Wnt signalling is a key regulator of vertebrate heart development yet the exact requirements of the Wnt signalling components remains unclear. The endogenous Wnt ligand Wnt6 has been identified as a regulator of cardiogenesis required for controlling heart muscle differentiation via canonical Wnt/β-catenin signalling. We show for the first time a requirement for an endogenous Wnt signalling inhibitor for normal heart muscle differentiation. Expression of sfrp1 is strongly induced in differentiating heart muscle. We show that sfrp1 is not only able to promote heart muscle differentiation but is also required for the formation of a normal sized heart muscle in the developing embryo. sfrp1 is functionally able to inhibit Wnt6 signalling and its requirement during heart development relates to relieving the cardiogenesis-restricting function of endogenous wnt6. In turn, we discover that sfrp1 gene expression in the heart is regulated by wnt6 signalling, which for the first time indicates that sfrp genes can function as part of a negative Wnt feedback regulatory loop. Our experiments indicate that sfrp1 controls the size of the differentiating heart muscle primarily by regulating cell fate within the cardiac mesoderm between muscular and non-muscular cell lineages. The cardiac mesoderm is therefore not passively patterned by signals from the surrounding tissue, but regulates its differentiation into muscular and non-muscular tissue using positional information from the surrounding tissue. This regulatory network may ensure that Wnt activation enables expansion and migration of cardiac progenitors, followed by Wnt inhibition permitting cardiomyocyte differentiation.

No description available.

Optimisation of the conventional tripartite of pancreatic cancer (PC) treatment have led to significant improvements in mortality, however further knowledge of the underlying molecular processes is still required. Transcript profiling of mRNA expression of over 44K genes with microarray technology demonstrated upregulation of secreted frizzled related protein 4 (sFRP4) and ??-catenin in PC compared to normal pancreata. Their pathway ??? Wnt signalling is integral to transcriptional regulation and aberrations in these molecules are critical in the development of many human malignancies. Immunohistochemistry protocols were evaluated by two independent blinded examiners for antigen expression differences associated with survival patterns in 140 patients with biopsy verified PC and a subset of 23 normal pancreata with substantial observer agreement (kappa value 0.6-0.8). A retrospective cohort was identified from 6 Sydney hospitals between 1972-2003 and archival formalin fixed tissue was collected together with clinicopathological data. Three manual stepwise regression models were fitted for overall, disease-specific and relapse-free survival to determine the value of significant prognostic variables in risk stratification. The models were fitted in a logical order using a careful strategy with step by step interpretation of the results. Immunohistochemistry demonstrated increased sFRP4 membranous expression (&gt 10%) in 49/95 PC specimens and this correlated with improved overall survival (HR:0.99;95%CI:0.97-6.40;LRchi2=134.75; 1df; ??&lt 0.001). Increased sFRP4 cytoplasmic staining (&gt 2/3) in 46/85 patients increased the disease-specific survival (HR:0.52;95%CI:0.31-0.89;LR test statistic =248.40;1df;??&lt 0.001). Increasing ??-catenin membranous expression (&lt _60%) in 26/116 patients was associated with an increased risk of overall death (HR:3.18;95%CI:1.14-8.89;LR test statistic =4.61;1df,??&lt 0.05). Increasing cytoplasmic expression in 65/114 patients was protective and was associated with prolonged survival on univariate, but not multivariate analysis (Disease specific survival HR:0.75;95%CI:0.56-1.00;logrank chi2=3.91;1df; ??=0.05). Increased nuclear ??-catenin expression in 65/114 patients was associated with prolonged survival (disease-specific HR:0.92;95%CI:0.83-1.02; LR test statistic= 49.72;1df;??&lt 0.001). At the conclusion, 12 patients (8.6%) remained alive, 122 died of their disease (68 males versus 54 females). They were followed for a median of 8.7 months (range 1.0-131.3) months. The median age was 66.5 years (range 34.4-96.0, standard deviation 10.9) years. Pancreatic resection was achieved in 79 patients with 46.8% achieving RO resection. The 30 day post-operative mortality was 2.1%. The overall 1 year survival rate was (33.7% ; 95%CI: 25.78-33.79) with a 5 year survival of (2.87%, 95%CI: 2.83-6.01) and a median survival of (8.90 months; 95%CI: 7.5-10.2). The median disease-specific survival was (9.40; 95%CI: 7.9-10.5 months) and the median time to relapse was 1.2 months (95%CI 1.0-1.2 months). A central tenet of contemporary cancer research is that an understanding of the genetic and molecular abnormalities that accompany the development and progression of cancer is critical to further advances in diagnosis, treatment and eventual prevention. High throughput tissue microarrays were used to study expression of two novel tumour markers in a cohort of pancreatic cancer patients and identified sFRP4 and ??-catenin as potential novel prognostic markers.

