Thomas B. Kornberg - US grants

DESCRIPTION (provided by applicant): Our discovery of cytonemes, specialized filopodia that orient toward cells that express signaling proteins, suggest a novel mechanism to move signaling proteins between producing and target cells. This model postulates that signaling proteins move between cells in a manner similar to the way neurotransmitters move at neuronal synapses, that fundamentally, neurons and non-neuronal cells communicate in similar ways. Recent work from my lab now provides direct and convincing experimental evidence that cytonemes ferry signaling proteins between producing and receiving cells, strongly supporting the cytoneme model. Our work also identified several unexpected properties of cytonemes that have significant implications for mechanisms of signal transduction. The work proposed in this application will develop new tools for imaging cytonemes and will build upon our previous findings to determine the roles, composition and functions of these remarkable organelles.

The developmental mutants of Drosophila melanogaster have proven to be a rich and productive area of study for investigations into basic mechanisms of development. Mutants have been isolated that transform one segment into another - for example whole legs replace antennae in the heads of Antennapedia flies - suggesting that it is the function of such genes to decide whether to adopt a particular developmental pathway. Other mutants have been isolated that reduce the number of segments, that alter the polarity of the segments, or in the case of the maternal-effect bicaudal alleles, that alter the polarity of the egg and create an embryo with two posterior ends. Such phenotypes suggest processes that organize and subdivide the young embryo. How the products of these many different genes cooperate to fashion the normal embryonic patterns is not known. However, it is commonly believed that the maternal-effect genes function during oogenesis and during the early, pre-cellular stages of embryogenesis to organize the embryo. It is believed that these genes operate to define embryonic polarity and positional coordinates and to provide sufficient information for cellular blastoderm formation. Since very little gene expression has been detected prior to cellularization, it has been assumed that the role of the zygotically active genes in determining segment number and type is important only after cellularization. In the work proposed here, we will challenge this assumption: we will directly determine the stage at which zygotically active genes have a controlling role in development. Based upon our recent discovery that a number of mutations in zygotically-acting genes affect morphogenesis during the pre-cellular stages, we already know that our previous conceptions about the relative contributions of the maternally- and zygotically-acting genes must be re-evaluated.

Funds are sought to purchase an instrument to facilitate analysis of radioactive materials after fractionation by electrophoresis or chromatography. Although autoradiography has for many years been the method of choice to quantitate Southerns, Northerns, Westerns, and chromatograms, X-ray films suffer from poor sensitivity and limited dynamic range. Recent advances in other technologies now make alternatives attractive, because their use can cut exposure times significantly and provide more accurate quantitation. Among the several competing technologies we have tested, the best in terms of versatility, performance , and adaptability for use by many investigators is the Molecular Dynamics PhosphorImager. In our experience, use of this machine not only improves efficiency by reducing the time wasted while waiting for gels, blots, and chromatograms to be exposed, but the precise quantitation of the PhosphorImager opens up possibilities for new assays and applications that would otherwise be overly cumbersome or too imprecise.

DESCRIPTION: (provided by applicant) The aim of this predoctoral training program in Genetics and Cell Biology is to train scientists to conduct research and prepare for careers in modern molecular genetics and cell biology and, in general, in the biomedical sciences. This program represents the merger of two existing successful training programs, "Genetics of Prokaryotic and Eukaryotic Organisms" with "Cell and Molecular Biology," which recognizes how they have operated to a considerable extent in the past. The new training program will bring together individuals who are studying central problems in genetics and cell biology, including regulation of the cell division cycle, control of DNA distribution in mitotic and meiotic cells, protein trafficking, and cytoskeletal organization using the tools of genetics and biochemistry. These studies exploit a wide range of systems, including bacteria, yeast, nematodes, zebrafish, and humans. The goals of the program are accomplished through coursework and other activities in genetics and cell biology and related areas and through the student's research. The features of our training program that are especially attractive to incoming students are (a) a large number of active, excellent laboratories from which students can choose for their thesis research, (b) a laboratory rotation system, which provides meaningful research experience in different laboratories, (c) an excellent set of courses that enables students with little prior training in genetics and cell biology to acquire a solid foundation in these areas, (d) tutorial training with faculty on how to present a seminar, (e) a highly collegial and interactive atmosphere at UCSF, (f) a high faculty to student ratio, and (g) an awareness that training graduate students is important to the UCSF faculty.

