Date of Graduation


Document Type


Degree Name

Master of Science in Biology (MS)

Degree Level



Biological Sciences


Timothy Evans

Committee Member

Jeffrey Lewis

Second Committee Member

Michael Lehmann


Axon Guidance, Drosophila, Midline Repulsion, Neurodevelopment, Robo1, Slit


The repellant ligand Slit and its Roundabout (Robo) family receptors regulate many aspects of axon guidance in bilaterians, including midline crossing of axons during development of the embryonic CNS. Slit proteins are produced by midline cells and signal through Robo receptors expressed on the surface of axonal growth cones to repel axons from the midline. Disruption of Slit-Robo signaling causes ectopic midline crossing phenotypes in the CNS of a broad range of animals, including insects and vertebrates.

Drosophila Robo1 has a conserved ectodomain structure of five immunoglobulin-like (Ig) domains plus three fibronectin (FN) repeats. By utilizing a genomic rescue construct based on endogenous robo1 regulatory sequences, we investigate which of these ectodomain elements are individually dispensable as well as how much of the receptor is required for its midline repulsive function. By restoring expression of variant Robo1 constructs in embryonic neurons of robo1 mutants, we found that the Ig1 domain is the only individual ectodomain element essential for midline repulsion in vivo. Additionally, the combinatorial deletions of either the Ig domains (Ig2-5) or the FN repeats (FN1-3) do not disrupt slit binding or midline repulsion. However, when these two deletions are combined (Ig2-FN3), so that only the Ig1 domain remains, midline repulsion is not completely restored to that of wild-type embryos. Interestingly, Robo1Ig2-FN3 is still able to bind Slit, indicating that Ig1 alone is both necessary and sufficient for Slit binding by Robo1, but not sufficient on its own for Robo1’s in vivo function. Furthermore, we find that while the Ig2-5 variant is sensitive to downregulation in vivo, the FN1-3 and Ig2-FN3 variants are insensitive to the Robo1 antagonists Commissureless (Comm) and Robo2, revealing a novel regulatory role for Robo1’s FN repeats.

This partial rescue phenotype of Robo1Ig2-Fn3 suggests that additional ectodomain elements of Robo1 apart from Ig1 may play a permissive role in repulsive signaling, and that a minimal number of domains, rather than a specific set, may be necessary for Slit-dependent signaling by Robo1. To that end, we further investigate steric hindrance as a potential mechanism to explain this partial rescue phenotype.