The role of direct heparan sulfate interactions and Ca2+ complexation in Hedgehog gradient formation in vivo

• Applicants: Professor Dr. Kay Grobe; Dr. Georg Steffes
• Subject Area: Developmental Biology
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 273964293

A major question in developmental biology is how cells coordinate their behaviors with that of their neighbors. As a central model for cellular communication during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in distant responding cells and tissues in Drosophila. Unimpaired Hh transport and gradient formation require extracellular expression of linear and highly negatively charged sugar chains called heparan sulfate (HS). However, HS binds and immobilizes most other extracellular soluble proteins, raising the question of how interactions with HS contribute to Hh spread instead of slowing it down. In the last funding period, we resolved this paradox by showing that direct electrostatic Hh interactions with cell-surface HS initiate and guide Hh transport in a similar way to electrostatically constrained DNA-binding protein movement in the nucleus. DNA-binding proteins preferably move along the axis of the double helix, and two DNA binding sites allow for direct transfer between sugar-phosphate backbones to increase search speed. Using a combination of in silico, advanced in vitro and in vivo techniques, we showed that Hhs do also switch directly between the many sugar-sulfate chains in the gradient field via two sites of densely arranged positive charge, and that positive charge reduction in one site severely impairs Hh switching in vitro and its transport to distant targets in vivo. Thus, we revealed that the ability to directly switch between HS chains represents a previously unknown determinant of temporally encoded morphogen movement with important implications for other HS-binding proteins. In this proposal, we aim to perturb the composition and position of charged amino acid residues in the disordered N-tail of Hh, using our established in vitro and in vivo protocols. We will also investigate dynamic HS expression and sulfation in developing tissues that determines accessible “microspaces” for morphogens. We expect that HS microspaces of increased HS sulfation over their surrounding tissue accumulate Hh, whereas neighboring microspaces expressing HS with lower sulfation will not be entered. We will visualize dynamic HS expression and sulfation changes in developing wing and eye disc microspaces by using single chain variable fragment (scFv) antibodies directed against defined HS structures. Our third aim is the continued characterization of fly lines made deficient in Ca2+-coordinating Hh amino acids. These lines were generated in the last funding period using our established protocol. Their phenotypes revealed that Hh Ca2+-coordination is non-essential for fly development, but is specifically required for stem cell proliferation and fly gametogenesis. In the next funding period, we therefore aim to further characterize this important yet poorly understood aspect of Hh biology.

• DFG Programme: Research Grants