Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Induction of vertebrate heart development requires inhibition of canonical/<i>β</i>-catenin dependent Wnt signalling, activation of non-canonical/<i>β</i>-catenin independent Wnt signalling and transcription factor activation. Wnt/<i>β</i>-catenin signalling may also have a later regulatory role in cardiogenesis. The recent discovery of Wnt6 expression next to and within the developing heart during the relevant stages of cardiomyogenesis, combined with knockdown and over-expression data suggests that Wnt6 may have a role in the regulation of this process. Inhibition of canonical signalling leads to increased expression of cardiac associated transcription factors such as members of the Nkx2 and GATA family. These families are expressed in overlapping regions which specify the early heart field prior to the expression of the later cardiomyocyte-specific genes. This study demonstrates the ability of <i>β</i>-catenin to inhibit cardiogenesis during later developmental stages, before the cardiac mesoderm begins to differentiate into myocardium (heart muscle) and that the newly discovered Wnt6 exerts inhibition of cardiogenesis in a <i>β-</i>catenin<i> </i>dependent manner. This inhibition of cardiogenesis by <i>β-</i>catenin can occur in a cell-autonomous manner and is a result of direct inhibition of cardiac transcription factors of the GATA family. Over-expression of these pro-cardiogenic transcription factors GATA4 and GATA6 can restore the cardiomyogenic differentiation programme in embryos where it has previously been inhibited by <i>β</i>-catenin. In conclusion GATA factors are the relevant targets of Wnt/<i>β-</i>catenin signalling in the inhibition of normal cardiac development. The subsequent loss of cardiac gene expression observed is therefore a result of insufficient GATA expression and function.

No description available.

In vertebrates, the urogenital sinus and the hindgut are connected at a hollow region called cloaca. A midline mesenchymal structure known as urorectal septum (urs) descends from the ventral body wall to separate the urogenital sinus from the hindgut before the formation of an anal opening. Subsequent cloaca membrane regression at the ventral midline of the genital tubercle (GT) is crucial for the formation of an anal opening. These two events are important during cloaca septation in urorectal development. Mice with defective Shh or Wnt signaling displayed similar urorectal defects such as GT agenesis, un-partitioned cloaca (persistent cloaca) and proximal urethral opening that are attributable to increased cell apoptosis. Furthermore, Shh and Wnt signal transduction coordinate with each other and regulate cell survival of the developing urorectum. However, the molecular mechanisms by which these two signaling pathways coordinate in urorectal development remain unclear. We previously identified Wnt inhibitory factor1 (Wif1) from Affymetrix array analysis for genes/pathways that is implicated in urorectal development. Wif1 is a secreted protein that binds directly to Wnt ligands preventing Wnts from binding to receptors. This leads to -catenin degradation and thereby inhibits their activities. It is known that Wif1 binds to Wnt3a and Wnt5a with high affinity and deletion of Wnt3a, Wnt5a and -catenin in mice caused GT agenesis, persistent cloaca and proximal hypospadias. Using ETU-induced anorectal malformations model, I found out that Wif1 is ectopically expressed in the un-tubularized and un-septated urorectum. Wif1 is mainly expressed at the fusing endoderm that associates with programmed cell death during cloaca septation. Exogenous addition of Wif1 protein in urorectum culture also caused cloaca membrane disintegration, and proximal urethral opening that may be due to aberrant apoptosis. Shh and Wif1 are differentially expressed at the cloaca endoderm. In normal mice, Shh is highly expressed at the cloaca endoderm except those Wif1-expressing endodermal cells. Blockage of Shh pathway by cyclopamine in urorectum culture induced ectopic expression of Wif1, concomitant with genital tubercle hypoplasia and un-septated cloaca. More importantly, deletion of Shh in mice hastened Wif1 expression at the cloaca membrane endoderm and elicited increased cell death in the Wif1 expressing endoderm. Wif1-/- embryos display urorectal defects including delayed genital outgrowth and proximal hypospadias. Therefore, disruption of spatiotemporal expression of Wif1 could lead to defective Wnt signaling and contributes to abnormal urorectal development in Shh-/- mutant. Current study revealed that Wif1 is involved in urorectal development and is implicated in urorectal defects. It may function as a pro-apoptotic factor to regulate endodermal cell death which is essential for the septation process. Its specific expression is restricted at the midline cloaca endoderm by Shh signaling to inhibit local Wnt--catenin activities during cloaca septation. I proposed novel hypothetical models to explain (1) the significance of the tempo-spatial expression of Wif1; (2) the significance of cell death; and (3) the molecular mechanism that Shh signaling regulates Wnt signaling activities through Wif1 in urorectal development. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

