Wnt signaling is an evolutionarily conserved pathway that is essential for the development of the metazoan embryo. Wnt signaling controls essential developmental processes including cell fate, cell polarity, dorsal-ventral patterning and tissue movement. Misregulated Wnt signaling can have disastrous effects on the developing human embryo, leading to potentially fatal congenital malformations including anencephaly and spina bifida. In addition to embryonic development, misregulated Wnt signaling has been implicated in human pathologies including colon and breast cancers and skeletal malformations. Wnt signaling is divided into two main pathway branches, canonical or beta-catenin dependent, and non-canonical, sometimes referred to as the planar cell polarity (PCP) pathway. The former branch activates the transcription of the downstream target genes leading to the patterning the dorsal-ventral axis of the developing embryo, whilst the latter has no downstream transcriptional targets but rather acts upon the cytoskeleton to control cell and tissue polarity and movement. Wnt signaling bifurcates into these two branches at the level of the protein Dishevelled (Dvl). The Dishevelled-associated activator of morphogenesis 1 (Daam1) protein was identified via a yeast-two hybrid screen using Dvl as bait. Daam1 interacts directly with Dvl and mediates activation of the small GTPase Rho, a key player in non-canonical Wnt signaling necessary for proper gastrulation in the Xenopus (frog) embryo. In addition to Daam1, vertebrates possess a second Daam, Daam2, originally identified via an in silico screen in humans. Similar to Daam1, frog Daam2 participates in non-canonical Wnt signaling, contributing to proper formation of the embryonic neural tube. However, conflicting opinions on the function of Daam2 have led to discrepancies regarding its position in Wnt signaling and function in development. Daam1/2 have not been extensively investigated at the genetic level, therefore, I employed the genetic model zebrafish (Danio rerio) to further clarify their role in Wnt signaling. Using techniques such as the latest gene-editing system CRISPR/Cas9 and other well-established molecular methods including in situ hybridization, RT-PCR and knockdown using morpholino oligonucleotides, I sought to further establish the role of the Daam protein family in vertebrate embryonic development. Together, my results indicate that the zebrafish Daam1a/b and Daam2 behave similarly to Daam1 and Daam2 in frog, respectively, by participating in the non- canonical Wnt signaling pathway and mediating morphology in the developing zebrafish embryo. / Biology

