Dally-like core protein and its mammalian homologues mediate stimulatory and inhibitory effects on Hedgehog signal response

Abstract

The distribution and activities of morphogenic signaling proteins such as Hedgehog (Hh) and Wingless (Wg) depend on heparan sulfate proteoglycans (HSPGs). HSPGs consist of a core protein with covalently attached heparan sulfate glycosaminoglycan (GAG) chains. We report that the unmodified core protein of Dally-like (Dlp), an HSPG required for cell-autonomous Hh response in Drosophila embryos, alone suffices to rescue embryonic Hh signaling defects. Membrane tethering but not specifically the glycosylphosphatidylinositol linkage characteristic of glypicans is critical for this cell-autonomous activity. Our studies further suggest divergence of the two Drosophila and six mammalian glypicans into two functional families, an activating family that rescues cell-autonomous Dlp function in Hh response and a family that inhibits Hh response. Thus, in addition to the previously established requirement for HSPG GAG chains in Hh movement, these findings demonstrate a positive cell-autonomous role for a core protein in morphogen response in vivo and suggest the conservation of a network of antagonistic glypican activities in the regulation of Hh response.

Fig. 1.

Dlp core protein is uniquely required for Hh response. (A) A ptc-luciferase Hh reporter assay shows that dlp or dlpΔGAG fully rescues Hh response in the presence of dlp UTR dsRNA, whereas gfp and dally do not. Control dsRNA treatments for neutral (yfp), negative (ptc), and positive (smo) effectors are shown. Red and green bars show ptc-luciferase reporter levels with control and HhN-conditioned medium treatments, respectively, normalized to a constitutive copia-Renilla luciferase reporter. Transfections were done in triplicate, and the values were averaged. Error bars show variability within each triplicate. A representative experiment is shown. (B) DlpΔGAG-Fc shows no detectable heparan sulfate (HS) modification. Fc fusion proteins or control GFP expressed in COS-1 cells was incubated with (+Enz) or without (−Enz) heparitinase I and III and chondroitinase ABC and analyzed by reducing SDS/PAGE and Western blotting to detect Fc (Left, Fc) or the HS core fragment produced by enzymatic treatment (Right, 3G10). Molecular weight markers are indicated.

Fig. 2.

Structure-function studies delineate a truncated domain of Dlp that is active in Hh response but fails to bind HhN in vitro. (A) (Right) Schematic diagram shows Dally-Dlp ΔGAG chimeras and Dlp truncations generated. White, Dally; black, Dlp; vertical lines, GAG attachment sites (mutated); blue lines, domains containing the 14 conserved Cys residues (69–474 in Dally and 80–553 in Dlp); v-shaped lines, deleted domains. (Left) A ptc-luciferase Hh reporter assay shows the relative activities of the chimeras in rescuing Hh response in the presence of dlp UTR dsRNA over a 500-fold range of expression construct transfected. Values shown are normalized ptc-luciferase levels in the presence of HhN-conditioned medium. Rescue by 0.01 μg of DNA is shown in Fig. S2B. (B) A ptc-luciferase Hh reporter assay shows the relative activities of Dlp derivatives in rescuing Hh response in the presence of dlp UTR dsRNA. −GPI, deletion of the GPI attachment domain; +Sdc TM, replacement of the GPI attachment domain with an Sdc TM domain; −furin, furin cleavage site RERR mutation to GEGG. Reporter assay details are presented in Fig. 1. (C) A ptc-luciferase Hh reporter assay shows the relative activity of Dlp_ΔNCF::Dally (blue) in rescuing Hh response in the presence of dlp UTR dsRNA over a 100-fold range of expression construct transfected. Values shown are normalized ptc-luciferase levels in the presence of HhN-conditioned medium. (D) Purified immobilized HhN specifically retains Ihog FN1 but not Dlp_ΔNCF in the presence of deca-heparin, based on SDS/PAGE and Coomassie blue staining of eluted proteins. Molecular weight markers are indicated. (E) No significant HhN-Ren binding to cultured cells expressing Dlp or DlpΔGAG with or without Ptc is detected, whereas Ihog alone or in combination with Ptc shows significant binding. Values shown are actual bound Renilla counts.

Fig. 3.

Dlp core protein is active in vivo. (A) Zygotic null dlp^A187 embryos were rescued to adulthood equivalently by arm-GAL4-driven expression of UAS-dlp⁺ or dlpΔGAG based on the percentage of viable non-Antp^Hu adult flies. Full rescue is expected to generate 20% non-Antp^Hu progeny. Error bars depict variability between two to three independent crosses per genotype. (B) Double labeling of stage 10 embryos by in situ (purple) and with anti-GFP antibody (brown) showed rescue of bap, wg, and hh transcription by dlpΔGAG expression in dlp^A187 GLC embryos. Anti-GFP reactivity distinguishes embryos with a paternal dlp⁺, Ubi-GFP chromosome. dlp⁺, w¹¹¹⁸ embryos labeled only by in situ. dlpΔGAG-expressing embryos representing the worst (Upper) and best (Lower) phenotypes observed are shown. (C) Cuticle preparations show partial rescue of naked cuticle formation by dlpΔGAG expression in dlp^A187 GLC embryos. Four cuticles representing the range of phenotypes observed with dlpΔGAG expression are shown. dlp⁺, w¹¹¹⁸ embryos.

Fig. 4.

Mammalian glypicans show conserved activating and inhibitory activities toward Drosophila Hh response. (A) A cladogram shows the phylogenetic relationship between the mammalian and Drosophila glypicans. (B) A ptc-luciferase Hh reporter assay using a 500-fold range of transfected expression constructs shows that dlp, GPC4, and GPC6 rescue Hh response in the presence of dlp UTR dsRNA; GPC1 partially rescues Hh response; and GPC2, GPC3, and GPC5 do not rescue Hh response. Rescue by 0.01 μg of DNA is shown in Fig. S8B. (C) A ptc-luciferase Hh reporter assay using a 100-fold range of expression construct transfected shows that dlp has trans-dominant positive activity toward Hh response, whereas dally, GPC2, GPC3, and GPC5 have trans-dominant negative activity. The dally construct is tagged with 3× FLAG, whereas the dlp and glypican constructs have a BM40 signal peptide and 1× HA tag. Results for all glypican with 0.1 μg of DNA transfected are shown in Fig. S8C. Reporter assay details are presented in Fig. 1. Values shown in B and C are the normalized ptc-luciferase levels in the presence of HhN-conditioned medium.