Isolation of BMP-induced neural genes
We used a PCR-based differential display assay (Liang et al., 1993; Liang and Pardee, 1992) to identify genes that define early stages of neural crest cell differentiation. This assay was designed on the basis of previous studies showing that cells in neural plate explants can be induced to differentiate into neural crest cells upon exposure to BMPs (Basler et al., 1993; Liem et al., 1995). Caudal neural plate tissue from stage 10 chick embryos was dissected into dorsal [d] and intermediate [i] regions (Liem et al., 1995; Yamada et al., 1993) and cultured in serum-free medium on a fibronectin substratum (Fig. 1A). Under control conditions, [i] explants did not give rise to migratory neural crest cells but when cultured in the presence of Dsl1 (a member of the BMP family) or other BMPs, many migratory neural crest cells were generated (Basler et al., 1993). In contrast, [d] explants grown alone express BMP genes (Liem et al., 1995) and thus generate migratory neural crest cells in the absence of exogenous BMP protein (Basler et al., 1993). RNA was isolated separately from [i] explants cultured alone or with Dsl1 and from [d] explants cultured alone (Fig. 1A). cDNAs derived from these RNA samples were then subjected to differential display PCR analysis (see Materials and Methods). We focus here on one clone obtained from this screen, A19-1, a gene that is expressed in a highly selective pattern by cells in the dorsal neural tube and by neural crest cells.
Sequence analysis revealed that clone A19-1 encodes a member of the rho family of small GTP-binding proteins (Madaule and Axel, 1985) and is most closely related to mammalian rhoB (Fig. 1B). Vertebrates contain two other closely related rho genes, rhoA and rhoC (Chardin, 1988). To establish the identity of clone A19-1, we isolated two additional chick rho genes. Comparison of the sequence of these three chick genes indicated that clone A19-1 encodes chick rhoB and that the two additional genes encode chick rhoA and rhoC (Fig. 1B). The sequences of these chick rho genes have recently been reported in an independent study (Malosio et al., 1997).
rhoB expression defines early stages of neural crest differentiation
We first examined the pattern of rhoB expression during early neural development with reference to other markers of neural crest differentiation. In stage 10 chick embryos, the expression of rhoB was detected in cells at the dorsal tips of the neural folds (Fig. 2A). After neural tube closure, a high level of rhoB expression was detected in cells at the dorsal midline of the neural tube (Fig. 2B), in a domain that overlapped with that of the genes encoding the zinc finger transcription factor slug (Fig. 2M,N) and BMP4 (Fig. 2V,W). The expression of cadherin6B, in contrast to that of slug and rhoB, was detected initially throughout the neural plate with the exception of the ventral midline and became restricted to the dorsal neural folds just before neural tube closure (Fig. 2P,Q). At a more rostral level of stage 10 embryos, where the emigration of neural crest cells had commenced, rhoB expression was detected in migratory neural crest cells located close to the neural tube (Fig. 2C,D). The expression of rhoB by cells at the dorsal midline of the spinal cord persisted at stage 20 (Fig. 2E) but was absent by stage 24 (Fig. 2F), a time at which the vast majority of neural crest cells have migrated from the neural tube. Thus, the expression of rhoB appears to define an early stage of neural crest cell differentiation.
In contrast to the restriction in rhoB expression to regions of neural crest cell generation, the expression of rhoA was detected throughout the neural plate and neural folds as well as in surrounding mesodermal tissues (Fig. 2G,H). After neural tube closure, the level of rhoA expression in the ventral neural tube was markedly decreased (Fig. 2I). The highest level of expression of rhoA dorsally was detected in lateral regions of the neural tube and not, as with rhoB, at the dorsal midline (Fig. 2I arrow; data not shown). rhoC was expressed at a high level in the notochord but only at very low levels in the neural tube (Fig. 2J-L). Thus, of the three rho genes examined, the expression pattern of rhoB is most closely associated with the position of neural crest differentiation.
