Author

Thomas Coate

Date

April 2008

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Cell and Developmental Biology

Institution

Oregon Health & Science University

Abstract

In this thesis, I have examined the molecular mechanisms associated with neuronal guidance, a central problem in developmental neurobiology. Eph receptor tyrosine kinases and their cell surface-bound Ephrin ligands are known to modulate the guidance of many developing neurons in the vertebrate nervous system, but the overlapping expression patterns and promiscuous interactions among multiple ligandreceptor pairs have hindered a functional analyses in vivo. As an alternative strategy, I have investigated the role of Ephrin-Eph receptor interactions in the control of neuronal migration within the developing enteric nervous system (ENS) of the moth, Manduca sexta. The ENS is formed by a population of approximately 300 neurons (the EP cells) that migrate along a set of identified muscle band pathways on the midgut while avoiding the enteric midline. I have shown that the EP cells express a single class-A Ephrin (MsEphrin; a GPI-linked ligand), which can be detected in their filopodial processes as they explore the midgut surface. Concurrently, the midline interband regions of the midgut (which are inhibitory to migration) express MsEph, the sole Eph receptor homologue in Manduca. To investigate the role of MsEphrin-MsEph interactions in the ENS, I manipulated their interactions during EP cell development using a combination of Fcand 6His-tagged fusion proteins, and knocked down MsEphrin expression levels using antisense Morpholino oligonucleotides. The results of these experiments suggested that normal MsEphrin-MsEph receptor interactions mediate cell-cell repulsion between the EP cells and the midline cells, a mechanism that restricts migration and outgrowth to the muscle band pathways. In addition, the results from these experiments suggested a novel role for reverse signaling via a GPI-linked Ephrin ligand in the control of neuronal guidance. Reverse signaling mediated by the Ephrin-B subclass has been well documented. Ephrin-B ligands have a transmembrane domain and short cytoplasmic domain, which permit direct communication with downstream effectors. In contrast, little is known about the mechanisms associated with Ephrin-A-mediated reverse signaling; their GPI anchorage to the membrane does not provide them direct communication into the cell. Using a candidate approach, I discovered that a Manduca Src ortholog (in its active form; phospho-Src) colocalizes with MsEphrin during migration and outgrowth. Pharmacological manipulations to Src in the developing ENS showed that Src activation functions to prevent midline crossing in a manner similar to MsEphrin-dependent reverse signaling. Using single-gut explant assays and high-resolution confocal imaging of manipulated EP cells, I discovered that MsEphrin-mediated reverse signaling promotes the local phosphorylation of Src. Additional experiments combining MsEph-Fc (to overstimulate reverse signaling) and PP2 (to inhibit Src activation) demonstrated that Src phosphorylation is necessary for MsEphrin-mediated reverse signaling in the developing ENS. This investigation represents the first in vivo demonstration of a signaling mechanism associated with reverse signaling via a GPI-linked Ephrin ligand. This work provides a platform for further investigations into the molecular and developmental mechanisms related to MsEphrin-MsEph receptor interactions in the developing ENS.

Identifier

doi:10.6083/M4DJ5CK4

School

School of Medicine

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