Marek Mlodzik - US grants

DESCRIPTION (provided by applicant): Epithelial cells generally require polarization in two axes for their function, the ubiquitous apical-basal polarity and a second axis within the plane of the epithelium, called Planar Cell Polarity (PCP). Both polarity and ordered patterning are established during organogenesis. Typical PCP examples include aspects of mammalian skin or internal organs, like the inner ear epithelium with its sensory cilia. In Drosophila, and insects in general, all cuticular structures show PCP features. The establishment of PCP in Drosophila serves as a paradigm to study PCP determination in development and disease. PCP is thought to be coordinated by long-range signals, resulting in the graded activity of the Frizzled (Fz) receptor (with Wnt family members as their ligands) and its associated signaling cascade (Fz/PCP signaling). This Fz/PCP signaling pathway is conserved throughout evolution and regulates many aspects of coordinated cellular polarization, including directed cell migration during mammalian gastrulation. Wnt-Fz/PCP signaling is distinct from the canonical Wnt-Fz/2-catenin pathway. The regulation of signaling specificity between the two pathways activated by the same receptor(s) is critical for development or disease but not yet understood. Similarly, the link between Fz/PCP signaling and the resulting cellular responses, including cytoskeletal organization or specific transcriptional events are only poorly dissected. The scope of this application is to investigate the mechanistic and functional interactions among the core Fz/PCP determinants, and the integration of newly identified PCP genes (isolated through ongoing genomic screens). Their function in Fz/PCP signaling and in general cellular polarization events will be investigated genetically, biochemically, and at the cell biological level. A combination of in vivo studies in Drosophila, cell culture analyses in mammalian and Drosophila cells, and biochemical experiments will be performed to achieve these goals. The global long-range polarization events will be given special attention. The processes of PCP establishment and Wnt/Fz signaling have been linked to several medical abnormalities, including deafness, cancer (several components of the pathway are proto-oncogenes or tumor suppressors), poly cystic kidney disease, and ciliopathies in general. Thus the information acquired in this application will both advance our understanding of cellular polarization and patterning, and will be of medical relevance in several disease contexts. PUBLIC HEALTH RELEVANCE: Cellular polarity is an essential feature of organogenesis and development and tissue function in general. The regulation and maintenance of the epithelial planar cell polarity (PCP) feature is linked to many diseases, ranging from ciliopathies and deafness, to cancer (e.g. several polarity determinants are tumor suppressors or proto-oncogenes). This application addresses the molecular regulation of PCP establishment;the information acquired here is of high significance for many medical disorders.

DESCRIPTION (Applicant's Description): The function of the visual system is to form images. Correct patterning of photoreceptor neurons in the retina is critical for their precise retinotopic axonal projections onto the optic lobes in the brain. Precise photoreceptor arrangement within the retinal epithelium is thus important. The Drosophila retina is a stereotyped pattern of about 800 ommatidia, or unit eyes, and the photoreceptor neurons within each ommatidium. The establishment of this precise arrangement serves as a paradigm to study epithelial planar polarity (often present in neuro-epithelial structures). It requires the Frizzled (Fz) receptor and its associated signaling pathway. Fz proteins are receptors for the Wg/Wnt growth factor family. Fz signaling regulates both early eye development and eye size, and the ommatidial arrangement within the retina. Fz uses distinct signaling pathways to achieve these different tasks. Wg/Fz signaling regulates eye size, whereas a distinct Fz-mediated signaling pathway regulates photoreceptor arrangement. It is important to achieve signaling specificity as these Fz functions lead to opposite responses: Wg/Fz signaling inhibits photoreceptor differentiation, whereas Fz/planar polarity signaling regulates the precise photoreceptor arrangement. The scope of this application is to identify the Fz domains that select between the two signaling cascades, and to identify the proteins that interact with the respective sequences. A combination of Drosophila in vivo studies and biochemical experiments will be used to achieve this goal. Several effectors of Fz have been identified and will be analyzed for their role in the selection between the two Fz signaling cascades. Wnt/Fz signaling has also been implicated in many forms of cancer and several of its components are proto-oncogenes. Thus the information acquired in this application will both advance our understanding of retinal patterning in the eye, and will also be of importance for the study of carcinogenesis.

