Upon sperm entry, the vertebrate egg undergoes a series of cell divisions that create a number of smaller cells without increasing the embryonic mass. This induces an elevation of intracellular calcium transient that is conserved across species. In zebrafish, fertilization occurs through an opening in the chorion, the micropyle and in Xenopus it can occur anywhere in the animal hemisphere. Wnt signaling activation is required during dorsal-ventral axis specification and it needs to be suppressed during the regionalization of the brain. Axin is a negative regulator of Wnt signaling and contains an RGS (Regulator of G Protein Signaling) domain. RGS domains are typical of RGS proteins, which are involved in a distinct signaling pathway, G-protein signaling. RGS proteins exert a negative effect of G-protein signaling by accelerating the GTPase activity (GAP) of the Gα subunit, thus turning off the signaling. Axin contains an RGS domain, however, it is not clear whether Axin is directly involved in G-protein signaling. We will also present a work performed using another negative regulator of the Wnt signaling network called naked cuticle (Nkd). Nkd has been shown to modulate β-catenin dependent and independent Wnt signaling. In chapter 2, we will show that the Axin-RGS like function is dispensable during the formation of the dorsal-ventral axis. We manipulated this protein by creating a point mutation in a critical aminoacid within the Axin-RGS domain, known to be detrimental for the GAP function of RGS proteins, Axin1Q162A. Maternal depletion of Axin1 in Xenopus oocytes causes hyperactivation of Wnt signaling and results in dorsalization. Axin1Q162A is able to suppress the dorsalization of maternally depleted embryo and restore normal dorsa-ventral axis formation. In chapter 3, we will describe the role of Axin during the patterning of the vertebrate brain. We show that the point mutant is not able to restore normal brain development in zebrafish embryos after Axin knockdown. We hypothesize that Axin-RGS like function is necessary during the patterning of the vertebrate brain that occurs after zygotic transcription has been initiated. Moreover, we show that Axin-RGS like activity may be dispensable during this stage of development. Finally, we demonstrate that Axin1Q162A localization differs from the wildtype Axin1 and Axin1 but not Axin1Q162A is localized to the plasma membrane upon Gα overexpression in zebrafish embryos. Embryonic organ laterality is preceded by molecular and physiological asymmetries. In chapter 4 we describe the role of another Wnt antagonis, Nkd cuticle, during left-right patterning. Prior to organogenesis, a group of cells called Dorsal Forerunner Cells, (DFCs), migrate ahead of the dorsal blastoderm during gastrulation to form the Kupffer's vesicle (KV). This vesicle will trigger a signaling cascade that will culminate with left-right determination. We show data that support the requirement of Nkd in organ laterality and convergence and extension movements using zebrafish and Xenopus laevis.

Wnt/β-catenin signaling and asymmetric cell division are essential to development and homeostasis in metazoans; these two mechanisms join into one in the Wnt/β-catenin Asymmetry (WβA) pathway in the nematode C. elegans. In WβA, nuclear asymmetry of two β-catenins, SYS-1 and WRM-1, is achieved by two parallel pathways that reduce SYS-1 and WRM-1 levels in the anterior daughter and increase their levels in the posterior daughter. While it is known that many conserved regulators of Wnt signaling are involved in WβA, how these components interact to achieve SYS-1 and WRM-1 asymmetry is not well understood. In this thesis, genetics, transgenics, and live-imaging are used to demonstrate how WβA regulates it’s multiple outputs. It is shown that APR-1/APC and PRY-1/Axin control asymmetric localization of both SYS-1 and WRM-1, and that Wnt signaling explicitly controls APR-1 regulation of either β-catenin via the kinase KIN-19/CKIα. Additionally, it is demonstrated that the Dishevelled proteins DSH-2 and MIG-5 are positive regulators of SYS-1, but negative regulators of WRM-1. Additionally, data from a screen designed to identify novel kinase regulators of Wnt signaling/asymmetric cell division is presented. Overall, this thesis takes current knowledge of conserved Wnt signaling component function and provides a compelling model of how those components are adapted to asymmetric cell division.