rhoB is expressed transiently in migrating neural crest cells
To define the pattern of rhoB protein expression, we generated a monoclonal antibody (4H7) directed against the carboxyterminus of chick rhoB. The selectivity of this reagent for detection of rhoB was established by western blot analysis (see Fig. 3A,B). The expression pattern of rhoB in the spinal cord of stage 14-17 chick embryos was compared with that of the neural crest marker HNK-1 (Tucker et al., 1984). rhoB was detected both in cells at the dorsal midline of the neural tube and in migrating neural crest cells (Fig. 3C). The expression of HNK-1 by neural crest cells, however, was evident only after their emigration from the dorsal neural tube (Fig. 3D). The coexpression of rhoB and HNK-1 was detected only in neural crest cells that had recently emigrated from the neural tube (Fig. 3E). The expression of rhoB was lost in neural crest cells that had migrated further from the neural tube, whereas the expression of HNK-1 persisted (Fig. 3E). Thus, rhoB expression defines the premigratory and early migratory phases of neural crest differentiation.
rhoB expression is induced by BMPs
The differentiation of neural crest cells appears to be initiated by BMP-mediated signals from the epidermal ectoderm (Liem et al., 1995). We therefore examined the effect of BMPs on the expression of rhoB and other early markers of neural crest differentiation. The expression of rhoB was detected at low levels in [i] explants grown alone for 24 hours but the level of expression was increased markedly (~7 fold) in [i] explants exposed to BMP4 (Fig. 4A), as expected from the design of the original screen. In contrast, the level of expression of rhoA was not significantly elevated by BMP4 exposure (Fig. 4A). The selective induction of rhoB by BMPs is consistent with the preferential association of rhoB expression in vivo with sites of neural crest differentiation. A marked induction in the level of expression of slug, cadherin6B and cadherin7 was also detected in [i] explants exposed to BMP4 (Fig. 4A). High levels of expression of rhoB, slug, cadherin6B and cadherin7 were also detected in [d] explants grown in the absence of added BMPs (Fig. 4A).
We next examined the temporal sequence of gene expression during the initial specification of premigratory neural crest cells, focusing on rhoB and slug, the two most selective markers of this step of neural crest cell differentiation. Marker gene expression was analyzed 3 hours, 7 hours and 9 hours after exposure of [i] explants to BMPs. After 3 hours, there was no induction of either slug or rhoB expression (data not shown). After 7 hours, induction of slug but not rhoB expression was evident (Fig. 4B). The induction of rhoB was detectable after 9 hours exposure to BMP4 (Fig. 4B). These results provide evidence that slug expression is induced prior to that of rhoB during the specification of premigratory neural crest cells.
Inhibition of rho activity perturbs neural crest development
To examine the involvement of rhoB in the early differentiation of neural crest cells, we attempted to block rho activity through the use of the Clostridium botulinum exotoxin C3. The C3 protein blocks rho activity through ADP-ribosylation of the Asn-41 residue conserved in all rho proteins (Aktories and Hall, 1989; Sekine et al., 1989). Importantly, C3 inactivates rho proteins without inhibiting rac or cdc42 activity (Ridley and Hall, 1992). Moreover, the inhibition of rho activity in intact cells can be achieved in vitro by exposure of intact cells to medium containing C3 (Jalink et al., 1994; Yamamoto et al., 1993).
We used an ADP-ribosylation assay (Ridley and Hall, 1992) to monitor the time course of inhibition of rho function in neural plate explants after addition of C3 to the culture medium. In [i] explants cultured in the presence of BMP (Dsl1) and in [d] explants, ~50% of rho proteins were ADPribosylated by C3 (~50 mg/ml) at 4.5 hours and nearly complete inactivation was evident by 9 hours (Fig. 5A). These results indicate C3 treatment efficiently ADP-ribosylates endogenous rho proteins in neural plate tissue in vitro.
