A Molecular Genetic Analysis of the Role of the Guanine Nucleotide Exchange Factor Trio During Axon Pathfinding in the Embryonic CNS of Drosophila Melanogaster

Download or read book A Molecular Genetic Analysis of the Role of the Guanine Nucleotide Exchange Factor Trio During Axon Pathfinding in the Embryonic CNS of Drosophila Melanogaster written by David J. Forsthoefel and published by . This book was released on 2005 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: The Drosophila melanogaster embryo is an ideal system in which to study axon guidance, because of the relative simplicity of the nervous system and the evolutionary conservation of the molecules utilized during development. The Abelson cytoplasmic tyrosine kinase regulates actin cytoskeletal dynamics in Drosophila, mice, and humans. In Drosophila, Abl is expressed in the developing central and peripheral nervous systems (CNS and PNS). In a genetic screen for modifiers of the Abl mutant semilethality phenotype, we identified trio, a cytoplasmic guanine nucleotide exchange factor that is also expressed in the CNS and regulates actin dynamics through Rho GTPases. Mutations in Abl and trio interacted genetically, leading to dramatic disruption of axon pathways at the CNS midline. Building upon these initial observations, we analyzed interactions between Abl, trio, and the attractive Netrin receptor frazzled (fra)/Deleted-in-Colorectal-Cancer (DCC). In fra;Abl and fra;trio double mutants, few axons crossed the midline, similar to the phenotype in trio, Abl mutants. Furthermore, mutations in Abl and trio suppressed the inappropriate midline crossover phenotype in embryos expressing the chimeric Robo-Fra receptor, consistent with an in vivo role for these molecules as Fra effectors. Fra bound Abl and Trio in coimmunoprecipitation and GST pulldown experiments, and tyrosine phosphorylation of Fra and Trio was elevated in cultured cells overexpressing Abl. Mutations in enabled (ena), another Abl substrate, suppressed the loss-of-commissure phenotype in fra, Abl, and trio mutants, as well as the Robo-Fra receptor phenotype. Together, these results suggest that Abl and Trio are effectors for multiple attractive receptors at the CNS midline, and that Ena may function during both attractive and repulsive signaling. Finally, a functional analysis of the requirement for Trio's conserved domains has been initiated. In transgenic rescue and overexpression experiments, TrioGEF1 was required for axon guidance across the CNS midline, while TrioSH3 inhibited midline crossing. Coexpression experiments with the Robo-Fra receptor and assays in other tissues and cultured cells suggest that the conserved N-terminal domain, spectrin-like repeats, and GEF2 domain may modulate GEF1 signaling in specific contexts. Future experiments must elucidate the mechanistic details of cytoskeletal control by Trio and Fra.