Eric C. Liebl

Dosage-Sensitive, Reciprocal Genetic Interactions between the Abl Tyrosine Kinase and the Putative GEF trio Reveal trio's Role in Axon Pathfinding

The Abelson tyrosine kinase (Abl) is integrated into signal transduction networks regulating axon outgrowth. We have identified the Drosophila trio gene through a mutation that exacerbates the Abl mutant phenotype. Drosophila Trio is an ortholog of mammalian Trio, a protein that contains multiple spectrin-like repeats and two Dbl homology (DH) domains that affect actin cytoskeletal dynamics via the small GTPases Rho and Rac. Phenotypic analysis demonstrates that trio and Abl cooperate in regulating axon outgrowth in the embryonic central nervous system (CNS). Dosage-sensitive interactions between trio and Abl, failed axon connections (fax), and enabled (ena) indicate that Trio is integrated into common signaling networks with these gene products. These observations suggest a mechanism by which Abl-mediated signaling networks influence the actin cytoskeleton in neuronal growth cones.

The Abelson tyrosine kinase, the trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. We detect genetic and/or physical interactions between Fra and these effector molecules that suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline.

Interactions between the secreted protein Amalgam, its transmembrane receptor Neurotactin and the Abelson tyrosine kinase affect axon pathfinding.

Two novel dosage-sensitive modifiers of the Abelson tyrosine kinase (Abl) mutant phenotype have been identified. Amalgam (Ama) is a secreted protein that interacts with the transmembrane protein Neurotactin (Nrt) to promote cell:cell adhesion. We have identified an unusual missense ama allele, amaM109, which dominantly enhances the Abl mutant phenotype, affecting axon pathfinding. Heterozygous null alleles of ama do not show this dominant enhancement, but animals homozygous mutant for both ama and Abl show abnormal axon outgrowth. Cell culture experiments demonstrate the AmaM109 mutant protein binds to Nrt, but is defective in mediating Ama/Nrt cell adhesion. Heterozygous null alleles of nrt dominantly enhance the Abl mutant phenotype, also affecting axon pathfinding. Furthermore, we have found that all five mutations originally attributed to disabled are in fact alleles of nrt. These results suggest Ama/Nrt-mediated adhesion may be part of signaling networks involving the Abl tyrosine kinase in the growth cone.

A host-dependent temperature-sensitive mutant of Rous sarcoma virus: evidence for host factors affecting transformation.

We have characterized a host range mutant of Rous sarcoma virus in order to identify host cell factors involved in transformation. This mutant, tsLA33-1, which was isolated from a stock of the temperature-sensitive mutant tsLA33, is not temperature-sensitive for transformation of chicken embryo fibroblasts, as judged by its ability to induce morphological changes and agar colony formation at both 36 and 41.5 degrees. In Rat-3 cells, however, this mutant induced a temperature-dependent transformation: infected Rat-3 cells were transformed at 34 degrees but not at 39.5 degrees. Retransformants were isolated from tsLA33-1-infected Rat-3 cells by growth in agar suspension at 39.5 degrees. Virus rescued from these retransformants induced a temperature-dependent transformation when reintroduced into rat cells. The level of expression of pp60v-src at 39.5 degrees was unchanged in the retransformants. When the retransformants were treated with herbimycin, an antibiotic which induces turnover of certain protein-tyrosine kinases, they reverted to a normal phenotype, indicating that the transformed phenotype of the retransformants was dependent on continued expression of pp60v-src. The retransformants are therefore pseudorevertants in which a cellular alteration has occurred that allows transformation at 39.5 degrees by the mutant pp60v-src. Thus the temperature-dependence of transformation by tsLA33-1 is affected by the cellular environment, and is suppressed or complemented both in chicken cells and in the rat cell pseudorevertants. No clear correlation between levels of phosphorylation at tyrosine and transformation was observed. In Rat-3 cells the pp60v-src encoded by tsLA33-1 may be defective in its interaction with low abundance substrates that are critical for transformation; alternatively the nonpermissive cells may require a higher threshold dose of pp60v-src for transformation.

The function of Drosophila larval class IV dendritic arborization sensory neurons in the larval-pupal transition is separable from their function in mechanical nociception responses

The sensory and physiological inputs which govern the larval-pupal transition in Drosophila, and the neuronal circuity that integrates them, are complex. Previous work from our laboratory identified a dosage-sensitive genetic interaction between the genes encoding the Rho-GEF Trio and the zinc-finger transcription factor Sequoia that interfered with the larval-pupal transition. Specifically, we reported heterozygous mutations in sequoia (seq) dominantly exacerbated the trio mutant phenotype, and this seq-enhanced trio mutant genotype blocked the transition of third instar larvae from foragers to wanderers, a requisite behavioral transition prior to pupation. In this work, we use the GAL4-UAS system to rescue this phenotype by tissue-specific trio expression. We find that expressing trio in the class IV dendritic arborization (da) sensory neurons rescues the larval-pupal transition, demonstrating the reliance of the larval-pupal transition on the integrity of these sensory neurons. As nociceptive responses also rely on the functionality of the class IV da neurons, we test mechanical nociceptive responses in our mutant and rescued larvae and find that mechanical nociception is separable from the ability to undergo the larval-pupal transition. This demonstrates for the first time that the roles of the class IV da neurons in governing two critical larval behaviors, the larval-pupal transition and mechanical nociception, are functionally separable from each other.

An Allele of Sequoia Dominantly Enhances a Trio Mutant Phenotype to Influence Drosophila Larval Behavior

The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, we have screened for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency.

Testing for Mutagens Using Fruit Flies.

Students often breed the fruit fly, Drosophila melanogaster, to explore basic principles of Mendelian inheritance (Jeszenszky 1997). However, flies are extremely versatile model organisms with a rich scientific history, and their use need not be confined to just this role. By simply introducing a few new fly cultures, the vials, incubators, and sorting microscopes already in our laboratories can be used by our students to investigate and understand a wider variety of genetic concepts. An experiment that has proven especially successful in my undergraduate teaching laboratory uses fruit flies to test student-selected compounds for their ability to cause mutations. This laboratory is easy to do, requires no prior experience with fruit flies, incorporates a student design component, and employs both rigorous controls and statistical analyses.