Specific and flexible roles of heparan sulfate modifications in Drosophila FGF signaling

Abstract

Specific sulfation sequence of heparan sulfate (HS) contributes to the selective interaction between HS and various proteins in vitro. To clarify the in vivo importance of HS fine structures, we characterized the functions of the Drosophila HS 2-O and 6-O sulfotransferase (Hs2st and Hs6st) genes in FGF-mediated tracheal formation. We found that mutations in Hs2st or Hs6st had unexpectedly little effect on tracheal morphogenesis. Structural analysis of mutant HS revealed not only a loss of corresponding sulfation, but also a compensatory increase of sulfation at other positions, which maintains the level of HS total charge. The restricted phenotypes of Hsst mutants are ascribed to this compensation because FGF signaling is strongly disrupted by Hs2st; Hs6st double mutation, or by overexpression of 6-O sulfatase, an extracellular enzyme which removes 6-O sulfate groups without increasing 2-O sulfation. These findings suggest that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts.

Figure 1.

Tracheal phenotypes of Hs2st and Hs6st mutants. (A–F) Embryonic tracheal phenotypes in Hs2st and Hs6st mutants. Tracheal phenotypes were observed in stage 14 embryos using enhancer trap activity for the trachealess (trh) gene. (A) Wild-type embryos. Hs2st^d267 (B) or Hs6st^d770 (C) zygotic mutations do not affect tracheal development. (D) Hs2st^d267 maternal and zygotic null mutations have little effect on tracheal morphology except for migration defects of the dorsal branch (arrowhead) in 9% of these embryos. (E) 39% (20 out of 51) of Hs6st^d770 null embryos showed tracheal migration defects. Abnormalities were commonly observed in the dorsal trunk (arrows) and the dorsal branch (arrowheads). The other embryos (61%) showed normal tracheal morphology (F). (G–M) Tracheoblast formation in Hs2st^d267 and Hs6st^d770 wing discs. Tracheoblasts were visualized by UAS-GFP driven by btl-Gal4 (G, I, J, K, L, and M) or trh enhancer trap expression (H). (G) Wild-type tracheoblasts. sfl^9B4 mutation causes loss of tracheoblasts (H). Overall morphology of the tracheoblasts was not affected in all Hs2st^d267 (I) and most Hs6st^d770 (J) mutants. Two examples are shown for Hs6st^d770 mutant discs with smaller tracheoblasts (K and L). This phenotype was completely rescued by Hs6st expression in btl-Gal4/UAS-Hs6st; Hs6st^d770 (M).

Figure 2.

HS disaccharide profiling of Hs2st and Hs6st mutants. (Left) Representative HPLC chromatograms of wild-type (black), Hs2st^d267 (pink), and Hs6st^d770 (green) mutant HS. (Middle) Graphical depiction of disaccharide composition in these mutants, represented as percentage of total HS. (Right) Total levels of sulfate groups in Hs2st^d267 and Hs6st^d770 mutants. The value indicates the ratio of total sulfate groups in mutants to that in wild type.

Figure 3.

Tracheal phenotypes and FGF-dependent MAPK activation in Hs2st; Hs6st double mutants. (A–I) Embryonic tracheal phenotypes in Hs2st; Hs6st zygotic double mutants. Note that these Hs2st; Hs6st double mutants received some maternal contribution of both gene products. Embryonic tracheae were observed at stage 11 (A–C), 12 (D–F), and 14 (G–I) using trh enhancer trap. (A, D, and G) Wild-type embryos. (B, C, E, F, H, and I) Zygotic Hs2st^d267; Hs6st^d770 double mutants. (J–M) MAPK activation in wild-type and Hs2st; Hs6st double mutants. Tracheal cells and MAPK activation were marked by trh enhancer trap (green) and anti–diphospho-MAPK antibody (red), respectively, in wild-type (J and L) and the double mutant (K and M) embryos at stage 10 (J and K) and 12 (L and M). (N–P) Tracheoblast phenotypes in Hs2st wing discs expressing Hs6st RNAi. Tracheoblasts in wing discs were labeled by expression of UAS-GFP driven by btl-Gal4 (N and O) or by trh enhancer trap (P). (N) Wild-type wing disc. (O) Hs2st^d267 UAS-GFP/Hs2st^d267; btl-Gal4/UAS-IR-Hs6st wing disc. (P) Tracheoblasts (red) in Hs2st^d267 UAS-GFP/Hs2st^d267; bnl-Gal4 1-eve-1/UAS-IR-Hs6st wing disc. Expression of bnl-Gal4 is shown by GFP (green).

Figure 4.

Overexpression of Sulf1 reduces the 6-O sulfate groups on HS and causes tracheal defects. (A) HS disaccharide profiling of wild-type (black) and actin-Gal4/+; UAS-Sulf1/+ (blue) animals. (B) Tracheoblast phenotypes in wild-type and btl-Gal4 UAS-GFP/btl-Gal4; UAS-Sulf1 wing discs.