2003 |
Reiter, Jeremy F |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Semaphorin-Plexin Signaling &Vertebrate Cell Movement @ University of California Berkeley
DESCRIPTION (provided by applicant): Historically, cell movement has been studied using in vitro or invertebrate systems. This proposal describes the application of the tools of mouse developmental genetics and biochemistry to understanding the regulation of vertebrate cell movement. It focuses on the role of plexin-A1, a semaphorin co-receptor previously implicated in axon guidance. Preliminary evidence suggests that plexin-A1 is required for the regulation of diverse developmental events including ventral folding, heart morphogenesis and angiogenesis. This proposal describes four sets of experiments designed to explore how plexin-A1 directs these morphogenetic processes. First, detailed molecular and cellular analysis of plexin-Al-deficient mouse embryos will clarify the role ofplexin-A1 in ventral folding. Second, chimera analysis will be used to identify which cells use plexin-A1 to direct morphogenesis. Third, analysis of other mutants with similar ventral folding defects will reveal how other molecules interact with plexin-A1 to execute ventral folding. Fourth, a combination of biochemical and developmental genetic approaches will address the mechanism of plexin signaling. By illuminating the role of plexin signaling in development, these studies may provide understanding into congenital disease as well as how cell movement is co-opted by pathologic processes such as tumor invasion and tumor angiogenesis.
|
0.976 |
2004 — 2005 |
Reiter, Jeremy F |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Embryonic Stem Cell Formation of Definitive Endoderm @ University of California San Francisco
[unreadable] DESCRIPTION (provided by applicant): The goals of this proposal are twofold: the creation of a versatile genetic system for manipulating the fate of mouse embryonic stem cells and the use of this system to begin to investigate the signaling pathways that regulate early definitive endoderm development. Stem cells have received much attention as potential sources for novel therapies. By virtue of their ability to differentiate into a wide variety of different cell types, embryonic stem (ES) cells may also represent an opportunity to learn about the genetic regulation of normal embryo development. To investigate the genetic control of ES cell differentiation, tools permitting regulated gene expression in ES cells are required. To this end, we are developing a novel gene expression system called TetTarget for use in mouse ES cells. TetTarget builds upon the well-described Tet regulatory system to confer robust gene expression and, unlike expression systems currently employed in ES cells, provides highly specific regulation of the level of induction, the timing of induction and the cell types in which gene induction occurs. Additionally, we make use of recombination mediated cassette exchange technology to allow new genes of interest to be efficiently introduced into the TetTarget expression cassette. Thus, TetTarget is a versatile genetic system that we anticipate will be helpful to members of the developmental biology community seeking to study varied aspects of stem cell biology. We propose to use the TetTarget system to study the development of definitive endoderm. We have chosen to concentrate on endoderm development for two reasons: less is known about endoderm development than mesoderm or ectoderm development, and a better understanding of endoderm development will help to establish approaches to producing stem cell-derived hepatic and pancreatic cells. Our initial efforts will focus on evaluating the function of genes previously implicated in early endoderm development. Specifically, we will use TetTarget to express candidate endodermal regulators such as activators of the Nodal signaling pathway and members of the Sox, Gata, Forkhead and Mix transcription factor families in embryonic stem cells. We will assess whether these regulators promote definitive endoderm formation and how changing the level and timing of expression affects endoderm production. Ultimately, this approach seeks to provide novel insights how ES cell differentiation into definitive endoderm is regulated, and by extension, how the definitive endoderm arises and develops in vivo. [unreadable] [unreadable]
|
1 |
2007 — 2021 |
Reiter, Jeremy F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Hedgehog Signaling At the Cell's Antenna: Smoothened and the Primary Cilium @ University of California, San Francisco
ABSTRACT Hedgehog signaling is a means of intercellular communication that, in vertebrates, relies on immotile cellular appendages called primary cilia. In our previous work, we discovered that vertebrate Hedgehog signals move Smoothened to primary cilia, that this movement is necessary for Smoothened activity, and that certain cancers depend on their cilia for constitutively active Hedgehog pathway activity. Despite these insights into Hedgehog signaling, how Smoothened movement to cilia is regulated and how ciliary Smoothened activates the downstream pathway remain unclear. In this renewal application, we examine the molecular mechanisms by which the Hedgehog receptor, Patched1, controls Smoothened activity (Aim 1), how Smoothened is activated within the cilium (Aim 2), and how Smoothened activates its downstream effector, the transcription factor GLI2 (Aim 3). We have discovered that the ciliary membrane has a unique lipid composition, and that specific ciliary lipids are necessary and sufficient to activate mammalian Hedgehog signaling. Therefore, the first two aims focus, in part, on how Patched1 regulates ciliary lipids and how ciliary lipids control Smoothened activity. These experiments will help reveal how lipids control signaling, and may identify new therapeutic strategies for blocking Hedgehog pathway-related cancer formation. How Smoothened communicates to GLI2 remains unknown. We have created a biochemically tractable knock-in Gli2 allele that will allow us to uncover mechanisms by which cilia regulate GLI2 activity in embryogenesis and oncogenesis. Thus, the proposed experiments use a combination of mammalian genetic, cell biological, imaging and biochemical approaches to reveal how the Hedgehog signal transduction pathway uses cilia to transmit information, both in development and disease.
