Article |
Address correspondence to Marianne Bronner-Fraser, Division of Biology, 139-74, California Institute of Technology, 1200 East California Blvd., Pasadena, CA 91125. Tel.: (626) 395-3355. Fax: (626) 395-7717. E-mail: mbronner{at}caltech.edu
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Abstract |
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Key Words: Slit2; neural crest; vagal; chemorepellent; gut
* Abbreviations used in this paper: CM, conditioned medium.
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Introduction |
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Several inhibitory molecules have been shown to play important roles in neural crest migration. For example, ephrinB family members prevent neural crest entry into the caudal portion of each somite, resulting in segmental migration in the trunk region (Krull et al., 1997; Wang and Anderson, 1997). Other molecules implicated in neural crest cell guidance include chondroitin sulfate proteoglycans (Oakley et al., 1994) and Semaphorin3A and 3C (Eickholt et al., 1999; Brown et al., 2001; Feiner et al., 2001). However, none of these explain differences between the ability of vagal and trunk neural crest populations to enter the gut.
Slit proteins play important roles in axonal guidance in both vertebrates and invertebrates (Brose et al., 1999; Kidd et al., 1999; Li et al., 1999). These glycoproteins are known to be potent chemorepellents for midline axons in Drosophila as well as olfactory, forebrain, and dentate gyrus axons in mammals (Brose et al., 1999; Kidd et al., 1999; Li et al., 1999; Nguyen Ba-Charvet et al., 1999; Bagri et al., 2002). Slits function as repulsive factors during migration of neurons and glia (Hu, 1999; Wu et al., 1999; Zhu et al., 1999; Kinrade et al., 2001) and also can regulate axon elongation/branching in mammals (Wang et al., 1999).
In this study, we examine the potential role of Slit in neural crest migration. Slits are expressed in places where they could influence migrating neural crest cells (in the dorsal neural tube from which neural crest cells emigrate and near the entrance to the gut, which is selectively invaded by vagal, but not trunk, neural crest cells). Consistent with this, we find that only trunk neural crest cells possess Robo receptors for Slit. We tested the possible functional role of Slit on neural crest migration in vitro and in vivo by confronting neural crest cells with Slit2-producing cells or soluble Slit2. The results reveal for the first time a dual role for Slit2 in a migratory cell type, both inhibiting movement when in direct confrontation and enhancing motility when in solution. Our results at least partially account for the differential ability of vagal, but not trunk, neural crest cells to invade and innervate the gut.
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Results |
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We found that the patterns of expression for Slit2 (Fig. 1, a, d, and e), Slit1 (Fig. 1, b and c), and Slit3 (Fig. 1 f) largely overlapped. All were expressed prominently in the splanchnic mesoderm that marks the entrance to the gut. In addition, there was marked expression in the ventral neural tube, notochord, and dorsomedial dermomyotome. Slit1 and Slit2 were expressed strongly in the roof plate, whereas Slit3 had little or no expression in this site. Interestingly, Slit1 and Slit2 were expressed in both the floor plate and in forming motor neuron pools. In contrast, Slit3 was expressed in motor neurons but had low expression in the floor plate. These expression patterns are similar to those noted previously in the mouse (Yuan et al., 1999).
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Cells expressing Slit2 were introduced onto neural crest migratory pathways. HEK293 cells transfected either with human Slit2 or control vectors (Li et al., 1999) were labeled with DiI and microinjected onto trunk and/or vagal neural crest migratory pathways in early chick embryos. Vagal level injections were performed into somites 17 of stage 1012 chicken embryos (Hamburger and Hamilton, 1951). When injected into this location, the labeled cells appeared to localize in ventral sites around the dorsal aorta, as has been previously described for injections of cells or latex beads (Bronner-Fraser and Cohen, 1980; Bronner-Fraser, 1982). Vagal neural crest cells migrate from the neural tube and proceed ventrally to populate the dorsal root and sympathetic ganglia. Other vagal crest cells migrate further ventrally to penetrate the gut. When encountering Slit2-secreting cells at the level of the aorta, the vagal neural crest cells appeared to mix and closely associate with Slit2-secreting cells (Fig. 4, ce). Their distribution pattern was similar to that observed after injection of parental control cells (Figs. 4, a and b). Little or no avoidance behavior was observed after either injection of Slit2 cells (n = 1/8 embryos) or control cells (n = 0/6 embryos) at vagal levels.
