In vivo function of differentially processed Hedgehog morphogens

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A major challenge in developmental biology is to understand how cells coordinate developmental behaviors with that of their neighbours. Cells often employ secreted signaling molecules such as the Hedgehog (Hh) morphogens to control developmental growth and patterning. Hh is an unusual signaling molecule, however. It is synthesized in dually lipid-modified form (N-terminally palmitoylated, C-terminally cholesterol-linked), resulting in morphogen multimerization and firm multimer tethering to the surface of producing cells. This raises the question of how Hh release and signaling is achieved. Using the recombinantly expressed vertebrate Hh family member Sonic hedgehog (Shh), we found that A Disintegrin And Metalloprotease (ADAM) family members mediate processing from its lipidated N-and C-termini on the surface of transfected cells, resulting in Shh core protein release. In this scenario, N- and C-terminal lipids are essential for the membrane-proximal positioning of ADAM cleavage sites and their subsequent processing. We further suggested that Shh processing and solubilization are coupled with its simultaneous activation. We showed that the ability of unprocessed multimeric Shh to bind to its receptor Patched on receiving cells is autoinhibited by N-terminal peptides that block the Patched-binding site in trans. However, proteins are truncated into Patched-binding competent soluble multimers upon palmitoylation-dependent cleavage and removal of the inhibitory N-terminal peptides. Based on this model, we explain biological inactivity of soluble Shh expressed in palmitoyl-acyltransferase-deficient mutants by the lack of N-terminal processing and the resulting blockade of the Patched-binding site. Here we propose to test this hypothesis in vivo, employing the Drosophila system. We will express variably N-truncated, non-palmitoylated Hh protein variants in cell clones lacking endogenous Hh function and determine their biolgical activities in vivo. A phenotypic rescue would confirm the indirect role of N-palmitoylation in Hh function and the requirement for N-terminal peptide processing for Hh activation. Moreover, we will use the power of Drosophila genetics to address the function of the presumably redundant proteases involved in Hh processing.