Understanding how migrating neurons navigate the complex environment of the embryonic brain is a fundamental question in central nervous system development. Although progress has been made in understanding how migrating cells move and change their direction in vitro, the mechanisms migrating cells employ as they are directed through tissue by multiple guidance factors are largely unknown.
We have examined interneuron stream migration in the cortex and tested SDF1’s role in stream maintenance and interneuron stream exit. Although SDF1 had previously been identified as a modulator of migration rate in vitro, data we were able to confirm, we also determined that SDF1 signaling reduces interneuron branching frequency both in vitro and in brain slices. In this context, inhibiting SDF1 signaling results in interneurons prematurely exiting from migratory streams and shifting the interneuron distribution from the SVZ/IZ to the cortical plate. We found that inducing branching in migrating interneurons is sufficient to cause cells to exit their migration stream and invade the cortical plate in brain slices. While treatment with AMD3100 will act autonomously on interneurons, we have not yet excluded the possibility that treatment with forskolin could act non-autonomously to cause CP invasion. Taken together our data suggest that SDF1 signaling suppresses branching frequency to allow cells to maintain a simpler morphology better suited to rapid migration within a defined stream. Interestingly, these treatments did not cause a measurable amount of interneurons to exit the MZ stream where SDF1 is also highly, but more uniformly expressed (,, bin 10). This could suggest intrinsic differences between the SDF1 response of interneurons occupying the two different migration streams, external substrate differences, or a more complex guidance switch with additional signaling factors to induce interneurons to exit the MZ to invade the cortical plate.
To characterize the intracellular signaling pathway used by SDF1 in interneurons, we interrogated known components of the pathway. We expected CXCR4 signaling to occur through Gi as in other cell types. We found that Gi regulation of cAMP concentration is important in defining interneuron speed and branching frequency. We also found that protein kinase A may transduce this 2nd messenger signal to further signaling pathways to create the complex migration behavior of interneurons.
Our data imply an inverse relationship between branching and the rate of migration. Treating interneurons with SDF1 resulted in both an increase in speed and a decrease in leading process branching. We next asked if regulation of these two aspects of cell behavior diverge farther down the SDF1 signaling pathway. Treatment over a range of forskolin concentrations, which increases cAMP through adenylyl cyclase sensitization, determined that increasing cAMP concentration increases branching while reducing speed. Taken together with our SDF1 treatment results, cAMP concentration is a central mediator of both speed and branching frequency. Whether cAMP affects speed and branching through separate downstream signaling pathways or through shared cytoskeletal regulation remains to be determined.
The inverse relationship between branching and speed has led us to propose a new model to describe interneuron stream migration (). In this model, interneurons migrating in streams, under the influence of SDF1, migrate faster as a result of a reduced branching frequency. This allows interneurons to spread from their ganglionic eminence origins through the cortex and achieve a proper interneuron distribution. As individual interneurons encounter lower SDF1 levels, and consequently reduce SDF1 signaling, they increase their branching and reduce their speed. This permits them to sample as yet unknown cues, change direction and migrate into the cortical plate to achieve their final position.
One possible cortical plate invasion cue could be a time-dependent reduction in SDF1-signaling levels. It is likely that SDF1 signaling within individual interneurons is reduced as they exit migration streams (Liapi et al., 2008
; Lopez-Bendito et al., 2008
). Consistent with our observation of increased branching and stream exit when treating brain slices with SDF1-signaling inhibitor, migratory interneurons in the CXCR4 knock out mouse exit their migration streams prematurely, demonstrating that a reduction in SDF1-signaling allows cells to exit migration streams but also that SDF1 alone cannot define and maintain stream migration (Li et al., 2008
). Whether this reduced SDF1-signaling is the result of downregulation of CXCR4 or internally initiated interference with the SDF1-signaling pathway, perhaps at the cAMP level, remains to be elucidated. One external cue for interneuron-stream exit is likely decreasing SDF1 concentration as interneurons migrate down the gradient of SDF1 expression (Stumm et al., 2007
). Consistent with this hypothesis, our measurement of interneuron branching in brain slices shows that interneuron branching is lower near the cortical notch, an area of high SDF expression, but farther up the cortical arch where SDF expression is reduced, interneuron branching increases ().
According to our data and model, as SDF1 signaling is reduced, the branching frequency of interneurons increases (). We speculate that this increase in branching allows interneurons to probe greater space and come into contact with additional guidance factors. A cortical plate attractant has been biologically demonstrated, but is yet to be identified (Lopez-Bendito et al., 2008
). In addition to the cortical plate attractant, it is possible that repellents could direct interneuron migration in the cortex. There is some evidence that semaphorins may be expressed in the dorsal VZ/SVZ (Tamamaki et al., 2003
) and SDF1 has been shown to reduce the response of pathfinding axons to repellents in the Slit and Semaphorin families (Chalasani et al., 2003
). In the cortex, interneurons migrating in SDF1-rich streams would be insensitive to any repellents. Upon reduction in SDF1-signaling, interneurons could then respond to the repellents and exit migration streams toward the cortical plate. Interestingly, we provide some evidence that reduction of SDF1 signaling with AMD3100 can slightly increase interneuron localization in the VZ (, bin 1), suggesting that blocking SDF1 signaling may leave interneurons free to explore the VZ. In contrast, if SDF1 signaling is intact but interneuron branching increased by forskolin treatment, fewer cells localize to the VZ, even though cells exit the SVZ/IZ stream on both sides, possibly displaying a balance between SDF1’s attractive ability and interneuron branching causing more exploratory migration (, bins 1 and 4,5,6). Finally, in our model increased branching would shift the balance of stimulation from SDF1 to the cortical plate attractant or a VZ localized repellent, resulting in slower migration and stream exit. The role of SDF1 in this model is to affect pathfinding by modulating cell morphology, not necessarily for SDF1 to function as an attractant or a motogen, both additional possible functions (Li et al., 2008
; Lopez-Bendito et al., 2008
Our data provides a new model as to how SDF1 controls interneuron stream migration through signal transduction and modification of cell migratory morphology and behavior. Because we have shown that changes in branching frequency are relevant in a tissue context, we expect future studies to further define the role of migrating interneuron branching in brain development.