The long-term goal of this project is to understand the molecular mechanisms that program the events of yeast sporulation -- the process by which yeast forms gametes. This process begins with a diploid vegetative cell and yields an ascus containing four haploid meiotic products derived from that cell. Because the steps of yeast meiosis are like those of higher eukaryotes, it is expected that the mechanisms and machinery used in yeast may shed light on meiosis in higher organisms and thus provide insights into the molecular basis of infertility and birth defects in humans. Prior studies by others led to the hypothesis that the events of sporulation are determined by a transcriptional cascade involving sequential transcription of early, middle, and late genes. Prior studies by others also identified what appears to be a key decision-making point at pachytene, after replication and recombination but before the first meiotic division, which monitors completion of recombination. We have recently identified the regulatory protein, Ndt80p, that governs transcription of many of the middle sporulation genes and proposed that it may play a central role in the decision of cells to initiate meiotic divisions after completion of replication and recombination. In addition, we have carried out whole-genome expression studies using DNA microarrays which reveal that Ndt80p controls transcription of more than 150 genes during sporulation. The goal of this proposal is to determine how the program of sporulation is controlled using Ndt80p as a jumping off point. In particular, these studies seek to understand how Ndt80p synthesis and activity are regulated and how it functions to activate transcription of a large group of genes involved in chromosome segregation and spore morphogenesis. Learning about Ndt80 protein is likely to yield enormous insight on the decision to exit meiotic prophase and on the overall program of sporulation.

PROJECT SUMMARY

This is a proposal to study how the dorsal air sacs of Drosophila adult develop. The dorsal air sacs are the major tracheal organs of the adult fly. They are complex, multi-lobed structures that function to fulfill the extreme demands of the indirect flight muscles for oxygen. Previous work in the Kornberg laboratory showed that dorsal air sac development begins during the third larval instar when a small group of cells in the wing imaginal disc produce FGF and this signaling protein induces nearby tracheal cells to embark on a program of growth and tubulogenesis. Their work also showed that long, cytoneme-like filopodia extend from the tracheal cells to the wing disc cells and that these cell extensions may serve as conduits for the movement of FGF between disc and tracheal cells. The studies proposed in this application delve further into the genesis of the dorsal air sacs, exploring how the cells of the wing disc and tracheal system interact to control the cell divisions, movements and growth during development of the dorsal air sacs.

Key issues addressed by this work are:
1) Experiments are proposed that will test the possibility that the progenitor cells of the dorsal air sac are the products of a developmental program that replaces larval tracheal cells with imaginal tracheoblasts during the third instar. Preliminary data obtained in the Kornberg lab suggests that this program involves regulated cell proliferation, death and migration and that it is triggered locally by the molting hormone ecdysone. Surprisingly, the tracheal cells themselves may synthesize the ecdysone that initiates this program. The role of ecdysone in regulating this program will be examined.
2) The genesis of most tubular organs in vertebrates involves extensive cell proliferation, but most tubular organs in Drosophila develop without cell proliferation. The dorsal air sacs are an exception, and experiments are proposed that will provide a better understanding of how cell proliferation is regulated and how it contributes to air sac development.
3) Secreted proteins provide instructional signals during development, but we do not yet understand how they move over long distances to reach their target cells. The interactions between the wing disc and tracheae that regulate dorsal air sac development present an excellent system to study the mechanism of long distance signaling. The structures that are postulated to mediate signaling will be examined.

