Mark A. Seeger

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.

Identification and characterization of roundabout orthologs in zebrafish

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.

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.

Attraction versus Repulsion: Modular Receptors Make the Difference in Axon Guidance

Growth cones navigate along their pathways with remarkable speed and fidelity. Clearly a complex set of attractive and repulsive cues are present in the extracellular environment and are being interpreted by the growth cone. These papers demonstrate the critical role that receptors and receptor complexes play as effectors for these different guidance cues. As is often the case, while questions have been answered, new ones have been raised. Is growth cone guidance a continual battle between these different signals, or a balance between attraction and repulsion, as suggested by Bashaw and Goodman? Is the growth cone a democracy where the majority rules and every signal has an equal voice? Or have systems evolved that allow some signals to dominate at particular times, mechanisms that can switch a growth cone from an attractive to repulsive response rapidly, as is suggested by Hong and colleagues? Most likely, we will find that each of these scenarios is correct and that axon guidance in vivo utilizes both mechanisms.The next challenge will be to decipher what lies between the cytoplasmic domains of these axon guidance receptors and the cytoskeleton. What are the proteins that bind to these different cytoplasmic domains, and how are they regulated? Bashaw and Goodman point out that several families of repulsive guidance receptors have been identified and yet their cytoplasmic domains share no motifs in common. Does this suggest that these various classes of receptors will utilize distinct sig-naling pathways? Are there real differences between a Robo-mediated repulsive signal versus an UNC5-DCC repulsive signal? At some point all of these signaling pathways must converge on the proteins that directly regulate assembly and disassembly of the growth cone cytoskeleton. Unraveling how all of this is orchestrated will certainly be fascinating.Some initial insights are already being generated with the Xenopus axon turning assay. Using different pharmacological inhibitors, Poo, Tessier-Lavigne, and colleagues have identified signaling pathways that are required for axon turning to gradients of various attractants and repellents (11xMing, G.L, Song, H.J, Berninger, B, Holt, C.E, Tessier-Lavigne, M, and Poo, M.M. Neuron. 1997; 19: 1225–1235Abstract | Full Text | Full Text PDF | PubMed | Scopus (412)See all References, 12xMing, G.L, Song, H.J, Berninger, B, Inagaki, N, Tessier-Lavigne, M, and Poo, M.M. Neuron. 1999; 23: 139–148Abstract | Full Text | Full Text PDF | PubMed | Scopus (214)See all References, 13xSong, H.J, Ming, G.L, He, Z, Lehman, M, McKerracher, L, Tessier-Lavigne, M, and Poo, M.M. Science. 1998; 281: 1515–1518Crossref | PubMed | Scopus (661)See all References). Cyclic nucleotide signaling pathways (both cAMP and cGMP) have dramatic effects on attractive and repulsive turning responses to distinct guidance cues. Phospholipase C-γ, phosphoinoitide 3-kinase, and Ca2+ also can play critical roles. These in vitro studies have begun to identify common signaling pathways that are utilized by different classes of guidance cues. We are now poised to ask how these signaling pathways are linked with specific receptors in vivo during the complex process of axon guidance.§E-mail: seeger.9@osu.edu (M. A. S.), beattie.24@osu.edu (C. E. B.).

A targeted gain of function screen in the embryonic CNS of Drosophila

In order to identify genes involved in the development of the central nervous system (CNS) we have undertaken a gain of function screen in the embryonic CNS of Drosophila. Transposable P-elements and the UAS/GAL4 system were used to initiate transcription of genes in a pan-neural pattern using scaGAL4. Over 4100 individual P-element insertion lines were screened with monoclonal antibodies BP102 and 1D4 to visualize axon pathways. Twenty-five P-element insertions corresponding to 18 genes resulted in aberrant CNS axon pathfinding when misexpressed with scaGAL4. Genes involved in axon guidance, embryonic patterning, and cell cycle regulation were isolated. In addition, we identified several zinc finger transcription factors not previously implicated in axon guidance or CNS development. This group includes Squeeze, Kruppel homolog-1, Hepatocyte nuclear factor 4, and two uncharacterized genes, CG11966 and CG9650. Calnexin99A, a putative molecular chaperone, was isolated as well.

In the Absence of Frazzled Over-Expression of Abelson Tyrosine Kinase Disrupts Commissure Formation and Causes Axons to Leave the Embryonic CNS

Background In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abls role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures. Methodology/Principal Findings The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS. Conclusion/Significance Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.

Mosaic analysis reveals a cell-autonomous, neuronal requirement for Commissureless in the Drosophila CNS

Commissureless (Comm) is a key regulator of axon guidance at the midline of the Drosophila CNS. Here we report results from mosaic analysis experiments that demonstrate a cell-autonomous, neuronal requirement for Comm. Using the MARCM system to positively label clonal patches, we find that neurons which are not deficient for Comm function make commissural projections 85% of the time. Clones of neurons that are homozygous mutant for comm make commissural projections at a statistically significant reduced frequency: 70% for comm1, a hypomorphic mutation, and 58% for comm5, a near null mutation. These data suggest that commissural axon guidance is dependent upon regulated expression of Comm in neurons.