The anterior pituitary gland is the major hormonal regulator in the body. The gland contains five secretory cell types whose emergence during development is defined by a tightly regulated array of transcription factors. In adult life, the gland is plastic with the relative proportions of cells varying depending on physiological context. Tumours of the pituitary gland account for 15% of all intracranial tumours in man and are caused by the selective proliferation of one of the secretory cell types. The majority of these (60%) are prolactinomas which consist of very slowly proliferating lactotroph cells, which produce the hormone prolactin. Pituitary tumours are almost never malignant and do not express common genetic markers for cancer, suggesting endogenous proliferative stimuli in the pituitary are the cause of tumour development. Oestrogen causes lactotroph hyperplasia during pregnancy and increases prolactin secretion. Our group previously showed that Wnt-4 mRNA was upregulated during oestrogen-induced lactotroph hyperplasia in Fischer 344 rats. Wnt molecules are key regulatory proteins controlling differentiation, proliferation and migration in development and adult life. Wnt-4 is involved in the emergence of lactotrophs during development, and has been implicated in pituitary tumour formation. Wnt molecules signal through three pathways. The well studied canonical pathway has been implicated in numerous cancers and centres around gene transcription initiated by translocation of β-Catenin into the nucleus. There are two non-canonical pathways: the Wnt-planar cell polarity (PCP) pathway and the Wnt-calcium pathway which are both poorly understood. In this thesis, the expression of Wnt-4 was studied in the pituitary, and effects of downstream signalling pathways in response to oestrogen were assessed. Wnt-4 was expressed in all secretory cell types of the pituitary, as well as the marginal zone (MZ), a region of the pituitary that may harbour stem cells. Oestrogen upregulated Wnt-4 protein in the somatolactotroph GH3 cell line, though this could not be replicated in primary tissue. A number of approaches (western blotting, immunofluorescence, reporter gene assays and mutant β-Catenin overexpression) were utilised to show that the canonical pathway was not activated in the pituitary. Wnt-4 had a clear inhibitory effect on calcium oscillations in GH3 cells, showing for the first time a non-canonical effect in the pituitary, though the downstream effects are currently unknown. Attempts made to study the activation of the PCP pathway were inconclusive. Efforts focused on the distribution of key structural and regulatory proteins in the anterior pituitary and the MZ. The MZ was characterised by a single layer of cells at the border of the anterior and intermediate lobes of the pituitary, with high expression of E-Cadherin and Sox 9, though no change in distribution was observed with oestrogen treatment. In the anterior lobe, oestrogen treatment decreased N and E-Cadherin expression, which could be an indicator of PCP pathway activation during oestrogen induced-lactotroph hyperplasia. Overall, data suggest that Wnt-4 does not directly cause oestrogen-induced lactotroph proliferation, but is likely to play a role in regulating tissue plasticity in the adult gland, as well as in the pathogenesis of pituitary tumours.

Wnt11 is a key signalling molecule that regulates cell polarity/migration during vertebrate development and also promotes the invasive behaviour of adult cancer cells. It is therefore essential to understand the mechanisms by which Wnt11 signalling regulates cell behaviour. The process of vertebrate gastrulation provides an excellent developmental system to study Wnt11 function in vivo. It is known that Wnt11 mediates coordinated cell migration during gastrulation via the non-canonical Wnt pathway that shares several components with a the planar cell polarity pathway (PCP) in Drosophila. However, the mechanisms by which these PCP components facilitate Wnt11 function in vertebrates is still unclear. While in Drosophila, the asymmetric localization of PCP components is crucial for the establishment of cell polarity, no asymmetric localization of Wnt11 pathway components have so far been observed in vertebrates. To shed light on the cellular and molecular mechanisms underlying Wnt11 signalling, I developed an assay to visualize Wnt11 activity in vivo using live imaging of Wnt11 pathway components tagged to fluorescent proteins. This allowed me to determine the sub-cellular distribution of these components and to correlate the effect of Wnt11 activity with the behaviour of living embryonic cells. I found that Wnt11 locally accumulates together with its receptor Frizzled7 (Fz7) at sites of cell-cell contacts and locally recruits the intra-cellular signalling mediator Dishevelled (Dsh) to those sites. Monitoring these apparent Wnt11 signalling centres through time-lapse confocal microscopy revealed, that Wnt11 activity locally increases the persistency of cell-cell contacts. In addition, I found that the atypical cadherin Flamingo (Fmi) is required for this process. Fmi accumulates together with Wnt11/Fz7 at sites of cell-cell contact and locally increased cell adhesion, via a mechanism that appears to be independent of known downstream effectors of Wnt11 signalling such as RhoA and Rok2. This study indicates that Wnt11 locally interacts with Fmi and Fz7 to control cell-contact persistency and to facilitate coherent and coordinated cell migration. This provides a novel mechanism of non-canonical Wnt signalling in mediating cell behaviour, which is likely relevant to other developmental systems. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 50 MB: Movies - Nutzung: Referat Informationsvermittlung der SLUB)

Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a common location for breast cancer cell metastasis, and progression of tumor in bone can lead to bone loss and affect human health. Osteocytes have important roles in bone homeostasis and osteogenesis, and their interaction with metastasized cancer cells are known to affect progression of metastasized tumor. However, the potential role of metabolic signaling and actin- cytoskeleton-associated moesin in the interaction of osteocytes and tumor cells remain poorly understood. In this study, we first examined the roles of metabolic signaling, specifically global AMPK modulators and mitochondria-specific AMPK inhibitor (Mito-AIP), as well as mechanical force in beta catenin signaling through interaction between osteocytes and precursor osteoblasts as well as osteocytes and breast cancer cells. We also evaluated the role of metabolic signaling in Rho GTPases including RhoA, Rac1 and Cdc42. We observed that AMPK activator (A769662) and Mito-AMPK stimulated beta catenin translocation to the nucleus, indicating the activation of Wnt signaling, while Mito-AIP did not significantly affect beta catenin activation in osteoblasts. We also observed that osteocyte conditioned medium (CM) treated with Mito-AIP substantially increased beta catenin signaling in osteoblasts, while decreasing beta catenin signaling in breast cancer cells. CM of osteocytes treated with fluid flow increased beta catenin signaling in breast cancer cells. A769662 and Mito-AIP also decreased the activities of RhoA, Rac1, and Cdc42 in cancer cells which are known to regulate cancer cell migration. Additionally, we evaluated the roles of intracellular and extracellular moesin (MSN) protein in well-established oncogenic signaling proteins, such as FAK, Src, and RhoA as well beta catenin signaling. Constitutively active MSN (MSN+) significantly increased FAK and Src activities in cancer cells, but decreased the activity of RhoA. Surprisingly, CM of mesenchymal stem cells treated with MSN+ decreased the activities of FAK, Src, and RhoA, suggesting the inhibitory role of extracellular MSN in tumor-promoting signaling. Our results suggest the distinct role of AMPK signaling, specifically at mitochondria of osteocytes, in the activities of beta-catenin signaling in osteoblasts and breast cancer cells and the distinct role of intracellular and extracellular MSN in these two types of cell.

No description available.

Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a well-studied model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the molecules governing opV placode development have not been well described. This study identifies the canonical Wnt signaling pathway as a regulator of opV trigeminal placode development. Introducing dominant-negative TCF and dominant-active β-catenin expression constructs by in ovo electroporation, we have manipulated the canonical Wnt pathway within the opV placode domain and surrounding ectoderm of chick embryos. Inhibition of canonical Wnt signaling results in the failure of targeted cells to express or maintain Pax3 protein, the earliest known specific molecular marker of opV placode cells. Misexpression of dominant-active β-catenin as an activator of canonical Wnt signaling, however, is not sufficient to promote the opV placode cell fate. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells to an opV placode fate. Strategies for manipulating the Wnt pathway at the level of ligand and receptor are also reviewed. Because it is clear that Wnt signaling is not acting alone in early development of the opV placode, we have also begun to investigate additional signaling pathways, such as FGFs, that may be involved in these developmental processes.

