Date of Award
Doctor of Philosophy (PhD)
The assembly of neural circuits requires a complex choreography of developmental events: neurons must be generated, extend neurites at the correct time and location, and then integrate extracellular information, like long-range guidance cues or cellular contacts, with an internal developmental program to make correct wiring decisions. Visualizing neural-circuit assembly in vivo can provide insight into how these events are coordinated. The C. elegans embryo, which contains only 222 neurons and 56 glia, is an attractive setting to study nervous system development comprehensively in an intact, living organism. However, methods to label and track optically-resolvable neurites or manipulate single neurons through gene expression do not exist, as most embryonic reporters are broadly expressed. Here, I present a method for expressing fluorescent reporters or any gene of interest in specific C. elegans embryonic neurons, glia, or other cell types, without cell specific drivers. Our method is based on a previous setup (Kamei et al., 2009), and uses an infrared (IR) laser to localize heat to the volume of a single precursor cell in the embryo. This induces gene expression in the progeny of that cell (1-4 cells/embryo) through heat-shock-response regulatory elements. I perform significant optimizations to adapt this strategy to cells in the C. elegans embryo, which are highly sensitive to heat toxicity. Direct temperature measurements of IR heating in the embryo reveal that cells are heated to physiological temperatures (320C) for 5 minute durations using our modified irradiation protocol. These conditions lead to high rates of gene induction (>60%) with no signs of damage. First, I use our system to label and track single neurons during early nervous system assembly. These studies reveal a retrograde extension mechanism for axon growth in specific interneurons. I also study the etiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants; these studies suggest that a timing/competence mechanism controls axon-outgrowth dynamics in the nerve ring. Next, I demonstrate the versatility of IR irradiation by performing cell-specific rescues, determining DAF-6/Patched-related site of action during sensory-organ development. Finally, I demonstrate that IR cell irradiation can be used to perform simultaneous ablation and labeling of cells in the same embryo. I use this system to uncover a role for the amphid sheath glia in dendrite extension. As IR induction can be used for targeted labeling, gene expression, and ablation without the need for cell-specific drivers, this tool opens to door to high resolution systematic analyses of C. elegans morphogenesis.
Singhal, Anupriya, "IR Laser-Induced Gene Expression for Tracking Development of Single Embryonic Neurons and Glia in C. Elegans" (2017). Student Theses and Dissertations. 389.