dissertationThe nervous system is comprised of an estimated 100 billion individual neurons, which are connected to one another to form a network that senses environmental stimuli and coordinates the organism's behavior. Because of the complexity of the nervous system, deciphering the developmental processes and adult wiring diagram has proved challenging. A number of axon guidance molecules have been identified; however, the means by which they guide billions of axons to their target cells in vivo remains poorly understood. Several axon guidance molecules have been found to be bifunctional, meaning they can elicit different growth cone responses depending on the presence or absence of other molecules, such as growth cone receptors, intracellular signal transduction molecules, or extracellular modulators. Axon sorting within axon tracts is perhaps a means by which axons are presorted to make a precise connection on their target cells. The zebrafish, Danio rerio, is an ideal model organism to study vertebrate axon guidance and axon sorting due to its external fertilization, optical transparency, amenability to forward genetics, and ease of making transgenic lines. In order to study axon guidance within the zebrafish retinotectal system, I developed a new method of misexpressing genes. Local misexpression can be induced by using a modified soldering iron in transgenic zebrafish in which a gene of interest is driven by a heat shock promoter. This method allowed me to examine the mechanisms by which Slit1a and Slit2 guide axons from the retina to the optic tectum. I determined the expression pattern of Slits in the zebrafish and used antisense morpholino technology to knock down Slit1a. The iv resultant axon guidance errors indicated that Slit1a acts to guide retinal axons through the optic tract. I then misexpressed Slit1a and Slit2 near the optic tract to observe their effect on axons. I found that both proteins appeared to attract retinal axons. Additionally, I saw that Slit2 seems to attract retinal axons earlier in the retinotectal pathway, at the optic chiasm. I also report on a new method, to whose development I contributed, for automated tracking of axons through electron microscopy datasets. Taken together, my results add new methods to the endeavor of mapping neural connectivity and development, and suggest a new role for Slits in axon guidance