|
1 |
2010 |
Arkin, Michelle R. (co-PI) [⬀] Reiter, Jeremy F |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Novel High Content Screen For Regulators of Ciliogenesis and Hedgehog Signaling @ University of California, San Francisco
DESCRIPTION (provided by applicant): The Hedgehog pathway is one fundamental means of controlling mammalian cell behavior and is used to regulate a wide variety of disparate biological events including tissue patterning, stem cell renewal, and cell proliferation. All Hedgehog signaling relies on the proto-oncogene Smoothened, mutations in which can cause basal cell carcinoma, the most common cancer in North America, and medulloblastoma, the most common solid cancer among children. Tumors have displayed resistance to Smoothened antagonists currently in clinical trials, indicating a need for other inhibitors of the Hedgehog pathway. We have discovered that mammalian Hedgehog signals move Smoothened to an organelle called the primary cilium, and that this movement is necessary for pathway activation. Although it is known that almost all mammalian cells possess a single primary cilium that extends into the extracellular environment, the functions of this organelle are poorly understood. We hypothesize that the primary cilium acts as a cellular antenna, through which Hedgehog signals are transduced. Despite their importance to both development and disease, the molecular mechanisms by which the cilium is generated and by which Smoothened functions remain unclear. To address these gaps in our understanding, we seek to conduct a high-throughput screen to identify novel compounds that can inhibit cilium formation or Smoothened movement to cilia. Using a combination of high-throughput microscopy and high-content analysis, we will visualize and quantitate Smoothened localization to the cilium in cultured cells. We will subject novel compounds that inhibit Smoothened localization to the cilium to secondary assays that reveal whether the compound affects cilium biogenesis, Smoothened trafficking, and Hedgehog pathway activity, among other parameters. This work will produce the chemical tools to pharamacogenetically dissect the molecular mechanisms of ciliogenesis and Smoothened regulation. Additionally, given the central role misregulated Hedgehog signaling plays in diverse human cancers, these compounds may provide the bases for the development of novel chemotherapeutic drugs. PUBLIC HEALTH RELEVANCE: Problems in cell-cell communication can underlie diseases as diverse as birth defects and cancer. One mechanism of cell-cell communication uses Hedgehog proteins, secreted signaling proteins. Defects in Hedgehog signaling can cause birth defects, such as holoprosencephaly, and several types of cancer, including medulloblastoma. Recent evidence has revealed that Hedgehog signaling relies on the primary cilium, a poorly understood organelle present on most cells. To be able to better understand how Hedgehog signaling and the primary cilium function, we propose to conduct a screen for small molecules that perturb these processes. These chemicals may provide the basis for the development of novel therapies for Hedgehog-related cancers.