Epithelial cells-require correct polarization and patterning for their function. Epithelial tissues are often polarized in two axes, in the apical-basolateral axis and in the plane of the epithelium. Planar polarization in vertebrates is evident in the skin or internal organs, like the inner ear epithelium with its sensory cilia, and in insects in the cuticular structures and the compound eye. The precise polarization patterns in Drosophila, and the photoreceptor arrangement in the retina in particular, serve as a paradigm to study epithelial planar polarity determination and coordination of polarized cell shape changes in general. The Frizzled (Fz) receptor and its signaling cascade serve as an entry point to study the molecular aspects controlling these processes. The underlying Fz signaling pathway is conserved throughout evolution and also regulates several other aspects of coordinated cellular polarization in vertebrates and invertebrates. Fz mediated planar polarity signaling is distinct from the canonical Wnt/Fz/beta-catenin pathway and its components are only now being identified. Moreover, the link between Fz/planar polarity signaling and the resulting cellular responses including specific transcriptional events are not understood. Whereas the canonical Wnt/Fz/beta-catenin pathway inhibits photoreceptor differentiation, the distinct Wnt/Fz/planar polarity signaling is essential for the correct arrangement of the photoreceptors. The scope of this application is to (1) identify genes required in Fz mediated photoreceptor polarization and (2) define their specific roles in planar polarity determination. Their function in Fz signaling and in cellular polarization events will be investigated. A combination of genetic and functional in vivo studies in Drosophila will be utilized to achieve these goals. Planar polarization and Fz signaling have been linked to medical abnormalities (including deafness/inner ear epithelium) and cancer (as components of the pathway are proto- oncogenes or tumor suppressors). The information acquired in this proposal will both advance our understanding of eye patterning, and will also be of medical relevance.

[unreadable] DESCRIPTION (provided by applicant): The function of the visual system is to form images. As correct patterning of photoreceptor neurons in the retina is critical for the precise retinotopic axonal projections onto the optic lobes, precise photoreceptor arrangement within the retinal epithelium is important. The Drosophila retina is a stereo-typed pattern of several hundred ommatidia, or unit eyes, and the associated photoreceptor neurons. The establishment of this precise arrangement serves as a paradigm for neuronal photoreceptor cell fate induction and patterning. This process requires first sequential waves of EGF-receptor (Egfr)/Ras-signaling activation to induce the respective neuronal fates, simultaneously with Frizzled (Fz)/planar polarity signaling to pattern the photoreceptor neuron clusters correctly. An interesting aspect of the final photoreceptor patterning and arrangement is governed by a process called ommatidial rotation. Fz signaling regulates the direction of rotation during eye development, but a distinct mechanism/pathway is required for the actual execution of the rotation. We have identified the Egfr pathway as critical for the rotation process per se. Interestingly, the downstream effector cascade is distinct from the one used by Egfr for photoreceptor neuron induction and survival. The scope of this application is to dissect the specific Egfr signaling requirements for photoreceptor motility and ommatidial rotation, and to identify the specific effector cascade(s) required downstream of Egfr and Ras in this context. A set of newly identified rotation specific genes will be integrated with the rotation specific Egfr signaling aspects. A combination of Drosophila in vivo studies and biochemical experiments will be used to achieve this goal. Several effectors of Egfr have been identified genetically or molecularly and will be analyzed for their role in ommatidial rotation. Egfr/Ras signaling has also been implicated in many forms of cancer and several of its signaling components (e.g. the receptor and Ras themselves) are proto-oncogenes. Thus, the information acquired in this application will both advance our understanding of retinal cell motility and patterning, and will also be of importance for the study of carcinogenesis. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Mount Sinai School of Medicine (MSSM) proposes to continue and expand a highly successful training program in Cellular and Molecular Biology (CMB) for predoctoral fellows. This program reflects an expansion of CMB relevant research at MSSM, which has experienced a large growth in NIH funding over the past 14 years. The training program reflects an integral component of the institution's Graduate Program, in which training mentors play key leadership roles. The program encompasses a laboratory-based, multidisciplinary predoctoral training in cellular and molecular biology. The current grant supports four predoctoral trainees. The program has demonstrated the ability to attract and develop a cadre of outstanding PhD and MD/PhD students in cellular and molecular biology focused research and attracts an increasing number of students to the laboratories of the CMB training faculty to conduct their predoctoral thesis research. The new establishment of the Stem Cell Institute at MSSM provides further strength to this training grant. The additional component would include 2 predoctoral fellows to be added over the course of the first 2-3 years of the new funding period. The training faculty consists of 50 preceptors from across several departments. All training faculty have peer-reviewed support from NIH and/or other extramural agencies. The curriculum for the predoctoral trainees involves some core elements including advanced course work in developmental, cellular, and molecular biology with focus on stem cell biology, organismal development, and cancer cell biology, as well as related structural studies. The training program includes a rigorous evaluation and selection process for trainees, and "the program is both cognizant of and actively involved in minority recruitment. The CMB training program combines cutting edge research with a high standard academic curriculum, and many interfaces with the clinical side to highlight the relevance of the basic research training to medical applications. The trainees will work closely with faculty drawn from throughout Mount Sinai and ensuring that the trainees'research is both comprehensive in scope and broad to cover all relevant areas of modern Cellular Biology research.

