Drosophila hedgehog signaling range and robustness depend on direct and sustained heparan sulfate interactions

Abstract

Morphogens determine cellular differentiation in many developing tissues in a concentration dependent manner. As a central model for gradient formation during animal development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in the Drosophila wing disc. Although heparan sulfate (HS) expression in the disc is essential for this process, it is not known whether HS regulates Hh signaling and spread in a direct or in an indirect manner. To answer this question, we systematically screened two composite Hh binding areas for HS in vitro and expressed mutated proteins in the Drosophila wing disc. We found that selectively impaired HS binding of the second site reduced Hh signaling close to the source and caused striking wing mispatterning phenotypes more distant from the source. These observations suggest that HS constrains Hh to the wing disc epithelium in a direct manner, and that interfering with this constriction converts Hh into freely diffusing forms with altered signaling ranges and impaired gradient robustness.

The role of glycosaminoglycan modification in Hedgehog regulated tissue morphogenesis

“…Both recent studies [ 62 , 63 ] provide evidence that electrostatic interactions between negatively charged HS and two basic HS-binding sites guide extracellular Hh transport in a similar way to maximize Hh search efficiency for its target Ptc ( Figure 2 ). The experimental strategy used in these studies consisted of site-directed mutagenesis of one of two established HS-binding sites of Hh [ 70 , 71 ] and Shh [ 70 , 72 ] into neutral alanines or acidic glutamates to impair the proteins’ capacity for intersegmental protein transfer and to convert Hh into a classic on/off binder with only one fully functional HS-binding site.…”

“…As described before, the apparent paradox that HSPG-interactions facilitate extracellular Hh transport instead of slowing it down — as is the case for Fgfs or BMP4 — does not support passive or hindered Hh diffusion as a physiologically relevant Hh transfer mechanism. However, two publications recently proposed an HSPG-dependent apical Hh transport mode that is independent of cytonemes and can also solve most of the problems associated with (hindered) diffusion [ 62 , 63 ]. The main hypothesis in these studies is that tradeoff problems of hindered diffusion, as described before, apply only to proteins with one binding site for the HSPG cell-surface interactor.…”

“…In the second study [ 63 ], the authors used the Gal4/UAS overexpression system to show that Hh variants with one HS-binding site exchanged for neutral alanines caused highly variable, and often striking, wing mispatterning phenotypes caused by altered gradient range and ectopic signalling in vivo . These findings are consistent with Hh conversion into a classic on/off binder with only one fully functional HS-binding site that unbinds more easily from HSPGs and undergoes passive diffusion in the bordering fluid-filled space.…”

Hedgehog on the Move: Glypican-Regulated Transport and Gradient Formation in Drosophila

“…The model of direct morphogen transfer from one Glp–HS chain to the next may apply to Hh associated with Shf, to delipidated Hh released as a consequence of double processing of the two terminal lipidated Hh peptides, or to Hh attached to LPPs or other lipid carriers ( Figure 5 ). One mechanism by which all these forms of Hh could be distributed on developing epithelial surfaces is through electrostatic interactions between the negatively charged Glp–HS sugar–sulfate chains and two basic HS-binding sites on the Hh morphogen [ 118 , 119 ]. These direct electrostatic interactions guide and constrict extracellular Hh transport to the HS-rich epithelial surface to maximise the efficiency with which Hh searches for its receptor, a process termed “sliding” ( Figure 5 ).…”