Collective nuclear behavior shapes bilateral nuclear symmetry for subsequent left-right asymmetric morphogenesis in Drosophila.

Proper organ development often requires nuclei to move to a specific position within the cell. To determine how nuclear positioning affects left-right (LR) development in the Drosophila anterior midgut (AMG), we developed a surface-modeling method to measure and describe nuclear behavior at stages 13-14, captured in three-dimensional time-lapse movies. We describe the distinctive positioning and a novel collective nuclear behavior by which nuclei align LR symmetrically along the anterior-posterior axis in the visceral muscles that overlie the midgut and are responsible for the LR-asymmetric development of this organ. Wnt4 signaling is crucial for the collective behavior and proper positioning of the nuclei, as are myosin II and the LINC complex, without which the nuclei fail to align LR symmetrically. The LR-symmetric positioning of the nuclei is important for the subsequent LR-asymmetric development of the AMG. We propose that the bilaterally symmetrical positioning of these nuclei may be mechanically coupled with subsequent LR-asymmetric morphogenesis.

Maternal almondex, a neurogenic gene, is required for proper subcellular Notch distribution in early Drosophila embryogenesis.

Notch signaling plays crucial roles in the control of cell fate and physiology through local cell-cell interactions. The core processes of Notch signal transduction are well established, but the mechanisms that fine-tune the pathway in various developmental and post-developmental contexts are less clear. Drosophila almondex, which encodes an evolutionarily conserved double-pass transmembrane protein, was identified in the 1970s as a maternal-effect gene that regulates Notch signaling in certain contexts, but its mechanistic function remains obscure. In this study, we examined the role of almondex in Notch signaling during early Drosophila embryogenesis. We found that in addition to being required for lateral inhibition in the neuroectoderm, almondex is also partially required for Notch signaling-dependent single-minded expression in the mesectoderm. Furthermore, we found that almondex is required for proper subcellular Notch receptor distribution in the neuroectoderm, specifically during mid-stage 5 development. The absence of maternal almondex during this critical window of time caused Notch to accumulate abnormally in cells in a mesh-like pattern. This phenotype did not include any obvious change in subcellular Delta ligand distribution, suggesting that it does not result from a general vesicular-trafficking defect. Considering that dynamic Notch trafficking regulates signal output to fit the specific context, we speculate that almondex may facilitate Notch activation by regulating intracellular Notch receptor distribution during early embryogenesis.

Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking.

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.

O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation.

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-fut1(R245A knock-in)), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1(R245A 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.

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.

Rescue of Notch signaling in cells incapable of GDP-L-fucose synthesis by gap junction transfer of GDP-L-fucose in Drosophila.

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.

A gain-of-function screen to identify genes that reduce lifespan in the adult of Drosophila melanogaster.

BACKGROUND: Several lines of evidence associate misregulated genetic expression with risk factors for diabetes, Alzheimer's, and other diseases that sporadically develop in healthy adults with no background of hereditary disorders. Thus, we are interested in genes that may be expressed normally through parts of an individual's life, but can cause physiological defects and disease when misexpressed in adulthood. RESULTS: We attempted to identify these genes in a model organism by arbitrarily misexpressing specific genes in adult Drosophila melanogaster, using 14,133 Gene Search lines. We identified 39 "reduced-lifespan genes" that, when misexpressed in adulthood, shortened the flies' lifespan to less than 30% of that of control flies. About half of these genes have human orthologs that are known to be involved in human diseases. For about one-fourth of the reduced-lifespan genes, suppressing apoptosis restored the lifespan shortened by their misexpression. We determined the organs responsible for reduced lifespan when these genes were misexpressed specifically in adulthood, and found that while some genes induced reduced lifespan only when misexpressed in specific adult organs, others could induce reduced lifespan when misexpressed in various organs. This finding suggests that tissue-specific dysfunction may be involved in reduced lifespan related to gene misexpression. Gene ontology analysis showed that reduced-lifespan genes are biased toward genes related to development. CONCLUSIONS: We identified 39 genes that, when misexpressed in adulthood, shortened the lifespan of adult flies. Suppressing apoptosis rescued this shortened lifespan for only a subset of the reduced-lifespan genes. The adult tissues in which gene misexpression caused early death differed among the reduced-lifespan genes. These results suggest that the cause of reduced lifespan upon misexpression differed among the genes.

