Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling

<p>Abstract</p> <p>Background</p> <p><it>O</it>-fucosyltransferase1 (OFUT1) is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the <it>N</it>-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in <it>Drosophila</it>.</p> <p>Results</p> <p>We show that expression of an isoform lacking fucosyltransferase activity, <it>Ofut1</it>^{<it>R</it>245<it>A</it>}, rescues the requirement for <it>Ofut1 </it>in embryonic neurogenesis. Lack of requirement for <it>O</it>-fucosylation is further supported by the absence of embryonic phenotypes in <it>Gmd </it>mutants, which lack all forms of fucosylation. Requirements for <it>O</it>-fucose during imaginal development were evaluated by characterizing clones of cells expressing only <it>Ofut1</it>^{<it>R</it>245<it>A</it>}. These clones phenocopy <it>fringe </it>mutant clones, indicating that the absence of <it>O</it>-fucose is functionally equivalent to the absence of elongated <it>O</it>-fucose.</p> <p>Conclusion</p> <p>Our results establish that Notch does not need to be <it>O</it>-fucosylated for <it>fringe</it>-independent Notch signaling in <it>Drosophila</it>; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.</p

Yki/YAP, Sd/TEAD and Hth/MEIS Control Tissue Specification in the Drosophila Eye Disc Epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. We show here that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc epithelium at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. We also show that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. Our results support a critical role for Yki- and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival

Yki/YAP, Sd/TEAD and Hth/MEIS Control Tissue Specification in the Drosophila Eye Disc Epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. We show here that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc epithelium at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. We also show that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. Our results support a critical role for Yki- and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival

Chronic Hypoxia Impairs Muscle Function in the Drosophila Model of Duchenne's Muscular Dystrophy (DMD)

Duchenne's muscular dystrophy (DMD) is a severe progressive myopathy caused by mutations in the DMD gene leading to a deficiency of the dystrophin protein. Due to ongoing muscle necrosis in respiratory muscles late-stage DMD is associated with respiratory insufficiency and chronic hypoxia (CH). To understand the effects of CH on dystrophin-deficient muscle in vivo, we exposed the Drosophila model for DMD (dmDys) to CH during a 16-day ascent to the summit of Mount Denali/McKinley (6194 meters above sea level). Additionally, dmDys and wild type (WT) flies were also exposed to CH in laboratory simulations of high altitude hypoxia. Expression profiling was performed using Affymetrix GeneChips® and validated using qPCR. Hypoxic dmDys differentially expressed 1281 genes, whereas the hypoxic WT flies differentially expressed 56 genes. Interestingly, a number of genes (e.g. heat shock proteins) were discordantly regulated in response to CH between dmDys and WT. We tested the possibility that the disparate molecular responses of dystrophin-deficient tissues to CH could adversely affect muscle by performing functional assays in vivo. Normoxic and CH WT and dmDys flies were challenged with acute hypoxia and time-to-recover determined as well as subjected to climbing tests. Impaired performance was noted for CH-dmDys compared to normoxic dmDys or WT flies (rank order: Normoxic-WT ≈ CH-WT> Normoxic-dmDys> CH-dmDys). These data suggest that dystrophin-deficiency is associated with a disparate, pathological hypoxic stress response(s) and is more sensitive to hypoxia induced muscle dysfunction in vivo. We hypothesize that targeting/correcting the disparate molecular response(s) to hypoxia may offer a novel therapeutic strategy in DMD

Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling-4

<p><b>Copyright information:</b></p><p>Taken from "Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling"</p><p>http://www.biomedcentral.com/1741-7007/6/1</p><p>BMC Biology 2008;6():1-1.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2242781.</p><p></p>dicated. Panels marked prime show separate channels of the same embryo. (A)Wild-type embryo. (B) mutant embryo from germline clone. A neurogenic phenotype is revealed by the expansion of ELAV staining. (C) mutant embryo from germline clone expressing from a genomic rescue construct(). OFUT1expression is visible. ELAV staining shows the absence of a neurogenic phenotype. (D) mutant embryo derived from germline clone. Absence of fucosylation was confirmed by the absence of the HRP epitope; neurogenesis appears normal

Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling-0

<p><b>Copyright information:</b></p><p>Taken from "Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling"</p><p>http://www.biomedcentral.com/1741-7007/6/1</p><p>BMC Biology 2008;6():1-1.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2242781.</p><p></p>dicated. Panels marked prime show separate channels of the same embryo. (A)Wild-type embryo. (B) mutant embryo from germline clone. A neurogenic phenotype is revealed by the expansion of ELAV staining. (C) mutant embryo from germline clone expressing from a genomic rescue construct(). OFUT1expression is visible. ELAV staining shows the absence of a neurogenic phenotype. (D) mutant embryo derived from germline clone. Absence of fucosylation was confirmed by the absence of the HRP epitope; neurogenesis appears normal

Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling-1

<p><b>Copyright information:</b></p><p>Taken from "Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling"</p><p>http://www.biomedcentral.com/1741-7007/6/1</p><p>BMC Biology 2008;6():1-1.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2242781.</p><p></p>type. (B) Schematic drawing. WG expression is indicated in red. D-V indicates dorsal-ventral boundary. is expressed in both compartments whereas is expressed only dorsally. (C) mutant clones, marked by absence of GFP (green). Ectopic WG is indicated (arrow). (D) mutant clones, marked by presence of GFP (green, using the MARCM technique [16]). Loss of WG is indicated (arrowhead). (E), (F) Rescue experiments, with clones positively marked by GFP (green) using MARCM. OFUT1was expressed in clones either by expressing a construct under -Gal4 control (E) or employing the genomic construct recombined onto an chromosome (F). Ectopic WG is indicated (arrow). OFUT1 expression was confirmed by anti-OFUT1 staining (not shown). The inset depicts a high magnification image of the boxed area with WG expression inside (yellow) and outside (red) of the clone border evident. (G) clones. Large clones mutant for (marked by loss of GFP), occupying nearly the entire disk, were generated using the technique. WG expression appears normal in cells surrounding the cells, but further away loss of WG expression is evident (arrowhead)

Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling-3

<p><b>Copyright information:</b></p><p>Taken from "Contributions of chaperone and glycosyltransferase activities of -fucosyltransferase 1 to Notch signaling"</p><p>http://www.biomedcentral.com/1741-7007/6/1</p><p>BMC Biology 2008;6():1-1.</p><p>Published online 14 Jan 2008</p><p>PMCID:PMC2242781.</p><p></p>ain (magenta). (A) Horizontal and (B) vertical sections of a disk stained after detergent treatment. Apical is up. Increased and mis-localized Notch protein is observed within mutant cells (green), as reported previously using antibodies against the intracellular domain [10]. (C) Horizontal and (D) vertical sections of a disk stained without detergent treatment. An mutant clone is devoid of cell surface Notch (arrow), but Notch is readily detected along the surface of wild-type cells. (E),(F) Correlations in localization among ER markers. Vertical sections of a wing disk doubly stained with KDEL (magenta) and with either (E) Boca or (F) Calnexin (green). Additional examples are shown in additional file