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Hedgehog Signaling Pathway
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Hedgehog Signaling Pathway
Hedgehog Signaling Pathway
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Articles linked to this work by shared authors, journal, and citation graph.
Updated: Jul 11, 2026

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients
Published on: December 14, 2015
Vivian F Su1, Kelly A Jones, Michael Brodsky
1Program in Gene Function and Expression, Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. vsu@crch.hawaii.edu <vsu@crch.hawaii.edu>
This study investigates how the Hedgehog (Hh) signaling protein spreads during development in Drosophila. Using a system that allows tracking of newly made Hh tagged with a fluorescent marker, the researchers observed how Hh moves from source cells to target cells. They found that cholesterol modification is important for Hh to reach certain areas of the cell, and that endocytosis is not required for Hh movement across cell surfaces. The findings suggest that Hh can travel through both the top and bottom sides of cells, and that a specific type of Hh particle forms only when cholesterol is present. These results help clarify how Hh gradients form during development and what factors influence their shape.
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Area of Science:
Background:
Morphogens like Hedgehog (Hh) are essential for directing tissue patterning during development. Prior research has shown that Hh signaling is influenced by cholesterol modification and receptor-mediated endocytosis. However, the precise mechanisms governing Hh gradient formation remain unclear. Existing studies suggest that cholesterol enhances Hh signaling range, but the role of endocytosis in gradient formation is debated. This gap motivated the current investigation into how Hh is distributed in developing tissues. Understanding how Hh spreads from source to target cells is critical for modeling developmental processes. Prior work has not resolved whether transcytosis is necessary for gradient formation. This uncertainty drove the need for a system that allows precise tracking of Hh movement. The current study builds on these findings to provide new insights into Hh distribution dynamics.
Purpose Of The Study:
The aim of this study is to characterize the three-dimensional distribution of newly synthesized Hedgehog (Hh) during gradient formation in the Drosophila wing. The specific problem addressed is how Hh spreads from its source to target cells and how cholesterol modification and endocytosis influence this process. The motivation stems from gaps in understanding whether transcytosis is required for Hh movement. By using an inducible expression system, the researchers sought to track Hh-GFP in real time. The study focuses on how Hh particles localize in different cell regions and whether receptor binding is necessary for gradient formation. The goal is to determine if endocytosis is essential for Hh signaling. The findings could clarify the role of cholesterol and endocytosis in gradient formation. This work contributes to broader efforts in developmental biology to model morphogen signaling.
Main Methods:
The researchers employed an inducible, cell type-specific expression system to generate and track newly synthesized Hh-GFP in the Drosophila wing. They used posterior-producing cells to express Hh-GFP and monitored its distribution in anterior target cells. Fluorescent imaging was used to identify punctate structures containing Hh-GFP. The distance of these particles from the source was measured to map the gradient over time. The study compared cholesterol-modified and unmodified Hh-GFP to assess their distribution patterns. Dynamin-dependent endocytosis was inhibited to determine its effect on Hh localization. The Hh receptor Ptc was stained to categorize Hh particles based on their location and receptor association. Quantitative analysis of particle distribution and localization was used to infer functional roles of cholesterol and endocytosis.
Main Results:
Cholesterol-modified Hh-GFP was predominantly found near the anterior/posterior (A/P) boundary, where Hh target genes are highly expressed. In contrast, unmodified Hh-GFP formed a flatter gradient with particles distributed farther from the source. Inhibition of Dynamin-dependent endocytosis blocked intracellular Hh particle formation but did not prevent Hh movement to apical or basolateral surfaces. Without cholesterol and endocytosis, Hh particles accumulated extracellularly. Ptc staining identified four categories of Hh particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Cytoplasmic particles lacking Ptc were mainly cholesterol-modified. These findings suggest that cholesterol is necessary for forming a specific subset of cytoplasmic Hh particles. The results indicate that transcytosis is not required for Hh gradient formation.
Conclusions:
The study demonstrates that cholesterol modification is required for forming a subset of cytoplasmic Hh particles not associated with Ptc. The findings indicate that Dynamin-dependent endocytosis is not essential for Hh movement across target cells. The localization of Hh suggests that it can move through both apical and basolateral regions of target cells. The absence of endocytosis leads to extracellular accumulation of Hh particles. These conclusions are based on the observed distribution patterns and receptor staining. The study provides direct evidence that transcytosis is not required for gradient formation. The role of cholesterol in Hh signaling is clarified by the localization of cytoplasmic particles. These results contribute to understanding how morphogens like Hh are distributed during development.
The study shows that cholesterol modification is required for a specific subset of cytoplasmic Hh particles not associated with Ptc.
They used an inducible expression system with Hh-GFP to monitor distribution in posterior and anterior cells.
Inhibiting it blocked intracellular Hh particle formation but did not prevent Hh movement to cell surfaces.
Ptc staining categorized Hh particles into cytoplasmic and cell surface types with and without receptor association.
Hh particles accumulate extracellularly, suggesting both factors are needed for proper localization.
The authors conclude that transcytosis is not required for Hh gradient formation.