A negative modulatory role for rho and rho-associated kinase signaling in delamination of neural crest cells
© Groysman et al.; licensee BioMed Central Ltd. 2008
Received: 22 February 2008
Accepted: 22 October 2008
Published: 22 October 2008
Neural crest progenitors arise as epithelial cells and then undergo a process of epithelial to mesenchymal transition that precedes the generation of cellular motility and subsequent migration. We aim at understanding the underlying molecular network. Along this line, possible roles of Rho GTPases that act as molecular switches to control a variety of signal transduction pathways remain virtually unexplored, as are putative interactions between Rho proteins and additional known components of this cascade.
We investigated the role of Rho/Rock signaling in neural crest delamination. Active RhoA and RhoB are expressed in the membrane of epithelial progenitors and are downregulated upon delamination. In vivo loss-of-function of RhoA or RhoB or of overall Rho signaling by C3 transferase enhanced and/or triggered premature crest delamination yet had no effect on cell specification. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced crest emigration without affecting cell proliferation. These treatments altered neural crest morphology by reducing stress fibers, focal adhesions and downregulating membrane-bound N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid inhibited emigration while enhancing the above. Since delamination is triggered by BMP and requires G1/S transition, we examined their relationship with Rho. Blocking Rho/Rock function rescued crest emigration upon treatment with noggin or with the G1/S inhibitor mimosine. In the latter condition, cells emigrated while arrested at G1. Conversely, BMP4 was unable to rescue cell emigration when endogenous Rho activity was enhanced by lysophosphatidic acid.
Rho-GTPases, through Rock, act downstream of BMP and of G1/S transition to negatively regulate crest delamination by modifying cytoskeleton assembly and intercellular adhesion.
The neural crest (NC) has long been a model for understanding cell migrations during development [1–5]. Nonetheless, the molecular network underlying the generation of cellular movement remains incompletely understood [6, 7]. This process involves an epithelial-to-mesenchymal transition (EMT) of the premigratory NC cells residing in the dorsal neural tube (NT) followed by delamination.
Bone morphogenetic protein (BMP), Wnt and fibroblast growth factor (FGF) signals were implicated in NC specification and lineage segregation [8–13] and evidence illustrates the involvement of BMP and Wnt in subsequent NC delamination and/or migration [14–20]. Our studies showed that a balance between BMP and its inhibitor noggin underlies the emigration of trunk-level NC independently of earlier cell specification . A decreasing rostrocaudal gradient of BMP4 activity is established along the NT by a reciprocal gradient of noggin. Noggin downregulation is, in turn, triggered by the developing somites, which thus determine the timing of NC emigration [18–20]. BMP then induces EMT of NC by triggering Wnt1 transcription. The latter promotes G1/S transition, which is a necessary step for delamination of trunk NC [14, 21].
Acting downstream of BMP and/or Wnt, transcription factors such as Sox9, FoxD3 and Slug were found to be sufficient for inducing some properties of NC differentiation when ectopically expressed in the neuroepithelium [22–24]. Moreover, when expressed in combination they induced some features of EMT  whereas when separately provided they were unable to promote NC delamination in the trunk [23–26]. Successful delamination also requires the activity of effector genes that act on re-organization of the actin cytoskeleton, alterations in adhesive properties and consequent loss of epithelial polarity [13, 27–30]. In this context, N-cadherin was found to be a component of the BMP-dependent network leading to NC EMT. N-cadherin inhibits the onset of NC delamination both by a cell adhesion-dependent mechanism as well as by repressing canonical Wnt signaling. Relief from N-cadherin-mediated inhibition is attained in the dorsal NT during the onset of cell emigration. This is accounted for by an ADAM10-dependent cleavage of the full-length molecule into a soluble domain with pro-delamination properties, a process triggered by BMP .
