Slit chemorepellant molecules were initially described by their involvement in axon and cell guidance. However, recently Slit molecules have also been implicated in trigeminal condensation and cancer metastasis (Dallol et al., 2003b; Shiau et al., 2008). We examined whether or not Slit molecules play a different role in neural crest cell development, similar to its inhibition of migration in metastatic cells. Our findings demonstrate that in addition to being neural crest chemorepellants, Slit molecules are capable of impairing neural crest cell migration.
Slit Molecules Impair Neural Crest Migration
The process of cell migration is extremely complex because it entails coordination of many seemingly opposing cell functions. Cells need to become polarized, make adequate plasma membrane protrusions, and generate traction while simultaneously retracting the cell body. Our findings herein demonstrate that Slit expression significantly impaired the movement of actively migrating neural crest cells. Slit molecules not only command where neural crest cells will migrate (De Bellard et al., 2003), but also impair the process of initial migration from the neural tube thereby becoming a highly migratory cell.
Our findings highlight a critical and highly debated question regarding neural crest cell migration: how can one distinguish a simple guidance phenomenon from a migratory or motility phenomenon in a highly migratory mesenchymal cell? Neural crest cells begin to migrate rapidly and persistently along set pathways after delaminating from the neural tube, suggesting a carefully guided developmental process (Kulesa et al., 2004; Hall, 2009; Kulesa and Gammill, 2010). Our present results support a new role for Slit molecules during neural crest cell development. One, Slit molecules provide guidance by preventing neural crest cells from entering regions with high concentrations of Slit molecules (De Bellard et al., 2003). Two, Slit molecules can delay and impair the initiation of neural crest migration (see cartoon in Fig. 11). Thus, Slit molecules not only behave as guidance ligand molecules (telling cells where to go), but are also capable of affecting cell motility (how/if cells reach those targets).
Several key observations support this hypothesis. First, during Slit GOF, neural crest cells migrated less efficiently away from the Slit-expressing neural tube, undermining the idea that Slit solely has a role in guidance in a gradient-dependent manner. Instead, during Slit GOF cells migrated very poorly along their regular ventral pathway or away from the neural tube in culture. However, not all the Slit GOF neural crest cells showed impaired migration. Some Slit-expressing cells still reached their targets in the embryo or the halo edge in culture. Second, we also observed that after Slit GOF, cells were dynamically changing their shape and rounding up, which is a less motile phenotype. Together, these observations point towards a role for Slits during the initiation of migration separate from their role in guidance. While some authors think that cells first undergo guidance then migration (Chodniewicz and Klemke, 2004), others have shown that many cells can polarize spontaneously in the absence of concentration gradients (Paliwal et al., 2004). In fact, neural crest cells will vigorously migrate in culture despite lack of guidance cues (even demonstrating spontaneous migration after neural tube removal). Thus, neural crest cells are capable of migrating without guidance cues. In other words, neural crest cell migration very likely entails separable guidance and motility mechanisms.
More relevant to our present findings is that recently Slit2 was found to be expressed in solid tumors (Wang et al., 2003) and unmethylated-Slit2 can behave as a tumor suppressor and impair cell migration in some human cancers (Prasad et al., 2008). It looks as if Slits molecules are important in the transition towards a migratory type in some mesenchymal cells. In other words, in the same way that Slits impair, or better, restrain cancer cell migration, they can also restrain the process of neural crest migration. Although the data presented here cannot determine if it is at the initiation of cell migration when neuroepithelial cells change their phenotype or after this step, we showed that Slit-Robo interactions are part of this transition.
Slit Molecules Affect Neural Crest Cytoskeleton
Cellular morphology and cell shape are highly dependent on cytoskeletal elements such as filamentous actin (F-actin). In vitro, epithelial neural crest cells display significant levels of F-actin fibers, with minimal cytosolic globular actin (Newgreen and Minichiello, 1995). Upon initiation of EMT, levels of F-actin decrease while cytosolic globular actin levels increase (Newgreen and Minichiello, 1995). Data presented here suggest that expression of Slit2 affects the pattern of actin distribution in neural crest cells. In highly motile neural crest cells, actin localized to areas of membrane blebs and sites of filopodial extension, with the greatest actin staining visible when filopodial retraction occurred (Berndt et al., 2008). Here, we found when neural crest cells expressed Slit2, fewer stress fibers were visible and, in general, actin distribution did not follow the classic patterns observed in mesenchymal cells. In contrast, during Slit LOF more stress fibers were observed and cortical actin increased on cellular extensions compared with controls, reminiscent of highly motile cells (Pellegrin and Mellor, 2007). These data suggest that decreases in Slit-expression result in alterations in actin more reminiscent of a mesenchymal phenotype.
We also observed that expression of Slit could affect the distribution pattern of another important cytoskeletal protein: tubulin. The pattern of cytoplasmic distribution of post-translationally modified tubulins, acetylated tubulin (Ac-tubulin) and tyrosinated tubulin (t-tubulin), were altered upon ectopic expression of mSlit1 and hSlit2. Similar to our observations for actin, mSlit1 expression in neural crest cells resulted in condensed fibers of Ac-tubulin, as found in less motile cells. Although the precise roles of Ac-tubulin in development have yet to be defined, the developmental regulation of post-translationally modified tubulin has been previously described (Fukushima et al., 2009). Ac-tubulin is observed in highly stable microtubules, such as those in the proximal sites of mature neurons and non-mesenchymal cells (Brown et al., 1993) as well as in non-motile types of cells (Geuens et al., 1986; Gundersen et al., 1987). One explanation for the observed changes in distribution of Ac-tubulin after Slit GOF is that Slits may promote stability of the cell cytoskeleton in pre-migratory neural crest. Complementing these results, Slit2 LOF reduced and redistributed the total number of cytoplasmic Ac-tubulin fibers, concomitantly increasing the concentration of acetylated tubulin in the MTOC.
