Quantitative Analysis of Hedgehog Gradient Formation Using an Inducible Expression System: a Dissertation
AuthorsSu, Vivian F.
Faculty AdvisorInge The PhD
Academic ProgramBiochemistry and Molecular Pharmacology
UMass Chan AffiliationsDepartment of Molecular Medicine
Document TypeDoctoral Dissertation
Embryonic and Fetal Development
Amino Acids, Peptides, and Proteins
Animal Experimentation and Research
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AbstractThe Hedgehog (Hh) family of proteins are secreted growth factors that play an essential role in the embryonic development of all organisms and the main components in the pathway are conserved from insects to humans. These proteins affect patterning and morphogenesis of multiple tissues. Therefore, mutations in the Hh pathway can result in a wide range of developmental defects and oncogenic diseases. Because the main components in the pathway are conserved from insects to humans, Drosophilahas been shown to provide a genetically tractable system to gain insight into the processes that Hh is involved in. In this study, the roles of Hh cholesterol modification and endocytosis during gradient fonnation are explored in the Drosophila larval wing imaginal disc. To exclude the possibility of looking at a redistribution of preexisting Hh instead of Hh movement, a spatially and temporally regulated system has been developed to induce Hh expression. Functional Hh-GFP with and without the cholesterol-modification was expressed in a wild-type or shi-tslendocytosis mutant background. The Gal80 system was used to temporally express (pulse) the Hh-GFP transgenes to look at the rate of Hh gradient formation over time and determine whether this process was affected by cholesterol modification and/or endocytosis. Hh with and without cholesterol were both largely detected in punctate structures and the spreading of the different forms of Hh was quantified by measuring distances of these particles from the expressing cells. Hh without cholesterol showed a greater range of distribution, but a lower percentage of particles near the source. Loss of endocytosis blocked formation of intracellular Hh particles, but did not dramatically alter its movement to target cells. Staining for Hh, its receptor Ptc and cortical actin revealed that these punctate structures could be classified into four types of Hh containing particles: cytoplasmic with and without Ptc, and cell surface with and without Ptc. Cholesterol is specifically required for the formation of cytoplasmic particles lacking Ptc. While previous studies have shown discrepancies in the localization of Hh following a block in endocytosis, Hh with and without cholesterol is detected at both apical and basolateral surfaces, but not at basal surfaces. In the absence of cholesterol and endocytosis, Hh particles can be observed in the extracellular space. Through three-dimensional reconstruction and quantitative analysis, this study concludes that the cholesterol modification is required to restrict Hh movement. In addition, the cholesterol modification promotes Ptc-independent internalization. This study also observes that Dynamin-dependent endocytosis is necessary for internalization but does not play an essential role in Hh distribution. The data in this thesis supports the model in which Hh movement occurs via planar diffusion.
Permanent Link to this Itemhttp://hdl.handle.net/20.500.14038/31619
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