Additional candidates for orchestrating NC delamination include the RhoGTPases. RhoGTPases are molecular switches that control a variety of signal transduction pathways; they are known primarily for their pivotal role in regulating the actin cytoskeleton, but not less significant are their effects on cell polarity, gene transcription, G1 cell cycle progression, membrane transport, and so on (reviewed in [32–34]). Rho proteins cycle between two conformational states, one bound to GTP, the active form, and the other bound to GDP, the inactive state. This switch is regulated by several activators and inactivators [35, 36]. The role of Rho GTPases in NC development remains virtually unexplored. Association between ectoderm and intermediate neural plate, a paradigm known to induce NC features [37, 38], activates transcription of Rho-related proteins . Transcription of rhoA and rhoB, but not of rhoC, was detected in the avian NT with rhoB being expressed in premigratory and early migrating NC . Initial rhoB mRNA is induced by BMP signaling , which is also required for its continuous transcription . In contrast, Wnt3a appears to have no effect on the onset of rhoB expression , yet Wnt6 was sufficient to stimulate prematurely both rhoA and rhoB mRNAs . Furthermore, inhibition of either canonical or non-canonical Wnt signaling had no effect on maintenance of rhoB in the dorsal NT . In contrast to avians, in mouse and Xenopus, rhoB mRNA is expressed in migrating NC but not in the NT [43, 44]; another, unconventional RhoGTPase termed RhoV was found instead to be expressed in the early Xenopus NC and to be required for its specification . The first functional study performed in avians in vitro suggested that Rho activity was required for NC delamination but not for subsequent cell migration . However, loss of RhoB function in mice exhibited no apparent morphogenetic defects, although the development of the NC was not directly monitored . Furthermore, in different cellular contexts, Rho signaling has been shown to promote cell migration or, conversely, to maintain the epithelial state [47–49]. Based on the above, it was important to investigate its function in the NC, a bona fide model for generation of cellular movement, and to begin understanding how it integrates within the already known molecular network leading to NC delamination.
Here we show that Rho signaling, through Rho-associated kinase (Rock) activity, negatively modulates the onset of NC emigration both in explants and in vivo. In vivo loss-of-function of either RhoA or RhoB enhanced emigration of NC cells. Likewise, inhibiting Rho signaling by C3 transferase stimulated the process. Consistently, treatment of explanted neural primordia with membrane-permeable C3 or with the Rock inhibitor Y27632 both accelerated and enhanced NC emigration. None of the above affected NC cell proliferation. Furthermore, they altered crest morphology by reducing stress fibers and focal adhesions. They also caused premature downregulation of membrane-associated N-cadherin. Reciprocally, activation of endogenous Rho by lysophosphatidic acid (LPA) inhibited emigration while enhancing stress fibers, focal adhesions and N-cadherin. The effect of LPA was specific to Rho as Y27632 rescued the observed phenotypes. Since NC delamination is triggered by BMP and depends upon successful G1/S transition, we examined possible interactions between these pathways. Blocking Rho or Rock rescued NC delamination in explants treated with noggin or with a G1/S inhibitor. In the latter case, G1-arrested cells successfully emigrated. Reciprocally, BMP4 was unable to rescue cell emigration upon inhibition with LPA. Together, our findings suggest that Rho-GTPases, acting through Rock, negatively regulate NC delamination by modifying cytoskeleton assembly and cell-cell adhesions and acting downstream of BMP and of G1/S transition.
Loss of the membrane-bound, active form of RhoA and RhoB is associated with the EMT of NC cells
To further confirm that the membrane-associated staining reflects active Rho proteins, explants were incubated with the Rho-binding domain of Rhotekin fused to glutathione S-transferase (RBD-GST), which specifically recognizes the GTP-bound form of Rho proteins, followed by indirect immunofluorescent detection with an antibody to GST. Consistent with the expression data described above, epithelial progenitors exhibited active Rho proteins in a membrane-associated pattern that colocalized with membranous green fluorescent protein (GFP), and this surface staining disappeared upon cell dissociation (Figure 2C and inset, and data not shown). To control for the specificity of this reaction, explants were pretreated with C3 transferase, which inhibits ADP-ribosylation of all Rho proteins  but not of Cdc42 or Rac . No membranous staining was detected under these conditions (Figure 2D and inset). Taken together, these results suggest that Rho signaling is active in the epithelial NC progenitors prior to EMT.
