Judith Lengyel - US grants

The goal of this proposal is to understand the function of the Drosophila segmentation gene tailless ( tll ). The mutant phenotype consists of characteristic defects or absence of structures of segmental origin located near the anterior and the posterior of the ectodermal region of the blastoderm. The remaining segments appear larger. Like the identified "gap" segmentation genes, tll mutants alter the ftz blastoderm striping pattern. Hence, tll acts early in development. However, it appears to serve a unique function, as there are no other zygotically active genes altering both poles of the embryo. While the development of a segment pattern in insects has been explained on the basis of a double gradient resulting from the action of two separate systems, it has not been predicted that the products of a single gene might be involved in establishing the two opposite poles. The objectives are to use molecular techniques to isolate the tll gene and to characterize its gene product. In situ hybridizations will be carried out to early embryos to determine whether the transcript is localized to the anterior and posterior regions altered in the mutants. The transcription unit will be defined and the transcription pattern determined. The gene will be sequenced, antibodies will be raised against fusion proteins, and the effect of mutations in other segmentation genes on expression of tll will be assessed. The experiments will provide insight into a unique gene which controls segmentation at the two ends of an organism, and so into the process of cell determination at the onset of development.

We are investigating the contributions of transcription, processing and turnover to the expression of specific genes in Drosophila cultured cells and embryos. We have evaluated these parameters in detail for the RNA transcribed from the copia sequence, a mobile DNA sequence in the Drosophila genome. In order to understand the role which the copia sequences and mobile DNA elements in general play in the cell, we are now investigating the translation product of copia RNA. In order to compare the control of expression of the copia sequence, which is expressed continuously in the cultured cells, with the control of expression of genes which are induced, we are currently measuring the parameters of synthesis, processing and turnover for two genes which are induced to a high level of expression by the stimulus of heat shock. Finally, we have demonstrated that the level of histone gene expression in Drosophila embryos is controlled both by changing rates of synthesis and by changing rates of turnover. We are currently investigating different mechanisms by which the control of histone mRNA turnover might be controlled.

The blastoderm stage in Drosophila melanogaster is a time of cell determination and dramatic transitions in the cell cycle and in transcription. The similarities between this stage and the midblastula transition in amphibians suggests that there is a constellation of events involving the cell cycle, transcription and cell movement that is common to the early development of many organisms. We are studying these events in Drosophila because of the potential for integrating molecular with genetic analyses in this system. We have succeeded in identifying four Drosophila blastoderm specific genes: terminus (ter), polebud (pod), serendipity (sry) and bsg25A. Using standard genetic screening techniques, as well as restriction fragment length polymorphism mapping, in situ hybridization to polytene chromosomes, and P- factor mediated transformation, we will identify mutant alleles in each genes, as we have done for the delta gene of the sry locus. In so doing, we will test the relationship between ter, pod and bsg25A, and hid, slater and schlaff, respectively, all embryonic lethal mutations which map close to these genes and affect embryonic morphology. We will characterize the mutant phenotype of each gene by light and electron microscopy. The ter gene encodes a TFIIIA-like, putative DNA-binding finger. We will use antibodies to ask whether the protein is present in the nucleus and will use recombinant DNA methods to engineer the production of fusion and native ter proteins to ask whether it binds DNA in vitro. If so, we will identify specific DNA-binding sequences. We will identify the cis-regulatory sequences of the ter gene, and test the hypothesis that the gene is autoregulatory and that the finger plays a role in this regulation. We will test the significance of domains of structural similarity between the pod protein and fos oncogene protein by characterizing the spatial expression of the gene (by in situ hybridization to whole embryos and antibody staining) and asking whether the pod protein is nuclear or associated with the cytoskeleton. We will prepare a molecular map of the bsg25A locus, sequence its mRNA, and examine the spatial expression of the gene in the embryo. The analysis of these blastoderm-specific genes will provide insight into the unique events in regulation of the cell cycle, cell movement and cell commitment that occurs during early embryogenesis. The regulation of these processes is relevant to similar events and their derangement, as they occur at later times during development and in the adult organism.

This project involves the preparation and publication of a catalog of the cloned DNA of Drosophila. Previous editions of "Cloned DNA by Chromosome Location," "Chromosome Rearrangements available for jumping, with one breakpoint known within a cloned sequence." and "Transformed lines available for cloning or deleting DNA at specific chromosome sites" are in press in the Drosophila Information Service. We anticipate that information about molecular genetics in Drosophila and the number of clonal genes will continue to increase dramatically. Based on that additional information future editions of the catalog of cloned DNA will be prepared and published. This catalog makes it possible for a worker interested in a particular chromosome site or gene to see if it has already been cloned or if a nearby site has been cloned or transformed. Such examples may be useful as the starting points for "walks" or "jumps" to clone adjacent sequences. We will prepare a computer operation to maintain and update this information. We will also establish the relational aspects of the three present lists by means of appropriate software. This will facilitate making the information available for inclusion in the future editions of "Genetic Variants of Drosophila melanogaster." We anticipate publications of the catalog of cloned DNA in 1986 and 1988 in the D.I.S.

