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

Abstract

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.

Fig. 1.

Gmd and Gmer are required for the fucosylation of bulk proteins and for Fng-dependent Notch signaling. (A) Schematic of GDP-l-fucose biosynthesis showing the roles of Gmd and Gmer, which are essential enzymes for its de novo synthesis in Drosophila. (B) Genomic organization of the Drosophila Gmd locus. The exons of the Gmd gene are shown as boxes, and the predicted coding regions are shaded black. A 0.8-kb deletion in Gmd^H78 is indicated by parenthesis. (C) Genomic organization of the Drosophila Gmer^SH locus. The exons are shown as boxes, with the predicted coding regions shaded black. A P-element, l(2)SH1096, is inserted 29-bp downstream of the predicted initiation codon. (D–G) AAL staining and (D′–G′) bright-field images of late third-instar wing imaginal discs. (D and D′) Wild-type wing disc. (E and E′) Gmd^H78 homozygous wing disc. (F and F′) Gmer^SH homozygous wing disc. (G and G′) Gmer^SH/Df(2R)BSC783 wing imaginal disc. (H–J) Anti-Wg antibody staining of late third-instar wing imaginal discs. (H) Wild-type wing imaginal disc. D, dorsal compartment; V, ventral compartment. Wg expression along the D/V boundary is indicated by a square bracket. (I) Gmer^SH homozygous wing imaginal disc. (J) Gmer^SH/Df(2R)BSC783 wing imaginal disc. White arrowheads indicate the D/V boundary in I and J. (Scale bar in D, 50 μm, applicable to D–J.)

Fig. 2.

Gmd and Gmer function cell-nonautonomously. (A–I) Late third-instar wing imaginal discs carrying somatic clones homozygous for Gmd^H78 (A–C), Gmer^SH (D–F), or Gfr¹ (G–I) were stained with AAL. (A, D, and G) Regions lacking GFP (green) are Gmd^H78 (A), Gmer^SH (D), or Gfr¹ (G) homozygous cells. (B, E, and H) AAL-staining (magenta) of wing imaginal discs carrying somatic clones homozygous for Gmd^H78 (B), Gmer^SH (E), or Gfr¹ (H). (C, F, and I) Merged images of A and B, D and E, and G and H, respectively. (J–R) Late third-instar wing imaginal discs carrying somatic clones homozygous for Gmd^H78 (J–L), Gmer^SH (M–O), or Efr¹ and Gfr¹ (P–R) and stained with an anti-Cut antibody. (J, M, and P) Regions lacking GFP (green) are Gmd^H78 (J), Gmer^SH (M), or Efr¹ and Gfr¹ (P) homozygous cells. (K, N, and Q) Anti-Cut antibody staining (magenta) of wing imaginal discs carrying somatic clones homozygous for Gmd^H78 (K), Gmer^SH (N), or Efr¹ and Gfr¹ (Q). (L, O, and R) Merged images of J and K, M and N, and P and Q, respectively. Insets: Higher magnifications of the regions indicated by white squares (K, N, and Q). The boundaries of the somatic clones are indicated by white broken lines. (Scale bar in A, 50 μm, applicable to A–R.)

Fig. 3.

GDP-l-fucose is delivered intercellularly within an organ. (A) GFP labeling showing the UAS-GFP expression pattern along the A/P boundary of third-instar wing imaginal discs under the dpp-Gal4 driver, which was used in B and C. (B, C, and E) Wg expression along the D/V boundary of the third-instar wing imaginal discs (square brackets in B, C, and E). (B) Wing disc of a Gmd^H78 homozygote overexpressing UAS-Gmd along the A/P boundary. (C) Wing disc of a Gmer^SH homozygote overexpressing UAS-Gmer along the A/P boundary. (D) Expression pattern of UAS-GFP in third-instar wing imaginal discs (white arrow), under control of a btl-Gal4 driver. (E) Wing disc of a Gmd^H78 homozygote overexpressing Gmd driven by btl-Gal4. (Scale bar in A, 50 μm, applicable to A–E.)

Fig. 4.

GDP-l-fucose is not supplied through body fluids. (A–C) AAL staining and (A′–C′) bright-field images of late third-instar eye imaginal discs (indicated by white square brackets). (A and A′) Wild-type eye imaginal disc. (B and B′) Gmd^H78 homozygous eye imaginal disc. (C and C′) Eye imaginal disc of a Gmd^H78 homozygote overexpressing UAS-Gmd driven by GMR-Gal4. (D and E) Anti-Wg antibody staining of late third-instar wing imaginal discs. AD, antenna imaginal discs. (D) Wing imaginal disc isolated from the larva of a Gmd^H78 homozygote overexpressing UAS-Gmd in the eye imaginal disc driven by GMR-Gal4. (E) Wing imaginal disc isolated from the larva of a Gmd^H78 homozygote overexpressing UAS-Gmd in the fat body driven by Lsp-Gal4. White arrowheads indicate the D/V boundary. (Scale bar in A, 50 μm, applicable to A–C′; in D, 50 μm, applicable to D and E.)

Fig. 5.

inx2 is required for the intercellular delivery of GDP-l-fucose. (A–O) Late third-instar wing imaginal discs stained with an anti-GFP antibody (A, E, and L, green), AAL (B, F, J, and M, magenta), and an anti-Cut antibody (C, G, K, and N, turquois). (A–D) ptc-Gal drove the expression of two UAS-GFP transgenes where a hairpin dsRNA of Gmd (Gmd IR) was produced. (E–H) ptc-Gal drove the expression of two UAS-GFP transgenes where a hairpin dsRNA of inx2 (inx2 IR) was produced. (I–L) ptc-Gal drove the expression of a UAS-GFP transgene where hairpin dsRNAs of Gmd and inx2 were produced. (M–O) ptc-Gal drove the expression of UAS-inx2 where hairpin dsRNAs of Gmd and inx2 were produced. The total number of UAS promoters in each experiment was adjusted to be the same by introducing UAS-GFP. D, H, L, and O are merged images of B and C, F and G, J and K, and M and N, respectively. White arrowheads indicate the regions of reduced AAL staining (J and K) and cut expression (K and L). (P–S) Adult wings. (P) Wild-type. (Q–S) Wings producing hairpin dsRNAs targeting Gmd (Q), inx2 (R), or Gmd and inx2 (S) under the control of ptc-Gal4. Square bracket and black arrow indicate a wing blade notch and wing vein-thickening, respectively, in S. (Scale bar in A, 50 μm, applicable to A–O.)