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To visualize the interaction between neural crest cells and Slit2-expressing cells, we turned to an in vitro system in which cultured neural crest cells were confronted with Slit2-expressing cells. Neural tubes containing premigratory neural crest cells were isolated from stage 1316 embryos and plated onto tissue culture dishes in close proximity to control or Slit2-expressing cells. In this way, delaminated neural crest cells emerged from the neural tube and migrated toward the Slit2-expressing cells in a two-dimensional environment. After each experiment, cultures were stained with the HNK-1 antibody to confirm that the migrating cells were neural crest cells. Under these conditions, 90% of the migrating cells were HNK-1 positive.
Whereas trunk neural crest cells freely approached and intermixed with control HEK cells (Fig. 5 a), a sharp border was observed between trunk neural crest cells and Slit2-expressing cells (Fig. 5 b). A similar experiment was performed for vagal neural crest cells, using neural tubes explanted from the levels of somites 17 of stage 1012 embryos. In this case, the vagal cells intermixed equally well with Slit2-expressing cells and control cells (Figs. 5, c and d). Thus, analogous differences in the response of trunk versus vagal neural crest cells were noted both in vivo and in vitro, suggesting that the former, but not the latter, avoided an exogenous source of Slit2. Table I summarizes the results for in vivo and in vitro assays.
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Slit2 repulsive effect on trunk neural crest is contact mediated
Although Slit2 is soluble and can be secreted, it is most commonly found in membrane extracts and tightly associated with the cell surface (Brose et al., 1999; Nguyen Ba-Charvet et al., 2001a). To determine if the effects of Slit2 on trunk neural crest cells were caused by soluble Slit2 present in the media (Wu et al., 1999) or by cell membraneassociated Slit2 (Hu, 2001; Nguyen-Ba-Charvet et al., 2001b), we repeated the in vitro assay above with cells that were dried and dead but retained membrane-bound Slit2 (not depicted). Under these conditions (Figs. 5, eh), trunk, but not vagal, neural crest cells avoided Slit2 membrane ghosts, confirming that the effects of Slit2 are cell surface dependent. These results are summarized in Table I.
Soluble Robo reverses Slit2 repulsion of trunk neural crest
Slit2 is a known ligand for the Robo1 and Robo2 transmembrane receptors (Brose et al., 1999). We tested the ability of a soluble form of the receptor to neutralize the effects of exogenous Slit2. RoboN consists of the extracellular portion of Robo1 (Wu et al., 1999). When trunk neural crest cells were grown in media conditioned by the RoboN-secreting cells, the repulsive effect caused by Slit2 was reversed (Table II), suggesting that the effect was indeed Slit mediated. In contrast, RoboN media had no significant effect on trunk crest encountering control HEK cells. Similarly, trunk neural crest cells showed no altered behavior when encountering RoboN-secreting cells alone (not depicted).
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To examine the effects of soluble Slit2, migrating neural crest cells were placed on fibronectin-coated substrates transfilter to Slit2-expressing cells, control cells, Slit2-conditioned medium (CM),* or control CM (Wu et al., 2001). This assures that crest cells under experimental conditions would be exposed only to the soluble form of Slit2. With either transfilter Slit2 cells or Slit2 CM, the maximum distance traveled by neural crest cells was an average of 21% more than that observed under control conditions (n > 100 neural tubes in a total of 12 separate experiments; Table III; Fig. 6). In contrast, vagal neural crest cells failed to exhibit enhanced migration under identical conditions (n = 12 neural tubes in a total of three separate experiments; Table III).
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Slit2 increases trunk neural crest cell motility
The results suggest that soluble Slit2 enhances neural crest migration, as the distance traveled by the cells as a function of time increases significantly. To view this at higher resolution, we tracked individual cell movement by imaging neural crest cells in the presence or absence of soluble Slit2 by time-lapse video microscopy. To aid visualization, trunk neural tubes were electroporated with a GFP expression vector in vivo (Megason and McMahon, 2002), and then the tubes were explanted in vitro. Cultures were primed with Slit2 or control CM and filmed under a confocal microscope. The resultant movies were analyzed to examine the total and net distance navigated, speed, directionality, and persistence of movement of individual, GFP-labeled neural crest cells.
The results confirmed static pictures showing that trunk neural crest cells moved more dynamically and for greater distances in Slit2 than in control medium (Fig. 8; see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200301041/DC1). With or without Slit2, neural crest cells moved in a somewhat erratic manner, with frequent changes in direction (Kulesa and Fraser, 1998, 2000). The cells appeared to collide more frequently in the presence of Slit2, raising the possibility that cellcell collision may effect their degree of movement.