Ulm, Univ., Diss., 2008.

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated, and stratified valves. Wnt signaling and its canonical effector β-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/β-catenin signaling by chemically-inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Further, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/β-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/β-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects. This work suggests that Wnt/β-catenin maintains a subpopulation of valve mesenchyme in a less-differentiated, progenitor-like state that allows these cells to respond to mitogens and morphogens. The coordinated interplay of signals with distinct effects on a “progenitor cell” pool is a common logic mechanism for balanced tissue growth and differentiation in many biological contexts. Inspired by epithelial organ homeostasis processes, we identified specific and dynamic expression of the well-established quiescent stem cell marker Lrig1 in the developing valves. Endocardial Lrig1 likely moderates ErbB2 levels and thereby signaling output to prevent excessive EMT and resulting pathologically enlarged valves. Finally, we use Cre-mediated lineage labeling to show that the cusps of the semilunar valves have differential mesenchymal origins and that the localization of said distinct mesenchyme may account for the discretely patterned extracellular matrix of mature valves. This dissertation includes previously published and unpublished coauthored material. / 10000-01-01

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease that can be subdivided into chronic rhinosinusitis sans nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD). Nasal polyps are a hallmark feature of CRSwNP and AERD. Although, the pathophysiology of polypogenesis is thought to be multifactorial, type 2 (allergic-type) immune responses and epithelial remodeling are considered to be central features. In polyp disease, epithelial remodeling and dysfunction leads to decreased epithelial diversity, with increased basal cells and decreased goblet and ciliated cells. A role for Wnt signaling is well established in cancer and fibrotic diseases, and recent studies have indicated that Wnt signaling is also upregulated in sinonasal polyposis, a non-cancerous disease. Wisp1, a downstream mediator of the Wnt pathway, is significantly increased in polyp disease compared to non-polyp disease. Wnt pathway ligands have been reported to induce the proliferation and impair differentiation of airway basal cells, but the direct effect of the downstream Wnt pathway gene, Wisp1, on respiratory basal epithelial cells is unknown. This study aims to investigate the role of Wisp1 on the proliferation, cell migration, and differentiation of human respiratory epithelial cells. In the current study, we found increased expression of WISP1 in nasal polyps examined directly ex vivo. Histological analysis of nasal polyps and WISP1 expression in primary basal epithelial cell cultures suggested epithelial production of Wisp1 as a feature of nasal polyposis. Using submerged and air-liquid interface (ALI) cell cultures, the effect of Wisp1 on proliferation, cell migration and differentiation was investigated. Increased basal cell accumulation and loss of differentiation are hallmarks of sinonasal polyposis. The chronic inflammatory environment present in polyp disease includes chronic increased WISP1 expression, which endorses a role for this protein in disease. The results of this study suggest that prolonged exposure to Wisp1 may lead to an increase in the cellular mass of respiratory basal epithelial cells, while limiting their ability to differentiate, thus allowing for unchecked accumulation. Taken together, our results suggest that Wisp1 may contribute to central features of nasal polyposis, thus identifying this protein as a potential target for future therapeutic intervention.

Wnt signaling regulates critical cellular interactions throughout normal development and directs cell fate decisions of stem cells. Previous work by our lab implicates -catenin as an essential modulator of embryonic stem cell self-renewal and differentiation. Genetic studies in mice have demonstrated broad functional redundancies between the most downstream effectors of the Wnt signaling cascade, the T-cell factor / Lymphoid enhancer factor (TCF/LEF) family of transcription factors. Despite this, loss-of-function experiments suggest that -catenin reinforces the pluripotent state by mediating a TCF switch in which repressive TCF7L1 is replaced with activating TCF7. However, these experiments do not account for potential confounding functional compensation by other TCF/LEF factors. As such, I hypothesized that TCF7 and TCF7L1 are functionally redundant in mouse embryonic stem cells and bind a largely overlapping set of target genes and interacting proteins. In support of this notion, we demonstrated that both TCF7 and TCF7L1 were similarly able to restore the altered transcriptomic profile and differentiation deficits observed in mouse embryonic stem cells (mESCs) lacking all full-length TCF/LEFs. With the expectation that TCF7 and TCF7L1 recruit similar transcriptional co-regulators to a broadly overlapping set of target genes, we employed the unbiased techniques, ChIP-seq and BioID to test our hypothesis. We observed that regardless of the degree of Wnt signaling activity, TCF7L1 was more abundantly associated with chromatin than TCF7, and TCF7 and TCF7L1 regulate distinct target genes. We demonstrated that Wnt stimulation, simulated by GSK-3 inhibition, facilitates TCF7L1 interactions with transcriptional modulators such as the BAF and nuclear receptor co-repressor complexes, despite a reduction in TCF7L1 levels. Taken together, the work in this thesis provides new insights into the mechanisms of Wnt target gene regulation by the TCF/LEF factors. / Thesis / Doctor of Philosophy (PhD) / Stem cells are capable of giving rise to multiple different cell types and thus are able to generate all adult tissues. The identity of a cell is controlled by external signals that regulate internal programs encoded by our genes. The execution of the instructions in genetic programs is conducted by proteins called transcription factors that can turn different genes on or off, giving rise to distinct cell types. The T-Cell Factors and Lymphoid Enhancer Factor (TCF/LEFs) are a family of four transcription factors regulated by external signaling molecules called Wnts. By using the TCF/LEFs, Wnts establish gene outputs that determine the identity of cells throughout embryonic development and in adult tissues. However, the mechanisms used by this family of transcription factors to establish the programs controlling cellular identity remain poorly understood. Using genetically engineered mouse embryonic stem cells, we have uncovered new information about the mechanisms TCF/LEFs use to regulate gene function, identified programs controlled by TCF/LEFs, and discovered potential protein partners that work with TCF/LEFs to implement genetic programs. This thesis provides novel insights into the control of cell identity by the TCF/LEFs, which has implications for the numerous human diseases linked to abnormal Wnt-mediated signaling.