To begin to determine the effect of inhibition of rho activity on the early differentiation of neural crest cells, we assayed the emergence of neural crest cells from [i] explants in the presence of BMP4, with or without C3. In control explants, the migration of neural crest cells from [i] explants exposed to BMP4 was first detected at 18 hours and by 24 hours, many cells had migrated from the explants (Fig. 5C). In the presence of C3 (~50 mg/ml), the number of migratory neural crest cells induced by 24 hours BMP4 exposure was reduced by over 80% (Fig. 5D,E). This result suggests that rho activity is required for the generation of migratory neural crest cells in response to BMP signaling. However, it does not resolve whether rho activity is required for the specification of premigratory neural crest cells, for the subsequent delamination of these cells or for the later migration of neural crest cells. We examine the potential involvement of rho proteins in each of these three steps of neural crest differentiation in the following sections.
Inhibition of rho activity does not prevent the specification of premigratory neural crest cells
The BMP-mediated induction of rhoB expression occurs later than that of slug (Fig. 4B), suggesting that rho activity may not be involved in the initial specification of premigratory neural crest cells. To address this issue more directly, we used RTPCR analysis and immunohistochemistry to examine if the C3- mediated blockade of rho activity affects the induction of slug expression by BMPs in [i] explants. Since rho function has been implicated in cell cycle progression (Olson et al., 1995), we also monitored the expression level of the gene encoding the s17 ribosomal protein and counted DAPI-stained nuclei to assess total cell number in these explants. In the presence of C3, there was a ~20% decrease in the level of s17 expression (Fig. 6A, legend). Nevertheless, the level of expression of slug in [i] explants grown in the presence of both BMP4 and C3 was ~80% of that in [i] explants cultured in BMP4 alone (Fig. 6A,B). Similarly, the BMP-mediated induction of slug protein expression persisted in [i] explants grown in the presence of C3 (Fig. 6C-F). Since slug is an early marker of avian neural crest cell differentiation (Nieto et al., 1994), these experiments support the idea that rho proteins are not required for the initial specification of neural crest cell fate.
We also analyzed the effects of C3 on cadherin7, rhoB and rhoA expression in [i] explants grown for 24 hours in the presence of BMP4. In explants grown in the presence of C3 the expression of cadherin7, a gene expressed at high levels only by migrating neural crest cells, was reduced to ~40% of controls (Fig. 6A,B). This result provides evidence that the expression of markers of later steps in neural crest differentiation is sensitive to the blockade of rho activity. The level of expression of rhoB itself was reduced to ~30% of controls in the presence of C3 (Fig. 6A,B). The basis of the reduction in rhoB expression is not clear but this observation raises the possibility that C3 may reduce rhoB function in neural cells both by inhibiting rho protein activity and by reducing the level of rhoB transcript. In contrast, the level of expression of rhoA was not reduced by the presence of C3 (data not shown).
Inhibition of rho activity blocks the delamination of neural crest cells
We next determined whether rho protein activity is required for later steps in neural crest cell differentiation, focusing first on the delamination of neural crest cells from the dorsal neural epithelium. To test this, neural tube explants were isolated from stage 10-11 embryos at the level of the five most recently formed somites (pNT explants). At this axial level, the emigration of neural crest cells has not yet started (Delannet and Duband, 1992; Loring and Erickson, 1987; Tosney, 1978). The time course of rhoB and HNK-1 expression by emigrating neural crest cells was first examined in control pNT explants. Neural crest cell emigration was detected by 3 hours in culture (data not shown) and extensive migration was observed by 6 hours (Fig. 7A,E). At this time, rhoB was expressed at high levels by cells close to the border of the neural tube explant (Fig. 7A). In contrast, HNK-1 was not expressed by neural crest cells at this stage (data not shown). Over the period from 6-21 hours in vitro, the expression of rhoB was progressively downregulated in neural crest cells as they migrated away from the border of the neural tube explant. In a complementary manner, the expression of HNK-1 increased with time and the extent of neural crest cell migration (Fig. 7B,C, data not shown).