In mammals, the external genitalia, urinary tract and anorectal tract are developed from a common embryonic primordium, the urorectum. Cloaca is the hollow space inside the urorectum that connects the hindgut and the urogenital sinus. During the urorectal development, the external genitalia is formed from the outgrowth of genital tubercle (GT) protruding from the urorectum, while the future urinary tract and anorectal tract are formed by the partition of cloaca during cloacal septation. GT outgrowth and cloacal septation are important developmental events for the formation of genitourinary and anorectal system. In human, dysregulation of these developmental events results in congenital anorectal malformations (ARM). Wnt signaling is one of the key signaling pathways that regulates urorectal development. The activity of Wnt signaling is initiated by the binding of Wnt ligands to cell surface receptors, which can be antagonized by secretory Wnt inhibitors. Dickkopf1 (Dkk1) and Wnt inhibitory factor 1 (Wif1) are secretory Wnt inhibitors implicated in urorectal development. However, the functions of other secretory Wnt inhibitors during urorectal developments remain to be elucidated. In this study, expression analyses showed that Dkk1, Dickkopf2 (Dkk2), Dickkopf4 (Dkk4), Secreted Frizzled-related Protein 1 (Sfrp1) and Wif1 were expressed in the developing urorectum. The dynamic, overlapping and restricted expression patterns of these Wnt inhibitors were closely associated with the GT outgrowth and the cloacal septation events, implying that these Wnt inhibitors functioned in a coordinated manner in defining the field of Wnt signaling activities in the developing urorectum. Wif1 knockout mice (〖Wif1〗^(-/-)) was used as the model to investigate the functions of and the interplay between secretory Wnt inhibitors in urorectal development. GT outgrowth and cloacal septation defects were observed in 〖Wif1〗^(-/-) embryos. Most of the 〖Wif1〗^(-/-) embryos displayed varying degrees of GT outgrowth defects, while septation defects were only occasionally observed. This suggested that GT outgrowth and cloacal septation were regulated by Wif1 via different regulatory mechanisms. In the urorectum of 〖Wif1〗^(-/-) embryos, Dkk1 was significantly upregulated in the peri-cloacal mesenchyme. Further expression analysis suggested that Dkk1 was sufficient to rescue cloacal septation defects but not GT outgrowth defects in 〖Wif1〗^(-/-)embryos. In the 〖Wif1〗^(-/-) embryos with severe GT outgrowth defects, the Fgf8-expressing distal urethral epithelium, the signaling center in the urorectum, was absent, suggesting that the GT outgrowth defects could be contributed by the loss of dUE-expressing signals such as Fgf8. This study demonstrated the importance of secretory Wnt inhibitors in the GT outgrowth and cloacal septation and suggested that secretory Wnt inhibitors played partially overlapping roles in urorectal development. A rescue mechanism for cloacal septation performed by Dkk1 upon Wif1 deletion was proposed. Such auto-regulatory mechanism within the Wnt signaling pathway indicated that Wnt inhibitors play essential regulatory roles in the urorectal development and a balanced Wnt signaling activity modulated by Wnt inhibitors is crucial to the development of urorectum. / published_or_final_version / Surgery / Master / Master of Philosophy

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr Virus (EBV).Early diagnosis of NPC will improve the overall survival. However, traditional EBV markers do not perform well in high-risk individuals or for early detection of NPC. Aberrant promoter hypermethylation of tumor suppressor genes (TSGs) is an important epigenetic change in early tumorigenesis. This study identified a promising panel of methylation markers for early detection of NPC and assessed the clinical usefulness of these markers using nasopharyngeal (NP) brushing and blood specimens. Methylation-sensitive high resolution melting (MS-HRM) assays were carried out to assess the methylation status of a selected panel of four TSGs (RASSF1A, WIF1, DAPK1, RAR2)in biopsies, NP brushings and cell-free plasma from NPC patients. NP brushing and blood samples from high-risk and cancer-free groups were used as controls. The DNA methylation panel showed higher sensitivity and specificity than the EBV DNA markerincell-free plasma for early stage (Iand II) NPC (sensitivity: 64.6% vs. 51.2% and specificity: 96.0% vs. 88.0%, respectively). In combination with plasma EBV DNA, testing for DNA methylation in plasma and NP brushings using the four-gene MS-HRM test significantly increased the detection rate for all stages of NPC(94.1% for stages I-II, 98.4% for stages III-IV) as well as recurrence(93.5%). Aberrant activation of the Wnt signaling pathway is a common mechanism for cell transformation and tumor development in a variety of human cancers. A high frequency of promoter hypermethylation