Inhibition of Rho/Rock activities promotes premature and enhanced NC delamination
Likewise, the specific Rock inhibitor Y27632 [55–57] was similarly applied to neural primordia excized from the level opposite the unsegmented mesoderm. At 16 h of incubation, the number of cells present on the substrate of Y27632-treated cultures was 3.2-fold higher compared to that monitored under control conditions (N = 6 counted cultures for control and Y27632 out of 10 and 16 showing a similar phenotype, respectively' Figure 3J). Notably, NC delamination in the presence of the Rock inhibitor was already evident as early as 2 h after drug addition when compared to control tubes in which the first delaminating cells became apparent about 9 h following explantation (Additional file 1A,C), indicating that premature delamination is not accounted for by increased cell proliferation. Cells delaminating in the presence of either C3 or Y27632, like their control counterparts, expressed HNK-1, confirming their NC identity (Figure 3B,E,H). In contrast to controls, however, they directly transited into a mesenchymal phenotype not exhibiting the characteristic dense intermediate epithelioid conformation (Figure 3A,D,G; Additional file 1B,C,D). Consequent to both treatments, the phenotype of cells was dramatically altered, with remarkable stellate morphologies and many long protrusions. This was accompanied by, and likely to result from, a loss of F-actin+ stress fibers (Figure 3C,F,I; Additional files 2 and 3). In spite of enhancing cell emigration, the proportion of proliferating cells was not increased upon treatment with either C3 or Y27632 (Figure 3K, N = 5 for each treatment). Hence, inhibition of Rho/Rock signaling in explants both enhances and accelerates delamination of NC progenitors while disrupting the F-actin cytoskeleton but without affecting their proliferation.
In contrast to both constructs that inhibited RhoB activity, treatment with N19-RhoA caused a dissociation of neuroepithelial cells when examined after 16 h, consistent with its broader expression pattern in the NT . This confirms the relative specificities of the constructs used to either RhoA or RhoB. In spite of hemi-NT dissociation (note round cells in Figure 5E, F compared to pseudostratified progenitors in N19-RhoB in Figure 5D), the emigrating N19-RhoA/GFP+ progenitors co-expressed both HNK-1 and FoxD3, confirming they are NC cells and also suggesting that labeled central nervous system progenitors that dissociated did not contribute to the NC migratory pathway. Likewise, no central progenitors were found to join the NC migratory pathways upon treatment with C3 transferase (Figure 4), altogether suggesting that dissociation of central progenitors is not sufficient for inducing their migration. Because of its adverse effect on NT progenitors, counting the number of emigrating cells in N19RhoA-treated embryos at advanced stages was less compelling; therefore, electroporations were performed for a shorter period at a very caudal level of the axis and embryos were fixed 8–10 h later, corresponding to the levels of the caudal or rostral segmental plate or early epithelial somite, respectively. In these regions, control-GFP+ NC cells were still confined to the NT, with no measurable cell delamination (data not shown). In contrast, premature NC delamination occured in neural primordia that received N19-RhoA while transfected central nervous system progenitors were still pseudostratified (Figure 5G–J). Furthermore, the early delaminating cells co-expressed NC-specific markers such as Snail2 (Figure 5I, inset), FoxD3 (see later figure) and Sox9 (not shown). No such effect was observed when transfecting either N19-RhoB or GAP-RhoB, further substantiating the specificity of the tools employed.
Taken together, our data demonstrate that loss of Rho function both in vivo and in explants facilitates the onset of NC emigration, suggesting that endogenous Rho plays a negative role in the process. The observation that both enhanced and accelerated delamination also occurs when inhibiting Rock further suggests that Rho acts via Rock signaling to maintain NC cells in an epithelial state.
Activation of Rho signaling with lysophosphatidic acid inhibits the onset of NC emigration
Next, small pieces of LPA-containing pluronic gel or of control gel were grafted dorsal to the NT in ovo and the number of Hoechst+ NC cells apparent dorsal to the neural primordium and up to the dorsomedial border of the somites was monitored. A bilateral decrease in the extent of NC emigration was observed opposite both epithelial and dissociating somite levels in NTs that received LPA over controls (77.2% and 42.6%, respectively; N = 4 for each treatment; Figure 6H). No apparent difference in cell survival was observed between the above treatments (data not shown). To further visualize this effect, control GFP was electroporated unilaterally into NTs immediately before application of the pluronic gel. As shown in Figure 6E–G, labeled cells emigrated from control neural primordia, but not from tubes that received LPA. Hence, stimulation of endogenous Rho function inhibits NC delamination in vivo and in explants, in further support of a negative regulatory role of Rho proteins.
