This thesis investigates the applicability of novel approaches designed to study the molecular mechanisms required for the initiation of organogenesis within the early endoderm. The endoderm is the germ layer that gives rise to the gut-tube and associated organs including the thyroid, lung, liver and pancreas. Our laboratory focuses on understanding the molecular mechanisms governing the developmental transition from endoderm to liver and pancreas. Several signaling pathways including Wnt, Retinoic Acid (RA), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor-β (TGFβ) have been implicated in the emergence of the liver bud from the endoderm in the mouse or other vertebrate species. However, neither the exact signals nor the precise roles during budding process have been identified, due to the complexity of specifically altering these essential pathways using traditional genetic approaches during the earliest stages of endoderm organogenesis. These traditional techniques include transgenic, knockout or conditional knockouts strategies. To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture. The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis. The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos.

Patients on peritoneal dialysis (PD) are reliant on the peritoneum to provide a semi-permeable barrier to allow for dialysis (solute clearance), salt and water removal (ultrafiltration). PD patients are at risk of developing peritoneal fibrosis and angiogenesis which can lead to a decline in peritoneal membrane function. Specifically, PD patients develop increased solute transport and decreased osmotic conductance leading to ultrafiltration failure. Peritoneal angiogenesis is the leading factor that results in augmented peritoneal membrane solute transport which is associated with worse outcomes – increased risk of mortality and PD technique failure. Transforming growth factor beta (TGFB) is one of the primary cytokines involved in inducing epithelial to mesenchymal transition (EMT) and fibrosis. We hypothesize that PD leads to injury of the epithelial lining of the peritoneum – the mesothelial cells. These cells undergo a transition process and transitioned mesothelium are a source for angiogenic and fibrogenic growth factors. Matrix Metalloproteinase (MMP) 9 is an angiogeneic factor and has been observed to correlate with increased expression of vascular endothelial growth factor (VEGF). MMP9 has the ability to cleave and activate membrane bound factors such as E-cadherin and b-catenin respectively. There is substantial evidence that the canonical WNT/b-catenin pathway is active during fibrosis, and angiogenesis in different biological contexts. Thus, we investigated the role of MMP9 and WNT signaling in peritoneal angiogenesis. Limited evidence exists describing the role of noncanonical WNT signaling but some reports suggest that non-canonical WNT signaling inhibits WNT/b-catenin signaling. Non-canonical WNT5A has differential effects based on receptor context and has been shown to block WNT/b-catenin signaling in the presence of Receptor Tyrosine Kinase Like Orphan Receptor 2 (Ror2). The overall hypothesis of this PhD thesis is that MMP9 and WNT signaling play a key role in inducing peritoneal angiogenesis and are associated with changes in peritoneal membrane function. We expect WNT5A and Ror2 to protect against peritoneal membrane injury. From the overnight effluent of stable PD patients, we cultured mesothelial cells and assayed these for expression of MMP and WNT related genes. MMP9 and WNT1 gene expression were observed to be strongly correlated with peritoneal membrane solute transport in patients on PD. WNT2 mRNA was also positively correlated with peritoneal solute transport. We overexpressed MMP9 in the mouse peritoneum to demonstrate its role in angiogenesis and confirmed these findings using MMP9 -/- mice. In addition to this, we have shown a novel mechanism by which MMP9 induces angiogenesis by E-cadherin cleavage and b-catenin mediated signaling. The observed cross-talk between MMP9 and b-catenin prompted investigation of the activation of canonical WNT/b-catenin signaling in development of peritoneal membrane injury. In an experimental model of TGFB induced pertioneal injury, we confirmed the activation of WNT/b-catenin signaling. In addition to this we, we blocked the WNT pathway and observed that WNT/b-catenin signaling is required to induce peritoneal angiogenesis. WNT5A mRNA was downregulated during TGFB induced injury suggesting a more protective role. Furthermore, several studies have demonstrated its ability to antagonize the WNT/b-catenin signaling pathway. We demonstrated that WNT5A protected against angiogenesis by blocking the canonical WNT pathway. WNT5A is thought to antagonize the WNT/b-catenin signaling pathway by signaling through receptor Ror2. In cell culture, we overexpressed TGFB and blocked Ror2. This resulted in elevated levels of VEGF and fibronectin suggesting that Ror2 is involved in mediating protection. Therefore, Ror2 possesses the ability to regulate VEGF and may be a potential candidate by which WNT5A mediates its protective effects. In conclusion, our findings identified MMP9 and WNT1 as potential biomarkers of increased peritoneal solute transport in patients that are on PD. We have also found a novel mechanism by which MMP9 interacts with b-catenin to induce peritoneal angiogenesis and have provided a first look at WNT/b-catenin signaling in peritoneal angiogenesis. Lastly, we have shown WNT5A to protect against peritoneal angiogenesis. Taken together, our findings are not only significant to the realm of PD research but hold wide applicability to research in the biomedical sciences. / Thesis / Doctor of Philosophy (PhD)

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Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.