An in-silico genomic survey to annotate genes coding for early development-relevant signaling molecules in the pearl oyster, Pinctada fucata.

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, TGFß, 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.

TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

Notch signaling is an evolutionarily conserved mechanism required for numerous types of cell fate decisions in metazoans. It mediates short-range communication between cells with receptors and ligands, both of which are expressed on the cell surfaces. In response to the ligand-receptor interaction, the ligand and the extracellular domain of the Notch receptor (NECD) in the complex are internalized into ligand-expressing cells by endocytosis, a prerequisite process for the conformational change of the membrane proximal region of Notch to induce critical proteolytic cleavages for its activation. Here we report that overexpression of transmembrane 2 (TM2) domain containing 3 (TM2D3), a mammalian homologue of Drosophila melanogaster Almondex (Amx), activates Notch1. This activation requires the ligand-binding domain in Notch1 and the C-terminal region containing TM2 domain in TM2D3. TM2D3 physically associates with Notch1 at the region distinct from the ligand-binding domain and enhances expression of Notch1 on the cell surface. Furthermore, cell surface expression of Notch1 and Notch2 is reduced in Tm2d3-deficient cells. Finally, amx-deficient Drosophila early embryos exhibit impaired endocytosis of NECD and Delta ligand, for which surface presentation of Notch is required. These results indicate that TM2D3 is an element involved in Notch signaling through the surface presentation.

Metabolism and transportation pathways of GDP-fucose that are required for the O-fucosylation of Notch.

Notch is a single-pass transmembrane receptor that mediates the local cell-cell interactions necessary for many cell-fate decisions. The extra cellular domain of Notch contains a tandem array of epidermal growth factor-like (EGF-like) repeats. Some of these EGF-like repeats are O-fucosylated by protein O-fucosyltransferase 1 (O-fut1), which is essential for Notch signaling in Drosophila and mouse. This O-fucose is further modified by Fringe, a GlcNAc transferase and other glycosyltransferases (O-fut1 in Drosophila and Pofut1 in mouse), to form an O-linked tetrasaccharide, which modulates Notch's selective binding to its ligands.

Notch Signalling Under Maternal-to-Zygotic Transition.

The development of all animal embryos is initially directed by the gene products supplied by their mothers. With the progression of embryogenesis, the embryo's genome is activated to command subsequent developments. This transition, which has been studied in many model animals, is referred to as the Maternal-to-Zygotic Transition (MZT). In many organisms, including flies, nematodes, and sea urchins, genes involved in Notch signaling are extensively influenced by the MZT. This signaling pathway is highly conserved across metazoans; moreover, it regulates various developmental processes. Notch signaling defects are commonly associated with various human diseases. The maternal contribution of its factors was first discovered in flies. Subsequently, several genes were identified from mutant embryos with a phenotype similar to Notch mutants only upon the removal of the maternal contributions. Studies on these maternal genes have revealed various novel steps in the cascade of Notch signal transduction. Among these genes, pecanex and almondex have been functionally characterized in recent studies. Therefore, in this review, we will focus on the roles of these two maternal genes in Notch signaling and discuss future research directions on its maternal function.

Notch Missense Mutations in Drosophila Reveal Functions of Specific EGF-like Repeats in Notch Folding, Trafficking, and Signaling.

Notch signaling plays various roles in cell-fate specification through direct cell-cell interactions. Notch receptors are evolutionarily conserved transmembrane proteins with multiple epidermal growth factor (EGF)-like repeats. Drosophila Notch has 36 EGF-like repeats, and while some play a role in Notch signaling, the specific functions of most remain unclear. To investigate the role of each EGF-like repeat, we used 19 previously identified missense mutations of Notch with unique amino acid substitutions in various EGF-like repeats and a transmembrane domain; 17 of these were identified through a single genetic screen. We assessed these mutants' phenotypes in the nervous system and hindgut during embryogenesis, and found that 10 of the 19 Notch mutants had defects in both lateral inhibition and inductive Notch signaling, showing context dependency. Of these 10 mutants, six accumulated Notch in the endoplasmic reticulum (ER), and these six were located in EGF-like repeats 8-10 or 25. Mutations with cysteine substitutions were not always coupled with ER accumulation. This suggests that certain EGF-like repeats may be particularly susceptible to structural perturbation, resulting in a misfolded and inactive Notch product that accumulates in the ER. Thus, we propose that these EGF-like repeats may be integral to Notch folding.

