Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

The focus of this study was to elucidate the molecular and cellular mechanisms whereby fibroblast growth factors (FGFs) mediate outgrowth of the limb. Specifically, we examined the epistatic relationship between FGF and Wnt/Planar cell polarity (PCP) signaling in establishing cell polarity as a mechanism for outgrowth. By implanting beads into embryonic limbs and lateral plate mesoderm, we established that FGF activates Wnt5a in a gradient fashion. Once it was established that Wnt5a was expressed at the right time and place to turn on PCP signaling, we investigated the ability of Wnt5a to influence cell migration and/or cell polarity. Our analysis revealed that there was no difference in cell migration when cells were exposed to an exogenous Wnt5a source. However, this did not rule out the possibility that cells were responding in a more mild fashion and polarizing toward a Wnt5a source. Live cell imaging was performed to observe the movement and morphology of limb mesenchyme cell cultures in the presence or absence of a Wnt5a expressing cell bolus. It appears as though the cells orient and move in a random fashion regardless of Wnt5a. However, this in vitro method may not truly recapitulate in vivo events. Future studies aim to develop better methods of observing cell polarization in vitro, including developing better methods to tract the movement of cells and observe “PCP” events. Due to the lack of information gathered from our in vitro studies, an in vivo study was conducted to test if FGF is necessary to polarize limb mesenchyme cells. If FGF is turning on Wnt5a and Wnt/PCP signaling is directing cell polarization, then FGF mutant clones will not migrate toward the AER. Therefore, it is expected that these mutant clones would be unable to undergo directed cell movement and/or cell divisions. Early clonal analysis indicates that a response to FGFs appears to be necessary to direct polarized outgrowth of limb mesenchyme.

Epithelial morphogenesis is contingent upon cell shape changes. Cell shape changes are the driving force for the metamorphosis of the adult Drosophila leg from the leg imaginal disc precursor. Genetic analysis has identified several Drosophila genes involved in regulating cell shape changes during leg disc morphogenesis. These include members of the RhoA signaling pathway and the product of the Stubble-stubbloid (Sb-sbd) locus, a transmembrane serine protease. Mutations in the Sb-sbd gene interact genetically with the members of the RhoA signaling pathway, however the nature of the relationship between Sb-sbd serine protease activity and RhoA signaling is not understood. To identify additional components of the RhoA signaling pathway that may help us to understand the role of the Sb-sbd protease in RhoA signaling the Drosophila genome was systematically scanned for genes that interact with Sb-sbd and RhoA mutations using deletions/deficiencies of specified regions of each chromosome. A total of 201 deficiencies uncovering approximately 84.9-91% of the euchromatic genome and spanning the X, second, and third chromosoms were tested. Of the 201 deficiencies tested, five putative interacting genetic regions and one gene within these deficiencies were identified. The candidate gene Eip78C encodes a nuclear steroid hormone receptor previously identified as having an important role in metamorphosis.

Hormonally-triggered regulatory hierarchies play a major role in organismal development. Disruption of a single member of such a hierarchy can lead to irregular development and disease. Therefore, knowledge of the members involved and the mechanisms controlling signaling through such pathways is of great importance in understanding how resulting developmental defects occur. Type II transmembrane serine proteases (TTSPs) make up a family of cell surface-associated proteases that play important roles in the development and homeostasis of a number of mammalian tissues. Aberrant expression of TTSPs is linked to several human disorders, including deafness, heart and respiratory disease and cancer. However, the mechanism by which these proteases function remains unknown. The ecdysone-responsive Stubble TTSP of Drosophila serves as a good model in which to study the functional mechanism of the TTSP family. The Stubble protease interacts with the intracellular Rho1 (RhoA) pathway to control epithelial development in imaginal discs. The Rho1 signaling pathway regulates cellular behavior via control of gene expression and actin cytoskeletal dynamics. However, the mechanism by which the Stubble protease interacts with the Rho1 pathway to control epithelial development, in particular leg imaginal disc morphogenesis, has yet to be elucidated. The Stubble protein consists of several conserved domains. One approach to a better understanding of the mechanism of action of Stubble in regulating Rho1 signaling is to define which of the conserved domains within the protease are required for proper function. Sequence analysis of twelve recessive Stubble mutant alleles has revealed that the proteolytic domain is essential for proper function. Alleles containing mutations which disrupt regions of the protease domain necessary for protease activation or substrate binding, as well as those with deletions or truncations that remove some portion of the proteolytic domain, result in defective epithelial development in vivo. In contrast, mutations in other regions of the Stubble protein, including the disulfide-knotted and cytoplasmic domains, were not observed. Another important step for defining the connection between Stubble and Rho1 signaling is to identify a Stubble target that acts as an upstream regulator of the Rho1 pathway. We performed a genetic screen in which 97 of the 147 Drosophila non-olfactory and non-gustatory G-protein-coupled receptors (GPCRs), a family of proteins that has been shown to be protease-activated and to activate Rho1 signaling, were tested for interactions with a mutant allele of Stubble. We found 4 genomic regions uncovering a total of 7 GPCRs that interact genetically when in heterozygous combination with a Stubble mutant. Further analysis of these genes is necessary to determine if any of these GPCRs is targeted by Stubble during activation of the Rho1 pathway.

