A long-term goal of this research is to elucidate the molecular mechanisms controlling contact-dependent axon guidance in the developing mammalian central nervous system (CNS). The ventral midline/floor plate of the rodent CNS represents an important intermediate target for pathfinding of commissural axons. Commissural axons are directed along a circumferential path from the dorsolateral spinal cord to the floor plate by long-range diffusible guidance cues. Short-range cues mediate contact-dependent interactions between commissural growth cones and floor plate cells that allow commissural axons to cross the midline and turn rostrally to form the longitudinally projecting ventral funiculus. Strikingly, these axons appear to ignore guidance cues present on the ipsilateral, but not the contralateral side of the spinal cord, since they turn into the longitudinal plane only after crossing the midline, and do not recross the midline. One interpretation of these observations is that commissural axons become responsive to an unidentified midline repellent only after they cross the midline. Receptor protein tyrosine kinases (RPTKs) of the Eph class are attractive candidates for participating in short-range guidance events at the midline since they interact exclusively with membrane-associated ligands or ephrins, and exhibit dynamic, spatially-restricted expression patterns in the developing CNS. Furthermore, transmembrane B-class ephrins mediate repulsive axon guidance in vitro. We have shown by in situ hybridization that all three B-class ephrins are localized to the floor plate, and that the B-class Eph receptor, EphB1, is likely to be expressed by commissural neurons in the developing mouse spinal cord. Strikingly, EphB1 protein is present on commissural axons only after they cross the ventral midline. Furthermore, preliminary studies demonstrate that B-class ephrins promote the collapse of, and repel, commissural growth cones/axons in vitro. These findings suggest that commissural axons specifically upregulate the expression of EphB1 after crossing the floor plate, and gain responsiveness to inhibitory B-class ephrins at the ventral midline. To test this hypothesis, the stripe/choice assay will be used to determine whether floor plate contact alters the responsiveness of commissural axons/growth cones to B-class ephrins in vitro. A novel spinal cord explant culture system will also be used to determine whether blockade of interactions between B-class ephrins and EphB1 leads to commissural axon recrossing of the ventral midline. Furthermore, ephrin-B3 null mice will be examined for defects in commissure formation in the developing spinal cord and brain.
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