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Camp Promotes Retinal Midline Crossing at the Zebrafish Optic Chiasm University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 cAMP Promotes Retinal Midline Crossing at the Zebrafish Optic Chiasm Alison Dell University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, Developmental Biology Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Dell, Alison, "cAMP Promotes Retinal Midline Crossing at the Zebrafish Optic Chiasm" (2013). Publicly Accessible Penn Dissertations. 626. https://repository.upenn.edu/edissertations/626 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/626 For more information, please contact [email protected]. cAMP Promotes Retinal Midline Crossing at the Zebrafish Optic Chiasm 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 etintr o-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 eductionr 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. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Jonathan A. Raper Second Advisor Michael Granato Keywords axon guidance, cAMP, chiasm, GPCR, neuron, RGC Subject Categories Cell Biology | Developmental Biology | Neuroscience and Neurobiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/626 CAMP PROMOTES RETINAL MIDLINE CROSSING AT THE ZEBRAFISH OPTIC CHIASM Alison L. Dell A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation _____________ Jonathan A. Raper, Ph.D Professor, Department of Neuroscience Graduate Group Chairperson _________________ Daniel S. Kessler, Ph.D. Associate Professor, Department of Cell and Developmental Biology Dissertation Committee Michael Granato, Ph.D., Professor, Department of Cell and Molecular Biology Greg Bashaw, Ph.D., Associate Professor, Department of Neuroscience Peter Klein, M.D, Ph.D., Associate Professor, Department of Medicine David Manning, Ph.D., Professor, Department of Pharmacology ! ! CAMP PROMOTES RETINAL MIDLINE CROSSING AT THE ZEBRAFISH OPTIC CHIASM COPYRIGHT 2013 Alison L. Dell This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ ACKNOWLEDGMENT So many people have helped me on this stage of my journey in scienceland. I’ve really benefited from both the cell/developmental and neuroscience communities here at Penn. I first want to thank my mentor, Jonathan Raper, for his enthusiasm for science, breadth of thought, and willingness to listen to crazy ideas. From the time I started my rotation, I was struck by how many different techniques and systems we put to use in the lab, doing whatever approach is the most decisive way to answer an experimental question. I have learned a tremendous amount; and working with Jonathan has changed the way I think, and approach problems, scientific and otherwise. My thesis committee: Greg Bashaw, Michael Granato, Peter Klein and Dave Manning really helped me when I was stuck in my project - suggesting I look for a link with existing guidance pathways, which helped lead to the work presented in Chapter 3, and I thank them for that. I want to thank the members of the Raper Lab too, both past and present. Hong Xu, a former postdoctoral fellow deserves particular thanks. We have now worked together on two projects. He was already working on Neuropilins in retinal guidance when I discovered that cAMP signaling impacts its expression. He has been very supportive and gracious about the dovetailing of our projects. Emma Fried-Cassorla also contributed a huge amount and has become a good friend, as well as collaborator. I also want to thank Naomi Twery, Vanisha Lakhina, and Alemji Taku fellow graduate students – we have struggled, and laughed a lot together, as well as Christy, Eugene and Puneet for being generally wonderful. Members of the Bashaw and Granato labs have been great for helpful discussions, fun times, late night reagents, CSHL dance parties, and general science solidarity. Finally, I want to thank Jane Dodd, Tom Jessell, Sara Wilson, and Peter Scheiffele, who strongly encouraged me to go to graduate """! ! school – and Samantha Butler who sort of warned me against it. It hasn’t been the easiest trip, but I’m glad that I took it. My family and friends have also been tremendously supportive. I thank my parents Penny and David, who have been unwavering in their belief in me, my brother Nathaniel for his poke/zap jokes when electroporation experiments were challenging, and for my sister, who inspires me everyday. I thank my life long friends Karen and Aeolan, Rajiv Sainath for showing me that googley eyes can fix almost any sadness, and Eva Wylie and Roxana Perez-Mendez. And I thank my husband Rob, who’s been quite understanding about my decision to flee Brooklyn and spend seven years poking fish in the eye. Thanks! ! "# ! ABSTRACT CAMP PROMOTES RETINAL AXON CROSSING AT THE ZEBRAFISH OPTIC CHIASM Alison L. Dell Jonathan A. Raper 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 G!i/o, G!q/11, G!s/olf, and G"#. We find that reduction of G!S 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
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