Rho-GTPase activity does not affect specification to the NC lineage
Interactions between Rho/Rock, N-cadherin and the actin cytoskeleton underlie NC delamination
To examine the mechanism by which Rho signaling negatively affects NC delamination, explants were stained for F-actin, N-cadherin and vinculin. While epithelial cells in control explants exhibited a dense array of stress fibers, the delaminating NC cells expressed significantly fewer yet they retained cortical actin bundles circumscribing the cell periphery. Treatment with Y27632 that stimulated NC delamination caused a total loss of stress fibers and a significant change in cell morphology (Additional file 3A,B; Figure 3). Reciprocally, LPA-induced inhibition of NC EMT was associated with a dramatic increase in stress fiber density and both effects were reversed by co-treatment with Y27632 (Additional file 3C,D).
Next, we examined the presence of focal contacts by immunostaining for vinculin. Control delaminating cells exhibited vinculin-containing focal attachment points. While treatment with Y27632 markedly reduced their number, LPA stimulated them (Additional file 4). This indicates that the number of cell-substrate contacts depends on Rho/Rock activity and is inversely related to the extent of NC emigration. Together, these results suggest that stimulation or inhibition of NC delamination by Y27632 or LPA, respectively, is associated with, and likely to operate via, regulation of cell adhesion and cytoskeletal properties. Therefore, to delaminate successfully, epithelial NC cells need to downregulate Rho/Rock activities, which, in turn, diminish the number of stress fibers and abrogate N-cadherin-mediated adhesion.
Rho/Rock signaling acts downstream of BMP/noggin in the regulation of NC delamination
Downregulation of noggin in the dorsal NT relieves BMP activity from the inhibition to which it is subjected along the caudal NT . BMP4 then triggers NC delamination via the canonical Wnt pathway . Moreover, N-cadherin, which inhibits NC delamination, is part of this molecular cascade . Since we show that Rho activity negatively modulates NC delamination and also alters N-cadherin expression, we asked whether Rho/Rock signaling are part of the BMP-dependent network leading to NC emigration.
Electroporation of noggin-DNA along with GFP-DNA into hemi-NTs in ovo inhibited delamination of NC cells following explantation of the neural primordia when compared to control epithelia in which GFP+ NC cells normally delaminated (Figure 9A,B; N = 12 for each treatment). This was accompanied by an increase in stress fiber density similar to that observed in LPA and GI254023X-treated explants (Figure 9D,E). To examine whether this was caused by an increase in Rho/Rock activity, noggin-transfected hemi-NTs were treated with Y27632, which dramatically reduced the amount of stress fibers and rescued NC delamination (Figure 9C,F; N = 16). Similar results were obtained upon treatment with C3 transferase (data not shown).
Next, we examined the ability of BMP4 to rescue cell delamination in explants in which the process was inhibited by LPA. As previously described, continuous treatment with LPA for 16 h (with one change to fresh LPA 8 h after explantation) virtually prevented NC delamination and the maintenance of epitheliality was confirmed by positive staining of membranous N-cadherin (Figure 9G; N = 12). When LPA was washed away and replaced by BMP4, cell emigration with concomitant loss of N-cadherin immunoreactivity was apparent within a few hours (Figure 9H; N = 7). In contrast, when LPA was replaced by a mixture of LPA and BMP4, the latter was unable to rescue cell delamination and the pattern of N-cadherin very much resembled that of LPA only-treated explants (Figure 9I; N = 14). These results suggest that Rho/Rock act downstream of BMP/noggin in the dorsal NT.
