The protein Hedgehog (Hh) is a highly conserved, secreted ligand (and morphogen) capable of patterning many different tissues during development. Recently, Sonic Hedgehog (SHH) a human homolog of Drosophila Hh was found to be a causative agent in certain cancers. While several drugs are being developed to combat the binding of SHH to its receptor Patched or the Patched-target Smoothened, very little is known about how SHH is secreted from the producing cell, another site for therapeutic targeting. We report here the characterization of a novel proteolytic event and genetic pathway that controls Hh intracellular sorting and axon transport using the Drosophila eye imaginal disc as our model system. In fly larval photoreceptor neurons the developmental signal Hh is guided to the apical (retina) and basal (growth cone, GC) ends where secretion of the morphogen is an inductive factor in photoreceptor differentiation and establishment of eye/brain neural connections. The Hh secreted from the basal side induces lamina development while Hh secreted at the retina induces ommatidial development. Hedgehog processing consists of autocleavage from its 46 kDa form (HhU) to become a lipid-modified N-terminal signaling molecule (HhN; 19kDa) and a C-terminal molecule (HhC24; 24 kDa). Following autocleavage, a fraction of the C-terminal auto-cleavage product then undergoes a second cleavage event leading to 16 kDa (HhC16) and 9 kDa products. Nothing is known about the significance of the C-terminal “2nd cleavage” other than its occurrence in both fly and human tissue. In an effort to identify regulators of Hh sorting, we discovered that the HhC “2nd cleavage” is a determining factor in the sorting of the HhN signaling domain. That is, if a cell induces more cleavage (more HhC16) we observe more HhN in the apical domain. Likewise, if a cell inhibits 2nd cleavage (less HhC16) we see more basal HhN. Creation of a “2nd cleavage mutant” shows that this process has developmental significance. Further, biochemical characterization of the 2nd cleavage suggests it occurs in the ER after autocleavage and that HhC24 can exit the cell in a Golgi independent manner (via lipid droplets) while HhC16 remains intracellular. The ER exit of HhC24 appears to be controlled by a conserved PP2A (Mts) /PKB (Akt) kinase pathway which potentially regulates the size and number of lipid droplets produced. These findings are an important first step in understanding the intracellular sorting and transport of Hh and highlight new targets for the treatment of SHH-related cancers. The discovery of divergent modes of Hh secretion and the “2nd cleavage” open novel avenues for Hh research by offering an alternative, and very direct, line of attack in the treatment of Hh-related cancer.

<p>Diverse mechanisms have been proposed to explain natural pattern formation processes, such as slime mold aggregation, feather branching, and tissue stratification. Regardless of the specific molecular interactions, the vast majority of these mechanisms invoke morphogen gradients, which are either predefined or generated as part of the patterning processes. However, using E. coli programmed by a simple synthetic gene circuit, I demonstrate here the generation of robust, self-organized ring patterns of gene expression in the absence of an apparent morphogen gradient. Interestingly, modeling and experimental tests show that the temporal dynamics of the global morphogen concentration serve as a timing mechanism to trigger formation and maintenance of these ring patterns, which are readily tunable by experimentally controllable environmental factors. This mechanism represents a novel mode of pattern formation that has implications for understanding natural developmental processes. In addition, the system can be coupled with inkjet printing technology and metabolic engineering approaches to develop future complex patterned biomaterials.</p> / Dissertation

In an attempt to describe how patterns emerge in biological systems, Alan Turing proposed a mathematical model encapsulating the properties of such processes. It details a partial differential equation governing the dynamics of two or more substances, called morphogens, reacting and diffusing in a specific manner, in turn generating what has now come to be denoted as Turing patterns. In recent years, evidence has accumulated to support Turing's claim and it has been proposed that it is responsible for the dynamical characteristics of phenomena such as skin pigmentation and branching of lungs in vertebrates. The aim of this paper is to study how the choice of model parameters and reaction kinetics influence the nature of patterns generated, as well as explore how boundary control can be employed to generate pre-defined patterns and the efficiency of this procedure. To simulate the patterns, the differential equation is solved in Python by means of a spectral method using discretized space and time domains. The model parameters were then studied to try to gain insight in their effects on the patterns yielded. The boundary control was implemented in MATLAB using a difference method. The metric used for efficiency was taken to be the energy expenditure of the boundary cells. The complex dynamics of the studied systems make it difficult to draw valuable conclusions on the influence of the parameters, but the results support the expected characteristics of the models used. The efficiency of the pattern generation is deemed to be closely related to the amount of boundary control utilized.

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

No description available.

The development of a multicellular organism, such as a human or an animal, begins with the fertilization of an egg cell. Thereupon the organism grows by repeated cell divisions until the adult size is reached and growth stops. Although it is known that intrinsic mechanisms determine the final size of developing organs and organisms, the basic principles of growth control are still poorly understood. However, there is strong evidence that certain morphogens, which are a special class of signaling molecules, act as growth factors and play a key role in growth control. In this work, growth control is studied from a mainly theoretical viewpoint. A discrete vertex model describing the organization of cells by a network of polygons is used, including a description of the cell cycle and a description of dynamical morphogen distributions. Self-organized growth is studied by introducing growth rules that govern cell divisions based on the local morphogen level. This discrete description is complemented by a continuum theory to gain further insight into the dynamics of self-organized growth processes. The theoretical description is applied to the developing wing of the fruit fly Drosophila melanogaster. In the developing wing, which is an epithelium consisting of single-layered cell sheets, the morphogen Decapentaplegic (Dpp) acts as a key growth factor. Experimental data shows that the Dpp distribution is dynamic and adapts to the size of the developing wing. Two mechanisms that rely on a regulatory molecule species and lead to such a dynamic behaviour of the Dpp distribution are studied. Several growth rules are tested and the resulting growth behaviour is quantitatively compared to experimental data of the developing wing. A particular growth rule, that triggers a cell division when the local morphogen level has increased by a certain relative amount, is found to be consistent with experimental observations under normal and several perturbed conditions. It is shown that mechanical stresses that arise due to spatial growth inhomogeneities can have a stabilizing effect on the growth process.

