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

Dermatophytoses represent a major health burden in animals and man. Zoophilic dermatophytes usually show a high specificity to their original animal host but a zoonotic transmission is increasingly recorded. In humans, these infections elicit highly inflammatory skin lesions requiring prolonged therapy even in the immunocompetent patient. The correct identification of the causative agent is often crucial to initiate a targeted and effective therapy. To that end, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) represents a promising tool. The objective of this study was to evaluate the reliability of species identification of zoophilic dermatophytes using MALDI-TOF MS. The investigation of isolates from veterinary clinical samples suspicious of dermatophytoses suggests a good MALDI-TOF MS based identification of the most common zoophilic dermatophyte Microsporum canis. Trichophyton (T.) spp. usually achieved scores only around the cutoff value for secure species identification because of a small number of reference spectra. Moreover, these results need to be interpreted with caution due to the close taxonomic relationship of dermatophytes being reflected in very similar spectra. In our study, the analysis of 50 clinical samples of hedgehogs revealed no correct identification using the provided databases, nor for zoophilic neither for geophilic causative agents. After DNA sequencing, adaptation of sample processing and an individual extension of the inhouse database, acceptable identification scores were achieved (T. erinacei and Arthroderma spp., respectively). A score-oriented distance dendrogram revealed clustering of geophilic isolates of four different species of the genus Arthroderma and underlined the close relationship of the important zoophilic agents T. erinacei, T. verrucosum and T. benhamiae by forming a subclade within a larger cluster including different dermatophytes. Taken together, MALDI-TOF MS proofed suitable for the identification of zoophilic dermatophytes provided fresh cultures are used and the reference library was previously extended with spectra of laboratory-relevant species. Performing independent molecular methods, such as sequencing, is strongly recommended to substantiate the findings from morphologic and MALDI-TOF MS analyses, especially for uncommon causative agents.

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<p>The generation of hematopoietic progenitors from human pluripotent cell sources for use in personalized medicine is an attainable goal for the ease of clinical intervention using these cells. Furthermore, generated platelets and mature red blood cells are enucleated which allows for the use of induced pluripotent stem cells as a starting source or other sources of genetic manipulation. Generating these cells has proven difficult as the cells appear to be stuck in a primitive state of differentiation and do not mature into an adult phenotype. This thesis shows that inhibition of the hedgehog signaling pathway early in the differentiation of pluripotent stem cells induces a maturation towards definitive hematopoiesis. Generated erythroid cells were shown to express beta globin at the transcript as well as protein level. This maturation effect was confirmed to occur through central hedgehog repressor, Gli3R, through genetic manipulation. Further interrogation of this mechanism showed that globin regulation was not mediated by chromatin methylation by the polycomb repressive complex. Finally, Gli3R was also shown to not act as a transcription factor influencing globin expression directly and is therefore engaging separate regulatory mechanisms. This data provides great strides towards the generation of clinically relevant hematopoietic populations from pluripotent sources, however Gli3R’s direct mechanism of action remains to be determined.</p> / Master of Science (MSc)

<p>Lens development is an intricate process governed by growth factor signaling and a hierarchy of transcription factors that regulate important processes required for normal lens development.</p> <p>Midline hedgehog (Hh) signaling has been implicated in lens defects including cyclopia and lens degeneration in rodents and fish. A lens specific model of hedgehog signaling has not been examined, and it was unknown whether the lens is able to respond to Hh signals. To investigate this question, and to determine any consequences of abnormal Hh signaling on lens development, a mouse model of constitutively active smoothened in the surface ectoderm and derivatives, (including the lens), was created. These mutants exhibited ectopic expression of FoxE3 by E12.5, and ectopic Pax6 expression by E15.5, along with deregulation of the lens cell cycle and lens degeneration.</p> <p>Similar to the Hh signaling pathway, normal expression of the transcription factor Activating Protein-2 (AP-2, <em>tcfap2</em>), in the lens, was shown to be essential for the maintenance of an epithelial cell phenotype, and the regulation of the lens cell cycle. AP-2α has been shown to be important at the placode stage of development for correct separation of the lens vesicle away from the overlying surface ectoderm. Defects resulting from the loss of AP-2α at this stage do not manifest until E12.5, at time at which AP-2β expression is lost in the lens, suggesting possible redundancy between the two AP-2 family members in early lens development.</p> <p>To investigate this possible redundancy, <em>Tcfap2a </em>and <em>Tcfap2b</em> were conditionally deleted from the lens at E9.5 (AP-2α/β DKOs). These family members were shown to play redundant roles during early lens development, with the double mutants exhibiting more severe defects than those seen in the AP-2α single knockout model A more nasally positioned lens stalk and a rotated lens were observed. Severe corneal defects and deregulation of the lens cell cycle were also evident.</p> <p>Roles for AP-2α in later lens development were unknown. To examine whether or not this transcription factor continues to play a role in lens epithelial cell maintenance subsequent to lens vesicle separation, a mouse model with <em>Tcfap2a</em> conditionally deleted from the lens during these later stages of development was created (MRL10-AP-2α). These mutants displayed a disorganized and multilayered lens epithelial cell layer with elongated epithelial cells that abnormally expressed fiber cell specific β/γ crystallins. These mutants also exhibited defects in cell adhesion between the epithelium and fiber cells, as well as between the epithelium and capsule, and exhibited fiber cell defects including vacuoles.</p> <p>Together, the work presented in this thesis outline previously unknown roles for Hh and AP-2 signaling in lens development. Both Hh and AP-2 are required for the maintenance of a normal lens epithelial cell phenotype and regulation of the cell cycle. This thesis also illustrates the requirement (and redundant roles) for AP-2α and AP-2β at the lens placode stage of development.</p> / Doctor of Philosophy (Medical Science)