|
1 |
2011 — 2014 |
Reiter, Jeremy F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Tissue-Specific Regulation of Ciliary Function by the Transition Zone @ University of California, San Francisco
DESCRIPTION (provided by applicant): Many cells in the human body possess a singular projection from their surface called a primary cilium. Although the existence of primary cilia has been recognized for over a century, only recently has it become clear that they function in the detection and interpretation of important intercellular cues. Some of these cues, such as Hedgehog signals, are key regulators of embryonic patterning and adult tissue homeostasis. Consequently, defects in Hedgehog signaling can cause birth defects and some forms of cancer. Similarly, defects in primary cilia can cause rare congenital syndromes such as Meckel and Joubert syndromes, may underlie more common human diseases such as polycystic kidney disease, and are important for the progression of some cancers. Mammalian cells with ciliary defects fail to respond to Hedgehog signals. We have found that some tissues are ciliated at specific times in development, and within adult tissues, some cells can be ciliated and others not. We hypothesize that the control of which cells are ciliated shapes how tissues respond to cilium-interpreted signals. We have recently identified a class of novel genes called the Tectonics, which support ciliogenesis in some tissues and control ciliary membrane composition in others. The Tectonics interact and co-localize with ciliary disease proteins at a subdomain of the cilium called the transition zone. We propose to study Tectonics and their interactors to understand the tissue-specific regulation of ciliary functions. Specifically, we will answer three complementary questions: 1) Do different Tectonics regulate ciliogenesis in different tissues? 2) Do Tectonics and other transition zone components promote ciliogenesis by regulating protein transport to the cilium? 3) Do Tectonics function with human disease genes, such as those underlying Meckel syndrome and polycystic kidney disease, to regulate ciliogenesis? The proposed experiments combine genetic, imaging and biochemical approaches to provide answers to these questions. This work will elucidate the mechanism by which Tectonics and associated proteins contribute to ciliary function, providing molecular and cell biological insights into how cilia function in development and how they misfunction in human ciliopathies. PUBLIC HEALTH RELEVANCE: Primary cilia are small projections found on many human cells involved in receiving and interpreting signals from other cells. Disruption of these ciliary signaling events contributes to birth defects, cancer, polycystic kidney disease, and other human disorders. We propose to investigate the mechanisms controlling cilium formation and protein composition to provide a molecular understanding of cilia-related diseases.)
|
1 |
2014 — 2017 |
Reiter, Jeremy F |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Predoctoral Training in Developmental Biology @ University of California, San Francisco
DESCRIPTION (provided by applicant): We seek to renew support for training of up to 10 predoctoral students per year in the Developmental and Stem Cell Biology (DSCB) Graduate Program at UCSF. The DSCB program builds off of a foundation of cell biology, biochemistry and genetics to instill deep understanding of development and developmental disorders. As the field of developmental biology is rapidly advancing, we provide our students a dynamic, interdisciplinary education that incorporates the most recent conceptual and experimental advances. Our students acquire the concepts and skills to make groundbreaking contributions to developmental biology, as evidenced by their pioneering discoveries and successful scientific careers. DSCB is a degree-granting, cross-campus program governed by an Executive Committee and run by a Director and an Associate Director. 61 faculty members in 18 basic science and clinical departments participate in the program. The faculty are leaders in their respective fields, have active research laboratories, and provide extensive mentorship to DSCB students to maintain membership. New interactions among laboratories and trainees are facilitated by an annual retreat, seminars, an annual student-run symposium, two weekly journal clubs, faculty research talks, joint research meetings, and shared student supervision. Previously (1994-2010), this grant supported students who studied developmental biology through two broader graduate programs. During the last funding period, the DSCB program instituted a wide range of improvements to make the training more dynamic and interdisciplinary. These changes culminated, in 2011, with the DSCB program conducting its own admissions and creating its own curriculum, allowing us to attract students from diverse backgrounds and of exceptionally high caliber who choose to join the DSCB program for its broad range of thriving thesis research laboratories and a faculty dedicated to excellence in graduate education. Other improvements that we have enacted include: (1) introducing an effective system of six-week laboratory rotations that accelerates selection of a thesis laboratory and helps reduce the years to degree; (2) completely overhauling the core developmental biology course and integrating it with revised courses in cell biology and genetics; (3) establishing literature-intensive, small group mini-courses; (4) expanding instruction on grant-writing, oral presentation, peer-review, ethics and the responsible conduct of research; (5) providing additional opportunities for teaching and leadership experience; (6) ensuring access to state-of-the-art equipment and facilities; and (7) expanding our cooperative, interactive faculty. The training grant supports students during their 1st and 2nd years of study. Student progress is monitored through classes, program requirements such as the qualifying exam, and regular thesis committee meetings. Students have been very successful at acquiring independent funding to cover subsequent years. Renewal of this training grant will support the intellectual growth of DSCB students, ensuring that each graduate is an independent scientist who helps lead the field of developmental biology for decades to come.