In 1952 Alan Turing published his paper "The Chemical Basis of Morphogenesis", which described a model for how naturally occurring patterns, such as the stripes of a zebra and the spots of a leopard, can arise from a spatially homogeneous steady state through diffusion. Turing suggested that the concentration of the substances producing the patterns is determined by the reaction kinetics, how the substances interact, and diffusion.  In this project Turing's model with linear reactions kinetics was studied. The model was first solved using two different numerical methods; the finite difference method (FDM) and the finite element method (FEM) with different boundary conditions. A parameter study was then conducted, investigating the effect on the patterns of changing the parameters of the model. Lastly the controllability of the model and the least energy control was considered. The simulations were found to produce patterns provided the right parameters, as expected. From the investigation of the parameters it could be concluded that the size/tightness of the pattern and similarity of the substance concentration distributions depended on the choice of parameters. As for the controllability, a desired final state could be produced thorough simulations using control of the boundary and the energy cost of producing the pattern increased when decreasing the number of controls.

The silica-based cell walls of diatoms are prime examples of genetically controlled, species-specific mineral architectures. The physical principles underlying morphogenesis of their hierarchically structured silica patterns are not understood, yet such insight could reveal novel routes towards synthesizing functional inorganic materials. Recent advances in imaging nascent diatom silica allow rationalizing possible mechanisms of their pattern formation. Here, we combine theory and experiments on the model diatom Thalassiosira pseudonana to put forward a minimal model for morphogenesis of branched rib patterns – a widespread feature of diatom cell walls. To this end, we developed an automated image analysis algorithm that enabled quantitative assessment of the morphological discrepancy between the experiments and model predictions. The model proposed here quantitatively recapitulates the time-course of rib pattern formation by accounting for silica biochemistry with autocatalytic formation of diffusible silica precursors followed by conversion into solid silica. We propose that silica deposition releases an inhibitor that slows down up-stream precursor conversion, thereby implementing a self-replicating reaction-diffusion system, recapitulated by a non-classical Turing mechanism. The proposed mechanism highlights the role of geometrical cues for guided self-organization, rationalizing the instructive role for the single initial pattern seed known as primary silicification site present in diatoms. The model features a wide spectrum of possible pattern morphologies depending on the model parameters, suggesting that this model may be applicable in other diatom species. Moreover, due to the generic nature of the proposed model for branching morphogenesis, the mechanism identified here may be relevant also in other biological systems known to exhibit.