DESCRIPTION (provided by applicant): The function of the visual system is to form images in the brain. Correct specification and patterning of the photoreceptor neurons within the retina are a prerequisite for precise retinotopic axonal projections and image formation. Many aspects of photoreceptor orientation and morphogenesis are generated through regulated cell adhesion behavior of individual neurons or groups of neurons. The Drosophila eye serves as a paradigm for most aspects of eye development, retinal biology and disease. The Drosophila retina is composed of a stereo-typed array of several hundred unit eyes, or ommatidia, each containing a precise arrangement of 8 photoreceptor neurons, which are oriented with respect to each other and also the whole retinal field and body axes. The establishment of this precise arrangement requires the interplay of several signaling pathways, transcription factors, and cell adhesion molecules. These are conserved and share equivalent functions in the mammalian eye. The correct specification of different photoreceptor subtypes requires an interplay of the Wnt/Frizzled-planar cell polarity (Fz-PCP), Egfr and Notch signaling pathways. A critical aspect that follows the cell fate specification is a cell adhesion/cell motility process called ommatidial rotation. It leads to a 90 degree rotation of clusters of photoreceptor precursors and places them in their proper position to allow subsequent photoreceptor morphogenesis. The scope of this application is to dissect the mechanistic regulatory interactions between the signaling pathways and the "executing" cell adhesion factors (and associated cytoskeletal elements). Based on our preliminary studies we hypothesize that the Nemo kinase (a MAPK family member) integrates regulatory input from Fz/PCP, Notch and receptor tyrosine kinase (RTK)/Ras signaling to itself regulate the behavior of several cell adhesion molecules and in particular the cadherin/catenin complexes. Strikingly, in all involved signaling pathways (Wnt/Fz-PCP, Notch and RTK/Ras) a novel non-canonical pathway branch is employed. The components of these cell adhesion specific signaling branches are only being discovered now, and thus an exciting new signaling network is emerging. We will use a combination of Drosophila in vivo studies and biochemical experiments to define the mechanistic regulatory interactions between the signaling components and the cell adhesion factors. In the human eye, Fz and Notch signaling are associated with many diseases, like Familial Exudative Vitreoretinopathy (FEVR) or Norrie Disease. Several components of the Wnt/Fz and Notch pathways are also critically linked to cancer and are associated with stem cell biology. Thus the information acquired in this application will advance our understanding of photoreceptor morphogenesis and eye diseases, and will also be of medical relevance in other areas, including stem cell biology and cancer. PUBLIC HEALTH RELEVANCE: RTK/Ras, Notch and Wnt/Fz-planar cell polarity signaling pathways have been implicated in many growth and patterning contexts, and are linked to many diseases, ranging from retinal degeneration and angiogenesis defects to cancer. This application addresses the regulatory input of these pathways that is specific to cell adhesion, motility and morphogenesis in the patterning of photoreceptor neurons and epithelial cells in general. The information acquired here will be of importance for several eye diseases and other medical disorders.

