Thermogenic fat is a promising target for new therapies in diabetes and obesity. Understanding how thermogenic fat develops is important to develop rational strategies to treat obesity. Previously, we have shown that Tyk2 and STAT3, part of the JAK-STAT pathway, are necessary for proper development of classical brown fat. Using primary preadipocytes isolated from newborn mice we demonstrate that STAT3 is required for differentiation and robust expression of Uncoupling Protein 1. We also confirm that STAT3 is necessary during the early induction stage of differentiation and is dispensable during the later terminal differentiation stage. Without STAT3, the brown preadipocytes have increased apoptosis early in the terminal differentiation phase. We also show that the block in differentiation is caused by an inability of STAT3 knockouts to down regulate β-catenin by the end of the induction phase. Application of Wnt/β-catenin inhibitors or knockdown of β-catenin during the induction phase is sufficient to fully rescue differentiation of brown adipocytes from the Myf5+ lineage, including reduction in apoptosis, restoration of histone acetylation in the UCP1 promoter and enhancer regions, and full restoration of the expression of brown fat genes. Finally, we show that in the beige lineage, STAT3 is also necessary during the induction phase and can be rescued by Wnt/β-catenin inhibitors, although the rescue is not as robust as it is in the Myf5+ lineage.

The Wnt signaling network has critical roles in development and disease. Simplified, this complex network has two distinct outputs: the Wnt/β-catenin module activates the phosphoprotein Dishevelled (Dvl) and leads to transcriptional activation while the Wnt/Planar Cell Polarity (PCP) module activates Dvl and leads to calcium release and directed cell movement. Wnt/β-catenin and Wnt/PCP share signaling components like Frizzled receptors, Dvl, and Naked (Nkd). It is an open question how converging Wnt signals diverge into separate outcomes. In this thesis, I used molecular techniques, functional studies in the zebrafish, and biochemical approaches to determine the role of Nkd in Wnt signaling. Nkd contains and EF-hand, a putative calcium binding domain, and is known to antagonize Wnt/β-catenin and disrupt Wnt/PCP signaling. We utilized a tissue that requires both Wnt/β-catenin and Wnt/PCP signaling to properly pattern the left/right axes of the embryo; the dorsal forerunner cells (DFCs). The DFCs exhibit aperiodic calcium release as they migrate to form the Kupffer's Vesicle (KV), the organ of asymmetry. Calcium inhibition in the DFCs disrupts their migration, alters KV formation, and disrupts left/right patterning. Nkd is enriched in the DFCs during migration and KV formation and endogenous Nkd knockdown in the DFCs produces the same phenotypes as calcium inhibition, making Nkd a candidate molecule for directing converging Wnt signals to distinct outcomes. To assess the role of the EF-hand in Nkd function, I created point mutations predicted to disrupt EF-hand affinity for calcium. Through functional studies in zebrafish embryos, I determined that Nkd EF-hand is necessary for Nkd function in Wnt/PCP signaling, but dispensable for Wnt/β-catenin signaling. Although Nkd has not been shown to bind calcium, our functional data with the Nkd EF-hand point mutant provides compelling evidence for a role for calcium in Nkd function in directing Wnt signaling output. EF-hand affinity for calcium is influenced by binding partners, and since Nkd binds to Dvl in the Dvl PDZ domain, we screened the domain for a region rich in amino acids that facilitate ion binding. We identified a 12-amino acid sequence in the Dvl PDZ domain with potential to create a negatively charged pocket to help coordinate calcium binding. We expressed the Nkd EF-hand (EFX) Dvl basic domain and PDZ domain (bPDZ). The purified EFX and bPDZ constructs were used to investigate the interaction between Nkd, Dvl, and calcium. I show, by circular dichroism, that the Nkd/Dvl complex undergoes a calcium-induced change in secondary structure. This reveals the mechanism by which Nkd directs Dvl from the default Wnt/β-catenin signaling module to the Wnt/PCP module in response to calcium.