|
1 |
2015 — 2021 |
Reiter, Jeremy F |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core C: Genetics and Genomics @ University of California, San Francisco
Biomedical Core C (Genetics & Genomics): Summary Twenty-four of the 58 laboratories within the UCSF-NORC employ genetics or genetic manipulation in their research. Some focus on uncovering genetic alterations regulating feeding behavior, metabolism or nutritional homeostasis associated with obesity and its health complications in humans. Others use genetically-defined animal models to study the mechanisms underlying those processes. The targeted alteration of genes within those model organisms is a tractable method to test molecular hypotheses about the involvement of those pathways. Still others use expression profiling of samples from humans, model organisms or cell lines to establish candidate pathways involved in energy balance. The UCSF NORC Genetics and Genomics Core provides access to, assistance with, and training in the use of an array of sophisticated methods and instruments for genetic and genomic assessment of molecular pathways underlying nutrition and obesity. As the Genetics and Genomics Core facilities are overseen by five administrative units at UCSF, this Core provides the infrastructure through which NORC users make use of those facilities. The Genetics and Genomics Core assists NORC users to identify and access those facilities. The NORC also supports the development of novel genetic and genomic technologies that are determined via a well-defined process to be of emerging need to the NORC research base. The NORC Genetics and Genomics Core provides tools and facilities for: 1. Global expression analyses, including single cell sequencing, to uncover relevant pathways in laboratory interventional studies. 2. Functional studies involving genetic alteration of model organisms or cell lines, including targeted knock-outs/ins and transgenics. 3. Maintaining and generating animal models required for NORC research. Overall, this Core lowers methodological barriers to help NORC researchers achieve the efficient and proper application of genetics and genomics tools. These capabilities accelerate a variety of diverse and interrelated studies of obesity, nutrition, food intake and metabolism.
|
1 |
2015 — 2021 |
Reiter, Jeremy F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transition Zone Control of Ciliary Signaling @ University of California, San Francisco
? DESCRIPTION (provided by applicant): Many cells in the human body possess a singular projection from their surface called a primary cilium. Although the existence of primary cilia has been recognized for over a century, only recently has it become clear that they function in the detection and interpretation of important intercellular cues. Some of these cues, such as Hedgehog signals, are key regulators of embryonic patterning and adult tissue homeostasis. Consequently, defects in Hedgehog signaling can cause birth defects and some forms of cancer. Similarly, defects in primary cilia cause congenital syndromes such as Meckel and Joubert syndromes, can underlie more common human diseases such as polycystic kidney disease, and are essential for the progression of some cancers. To function in signaling, primary cilia need to maintain a different composition than surrounding parts of the cell. We identified the transition zone, a region of the ciliary base, as a critical regulator of ciliary composition. To understand how the transition zone controls which proteins localize to cilia, we will answer three complementary questions. First, given that the transition zone is a complex and highly structured region of the cilium, we will determine how it is built. Identifying the architecture of the transition zone and how it is disrupted by ciliopathy mutations will provide structural insights into the origins of ciliary signaling defects. Second, we will examine whether the transition zone regulates protein entry into the cilium, exit from the cilium, or acts as a diffusion barrier at the ciliary base. Understanding how different components impart different characteristics to the transition zone will help reveal how this gate controls ciliary composition. Third, we will examine how different complexes cooperate within the transition zone to support ciliogenesis and ciliary signaling. These experiments will help elucidate how mutations in different transition zone components result in different developmental phenotypes, both in mice and humans. By elucidating the mechanisms by which the transition zone controls ciliary composition, we will understand how the cell compartmentalizes this organelle to perform critical signaling functions during mammalian development.