DESCRIPTION (provided by applicant): Frizzled (Fz) receptors act as signal transducers of Wnt growth factors via several effector pathways, notably Wnt/2-catenin (2-cat) and Fz/planar cell polarity (PCP) signaling. Fz/PCP and Wnt-Fz/2-cat signaling diverge downstream of the cytoplasmic factor Dishevelled (Dsh). Often both pathways act in the same tissues, and a tight regulation of signaling specificity is essential for normal development and cellular homeostasis. In deregulated scenarios, selection of the wrong Fz-pathway often leads to disease (i.e. PKD or cancer). Although a molecular framework for both pathways is established, very little is known about the regulation of Wnt/Fz-Dsh signaling specificity. This application investigates the molecular aspects of signaling specificity distinguishing between Wnt-Fz/2-Cat and Fz/PCP signaling. We will address this in Drosophila (the model organism where both pathways were discovered) and in mammalian cell culture. The specific focus is the regulation of Dsh by phosphorylation. Dsh is a cytoplasmic factor shared between both pathways and recruited to the membrane by Fz receptors. Based on preliminary data we hypothesize that Dsh is differentially phosphorylated in a pathway specific manner. A combination of cell culture, in vivo, and biochemical studies will dissect the phosphorylation events on Dsh that switch its function between the Wnt- Fz pathways. The Specific Aims are: 1. To determine specific phosphorylation event(s) on Dsh and their role in the selection of the signaling outcome;and 2. To identify and functionally define Tyrosine kinases acting in Dsh regulation and pathway specific responses. Our mass-spectrometry studies indicated that differential phosphorylation of Dsh contributes to pathway selection and we have identified several kinases acting on Dsh. Physiological and medical relevance of the phosphorylation events will be established in vivo. This will be complemented by the generation of specific antisera to the relevant phosphorylated sites in Dsh. These are likely to have diagnostic potential. PUBLIC HEALTH RELEVANCE: The regulation and selection of the correct Wnt signaling pathways is an essential feature of development and tissue function and homeostasis. Deregulation of Wnt is linked to many diseases, ranging from ciliopathies and angiogenesis defects, to cancer (e.g. several pathway components are tumor suppressors or proto-oncogenes). This application addresses the molecular regulation of Dishvelled a key component in Wnt/Frizzled-signaling specificity;the information acquired here will be of high significance for several medical disorders.

DESCRIPTION (provided by applicant): The function of the visual system is to form images in the brain. Correct specification and patterning of the photoreceptor neurons within the retina are a prerequisite for precise retinotopic axonal projections and image formation. The Drosophila eye serves as paradigm for many aspects of eye development, retinal biology and disease. The Drosophila retina is composed of a stereo-typed array of 700-800 ommatidia, or facets, each containing a precise arrangement of 8 photoreceptor neurons. The establishment of this precise arrangement requires the interplay of several signaling pathways and transcription factors, all of which are conserved and share equivalent functions in the mammalian eye. In particular, the correct distinction of two subtypes of photoreceptors, R3 and R4, requires a precise regulation of the Wnt/Frizzled (Fz)/planar cell polarity (PCP) pathway. Strikingly, the second Wnt-Fz mediated signaling pathway, canonical 2-catenin signaling, causes photoreceptor cell death and reduction of eye size. The Fz receptor and its immediate downstream effector Disheveled (Dsh, Dvl in mammals) are shared between Fz/2-catenin and Fz/PCP signaling but activate distinct effectors pathways downstream of Dsh, and thus correct regulation of Fz-Dsh signaling specificity is critical for normal eye development. The regulation of this signaling specificity is poorly understood. The scope of this application is (1) to define regulatory kinase input at the level of Fz-Dsh mediating the correct and specific signaling outcome, (2) to determine the role of Tyrosine phosphorylation of Dsh in pathway selection, (3) to establish the functional requirements of new transcription factors identified to act in the R3/R4 Fz/PCP- mediated cell fate specification, and (4) to define the role of anaphase promoting complex (APC/C) subunits and a Ubiquitin-like peptide in Fz/PCP signaling and R3/R4 specification. These Aims are based on exciting preliminary data. A combination of in vivo studies in the eye, and cell culture and biochemical experiments will be utilized to achieve these goals. In the human eye, Fz signaling is associated with diseases like Familial Exudative Vitreoretinopathy (FEVR) and the Norrie Disease. Several components of the Fz pathway(s) are also critically linked to cancer and are associated with stem cell biology. Thus the information acquired in this application will advance our understanding of retinal patterning and eye disease, and will also be of medical relevance in other areas, including cancer and stem cell biology.

