Eric Liebl - US grants

This research will use genetic analysis to identify and molecularly characterize proteins that are on signal transduction pathways influenced by the Abelson tyrosine kinase (Abl) in Drosophila. Activated Abl kinase is a human oncogene, resulting in both chronic myelogenous leukemia and acute lymphocytic leukemia. As tyrosine kinase signal transduction pathways are often conserved between insects and mammals, insights into Abl kinase signal transduction pathways gained in the Drosophila model system may result in new modes of treatment for these human leukemias. Flies which are homozygous mutant for Abl develop to pupation. New mutations which enhance, or worsen this phenotype, resulting in pre-pupal lethality, have been recovered. In other words animals which are heterozygous mutant for an enhancer mutation and homozygous mutant for Abl die before pupation. As such these new enhancer mutations are in genes which are functionally redundant to the Abl kinase. Two of these new enhancer mutations (M89 and M109) will be mapped to an exact chromosomal location, and cDNAs will be recovered, cloned and sequenced. The expression patterns of these genes will be characterized. New mutant alleles of these genes will be generated and mutant phenotypes will be characterized. cDNAs cloned will be re-introduced into transgenic flies and the rescue of mutant phenotypes will be tested as confirmation that the correct cDNAs have been cloned. A novel genetic screen is also proposed. In a genetic background with sub-optimal Abl tyrosine kinase activity, new mutations will be recovered that alter this background in a kinase-dependent manner. As these mutations depend on Abl kinase activity for their effects, they will likely be in genes which code for proteins which are biochemically associated with Abl's kinase activity, such as direct substrates, kinase regulators or specific phosphatases. These mutations will be mapped to an exact chromosomal location, and cDNAs recovered, cloned and sequenced. The expression patterns of these genes will be characterized. cDNAs cloned will be re-introduced into transgenic flies and rescue of mutant phenotypes will be tested to confirm that the correct cDNAs have been cloned.

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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.

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.

DESCRIPTION (provided by applicant): Normal development requires that nerves grow to make proper contacts with other nerves or muscles. This project contributes to our knowledge of this stage of nervous system development, so that a clearer understanding of the etiology of human developmental defects arising from abnormalities of this process can be gained. As the wiring up of the nervous system occurs early in development, this process is often studied in model organisms whose embryos develop externally. In addition, as these processes have been highly conserved across the animal kingdom, model organisms amenable to genetic analysis such as fruit flies offer ideal experimental models, with knowledge gained in flies often directly applicable to humans. As a nerve cell extends its axon to connect to another nerve or muscle cell, the tip of that axon must properly navigate through three-dimensional space. Receptor proteins that extend across the tip of the axon's membrane sense both attractive and repulsive guidance cues, and the presence of those cues is translated into changes in the axon's cytoskeleton, causing the axon to extend, retract, or turn as appropriate. This project centers on the signal transduction networks linking trans-membrane guidance receptors to cytoskeletal dynamics, focusing on two key players in these networks, the Abl kinase protein and the Trio protein. Previous work in fruit flies has shown these two to have a strong genetic interaction, and Specific Aim 1 of the research is to determine the detailed biochemical underpinnings of that genetic interaction. The application's two other aims use the power of fruit fly genetics as a tool of discovery, as one way to uncover novel biochemical interactions is to find mutations that have synergistic interactions. A novel genetic mutation that exacerbates the trio mutant phenotype has been discovered, and the specific gene responsible for this effect will be cloned and characterized. Also, a systematic search encompassing more than half of the fruit fly genome will be undertaken to identify and catalog other, novel mutations with the ability to modify either the trio or the abl mutant phenotypes. Through these aims a more complete picture of the proteins involved in linking axon guidance receptors to cytoskeletal dynamics will be gained. A fuller comprehension of the signals required for normal axon guidance will set the stage for an understanding of abnormalities that may arise in these critically important processes.