Brugia Malayi - Clark Science Center - Smith College

Function of Slit and Robo in Axon Guidance
Abigail Antoine and Jin Sook Park
Axon-glia interactions in the developing brain are caused by Roundabout receptors detecting the presence of Slit proteins. This
signal-receptor pathway guides them to form proper connections within the central nervous system. What happens when one or
more of these signals are missing? Using zebrafish as our model system, we were able to study embryonic forebrains to determine
if specific mutations resulted in particular phenotypes, specifically in the formation of the post-optic commissure. Out of the
many different mutant fish lines we had available to us, we focused on the Slit 2 and Slit 3 mutants, as well as Astray/Twitch-twice
double mutants.
As the embryo brain develops, glia act as a substrate on which axons projecting from the retina move across in order to form
the post-optic and anterior commissures. The Slit 1a protein acts as a chemoattractant, positively guiding the axons along the right
path, while Slit 2 and Slit 3 act in the opposite manner, as a chemorepellant, preventing axons from straying too far from where
they are supposed to be. 1 In a mutant fish, one or more of these proteins or receptors are missing or nonfunctional, and this
would affect formation of the commissures.
To pinpoint a specific phenotype within each mutant group, we examined developing embryos and tried to observe if there
were any abnormal behaviors or physical characteristics. At about 32 hours post fertilization, specific proteins were labelled within
the embryo using immunohistochemistry, which would fluorescently label for axons and glia. This labeling allows us to examine
the commissure formations and compare the forebrain images to a control group in order to determine the existence of mutant
phenotype.
Since Slit 2 and Slit 3 were believed to have similar roles in axon guidance, it was unclear whether or not a loss of function in
only the Slit 3 protein would show any distinct abnormalities or whether Slit 2 proteins would be able to compensate for the loss
of the other chemorepellant. This examination process was made more difficult by the fact that the genotyping protocols for the
Slit 2 and Slit 3 mutants were not working properly at this time, which inhibited us from processing a group of confirmed mutants
for either fish line. In groups of potential Slit 3 mutants, there were instances of defasciculation of the commissure. Potential
Slit 2 mutant embryos were separated by observed behaviors, such as a circular swimming pattern or deafness, and analysis of the
commissures will be continued during the semester. Genotyping would be the next step in order to identify confirmed mutants.
However, further examination of both mutant lines must be completed in the year to follow in order to confirm phenotypes.
(Supported by the Blakeslee Fund in the Biological Sciences)
Advisor: Michael Barresi
References:
1
Barresi, M.J. 2005. Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain. Development 132, 3643-3656.
2012
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