INBRE Supported Research

Longitudinal axons of the central nervous system connect the brain and the spinal cord, transmitting signals between brain regions. To establish longitudinal tracts during embryonic development, longitudinal axons must precisely navigate along specific pathways. However, the molecular guidance mechanisms for longitudinal axons remain largely unknown. Our experiments test the possibility that longitudinal axon growth is influenced by the Slit family of repulsive signaling proteins.

I am investigating the role of the chemorepellent Slit proteins and their Robo receptors in the dorsal/ventral (D/V) positioning of pioneer longitudinal axons using mutant mice as well as mis-expression experiments in chick embryos. Mis-expression experiments are performed by in ovo electroporation of expression plasmids in the developing brain. Transfected axons are visualized using GFP, while non-transfected axons are labeled with antibodies against the ßIII-tubulin protein.

Analysis of Slit1/2 double knockout mice revealed significant defects in longitudinal axon guidance, as LLF tract axons of the hindbrain invaded a previously Slit positive region. Slit misexpression in the developing chick hindbrain showed that Slit is sufficient to alter the guidance of the LLF axon tract, as indicated by axon guidance errors in the region of ectopic Slit expression. In addition, Robo1 loss-of-function experiments in the MLF tract using a dominant negative Robo receptor revealed a phenotype similar to that observed in Slit1/2 knockout mice, indicating that the Robo1 receptor is necessary for the proper guidance of the MLF longitudinal tract.

Slit/Robo signaling thus plays an important role in D/V positioning of tracts during development. Our results support a model in which the repulsive effects of Slit/Robo signaling steer longitudinal axons to specific D/V positions. A better understanding of the signals that guide axons to proper brain regions may lead to improved treatments for neural degenerative disorders through the use of neural stem cells along with the correct manipulation of these guidance cues.

1. Wildtype mouse embryo showing proper guidance of the LLF tract through the hindbrain (indicated by arrow). Axons avoid the Slit positive region as they grow longitudinally.


2. Slit1/2 knockout embryo showing LLF axons invading the dorsal hindbrain region when Slit is not present (indicated by arrows).

1. LLF tract in chick hindbrain. Axons are labeled with B-tubulin (red), and ectopic Slit is indicated in green.


2. Control side showing normal growth of LLF tract axons.


3. Electroporated side showing axons wandering into the dorsal hindbrain (arrowhead).