Seeking further knowledge of Robo signaling with forward genetics

How the nervous system generates its complex connectivity has intrigued scientists for over a century. The growing axons of developing neurons receive guidance information from their environment through receptors on the surface of their growth cones. The roundabout genes (or robos) represent one major receptor family, and Slits are their ligands. In Chapter 1, I summarize what is known about Slit-Robo signaling in both the Drosophila ventral nerve cord and the vertebrate visual system. I also review the molecules that are known to participate in Slit-Robo signaling. Slits are generally thought to act as repellents for growth cones, and have been demonstrated biochemically to bind to Robo receptors. Although Slits are known to act through Robo receptors in Drosophila, this has not been formally tested in vertebrates. This distinction is important due to several differences between these two systems. In Chapter 2, I use an in vitro protocol that I have developed to culture zebrafish retinal ganglion cells (RGCs). Adding either human Slit2 (in conditioned media) or partially purified zebrafish Slit2 to these explant cultures confirms that Slit2 acts to collapse zebrafish RGCs. By performing similar collapse assays on explants that lack Robo2 receptor, I show that this Slit2 induced collapse of RGCs requires the Robo2 receptor. The astray mutant (defective in the zebrafish homolog of Robo2) has provided much insight into Slit-Robo signaling in the vertebrate visual system. How this receptor transduces its signal to elicit changes in growth cone behavior is, however, poorly iv understood. In Chapter 3, I present the results of a noncomplementation screen for astray that was designed to help understand Slit-Robo signaling. We screened 21,649 mutagenized haploid genomes and recovered 9 new alleles of astray. We sequenced these mutations and characterized their phenotypic strengths. Two new alleles of astray display a novel phenotype in which one or both optic tecta are innervated. In Chapter 4, I discuss the results of the in vitro experiments and the new alleles from the screen. I also propose some future directions that could further expand our understanding of Slit-Robo signaling.