We next examined the influence of C3 on the delamination of neural crest cells from such pNT explants. In order to block rho activity before the onset of neural crest emigration from neural tube explants, a higher concentration of C3 (150-200 mg/ml) was used. At this concentration, the inhibition of rho proteins in pNT explants was essentially complete by 3 hours (Fig. 7D). At 6 hours, there was an 87% reduction in the number of neural crest cells that emerged from pNT explants cultured in the presence of C3 (Fig. 7F). These findings provide evidence that the inhibition of rho activity results in a blockade of the delamination of neural crest cells from the dorsal neural tube.
rho proteins have been implicated in the assembly of the actin cytoskeleton (Ridley and Hall, 1992). We therefore used fluorescently labelled phalloidin to examine whether C3 modifies the actin cytoskeleton of neural crest cells located at the border of pNT explants. In control explants, emerging neural crest cells were flattened and exhibited a well-organized filamentous actin cytoskeleton, (Fig. 7G). In contrast, in the presence of C3 (~150 mg/ml), prospective neural crest cells at the margin of the neural tube explants did not exhibit prominent actin stress fibers and many cells had an elongated morphology (Fig. 7H). Thus, inhibition of rho activity appears to perturb the actin cytoskeleton of emerging neural crest cells and produces a marked change in their morphology. These cytoskeletal and morphological changes may contribute to the failure of neural crest delamination when rho activity is inhibited.
The later migration of neural crest cells persists when rho activity is inhibited
The rapid downregulation of rhoB expression after the emergence of neural crest cells from the neural tube in vivo (Fig. 3C,E) and in vitro (Fig. 7A-C) suggests that rhoB activity is not required for the later migration of neural crest cells. It remains possible, however, that other rho proteins contribute to the migration of neural crest cells. To assess this, we examined the influence of C3 treatment on neural crest cell migration in vitro. In these assays, we isolated regions of neural tube from a more rostral axial level at which neural crest emigration has commenced, to permit a more rapid initial emergence of neural crest cells. Segments of the neural tube were therefore isolated from the level of the 6th to the 10th most recently formed somites in stage 10-11 chick embryos (aNT explants) (Fig. 8A), plated on a fibronectin substratum and grown in vitro for 13.5 hours. C3 (~100 mg/ml) was added to experimental cultures with a delay of 6 hours after plating to permit the delamination and initial migration of neural crest cells. At this concentration, there was a >90% inactivation of rho proteins 3 hours after the addition of C3 (t=9 hours from the onset of culture) and a complete inactivation by 6 hours (Fig. 8B).
Phase-contrast images of the same culture fields were obtained 6 hours, 9 hours and 13.5 hours after the onset of culture and the extent of migration of neural crest cells was compared in the presence and absence of C3 (Fig. 8D-I). We focused on those cells that had migrated from aNT explants at early stages and thus were located furthest from the explant. In these neural crest cells the expression of rhoB has been downregulated (Fig. 7A-C, data not shown). In control cultures, the rate of neural crest cell migration was relatively constant over the entire 13.5 hours culture period, with a mean migration rate of ~30 mm/hour (Fig. 8C). In cultures exposed to C3 from 6 hours, neural crest cell migration persisted over the subsequent 7.5 hours culture period. Moreover, the mean rate of migration in the presence of C3 was not significantly different from the rate detected prior to C3 addition, or from the rate of cell migration in control cultures (Fig. 8C). Thus, the later migration of neural crest cells appears not to be markedly affected by C3. These data, taken together with the downregulation of rhoB expression in neural crest cells soon after their migration in vitro and in vivo indicate that rhoB activity is not required for the later migration of these cells. They also argue against a critical role for other rho proteins in this migratory step, at least as assayed in vitro on a fibronectin substratum.
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