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First published online 13 August 2003
doi: 10.1242/dev.00679

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neurotic, a novel maternal neurogenic gene, encodes an O-fucosyltransferase that is essential for Notch-Delta interactions

¹ PRESTO, Japan Science and Technology Corporation
² Department of Biological Science and Technology, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan
³ Genome and Drug Research Center, Tokyo University of Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan
⁴ Department of Nutrition, School of Medicine, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan
⁵ Department of Molecular and Tumor Pathology, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
⁶ Department of Biology, Developmental, Cell and Molecular Biology Group, Duke University, Durham, NC 27708, USA
⁷ Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (56K): [in a new window]   Fig. 3. Epistatic analyses between nti, Notch and fng. In all figures, Wg expression is shown in purple and somatic clones that lack nti function, and/or that overexpress specific genes, are shown in green. Anterior is towards the left and dorsal is upwards. (A) nti somatic clones showed cell-autonomous loss of Wg expression. Higher magnifications of the clones at the DV border are shown in the insets. y w hs-flp/+; FRTG13 nti/FRTG13 tubP-GAL80; tubP-GAL4/UASGFP larva wing imaginal disc is shown. nti homozygous clones are shown by GFP fluorescence (green). (B) Cell-autonomous Wg ectopic expression was detected when an activated form of Notch was expressed in nti mutant cells (arrowhead) as in wild-type cells (data not shown), indicating that nti is not needed for NotchICD function. Genotype of the wing imaginal disc is y w hs-flp/+; FRTG13 nti/FRTG13 tubP-GAL80; tubP-GAL4/UAS-GFP UAS-Nact. Thus, GFP indicates nti homozygous cells overexpressing NotchICD. (C) Overexpression of Notch is associated with ectopic Wg expression near the DV boundary. The genotype is y w hs-flp/+; FRTG13/FRTG13 tubP-GAL80; tubP-GAL4/UAS-GFP UAS-Notch. GFP shows full-length Notch overexpressing cells. (D) Full-length Notch effect was suppressed in nti mutant cells, indicating that nti is epistatic to full length Notch receptor. The genotype is y w hs-flp/+; FRTG13 nti/FRTG13 tubP-GAL80; tubP-GAL4/UAS-GFP UAS-Notch. GFP shows nti homozygous cells overexpressed for full-length Notch. (E) Ectopic expression of fng in the ventral compartment of the wing imaginal disc is associated with ectopic Wg expression near the margin. The genotype is y w hs-flp/+; FRTG13/FRTG13 tubPGAL80; tubP-GAL4/UAS-GFP UAS-fng. GFP shows Fngoverexpressing cells. (F) Ectopic expression of fng in nti mutant cells had no effect on Wg expression, showing that Nti is essential for Fng function. The genotype is y w hs-flp/+; FRTG13 nti/FRTG13 tubPGAL80; tubP-GAL4/UAS-GFP UAS-fng. GFP shows nti homozygous cells overexpressing Fng.

View larger version (102K): [in a new window]   Fig. 1. nti mutation gives similar phenotypes to those defective in Notch signalling. (A-D) nti is a maternal neurogenic gene. Embryos were stained with anti-Elav to detect all differentiated neurons (Robinow and White, 1988). (A) Wild-type embryo shows normal nervous systems. (B) nti mutant embryos derived from heterozygous mothers also have a normal embryonic nervous system. (C) Embryos that lack both maternal and zygotic nti function show a severe neurohyperplasia, and their germ band fails to shorten. (D) Paternally supplied nti gene could hardly rescue the maternal effect neurogenic phenotype; however, germ band retraction occurred in these embryos. (E) Wild-type wing. (F) nti mutant clones are associated with wing nicking and vein thickening (arrowhead). Both are typical phenotypes associated with defects in Notch signalling. (G) Wg is expressed at the DV boundary in wing imaginal disc. (H) Wg expression (purple) is lost in nti mutant clone (arrowhead). nti clones are detected by lack of GFP expression (green). (A-D, G-H) Anterior is towards the left and dorsal is upwards.

