Mark Seeger - US grants

DESCRIPTION (Verbatim from the Applicant's Abstract): The midline of the CNS is an interesting boundary for neuronal growth cones and provides an ideal system for dissecting molecular mechanisms of axon guidance. In vertebrates and invertebrates, the CNS midline produces both attractive and repulsive guidance cues and regulates the ability of axons to cross or not to cross. Our studies focus primarily on the Drosophila embryonic CNS where a powerful combination of genetic, molecular, and cellular approaches can be utilized to address these fundamental issues of axon guidance. This application addresses two key regulators of guidance at the midline: Commissureless (Comm) and Roundabout (Robo). Robo is the receptor for the midline chemorepellent Slit and functions to prevent axons from crossing the midline boundary. Comm encodes a novel transmembrane protein that is required for axons to cross the midline. Comm functions, at least in part, by regulating the surface accumulation of the Robo receptor. Comm and Robo form a coimmunoprecipitable complex that somehow leads to the endocytosis or membrane clearance of the Robo receptor. We have begun to map regions of Comm that are required for this effect on Robo and find that a Comm protein consisting of the transmembrane domain and the first 97 amino acids of the cytoplasmic domain is necessary and sufficient for this activity. The experiments outlined in this application are designed to test and extend our hypotheses about Comm function. We will further characterize the Comm-Robo interaction, defining amino acids required for this interaction . These structure-function studies will provide insights into the molecular mechanisms by which Comm regulates Robo. We will define where Comm activity is required during embryonic development. Complexities regarding Comm mRNA and protein distribution make this a critical issue to resolve. Comm is required for proper neuromuscular synatogenesis; as a first step in understanding this aspect of Comm function we will determine what regions of Comm are required for synaptogenesis. Finally, we propose to identify other components of the Comm and Robo pathways by using genetic screens to isolate enhancers and suppressors of specific comm mutant phenotypes. While inhibitory guidance receptors like Robo are essential for neural development, their presence at latter stages is problematic for axon regeneration. Understanding how these proteins function and are regulated is essential for the development of treatments for the devastating consequences of spinal cord injury.

0090239
Liebl

Liebl and Seeger will use the fruit fly model to detail some of the
molecular machinery involved in creating a functional, correctly wired
central nervous system during embryogenesis. They are investigating
molecular events that occur at the tip of growing axons as they navigate
through the central nerve cord and out to innervate the body. Specifically
they will address whether an established key player, the Abl protein, works
in concert with two other newly identified proteins, Trio and Amalgam.
Genetic experiments from the Liebl and Seeger labs have shown suggestive
interactions between Abl and Trio, and Abl and Amalgam. Using cell
biological, biochemical and further genetic methods, they ask whether these
proteins have any direct or indirect physical and/or biochemical
interaction.

This work will clearly deepen our understanding of the different roles the
Abl protein plays in axon tips as the central nervous system is first
forming. Much similar work from fruit fly studies has been shown to be
directly analogous to processes that occur in mammalian nervous system
development. Thus by using fruit flies to pursue such fundamental
questions as establishing the key players in central nervous system
development and understanding their interactions, these researchers are
likely laying the groundwork for a fuller understanding of the complex
processes that occur during mammalian central nervous system development.

During development of multicellular organisms, regulatory systems governing cell proliferation and developmental programs regulating the formation of specific organs, tissues or cell types, have to interface to ensure that an appropriate number of cells are generated for every tissue and organ. Equally important is the regulated termination of cell proliferation and initiation of lineage appropriate differentiation. A substantial body of work has provided a rather detailed picture of the regulatory systems controlling cell proliferation and cell cycle arrest. In contrast rather little is known, how tissue or lineage specific developmental programs functionally interact with cell cycle regulatory system. In the fruit fly Drosophila melanogaster, the pan-neural expressed neuronal precursor gene prospero (pros) is critical for proper termination of cell proliferation and initiation of neuronal differentiation during embryonic neurogenesis. In this role, pros activity is required for the proper transcriptional regulation of multiple key cell cycle regulatory genes, including the cyclin dependent kinase (cdk) inhibitor gene dacapo, the cdc25 gene string, E2F and cyclin E. Two additional pan-neural neuronal precursor genes, deadpan (dpn) and asense (ase), have been shown to be critical for cell proliferation during larval optic lobe development and proper expression of the cdk inhibitor gene dacapo. This group of pan-neural transcription factor encoding genes represent, or are part of, a critical regulatory link between neuronal lineage specific developmental programs and cell proliferation and/or neuronal differentiation. Experiments conducted under this project will firstly determine, using a range of developmental-genetic approaches, the regulatory capacity and genetic interactions of Pros in the regulation of cell proliferation and differentiation during embryonic and larval neurogenesis. Secondly, functional in vivo and in vitro analysis of transcriptional regulatory regions of dacapo, string and cyclinE will be performed to determine the specific sequence motifs involved in the pros mediated transcriptional regulation of these genes. To this end reporter gene constructs will be used to map and functionally define Pros response elements in transgenic flies. In vitro DNA binding analysis and in vitro mutagenesis experiments will complement the in vivo approaches. These experiments will provide an initial understanding of the functional mode and interactions of this newly emerging regulatory system. As all genes involved in this study are evolutionarily conserved from Drosophila to humans, the information gained from this analysis should have direct relevance for the understanding of similar developmental processes in a number of other organisms.

