Neuronal [Ca²⁺]_i at various intervals (0 to 18 s) after application of Slit-2.

Image courtesy of Dr. Mu-ming Poo, University of California, Berkeley, USA.

Extracellular signals guide the migration of immature neurons from their birthplace to their specific final destination in the developing brain. Although the leading growth cone and soma of the migrating neuron have coordinated motility, the mechanisms that underlie this coordination are still largely unknown. Are extracellular cues sensed solely by the growth cone and then transmitted to the soma for coordinated movement, or do the soma and leading growth cone perceive guidance cues independently? Now in Cell, Mu-ming Poo and colleagues address these questions and report that the response to the guidance cue depends upon the leading growth cone.

The authors examined the behaviour of growth cones and soma in cultured cerebellar granule cells following exposure to Slit-2 — a repulsive factor that stops or reverses neuronal migration. Application of a Slit-2 gradient to the front of the leading growth cones inhibited their motility and caused them to either collapse or retract. The soma then reversed its translocational direction and the trailing neurite acquired a reversal in motility direction. This directional change was not observed upon application of Slit-2 to the front of the soma or trailing neurite, indicating that only the leading growth cone responds to the guidance signal. The authors attributed this response to the Slit-2 receptor Robo2, which is preferentially located at the growth cone.

Monitoring intracellular Ca²⁺concentration by fluorescence ratio imaging revealed that frontal — but not rear — exposure to Slit-2 triggered a propagating Ca²⁺ wave from the growth cone to the soma. The authors showed that this wave was necessary and sufficient for the reversal of soma translocation. Preventing Ca²⁺ release in the soma abolished reversal, and triggering a Ca²⁺ wave by releasing Ca²⁺ from internal stores with ryanodine induced a reversal of migration.

As cytoskeletal rearrangements are likely to be associated with the reversal process, Mu-ming Poo and colleagues investigated the involvement of Rho GTPases, which are known downstream effectors of Ca²⁺ and regulators of cytoskeleton organization. Inhibition of Rho GTPases allowed growth cone collapse but inhibited the reversal of soma translocation. The transfection of dominant-negative forms of Rho GTPases into granule cells and treatment with RhoA activity inhibitors showed that RhoA was required for Slit-2-induced migration reversal even though RhoA did not affect motility. The presence of Slit-2 and increased Ca²⁺ levels also induced RhoA inhibition, suggesting that migration reversal may be associated with a gradient of RhoA inhibition, created by the Slit-2-induced-Ca²⁺ wave.

RhoA was visualized using RhoA-GFP fusion proteins and appeared to accumulate in a polarized manner toward the leading front. Application of Slit-2 and a frontal Ca²⁺ wave induced the redistribution of RhoA in the soma. RhoA activity was monitored by fluorescence resonance energy transfer (FRET) and was also shown to be asymmetrically distributed. Slit-2 application induced a front-to-rear translocation of active RhoA, indicating that a reversal in soma migration is associated with a redistribution of active RhoA.

Taken together, these results show that the leading growth cone of migrating neurons is responsible for sensing the repulsive factor Slit-2. The long-range signalling between the growth cone and the soma that triggers neuronal migration reversal is a propagating Ca²⁺ wave that leads to the redistribution of RhoA towards the rear of the soma. How the Ca²⁺ wave induces RhoA redistribution is still unknown. Downregulation of RhoA appears to alter cortical myosin activity, which is involved in the reorganization of F-actin. The authors speculate that RhoA redistribution may rely on a myosin-dependent flow of cortical F-actin that pulls RhoA to the rear.

Author: Kim Baumann