Identification of Genes Involved in the C. elegans VAB-1 Eph Receptor Tyrosine Kinase Signaling Pathway

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex.

The role of a kinesin, KLP-20, on neural development and epidermal morphogenesis in Caenorhabditis elegans

The heterotrimeric kinesin-II motor in Caenorhabditis elegans consists of KLP-20, KLP-11, and KAP-1 subunits and broadly functions in cellular transport for the development of biological structures including cilia and axons. The results of this paper support the ubiquitous and necessary role kinesin-II motors have in development, particularly the KLP-20 microtubule-associating subunit. Mutations in klp-20 result in a variable abnormal (vab) phenotype characterized by observable epidermal defects, although the role of this gene in development and the mechanism by which the vab phenotype is produced is largely unknown. The vab phenotype is highly penetrant in the first larval stage (L1) of C. elegans, which supports that klp-20 functions in early development. Ciliated amphid sensory neurons can be stained with a fluorescent dye, DiI, to simultaneously test cilia structure and function, as well as the morphology of the amphid sensory organ. Reduced dye uptake in klp-20 mutant L1s suggests that the microtubule-based cilia are under-developed as a result of defective kinesin-II function. Consistent observations of the PLM mechanosensory neuron using the zdIs5 reporter suggest that klp-20 has an essential role in neuron development, as mutations to klp-20 result in under-developed PLM axons. Qualitative observations suggest there may be an interaction between the development of the overlying epidermis and the underlying nervous system, as a more severe vab phenotype is observed simultaneously with reduced dye uptake, and hence amphid sensory cilia under-development. Furthermore, a more severe vab phenotype manifested as large bumps on the posterior epidermis appears to be spatially correlated with PLM defects. The results presented and discussed in this paper suggest that KLP-20 has a necessary role in neurodevelopment and epidermal morphogenesis in C. elegans during embryogenesis.

The roles of EFN-1 and seam cell V2 in the transmission of a stop cue for the posterior lateral microtubule in Caenorhabditis elegans

A functional nervous system requires the precise arrangement of all nerve cells and their neurites. To achieve this correct assembly, a myriad of molecular guidance cues works together to direct the outgrowth of neurites to their correct positions. The small nematode C. elegans provides the ideal model system to study the complex mechanisms of neurite guidance due to its relatively simple nervous system, composed of 302 neurons. I used two mechanosensory neurons, called the posterior lateral microtubule (PLM), to investigate the role of the ephrin and Eph receptor protein family in neurite termination in C. elegans. Activation of the C. elegans Eph receptor VAB-1 on the PLM growth cone is sufficient to cause PLM termination, but the identity and location of the activating ligand has not been established. In my thesis I investigated the ability of the ephrin ligand EFN-1 to activate VAB-1 to cause PLM termination when expressed on the same cell (in cis) and on opposing cells (in trans) to the receptor. I showed that EFN-1 is able to activate VAB-1 in cis and in trans to cause PLM termination. I also assessed the hypodermal seam cells as the source of the ephrin stop cue using fluorescently labelled and seam cell mutant transgenic worms. I found that although the PLM shows consistent termination on the seam cell V2 in wild type worms independent of PLM length, this process is not significantly disrupted in seam cell mutants. With this information I have created a new hypothesis that the PLM neurite is able the provide a positional cue for the developing seam cells, and have created a new transgenic strain which can be used to assess the impact of PLM and ALM cell ablation on seam cell position. My research is the first to demonstrate the ability of an ephrin ligand to activate its ephrin receptor in cis, and further research can investigate if this finding has in vivo applications.