From fats to fluorescent fish: lipid modifications of sonic hedgehog ligand dictate cellular reception and signal response
Grover, Vandana Kaminie
Sonic hedgehog (Shh) signaling regulates cell growth during embryonic development, tissue homeostasis and tumorigenesis. Concentration-dependent cellular responses to secreted Shh protein are essential for tissue patterning. Shh ligand is covalently modified by two lipid moieties, cholesterol and palmitate, and their hydrophobic properties are known to govern the cellular release and formation of soluble multimeric Shh complexes. The influences of the lipid moieties on cellular reception and signal response, however, are not well understood. To study the influences of the lipid moieties on signal response, recombinant forms of Shh ligand, that were either fully lipidated or had one or both lipid adducts eliminated, were analyzed in NIH3T3 mouse embryonic fibroblasts. Quantitative measurements of recombinant Shh protein concentration, cellular localization, and signaling potency were integrated to determine the contributions of each lipid adduct on ligand cellular localization and signaling potency. We showed that either adduct is sufficient to confer cellular association, but the cholesterol adduct anchors ligand to the plasma membrane and the palmitate adduct augments ligand internalization. Additionally, we determined that lipid modification augments Shh concentration, and that signaling potency correlates directly with cellular concentration of Shh ligand. These studies, thus reveal complementary functions of hydrophobic modification in morphogen signaling by attenuating cellular release and enhancing Shh protein reception in target tissues. To explore these questions in vivo, we generated a visualizable form of Shh ligand for expression in zebrafish embryos. This fluorescent form of Shh retains autoprocessing and lipid modification, receptor binding, and signaling effeciency. Employing this visualizable form of Shh and a binary Gal4-UAS system, transgenic lines of zebrafish were created that regulate temporal and spatial expression of Shh-EGFP within the notochord and floor plate. We also generated transgenic lines of mutant forms of Shh protein lacking a palmitoyl or cholesteryl adduct. These tools will help further illuminate the roles of Shh lipid adducts on tissue patterning, ligand distribution, and signal potency.