DESCRIPTION (provided by applicant): Epithelial cells are often polarized in two axes, in the apical-basal axis and in a second axis within the plane of the epithelium, generally referred to as Planar Cel Polarity (or PCP). Classical examples of PCP in mammals include aspects of skin development with hair bud orientation or cellular arrangements in internal organs, like the inner ear epithelium with its sensory cilia. In Drosophila, all adult cuticular structures display PCP features. The study of PCP establishment in Drosophila serves as a paradigm for PCP establishment in developmental patterning and disease. Analyses in Drosophila have established a conserved molecular cassette and pathway anchored around the Frizzled (Fz) trans-membrane protein and associated regulatory factors. This Fz/PCP signaling pathway and its core regulatory components are conserved throughout evolution regulating many aspects of cellular polarization not only in epithelial organs, but also in directed cell migration of mesenchymal cells during mammalian gastrulation and neurulation. Although the molecular mechanisms of the intracellular interactions among the core Fz/PCP factors are beginning to be understood, very little is known about the mechanisms of long-range PCP regulation coordinating polarity across whole organs. The scope of this application is to follow-up on interesting observations that suggest that long-range PCP coordination requires that the Fz protein (generally a receptor for Wnt-family type ligands) acts as a ligand itself, activating a novel signaling pathway by binding to the four-pass trans-membrane receptor Van Gogh (Vang; Vang-like in mammals). Based on interesting preliminary data, we propose as Specific Aims to identify the components of and start assembling this novel pathway, acting downstream of Vang. We have established several assays that will allow us to address this: A combination of genome-wide genetic in vivo studies in Drosophila and cel culture biochemical analyses will be performed to achieve our goals. As the global long- range PCP-type polarization events are largely obscure, our application will provide the first insight(s) into the mechanism of a Vang-signaling pathway that acts downstream of Fz-binding interpreting long-range cues. PCP establishment and the Fz-Vang factors have been linked to several medical abnormalities, including deafness, cancer (several components of the pathway are proto-oncogenes or tumor suppressors), poly cystic kidney disease, and ciliopathies. As, such the information acquired in this application will both advance our understanding of PCP and organ patterning, and will also be of medical relevance in several disease contexts.

DESCRIPTION (provided by applicant): Epithelial cells often require polarization in two axes for their function, ubiquitous apical-basal polarity and a second axis within the plane of the epithelium, called Planar Cell Polarity (PCP). Typical mammalian PCP examples are highlighted by the organization of the skin and many internal organs, e.g. the inner ear with its sensory cilia and, importantly, also include directed cell migration during mammalian gastrulation and neural tube closure. In Drosophila, all adult cuticular structures show PCP features. The establishment of PCP in Drosophila serves as a paradigm to study PCP determination in development and disease. PCP is coordinated by the activity of the Frizzled (Fz) receptor (with Wnt family members as their ligands) and it's associated signaling cascade (Fz/PCP signaling), which is highly conserved throughout evolution and regulates many aspects of coordinated cellular polarization, including directed cell migration. Although the frame work of the signaling pathway(s) regulating PCP is beginning to be established, the specific links between PCP-signaling and the resulting cellular responses, including the regulation of cell adhesion and cell motility are only beginning to be dissected. Similarly, the cell adhesion effectors of the PCP pathway(s) are largely unknown. The scope of this application is to dissect the mechanistic regulatory interactions between PCP associated signaling pathways and the respective cell adhesion factors, using the Drosophila eye paradigm as a model. Based on our preliminary studies we hypothesize that regulatory input from Fz/PCP, Notch and receptor tyrosine kinase (RTK)/Ras signaling converges on E-cadherin/catenin, Nectin/Afadin, and Integrin/ECM mediated cell adhesion/cell motility regulation. Strikingly, all signaling pathways involved (Wnt/Fz-PCP, Notch and RTK/Ras-signaling) employ a non-canonical pathway branch. The components of these cell adhesion specific signaling branches are only being discovered now, and thus an exciting new signaling network is emerging. We will use a combination of Drosophila in vivo studies, live imaging in the fly eye, and biochemical experiments to define the mechanistic regulatory interactions between the signaling components and the cell adhesion factors, leading to a highly regulated cell motility process. Several components of the signaling pathways and cell adhesion modules are critically linked to cancer and other diseases, and are also associated with stem cell biology. Thus the information acquired in this application will not only advance our understanding of regulated cellular motility but will also be of medical relevance in several disease associated contexts.

DESCRIPTION (provided by applicant): Wnt canonical signaling plays critical roles in embryonic development and cell fate decisions and its constitutive activation has been directly implicated in human cancer. The noncanonical Wnt pathway, also importantly involved in development including planar cell polarity (PCP) and convergent extension, shares components with Wnt 2-catenin signaling including the Frizzled (Fz) receptors and Dishevelled (Dsh/Dvl). However, pathway signaling diverges downstream of Dsh/Dvl to activate strikingly different biochemical pathways and biological outcomes. This application investigates the molecular aspects of Wnt signaling specificity, taking advantage of complementary approaches and expertise involving both mammalian and Drosophila systems. Using a kinome shRNA screen we have identified two novel Dsh kinases, dWnk and dCDKL. Our preliminary studies indicate that these kinases specifically function in canonical and PCP pathways, respectively. In Aim 1, we propose to further characterize Drosophila Wnk kinase in Wnt-pathway specific responses. In Aim 2, we will investigate the role of mammalian Wnk kinases in Wnt canonical signaling and in Wnt-driven cancer cells. In Aim 3, we will characterize the role of CDKL kinases in Wnt-signaling pathway responses. These investigations should elucidate novel components of Wnt pathways, lead to a better understanding of the evolutionary conservation and/or divergence of Wnt pathway specification, and possibly identify new therapeutic targets for Wnt activated human tumor cells. Our efforts are enhanced by close collaborative interactions among investigators, whose expertise and interrelated efforts strongly complement and help to inform these investigations.