Traumatic brain injury (TBI) is a major cause of disability worldwide. No effective treatment is currently available to restore function to the injured brain. After injury, massive neuronal death occurs which can result in long-lasting cognitive dysfunction. Following immediate mechanical damage, a series of secondary effects of injury occur including evolving neuronal damage, inflammation, astrocyte reactivation, blood brain barrier disruption and other physiological effects. Additionally, neural stem cell (NSC) proliferation has been observed following TBI, suggestive of an endogenous attempt to repair the brain. Stimulating proliferation of NSCs is a promising strategy to facilitate recovery following TBI, but the mechanisms underlying NSC proliferation remain unknown. In this work, we have addressed the following specific aims. In the first aim, we determined the role of Shh signaling in NSC proliferation after TBI. Using a fluid percussion model of TBI and conditional transgenic animal models, we investigated the role astrocytes play in NSC proliferation. Using a Sonic hedgehog (Shh) pathway inhibitor, we found that NSC proliferation after TBI relies on Shh signaling. In the second aim, we determined the role of astrocyte activation in NSC proliferation after TBI. Using transgenic tools, we determined that astrocytes are a major cellular source of Shh and that astrocyte-specific deletion of Shh inhibited NSC proliferation. This indicates that NSC proliferation relies on Shh signaling and that astrocytes represent the key cellular source. In the final aim, we sought to define the functional requirement of Nestin in NSC proliferation. Recent studies in our lab found that Nestin, an intermediate filament protein predominantly expressed by NSCs, played a role in Shh signaling in the setting of medulloblastoma cells. Here, we found that knockdown of Nestin impaired Shh signal transduction and Shh-driven proliferation in NSCs. Further, we generated a new mouse model allowing conditional deletion of Nestin in NSCs to determine whether Nestin played a similar role a non-neoplastic setting. Conditional deletion of Nestin in NSCs abolished the proliferation of hippocampal NSCs after TBI. These findings reveal the critical role of Nestin in Shh signaling and proliferation in NSCs following TBI. Our studies elucidate the cellular and molecular basis for NSC proliferation after TBI, which pave the road for development of therapeutic approaches to treat TBI by augmenting endogenous NSC regeneration. / Cancer Biology & Genetics

Major depressive disorder (MDD) is one of the most common psychiatric illnesses worldwide, with pharmacotherapy as a first-line option for the management of this illness. The National Center for Health Statistics found that the use of antidepressants has increased by more than 4 fold in the last 20 years. While SSRI’s act centrally to treat MDD, their peripheral effects are often overlooked. Interestingly, components of the serotonergic system including the serotonin transporter (SERT), serotonin receptors, and enzymes important for serotonin synthesis (tryptophan hydroxylase 1 and 2; Tph1 and Tph2) are affected by SSRI treatment both centrally and peripherally. This disruption of serotonin signaling in the pancreas is of particular interest as there is a considerable link between the serotonin and hedgehog signaling pathways, both of which are important for pancreatic beta cell function. I hypothesize that pancreatic beta cell exposure to the SSRI fluoxetine in vitro will lead to altered hedgehog signaling ultimately resulting in a disruption in insulin secretion. / Thesis / Master of Science in Medical Sciences (MSMS)