David S. Stein - US grants

The dorsal-ventral axis in the Drosophila embryo is established by an inductive cue originates in the somatic follicle cells that ensheathe the developing oocyte. This inductive cue requires the expression of the maternal effect locus pipe in the somatic tissue of the female. The activity of this gene contributes to defining the polarity of a signal transduction pathway that results in graded nuclear uptake of the dorsal gene product, a transcription factor. The broad, long-term objective of this proposal is to elucidate the molecular nature of the somatic inductive cue and how it functions and is regulated. These investigations have medical relevance in two respects. Drosophila embryonic dorsal- ventral polarity appears to result, at least in part, from extracellular regulation of serine proteolytic activity, similar to what is seen during blood clot formation and complement fixation in humans. Second, regulation of nuclear localization is a general mechanism for the regulation of transcription factors and the dorsal gene shares amino acid sequence homology with a number of vertebrate transcription factors whose activities are also regulated in this way. One of these, the transcription factor NF kappa B is an important transcriptional regulator of the immune system. To investigate the mechanism by which the somatic epithelium of the egg chamber controls embryonic dorsal-ventral polarity polarity the pipe gene will be cloned and characterized. Its nucleotide sequence will be determined and temporal and spatial patterns of expression of the RNA will be investigated. Antibodies directed against the pipe protein product will be obtained and immunohistochemistry will be used to investigate spatial and subcellular localization of the protein during dorsal-ventral pattern formation. To determine how the prior determination of dorsal-ventral polarity in the egg chamber influences embryonic dorsal-ventral polarity, the pipe RNA and protein expression patterns will also be investigated in the ovaries of females carrying mutations which alter the polarity of the egg chamber.

Establishment of dorsal-ventral (DV) polarity in the Drosophila embryo has its origins in the egg chamber within which the oocyte develops. The dorsally-located oocyte nucleus regulates the polarity of the overlying follicular epithelium via an inductive interaction involving the Drosophila homologues of TGFalpha and the EGF receptor. DV polarity is then transmitted from the follicular epithelium to the developing embryo via a signal mediated by the products of the genes nudel, windbeutel and pipe. These genes regulate a serine proteolytic cascade that ultimately produces a ligand for the Toll receptor in the embryonic membrane. We have recently cloned the pipe gene, which exhibits a ventrally-restricted expression pattern that is altered in the background of maternal-effect mutants that affect egg chamber DV polarity. The pipe locus encodes two isoforms of a protein with amino acid sequence similarity to the previously identified heparan sulfate 2-O-sulfotransferase (HSST) from Chinese Hamster Ovary cells, an enzyme shown to be involved in glycosaminoglycan (GAG) modification. The overall aims of this proposal are to confirm that Pipe functions as an HSST and investigate how spatially-restricted activity of this protein is involved in the localized activation of the Toll receptor by its ligand. To determine which of the HSST isoforms fulfills the ovarian function of pipe, cDNAs encoding the different isoforms will be expressed in the ovary under inducible transcriptional control and their ability to define ventral cell fate assessed. Nucleotide sequence alterations in mutant alleles of the pipe gene will be identified to provide information about functionally important parts of the protein. Antibodies will be raised against the protein product to determine its subcellular localization in the egg chamber. A series of experimental approaches will be used to confirm the role of GAGs in the establishment of dorsal-ventral polarity, to determine which GAG side chain is/are likely to be the targets of Pipe and to determine whether the Nudel protein carries the GAG side chain(s) modified by Pipe. The Drosophila dorsal-ventral pathway is a model for a variety of medically-relevant vertebrate processes involving localized serine proteolytic activation or receptor/ligand interactions. A role for sulfated GAGs has been demonstrated for both of these processes in humans. Thus, the identification of Pipe as an HSST confirms and enhances the value of this pathway to serve as a model for studying human disease.