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Discussion |
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The expression of Slits in the mesentery is uniform throughout the rostrocaudal extent of the gut mesenchyme. Thus, the selective entry of vagal neural crest cells lies not in the distribution of the ligand but rather with expression of Slit receptors, Robo1 and Robo2, in different neural crest populations (Li et al., 1999; Holmes and Niswander, 2001; Vargesson et al., 2001). Migratory and condensing trunk neural crest cells express both Robo receptors, whereas neither receptor was detected on vagal neural crest. The absence of Robo receptors on vagal neural crest cells is consistent with their lack of response to Slit2. In previous publications that have examined the embryonic distribution of Robos, no expression on migrating neural crest cells was noted. By using higher resolution in situ hybridization and, in particular, in situs performed on tissue sections, we detected expression of both Robo1 and Robo2 on trunk neural crest cells. Not all of the HNK-1positive cells expressed detectable levels of Robo receptors; it is possible that some of the migrating cells expressed levels of receptors that were below the sensitivity of this method. Consistent with this possibility, older embryos during later stages of migration had more prominent Robo expression on migrating trunk neural crest cells, suggesting that receptor levels increase with developmental age.
Our data support the idea that Slits can function as direct chemorepellents for trunk neural crest cells. In vivo, we cannot rule out the possibility that Slits might also influence neural crest cells via indirect interactions with other tissues adjacent to neural crest migratory pathways, as the neural tube and dermomyotome also express Robo receptors. Our in vitro data, however, show that exogenous Slit2 and Slit1 can repel trunk neural crest cells in the absence of neighboring cell types. Furthermore, addition of RoboN specifically reverses the inhibitory effects of Slit2, suggesting a direct interaction.
Slit2 is likely not the only chemorepellent for trunk neural crest cells. Although Slit2 was used for most of the functional experiments performed here, we observed that multiple Slit isoforms, Slit1 and Slit3, have similar expression patterns in mesoderm at the entrance to the gut and that Slit1 can function similarly to Slit2. Furthermore, all three Slits share the highly conserved four leucine-rich repeats that have been shown to be responsible for Slit repellent activity (Battye et al., 2001; Chen et al., 2001; Nguyen Ba-Charvet et al., 2001a). Such redundancy has also been shown for olfactory neurons; these neurons that are repelled by endogenous Slit2 in the subventricular zone are also repelled by Slit1 (Wu et al., 1999). However, we cannot exclude the possibility that there are subtle functional differences in the different Slit proteins (Bagri et al., 2002; Plump et al., 2002).
The membrane-bound form appears to be responsible for Slit2's chemorepulsive effects on trunk neural crest cells. The 200-kD Slit2 glycoprotein is proteolytically processed into a 140-kD NH2-terminal fragment and an 60-kD COOH-terminal fragment in cell culture and in vivo (Brose et al., 1999; Nguyen-Ba-Charvet et al., 2001b). The NH2 terminus is more tightly bound to the membrane and has been shown to mediate repellent activities and stimulation of axon elongation (Brose et al., 1999; Wang et al., 1999; Niclou et al., 2000; Battye et al., 2001; Nguyen-Ba-Charvet et al., 2001b). As the COOH terminus does not appear to interact with Robo receptors (Nguyen Ba-Charvet et al., 2001a), the repulsion we observed on neural crest cells almost certainly involves only the NH2 terminus of the protein. In support of this, the leucine-rich repeats of the NH2 terminus appear to be sufficient to achieve trunk neural crest repulsion (Chen et al., 2001; unpublished data). Moreover, trunk neural crest cells exposed to diffusible Slit2 either in chemotaxis chambers or collagen gels showed no apparent repulsion (unpublished data).
In addition to being a chemorepellent, our data suggest that soluble Slit2 selectively enhances the distance migrated by trunk neural crest cells. Such dual function for Slit2 concurs with observations on axon guidance where Slit family members have been shown to play multiple roles, causing both faster axon growth and induction of branching (Wang et al., 1999; Ozdinler and Erzurumlu, 2002). For the case of trunk neural crest cells, Slit2 appears to have opposite effects of inhibition and promotion of motility in the same cell type. Our results are consistent with previous studies showing that Slit2 can affect cell motility independent of its repulsive activity (Mason et al., 2001).