|
1 |
2016 — 2018 |
Arkin, Michelle R. (co-PI) [⬀] Reiter, Jeremy F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A High Content Screen Dissecting Ciliogenesis and Oncogenic Hedgehog Signaling @ University of California, San Francisco
? DESCRIPTION (provided by applicant): This proposal seeks to identify and characterize drug-like molecules that inhibit ciliary Hedgehog (Hh) signaling. Cells orchestrate their behaviors by communicating through secreted signals such as Hh proteins. Unlike other intercellular signals, mammalian Hh proteins are transduced through primary cilia, microtubule-based projections on the surface of many cells. The Reiter lab discovered that a central component of the Hh pathway, Smoothened (Smo), moves to the primary cilium in response to Hh stimulation where it activates the downstream pathway. The cilium is essential for Hh signaling both in embryonic development and in oncogenesis. Activating mutations in Smo cause basal cell carcinoma (BCC), the most common cancer in the U.S., and medulloblastoma, the most common solid cancer in children. A Smo antagonist was recently approved for clinical use, although cancers can become resistant quickly. Smo antagonists that act through complementary mechanisms may prevent the emergence of resistance in Hh-associated cancers. To identify novel Hh pathway antagonists and gain insight into ciliary function, the Reiter and Arkin labs completed a pilot screen for inhibitors of Smo movement to cilia. This screen identified novel inhibitors, some of which block BCC cell growth by inhibiting Smo translocation and some of which block ciliogenesis. Investigating the mechanisms by which these inhibitors act revealed previously unknown aspects of ciliary Hh signaling. This project will expand the screen, identify the molecular mechanisms underlying inhibitor action, and understand ciliary signaling. Specifically, we will: 1) screen in-house and Chemical Biology Consortium libraries, substantially expanding the chemical space that has been assessed, 2) execute secondary screens to efficiently identify inhibitors that act through previously undescribed mechanisms, 3) use newly identified antagonists, together with unique genetic tools, to uncover unrecognized steps of ciliogenesis and Smo translocation. Despite their importance to both development and disease, the mechanisms underlying ciliogenesis and Hh signaling remain unclear. The proposed investigation will provide tools to reveal how cells communicate through cilia. These compounds may also provide leads for novel chemotherapeutics, critical for preventing the emergence of resistance and relapse in Hh pathway-associated cancers.
|
1 |
2016 — 2019 |
Reiter, Jeremy F Vaisse, Christian [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Obesity in Ciliopathies: How Neuronal Primary Cilia Control Appetite @ University of California, San Francisco
? DESCRIPTION (provided by applicant): Our studies address how neuronal primary cilia control obesity. The primary cilium is a cell surface projection that receives and transduces select extracellular signals. In humans, mutations that disrupt the function of primary cilia cause ciliopathies, pleiotropic diseases of which obesity is a cardinal manifestation. How ciliary dysfunction leads to obesity is unclear, but is thought to involve disruption of neuronal signaling pathways that regulate energy homeostasis. The Melanocortin-4 receptor (MC4R) is a central mediator of the regulation of long-term energy homeostasis and mutations in MC4R are the most common monogenic cause of severe human obesity. We have found that MC4R and the recently described MC4R associated protein MRAP2 localize to the primary cilia and that disruption of primary cilia abolishes the anorexigenic function of MC4R in mice. These results suggest three important hypotheses: 1) MC4R functions at the cilium. 2) The endogenous MC4R ligands, ?MSH and AGRP, are sensed by the second order MC4R-expressing neurons through their primary cilia, making this non- synaptic mechanism of modulating neuronal activity an important component of long-term energy homeostasis. 3) Disruption of MC4R signaling is the cause of obesity in ciliopathies. We will test these hypotheses by identifying how MC4R and MRAP2 are targeted to cilia, how ciliopathy-associated proteins participate in MC4R function, and how these proteins signal through cilia to indicate satiety. This work will define molecular and cellular steps of neuronal regulation of energy homeostasis and answer the long-standing question of how ciliary dysfunction causes obesity in humans.
