Stephen T. Crews - US grants

The central nervous system (CNS) consists of a diverse set of nerve cells. Neurogenesis is the process in which these nerve cells are formed and attain their unique cell fates. The subject of this proposal is to identify and analyse the function of genes involved in neurogenesis. Drosophila melanogaster is an excellent system to investigate the molecular mechanisms of CNS formation because of the powerful genetic, molecular, and cellular techniques that can be utilized. We will focus our attention on the development of a specific set of nerve cells, those that lie along the midline of the CNS, and identify and study the genes that function in their formation. Our previous molecular and cellular studies have shown that the single-minded (sim) gene plays an important role in determination of cell fate of the precursors of the midline group of cells. In this proposal, the structure, expression of the different sim transcripts and gene products, and their role in neurogenesis will be investigated. Experiments will also seek to understand the mechanisms that control the specificity of gene expression during formation of the CNS. Additional genes with sequence similarity to sim in both Drosophila and vertebrates will be identified by low-stringency hybridization techniques. Genetic and molecular methods will be used to isolate novel genes that are involved in the development of the midline group of cells. This will include a genetic screen in which mutant strains are directly examined for defects in the midline nerve cells. Another genetic screen will seek mutants that interact with the sim gene. A third method involves the use of genomic insertion elements to identify genes expressed along the midline. The work proposed will begin to reveal the molecular mechanisms that control the formation of the CNS. Thus, it is likely to be relevant in better understanding the development of the human CNS, and in the study of certain mental disorders and disease.

A grant has been awarded to Dr. Stephen Crews at the University of North Carolina at Chapel Hill to purchase a high speed Drosophila embryo sorter. The Sorter allows large numbers of mutant organisms to be isolated for biochemical and molecular experimentation. The overall goal of the research at UNC-Chapel Hill involves using genetics and the study of mutant strains of Drosophila to understand the molecular and cellular basis of animal development and physiology. By comparing biochemical and cellular phenomena in mutant vs. non-mutant Drosophila strains, it can be determined how observed differences contribute to the biological process being studied. In this way, it is possible to understand, in great detail, how molecules work together to generate complex biological structures and phenomena. However, one major problem that has hampered progress is the inability to isolate sufficient numbers of mutant embryos for molecular and biochemical analyses. The recent development of the embryo sorter, which combines the technologies of flow cytometry and the use of Green Fluorescent Protein transgenic Drosophila strains, now allows the isolation of sufficient numbers of embryos for biochemical studies. The embryo sorter also allows high-throughput screening of mutant and transgenic embryos for the identification of novel mutants and genes.

The Drosophila group at UNC-Chapel Hill consists of 8 labs focused on the molecular genetics of embryonic development and physiology. All of the labs are committed to understanding biological processes using a comprehensive array of technologies involving genetics, cell biology, biochemistry, and molecular biology. Labs are interested in nervous system development, cell adhesion, hormonal control, cell signaling, cell cycle control, RNA splicing, gene transcription, and DNA repair. Large numbers of embryos will be run through the COPAS Select to isolate pure populations of mutant embryos. These embryos will be analyzed for changes in: (1) RNA populations and gene expression, and (2) protein levels and modifications. This will provide important insight into the biological role that the mutant gene normally plays. In addition, the COPAS Select will be used to sort through tens of thousands of embryos looking for those that have exceptional properties indicating the existence of novel genes, transgenic insertions, cellular markers, and mutations. These new entities will allow the expansion of research efforts into promising new directions.

Drosophila research has a number of benefits to the public involving novel insight into basic biological processes, human health, and agriculture. One of the most important lessons learned during the past 20 years of biological research is that the genes carrying-out important biological processes in humans are conserved in Drosophila. This includes many disease genes. The same is true for insect pests. Because of the advanced genetics of Drosophila, it is advantageous to initially study basic biological processes in Drosophila and then continue study in other species, including humans. There are currently a number of biotechnology companies that are utilizing Drosophila to study problems in applied science. It should also be emphasized that besides applied research, work on Drosophila has proven to be one of the best experimental systems for understanding how biological organisms function. This basic knowledge is one of the cornerstones of 20th century science (5 Nobel prizes have been awarded to Drosophila researchers), and promises to provide spectacular results in the 21st century. Further significance of the funded work concerns the educational mission of the University of North Carolina. The Drosophila labs are active training grounds for postdoctoral fellows, graduate students, undergraduates, and high school students. Use of sophisticated equipment, such as the COPAS Select, to solve complex scientific problems will enhance the education and productivity of these students.

