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Gradients of morphogens, secreted signaling molecules, are crucial for providing cells with positional information during animal development. While the processes of forma- tion and interpretation of these gradients have been extensively studied, the impact of morphogenetic events on patterning through morphogen gradients remains largely unex- plored. This thesis aims to understand the interplay and feedback mechanisms between tissue shape and morphogen gradients formation. To address this, we developed an analysis pipeline using MATLAB to accurately measure morphogen gradients in curved epithelia. By computationally deforming confocal images of curved tissues, we quantified the levels of a protein of interest at a specified distance from a reference point along the apico-basal axis. Applying our pipeline to the Hedgehog morphogen gradient in the Drosophila eye and wing imaginal discs, which serve as model systems for folded and flat epithelial tissues, respectively, we made an intriguing discovery. Despite the distinct morphologies of these tissues, the decay rate of the Hedgehog gradient remained com- parable. This led us to investigate the robustness of Hedgehog gradient formation by manipulating the morphology of the wing and eye discs. We induced ectopic fold forma- tion at the boundary between the source and receiver tissue of Hedgehog in the wing disc. We found that the decay rate of the Hedgehog gradient remained unchanged even in the wing disc with perturbed morphology, supporting the notion that the Hedgehog gradient is robust towards variability in tissue shapes. Additionally, we locally flattened the eye disc by introducing a mutation that inhibited depolymerization of F-actin. This resulted in the inability of cells to form the morphogenetic furrow and in an expansion of the Hedgehog range compared to the wild-type. However, according to our quantifica- tion, the expansion in the Hedgehog range is to be attributed to a shift in its source rather than a change in decay rate of the gradient. Overall, by developing quantitative methods to analyze the distribution of signaling proteins in curved tissues, we contribute to the understanding of the interplay between tissue morphology and pattern formation through morphogen gradients. Our findings highlight the robustness of the Hedgehog gradient formation towards diverse tissue morphologies. This observation leads us to hypothesize that this property of robustness could extend to other morphogens that employ transport mechanisms similar to Hedgehog.:Contents Summary ... ... .... i 1 Introduction ... ... .... 1 1.1 Basic principles of animal development: an intricated story ... ... .... 1 1.2 Epithelial folds: a fundamental building block for morphogenesis ... ... .... 3 1.3 Patterning via morphogen gradients ... ... .... 4 1.4 Hedgehog gradient in Drosophila imaginal discs as a model system... ... .... 13 2 Aims of the Thesis ... ... .... 21 2.1 Developing an analysis pipeline to quantify morphogen gradients in curved epithelia... ... .... 21 2.2 Assessing the robustness of the Hedgehog morphogen gradient in naturally folded and flat tissues: the eye and wing imaginal discs ... ... .... 22 2.3 Testing the robustness of the Hedgehog gradient by perturbing the morphology of the wing and eye discs ... ... .... 22 3 Materials and methods ... ... .... 25 3.1 Fly stocks... ... .... 25 3.2 Immunohistochemistry... ... .... 28 3.3 Imaging ... ... .... 30 3.4 Data analysis... ... .... 30 4 Results ... ... .... 47 4.1 Analysis pipeline to computationally flatten curved epithelial tissues: limitations in applicability and comparison to other methodologies... ... .... 47 4.2 The Hedgehog gradient is comparable between wing and eye disc in Drosophila ... ... .... 54 4.3 The extracellular basal gradient of Hedgehog has a decay rate comparable to the one of the internalized morphogen ... ... .... 62 4.4 Folds in the wing do not affect the Hedgehog gradient... ... .... 66 4.5 Downregulation of ci leads to lower levels of the Hedgehog receptors Ptc, which in turn results in a longer Hedgehog gradient ... ... .... 71 4.6 Local flattening of the morphogenetic furrow expands the source of Hedge- hog but does not affect the decay rate of the gradient ... ... .... 74 5 Discussion ... ... .... 83 5.1 Developing quantitative methods to analyze morphogen gradients in curved epithelia opens new possibilities to study the interplay between morphogens gradients and morphogenesis ... ... .... 83 5.2 A methodological consideration: the decay rate as a relevant parameter for assessing the robustness of the Hedgehog morphogen gradient ... ... .... 85 5.3 The decay rate of the Hedgehog gradient is comparable between the wing and the eye disc ... ... .... 90 5.4 The transport mechanism underlying the formation of the Hedgehog gra- dient in the wing disc is robust towards deformations of the apical side of the tissue ... ... .... 91 5.5 The capt mutation in the eye disc affects the signaling for differentiation without affecting the decay rate of the Hedgehog gradient ... ... .... 94 5.6 Active transport and binding to heparan sulfate proteoglypicans allow the Hedgehog morphogen gradient formation to be robust towards variation in tissuemorphology ... ... .... 98 5.7 Tissue morphology: obstacle or aid to patterning via morphogens ... ... .... 99 6 Conclusion... ... .... 103 7 Acknowledgments ... ... .... 105 8 References ... ... .... 107 9 Declaration according to §5.5 of the doctorate regulations ... ... .... 117 |