DESCRIPTION (provided by applicant): Polarization of epithelial cells is evident in two axes, in the ubiquitous apical-basolateral axis and within the plane of the epithelium as a second axis, the latter generally referred to as Planar Cell Polarity (or PCP). Examples of PCP are present in almost all organs, and in mammals, for example, most obvious in aspects of skin development with hair follicle orientation or cellular arrangements in internal organs, like the inner ear epithelium with its sensory cilia. In Drosophila, all adult cuticular structures and organs display striking PCP features, which makes the study of PCP establishment in Drosophila serve as a paradigm for the process in developmental patterning and disease in general. Analyses in Drosophila have established a conserved molecular pathway anchored around the Frizzled (Fz)/PCP core factor cassette and associated regulatory factors. This core Fz/PCP pathway and its regulatory components are conserved throughout evolution regulating many aspects of cellular polarization not only in epithelial organs, but in mammals also in directed cell migration of mesenchymal cells during gastrulation and neurulation. Although a framework of the interactions among the core Fz/PCP factors is emerging, little is known about the actual molecular mechanisms underlying their interactions. The scope of this application is to follow-up on very interesting gene identifications from a genome wide screen for novel regulators of the core PCP factors. Based on interesting preliminary data, we propose as Specific Aims to (1) address the role of the Ubiquitin-ligase activity of the Anaphase Promoting Complex (APC/C) and define which components of this complex act in PCP and how, (2) define the function of the novel ubiquitin-like modifier encoded by CG15283 in PCP establishment and how it might regulate the activity of core Fz/PCP factors or APC/C components during PCP signaling, and (3) identify the PCP specific substrates of the APC/C and define their molecular regulation by the APC/C. We have established several assays that will allow us to address these aims via a combination of functional in vivo studies in Drosophila, biochemical experiments, and cell culture analyses. As the roles of the APC/C outside cell cycle control and a function of the conserved CG15283 peptide are largely obscure or completely unknown, respectively, our application will provide first insight(s) into the mechanism(s) of these. Fz/PCP establishment has been linked to several medical abnormalities, including deafness, cancer, polycystic kidney disease, and ciliopathies. Thus the information acquired in this application will both advance our understanding of PCP regulation and organ patterning, and will also be of medical relevance in several disease contexts.

Epithelial cells are polarized in two axes for their function, ubiquitous apical-basal polarity and a second axis within the plane of the epithelium, the latter referred to as Planar Cell Polarity (or PCP). Both, cell polarity and ordered cellular patterning during organogenesis depend on PCP mechanisms. Typical PCP examples include in Drosophila, and insects in general, all cuticular structures and the compound eyes. Similarly, in mammals aspects of PCP are evident in mammalian skin, the inner ear epithelium with its sensory cilia, or the respiratory system and almost all other internal organs. Moreover, convergent extension processes during gastrulation and neural tube patterning and closure requires PCP signaling. PCP establishment in Drosophila serves as a paradigm to study this type of polarity in development and disease. PCP is coordinated by long-range signals from Wnts, resulting in asymmetric localization of the Frizzled (Fz) receptor (with Wnt family members as their ligands) and its associated signaling cascade. The core Fz/PCP factors are required to interpret the polarity within the cell and relay it to neighboring cells. All members of the core Fz/PCP group are conserved throughout evolution and regulate all PCP aspects of coordinated cellular polarization. Wnt-Fz/PCP signaling is distinct from the canonical Wnt-Fz/?-catenin pathway (and correct regulation of signaling specificity between the two Wnt-pathways, activated by the same receptor(s), is critical for development and disease). In PCP- signaling Fz acts both, as the receptor for Wnts and a ligand for its intercellular binding partner Vang/Stbm (Vangl1/2 in mammals). The cellular mechanism(s) acting downstream of Vang/Vangl upon Fz binding are unknown. The scope and focus of this application is to investigate the mechanistic and regulatory interactions of Vang/Vangl as a result of these intercellular interactions, and their integration within the core PCP interaction framework. Based on exciting preliminary data, we propose as Specific Aims to (1) establish the physiological significance of Fz-induced Vang phosphorylation and associated kinase function, (2) to functionally dissect the molecular interactions of Vang/Vangl and its cytoplasmic effectors, a process that will also be aided by including patient data with Vangl associated neural tube closure defects, and (3) define a novel molecular and cellular mechanism as a response downstream of Vang to its intercellular Fz interaction. A combination of in vivo studies in Drosophila, cell culture analyses in mouse skin cells and Drosophila cells, and biochemical experiments will be performed to achieve these goals. Post-translational modification events will be given special attention. The processes of PCP establishment and Wnt/Fz signaling have been linked to several medical abnormalities, ranging from deafness to spina bifida/neural tube closure defects, and cancer, or poly-cystic kidney disease and ciliopathies in general. Information acquired here will both advance our understanding of cellular polarization, and provide medical relevance in many disease contexts.

