Further Development 15.19: How a Growth Cone Can Rapidly Change Its Responsiveness to Guidance Cues

Neural Crest Cells and Axonal Specificity

In Drosophila, this mechanistic change in responsiveness is controlled by an endosomal protein called Commissureless (Comm), which is expressed only in precrossing axons and functions to divert Robo proteins to the lysosome instead of permitting their expression at the membrane. In Drosophila, this endosomal trafficking mechanism for protein enables a faster change in the responsiveness of midline-crossing commissural axons than would be possible through regulation of robo gene expression (see Figure 15.31; Keleman et al. 2002; 2005; Yang et al. 2009).

Vertebrates also use Slit and Robo signaling for midline repulsion, but the switch in growth cone responsiveness between pre- and postcrossing axons is somewhat different. In vertebrates, there are several Slit (1–3) and Robo (1–4) proteins with Robo3 having two isoforms, Robo3.1 and Robo3.2, which are expressed in pre- and postcrossing commissural axons, respectively (Mambetisaeva et al. 2005). As neurons extend axons toward the midline, those axons destined to remain ipsilateral (i.e., on the same side of the CNS on which the soma resides) are expressing Robo1 and Robo2, so they are repelled from crossing the midline by Slit (see Figure 15.31). Axons expressing Robo3.1, however, are able to cross the midline. Although the precise mechanism is unclear, Robo3.1 appears to actively promote midline crossing, since knockdown of Robo3.1 results in the failure of commissural axons to cross the midline. Once the commissural neuron’s growth cone has crossed the midline, it downregulates Robo3.1 and upregulates Robo1, 2, and 3.2. This change in Robo repertoire expression allows the full force of Slit to act as a chemorepellent, which forces the growth cone away from the midline and prevents any re-crossing events (see Figure 15.31, mouse; Long et al. 2004; Sabatier et al. 2004; Woods 2004; Chen et al. 2008).

In addition to the downregulation of Robo3.1, the change in growth cone responsiveness at the midline is reinforced by altering how the neuron interprets gradients of the midline attractant Shh. Postcrossing axons upregulate 14-3-3 proteins that function through Protein kinase A (PKA) to alter the growth cone’s interpretation of the Shh signal from attractive to repulsive (see Figure 15.31; Yam et al. 2012). Therefore, the precise spatial and temporal regulation of Slit-Robo and Shh signaling in pre- and postcrossing axons enables commissure formation. Mutations in the human ROBO3 gene disrupt the normal crossing of axons from one side of the brain’s medulla to the other (Jen et al. 2004). Among other problems, people with this mutation are unable to coordinate their eye movements.

Developing Questions

How is the alternative splicing of Robo3 temporally regulated during midline crossing? Moreover, how are Robo3.1 and Robo3.2 functioning to mediate midline attraction and then repulsion?

Literature Cited

Chen, Z., B. B. Gore, H. Long, L. Ma, and M. Tessier-Lavigne. 2008. Alternative splicing of the Robo3 axon guidance receptor governs the midline switch from attraction to repulsion. Neuron 58: 325–332.
PubMed Link

Jen, J. C. and 35 others. 2004. Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis. Science 304: 1509–1513.
PubMed Link

Keleman, K., C. Ribiero, and B. J. Dickson. 2005. Comm function in commissural axon guidance: cell autonomous sorting of Robo in vivo. Nat. Neurosci. 8: 156–163.
PubMed Link

Keleman, K. and 7 others. 2002. Comm sorts robo to control axon guidance at the Drosophila midline. Cell 110: 415–427.
PubMed Link

Long, H. and 9 others. 2004. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron 42: 213–223.
PubMed Link

Mambetisaeva, E. T. W. Andrews, L. Camurri, A. Annan and V. Sundaresan. 2005. Robo family of proteins exhibit differential expression in mouse spinal cord and Robo-Slit interaction is required for midline crossing in vertebrate spinal cord. Dev. Dyn 233: 41-51.
PubMed Link

Sabatier, C. and 7 others. 2004. The divergent Robo family protein rig-1/Robo3 is a negative regulator of slit responsiveness required for midline crossing by commissural axons. Cell 117: 157–169.
PubMed Link

Woods, C. G. 2004. Crossing the midline. Science 304: 1455–1456.

Yam, P. T. and 9 others. 2012. 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing. Neuron 76: 735–749.
PubMed Link

Yang, L., D. S. Garbe, and G. J. Bashaw. 2009. A frazzled/DCC-dependent transcriptional switch regulates midline axon guidance. Science 324: 944–947.
PubMed Link