Project Summary

Intellectual Merit Criteria: The Drosophla model system has been instrumental in identifying many of the molecules and interactions involved in steering growth cones to their targets during nervous system development. Much work in this area has focused on the dynamic role of the cytoskeleton in directional growth cone movement; how axon guidance receptors signal to the cytoskeleton and how cytoplasmic proteins affect cytoskeletal dynamics. One of the key cytoplasmic regulators of cytoskelton dynamics is the Abelson tyrosine kinase (Abl). Genetic work from the Liebl and Seeger laboratories has shown that the guanine-nucleotide-exchange-factor protein Trio as well as the transmembrane neuronal cell adhesion molecule Neurotactin (Nrt) and Neurotactin's ligand Amalgam (Ama) are integrated into Abl-mediated signaling networks during growth cone outgrowth. Much of this proposal is designed to identify the molecular bases of these genetic interactions.
Throughout this proposal Dr. Liebl exploits the strengths of the Drosophila model system. For example, we have identified a protein:protein interaction between Trio and Abl involving these proteins' SH3 domains. He proposes a structure/function analysis of Trio, assaying the ability of Trio deletion constructs, such as an SH3-deleted version of Trio, to rescue aspects of the trio mutant phenotype. We have identified Trio as a phosphotyrosine containing protein. He proposes to both mapping the Trio's major tyrosine phosphorylation site(s), we propose and to determine the in vivo relevance of Trio's tyrosine phosphorylation by testing for the rescue of trio mutant phenotypes with a version of Trio in which tyrosine phosphorylation sites have been mutated to phenylalanine. We have also initiated a genetic screen in which proteins that interact with Trio may be identified via mutation. Under this proposal we will map and clone the genes for these proteins.
The cytoplasmic domain of Nrt is essential for its role as an adhesion molecule, and is Nrt's link into Abl signaling networks. A structure/function analysis of Nrt's cytodomain will be carried out integrating a Drosophila cell culture adhesion assay system, in vivo rescue of nrt mutant phenotypes, and comparative genomic approaches utilizing the Drosophila pseudoobsura and Anopheles gambiae (mosquito) genomes. The results of these and other proposed experiments will allow him to begin to understand the molecular underpinnings of the trio, Abl and nrt, Abl genetic interactions and the roles of these specific molecules in the growth cone. Through this work, a fuller, more accurate understanding of the molecular machinery controlling growth cone guidance will emerge.

Broader Impacts Criteria: This is a collaborative proposal between Dr. Liebl at Denison University, an undergraduate, liberal arts college, and Dr. Seeger at Ohio State University, a major research institution. This research collaboration has been active over the past five years. During this time undergraduate students at both Denison and Ohio State have been exposed to substantive research projects. The most recent publications resulting from this collaboration, in Neuron and Development, included ten undergraduate co-authors, seven of whom were women. Members of the Liebl and Seeger labs meet regularly to discuss data and exchange materials. Seeger's graduate students have "shadowed" Liebl to gain insight into a career at a liberal arts institution. Denison undergraduates are welcomed into Seeger's Ohio State lab in order to do experiments not technically possible in Liebl's Denison facilities. Thus this collaboration has proved to be a rich training ground for both graduate and undergraduate researchers, with nine undergraduates from Liebl's lab, and four undergraduates from Seeger's lab going on to post-graduate (M.D. and/or Ph.D., D.V.M.) study over the past six years.
Both Liebl and Seeger regularly teach undergraduate courses with laboratory sections. Having an active research program and ongoing exposure to the intellectual culture of a major research institution has kept Liebl's teaching up-to-date. Being exposed to the liberal arts culture where teaching excellence is of high priority has invigorated and informed Seeger's teaching.