Inhibition of Rho/Rock activity rescues delamination of G1-arrested NC cells
Growing evidence illustrates that EMT of NC cells followed by cell delamination are modulated by a complex network of interacting transcription factors, cell adhesion molecules and other regulatory proteins. A more complete knowledge of the component genes and their mechanisms of action is required for understanding such a basic developmental mechanism. A role for Rho signaling in this context was highly expected yet remained unclear. We report that Rho/Rock signaling helps maintain premigratory cells in an epithelial state. This is supported by several lines of evidence. The C3 exoenzyme is commonly used as an inhibitor of ADP ribosylation of Rho proteins, thus selectively preventing their activity [50, 51]. Addition of membrane-permeable C3, which inhibited the formation of actin stress fibers, resulted in enhanced NC emigration from explanted NTs and so did C3-encoding DNA when missexpressed in ovo. In contrast, previous data stemming from NT explants showed that soluble C3 inhibited NC delamination, hence implicating Rho proteins, in particular, RhoB, as a positive signal in the process . However, relatively high levels of the transferase were used in that study, perhaps to overcome its poor penetrance into intact cells. We reason that such high concentrations might have compromised some cellular functions related to the process of interest. Instead, recent availability of a membrane-permeable version of the enzyme enabled us to lower by 50- to 200-fold the concentration of drug applied to similar explants while still keeping its expected biological activity. To challenge our results by independent means, we overexpressed two specific dominant-negative forms of RhoB that also enhanced NC delamination. Furthermore, N19-RhoA stimulated premature cell emigration. Moreover, inhibition of Rock activity both advanced and enhanced the process dramatically. Reciprocally, stimulation of Rho activity by LPA prevented NC delamination. Furthermore, inhibition of NC delamination achieved with either LPA or the ADAM10 inhibitor GI254023X, the latter preventing N-cadherin degradation, was reversed by the Rock inhibitor Y27632 or by C3 (Figures 6 and 8; Additional file 3; MG and CK, unpublished results). Altogether, our loss and gain of function analyses, performed in ovo and in explants, demonstrate that Rho proteins act as negative signals in the process of EMT and delamination. This is consistent with our observaton that active Rho proteins are present in the epithelial progenitors and are lost from dissociating cells. It is important to stress that in different biological systems, Rho proteins were shown to act distinctly. Whereas in several instances Rho/Rock mediate the maintenance of the epithelial state, consistent with our data (see, for example, [49, 64]), in others, like in untransformed colon epithelial cells, Rho proteins maintain adherens junctions and epitheliality yet exert this effect via Dia rather than Rock, which instead disrupted intercellular adhesions . Furthermore, during endocardial cushion development, inhibition of Rock blocked EMT  rather than stimulating the process as observed in the NC. Missexpression of RhoA in post-otic NC cells was reported to alter patterns of NC migration even if virtually no active RhoA was detected in the intact migrating cells. Yet, RhoA missexpression had only a minor effect on cell delamination ; the level of active RhoA was, however, not assessed in the premigratory NC. These differences between post-otic and trunk NC are consistent with increasing data sustaining that cranial NC progenitors emigrate from the neuroepithelium as groups rather than individually and, therefore, use different mechanisms than those documented for the trunk [7, 68]. These and other results suggest that the multiplicity of activities mediated by Rho proteins and their downstream effectors should be carefully considered in a context-dependent fashion.
Which of the Rho proteins is active in this biological context? The two Rho proteins present in the avian NT are RhoA and RhoB; in contrast, RhoC was undetectable  (MG and CK, unpublished observations). Our functional assays show that loss of RhoA as well as of RhoB function enhanced emigration of NC cells in ovo. Consistent with previously published specificity data (see Results), we confirm that the dominant negative constructs used are selective as they had distinct effects on the integrity of the neuroepithelium. N19-RhoA caused a dissociation of the hemi-NTs, consistent with the broader expression of RhoA mRNA in the NT when compared to RhoB , whereas neither N19-RhoB nor GAP-RhoB altered general neuroepithelial morphology. In addition, N19-RhoA, but not N19-RhoB, caused a premature onset of NC delamination from segmental plate levels of the axis where all cells are still epithelial. Various reasons might account for the latter observation, such as differences in the half-life of the two proteins, differential plasmid efficiency, and so on. On the other hand, we cannot rule out the possibility that the effects monitored with each of the mutants represent an underestimate of the true effect as a partial compensation by endogenous RhoA of the N19RhoB or GAP-RhoB effect (and vice versa) might have occurred. The observation that the effect of C3 transferase in vivo was somewhat more significant agrees with such a notion. Consistent with the involvement of both proteins in maintenance of the epithelial state, we observed that in the epithelial and flattening epithelioid progenitors, the distribution of both RhoA and RhoB was predominantly membrane-associated, a pattern reflecting the expression of post-translationally modified proteins in their active state . Moreover, this pattern was gradually downregulated in cells about to delaminate and fully disappeared upon acquisition of a mesenchymal phenotype. Similar results were obtained upon direct visualization of active RhoGTPases with RBD-GST, which, together with results from the functional experiments, further strenghten the notion that active proteins relevant to NC EMT are membrane-associated. In addition to cell surface expression, RhoB-immunoreactive protein was expressed in the cytoplasm, likely in endosomal compartments  and this pattern did not change after NC cells delaminated, possibly reflecting the observed maintenance of RhoB mRNA and perhaps also of cytoplasmic RBD-GST binding in early migrating progenitors. Hence, vesicular RhoB could have other functions, not necessarily related to NC delamination, both before as well as during cell migration. Enhanced and premature delamination of NC cells is also observed upon inhibition of Rock signaling. The latter, although classically considered to act downstream of RhoA, was recently found to mediate activities of RhoB in various contexts [70–73]; hence, membrane-associated RhoA and RhoB might signal through Rock to affect EMT of NC cells, whereas in endosomes, RhoB pimarily interacts with mDia to affect protein trafficking (see, for example, ) and yet unknown functions in the NC.