View larger version (28K): [in a new window]   Fig. 2. (A) Sequence comparison of Nti and human GDP-fucose O-fucosyltransferase (O-FucT-1). The identical amino acids are reversed, conserved changes are shaded. The proteins are 42% identical and 75% similar. The position of the 4R6 mutation is shown in red (replace Lysine in Nti by stop in 4R6). The putative type II transmembrane domain is underlined. The positions of the potential N-glycosylation (*) site, and the EXD motif that is important for glycosyltransferase activity (#), are also shown. (B,C) In situ hybridisation to whole-mount embryos stained with a nti cDNA probe revealed that nti mRNA is uniformly expressed in stage 3 embryos (B), and gradually disappears by stage 11 (C). nti mRNA is not detectable after stage 14 (not shown). (D) Wg expression in wing imaginal disc was recovered in nti mutant cells with hs-CG12366 (hs-nti; lack of GFP shown by green, arrowhead), indicating that nti is CG12366. (E) Expression of a nti inverted repeat RNA driven by ptc-GAL4 generates wing phenotypes (vein thickening and wing nicking indicated by arrow and arrowhead, respectively) that resemble wings containing nti homozygous mutant clones (Fig. 1F), indicating that nti-IR is indeed blocking nti function. (B-D) Anterior is towards the left and dorsal is upwards. (F) A schematic diagram of the tetrasaccharide, Sia-2, 3-Gal-ß1, 4-GlcNAc-ß1, 3-Fuc, attached to a EGF repeat of Notch. Nti adds O-linked fucose, and O-fucose residues is further elongated by Fng, ß1, 3-N-acetylglucosaminyltransferase. The N-acetylglucosamine (GlcNAc) is further elongated by an endogenous ß1, 4-galactosyltransferase and a ß2, 3-sialyltransferase. (G) Model for Fng-dependent and Fng-independent glycosylation of a Notch EGF repeat. (Upper) O-fucose residue attached by Nti is elongated to yield the tetrasaccharide in Fng-dependent manner. (Lower) Without Fng function, EGF repeats of Notch is O-fucosylated by Nti.

View larger version (34K): [in a new window]   Fig. 4. Nti function is essential for Notch receptor to bind Delta ligand. (A) Co-expression of Nti and Fng with Notch synergistically increases the binding to Delta-AP. (Upper panel) Delta-AP binding to transfected S2 cells as measured by an enzymatic activity of alkaline phosphatase (Brückner et al., 2000). S2 cells were transfected with the expression vectors for the proteins indicated as + or the empty control vectors to keep the total amount of the plasmids constant. The relative binding of the ligand was expressed as an arbitrary unit that takes the normalised bound AP activity of the empty vector-transfected cells as zero, and that of the cells transfected with Notch alone as one. Means±s.d. of triplicate assays are shown. Lower panel, Western blots of S2 cell extracts from the cells prepared in parallel to those used for the binding assays presented in the upper panel. The blot was probed with the anti-Notch, anti-Myc, anti-HA and anti-tubulin antibodies. Two forms of Notch were recognised by the anti-Notch antibody (9C6) that directs a region of the intracellular domain of Notch. The upper band is the unprocessed form of Notch and the lower band is the C-terminal part of the cleaved form. (B) Co-expression of nti-IR (RNAi for nti) with Notch abolishes the binding to Delta-AP irrespective of Fng overexpression. The analysis was carried out and the results were presented as in A. (C) Expression of Notch on cell surface is not influenced by the changes in Nti expression. The cells transfected with the indicated expression vectors were analysed by flow cytometry with an antibody directed against the extracellular domain of Notch (Rat-1). The numbers denote the percentages of cells in the indicated squares.

View larger version (29K): [in a new window]   Fig. 5. Neither overexpression Nti nor co-expression of nti-IR with Delta-AP affects the ability to bind to Notch. Conditioned media were prepared from S2 cells transfected with Delta-AP and either the empty vector, Nti, Nti-myc or nti-IR in 1:1 ratio as indicated. Binding to the S2 cells co-transfected with Notch and Fng are measured.

View larger version (33K): [in a new window]   Fig. 6. The signature motif for glycosyltransferases is necessary for the Nti activity. Notch-Delta binding was assayed with Nti-G3-myc that was created from Nti-myc by inducing amino acid changes ERD (shown by # in Fig. 2A) to GGG. ERD is thought to be important for glycosyltransferase activity. The analysis was done and the results were presented as in Fig. 4A. Compared to the binding observed in Nti-myc, Notch and Fng co-expressing cells, Nti-G3-myc cotransfection with Notch and Fng does not enhance Notch-Delta binding. Instead Nti-G3-myc acts as dominant-negative form of Nti, because the Notch-Delta binding is lower than observed in Notch and Fng co-transfected cells.

View larger version (11K): [in a new window]   Fig. 7. Schematic drawing of Nti molecular function. O-fucosecontaining glycans attached to the specific EGF repeat of Notch is the tetrasaccharide: Sia-2, 3-Gal-ß1, 4-GlcNac-ß1, 3-Fuc-1-O-Ser/Thr. First, O-fucose is attached to a serine or threonine within a consensus sequence of Notch EGF repeat by Nti. O-fucosylation is essential for binding between Notch and Delta. Thus, if there is a lack of O-fucosylation, activation of Notch does not occur (lower panel). In the cells that do not express Fng, O-fucose on Notch does not elongate further (middle panel). However, Notch modified by O-fucose is competent to bind to the Delta and Serrate. Next, N-acetylglucosamine is added to the O-fucose residue by Fng, then O-fucose glycans are elongated by ß1, 4-galactosyltransferase and 2, 3-sialyltransferase (upper panel). Notch modified by these O-linked glycans has an enhanced activity to bind to Delta. Thus, in the cells expressing Fng and the other two endogenous glycosyltransferases, Notch become more potent to receive signal than neighboring cells that do not express Fng.