The Wnt pathway is a fundamental signalling pathway conserved in all animals, regulating growth, differentiation, embryonic development, and tissue homeostasis in adults. Wnt signalling is kept quiescent by ubiquitin-mediated degradation of the transcription factor β-catenin, orchestrated by a group of proteins called the Destruction Complex. Aberrant Destruction Complex activity is a common theme in many cancers, and is the primary cause of colon cancer. Through mass spectrometry analysis of Axin protein complexes (a key Destruction Complex component) we identified the deubiquitinating enzyme USP34 as an Axin-interacting protein. Functional studies showed USP34 functions to positively regulate Wnt signalling, acting downstream of β-catenin stabilization. While characterizing USP34 we also discovered a new positive regulatory role for Axin in promoting signalling that is dependent on its nuclear localization. Our results suggest that USP34 stabilizes the nuclear pool of Axin through regulating its ubiquitination and offers a potential strategy to target pathological Wnt signalling.

The Wnt pathway is a fundamental signalling pathway conserved in all animals, regulating growth, differentiation, embryonic development, and tissue homeostasis in adults. Wnt signalling is kept quiescent by ubiquitin-mediated degradation of the transcription factor β-catenin, orchestrated by a group of proteins called the Destruction Complex. Aberrant Destruction Complex activity is a common theme in many cancers, and is the primary cause of colon cancer. Through mass spectrometry analysis of Axin protein complexes (a key Destruction Complex component) we identified the deubiquitinating enzyme USP34 as an Axin-interacting protein. Functional studies showed USP34 functions to positively regulate Wnt signalling, acting downstream of β-catenin stabilization. While characterizing USP34 we also discovered a new positive regulatory role for Axin in promoting signalling that is dependent on its nuclear localization. Our results suggest that USP34 stabilizes the nuclear pool of Axin through regulating its ubiquitination and offers a potential strategy to target pathological Wnt signalling.

ABSTRACT Chibby (or PIGEA-14) is a novel antagonist of the Beta-catenin pathway in nucleus. However, the tumor-suppressing function of Chibby and the importance of nuclear targeting to the cellular functions of Chibby have not been validated. By fusion of Chibby cDNA with green fluorescent protein (GFP) or Flag-tag, it was found that exogenous Chibby expression was detected in the nucleus as well as cytoplasm of transfected HeLa cells, but with a preferential nuclear localization (more than 50% cells with nuclear Chibby expression). Chibby overexpression significantly abrogated the cellular Beta¡Vcatenin activities and induced apoptosis in HeLa cells. Moreover, Chibby gene delivery attenuated the proliferation, migration, and anchorage-independent growth of HeLa cells, supporting the tumor suppressor function of Chibby. Mutation or deletion of the predicted nuclear localization sequence (NLS), at residues 123-126, significantly promoted the cytoplasmic localization of Chibby, indicating residues 123-126 is the NLS domain of Chibby. Interestingly, ecotopic expression of Chibby NLS mutants remained capable of inducing apoptosis and inhibiting Beta¡Vcatenin activities in HeLa cells. Besides, overexpression Chibby NLS mutants effectively attenuated the viability, motility and colonies formation of HeLa cells. Expression analysis revealed that Chibby NLS mutants retained Beta-catenin in the cytoplasm and prevented its nuclear entry, thereby inhibiting the Beta-catenin transcriptional activities. In summary, Chibby shuttles between nucleus and cytoplasm, and possesses the functions of tumor suppressor and Beta-catenin antagonist.

My Ph.D. research project in the Bhagwati Gupta lab focuses on understanding the mechanism by which the Axin family of scaffolding proteins functions to regulate biological processes in multicellular eukaryotes. Towards this, I am using the nematode (worm) Caenorhabditis elegans as an animal model to investigate the role of one of the Axin homologs, PRY-1. Studies in various model systems and humans have shown that the Axin family of proteins plays crucial roles during cell proliferation, cell differentiation, and organ formation. Such a role of Axin depends on the negative regulation of the WNT signaling cascade. Consistent with these, alterations in Axin function are associated with developmental abnormalities and age-associated diseases such as axis duplication, neuroectodermal defect, and muscle degeneration. As a scaffolding protein, Axin family members bind to and recruit multiple protein partners that are both WNT dependent and independent. However, how Axin interacts with these factors to regulate molecular events is not well understood. While some Axin-interacting factors have been identified, many more remain to be discovered. My project deals with the identification and functional characterization of pry-1/Axin interactors in C. elegans. The key findings of my Ph.D. research are published in five peer-reviewed papers. Collectively, the results demonstrate that PRY-1 is necessary to regulate lipid metabolism, stress response, muscle health, and aging. I have shown that PRY-1 utilizes multiple pathways to control these diverse processes. Specifically, PRY-1 functions via the SREBP transcription factor homolog SBP-1 to regulate yolk lipoprotein expression to promote lipid synthesis. The analysis of pry-1’s role in aging and muscle health has revealed its interactions with the energy sensor AMPK homolog AAK-2, thereby affecting the function of the Insulin/IGF1 signaling (IIS) transcriptional regulator DAF-16/FOXO. Moreover, I have identified several mRNA genes and microRNAs that function downstream of PRY-1/Axin signaling to either suppress or enhance pry-1 mutant defects. All these novel interactors have mammalian homologs. Altogether, these findings form the basis to pursue future work to investigate the conserved mechanism of Axin signaling and hold the potential for effective intervention to delay aging and age-associated muscle deterioration. / Thesis / Doctor of Philosophy (PhD)