Considering that both cardiovascular disease (CVD) and congenital heart diseases (CHD) are still the leading cause of morbidity and mortality worldwide, there is a need for a robust and reliable cardiac model. Cardiac organoids are complex, three-dimensional cellular constructs that recapitulate the processes of the human embryonic heart. However, certain vital morphological features within the fetus are not yet replicable with cardiac organoids. Here we report our investigation to generate cardiac organoids with chamber formations. Our method involves modulating the Wnt pathway at two different instances while also implementing two cell seeding densities, all to determine the most optimized that to produce chamber formations within cardiac organoids.

No description available.

No description available.

Vulval development in C. elegans serves as powerful paradigm to understand the interplay of diverse signal transduction pathways during organogenesis. Previous studies have demostrated that the canonical Wnt signaling pathway plays a pivotal role in the development of the vulva in C. elegans and helps in establishing the 20-10-20 vulval induction pattern of the vulval precursor cells (VPCs). The main focus of my masters research project was to get an understanding of how this vulval induction pattern, established in response to Wnt signaling has evolved in other closely nematode species, particularly C. briggsae. We fmd that the Wnt signaling pathway has evolved to positively as well as negatively regulate the competence of VPCs in C. briggsae. We demonstrate that while mutations inpry-1/Axin in C. elegans result in Multivulva (Muv) phenotype, mutations in the C. briggsae pry-1 gene give rise to a novel MultivulvaVulvaless (Muv-Vul) phenotype. This phenotype is characterized by VPCs anterior to P6.p frequently adopting induced cell fates while those posterior to P6.p frequently adopt a non-induced fate. Furthermore, we also show that the functioning of the Wnt signaling pathway in C. briggsae is dependent upon the activity of key regulators of the Wnt pathway such as the TCFILEF-1 family member pop-1, the f3-catenin bar-] and the hox gene lin-39. Taken together, the fmdings from this study show that while a conserved canonical Wnt pathway confers competence on VPCs in both C. elegans and C. briggsae, the final outcome nonetheless seems to have diversified. / Thesis / Master of Science (MSc)

The nematode, Caenorhabditis elegans is an ideal animal model to study conserved mechanisms of developmental and postdevelopmental processes. Here, I describe the role of an Axin family member, pry-1, in aging, lipid metabolism, and seam cell development. Our analysis of pry-1 animals showed a catastrophic collapse of adult lifespan, which was accompanied with hallmarks of accelerated aging. Transcriptome profiling of pry-1 mutants revealed altered expression of genes associated with aging and lipid metabolism such as vitellogenins, fatty acid desaturases, lipases, fatty acid transporters and genes involved in cuticle synthesis. Consistent with this, pry-1 animals display significantly reduced levels of somatic lipids. Knockdowns of vitellogenins in the pry-1 background restored lifespan and lipid levels, suggesting that vitellogenins are necessary to mediate pry-1 function in aging and lipid metabolic processes. Additionally, lowered expression of desaturases and lipidomics analysis provided evidence of reduced fatty acid synthesis in pry-1 animals. In agreement with this, an exogenous supply of oleic acid restored depleted lipids in somatic tissues in addition to suppressing the short-lived phenotype of worms. In addition, transcriptome profiling for differentially expressed miRNAs in pry-1(mu38) identified heterochronic miRNAs (lin-4 and let-7 -family members) to act downstream of pry-1 /Axin. In C. elegans, these miRNAs are known to robustly regulate the stem-like, seam cell division. Loss of pry-1 function caused heterochronic defects such that the seam cells divide precociously to produce additional cells. The pry-1-miRNAs are involved in mediating silencing of the heterochronic gene, hbl-1, a C. elegans hunchback homolog, to regulate seam cell division. Furthermore, I report identification of novel miRNAs from C. elegans and C. briggsae. Overall, our findings demonstrate a novel role of the Wnt signaling regulator, pry-1/Axin, in the maintenance of adult lifespan that involves lipid homeostasis and regulation of heterochronic miRNA to control the developmental timing of seam cell division in C. elegans. / Thesis / Doctor of Philosophy (PhD)