No description available.

No description available.

El pàncrees és una glàndula secretora formada per teixit exocrí i endocrí. El compartiment endocrí està format per les cèl•lules alpha (productores de glucagó), les cèl•lules beta (productores d'insulina), les cèl•lules delta (somatostatina), les cèl•lules PP (polipèptid pancreàtic) i les cèl•lules epsilon (grelina). La Diabetis Mellitus és un grup de malalties metabòliques que es caracteritzen per mantenir nivells elevats de glucosa en sang com a resultat de la incapacitat de produir o utilitzar la insulina. Les cèl•lules productores d'insulina són les cèl•lules beta del pàncrees. Actualment, el tractament de la diabetis es basa en injeccions periòdiques d'insulina. Per solucionar i millorar la vida d'aquests pacients hi ha molts grups que treballen per trobar noves fonts de cèl•lules productores d'insulina que recuperin la massa de cèl•lules beta perdudes durant la diabetis. Per a això, és necessari conèixer detalladament els passos que se succeeixen per generar una cèl•lula productora d'insulina a partir d'una cèl•lula indiferenciada. Durant la organogènesi pancreàtica s'activen un conjunt de factors de transcripció que són essencials per a la correcta formació de l'òrgan, entre ells el factor proendocrí Neurogenina3 (Neurog3), així com també un seguit de senyals extrínsecs (vies de senyalització) que participen en aquest procés. Neurogenina3 és un factor de transcripció de la família bHLH que exerceix un paper essencial en la diferenciació endocrino-pancreàtica, ja que en la seva absència no hi ha formació de les cèl•lules endocrines del pàncrees. En el primer objectiu d'aquesta tesi hem identificat possibles noves dianes de Neurog3, mitjançant l'ús d'un model de diferenciació endocrina in vitro de cèl•lules ductals pancreàtiques, en el qual la sobreexpresió de Neurog3 indueix l'activació del programa transcripcional endocrino-pancreàtic. Mitjançant l'estudi de canvis globals en el perfil d'expressió gènica d'aquestes cèl•lules hem identificat un conjunt de gens relacionats amb la via de senyalització Wingless (Wnt) com a dianes potencials de Neurog3 in vitro. Entre aquests gens, hem centrat els nostres estudis en l'anàlisi del gen que codifica pel lligand de la via Wnt, Wnt9a. El segon objectiu d'aquesta tesi se centra a estudiar la possible participació del lligand Wnt9a en el procés de diferenciació endocrina del pàncrees. Així, vam demostrar per primera vegada la presència en pàncrees embrionari de ratolí del mRNA de Wnt9a, així com la regulació gènica d'aquest lligand per part de factors de transcripció que participen al programa de diferenciació endocrina. Estudiem també el paper de Wnt9a dins de la cascada endocrino-pancreàtica, demostrant un paper regulador de Wnt9a sobre els efectes promoguts per Neurog3 en alguns dels gens endocrins estudiats. Aquests resultats ens han portat al tercer objectiu d'aquesta tesi: caracterització de la diferenciació endocrino-pancreàtica en el model murí gen-anul•lat per Wnt9a. A estadi e18.5 (just abans del naixement) observem un augment generalitzat de les cèl•lules productores d'hormones del compartiment endocrí (cèl•lules β insulina-positives, cèl•lules α glucagó-positives i cèl•lules δ somatostatina-positives) en relació a l'àrea pancreàtica total, que correlaciona amb una major taxa de proliferació d'aquestes. L'anàlisi de l'expressió gènica realitzat a estadi e15.5 (moment de màxima expansió endocrina) no mostra diferències en els nivells del mRNA de Neurog3, indicant que l'augment en el compartiment endocrí observat a e18.5 no es deu a una major especificació endocrina. No obstant això, l'estudi de diferents factors integrants del programa transcripcional endocrí mostra un augment en l'expressió de Pdx1 en els animals deficients que podria explicar l'augment en cèl•lules beta observat a e18.5. Per tant, en aquesta tesi definim per primera vegada la relació directa entre factors de transcripció bHLH i components de la via Wnt en pàncrees, així com identifiquem la presència del mRNA de Wnt9a en pàncrees embrionari de ratolí i en illots adults. Demostrem la regulació gènica de Wnt9a per part de factors de la cascada de transcripció endocrina, així com la regulació d'aquests per part de Wnt9a sota l'acció de Neurog3. Identifiquem, mitjançant l'estudi del model animal gen anul•lat per Wnt9a, un augment del compartiment endocrí abans