The proposed activities will be carried out as part of a training program for graduate students and postdoctoral fellows at the University of California, San Francisco and will include the participation of undergraduate students recruited to UCSF through their Summer Research Training Program that provides undergraduates, including under-represented minorities, with opportunities to conduct research.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the mechanisms of long-distance cell signaling that are important to development and to disease. We address these important issues with a model system, applying its powerful genetic and molecular techniques to identify and analyze key genes and processes. Our work has shown that in Drosophila, the hedgehog (hh) gene plays a central role setting up and regulating both the segments in the embryo and the compartment borders and signaling centers in the wing disc. Our recent work has focused on the activities of its receptor, Patched (Ptc) and on the transcription factor that mediates Hh signal transduction, Cubitus interruptus (Ci). The human orthologs of both Ptc and Ci have been implicated in a number of human diseases, including cancer. Specific aims are proposed in this application to: (1) complete a forward genetic screen to identify additional components of the Hh signal transduction pathway; (2) identify novel targets of Hh signal transduction using complementary molecular approaches; (3) characterize the structure and regulation of the Patched receptor; (4) analyze the conversion of Ci to its repressor and activator forms; and (5) describe the mechanism by which Hh moves from its sites of synthesis to engage its receptor in neighboring or a distant cells. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goal of this research is to understand the processes that induce, regulate and pattern development and morphogenesis of the Drosophila dorsal air sacs (DAS). Our work has shown that Bnl-FGF induces this large, complex and multi-lobed tracheal organ which functions in ways that are analogous to the vertebrate lung. This organ ventilates the adult fly and supplies oxygen to its flight muscles. Its development offers a powerful system to study the pathways that lead to organ induction, growth and patterning. Specific aims are proposed to: (1) gain a detailed description of the development and morphology of the DAS; (2) identify and analyze genes expressed at various stages and at different locations in the DAS; and (3) identify and characterize mutants defective in DAS development. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytonemes are long filopodial processes we believe are used in long range signaling of growth factors during development. Cytonemes express growth factor receptors and extend up to 800 um before contacting their target cells in the imaginal disk. We wish to use electron microscopy to study the contact points between cytonemes and target cells in Drosophila larvae.

DESCRIPTION (provided by applicant): Cell cycle progression, cell migrations and gene expression that occur at the wrong time or place, or that are inaccurately regulated, can result in abnormal development and can lead to disease states. There are many systems that can be studied to improve our understanding of the cell cycle, cell movements and gene expression. One that has several unique advantages is the early Drosophila embryo when nuclear cycles produce approximately four thousand nuclei within the first two hours after fertilization. These early cycles offer one of the few tractable systems with both high uniformity and an abbreviated and apparently simplified cell cycle. This proposal describes an investigation of these nuclear divisions; findings that are the basis for the proposed experiments already reveal that our understanding of this early, critical stage of development must change radically.

DESCRIPTION (provided by applicant): Hedgehog signaling has been implicated in many cancers and has been shown to be a useful target for anti-cancer therapy. Previous work, including key contributions from my lab, have established that the Hedgehog protein is one of the major regulators of animal development, playing key roles in most tissues and organs in both invertebrates and vertebrates. Using newly developed reagents and methods that open new ways to study Hh signaling, the work proposed in this application will identify and characterize the processes that Hh-producing cells use to prepare and deliver Hh protein, the processes that Hh-receiving cells use to prepare for and handle its arrival, and the processes that regulate the form and activity of Ci, the transcription factor that is responsible for Hh pathway output. These issues are critical to understanding how new and better therapeutic regimens controlling Hh activity can be developed.

? DESCRIPTION (provided by applicant): Recent progress in tumor biology has revealed that stromal cells - the non-transformed neighbors of tumor cells - play essential roles for tumor stem cells, tumor progression and metastasis. These roles involve communication between tumor and stromal cells, but the signals that are exchanged and the mechanism by which these signals move and elicit responses remain obscure. This is a proposal to build on our recent discovery that specialized organelles called cytonemes move and exchange signaling proteins between epithelial and mesenchymal cells in Drosophila - revealing that paracrine signaling in these normal contexts is mediated by cytonemes that make direct synaptic contacts between signaling cells. Importantly, we also identified mutant genetic conditions that compromised cytonemes and eliminated the contacts cytonemes normally make to mediate signaling, and showed that signaling was abrogated if cytonemes did not make direct synaptic contacts with target cells. All the paracrine signaling was cytoneme-dependent. We also tested the prediction that tumor cells communicate with stromal neighbors by a similar mechanism, and when we examined cells in a Drosophila tumor model, we detected cytonemes that extend from tumor cells to their normal neighbors. The presence of these organelles is consistent with the idea that they mediate signaling between tumor and stromal cells. The objective of the work proposed here is to investigate the role of cytonemes in tumorigenesis in Drosophila and mouse models. The goals are to identify where and in what form cytonemes are present, discover how cytonemes link tumor cells with their non-transformed neighbors, and determine whether cytoneme-mediated signaling is essential for the tumor growth. Based on previous studies, the likelihood that cytonemes are present and have essential roles in the vertebrate contexts is high. The proposed work may open a new avenue for controlling tumor growth, and by bringing this novel mechanism of cell-cell communication to the attention of the broader community of cancer biologists, it may alert them to the importance of cytoneme-mediated signaling and to the practicality of harnessing it for studies and therapy.