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Frizzled (FZD) are highly conserved receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. They are involved in a great variety of processes during embryonic development, organogenesis, and adult tissue homeostasis. In particular, FZD5 is an important therapeutic target due to its involvement in several pathologies, such as tumorigenesis. Nevertheless, little is known regarding the activation of FZD receptors and the signal initiation, and their GPCR nature has been debated. In order to investigate the activation mechanism of these receptors, FRET (Förster Resonance Energy Transfer)-based biosensors for FZD5 have been developed and characterized. A cyan fluorescent protein (CFP) was fused to the C-terminus of the receptor and the specific FlAsH-binding sequence (CCPGCC) was inserted within the 2nd or the 3rd intracellular loop. Single-cell FRET experiments performed using one of these sensors, V5-mFZD5-FlAsH436-CFP, reported structural rearrangements in FZD5 upon stimulation with the endogenous ligand WNT-5A. These movements are similar to those observed in other GPCRs using the same technique, which suggests an activation mechanism for FZD reminiscent of GPCRs. Furthermore, stimulation of the FZD5 FRET-based sensor with various recombinant WNT proteins in a microplate FRET reader allowed to obtain concentration-response curves for several ligands, being possible to distinguish between full and partial agonists. This technology allowed to address the selectivity between WNTs and FZD5 using a full-length receptor in living cells. In addition, G protein FRET-based sensors revealed that WNT-5A specifically induced Gαq activation mediated by FZD5, but not Gαi activation. Other WNT proteins were also able to induce Gαq activation, but with lower efficacy than WNT-5A. In addition, a dual DAG/calcium sensor further showed that WNT-5A stimulation led to the activation of the Gαq-dependent signaling pathway mediated by FZD5, which outcome was the activation of Protein Kinase C (PKC) and the release of intracellular calcium. Altogether, these data provide evidence that the activation process of FZD5 resembles the general characteristics of class A and B GPCR activation, and this receptor also mediates the activation of the heterotrimeric Gαq protein and its downstream signaling pathway. In addition, the FZD5 receptor FRET-based sensor provides a valuable tool to characterize the pharmacological properties of WNTs and other potential ligands for this receptor. / Frizzled (FZD) sind hochkonservierte Rezeptoren welche zur Klasse F der G- Protein-gekoppelte Rezeptor Superfamilie gehören. Diese haben wichtige Funktionen in verschiedenen physiologischen Prozessen wie zum Beispiel Embryonalentwicklung, Organogenese und adulte Gewebe-homöostase. FZD5 ist aufgrund seiner Beteiligung an verschiedenen pathologischen Prozessen wie der Tumorgenese ein wichtiges therapeutisches Ziel. Jedoch ist über die Aktivierung und Signalauslösung der FZD Rezeptoren sehr wenig bekannt und deren GPCR Eigenschaften sind umstritten. Um den Aktivierungsmechanismus dieser Rezeptoren zu untersuchen, wurden FRET (Förster Resonance Energy Transfer)-basierte FZD5 Biosensoren entwickelt und charakterisiert. Ein cyan fluoreszierendes Protein (CFP) wurde an den C-Terminus des Rezeptors fusioniert und die FlAsH-bindende Sequenz (CCPGCC) wurde im 2. oder 3. intrazellulären Loop eingefügt. Einzel-zell FRET Versuche mit dem Sensor V5-mFZD5-FlAsH436-CFP haben gezeigt, dass Stimulation mit dem endogenen Ligand WNT-5A zur FZD5 Konformationsänderungen führt. Diese Konformationsänderungen sind ähnlich wie bei anderen GPCRs, was darauf hinweist, dass der FZD Aktivierungsmechanismus vergleichbar mit dem von GPCRs ist. Außerdem wurde der FZD5 FRET-basierter Sensor mit verschiedenen rekombinierten WNT Proteinen stimuliert und mit einem FRET-Platten Reader gemessen, was die Erstellung von Konzentrations - Wirkungskurven und die Unterscheidung zwischen Voll- und Partialagonisten ermöglichte. Diese Methode erlaubte es, die Selektivität zwischen WNTs und FZD5 mittels des Volllängenrezeptors in lebenden Zellen zu untersuchen. Zudem haben G-Protein FRET-basierte Sensoren gezeigt, dass WNT-5A die FZD5 vermittelte Gαq Aktivierung jedoch nicht die Gαi Aktivierung spezifisch induziert. Andere WNT Proteine können auch die Gαq Aktivierung induzieren aber mit geringerer Effizienz als WNT-5A. Ein doppelter DAG/Calcium Sensor hat zudem gezeigt, dass WNT-5A Stimulation zu einer durch FZD5 vermittelten Aktivierung der Gαq-abhängigen Signaltransduktionkaskade führt, was zur Aktivierung der Protein Kinase C (PKC) und zur Freisetzung intrazellulären Calciums führt. Zusammenfassend wurde in der vorliegenden Arbeit die Ähnlichkeit des FZD5 Rezeptors zur Klasse A und B der GPCRs bezüglich allgemeinen Eigenschaften und Aktivierung verdeutlicht. Zudem vermittelt dieser Rezeptor die Aktivierung der Gαq-abhängigen Signaltransduktionkaskade. Ein FZD5 Rezeptor FRET-basierter Sensor stellt ein wertvolles Werkzeug zur pharmakologischen Charakterisierung der WNTs und anderer potentiellen FZD5 Liganden dar.

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京都大学 / 新制・論文博士 / 博士(医学) / 乙第13573号 / 論医博第2299号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 藤田 恭之, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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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)

Cardiovascular disease is among the leading causes of death in the US. It stems from the pathological buildup of plaque within the vasculature known as vascular calcification. Medial calcification, or arteriosclerosis is the buildup of plaque within the medial layer of the arteries resulting in artery wall stiffening and reduction of blood flow. Evidence suggests that the vascular smooth muscles cells (VSMCs) that line the medial layer of the arteries, undergo a phenotypic switch to osteoblast-like cells to deposit calcium while in this pathological state. The Wnt/BETA-catenin pathway could potentially play a role in the phenotypic modulation. Inhibition of the Wnt signaling pathway could be a promising approach to combat vascular calcification. Sclerostin (SOST) has been shown to be upregulated during arteriosclerosis in a manner that is indicative of the possible therapeutic potential of the protein. Therefore, we propose to confirm the role of Wnt signaling in vascular calcification and investigate the effects of SOST treatment on vascular calcification.

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