Although many trunk neural crest cells exhibited enhanced migration in the presence of soluble Slit2, a subpopulation of cells in explant cultures moved relatively short distances away from the center of the explant, as assayed both by static visualization and by time-lapse video microscopy. One possibility is that there are two separate populations of cells in our cultures. The majority (90%) of cells was shown to be HNK-1positive neural crest cells, but a minority population fails to express this epitope and may represent neural tube mesenchymal cells rather than neural crest cells. Thus, it is possible that this small population of Slit2-unresponsive cells represents a nonneural crest population.
The dual functions of Slit2 in both repulsion and stimulation of migration in the same type of cell are not necessarily contradictory; an optimal chemorepellent might logically be expected to stimulate rapid movement away from its source. Previous studies have shown that Slit2 repels axons from motor, olfactory, dentate gyrus, and retinal neurons (Brose et al., 1999; Li et al., 1999; Erskine et al., 2000; Ringstedt et al., 2000), while it inhibits migration of olfactory bulb and cerebral cortical neurons as well as leukocytes (Hu, 1999; Wu et al., 1999, 2001). In addition to repulsive effects on axonal growth and cell migration, Slit2 is a potent stimulator of axonal elongation and dendrite branching (Wang et al., 1999; Ozdinler and Erzurumlu, 2002; Whitford et al., 2002). In the last few years, several axonal pathfinding and axonal regeneration inhibitor molecules have been shown to induce both chemorepulsion or chemoattraction, depending on the type or age of the cell (Lohof et al., 1992; Song et al., 1998; Cai et al., 2001; Qiu et al., 2002). These opposing effects appear to be due to the internal levels of cyclic nucleotides in the cells, leading to promotion or inhibition of axonal growth (Cai et al., 1999; Hopker et al., 1999).
In summary, we have shown that Slit2 is a true bifunctional molecule: it can both repel neural crest cells and increase their rate of migration. The presence of Slit family members at the entrance of the gut mesenchyme coupled with the presence of Robo receptors on trunk and absence from vagal neural crest provides the first molecular explanation for the differences in migratory behavior of these two subpopulations via Slit chemorepulsion. The results suggest that neural crest cells may not only avoid, but also rapidly move away from, sources of chemorepellents like Slit.
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Materials and methods |
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Section in situs were performed as follows. Embryos were fixed in modified Carnoy's and then dehydrated in 70%, 90%, 2 x 100% ethanol series, followed by two changes of histosol, and then paraffin. Sections were mounted on gelatin-covered superfrost glass slides and dried overnight. Before in situ, slides were dewaxed in histosol, rehydrated by passing through a series of ethanol (90, 70, and 50%), and rinsed in water, then PBS, and then in two changes of 2x SSC. Hybridization was done by diluting 1.5 ng/µl of slit2 and robo1 and 2 probes (Vargesson et al., 2001), incubated at 65°C overnight in 50% formamide, 2x SSChumidified chamber. Slides were washed for 15 min in 50% formamide, 1x SSC, 0.1% Tween-20 at 65°C and then in MABT. The slides were then blocked for 1.5 h in MABT with 2% Boehringer blocking reagent and 20% any goat serum. AntiDIG-AP was diluted 1:2,000 in the above solution and applied overnight at room temperature. The next day, slides were rinsed in MABT in agitated bath, 30 min each, and then equilibrated in NTMT (0.1 M NaCl, 0.1 M Tris, pH 9.5, 5 mM MgCl2, 0.1% Tween-20) before BM purple color reaction.
In vivo avoidance assay
Fertilized eggs were incubated at 38°C for 2856 h, until embryos reached HH1012 (for vagal neural crest) and HH1216 (for trunk neural crest). Occasionally, stage 12 embryos received injections at both vagal and trunk levels. Eggs were windowed and visualized by a sub-blastodermal injection of India ink (diluted 1:10 in PBS). Slit2-expressing or control HEK293 cells, labeled with DiI, were injected in the somites at trunk or vagal levels. The eggs were closed with Scotch tape and reincubated for an additional 24 h. Embryos were removed from the eggs, stripped of the membranes, and fixed in 4% paraformaldehyde overnight before being stored in PBS. Embryos were thoroughly washed in PBS and then blocked overnight with PBS containing 1% Triton X-100 and 10% FBS at 4°C. After 3 h at room temperature in washing buffer (PBS with 1% Triton-X100, 1% FBS), embryos were incubated with 1:300 HNK-1 supernatant in PBS overnight at 4°C. The next day, embryos were extensively washed and incubated with an antimouse IgM-specific Alexa 488conjugated antibody (Molecular Probes). The next day, the embryos were washed extensively, Z scanned with a 410 LSM confocal microscope, and projected into a single file with LSM 5 Image Browser by Carl Zeiss MicroImaging, Inc.