Epithelial cells often require polarization in two axes for their function, (1) ubiquitous apical-basal polarity and (2) a second axis within the plane of the epithelium, the latter called Planar Cell Polarity (PCP). Typical mammalian PCP examples are highlighted by the organization of the skin and many internal organs, e.g. the inner ear with its sensory cilia, and, importantly, also include directed cell migration during mammalian gastrulation and neural tube closure. In Drosophila, all adult cuticular structures show PCP features. The establishment of PCP in Drosophila serves as a paradigm to study PCP determination in mammalian development and human disease. PCP is coordinated by the activity of the Frizzled (Fz) receptor (with Wnt family members as their ligands) and its associated signaling cascade (Fz/PCP signaling), highly conserved throughout evolution. It not only regulates many aspects of cellular polarization, but also impacts coordinated and directed cell migration. Whereas the framework of PCP signaling is beginning to be established, its specific links to the regulatory feed into cell motility, cell migration, and associated biophysical changes in the responding cells remain largely unknown. The scope of this application is to establish the technology and analytical tools to allow an automated dissection and quantification of PCP regulated cell motility features at cell-biological levels. We will use the Drosophila eye as model system, as it provides unique advantages through its sophisticated genetic tools and available molecular markers. The goal is to establish imaging protocols and quantitative tools to allow the formulation of a biophysical model of PCP-regulated cell motility in the eye that then will serve as the paradigm for molecular disease oriented dissections of related processes. We will use a combination of Drosophila in vivo studies, a novel in vivo time-lapse imaging protocol for fly eye development, and mathematical quantitative analyses to define the critical parameters governed by PCP signaling that impact cell motility through, for example, junctional remodeling and associated cellular mechanic forces. Several components of PCP signaling and its associated downstream regulatory effectors are critically linked to neural tube closure defects (a prevalent child health development issue and frequent cause of late miscarriages or early embryonic death); in addition several other diseases, including cancer, and also stem cell biology features are linked to the molecules studied here. Thus, the information acquired in this application will not only advance our understanding of regulated cellular motility, but it will also be of medical relevance in many disease associated contexts.

