Proteolytic processing of palmitoylated Hedgehog peptides specifies the 3-4 intervein region of the Drosophila wing

Abstract

Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3–4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3–4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.

Disrupting Hedgehog Cardin–Weintraub sequence and positioning changes cellular differentiation and compartmentalization in vivo

“…Moreover, en-Gal4-regulated posterior overexpression of nonpalmitoylated Hh C85S under UAS control (en>Hh C85S ) apposed veins L3 and L4 ( Fig. 3C, arrows) (Crozatier et al, 2004;Lee et al, 2001), supporting the conclusion that unprocessed Hh C85S Npeptides block Ptc binding of endogenous Hh, as described previously (Schurmann et al, 2018) and as outlined in Fig. S4.…”

“…Therefore, extreme dominant-negative protein activities resulting from impaired cluster release are converted into milder forms resulting from partial blockade of Ptc-binding sites ( Fig. S4) Schurmann et al, 2018). This is exactly what we observed.…”

“…inactive), clusters. Most recently, by uncovering a dominant-negative, cell-autonomous function of overexpressed Hh C85A/S on endogenous Hh in the Drosophila melanogaster wing disc, we confirmed the above-described N-palmitate-dependent Hh processing and activation in vivo (Schurmann et al, 2018). We also observed that relocating a stretch of basic amino acids located close to the N-palmitate, called the Cardin-Weintraub (CW) motif, completely abolished palmitoylated Hh processing and bioactivation.…”

“…Incubation with peroxidase-conjugated donkey anti-rabbit IgG (Dianova) was followed by chemiluminescent detection (Pierce). Shh constructs were generated from murine cDNA (NM_009170) by PCR and expressed in Bosc23 cells (RRID: CVCL_4401), as previously described (Schurmann et al, 2018) The cells were obtained from D. Robbins (University of Miami, USA), and tested negative for mycoplasma. Genetic characteristics were authenticated by PCR-single-locus-technology (Eurofins).…”

Conserved cholesterol-related activities of Dispatched drive Sonic hedgehog shedding from the cell membrane

“…Second, with the continued membrane association of these N-terminal peptides, palmitate limits possible modes of morphogen solubilization to shedding and assures completion of this process. This is important, because unprocessed palmitoylated peptides sterically inhibit Shh binding sites for the receptor Ptc, both in vitro (Ohlig et al, 2011) and in vivo (Schürmann et al, 2018). Therefore, Nterminal proteolytic processing not only releases Shh but also unmasks Ptc binding sites and thereby couples Shh solubilization with its bioactivation.…”