DESCRIPTION: (Applicant's Description) Altered expression of the c-myc proto-oncogene has been observed in nearly one seventh of fatal cancers in the United States, implying a fundamental involvement of c-myc in cancer. c-myc encodes a transcription factor of the basic helix-loop-helix/leucine zipper type. A major unresolved dilemma in understanding the role of c-Myc in cancer is determining how its oncogenic function is related to its activity as a transcriptional regulator and to its function in normal cells. Drosophila melanogaster is a superb system in which to carry out functional and genetic studies of protein function in vivo. Further, the high degree of structural conservation of proteins in flies and vertebrates provides assurance that the mechanisms and genes discovered in Drosophila are likely to perform similar functions in humans. To initiate a genetic analysis of the function of Myc proteins in the Drosophila system, we and our collaborators isolated the Drosophila homologue of the human c-myc gene, d-myc, and demonstrated that it corresponds to the product of a known Drosophila gene, diminutive (dm). We have subsequently generated additional, lethal mutations in dm. To begin a structure/function analysis of the Drosophila c-Myc homolog, we will first determine the changes in the protein coding sequence that correspond to the new, lethal mutations in dm. Our second specific aim will determine the in vivo consequences of the loss of d-myc function using the newly generated lethal alleles of dm. Zygotic mutants for these alleles, which do not develop beyond the first larval instar, and homozygous mutant clones in the eye will be characterized with markers for cell size, cell division, DNA replication and apoptosis. The requirement for d-myc expression during oogenesis will be examined in females carrying germline or follicle cell clones of dm mutations. The third specific aim of this proposal will identify genes encoding proteins that either regulate d-Myc expression or function, or are themselves targets of d-Myc transcriptional activity by carrying out genetic screens for modifiers of d-myc-related phenotypes in the ovary and eye. We anticipate that this work will not only provide a means to test current hypotheses regarding the function of c-Myc, but will also identify novel genes and pathways involved in regulating the c-Myc network. In addition to providing valuable insights into the normal function of c-Myc, we are optimistic that new directions for therapeutic interventions of cancer will be suggested through the enhanced understanding of c-Myc regulators and effectors that will be achieved via the genetic analysis of the Drosophila c-Myc homolog.

Project Summary
The mechanisms through which the seemingly simple fertilized egg generates a complex multicellular organism is a topic of fundamental interest that has been pondered since the time of Aristotle. A major insight that has emerged from studies of early development in the fruitfly Drosophila melanogaster is that the generation of pattern in the embryo is dependent upon the prior establishment of pattern in the ovarian follicle in which the egg is formed. The mother fly provisions the developing egg with a set of localized determinants that will later direct the spatially- specific expression of the embryo's own genes. Among the structures that are specified by localized maternal determinants are the most terminally-positioned structures of the Drosophila embryo, the acron at the anterior and the telson at the posterior. Formation of the termini requires activation of the transmembrane receptor tyrosine kinase Torso, which is distributed throughout the membrane of the early embryo. This activation must occur specifically at the ends of the embryo. The ligand for Torso is thought to be the Trunk protein, which is secreted into the fluid-filled perivitelline space that lies between the embryonic membrane and the inner surface of the eggshell, the vitelline membrane. Spatially-restricted activation of Torso by Trunk is believed to be controlled by the product of the gene torsolike. During oogenesis, torsolike is expressed in two specialized groups of polar follicle cells at the anterior and posterior ends of the developing oocyte. Our laboratory has found that Torsolike is localized to the anterior and posterior ends of the eggshell. These observations suggest a mechanism whereby an inactive precursor form of Trunk is secreted uniformly into the perivitelline space and processed into an active ligand specifically at the ends of the egg where it then binds and activates the Torso receptor. Trunk activation requires the function of Torsolike in a process that has not been elucidated. The objectives of this work are to determine the molecular mechanism by which Trunk becomes activated, and to elucidate the role Torsolike protein plays in this process. The specific aims are: (1) To determine whether Trunk is processed from an inactive precursor into an active ligand. (2) To identify other effector molecules involved in the formation of the Trunk ligand by their abilities to interact with Torsolike, and through the characterization of new mutations that affect the specification of the termini. (3 ) To identify the determinants of Torsolike that mediate its localization in the vitelline membrane of the eggshell. Intellectual merit: In addition to their contribution to the understanding of pattern formation in early development these experiments have significance outside of the context of Drosophila early development. Signal transduction through receptor tyrosine kinases is required for a wide variety of cell/cell communication pathways that operate in numerous physiological and developmental contexts, including axonal pathfinding, cell migration, cell differentiation and proliferation. The role of the Drosophila Torso receptor in embryonic patterning is a particularly compelling paradigm because of the exquisite regulation of its activation, both spatially and temporally. The control of Torso activation by its ligand is likely to provide important insights applicable to a variety of other ligand/receptor interactions that are of intellectual, as well as medical interest. Broader impacts resulting from the proposed activity: (1) Discovery, understanding, teaching, training and learning. The maternal control of Drosophila embryonic anterior-posterior polarity is a textbook paradigm that is used in undergraduate teaching of developmental biology. The experiments described herein seek a deeper understanding of this process. Furthermore, the investigations proposed provide numerous opportunities for experimental contributions by undergraduate students at the University of Texas, which will contribute to "hands on" training of those students in a research setting. Interested undergraduate students will be encouraged to participate in this project. These students will have the opportunity to present their work at local and national scientific meetings. Both the graduate student researcher and faculty associates comprising the research team on this proposal will also engage in undergraduate teaching. (2) Participation of underrepresented groups. The University of Texas matriculates a large population of students from historically underrepresented groups. Opportunities for hands on training in laboratory research related to the proposed studies will be available to interested students from these groups. (3) Dissemination of Results. Results obtained will be published in the scientific literature and reported at scientific conferences. Moreover, segments of the proposed investigations involve the generation of reagents likely to be of use to other investigors. These will be disseminated freely to any who request them.