How nervous systems are assembled and properly wired during development remains a major question in biology. At the central nervous system (CNS) midline of all organisms with bilateral symmetry, neurons make the decision to cross or not to cross from one side of the midline to the other. The CNS midline has served as a model for the molecular genetic dissection of axon guidance mechanisms. Several signaling pathways have been identified that regulate this behavior of axons at the CNS midline, including the Slit-Robo signaling pathway. This pathway is highly conserved and functions in organisms ranging from worms to humans. While this core signaling pathway is highly conserved, the regulation of Slit-Robo signaling varies in different organisms. In the fruit fly, Drosophila, the Commissureless (Comm) protein is a key regulator of Slit-Robo signaling. Comm functions by regulating the sub-cellular distribution of the Robo receptor. While Comm is essential for proper CNS development in Drosophila, Comm-like genes are not found outside of insects. By examining the sequenced genomes of diverse insects, Dr. Seeger and his colleagues have found that Comm-like genes are present in only a subset of insects. Dr. Seeger will test the hypothesis that Comm-like genes in mosquitoes and body lice regulate Robo receptor distribution as in Drosophila. They will also address how Slit-Robo signaling is regulated in insects that lack a Comm-like gene, like beetles and wasps. What will emerge from these studies is a better understanding of the mechanisms that regulate the Robo-Slit signaling pathway as well as insights into the evolution of axon guidance mechanisms. Finally, these studies will provide extensive research experiences in molecular genetics, cell biology, and developmental neuroscience for numerous undergraduates and several graduate students within the Seeger laboratory, helping to provide hands-on training for our next generation of scientists.

A complete understanding of animal development and physiology requires examining many cells of the same type in culture. These cells allow detailed investigations that are not possible while the cells are within their tissue in an intact animal. This work is being performed in Drosophila melanogaster, which has been an important genetic model for over a hundred years and for which it has not been possible to make cell lines. A novel strategy is being used to generate Drosophila cell lines, which will benefit many types of investigations. These lines will be distributed to the scientific community through by the Drosophila Genomics Resource Center. In addition to enabling many new types of important research, this project will enhance the development of the scientific workforce by including students in the project. In particular, undergraduate interns in a summer research program, drawn from all over the country, will work on developing and characterizing the cell lines. The summer program is co-directed by the investigator and is targeted to students from other universities and colleges who have limited access to research and are from groups underrepresented in science.


This EAGER project is using an innovative strategy to produce Drosophila cell-type specific cell lines. This strategy involves manipulating the expression of tumor suppressor genes via a new drug-inducible system. It is expected that expressing RasV12 reversibly in a tissue-specific manner will enable the generation of cell-type specific cell lines. Accordingly, each cell line will be allowed to propagate with high RasV12 expression, and then allowed to differentiate by turning off RasV12. These methods will be used on starter cells derived from different embryonic tissues to produce cell lines of three major cell types: epithelium, muscle and neuron. The lines will be validated, characterized, and distributed to investigators. They will enable many novel and essential experiments, such as genome-wide screens for new genes involved in epithelial cell polarity, myoblast fusion, and neurite outgrowth.

During animal development, cells increase in number as the overall body and each part of the body grow in size and complexity. The processes that coordinate growth, shape generation, and final size are essential to ensure the correct body pattern is made and key features such as limbs are properly formed. In this project, the fruit fly wing will be used as a model to study limb growth. By studying the wing at the beginning of development, when the tissue is simpler, the direct effects of genes that regulate cell growth can be tested. The research will use important resources that have been developed in the nationally funded genome projects and will involve molecular biology, genetics and microscopy. It is expected that a comprehensive view of how a limb is established from the earliest stages will emerge. The genes used in limb development in fruit flies are conserved with other animals, including humans, so that general principals will be learned. To increase public understanding of science, an animated movie will be made in conjunction with the Advanced Computing Center for the Arts and Design. The movie will highlight the beginning of fruit fly genetics, which started a field of research that has fueled the fundamental understanding of animal biology for more than a century.

As animal body parts and organs grow in size and complexity, cell proliferation must be linked to cell fates so that the final structure has the correct pattern and proportions. A major goal in developmental biology is to understand the link between patterning and growth (scaling) at a mechanistic level. In the proposed work, the small group of cells comprising the wing disc, which become the fly thorax and wing, will be studied as they increase in number from about 30 to 150 cells. In this short time window the major pattern is established. The mechanisms that operate here encapsulate those typical of all growing systems and involve conserved growth factors in the Dpp and Egfr pathways. By studying the regulation of cell-cell signaling and determining how cell fate is linked to domains of cell proliferation, the work is expected to both fill a gap in knowledge about early development of the Drosophila wing and provide insight into fundamental processes shared in all animals. In an effort to broaden public understanding of genetics, the story of the pioneer Drosophila geneticist, Morgan, will be documented in a biographical film. In partnership with ACCAD (Advanced Computing Center for the Arts and Design), a state-of-the-art graphic film will be made that describes the discovery of the first Drosophila mutant - the famous white-eyed fly. The film will be posted on YouTube for broad dissemination.