Xylosylation of EGF16–20 negatively regulates <i>Drosophila</i> Notch signaling <i>in vivo</i>.

<p>(A) Schematic of the EGF repeats of wild-type and mutant <i>Notch</i> genomic transgenes. Blue boxes show EGF repeats with a consensus <i>O</i>-glucosylation site; orange boxes denote EGF repeats with a serine-to-alanine mutation in the <i>O</i>-glucosylation site, which prevents the addition of <i>O</i>-glucose and therefore xylose. (B–E) <i>N⁻/Y; N^gt-wt/+</i>, <i>N⁻/Y; N^gt-10_15/+</i>, and <i>N⁻/Y; N^gt-24_35/+</i> males exhibit no wing vein loss, but <i>N⁻/Y; N^gt-16_20/+</i> males (D) exhibit loss of L2, L4 and L5 veins (arrowheads). (F) At 25°C, <i>N⁻/Y; N^gt-wt/+</i> males expressing <i>GXYLT1-HA</i> in the <i>apterous-GAL4</i> domain show thickening of the distal wing veins. (G) In a <i>N⁻/Y; N^gt-10_15/+</i> background, <i>ap>GXYLT1-HA</i> becomes lethal at 25°C, and is not suppressed at 22°C. (H) In a <i>N⁻/Y; N^gt-16_20/+</i> background, the <i>ap>GXYLT1-HA</i> phenotype is fully suppressed. Note the presence of wing vein loss. (I) <i>N^gt-24_35</i> does not suppress the <i>ap>GXYLT1-HA</i> phenotype. (J) At 25°C, <i>N⁻/Y; N^gt-wt/+</i> males expressing <i>nub>XXYLT1-HA</i> show severe wing vein and margin defects. (K) The phenotypes are dramatically enhanced in <i>N⁻/Y; N^gt-10_15/+</i> males raised at 25°C (inset) and are comparable to (J) when raised at 18°C. (L,M) The <i>nub>XXYLT1-HA</i> phenotypes are fully suppressed in <i>N⁻/Y; N^gt-16_20/+</i> males (L), but are enhanced in <i>N⁻/Y; N^gt-24_35/+</i> males (M; compare to J). All wings, including the inset in M, are shown to scale.