Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Jonathan A. Raper

Second Advisor

Michael Granato

Abstract

During development, axons navigate a complex intracellular milieu, often over long distances to reach target tissues at stereotyped locations. The basis for this navigation lies in the growth cone - a motile structure at the tip of the growing axon. Studded with receptors for an array of guidance cues, the growth cone interprets a balance of attractive and repellant signals en route to its target. However, during pathfinding, axons often must extend along or near areas expressing repellents. We previously showed that growth cones can modulate their response to multiple repellents through a G protein coupled receptor, calmodulin, cAMP and PKA mediated pathway that ultimately abrogates the repellent response through inhibition of Rho. (Chalasani et al, 2003 PMID:12598624) We sought to further investigate the role of cAMP and G Protein signaling in guidance - and used the zebrafish retinto-tectal projection as our model system, due to its well characterized projection - all axons extend towards the ventral midline, cross at chiasm and project dorsally and posteriorly to the contralateral tectum. In Chapter 2, I describe my contributions to a paper recently published (Xu, et al 2010 PMID:20505109), in which we describe the role of the calcium/calmodulin adenylyl cyclase, ADCY8, in directing the trajectory of retinal ganglion cells. In Chapter 3 I describe a study that examined the role of G protein coupled receptor signaling in directing retinal trajectories. In this study, I took advantage of the UAS/GAL4 system to express dominant negative constructs targeting specific classes of heterotrimeric G proteins in retinal ganglion cells. We generated constructs targeting GNAi/o, GNAq/11, GNAs/olf, and Gbetagamma. We find that reduction of GNAS produces a consistent guidance error - misprojection to the ipsilateral tectum. These errors phenocopy the knockdown of ADCY8 as well as knockdown of the axon guidance molecules Semaphorin 3D, Semaphorin 3E and their co-receptor, Nrp1a. Using a combination of QPCR, morpholino injection, and in vivo manipulation we demonstrate that depression of cAMP signaling, either through the heterotrimeric G protein GNAS or ADCY8 reduces Neuropilin expression, and renders navigating axons insensitive to Semaphorin 3D and Semaphorin 3E signaling near the chiasm.

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