Ephrins off the beaten path

Abstract: The Orchestration of cell-cell interactions is crucial to the developing embryo and the adult organism. Intercellular communication is to a large extent controlled by signalling events downstream of receptor tyrosine kinases (RTKs). Ligands for RTKs are often soluble and confer long distance information to the receptor expressing cell. In contrast to this the Eph receptor tyrosine kinases bind membrane attached ligands, denominated ephrins, expressed in neighbouring cells. The main cellular response to Eph-ephrin signalling investigated so far has been repulsion of the receptor expressing cell. The establishment of topographic maps in the developing visual system as well as boundary formation in hindbrain rhombomeres are examples of this repulsive action. Recent research, however, has revealed a greater variety of non-repulsive outcomes of Eph signalling influencing such diverse processes as neural tube closure, synaptic plasticity and adult stem cell proliferation. The expression of ligands and receptors is not limited to the developing nervous system but exhibits an astounding temporal and spatial diversity. My initial work focused on a somewhat surprising phenotype in the ephrin-A5 null mutant. This mutant displays errors in the establishment of topographic maps in the developing retino-tectal system due to a lack of repulsive cues (ephrin-A5) for the growing axons. In 20% of the mutants another phenotype was obvious: anencephaly. Anencephaly is the result of improper closure of the anterior neural tube at embryonic day 8.5. Our analysis showed that the neural plate folds correctly in the mutants. The folds were properly juxtaposed in the dorsal midline but failed to fuse in some embryos and persistent cell proliferation led to eversion of the neural folds. This phenotype is not easily explained by lack of repulsion but rather by lack of an adhesive component allowing the neural folds to fuse and stay fused. Expression analysis of the neural tube revealed that ephrin-A5 was coexpressed with EphA7 at the dorsal tips of the closing neural tube. As other RTKs the Eph receptors are dimerized upon ligand binding, which allows for cross-phosphorylation between the receptors tyrosine kinase domains, initiating an intracellular signalling cascade. The Eph-mediated triggering of growth cone repulsion has been shown to be dependent on this mechanism. We found two additional splice forms of the EphA7 receptor to be expressed in the neural folds. These splice forms were identical to the full-length EphA7 receptor (EphA7-FL) with respect to the extracellular domain but they both lacked the major part of the intracellular domain of EphA7-FL containing the tyrosine kinase domain. We showed that the expression of the truncated splice form (EphA7-T1) silenced the tyrosine kinase signalling of EphA7-FL and thus shifted repulsion into adhesion. As ephrin-A5 binds EphA7 with high affinity it is possible that the ephrin-A5EphA7 complex could act as adhesion molecules when the repulsive signalling is shut off. The differential response to ephrinA5 showed, to our knowledge, the first example of how the response to a ligand can be modulated between repulsion and adhesion by alternative use of different splice forms of a receptor. Many organs of an adult mammal contain populations of stem cells which maintain self-renewing capacity and are able to generate the multiple cell types of the organ. The proliferation of neural stem cells can be increased both in vivo and in vitro by the addition of mitogens such as epidermal growth factor (EGF) and fibroblast growth factor (FGF). We have identified the pitiuary adenylate cyclase-activating polypeptide (PACAP) to be a positive regulator of stem cell proliferation in the neurogenic adult murine subventricular zone (SVZ) and hippocampus. Even though many extracellular factors have been shown to increase neural stem cell proliferation, an extracellular factor that negatively can regulate stem cell proliferation remains to be identified. The expression of ephrins and Eph receptors in the ventricular wall of adult mice led us to investigate whether these proteins could be involved in adult neurogenesis. The analysis of ephrin-A2 and EphA7 null mutant mice revealed an increase in proliferation in the SVZ. The increased proliferation was accompanied by a shortening of the cell cycle in the mutants. Cultured neural stem cells from the ephrin-A2 null mutants proliferated faster than wild type cells. Analysis of ephrin-A2 null mutant olfactory bulbs two weeks after BrdU injection, revealed an increase in BrdU positive cells. This shows that the cells born in the SVZ of the mutants migrate in a normal way. To determine if the mice lacking ephrin-A2 de facto had more cells the total amount of cells in defined areas of cortex and striatum was counted along with numbers of astrocytes and neurons. In the ephrin-A2 null mutants there was an increase in total cell number as well as in both neurons and astrocytes. There was however no shift in ratio between the astrocytic and the neuronal populations. Intracerebroventricular infusion of ephrin-A2-Fc proteins that block endogenous ephrin-Eph interactions without activating the receptor or ligand mimicked the ephrin-A2 mutant with increased proliferation and shortened cell cycle. Our data suggest that removal or blockage of Eph-ephrin signalling increases stem cell proliferation and thus establishes ephrin-A2 as an extracellular negative regulator of stem cell proliferation. When examining the adult hernatopoietic stem cell (HSC) niche we could confirm the role for ephrins as in vivo negative regulators of cell proliferation. Our analysis of ephrin function in stem cell proliferation in the small intestine and skin epidermis reveals an opposite role. Disruption of endogenous ephrin/Eph interactions in these tissues reduces the rate of proliferation, which copies the phenotype of the EphB2/B3 double null mutants. Thus ephrins are capable of both facilitating and attenuating cell proliferation in multiple tissues.