Evolutionary variations during vulval development in C. elegans and its related nematode species are well analyzed. The formation of C. elegans vulva involves many complex cell-cell interactions that are mediated through well conserved EGF/EGFR/RAS, LIN12/Notch and WNT signaling pathways. These pathways specify distinct cell fates of the six epidermal vulval precursor cells (VPCs), P(3-8).p. pry-1/Axin in C. elegans is identified as a part of destruction box complex that mediates (beta)-catenin degradation and is known to negatively regulate canonical Wnt signaling pathway during its development. I focused on the genetic analysis of pry-1 I Axin function in C. briggsae, sister species to C. elegans, to study inter-species comparisons of vulva formation. Three alleles, lin(sy5353), lin(sy5411) and lin(sy5270) were genetically mapped to LG I using standard genetic and in del mapping techniques. Interestingly, a unique simultaneous Multi vulva and Vulvaless (Muv-Vul) phenotype was observed during vulva formation in Cbr-pry-1 alleles, resulting from the varying induction potentials of the VPCs along the A/P axis, compared to Cel-pry-1 animals. In order to analyze these phenotypic differences between Cel-pry-1 and Cbr-pry-1 in greater detail, I dissected vulval development in sy5353 animals. VPC competence analysis was done through cell lineages and ablations studies, while the C. briggsae vulval cell fate markers were used for cell fate specification analysis. Cell ablations revealed that P7.p and P8.p in Cbr-pry-1 animals exhibited non-competence towards anchor cell signaling. Additionally, gonad-independent inductions was observed in P(3-8).p cells and they adopted 2° cell fate specifications. Using RNAi approach, Cbr-pry-1 interactions with other vulval pathway genes were dissected and it was observed that Cbr-lin-12 is involved in VPC competence of P7.p and Cbr-pop-1 exhibited different regulatory levels during vulval development compared to C. elegans. Thus, it can be inferred that the mechanisms of vulva formation in C. briggsae has evolved through changes in the competence of VPCs. / Thesis / Master of Science (MS)

Doctor of Philosophy / Department of Biology / Susan J. Brown / How animals polarize and establish the main axis during embryogenesis has been one of the most attractive questions in Biology. Increasing body of work in various model organisms implicates that most metazoans utilize the canonical Wnt signaling pathway to pattern the anterior-posterior (AP) axis, despite the limited evidence from arthropods. In Drosophila, a highly derived insect, canonical Wnt activity is not required for global AP patterning, but in typical insects including Tribolium castaneum, loss of canonical Wnt activity results in posterior truncation. To determine the eff ects of increased canonical Wnt levels, I analyzed the function of axin, encoding a highly conserved negative regulator of the pathway. Tc-axin transcripts are maternally localized to the anterior pole in freshly laid eggs. Parental RNAi for Tc-axin produced progeny phenotypes that ranged from mildly a ffected embryos with cuticles displaying a graded loss of anterior structures, to severely a ffected embryos lacking cuticles and condensing to the posterior pole of the egg without any de finable structures. Altered expression patterns of several blastodermal markers indicated anterior expansion of posterior fates. Epistasis analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the eff ects of Tc-axin depletion are mediated through this pathway and that canonical Wnt activity must be repressed for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt activity must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt signaling in de fining AP polarity and patterning in metazoan development. Additionally, as an anterior structure, the extraembryonic serosa is reduced in Tc-axin RNAi progeny. However, in Tc-pangolin (Tc-pan, a homolog of Wnt downstream component) RNAi progeny, an interesting phenotype was produced that serosa was not only reduced but also separated into distinct anterior and dorsal domains. I carefully recorded this phenomenon with live imaging using a Tribolium transgenic line that expresses GFP in each nucleus. Through careful examination with embryonic fate-map markers, I found that the tissue between separated serosa domains is dorsally extended head lobe. And I also found that in severe phenotype, dorsal serosa was completely gone while anterior serosa not, suggesting independent regulation mechanisms for anterior and dorsal serosa formation. This descriptive data will complement future study in the genetic mechanism underlying serosa formation by providing more details in morphogenesis.

