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Genetic Analysis of Drosophila Stomatogastric Nervous System
Biochemistry and Molecular Biology
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The enteric nervous system (ENS) is of critical importance to an organism, as it controls the function of the gastrointestinal system. The ENS is remarkably complex, and although sculpted early in development, has to maintain plasticity throughout adulthood in the face of changing food levels, intestine size and inflammatory insults. Compared to the central nervous system, the ENS is poorly understood, despite the impact of several medically significant conditions. Hirschsprung syndrome of colon and rectum (HSCR) is a multigenic congenital disease and occurs one in five thousand live births (0.02%). Affected children require corrective surgery due to a lack of neurons in the lower intestine. The main causative mutations for HSCR are in the Ret receptor tyrosine kinase, which responds to the Glial Cell Line-Derived Neurotrophic Factor (GDNF) ligand. Down syndrome patients suffer from a 100-fold increase in the incidence of HSCR (1-2%). Rather than loss-of-function mutations, HSCR in these patients is thought to be due to an extra copy of the Dscam gene (Down syndrome cell adhesion molecule). Loss of Ret signaling leads to an absence of neural precursors in the colon and rectum whereas Dscam is proposed to disrupt local axonal connections made by enteric neurons. The precise functions of both genes in ENS formation are still under debate.The Drosophila stomatogastric nervous system (SNS) is the invertebrate equivalent of the ENS and with the fly's powerful genetics offers the opportunity to shed light on the molecular and cellular mechanisms functioning in ENS development. Ret gene expression is conserved in the migrating neural precursors and we have found Dscam protein is present in the axonal connectives. Although the adult SNS is well characterized in the fly, the embryonic SNS required tool development to allow genetic manipulations.In Chapter 1, I present the initial experimental design, which employs the genetically tractable model organism, Drosophila. The original goal of my thesis was to clarify the precise role of the Ret receptor in cell migration, survival and cell death. An additional goal was to determine whether the Ret and EGFR receptors cooperate during development by signaling through the same pathway (MAP kinase). A parallel objective was to elucidate the role of the Dscam receptor in normal ENS development and the increased prevalence of HSCR in Down syndrome. The rationale behind all these aims is outlined.To develop the Drosophila embryonic SNS as a model system for the vertebrate ENS, I constructed and characterized tools for transgenic expression (Chapter 2). I cloned fragments of the Ret promoter to the GAL4 gene. Although expression was broader and weaker than hoped for, two lines RetP2A and P2B-GAL4 express in a large subset of SNS precursors and continue to be expressed throughout larval stages. I elaborate on a screen performed in collaboration with another graduate student, Logan Myers, which lead to the identification of the goosecoid (Gsc) promoter. The GscG-GAL4 driver is active during SNS development, but switches off at the end of embryogenesis. I then used these lines to demonstrate that the epidermal growth factor receptor (EGFR) plays a role during axon outgrowth as well as a previously documented role in precursor proliferation and migration. These results demonstrate that our genetic toolset may be employed for the manipulation of SNS precursors and functional analysis during larval stages.In Chapter 3, I establish that Dscam1 receptor is expressed in the developing stomatogastric nervous system. I present phenotypic analysis of Dscam1, Dscam1 frazzled and Dscam1 robo1 double mutants, showing that the subtle phenotypes observed in Dscam1 mutants alone are not enhanced nor suppressed by frazzled and/or robo mutations. Moreover, I aimed to model Down syndrome gut nervous system defects in order to elucidate the effect of Dscam trisomy on cell proliferation, migration and/or survival. To achieve this, I employed the tools described in Chapter 2, along with GFP tagged full length and dominant negative Dscam1 isoforms. Thorough phenotypic analysis of embryonic neuroanatomy revealed the SNS structure to be affected. An additional copy of Dscam1 causes overgrowth of the developing frontal nerve and motor neurons innervating the hindgut. We analyzed larval feeding behavior and, surprisingly, did not observe any obvious feeding problems. Consequently, we questioned whether the function of defecation was affected and developed an assay to evaluate possible defects. I found that Dscam1 overexpression in the SNS impairs the ability of larvae to clear food from their gut. We are currently trying to localize the origin of this defect.Finally, in Appendix 1, I expand on the results obtained using a high-throughput larval locomotion assay. This sensitive assay was employed to assess for additional functional consequences of increased and altered neural connectivity. While driving dominant negative Dscam1 clearly increased overall kinesis of second instar larvae, an added copy of full length Dscam1 adversely affected locomotion. Interestingly, behavioral outputs were dramatically altered in the presence of a typical food odor, ethyl acetate. In summary, genetic and functional analysis of Drosophila stomatogastric nervous system was facilitated by the new toolset described herein. These tools were used to dissect the role of EGFR and Dscam signaling in SNS formation. The results provide a mechanistic insight into two complex, multigenic congenital diseases by analyzing the link between genes, nerves and behavior.