Design and preparation of a novel temperature-responsive ionic gel. 3. Valence selective control of transport modes of ions in response to temperature.

We propose a novel model dialysis system that can valence-selectively control the transport modes of ions in response to temperature change. In a dialysis system consisting of an anionic gel membrane and mixed solutions containing a driving electrolyte and electrolytes with uni-, bi-, and trivalent cations, the dependence of the charge density of the gel and the valence of the ions on the transport modes of the ions through the gel membrane was investigated by computer simulations. The simulations show that the system has four transport types in the transport modes of the cations according to their valence [downhill (transport along their own concentration gradient in the system) and uphill (transport against their own concentration gradient)] in response to the charge density changes: (A) downhill transport of all the cations; (B) uphill transport of trivalent cations, downhill transport of the other cations; (C) uphill transport of bi- and trivalent cations, downhill transport of univalent cations; and (D) uphill transport of all the cations except for the driving cations. To examine the prediction of the simulations, a temperature-responsive anionic gel membrane was prepared from a modified poly(vinyl alcohol) (PVA) containing 2 mol % of sulfonic acid groups and another modified PVA prepared by in situ polymerization of N-isopropylacrylamide in a PVA solution. Permeation experiments in a dialysis system consisting of the membrane and mixed electrolyte solutions of NaCl, LiCl, CaCl2, and LaCl3 indicate that the system valence-selectively controls the transport modes of the cations in response to temperature change as predicted in our simulations.

Insight into Notch Signaling Steps That Involve pecanex from Dominant-Modifier Screens in Drosophila.

Notch signaling plays crucial roles in intercellular communications. In Drosophila, the pecanex (pcx) gene, which encodes an evolutionarily conserved multi-pass transmembrane protein, appears to be required to activate Notch signaling in some contexts, especially during neuroblast segregation in the neuroectoderm. Although Pcx has been suggested to contribute to endoplasmic reticulum homeostasis, its functions remain unknown. Here, to elucidate these roles, we performed genetic modifier screens of pcx We found that pcx heterozygotes lacking its maternal contribution exhibit cold-sensitive lethality, which is attributed to a reduction in Notch signaling at decreased temperatures. Using sets of deletions that uncover most of the second and third chromosomes, we identified four enhancers and two suppressors of the pcx cold-sensitive lethality. Among these, five genes encode known Notch-signaling components: big brain, Delta (Dl), neuralized (neur), Brother of Bearded A (BobA), a member of the Bearded (Brd) family, and N-ethylmaleimide-sensitive factor 2 (Nsf2). We showed that BobA suppresses Dl endocytosis during neuroblast segregation in the neuroectoderm, as Brd family genes reportedly do in the mesoderm for mesectoderm specification. Analyses of Nsf2, a key regulator of vesicular fusion, suggested a novel role in neuroblast segregation, which is distinct from Nsf2's previously reported role in imaginal tissues. Finally, jim lovell, which encodes a potential transcription factor, may play a role in Notch signaling during neuroblast segregation. These results reveal new research avenues for Pcx functions and Notch signaling.

Design and preparation of a novel temperature-responsive ionic gel. 2. Concentration modulation of specific ions in response to temperature changes.

We have designed a novel temperature-responsive dialysis system consisting of mixed electrolyte solutions and an ionic gel whose charge density changes in response to temperature changes. The system can modulate the time-concentration profile of just bivalent ions in arbitrary forms by changing the transport modes of the ions in response to the temperature, as long as the system has a sufficient concentration difference of the driving electrolyte for the modulation. The simulation in a model system consisting of mixed KCl and CaCl(2) solutions and the ionic gel shows that the system modulates the time-concentration profile of Ca(2+) ions as a sawtooth waveform and also keeps the concentration a prearranged value for a certain time period by controlling the transport modes of just Ca(2+) ions in two ways: downhill (transport along their own concentration gradient in a system) and uphill (transport against their own concentration gradient), in response to temperature changes. The simulations agree quantitatively with the experiments using a temperature-responsive ionic gel prepared in the previous paper.

Class I myosins have overlapping and specialized functions in left-right asymmetric development in Drosophila.