|
1 |
2017 — 2018 |
Dynlacht, Brian D [⬀] Reiter, Jeremy F |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Restoring Ciliogenesis as a Novel Approach to Blocking Breast Cancer Growth @ New York University School of Medicine
PROJECT SUMMARY The primary cilium, an antenna-like structure on the surface of most quiescent and differentiated cells, is essential for transducing signals required for growth control, including Hedgehog (Hh) signals. Consequently, loss of cilia alters responsiveness to extracellular cues. Many cancers, including breast cancer, exhibit near- complete loss of cilia during the early stages of oncogenic transformation. Novel strategies are urgently needed to treat the most lethal sub-type, triple-negative breast cancer (TNBC). TNBC is disproportionately observed in African-Americans and thus represents a key health disparity. We propose that restoring ciliogenesis to breast cancer cells will restore growth suppressive signaling and block tumor growth. Our preliminary data indicate that (1) cancer cells can regrow cilia; (2) single gene or pharmacological intervention can cause cancer cells to regrow cilia; and (3) ciliary regrowth inhibits proliferation. We will build on these findings by conducting an innovative functional screen to identify the genes that restrict ciliogenesis in breast cancer cells, and test whether restoring ciliogenesis limits tumor growth in vivo. The identified ciliogenesis inhibitors will help unravel the molecular mechanisms by which cancer cells suppress ciliogenesis, identify how cilia inhibit breast cell proliferation, and test, for the first time, whether reversing the loss of cilium-associated signaling restores growth regulation to transformed breast cancer cells. In Aim 1, we will perform a novel genome-wide screen to identify inhibitors of ciliogenesis in breast cancer. Prior screens have identified regulators that promote ciliogenesis. In contrast, our screen will identify negative regulators of cilium assembly. We will also test the innovative hypothesis that restoration of cilia in combination with use of clinically relevant inhibitors of ciliary signaling can block tumor growth. In Aim 2, we will test how restoring ciliogenesis impacts breast cancer growth in animal models. Together, these studies will reveal how cancer cells restrict ciliogenesis, an important, unanswered question in cancer cell biology. The successful demonstration that restoring ciliogenesis inhibits tumor growth will reveal a novel strategy for targeting breast cancer, which may be applicable to other cancer types. By taking advantage of the complementary strengths of the Dynlacht and Reiter laboratories, we will innovatively combine RNAi screening, state-of-the-art ciliary analysis, and organismal cancer modeling. While highly exploratory, in keeping with the R21 mechanism, discovering the means to promote ciliogenesis and restrain breast cancer growth will identify novel ways that tumors avoid growth control and may establish a novel paradigm for treating breast cancer. Furthermore, to address cancer health disparities, we will: (1) begin to lay the mechanistic foundation for understanding why TNBC disproportionately affects African-Americans and (2) acquire and characterize primary TNBC samples to determine whether tumor ciliation or expression of regulators of ciliogenesis differs in different ethnic populations. !
|
0.942 |
2017 — 2021 |
Reiter, Jeremy F Yoder, Bradley K. (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Understanding Ciliary Functions in Mammalian Development @ University of California, San Francisco
Project Summary/Abstract Many cells possess projections from their surfaces called cilia. Mouse genetics has been instrumental in uncovering novel requirements for cilia in development, such as left/right axis specification, and skeletal and neural tube patterning. During mammalian development, cilia generate and sense flow, and interpret intercellular cues, such as Hedgehog signals. Defects in ciliary function in humans cause diverse diseases known as ciliopathies. Despite the importance of the cilium, fundamental aspects of ciliary biology, including how cilia are constructed, how they signal, and how they coordinate diverse developmental events, remain poorly understood. To illuminate answers to these longstanding questions, we propose to make use of a unique resource, the knockout mouse mutant strains being created by the International Mouse Phenotyping Consortium (IMPC). We have developed an innovative algorithm and demonstrated that it can identify mutant lines for which ciliary analysis will be valuable. We will use this algorithm to select mutants, and use these mutants to answer three complementary questions about cilia: 1) How are the sophisticated structures and subdomains of cilia built and contribute to ciliary function? 2) How is ciliary motility established and regulated? 3) How do cilia transduce intracellular signals, such as Hedgehog signals, and how does loss of this intercellular communication contribute to the pathogenesis of ciliopathies? To address these questions, we will combine advanced imaging approaches, including super-resolution microscopy, micro-computed tomography, with novel genetic tools such as transgenes that label cilia with GFP. The expertise accrued during our combined 25 years working on cilia in mouse development will allow us to use the novel mouse mutants to uncover novel principles underlying ciliogenesis and ciliary signaling. These discoveries will help identify the causative genes for orphan diseases, and illuminate the developmental origins of human ciliopathies.