Previously, we showed that G1/S transition is necessary for NC delamination . Furthermore, this process is stimulated by BMP-dependent canonical Wnt signaling through stimulation of cyclin D1 transcription . Loss or gain of Rho function did not alter G1/S transition in the NC system in spite of reported effects of Rho GTPases on cell cycle dynamics in other systems [87–92]. Nevertheless, for the first time, we observed that EMT of NC cells that were arrested in G1 by treatment with either mimosine or noggin can be rescued by inhibiting Rho or Rock activities. Under both conditions, the emigrating cells failed to incorporate BrdU, showing that G1/S transition can be dissociated from EMT. These data suggest that Rho/Rock act downstream of G1/S. Consistent with this interpretation and with positive and negative roles of cyclinD1 and Rho in NC EMT, respectively, it was shown that cyclin D1 triggers cellular migration in a model of cell metastasis through the inhibition of Rho GTP and RockII activity and signaling .
Although this study focused on the function on Rho signaling, the possibility that additional GTPases like Rac and Cdc42 are involved in NC delamination awaits further testing. Antagonistic activities of Rho and Rac were reported , raising the option that downregulation of Rho would, among other events, activate Rac and favor NC EMT. Hence, our findings underscore the complexity of the process of NC delamination by unraveling the involvement of additional factors and functional interactions. Altogether, these establish a growing genetic network responsible for the generation of NC motility.
We demonstrate that Rho/Rock activity is necessary and sufficient for maintaining NC progenitors in an epithelial state without affecting their state of specification. In addition, Rho activity acts downstream of BMP and G1/S transition, two essential events for achieving successful NC delamination. Acting as a downstream effector, Rho via Rock maintains stable F-actin stress fibers through which it is likely to preserve N-cadherin associated to the membrane of NC progenitors. Together, we suggest that Rho activity negatively modulates NC delamination by stabilizing the actin cytoskeleton and intercellular adhesions mediated by N-cadherin.
Materials and methods
Fertile quail (Coturnix coturnix japonica) eggs from commercial sources were used.
Expression vectors and electroporation
DNA expression vectors employed were: pCAGGS-AFP, which served as control ; YFP-C1-Lyn (from Tobias Meyer); the specific Rho targeting construct p190-rhoB-C (GAP-RhoB; from Yi Zheng ); dominant-negative RhoB and RhoA lacking GTPase activity (N19-RhoA and N19-RhoB; from George Prendergast ); the C3-like ADP-Rybosyltransferase (from Klaus Aktories ); and xNoggin . DNAs were subcloned into pCAGGS and either fused in frame to a GFP-encoding sequence or co-electroporated with control GFP. Experimental details are available upon request. DNA (3–5 mg/ml) was microinjected into the lumen of the NT of 15–18 somite-stage embryos at the level of the segmental plate and/or two recently formed somites. A four parameter PulseAgile square wave electroporator (PA-4000, Cyto Pulse Sciences, Inc. Maryland, USA) was used to deliver three groups of sequential pulses as follows: 3 × 18 V, 20 ms each; 3 × 26 V, 15 ms each; 3 × 18 V, 20 ms each. Embryos were reincubated for an additional 16 h, some followed by a 1 h pulse of BrdU (10 mM). Other electroporated embryos were reincubated for 2 h followed by explantation of isolated neural primordia (see below).