No description available.

Orientador: Rebeca de Souza Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T23:04:36Z (GMT). No. of bitstreams: 1 Dutra_SabrinaNogueira_D.pdf: 4132202 bytes, checksum: 8d692501f423535818a567ec7a79ed11 (MD5) Previous issue date: 2015 / Resumo: A queilite actínica (QA) é uma desordem potencialmente maligna dos lábios, resultado da exposição crônica e excessiva ao raios ultravioleta, e que pode evoluir para um carcinoma de células escamosas de lábio. A via de sinalização Wnt/?-catenina atua em genes relacionados ao ciclo e a proliferação celular e está envolvida no desenvolvimento e progressão tumoral, e algumas de suas moléculas já foram identificadas em QA. O objetivo deste estudo foi correlacionar as características histopatológicas da QA com dois sistemas de classificação para gradação histopatológica, o da OMS e do sistema binário; e avaliar a participação de marcadores da via de sinalização Wnt/?-catenina por meio de reação imunoistoquímica contra os anticorpos Wnt1, Wnt5a, ?-catenina, axina, APC e ciclina D1 em casos de QA, e relacionar esta expressão com os dois sistemas de classificação. Os resultados mostraram correlação das características histopatológicas com o aumento da displasia epitelial de forma mais evidente pelo sistema binário do que pela classificação da OMS; positividade de Wnt 1, potente ativador da via canônica, em 96,7% dos casos; marcação anormal citoplasmática com ou sem marcação nuclear de ?-catenina em 81,9% dos casos; positividade de ciclina D1 em 75,4 % dos casos; negatividade de Wnt 5a, que possui potencial inibidor da via e pode contribuir indiretamente para ativação da via canônica; positividade de APC e axina em 96,7% e 95% dos casos de QA. Além disso, os anticorpos Wnt1, ciclina D1, APC e axina apresentaram aumento do índice de marcação imunoistoquímica de acordo com o aumento do grau de displasia epitelial de forma progressiva pelo sistema binário. Pode-se, assim, sugerir uma possível relação destas proteínas da via de sinalização Wnt/?-catenina com o desenvolvimento e a progressão da QA e que o uso do sistema binário de classificação de gradação histopatológica em QA apresentou melhor correlação com as características histopatológicas e com os índices de marcação imunoistoquímica da via de sinalização Wnt/?-catenina do que o sistema de classificação da OMS / Abstract: Actinic cheilitis (AC) is a potentially malignant disorder of the lips, resulting from chronic and excessive exposure to ultraviolet radiation, which can develop into a lip squamous cell carcinoma. Wnt/?-catenin signaling pathway acts on genes related to cell cycle and proliferation and is involved in tumor development and progression, and some of its molecules have already been identified in AC. Thus, the aim of this study was to correlate the histopathological features of AC with two grading histopathological systems, the WHO and the binary system; and to evaluate Wnt/?-catenin signaling pathway involvement by means of immunohistochemistry reaction against Wnt1, Wnt5a, ?-catenin, axin, APC and cyclin D1 in AC, and to relate this expression with the two histopathological grading systems. The results showed a correlation between the histopathologic features with the increased epithelial dysplasia mainly by using the binary system than using the WHO classification; Wnt 1 positivity, a potent canonical pathway activator, in 96.7% of the cases; abnormal ?-catenin cytoplasmic staining with or without nuclear staining in 81.9% of cases; cyclin D1 positivity in 75.4% of cases; Wnt 5a negativity, which has a potential for an inhibiting the pathway and may indirectly contribute to the activation of canonical pathway; APC and axin positivity in 96.7% and 95% of AC. In addition, Wnt1, cyclin D1, APC and axin showed a progressive increased immunohistochemical staining index according to the increasing degree of epithelial dysplasia by the use of the binary system. Therefore, we were able to suggest a possible relationship of these Wnt/?-catenin signaling pathway proteins with the AC development and progression and that the use of binary grading system in the histopathological classification of AC showed a better correlation with the histopathological features and the immunohistochemical staining indexes of Wnt/?-catenin signaling pathway than the use of WHO grading system / Doutorado / Patologia / Doutora em Estomatopatologia