DESCRIPTION (provided by applicant): Dorsal-ventral polarity in the Drosophila embryo is initiated during oogenesis by expression of the Pipe sulfotransferase in the ventral follicle cells which, by an unknown mechanism, leads to the localized activation of a serine protease cascade in the perivitelline space between the embryonic membrane and the eggshell. Our long-term goal is to understand how this serine protease cascade is spatially regulated. The objectives of this application are to elucidate the mechanism through which spatial restriction of the serine protease cascade is implemented and to identify the target of Pipe action in the follicle cell layer. Our central hypothesis is that Pipe activity in the ventral follicle cells results in the sulfation of a glycoprotein or glycolipid that is secreted into and localized within the ventral perivitelline space where it brings about the localized activation of the serine protease cascade. The rationale for the proposed research is that it will greatly expand our understanding of DV pattern formation and will also provide insight into mechanisms that regulate analogous localized serine proteolytic events that influence human health such as the ones involved in the formation and breakdown of blood clots. Blood coagulation and fibrinolysis are critical factors in the etiology of thrombotic diseases such as heart attack and stroke which represent leading causes of death worldwide. Thus, the proposed research is relevant to the mission of the NIH to obtain fundamental knowledge that will potentially reduce the burden of disease. We will pursue two Specific Aims: 1) To determine the extent to which the activity of the serine protease cascade is regulated by the localization of the proteases themselves, by their spatially restricted activation or activities, or by physical interactions with one another that modulate their activities. Functional, tagged versions of the proteases will be used to examine their processing and spatial distribution and to investigate protein-protein interactions in wildtype and various genetic backgrounds. 2) To identify genes involved in the synthesis of the target of Pipe sulfotransferase activity in the ventral follicle cells. Genetic screens will be carried out to identify mutations that lead to the production of dorsalized embryos by mutant females. The proposed research is significant as it will yield new insights into mechanisms that regulate serine protease action and thus may contribute to the development of improved therapeutic agents for the modulation of serine proteases that influence human health.