A cell signalling pathway, encoded by the maternal terminal system and bearing striking similarity to the PDGF-activated pathway in mammalian systems, is active at both poles of the Drosophila embryo. In the posterior, this pathway is required to establish a largely unsegmented domain, the telson. In the anterior, a morphogen gradient, consisting of the bicoid (bcd) transcription factor, interacts with the terminal system to establish the nonsegmented acron instead of a telson. The key zygotic gene activated in each of these domains is tailless (tll), which encodes a steroid receptor-like putative transcription factor. To understand the establishment of head at the anterior rather than tail, we will characterize bcd control of tll in the anterior. We will determine, using both in vitro and in vivo techniques, bcd protein binding sites in the tll promoter, and characterize their interaction with the terminal system. To understand how a nonsegmented domain is subdivided, we will investigate the hierarchy of gene activity in the posterior. This will first involve determination, in vitro and in vivo, of tll protein binding sites in presumed target genes (both those activated and those repressed by tll). Because so few genes are known that are required in the posterior, we will carry out genetic screens to identify additional members of the terminal hierarchy, and place described and newly discovered genes into a pathway. Understanding in greater detail the gene expression activated by a cell signalling pathway should contribute to our knowledge of genes that might be deranged in oncogenesis. Also, it is becoming evident that both general mechanisms of developmental control, and even specific genes required in embryogenesis, that have been identified in Drosophila have homologs in vertebrates. By characterizing a genetic hierarchy required to subdivide an embryonic field into smaller, nonsegmented domains (as occurs in the vertebrate dorsal-ventral axis, limb bud and retina) we will increase our knowledge of mechanisms by which positional information is interpreted and refined. Ultimately this will allow us to understand the genetic and cellular basis of human birth defects.

Many growth factors affect the decision of a cell to divide or differentiate by binding to a specific receptor tyrosine kinase (RTK) in the cell membrane. Activation of RTKs has been found to lead, through a series of common intermediates, to activation of MAP (mitogen-activated protein) kinases. The specificity of effect of different RTKs may be due, at least in part, to the diversity of transcription factors modulated by these MAP kinases. Very few transcription factors have been shown to be direct targets of an activated RTK pathway; even less is known about athe genes that these activated transcription factors regulate. Our goal is to identify and characterize the transcription factor target of a genetically well described RTK. The pathway activated by the torso (tor) RTK, which functions to establish fate at the two poles of the early Drosophila embryo, is uniquely suited for studies to identify a novel transcription factor modulated by an RTK. This pathway has been studied in detail genetically and most of its components identified; staged Drosophila embryos can be collected in quantities suitable for protein purification. We have identified a small (<14 bp) tor Response Element (tor-RE) in the promoter of the tailless (tll) gene (the key gene regulated by the tor pathway). The tor-RE is unusual in that it acts as a repressor element; its function is inactivated in cells in which the tor RTK is active, allowing transcription of tll. To identify the protein (the tor-REB) binding to the tor-RE, and characterize its function in the tor RTK pathway and possibly in other pathways, we propose to do the following; 1.) further determine the presumptive tor-RE by site-directed mutagenesis and transformation analysis, 2.) purify the transcription factor (the tor-REB) that binds to the tor-RE, 3.) clone and sequence the gene encoding the tor- REB, 4.) characterize the interaction between the tor-REB and components of the tor system (particularly mAP kinase), 5.) characterize tor-REB RNA and protein expression during development in Drosophila, 6.) carry out genetic studies on the tor-REB gene to confirm that it functions in the terminal pathway, and to assess its role in different pathways and developmental processes. 7.) Identify the mammalian homolog of the tor-REB and test whether it is modulated by RTK activated pathways in vertebrates. These studies are expected to provide novel insights into the molecular mechanisms by which RTK activated pathways affect cell differentiation and proliferation, and may identify genetic targets for diagnostic screening in cancer.

We are now in the position to be able to study molecular/genetic pathways controlling morphogenesis. Certain processes of morphogenesis, particularly convergent extension, which cells intercalate between each other to generate a longer, narrower structure, are common to many crucial events in development, such as gastrulation and tubulogenesis. The generality of these processes, as well as the numerous homologies recently discovered between Drosophila and vertebrate genes, virtually assures that genes controlling morphogenesis discovered and analyzed in the model organism Drosophila will have vertebrate homologs. Development of the posterior gut provides an excellent entree into the problem of convergent extension, since this process occurs in the Drosophila embryo after cell proliferation has already occurred, and at least for the hindgut, without the complicating factor of interaction with mesoderm. Additionally, earlier work from our and other laboratories on embryonic patterning has already characterized the early transcription factor hierarchy that establishes the gut primordia. By screening for mutants affecting posterior gut development, we have identified the arc gene, which controls convergent extension in the Malpighian tubules, and the drm gene, which controls elongation of the hindgut. The Arc protein contains a single PDZ domain and appears to associate with the apical membrane surface. We will characterize Arc function by localizing it in more detail, and determining what proteins it interacts with. We will look for Arc-related proteins in Drosophila and vertebrates to see if they play similar roles in morphogesis. We will carry out a molecular characterization of drm, to determine its expression pattern, and the molecular properties and cellular location of the Drm protein. Using genetic screens, we will identify additional previously undescribed genes that play a role in posterior gut morphogenesis. We will characterize phenotypes of all of these genes in more detail (including at the electron microscope level). We will use these data to generate a model for the genetic basis of convergent extension morphogenesis in gut tubules. Characterization of the Drosophila arc, drm and other genes will assist in identifying vertebrate genes controlling morphogenesis; defects in these vertebrate cognates are likely to contribute to human birth defects.