Epithelial tissues are one of the most abundant tissues in our body. They make up essential organs like the gut, heart and eyes. These organs take up their complex 3D shapes during normal development of the embryo. Our understanding of such large-scale 3D shape changes is limited mainly due to the technical difficulties of imaging and quantifying such developmental events. In this thesis, I study two events in which epithelial monolayers change their 3D shape. In both the projects, I use data from light-sheet microscopic images of developmental events. These data are provided by my collaborators. In this thesis, I further analyzed them using quantitative approaches and interpreted them using computational models. In the first project, I study a case of a developing tissue inside a rigid confinement. A perfect model system for this is the Drosophila embryo which consists of an epithelial monolayer (blastoderm) inside a rigid shell (vitelline membrane). During gastrulation, the blastoderm is under compressional stresses due to tissue proliferation and compression from the germband extension. During this time, an invagination separating the future head and the trunk region appears. This is known as the cephalic furrow (CF). As the CF disappears after some time, its relevance in the normal development of the embryo is unclear. To understand its role, my collaborators image the blastoderm in mutant embryos which lack CF. These mutant embryos have either of the genes even-skipped (eve) or buttonhead (btd) knocked down. In the absence of CF, temporary ectopic folds appear in the blastoderm in locations which vary between embryos. Unlike the CF, ectopic folds appear suddenly and hence look like buckling events. I hypothesize that ectopic folds appear because of the compressive stresses generated in the blastoderm during the germband extension or by the compression of tissues that are adjacent to mitotic domains. Moreover, in normal embryos, CF, which is a controlled invagination, acts as a sink for the compressive stresses and thus suppresses ectopic folds. To test this hypothesis, I modelled the blastoderm as a 2D elastic tissue which is confined inside a rigid boundary acting as the vitelline membrane. In my model, I show that the stresses generated by both the germband extension and the mitotic domains contribute to the formation of ectopic folds. I model the CF as a region with some preferred intrinsic curvature, thus acting as a programmed fold. I show that ectopic folds are inhibited in the presence of a CF. However, the efficiency of the CF depends on the strength of the CF and, interestingly, the timing of the CF. I observe that even a weak CF can inhibit ectopic folds if it appears before the appearance of mitotic domains. I speculate that this could explain why the CF appears before the mitotic domains in the Drosophila embryo. n the second project, I study a case of shape change associated with the development of the Drosophila wing. Here I focus on the wing disc pouch, an epithelial monolayer that forms the adult wing blade. During metamorphosis, the larval wing disc evaginates to form the pupal wing. This process is known as eversion. During late larval stage, the wing disc pouch looks like a spherical cap. I refer to this stage as wL3 (wandering larval stage 3) in this thesis. Four hours after pupariation (4hAPF), the spherical cap deforms to an asymmetric dome such that it has a higher curvature along one cross-section compared to its perpendicular cross-section. Using segmented outlines of the wing in the two cross-sections of multiple images at different developmental stages, I compute the mean shape and quantify the curvature along the arclength of these shapes. To model this shape change, I use a 3D spring lattice whose initial curvature is matched to the curvature of the wL3 stage. Next, using apical cell shape data, provided by my collaborator, I compute a quantity referred to as a “spontaneous deformation tensor”. This tensor quantifies the amount of deformation, at a specific location between two developmental stages, due to cell area changes, cell elongation changes, and neighbour exchanges. I input this deformation pattern in my model which then changes its 3D shape. I find that the deformation due to changes in cell area and elongation increase the size of the tissue globally without affecting its curvature. However, the deformation due to cell rearrangements enhances curvature along one cross-section more than its perpendicular cross-section. Overall, the quantifications and modelling shows how different cellular behaviours deform the tissue locally. Moreover, a spatial pattern of different cellular behaviours can explain essential aspects of the shape change observed during the development of the wing.

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<p>Renal dysplasia, a developmental disorder characterized by defective nephrogenesis and branching morphogenesis, ranks as one of the major causes of renal failure among the pediatric population. The molecular mechanisms underlying the pathogenesis of renal dysplasia are not well understood; however, changes in gene expression are a major contributing factor. In this study, we demonstrate that the levels of activated β-catenin, a transcriptional co-regulator, are elevated in the nuclei of ureteric, stromal, and mesenchymal cells within dysplastic human kidney tissue. To determine the mechanisms by which mesenchymal β-catenin over-expression leads to renal dysplasia, we generated a conditional mouse model in which β-catenin was stabilized exclusively in the metanephric mesenchyme. Kidneys from these mutant mice are remarkably similar to dysplastic human kidneys. In addition, these mutant mice also demonstrate the formation of 4 to 6 ectopic kidneys. While nephrogenesis appeared normal, investigation of ureteric branch pattern revealed ectopic ureteric budding off the Wolffian duct, ectopic branching off the initial ureteric bud stalk and a disorganization of branch patterning. In-situ hybridization of mutant kidneys revealed increased expression of Gdnf, Cret, and Wnt11, key factors that regulate ureteric branch patterning. We further demonstrate that β-catenin directly binds to TCF consensus binding sites within the Gdnf promoter region located 4.9kb, 2.25kb and 2.1kb upstream of the Gdnf transcriptional start site. Molecular cloning of the 4.9kb fragment upstream of a luciferase gene revealed that ß-catenin regulates gene transcription from the 4.9kb consensus site. Consistent with these findings, genetic deletion of β-catenin from the metanephric mesenchyme cell lineage lead to decreased Gdnf expression and a reduction in ureteric branching morphogenesis resulting in renal hypoplasia. Taken together, our findings establish that β-catenin is an essential regulator of Gdnf expression within the metanephric mesenchyme. Furthermore, we have identified a novel disrupted signalling pathway that contributes to the pathogenesis of renal dysplasia. In this pathway, an over-expression of β-catenin directly leads to an over-expression of Gdnf, causing ectopic and disorganized branching morphogenesis and, consequently, renal dysplasia.</p> / Master of Health Sciences (MSc)