as a model system for lophotrochozoan developmental biology research. Pinctada fucata is an important commercial resource, and a significant body ...of primary research on this species has emphasized its basic aquaculture biology such as larval biology and growth, aquaculture, pearl formation and quality improvement, shell formation, and biomineralization. Recently, a draft genome sequence of this species was published, and many experimental resources are currently being developed, such as bioinformatics tools, embryo and larva manipulation methods, gene knockdown technique, etc. In this paper, we report the results from our genomic survey pertaining to gene families that encode developmental signaling ligands (Fgf, Hedgehog, PDGF/VEGF, TGFβ, and Wnt families). We found most of the representative genes of major signaling pathways involved in axial patterning, as well as copies of the signaling molecule paralogs. Phylogenetic character mapping was used to infer a possible evolutionary scenario of the signaling molecules in the protostomes, and to reconstruct possible copy numbers of signaling molecule-coding genes for the ancestral protostome. Our reconstruction suggests that P. fucata retains the ancestral protostome gene complement, providing further justifications for the use of this taxon as a model organism for developmental genomics research.
Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth ...factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions.
Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the ...protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1R245A knock-in), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1R245A knock-in and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein's functions.The requirement of O-fucose monosaccharide on Notch is not fully understood.
Loss of O-fucose monosaccharide on Notch caused temperature-sensitive loss of Notch signaling.
O-Fucose monosaccharide of Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch signal activation.
Our findings elucidate how different forms of glycosylation on a protein influence protein functions.
► Canonical Wnt signaling is required for the LR asymmetric development of the midgut. ► Wnt signaling needs to be activated in the visceral muscle for normal gut laterality. ► Wnt signaling in the ...gut is activated with bilateral LR symmetry. ► Roles of Wnt signaling in LR asymmetric development may be conserved evolutionarily.
Many animals develop left–right (LR) asymmetry in their internal organs. The mechanisms of LR asymmetric development are evolutionarily divergent, and are poorly understood in invertebrates. Therefore, we studied the genetic pathway of LR asymmetric development in Drosophila. Drosophila has several organs that show directional and stereotypic LR asymmetry, including the embryonic gut, which is the first organ to develop LR asymmetry during Drosophila development. In this study, we found that genes encoding components of the Wnt-signaling pathway are required for LR asymmetric development of the anterior part of the embryonic midgut (AMG). frizzled 2 (fz2) and Wnt4, which encode a receptor and ligand of Wnt signaling, respectively, were required for the LR asymmetric development of the AMG. arrow (arr), an ortholog of the mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor of the Wnt-signaling pathway, was also essential for LR asymmetric development of the AMG. These results are the first demonstration that Wnt signaling contributes to LR asymmetric development in invertebrates, as it does in vertebrates. The AMG consists of visceral muscle and an epithelial tube. Our genetic analyses revealed that Wnt signaling in the visceral muscle but not the epithelium of the midgut is required for the AMG to develop its normal laterality. Furthermore, fz2 and Wnt4 were expressed in the visceral muscles of the midgut. Consistent with these results, we observed that the LR asymmetric rearrangement of the visceral muscle cells, the first visible asymmetry of the developing AMG, did not occur in embryos lacking Wnt4 expression. Our results also suggest that canonical Wnt/β-catenin signaling, but not non-canonical Wnt signaling, is responsible for the LR asymmetric development of the AMG. Canonical Wnt/β-catenin signaling is reported to have important roles in LR asymmetric development in zebrafish. Thus, the contribution of canonical Wnt/β-catenin signaling to LR asymmetric development may be an evolutionarily conserved feature between vertebrates and invertebrates.
Many bilaterally symmetric animals develop left–right (L–R) asymmetry in their internal organs. The mechanisms of L–R asymmetric development are well understood in vertebrates, whereas they are still ...elusive in invertebrates. Therefore, we decided to study the genetic pathway of L–R asymmetric development in
Drosophila. To identify the genes required for L–R asymmetric development in
Drosophila , we screened EMS (Ethylmethanesulfonate) induced mutations that affect stereotypical L–R asymmetry of the embryonic gut. From this screening, we isolated
arrow mutant that affected L–R asymmetry in the anterior midgut.
arrow is an ortholog of a mammalian gene encoding low-density lipoprotein receptor-related protein 5/6, which is a co-receptor in the Wnt signaling pathway. The anterior midgut consists of the visceral muscle and epithelial tube. Our analysis revealed that Wnt signal in the visceral muscle but not the epithelium of the midgut is reguired for the normal laterality development of this organ. However, the visceral muscle was formed normally in
arrow homozygous embryos, suggesting the Wnt signal probably functions in cell rearrangement, which is known to occur during L–R asymmetric development of the anterior midgut.
Drosophila genome encodes seven Wnt ligands. Therefore, we next determined ligands are required for the L–R asymmetric development of the anterior midgut. Homozygous embryos of
Wnt4 showed a similar laterality defects to those of
arrow mutants in the anterior midgut. These results suggest that Wnt signal activated by Wnt4 in the visceral muscle play an essential role in the L–R asymmetric development of the anterior midgut. Moreover, our analysis suggested that Wnt canonical pathway is required for the normal L–R asymmetric development of the anterior midgut.
A planar dot pattern was fabricated using the flat-patterning method involving nanoscale composition control. A small percentage of Fe and Pt atoms was locally diffused into an L1 0 FePtRh film by ...annealing, resulting in ferromagnetic dots with a diameter of 50 nm and a nonmagnetic spacing with a width of 100 nm. Nanoscale composition profiles around the dots were analyzed by an energy dispersive X-ray detector attached to a transmission electron microscope. Only the area in which the composition crossed the ferromagnetic-nonmagnetic threshold underwent a magnetic phase change. Magnetic force microscopy revealed ferromagnetic dots with a single-domain structure in the nonmagnetic matrix.
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