RUI: The Role of Slits and Robos During Axon and Glial Cell Guidance in the Forebrain

Smith College, Northampton MA

Investigators

Abstract

Communication between the left and right hemispheres of the brain requires precisely positioned commissures made up of neuronal processes called axons that cross the midline. The guidance of axons across the midline occurs during embryonic development, and this guidance is controlled by an elaborate communication system between the pathfinding axons and their growth environment. The growth environment provides both the necessary growth substrate as well as strategically positioned attractive and repellent cues that all together direct specific pathfinding axons across the midline in the correct position. The main goal of this research is to understand how the diversity of Slit-Robo signaling controls the positioning of commissures as well as their glial growth substrate in the vertebrate forebrain. To accomplish this goal the Barresi lab has characterized a simple system in the zebrafish forebrain to simultaneously assay commissural axons, their astroglial growth substrate, and the Slit-Robo guidance system. The experiments outlined in this proposal will fully exploit the embryological, molecular and genetic techniques available in zebrafish to breakdown the synergistic roles of the Slit-Robo guidance system during astroglial bridge assembly and commissure formation in the diencephalon. Recently Dr. Barresi and colleagues have shown that commissural axons grow along a bridge-like structure made of astroglial cells. In addition, both repellent and potentially positive Slit cues function not only to guide axons across the forebrain, but also to set up the correct positioning of the midline astroglial bridge. One aim of this proposal is to test whether a particular Slit family member, slit1a, is sufficient to attract commissural axons and astroglial cells to the midline. Assaying this novel function will be accomplished by temporally and spatially misexpressing slit1a in the diencephalon of wild type embryos as well as in you-too (Gli2) mutant embryos that have a reduction in slit1a expression. Interestingly, roundabouts (robo), the Slit receptors, are differentially expressed in both commissural neurons and the astroglial bridge. The remaining proposed experiments are designed to decipher which of the four Robos (robo1, 2, 3 or 4) individually or synergistically function to mediate Slit repulsion or attraction for either commissural axons or astroglial cells within the diencephalon. This will be accomplished through the use of anti-sense Morpholino oligonucleotide gene knock-down techniques designed specifically against the four different robos, and by utilizing two mutant fish lines that have a loss of astray (robo2) or twitch-twice (robo3) gene function. Completion of these aims will help us to better understand the molecular (Slit-Robos) and cellular (astroglial) cues that influence midline axon crossing. At Smith College, the Barresi lab is committed to involving undergraduate students in this research, in particular woman and woman from underrepresented groups will be the main contributors carrying out these studies.