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.

No description available.

<p>Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.</p>

Retinal degeneration is characterized by the irreversible loss of photoreceptors. A key research question is the identification and characterization of photoreceptor protective factors that prevent or delay vision loss. The Wnt pathway is a critical cellular communication pathway involved in development and diseases of the central nervous system (CNS). Recently, we discovered that multiple components of the Wnt pathway were differentially expressed in the rd1 mouse model of retinal degeneration. One of the most highly upregulated genes was Dickkopf3 (Dkk3), a secreted Wnt pathway protein of unknown function. Additionally, we demonstrated that Wnt signaling is neuroprotective in primary retinal culture (Yi et al., 2007). These data led to the hypothesis that Dkk3 is a regulator of Wnt-mediated neuroprotection during retinal degeneration. The role of Dkk3 in the retina and its activity in the Wnt pathway was identified in this dissertation project using a series of biochemical, molecular and cell biology methodologies. First, Dkk3 was shown to be expressed and secreted from Muller glia in mouse retinal tissue and primary Muller glia culture. I then demonstrated that Muller glia are a Wnt-responsive cell type and that Dkk3 potentiates Wnt3a-mediated signaling. Interestingly, the latter effect was not observed in other cell types in the retina such as retinal ganglion cells and retinal pigmented epithelial cells. Thus, Dkk3 may act on Muller glia to positively modulate Wnt signaling during retinal degeneration, which could potentially amplify the neuroprotective activity of the Wnt pathway. Next, the role of Dkk3 in cellular viability was explored. HEK293 cells stably expressing Dkk3 were shown to be significantly protected from staurosporine-induced apoptosis compared with vector control. This result suggests that Dkk3 may mediate a direct pro-survival effect onto photoreceptors during retinal degeneration. Protein interaction experiments demonstrated that Dkk3 formed a complex with the single pass transmembrane proteins Krm1 and Krm2 in the membrane, potentially in the endoplasmic reticulum (ER). Furthermore, Wnt signaling luciferase reporter assays demonstrated that Krm2, but not Krm1, abolished Dkk3-mediated Wnt3a potentiation. These data suggest that Dkk3 modulates Wnt signaling by antagonizing Dkk1-Krm dependent Wnt inhibition. Further studies will determine whether this activity is sufficient for the potentiation of Wnt signaling by Dkk3. Lastly, co-immunoprecipitation followed by mass spectrometry analysis was used to identify a novel interacting protein of Dkk3. Dkk3 was shown to interact with glucose response protein 78 (GRP78), an ER-resident chaperone. This suggested that Dkk3 protein is part of the unfolded protein response through GRP78 in the ER. In conclusion, these studies identified two novel functions of Dkk3 in regulating Wnt signaling pathway and cellular viability and suggest a physiological role for Dkk3 and Wnt signaling during retinal degeneration. Future studies will explore the significance of Dkk3-Krm and Dkk3-GRP78 interactions in the retina. Further, elucidation of the regulation of Dkk3 and other Wnt ligands in the ER and the consequence of ER stress on the biological activity of Wnt signaling will provide a better understanding of the role of the Wnt pathway during retinal degeneration.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

The USP4 is a deubiquitinating enzyme found elevated in certain human lung and adrenal tumours. USP4 has a very close relative, USP15, which has caused great difficulty in studying only one or the other. We have had generated two antibodies specific to USP4 and USP15, and have confirmed that the two do not cross react. Although there have been previous findings of interacting partners, possible substrates and pathways in which it is involved, the biological role of USP4 is mostly unknown. We have used these antibodies to determine that USP4 and USP15 expression differs across tissue and cell types, and that expression changes as the organism ages. We have shown that USP4 plays a role in canonical Wnt signaling, perhaps by stabilizing Beta-catenin, and identified GRK2 as a kinase, phosphorylating USP4. These data have provided enough information to form a hypothesis, implicating USP4 with the destruction complex in the Wnt signaling pathway.

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.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.