of WIF1was observed in NPC cell lines (100%), primary tumor biopsies(89.7%), NP brushings (80.2%), and cell-free plasma (51.8%),with no significant correlation with NPC stage. Simultaneously, expression of WIF1 was completely silenced in NPC cell lines (HONE1, HK1, HNE1, SUNE1, CNE1, CNE2, and C666),but not in immortalized NP epithelial cells (NP460 and NP69). These together suggested an important role of WIF1 in NPC development. In vitro and in vivo functional assays revealed a tumor suppressive role of WIF1in NPC. Restoration of WIF1expression in NPC cells significantly suppressed anchorage-independent growth, in vivo tumorigenicity, invasion, migration, and angiogenesis of NPC cells. A number of important angiogenesis-related genes were down-regulated by WIF1expression, including IL6,IL8,VEGF165,VEGFA, PDGFB, and MCP1. There is inhibition of the Wnt/β-catenin signaling pathway, manifested as decreased β-catenin expression and TCF/LEF Wnt promoter activity. These data indicated the important regulatory role of Wnt signaling pathway in NPC tumorigenicity, invasion, migration, and angiogenesis, by interacting with the complex signaling network in NPC cells. To conclude, the MS-HRM assay on the selected gene panel in combination with the EBV DNA test, increases the sensitivity for NPC detection at an early stage and detection of recurrence and has great potential to become a non-invasive test for early diagnosis and disease monitoring after treatment. Collectively, results from this study reveal that WIF1is not only a sensitive biomarker, but also a tumor suppressor gene in NPC. Understanding the molecular regulatory role ofWIF1in NPC will facilitate the diagnosis of NPC, and development of novel NPC therapeutic strategy. / published_or_final_version / Clinical Oncology / Doctoral / Doctor of Philosophy

Satellite cells are muscle stem cells that are responsible for the growth and repair of skeletal muscle tissue. Satellite cells typically exist in a quiescent state in their niche between the sarcolemma and basal lamina. In response to muscle tissue injury, activated satellite cells, otherwise known as myoblasts, migrate to the site of injury where they proliferate and subsequently differentiate and fuse to repair damaged myofibers. The success of muscle growth and repair is highly dependent on the speed and degree to which these myoblasts migrate, proliferate and differentiate. This overall process, referred to as myogenesis, is largely controlled by the myogenic regulatory factors, a group of basic helixloop- helix transcription factors including MyoD, Myf5, myogenin and Mrf4. It has recently been found that the Wnt family of secreted signalling proteins are highly involved in the regulation of developmental processes such as myogenesis. Wnt proteins are a family of 21 highly-conserved, secreted, cysteine-rich signalling molecules which are found in all multi-cellular organisms. Wnt signalling is highly versatile and is initiated by the binding of extracellular Wnt to cell-surface Frizzled receptors (Fz). It is highly dependent on both the Wnt isoform and Fz type and may initiate one of three known signalling pathways. Wnt3A and Wnt7A are of particular interest as they have previously been linked with myogenesis. C2C12 myoblasts over-expressing Wnt3A have been seen to have reduced levels of motility and terminal differentiation. Wnt7A is suspected to maintain a healthy satellite cell pool by regulating self-renewal; injection of recombinant Wnt7A into mouse leg muscle resulted in increased satellite cell numbers. In vitro Wnt studies have typically involved the treatment of mouse cells with conditioned medium containing Wnt, often at unknown concentrations. In our study we wished to test the effects of known concentrations of recombinant Wnt3A and Wnt7A on mouse C2C12 and donor-derived human skeletal muscle myoblasts (HSkM) in vitro. Wnt3A and Wnt7A were seen to increase the rate of C2C12 migration in a dose dependent manner. HSkM cells treated with 10 ng/ml Wnt3A also displayed increased motility. Neither Wnt3A nor Wnt7A were seen to have any significant effects on the proliferation of C2C12 or HSkM cells. Wnt3A (10ng/ml and 100 ng/ml) but not Wnt7A was seen to decrease C2C12 terminal differentiation as measured by expression of myosin heavy chain (MyHC). Subsequent confocal microscopy revealed that Wnt3A significantly reduced the percentage of MyoD+ C2C12 nuclei during differentiation. A reduction in nuclear MyoD would support the observed impaired commitment to differentiation. However, donor-derived human skeletal muscle myoblasts treated with 10 ng/ml Wnt3A were not seen to have significantly reduced nuclear MyoD levels or terminal differentiation; the reason for this is unclear but may relate to a number of factors including the concentration of Wnt, Fz and co-receptor profiles and the presence of specific extracellular matrix and serum factors. These studies provide new insight into the role of Wnts in myogenesis and lay the foundation for future work on Wnt3A and Wnt7A. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2014.

