Novel Non-Repulsive Outcomes From Ephrin-B Reverse Signaling

Abstract

In this dissertation, I present a detailed characterization of the physiological roles for reverse signaling through B-subclass ephrins during embryonic development. The stereotypical outcome of Eph-ephrin signaling has long been established as one of cell-cell repulsion, elicited through a localized breakdown of the actin cytoskeleton. In contrast to this dogma, I have found that ephrin-B reverse signaling is instead necessary for mediating cell-cell adhesion events during several critical midline closure events. I demonstrate that mice with germline mutations specifically disrupting the ability of ephrin-B2 to conduct cell autonomous signals present with defects in urorectal septation of the hindgut, tubularization of the urethra, tracheoesophageal septation of the foregut, closure of the palatal shelves, and closure of the embryonic eyelid, defining roles for ephrin-B2 reverse signaling in each of these developmental events. Further, I show that mice with germline mutations either deleting EphB2 or similarly impairing the ability of EphB2 to conduct cell autonomous forward signals indicate that EphB2 acts non-cell autonomously to activate ephrin-B1 reverse signaling in closure of the ventral body wall. The developmental malformations in these mutant animals are each hallmarked by the failure of lateral mesenchymal folds to properly adhere, typically at the midline, which is difficult to reconcile with the canonical outcome of cell-repulsion from Eph-ephrin signaling. Consistent with a role in eliciting cell-cell adhesion at these septation events, I show that EphB and ephrin-B molecules are expressed in the epithelia where adhesion will take place. Moreover, my data specifically localizes ephrin-B reverse signaling to these adherent epithelia. Finally, an in vitro palatal shelf fusion assay used to determine the role of Eph-ephrin signaling in these developmental events indicates clear roles for ephrin-B2 reverse signaling in cell-cell adhesion. Taken together, my data leads me to propose that ephrin-B reverse signaling is not only capable of cell repulsion, but is also able to elicit cell-cell adhesion responses, which are employed in a bevy of adhesion-based septation events during embryonic development. Finally, my analysis of reverse signaling-deficient ephrin-B2 mice also indicates an additional novel, non-repulsive role for ephrin-B reverse signaling in mediating ionic homeostasis within the inner ear. I show that these ephrin-B2 mutant mice present with a circling or "waltzing" phenotype due to severe defects within the vestibular apparatus. My data shows that the disruption of bidirectional signaling between EphB2 and ephrin-B2 result in malformed structures within the vestibular apparatus and abnormal endolymph fluid. Further, I show that EphB2 and ephrin-B2 are expressed on non-motile, secretory epithelia within the inner ear, suggesting that these molecules play important roles in maintaining the proper volume and ionic makeup of the endolymph fluid running through the vestibular apparatus. My data leads me to propose that ephrin-B2 reverse signaling is therefore not only capable of regulating the cytoskeleton to produce either cell repulsion or cell adhesion outcomes, but that these reverse signals can mediate cellular responses independent of cytoskeletal dynamics, such as ionic homeostasis.