One Pathway to Rule Them All: Wnt Signaling Guides Specification and Axon Growth in VD13

Abstract

The nervous system is highly complex, and its formation is regulated and guided from the moment a neuron is born to throughout its life. Neurons must undergo a series of steps throughout development, typically including a combination of the following: specification, migration, axon outgrowth, target recognition, synapse formation, and synapse maintenance and change. These developmental steps are largely driven by both extrinsic signaling cues and intrinsic programs, which work together to create the myriad of diversity that we see throughout the nervous system. Here, we will look at two such developmental regulators. The canonical Wnt signaling pathway is a signaling system with an extrinsic ligand, and the Hox genes are intrinsic molecules that work to pattern the anterior/posterior axis. We will specifically look at the role Wnt signaling and the Hox genes play in specification and axon growth, two critical parts of neuronal development. Specification is a hierarchical process in which neurons undergo a series of developmental changes, in which each step defines a more precise neuronal identity. Thus, identifying the genes required for proper specification is vital to understanding the development of the nervous system. The genes and receptors which a neuron express during the stages of specification will influence subsequent steps in its development, such as migration, axon growth, and synapse formation. Here, we will also examine axon growth, and the cues that function to provide both attractive and repulsive signals to guide the growing axon to its target location. We find that similar signals function in both specification and axon growth in the D-type GABAergic motor neuron, VD13. In Chapter III, we describe a novel transgenic marker, lhIs97, which utilizes a fragment of the plx-2 promoter to drive mCherry expression in the most posterior of the D-type GABAergic motor neurons, VD13, as well as in the bilaterally symmetrical LUA neurons, LUAL and LUAR. Using this marker, we provide evidence that the Wnt signaling pathway and the Hox gene egl-5 may function as terminal selectors for VD13, assigning its final, terminal identity as the most posterior D-type neuron. Additionally, we show the underlying morphology to the previously published under- and over-growth phenotype reported when Wnt pathway signaling members or egl-5 are mutated. In Chapter IV, we look further into specification, first by looking at known factors involved in the specification of all of the VD neurons. Here, we find that unc-55, which is involved in the differentiation of the VD neurons from the DD neurons, is not required for expression of mCherry via lhIs97, our VD13 specific marker. plx-2 and mab-20, which are, respectively, a transmembrane receptor and a semaphoring signaling molecular, are also not required for expression of RFP in VD13. We also show that when the heparan sulfate proteoglycan sdn-1 is lost, there is increased expression of lhIs97 in DD6, but no significant difference in expression in VD13. Thus, while sdn-1 may somehow be involved in differentiating DD6 from VD13, unc-55, plx-2, and mab-20 are not necessary for lhIs97 expression in VD13. Additionally, in Chapter IV, using lhIs97, we perform a genetic screen for temperature sensitive alleles involved in VD13 specification and, potentially, axon growth. We report eight candidate lines which all have varying loss of expression of lhIs97 mCherry in VD13. Five of the eight new alleles appear to be temperature sensitive. The lh40 line has a premature stop codon in sma-9. sma-9 is a known transcription factor that has been implicated in many different processes in the worm, including in dorsal-ventral patterning. Based on available evidence, we think it is a candidate to be involved in specification and axon growth in VD13. None of our lines completely lost expression of the RFP marker in VD13, which suggests that our screen has not reached saturation for potential candidates that are involved in specification and axon growth in VD13. Thus, further screens may well provide additional candidates that are involved in this system. Finally, in Chapter V, we look further into the role that the Wnt signaling pathway has in directional axon growth and repulsion. Here, we show that bar-1/-catenin, frequently an effector of Wnt signaling, is instead epistatic to the Wnt ligands egl-20 and lin-44 in axon growth. Additionally, we show that, of the three C. elegans Disheveleds, dsh-1 and mig-5, but not dsh-2, are necessary for axon growth. Finally, we find that the Hox gene egl-5 acts cell autonomously to regulate axon outgrowth. Overall, in this report we show that the Wnt signaling pathway and the Hox gene egl-5 regulate specification and axon growth in the most posterior D-type GABAergic motor neuron, VD13.