Explants of neural primordia
Intact or electroporated neural primordia containing premigratory NC were excized from segmental plate levels of 16–20 somite-stage embryos and then explanted onto 8-well chamber slides (Lab-Tek, Nunc, Rochester, NY, USA) pre-coated with fibronectin (50 μg/ml; Sigma, St. Louis, Mo, USA) as described . Culture medium consisted of CHO-S-SFM II (Gibco-BRL, Gaithesburg, MD, USA) to which membrane-permeable C3 (CT04, 1 μg/ml; Cytoskeleton, Inc. Denver, Co, USA), Y27632 (15 μM; Sigma), the amino-acid mimosine (600 μM; Calbiochem, San Diego, CA, USA), LPA (1 μg/ml, replaced once after 8 h in explants kept for a total of about 16 h; Cayman Chemical Co. Ann Arbor, Mi, USA), and the selective ADAM10 inhibitor GI254023X (12 μM) [98, 99], BMP4 (100 ng/ml; R&D Minneapolis, MN, USA), or combinations of the above were added.
Grafting of LPA-containing pluronic gel
Pluronic F-127 gel was prepared as previously described  and mixed with a concentration of 100 μg/ml LPA. Pluronic gel is liquid at low temperature but sets at room temperature, thus remaining stable over the site of application for several hours. Small pieces of control or LPA-containing gels were placed dorsal to NTs at the level of the segmental plate mesoderm and embryos were further incubated for 8 or 16 h.
Tissue processing, immunocytochemistry and in situ hybridization
Embryos were fixed with 4% formaldehyde, embedded in paraffin wax and sectioned at 5 or 10 μm. Rabbit anti-GFP (Molecular Probes, Eugene, OR, USA) was used at 1:500 in combination with HNK-1 or BrdU immunolabeling or combined with in situ hybridization for FoxD3, Snail2 or Sox9 [22–24]. Antibodies against intracellular or extracellular domains of N-cadherin were applied following methanol fixation as described . Vinculin antibodies were from the Hybridoma Bank. Filamentous actin was visualized with phalloidin. RhoA was visualized with two different antibodies: 26C4 (monoclonal SC418; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rat monoclonal Lulu51 . Likewise, RhoB was evidenced with polyclonal antibody 119 (SC-180; Santa Cruz Biotechnology) or with an anti-RhoB monoclonal antibody from the Hybridoma Bank . All antibodies were found to specifically recognize their respective antigens [40, 101]. For visualization of Rho proteins, explants were fixed in 10% trichloroacetic acid as previously described . The active, GTP-bound form of Rho was localized using GST-Rho-binding domain of Rhotekin (RBD-GST, Cytoskeleton, Inc.) as previously described . Nuclei were visualized with Hoechst. Embryo sections and explants were photographed using a DP70 (Olympus) cooled CCD digital camera mounted on a BX51 microscope (Olympus).
NC delamination was monitored in at least 5 embryos per treatment out of 8–21 embryos showing a similar phenotype, as described . Briefly, the number of GFP+ or Hoechst+ cells located up to the migration staging area was measured in 25 sections of control versus experimental hemi-NTs, and expressed as mean ± standard deviation of total cases monitored, respectively. The number of NC cells with mesenchymal morphology that exited explanted NTs was counted in 20–25 microscopic fields/explant, each comprising an area of 2,500 μm2. BrdU incorporation was measured as previously described . Results represent the average number of cells per explant (± standard deviation of 5–6 cultures counted out of at least 12–16 cultures/treatment showing a similar phenotype) normalized to the length of the NT fragment. Significance of results was determined using the unpaired Student's t-test.
bone morphogenetic protein
green fluorescent protein
Rho-binding domain of Rhotekin fused to glutathione S-transferase.
We thank Alexander Bershadsky and all members of our group for valuable discussions. We are indebted to Yi Zheng, G Prendergast, K Aktories, S Yonemura and A Ludwig for reagents. Anti-vinculin and a RhoB antibody were obtained from the Developmental Studies Hybridoma Bank. This work was supported by grants from DFG (SFB 488), Israel Cancer Research Fund (ICRF), Israel Science Foundation (ISF), March of Dimes, and EEU 6th Framework program Network of Excellence MYORES to CK.
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