del naixement, a causa d'una major taxa de proliferació, en absència de Wnt9a. Per tant, el conjunt d'aquests resultats indicaria que la via de senyalització Wnt juga un paper important durant el procés de diferenciació endocrina del pàncrees, suggerint una connexió entre el programa transcripcional endocrí i la via de senyalització intracel•lular Wnt. / The pancreas is a gland consisting of secretory exocrine and endocrine tissue. The endocrine compartment is formed by the alpha cells (glucagon producing), the beta cells (insulin producing), delta cells (somatostatin), PP (pancreatic polypeptide) cells and epsilon cells (ghrelin). Diabetes Mellitus is a group of metabolic diseases characterized by maintaining high levels of blood glucose resulting from the inability to produce or use insulin. Currently, the treatment for diabetes is based on regular injections of insulin. There are many groups working on finding new sources of insulin-producing cells to recover beta cells mass lost during diabetes development. For this reason, it’s necessary to detail the molecular steps that occur from an undifferentiated cell to an insulin-producing cell. During pancreatic organogenesis, a set of transcription factors that are essential for proper formation of the organ are activated, including the proendocrine factor Neurogenin3 (Neurog3), as well as a number of extrinsic signals (signalling pathways). Neurogenin3 is a transcription factor that belongs to bHLH transcription factors family. It plays an essential role in pancreatic-endocrine differentiation, since in its absence there is no formation of endocrine cells. In the first objective of this thesis we have identified potential new targets for Neurog3, using an in vitro model of endocrine differentiation process, pancreatic ductal cells (mPAC), in which adenoviral overexpression of Neurog3 induces the activation of the transcriptional differentiation program. Using whole genomic profile study, we identified a set of genes related to signalling Wingless (Wnt) pathway as potential targets of Neurog3 in vitro. Among these genes, we focused our studies on the analysis of the gene encoding the ligand of the Wnt pathway, Wingless- type MMTV integration site 9A (Wnt9a). The second objective of this thesis focuses on the study of the possible role of Wnt9a during endocrine differentiation. Thus, we demonstrate for the first time, the presence of Wnt9a mRNA in mouse embryonic pancreas, as well as its regulation by transcription factors involved in endocrine differentiation program. We studied the role of Wnt9a within the endocrine differentiation cascade, demonstrating a regulatory role of Wnt9a on some Neurog3 activated genes. Finally, we did the characterization of the endocrine differentiation process in the Wnt9a knock out animal mouse model. At embryonic stage (e) 18.5 (just before birth), we observed a general increase in the major hormone-producing cells of the endocrine compartment (β cell insulin-positive, α cells glucagon-positive cells and δ somatostatin- positive) in relation to the total pancreatic area, which correlated with a high rate of proliferation. The analysis of gene expression performed at (e) 15.5 (time of maximum endocrine expansion) shows no difference in levels of Neurog3 mRNA, indicating that the increase in the endocrine compartment observed at (e) 18.5 is not due to a greater endocrine specification. However, the study of transcriptional endocrine factors shows an increase in the expression of Pdx1 gene in the deficient Wnt9a animals; this could explain the increase in beta cells observed at (e) 18.5. Therefore, this thesis define for the first time the direct relationship between bHLH transcription factors and components of the Wnt pathway in the pancreas, as well as the identification of Wnt9a mRNA in embryonic mouse pancreas and in adult islets. We demonstrate the regulation of Wnt9a by transcription factors of the endocrine cascade and the regulation of some of them by Wnt9a. We have identified an increase of the endocrine compartment, just before birth, in Wnt9a deficient animals, probably due to a higher rate of proliferation in the absence of Wnt9a. Hence, all these results indicate that Wnt signalling pathway plays an important role during pancreatic endocrine differentiation, suggesting a connection between the endocrine transcriptional program and Wnt signalling.