Satellite cells are known as the definitive muscle stem cells and are responsible for skeletal muscle maintenance and repair. The capacity of these satellite cells to participate in myogenesis decreases with age and as a result, muscle repair and maintenance in an aging organism is characterized by fibrosis, lipid accumulation and atrophy, a process known as sarcopenia. Recent parabiotic studies have shown that satellite cells with reduced myogenic capability in aging muscle can be rejuvenated to undergo effective myogenesis when exposed to a young environment. Further analysis has suggested that the Wnt family of signaling proteins identified in serum is pivotal in regulating cell fate, proliferation and differentiation, during aging. Wnt3a is known to regulate fibrogenensis, Wnt10b adipogenesis and Wnt7 myogenesis. In the current study, we aim to determine the cytosolic and secreted expression profiles of the three Wnt isoforms, Wnt3a, 7 and 10b, during myogenesis of early and late passage C2C12 myoblasts. We then extend our analysis to determine whether conditioned media could improve the myogenic capacity of late passage cells. Late passage C2C12 cells had elevated Wnt3a cytosolic levels along with reduced differentiation capacity and a rapidly declining Wnt7 levels, in comparison to early passage cells. The elevated Wnt3a suggests an elevated fibrogenic predisposition, whereas the declining Wnt7 cytosolic levels, a decrease in myogenic capacity. Furthermore, analysis of the secreted vs. cytosolic ratio in Wnt7 levels revealed a more rapid decline in late vs. early passage cells during differentiation, supporting the observed decreased myogenic ability. Moreover, late passage cells also showed lower Wnt10b levels compared to early passage cells. This low level of Wnt10b is likely associated with an increase in adipogenic predisposition. The results obtained in the cross-over experiments indicated that conditioned media from early passage cells did not improve the differentiation of late passage cells by the low levels of Myogenin and MHC. However, early passage cells treated with conditioned media from late passage cells surprisingly showed a marginal increase in both Myogenin and MHC levels. Interestingly, cytosolic Wnt3a and 7 in late passage cells treated with ‘young media’ were increased compared to control whereas early passage cells treated with ‘old’ media showed significantly decreased levels of Wnt3a and 7. Furthermore, early passage cells acquired a declining expression when treated with ‘young’ media whereas late passage cells had an increasing level when treated with ‘old’ media. This indicates a possible improvement in differentiation in late passage cells. Taken together, our results support a role for Wnt7 and Wnt10b in promoting myogenesis while Wnt3a may decrease myogenesis. With the increase in passage numbers, the reduced myogenic predisposition is regulated by reduced Wnt10b, 7 and elevated Wnt3a levels, respectively. Moreover, we speculate that the lack of myogenic improvement in the cross-over experiment could be the presence of unknown secreted factors in ‘young’ media that impedes myogenesis. Finally, cell lines are known to be biologically different to primary myoblasts through the accumulation of mutations which could render the cells less sensitive to growth factors. Therefore, it is imperative that the current study is repeated with primary culture myoblasts. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Signaling molecules play essential roles in communication between cells. Wnt signaling molecules are critical for embryonic development of several organisms. I examined the involvement of Wnt signaling during two major developmental processes, namely embryo implantation and formation of the embryonic body axes. Using RT-PCR analysis, I showed that multiple Wnt genes are expressed in the blastocyst at the time of implantation. Moreover, expression of Wnt 11 requires both estrogen produced by the mother and the uterine environment. Using a transgenic approach, I showed that beta-catenin-regulated transcriptional activity, which is a major transducer of Wnt signaling, is activated in the uterus specifically at the site of implantation in an embryo-dependent manner. These results introduce Wnts as candidate signaling factors that may mediate the communication between the embryo and uterus that initiates implantation. / Wnt/beta-catenin signaling triggers axis formation in Xenopus and zebrafish embryos. I showed that, during embryonic development, beta-catenin-regulated transcriptional activity is first detected in the prospective primitive streak region prior to gastrulation. This demarcates the posterior region of the embryo. This activity then becomes restricted to the elongating primitive streak and to the node. In Xenopus embryos, beta-catenin participates in the formation of the organizer through the activation of the homeodomain transcription factors Siamois and Twin. I obtained evidence that a Siamois/Twin-like binding activity exists in mouse embryos and is localized in the node. These results strongly suggest that, as the case in Xenopus and zebrafish, the Wnt/beta-catenin pathway is involved in establishing embryonic body axes. / Furthermore, using the transgenic mouse line that I generated for these studies, I mapped the transcriptional activity of beta-catenin during mouse embryonic development. These results revealed when and where this activity, and presumably Wnt signaling, is active during the development of several organs and embryonic structures.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 133-173).