|
1 |
2018 — 2019 |
Kodani, Andrew Tadashi (co-PI) [⬀] Reiter, Jeremy F |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
How Zika Virus Disrupts Neuronal Cellular Architecture @ University of California, San Francisco
Project Summary Zika virus (ZIKV) is a flavivirus transmitted by mosquitoes and sex. Outbreaks are linked to congenital neurodevelopmental defects, including microcephaly. Recent studies have revealed that ZIKV can infect neural stem cells and inhibit neurogenesis in mice and humans. However, the cellular mechanisms by which ZIKV disrupts brain development remain largely mysterious. We have found that ZIKV disrupts centrosome organization, a phenotype associated with inherited forms of microcephaly (MCPH). ZIKV infection induces supernumerary Centrin foci that accumulate the MCPH- associated protein CEP63, a critical regulator of centriole duplication. The nonstructural protease/helicase produced by ZIKV called NS3, localizes to the centrosome and binds to CEP63 and its interacting protein, CCDC14. Like ZIKV infection, expression of NS3 alone induces supernumerary Centrin foci that accumulate CEP63, suggesting that ZIKV NS3 interacts with and alters the function of the MCPH-associated protein CEP63 to disrupt centrosome organization in neural precursor cells. As centrosome defects cause human microcephaly, we propose that ZIKV-produced NS3 causes microcephaly by interacting with and abrogating the centrosomal function of the microcephaly protein CEP63. To understand how NS3 disrupts centrosome organization and how centrosome disorganization contributes to the development of microcephaly, we will investigate how NS3 localizes to the centrosome, how it acts at the centrosome to change its architecture, how altered centrosomal architecture changes the identity of human neural precursor cells, and how ZIKV-associated changes in centrosomes disrupt the innate immune response. Together, these studies will provide insights into the parallels between ZIKV-associated microcephaly and inherited forms of microcephaly, shedding light on the fundamental mechanisms by which centrosomes function during neurogenesis and providing candidate targets for inhibiting the deleterious effects of ZIKV on cortical development.
|
1 |
2021 |
Reiter, Jeremy F |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Illuminating the Function of the Understudied Kinase Dyrk2 in Ciliary Hedgehog Signal Transduction @ University of California, San Francisco
ABSTRACT We defined the proteomes of cilia from diverse organisms, including sea urchins and sea anemones and identified DYRK2, a poorly studied kinase not been previously implicated in ciliary biology. Subsequent study confirmed that DYRK2 localizes to cilia and revealed that loss of DYRK2 disrupts ciliary morphology. We also found that DYRK2 participates in ciliary Hedgehog signal transduction, communicating between Smoothened and GLI transcription factors, two central components of the pathway. Mutation of mouse Dyrk2 resulted in skeletal defects reminiscent of those caused by loss of Indian hedgehog. Like Dyrk2 mutations, pharmacological inhibition of DYRK2 dysregulated ciliary length control and attenuated Hedgehog signaling. In this pilot project, we will examine the molecular mechanisms by which DYRK2 functions in ciliary morphology, Hedgehog signaling, skeletal development and cancer. More specifically, we will investigate how DYRK2 acts downstream of Smoothened to transduce Hedgehog signals (Aim 1), how DYRK2 functions in skeletal development (Aim 2), and whether pharmacological inhibition of DYRK2 may be a tractable therapy for Hedgehog pathway-associated cancers (Aim 3). The proposed experiments use a combination of mammalian genetic, cell biological, imaging and biochemical approaches to reveal how DYRK2 functions in ciliary Hedgehog signal transduction, both in development and disease.
|
1 |