Hedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient

Abstract

Cellular differentiation is directly determined by concentration gradients of morphogens. As a central model for gradient formation during development, Hedgehog (Hh) morphogens spread away from their source to direct growth and pattern formation in Drosophila wing and eye discs. What is not known is how extracellular Hh spread is achieved and how it translates into precise gradients. Here we show that two separate binding areas located on opposite sides of the Hh molecule can interact directly and simultaneously with two heparan sulfate (HS) chains to temporarily cross-link the chains. Mutated Hh lacking one fully functional binding site still binds HS but shows reduced HS cross-linking. This, in turn, impairs Hhs ability to switch between both chains in vitro and results in striking Hh gradient hypomorphs in vivo. The speed and propensity of direct Hh switching between HS therefore shapes the Hh gradient, revealing a scalable design principle in morphogen-patterned tissues.

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

“…Two models have been proposed on the role of Glp in long-range gradient formation: In the IST, or monkey bar model, the HS chains of Glps repeatedly transfer Hh directly from producing to receiving cells. Importantly, the degree of HS modification appears to control this movement, providing an explanation for the robustness of the Hh gradient and its ability to be scaled simultaneously (through the net charge differences between donor and acceptor HS chains) [ 118 ]. The model, therefore, proposes that Hh moves from HS chains with relatively low negative charge to more sulphated HS with an increased net negative charge, but not back, which would also give directionality to the movement.…”

“…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 ).…”

“…When expressed under the control of the endogenous Hh promotor in Drosophila eye and wing discs that have also been rendered null for endogenous Hh function [ 126 ], the mutant Hh protein caused the selective loss of eye and wing tissues known to require HSPGs for Hh long-range spreading. In contrast, other fly tissues from the same disc that depend on short-range Hh signalling developed normally, demonstrating that the mutant protein was selectively impaired in its ability to move between multiple HS chains for long-range relay, but less so in cell-to-cell signalling and in its ability to bind to its receptor on target cells in vivo [ 118 ] that could also be achieved by the solubilised diffusing ligand. Importantly, en -Gal4/UAS-controlled overexpression of the same proteins with only one fully functional HS binding site in the posterior wing disc compartment induced ectopic signalling at the peripodial membrane, which overlies the actual wing disc and is separated from this epithelium by a narrow, fluid-filled, closed compartment called the peripodial space ( Figure 2 ).…”

Sweet but Challenging: Tackling the Complexity of GAGs with Engineered Tailor‐Made Biomaterials

“…[ 59 ] HS participates in a wide range of functions, including cell differentiation, tissue morphogenesis, cell interactions and proliferation, and interaction with GFs, cytokines, and cell adhesion molecules. [ 2,4,60–67 ]…”

“…[59] HS participates in a wide range of functions, including cell differentiation, tissue morphogene-sis, cell interactions and proliferation, and interaction with GFs, cytokines, and cell adhesion molecules. [2,4,[60][61][62][63][64][65][66][67] Each GAG can exert various biological functions, which are often dictated by their interactions with specific proteins, as described elsewhere. [68] Hep and HA are the most advanced GAGs for biomaterial applications.…”