Abstract
BackgroundThe Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning. Mutations in human Hh and other Hh pathway components have been linked to human diseases. Analysis of Hh distribution during development indicates that cholesterol modification and receptor mediated endocytosis affect the range of Hh signaling and the cellular localization of Hh.ResultsWe have used an inducible, cell type-specific expression system to characterize the three-dimensional distribution of newly synthesized, GFP-tagged Hh in the developing Drosophila wing. Following induction of Hh-GFP expression in posterior producing cells, punctate structures containing Hh-GFP were observed in the anterior target cells. The distance of these particles from the expressing cells was quantified to determine the shape of the Hh gradient at different time points following induction. The majority of cholesterol-modified Hh-GFP was found associated with cells near the anterior/posterior (A/P) boundary, which express high levels of Hh target genes. Without cholesterol, the Hh gradient was flatter, with a lower percentage of particles near the source and a greater maximum distance. Inhibition of Dynamin-dependent endocytosis blocked formation of intracellular Hh particles, but did not prevent movement of newly synthesized Hh to the apical or basolateral surfaces of target cells. In the absence of both cholesterol and endocytosis, Hh particles accumulated in the extracellular space. Staining for the Hh receptor Ptc revealed four categories of Hh particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Interestingly, mainly cholesterol-modified Hh is detected in the cytoplasmic particles lacking Ptc.ConclusionWe have developed a system to quantitatively analyze Hh distribution during gradient formation. We directly demonstrate that inhibition of Dynamin-dependent endocytosis is not required for movement of Hh across target cells, indicating that transcytosis is not required for Hh gradient formation. The localization of Hh in these cells suggests that Hh normally moves across both apical and basolateral regions of the target cells. We also conclude that cholesterol modification is required for formation of a specific subset of Hh particles that are both cytoplasmic and not associated with the receptor Ptc.
Highlights
The Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning
We demonstrate that when endocytosis is blocked, newly synthesized HhNp-GFP is still detected in particles and can still move anteriorly, arguing against an essential role of transcytosis
Wings of flies ectopically expressing functional untagged HhNp [7] and wings expressing HhNp-GFP had similar phenotypes of merged wing veins L2 and L3 (Figure 1H and 1I). These results demonstrate that the Hh-GFP fusion proteins are properly synthesized, and that HhNp-GFP has the same properties as previously described for wild-type HhNp [39]
Summary
The Hedgehog (Hh) family of secreted growth factors are morphogens that act in development to direct growth and patterning. Hh directly induces different cell fates in a concentration dependent manner, and is classified as a morphogen. This concentration gradient is tightly regulated and any disruption can cause abnormal cell specification [2,3]. Models that have been proposed for morphogen distribution and gradient formation include extracellular movement by diffusion and intracellular movement by transcytosis [4,5]. In the free diffusion model, the morphogen is secreted from the producing cells into the extracellular space and diffuses in three-dimensions out to the target cells. In the restricted planar diffusion model, the morphogen moves directly from cell to cell, always remaining in the two-dimensional epithelial cell layer close to the cell surface. The transcytosis model proposes a unique mechanism where successive rounds of endocytosis and exocytosis move Hh through cells [4,5]
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