More relevant to our findings is that recently Slit2 has been found to increase E-Cadherin and decrease beta-catenin (Tseng et al., 2010), both key components of cell structural dynamics. Both of these findings were observed in the context of cancer metastasis transitions. It is known that the EMT transformation of the neural crest is accompanied by changes in cell adhesion molecules (Nakagawa and Takeichi, 1998), which are remarkably similar to those that occur during metastasis. In addition, when Okamoto and co-workers over-expressed Slit2 in one-cell zebrafish embryos, an impairment of the convergent-extension movement of the mesoderm was observed (Yeo et al., 2001). These findings are similar to the findings we present here. Slit molecules impaired/altered the transition of neural crest cells from a non-migratory to a migratory, mesenchymal cell.
One interesting finding from Slit GOF experiments was the observation that neural crest cells showed a higher incidence of membrane blebbing. This phenomenon is believed to be actively generated by alterations in the actin cytoskeleton during neural crest delamination. More importantly, membrane blebbing protrusive activity has been shown to be concomitant with neural crest cells EMT (Berndt et al., 2008). Our findings that Slit molecules increased cell blebbing suggest that Slit molecules may cause actin detachment from cell membrane as observed by Berndt and co-workers.
There are several findings that highlight the complexity and importance of Slit-Robo interactions. One came from screening for genes responsible for axonal regeneration. In this study, Chen and co-workers found that Slit-Robo interactions play a modulatory, critical step in the capability of neurons to regenerate by affecting downstream signals required for cytoskeletal assembly during neurite outgrowth (Chen et al., 2011). Also, it is known that Slit molecules via its Robo receptor are capable of altering the expression of small GTPases like Rho, Cdc42, and Rac, which are responsible for cytoskeletal re-arrangements (Wong et al., 2001; Yiin et al., 2009; Huang et al., 2011). These molecules are known to be important not only in the cytoskeletal re-arrangements necessary during migration but also importantly during the initiation of neural crest cell migration (Wong et al., 2001; Werbowetski-Ogilvie et al., 2006; Yiin et al., 2009). This data in conjunction with the more recent description of the role that FGF and RA have on the timely delamination of neural crest denote that the rostro-caudal complementary expression of Robo1 and Robo2 may be also play a role in the timely delamination of trunk neural crest (Martínez-Morales, 2011).
There are two possible mechanisms that could explain our results. One will be the “sandwich” model proposed by Kraut and Zinn, of an anti-migratory effect of Slit-Robo signaling on sensory neurons observed in the fly embryo (Kraut and Zinn, 2004). The model proposed might also apply to cells while still inside the neural tube, because it allows trans-signaling between two pre-migratory neural crest cells, with one presenting Slit to the other via binding to its Robo receptors (Fig. 11A). We think the role of Slits is more complex. One important reason is that although in the Kraut and Zinn (2004) article the Slit sandwich model is sufficient to prevent migration of sensory neurons in Drosophila, in neural crest it is not sufficient. This is because we observed that neural crest cells were capable of delaminating and migrating even while constitutively expressing high levels of Slits in vivo and in vitro, albeit their motility was restrained. The other mechanism will be the axon regeneration inhibition proposed by Chen et al. (2011) where they proposed a cell-autonomous role for Robo-Slit in inhibiting growth cone re-growth. We think this model explains better why neural crest expressing both Slit and Robo simultaneously could not move as freely as control cells, and corresponds to our preliminary findings that Slit GOF and LOF alter neural crest EMT transition and induce cytoskeleton re-arrangements (Fig. 11C and data not shown).
Figure 11. Graphic abstract of Slit function during trunk neural crest migration. Cartoon showing the expression of Slits (red) and Robo receptors (blue) during trunk neural crest development. A: A pre-migratory neural crest cells that simultaneously expresses Slit and its Robo receptors; these cells are not motile. B: Migrating neural crest cells that express Robo receptors but no Slit; these are highly motile cells avoiding dermomyotome (Jia et al., 2005). C: Neural crest cells expressing Robo receptors stopping by the dorsal aorta after encountering Slit molecules expressed at the entrance of the developing gut; these cells are non-motile, non-epithelial (De Bellard et al., 2003).
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The novelty of the work presented here is that it addresses an earlier aspect of neural crest cell migration, namely the time of delaminating from the neural tube, and it unravels a new function for Slit/Robo system in the control of neural crest EMT. In summary, we can propose two functions for Slit molecules during neural crest development. The first established effect of Slits is analogous to their effect on axonal pathfinding: cells avoid areas expressing Slit like the dermomyotome (Fig.11B with 1 vs. 2 paths depending on Slits) (Jia et al., 2005) and the gut (Fig. 11C) (De Bellard et al., 2003). The second effect pertains to its effect in impairing timely delamination of neural crest (Fig. 11A).