During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows. We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity. CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin. Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP. Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Hedgehog (Hh) est un morphogène conservé au cours de l’évolution, et qui est impliqué dans un grand nombre de processus développementaux. Ma thèse vise à comprendre comment la sécrétion, et la mise en place du gradient d’Hh sont régulées à partir de son site de production, en utilisant la drosophile comme modèle animal. Pour identifier les régulateurs positifs impliqués dans la maturation du signal Hh, nous avons conçu et réalisé un crible génétique couvrant l’ensemble du génome, par ARNs interférents (ARNi). Grâce à ce crible, nous avons identifié la petite protéine GTPase Rab8 qui serait impliquée spécifiquement dans le routage intracellulaire de Hh. Selon notre modèle proposé, la protéine Hh serait secrétée de 2 façons. Sa sécrétion du coté apical est nécessaire à l'activation de gènes cibles à longue distance, alors que sa sécrétion du coté baso-latéral permettrait l'activation de gènes cibles à courte distance. La façon par laquelle Hh est transportée de la membrane apicale à la membrane basale à l’intérieur des cellules productrices n’est pas connue. La perte de fonction de Rab8 dans les cellules productrices de Hh induit une augmentation de l’activation des gènes cibles à courtes distances, alors l’expression des gènes cibles activés à longues distances est réduite. De plus, en utilisant des expériences sur tissus vivant pour suivre la dynamique de l'internalisation de la protéine endogène d’Hh, nous avons constaté que la perte de Rab8 n'a pas d’effet sur sa sécrétion primaire, mais entraine des défauts dans l’endocytose de Hh, affectant, par la suite, la mise en place du gradient morphogénétique. / Hedgehog (Hh) is a conserved secreted morphogen involved in an array of developmental processes. Using Drosophila as a model, during my thesis we aimed to ask how the secretion, extraction and transport of Hh protein are regulated at the site of its production. To understand the positive regulators of Hh secretion and transport we designed and performed a genome-wide RNAi screen in Drosophila to identify new regulators of Hh transport and identified the small GTPase Rab8 as a novel component required for Hh trafficking. According to our proposed model, there are two pools of secreted Hh. The apical pool is needed for long range target gene activation, and basolateral pool for short range target gene activation. It is not clear how Hh is sorted apico-basally in the producing cells. Interfering with Rab8 function in the Hh producing cells extends Hh short range targets. Conversely, it reduces the long range Hh targets, suggesting that interfering with Rab8 function in the Hh producing cells impairs Hh trafficking, thus hampering the fine tuning between the two secreted pools of Hh. Moreover, using live assays to track the dynamics of endogenous Hh internalization, we observed that loss of Rab8 in Hh producing cells does not affect its primary secretion, but causes defects in Hh endocytosis, subsequently affecting its gradient activity. We hypothesize a model where Hh is targeted for primary secretion to the apical side of the wing disc, which then is internalized, and this internalized Hh is then directed for recycling which is essential for its long range activity.

No description available.

<p>Heparan sulfate (HS) is a ubiquitous polysaccharide on the cell surface and in the extracellular matrix. HS is an important actor in the regulation of cell signaling, especially in the developing embryo. In combination with cell culture and biochemical experiments, <i>in vivo</i> studies of genetically modified animals have pointed out the sulfation pattern of HS as highly important for binding of ligands, their receptors and other signaling modulators.</p><p>The sulfation pattern of an HS chain is gained by several modifying steps, performed by multiple enzymes during biosynthesis in the Golgi apparatus. By alterations of sulfation pattern, and the amount of sulfate groups, a cell can regulate the binding properties of its HS to different molecules. The most highly sulfated form of HS is called heparin, and can only be found intracellularly in mast cells.</p><p>This thesis describes the phenotypes and the alterations in HS/heparin biosynthesis of two genetically modified mouse strains deficient in N-deacetylase/N-sulfotransferase-1 (NDST1) and -2 (NDST2) respectively. We have found NDST1 to be important for correct sulfation of HS and that NDST2 is crucial in heparin biosynthesis. NDST2 deficient mice completely lack heparin and therefore have a severe mast cell phenotype. NDST1 deficient mice produce undersulfated HS and show several developmental disturbances. Some NDST1 embryos die in utero while the rest die neonatally due to breathing difficulties. Defect brain, eye and skeletal development has also been observed while some organs, such as the liver, appear to be largely unaffected. Several phenotypes are similar to defects seen in other mouse strains with impaired fibroblast growth factor and bone morphogenetic protein signaling, among others. This suggests the phenotypes of NDST1 deficient embryos to be of a multi factorial origin, in complete accordance to the many signaling pathways HS is suggested to modulate.</p>