Epithelial cells are polarized in two axes for their function, ubiquitous apical-basal polarity and a second axis within the epithelial plane, referred to as Planar Cell Polarity (PCP). Cell polarity and ordered cellular patterning during organ development and homeostasis depend on PCP mechanisms. Classical PCP examples include in Drosophila adult cuticular structures. Similarly, in mammals striking aspects of PCP are evident in the skin, the inner ear epithelium, or the respiratory system and most other internal organs. Moreover, convergent extension processes during gastrulation and neural tube closure requires PCP signaling, and the PCP pathway is linked to the regulation of asymmetric cell divisions in stem cells of many organs. Studies of PCP establishment in Drosophila serve as a paradigm to unravel this type of polarity in development and human disease. PCP is coordinated by long-range Wnt ligand signals, resulting in asymmetric localization of their receptors, the Frizzled (Fz) proteins, and associated signaling cascade. Core Fz/PCP factors are required to interpret polarity within the cell and relay this to neighboring cells. All core Fz/PCP members are evolutionarily conserved and regulate all PCP aspects. This Wnt-pathway is distinct from canonical Wnt-Fz/?-catenin signaling (and correct regulation of signaling specificity between the two Wnt-pathways, activated by the same receptor(s), is critical for development and disease). In Wnt-PCP-signaling Fz's act both, as receptors for Wnts and ligands for its intercellular binding partner(s) Van Gogh/Vang (Vangl1/2 in mammals). The cellular mechanism(s) affecting polarity downstream of either Fz or Vang upon polarized localization remain very poorly understood. The scope and focus of my lab's research and this application is to investigate the mechanistic interactions of long-range PCP signaling and the resulting cell biological read-outs and intracellular responses. Our recent focus has been/is on Vang/Vangl function, as a result of its intercellular interaction with Fz, and the associated cellular response. Our work is and will be also guided by patient derived data and the respective functional dissection of these mutations. Based on exciting ongoing experiments, we will address the physiological significance of Fz-induced Vang phosphorylation and associated kinase function, and how these affect Vang interactions with cytoplasmic effectors. These studies will be aided by including patient data with Vangl1/2 associated neural tube closure defects. In parallel, we are dissecting the intracellular cell biological responses to PCP signaling and how these affect positioning and the mechanistic interplay with cilia associated proteins, with the advantage of being able to do so in non-ciliated Drosophila cells. A combination of in vivo studies and cell culture biochemical experiments will be performed to achieve these goals. The processes of PCP establishment and Wnt/Fz signaling have been linked to several medical abnormalities, ranging from deafness to neural tube closure defects and cancer, or ciliopathies in general. Information acquired will advance our understanding of cellular polarization, and provide medical relevance in many disease contexts.

Project Summary Wnt/Wingless (Wg)-signaling plays important roles in intercellular signaling in all metazoan organisms. It functions in many critical processes, such as organ patterning, growth control, cell polarity, and stem cell maintenance. When deregulated it is linked to diseases ranging from congenital heart disease and aberrant vasculogenesis to cancer. Recent studies in my lab have shown that Intraflagellar Transport complex A (IFT-A) proteins modulate canonical Wnt/Wg-signaling independently of the ciliary role of IFTs. We demonstrated that they do so together with Kinesin2 (as they do in ciliary functions) and promote nuclear translocation of ?- catenin upon Wnt-pathway activation and act downstream of ?-catenin stabilization. Kinesin-2 and IFT-A proteins act as a complex during Wg-signaling in Drosophila and mammals. Mutants of both, Kinesin 2 and IFT-A, affect Wg/Wnt-signaling in the same manner, and they interact genetically and physically. Kap3, a kinesin associated protein that serves as the bridging factor between Kinesin 2 and IFT-A in ciliary function is also required in the same manner and is involved in the formation of a physical complex of Kinesin 2-Kap3- IFTs. The IFT-A protein IFT140 then directly binds to ?-catenin, called Armadillo/Arm in Drosophila. Upon pathway stimulation by Wg/Wnt and resulting pathway activation, all these factors co-localize with each other and ?-catenin, and bind together to micro-tubules (MTs). Single or double mutant cells for Kinesin-2, IFT-As, or Kap3 fail to properly activate Wg/Wnt-signaling targets in both Drosophila and MEFs. In addition, axin double mutant backgrounds with IFT-A or Kinesin-2 reveal high levels of cytoplasmic Arm/?-catenin but fail to activate Wg/Wnt targets, due to reduced nuclear ?-catenin localization. These data indicate that the Kinesin-2/IFT-A complex promotes nuclear localization of Arm/?-catenin by protecting it from a cytoplasmic tether/inhibitor. We have thus identified a mechanistic function of the Kinesin-2/IFT-A complex in Wnt-signaling, independent of their role in the cilium. As the associated mechanism(s) are conserved in mammalian cells, these observations are potentially amenable to drug treatment. However, several questions remain: (i) How is the Kinesin-2/IFT-A complex promoting nuclear localization; our data suggest it is by movement along microtubules, but this needs to be refined; and (ii) what is the factor that competes with IFT140 binding to Arm/?-catenin and serves an `inhibitory' function for Arm/?-catenin nuclear localization. We will address in Aim 1 the cell biology of this mechanism using an elegant ex vivo Wnt-signaling system, and Aim 2 is tailored to identify antagonistic factor(s) that compete with IFT140 binding. Our preliminary data suggest that this knowledge can serve as an entry-point for new drug development to inhibit overactive Wnt/?-catenin signaling. Thus, information acquired in this application will advance our mechanistic understanding of Wnt/?-catenin signaling, and potentially also lead to follow up studies to develop new drugs and thus benefit the treatment in disease associated contexts.