The class I myosin genes are conserved in diverse organisms, and their gene products are involved in actin dynamics, endocytosis, and signal transduction. Drosophila melanogaster has three class I myosin genes, Myosin 31DF (Myo31DF), Myosin 61F (Myo61F), and Myosin 95E (Myo95E). Myo31DF, Myo61F, and Myo95E belong to the Myosin ID, Myosin IC, and Myosin IB families, respectively. Previous loss-of-function analyses of Myo31DF and Myo61F revealed important roles in left-right (LR) asymmetric development and enterocyte maintenance, respectively. However, it was difficult to elucidate their roles in vivo, because of potential redundant activities. Here we generated class I myosin double and triple mutants to address this issue. We found that the triple mutant was viable and fertile, indicating that all three class I myosins were dispensable for survival. A loss-of-function analysis revealed further that Myo31DF and Myo61F, but not Myo95E, had redundant functions in promoting the dextral LR asymmetric development of the male genitalia. Myo61F overexpression is known to antagonize the dextral activity of Myo31DF in various Drosophila organs. Thus, the LR-reversing activity of overexpressed Myo61F may not reflect its physiological function. The endogenous activity of Myo61F in promoting dextral LR asymmetric development was observed in the male genitalia, but not the embryonic gut, another LR asymmetric organ. Thus, Myo61F and Myo31DF, but not Myo95E, play tissue-specific, redundant roles in LR asymmetric development. Our studies also revealed differential colocalization of the class I myosins with filamentous (F)-actin in the brush border of intestinal enterocytes.

An integrated medical image database and retrieval system using a web application server.

We developed an Integrated Medical Image Database and Retrieval System (INIS) for easy access by medical staff. The INIS mainly consisted of four parts: specific servers to save medical images from multi-vendor modalities of CT, MRI, CR, ECG and endoscopy; an integrated image database (DB) server to save various kinds of images in a DICOM format; a Web application server to connect clients to the integrated image DB and the Web browser terminals connected to an HIS system. The INIS provided a common screen design to retrieve CT, MRI, CR, endoscopic and ECG images, and radiological reports, which would allow doctors to retrieve radiological images and corresponding reports, or ECG images of a patient simultaneously on a screen. Doctors working in internal medicine on average accessed information 492 times a month. Doctors working in cardiological and gastroenterological accessed information 308 times a month. Using the INIS, medical staff could browse all or parts of a patient's medical images and reports.

Delivery of medical multimedia contents through the TCP/IP network using RealSystem.

We developed a low cost, user-friendly multimedia delivery system, to provide medical lectures saved as multimedia contents to persons engaged in medicine. This system was created using the RealSystem package with the TCP/IP network. Users can review lectures and medical meeting presentations with video and audio through the Internet, whenever convenient. Each medical source of video and slide has been clearly displayed on a screen. Members of medical associations or medical students can easily review the most interesting parts of these files. This system is being used efficiently in distance learning and aids the diffusion of the latest information and technology to busy physicians and medical students.

Web-based delivery of medical multimedia contents using an MPEG-4 system.

Moving picture expert group compression standard version 4 (MPEG-4) is a standard for video coding aimed at multimedia applications. MPEG-4 was developed to enable high compression rate in a low bitrate transmission via the Internet or mobile telecommunications. Although these characteristics of MPEG-4 are suitable for telemedicine, little is known about the possibility of using this technology in the field of telemedicine. We evaluated the quality of MPEG-4-encoded medical video streams and compared them with original analogue videos and audio-video-interleave (AVI) files. Although MPEG-4 video streams have the advantage of small file size, they were found to be inferior to original videos and AVI files in terms of smoothness of motion pictures, sharpness of images and clearness of sound. Illegibility of characters was a major problem in MPEG-4 files. The score for total impression of MPEG-4 files was significantly lower than those for AVI files. The results of this study suggest that the quality of MPEG-4-encoded video streams is not adequate for telemedicine.

A method for displaying two images on a screen in distance medical education.

This article describes a method for simultaneously displaying several images on a screen during an online multimedia presentation. Users with 'REALPLAYER' software and Web browsers can view images with synchronous audio on their personal computers via the Internet. Medical researchers and physicians often find it useful to compare images after treatment with those before treatment, by displaying several arranged images simultaneously. Medical care providers can browse this type of multimedia content in their offices and universities at their convenience. There presently exist two methods for creating multimedia content with voice and images using RealSystem technology. Electronic lectures of otorhinolaryngology explaining new surgical procedures for patients with chronic otitis media were created with this method and made available to otorhinolaryngologists through the Internet.