0316102
Crews

Multi-branched tubular organs, including the insect trachea, derive from repeated groups of precursor cells that migrate and fuse together. This process involves cell movement, recognition, adhesion, cytoskeletal rearrangement, and formation of adhesive junctions. Mediating this process are specialized cells that can mediate discrete events, such as branching and fusion. The long-term goals of this project are to investigate the molecular and cellular mechanisms that underlie Drosophila tracheal fusion, and understand how transcriptional regulatory proteins mediate this process. The proposed research involves studying the molecular genetics of the Drosophila dysfusion bHLH-PAS gene, which mediates tracheal fusion events. The aims of the project involve: (1) generating dysfusion mutants, (2) carrying out genetic and cellular experiments to investigate the precise role of dysfusion in controlling tracheal fusion and transcription, (3) exploring the biochemistry, genetics, and developmental consequences of Dysfusion interactions with other bHLH-PAS proteins, and (4) initial analysis of the roles of dysfusion in other cell types. Together these approaches will advance understanding of how regulatory proteins work collectively to carry-out complex developmental and cellular events. Given: (1) the functional similarities between insect tracheal development and (2) the formation of other vertebrate multi-branched tissues, and the high degree of conservation among invertebrate and vertebrate bHLH-PAS proteins, this work has broad significance.

One major component of the proposed research is educational. Funds will be used to support training of postdoctoral fellows, graduates students, and undergraduates. Strong emphasis is placed on undergraduate training. The projects are designed to provide a multidisciplinary learning experience for each student, and each project involves training in genetics, cell biology, and molecular biology. One applied aspect of this work concerns the application of this research to the agricultural industry regarding the generation of more efficacious agents for pest control.

DESCRIPTION (provided by applicant): The Zeiss LSM5 Pascal confocal microscope is a powerful instrument for imaging cellular behavior in wild-type and mutant Drosophila and C. elegans embryos. This instrument captures high-resolution images of fixed embryos and cells labeled with multiple fluorochromes, and can be utilized for specialized functions including fluorescence-photobleaching and time-lapse experiments. The 3 laboratories seeking funding for the Pascal are well-funded labs studying a diverse set of problems regarding embryonic development. 2 of the applicant labs are senior investigators that have shifted their lab's efforts predominantly into analyzing developmental events using microscopy. One (1) of the projects is studying neural development using a genome-wide approach that requires confocal microscopic analysis of hundreds of genes. The second project studies cell adhesion, cytoskeletal dynamics, and cellular signaling pathways at high cellular resolution. The third applicant is a highly experienced cell biologist who will utilize the Pascal to study the developmental dynamics of C. elegans development. All 3 labs are seriously hampered by lack of microscope time. The Pascal will allow the labs to expeditiously and comprehensively study the cellular basis of embryonic development in combination with existing bright-field and spinning disk microscopes.

: Glia perform a variety of important roles in controlling and supporting neurotransmission in the nervous system. The Drosophila midline glia play a unique role in CNS development by controlling the guidance of commissural axons that cross the midline. These glia are derived from bipotential glial/neuronal precursors that migrate in a characteristic manner to form the glial scaffold that ensheaths commissural axons. Over 50 genes have been identified that are expressed in midline glia. Thus, the midline glia represent an excellent system to comprehensively study the regulatory pathways that control glial development and function. The Drosophila midline glia share functional characteristics with the vertebrate floorplate cells that reside at the midline of the spinal cord. The floorplate cells also play important roles in controlling the development of specific neuronal cell types, directing axon migration, and forming a scaffold surrounding the commissural axons. Consequently, our studies have relevance to issues of human health due to the importance of glia and floorplate cells for proper nervous system development and function. The major goals of this proposal are to understand the functions of Drosophila midline glia, and how they migrate and interact with neurons and axons to form the mature glial scaffolding. Mechanistic cellular insight into midline glial migration will be determined using advanced imaging techniques we have developed, including live imaging to visualize midline glia in vivo. Midline migration and ensheathment are mediated by two cell adhesion proteins, Wrapper and Neurexin IV. Genetic experiments will screen for additional genes involved in adhesion, signaling, and other processes that regulate and mediate midline glial development. Another goal is to understand the transcriptional circuitry that controls midline glial development. The use of ChIP-Seq will identify the transcriptional target sites of Single-minded throughout the genome, thus revealing how this master regulatory protein controls transcription and development. The interactions between Single-minded and Suppressor of Hairless (another important regulator of midline glial development and the downstream effector of Notch signaling) will be analyzed using genetic and molecular techniques. Transgenic approaches will be employed to identify and analyze midline glial cis-regulatory modules to mechanistically explore how midline glial transcription is controlled. Together, this project will provide a comprehensive view regarding how transcriptional circuitry, signaling pathways, and adhesion proteins control the complex morphogenetic processes required for midline glia to interact with axons and form a glial scaffolding.