DESCRIPTION (provided by applicant): Sulfated carbohydrates such as glycosaminoglycans control many, diverse processes affecting human health including blood clotting, immune and neural cell migration, viral and microbial infection, and growth factor/receptor signaling. However, for many of these processes, how the sulfated carbohydrates mediate their biological effects at the mechanistic level is not understood, presenting a barrier to our ability to harness the properties of these molecules for therapeutic purposes. Drosophila embryo dorsal-ventral polarity is determined by a sulfated carbohydrate embedded in the eggshell that influences serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective (GD) processes and activates Snake, Snake processes and activates Easter, and Easter cleaves the Spaetzle precursor into a ligand for the transmembrane receptor Toll. Activation of Easter occurs solely on the ventral side of the egg under the control of Pipe, a homologue of vertebrate glycosaminoglycan sulfating enzymes that is expressed in ventral follicle cells of the developing oocyte. Pipe transfers sulfate to the carbohydrate side chains of glycoproteins that become incorporated into the inner vitelline membrane layer of the eggshell and constitute a ventral cue that promotes processing of Easter by activated Snake. The long-term goal of this project is to determine how the sulfated ventral cue controls the spatially restricted processing of Easter, and in turn, the activation of Toll. Th central hypothesis, which is based on the recent observation that GFP-tagged GD becomes ventrally localized in the perivitelline space, is that processed GD binds to the sulfated ventral cue and once concentrated there, facilitates an interaction between Snake and Easter that results in Easter processing. The proposed investigations pursue three specific aims: 1) To elucidate the mechanism that controls the ventral localization of GD within the perivitelline space of the egg by determining the extent to which GD processing is required for its localization and by identifying determinants within GD that are required for localization and processing. 2) To determine how localized GD mediates the cleavage of Easter by Snake by identifying the GD domains that are required for its interactions with Easter and Snake, and by determining the extent to which GD promotes Snake protease activity, Easter susceptibility to cleavage, or productive interaction between the two proteins. 3) To elucidate the structure of the Pipe-modified carbohydrates that comprise the ventral cue and determine how these molecules facilitate GD localization and Easter processing by isolating VML, a key eggshell protein modified by Pipe, examining its associated carbohydrates by mass spectrometric analysis, and identifying proteins that interact with it in a Pipe/ventral cue-dependent manner. The proposed studies are significant because in the course of elucidating the molecular mechanism underlying this paradigmatic developmental pathway, they will yield new insights into the ways in which serine proteases and other medically important proteins are influenced by their interactions with sulfated carbohydrates. PUBLIC HEALTH RELEVANCE: The goal of this work is to elucidate the mechanism through which a sulfate-modified carbohydrate molecule, present ventrally in the Drosophila egg, controls the formation of the Drosophila embryo dorsal- ventral axis. The proposed research is relevant to public health because many medically critical processes including blood clotting, cell migration, infection by viruses, bacteria and parasites, and signaling between ligands and their receptors are dependent upon sulfated carbohydrates. The proposed research is relevant to the part of NIH's mission that pertains to pursuing fundamental knowledge about the nature and behavior of living systems in order to reduce the burdens of illness in that it will provide insigh into the etiology and treatment of disease states that are influenced by sulfated carbohydrates.

DESCRIPTION (provided by applicant): For genes encoding proteins that are required for organismal or cell viability, an inability to generate and examine loss-of-function mutant phenotypes can be an impediment to elucidating protein function. Homozygous mutant individuals may die at an early stage in development, making it difficult to establish the differen roles that the relevant proteins play at later stages of development. Tissue-specific generation of homozygous mutant clones, or expression of dsRNA, can in some cases overcome this limitation. However, these strategies are subject to the problem of protein perdurance beyond the time at which gene expression ceases, which can complicate analyses in which rapid elimination of gene function is required. Temperature-sensitive (ts) mutations provide an alternative means of conditionally eliminating gene product function. However, ts mutants are laborious to isolate and cannot be applied to the study of protein function in homeothermic organisms such as mammals. The objective of the investigations described in this proposal is the development of a general method for rapid, spatially- and temporally-controlled light-induced elimination of proteins of interest for phenotypic analysis. Two specific aims will be pursued to develop and test two distinct experimental methods to achieve protein elimination. Specific Aim 1 will utilize a small protein tag that we refer to as the photodegron, which can be expressed as a genetic fusion to other proteins. The photodegron undergoes a light-dependent conformational change that exposes a degradation signal recognized by a ubiquitous class of ubiquitin ligases, the N-recognins. In preliminary studies carried out in yeast, the photodegron mediated light-dependent elimination of function of the heterologous proteins to which it was attached. Continuing work will examine the kinetics of photodegron-induced degradation in yeast and test the ability of the photodegron to direct protein degradation in Drosophila embryos. Additional studies will increase the versatility of the photodegron method by incorporating a signal that targets it for degradation via the Nedd4 family of ubiquitin ligases. The investigations in Specifi Aim 2 will pursue a second strategy to achieve light- induced protein degradation in vivo by making use of the light-dependent interaction between the Arabidopsis thaliana proteins Cryptochrome 2 (CRY2) and CIB1. Planned experiments will test the ability of CRY2-ubiquitin ligase fusion proteins to mediate the degradation of proteins fused to CIB1 in Drosophila embryos. Based on recent advances in the understanding of light-responsive proteins and of ubiquitin/proteasome-mediated protein degradation, we hypothesize that it will be possible to use the sophisticated genetics of yeast and Drosophila to develop facile experimental strategies for rapid, temporally- and spatially-controlled protein elimination. These methods will overcome several limitations associated with currently available strategies for generating protein loss-of-function phenotypes and will provide a powerful tool for the study of medically important proteins involved in a wide variety of biological questions and experimental organisms.