In vitro avoidance assay
Quail neural tubes from HH1012 (for vagal crest, using only the neural tubes from the first until the seventh somite) and HH1316 (for trunk, the posterior parts of the tube) were dissociated in 1.5 mg/ml of dispase and washed in Leibovitz-15 medium. The neural tubes were cut in small pieces (size of two to three somites) and pipetted in the center of wells coated with fibronectin (10 µg/ml) already surrounded by a monolayer of live or dried Slit2 or control HEK293 cells (Li et al., 1999). Neural tubes were cultured in DME, 10% FBS, and 100 mg/ml and 100 U of penicillin and streptomycin, respectively, for 18 h, after which they were fixed in 4% paraformaldehyde for 30 min and subsequently blocked for 30 min with PBS, 1% Triton X-100, 10% FBS. Primary antibody was HNK-1 for visualizing neural crest cells, followed by an antimouse IgMAlexa 488 secondary (Molecular Probes). At the end, slides were incubated with DAPI in PBS to visualize cell nuclei.
Neural crest response was determined as follows. If both types of cells (neural crest and HEK) either mixed and/or overlapped, it was scored as no repulsion. If, however, neural crest cells kept a marked distance between both cells, failed to overlap, and/or changed their shape from mesenchymal to slender, more collapsed, it was scored as repulsion. The number of neural tubes with such responses was counted, and the percentage was determined based on the total number of neural tubes with neural crest in close proximity to the control cells. Neural tubes with neural crest cells that were not in close proximity to the cells were not counted.
Transient transfections of mSlit1 (Wu et al., 1999) into HEK293 cells were done using Lipofectamine 2000TM (Invitrogen) according to the manufacturer's instructions. The next day, cells were lifted and plated onto fibronectin-coated slides overnight. The next day, a hole in the monolayer was created, neural tubes were added, and the rest was as described above.
In vitro migration assay
Quail neural tubes from HH1012 (for vagal crest) and HH1316 (for trunk, the posterior parts of the tube were obtained as described above) were cultured in DME/10% FBS in fibronectin-coated 24-transwells (Corning) above either Slit2 or control HEK cells or CM from the same cells. Cultures were grown overnight, and the next day, neural crest cells could be seen as a halo around the neural tubes. The cultures were fixed with ice-cold methanol for 20 min, cells were stained with toluidine blue, and the maximal distance of neural crest cell from the neural tube was measured and compared.
In vitro wound assay
Quail neural tubes from HH1316 (for trunk, the posterior parts of the tube were obtained as mentioned above) were cultured in DME/10% FBS in a fibronectin-coated 2-well chamber slide (Nunc) overnight, and the medium was changed to one conditioned for 5 d by Slit2 or control cells. For the priming experiments, neural crest cells were preincubated with CM for 2 h before making the wounds. Wounds were made by scraping cells (two to three cellswidth lanes) with a pulled glass needle, and lanes were scored over 11.5-h periods to count the cells crossing the wound space. The criteria used was to count the points at which the wound was sealed at any length of the wound lane (regardless of the number) as "sealed"; "nonsealed" was scored as the number of lanes that did not have cells contacting from the opposing sides of the wound.
Time-lapse video microscopy
Quail embryos stage 1315 were electroporated with pCIG vector carrying the GFP marker (Megason and McMahon, 2002) with two pulses of 100 mV each. After 12 h in a 38°C incubator, neural tubes were isolated as described above and cultured on glass slides coated with fibronectin in DME/10% FBS. The next day, cells were primed for 1 h with control or Slit2 CM as before, and labeled cells were imaged using a Carl Zeiss MicroImaging, Inc. 410 LSM every 90 s for 3 h. The average speed traveled by individual neural crest cells in control versus Slit2-containing medium was determined by comparing cells filmed on the same day during a set number of frames (each frame corresponding to 90 s). The captured images were converted into a QuickTime movie with NIH Image 3 and analyzed with the DIAS (Dynamic Image Analysis System; Solltech Inc.) program for cell tracking and measurements.
Online supplemental material
The supplemental material (Fig. S1 and Videos 1 and 2) is available at http://www.jcb.org/cgi/content/full/jcb.200301041/DC1. The movies show neural crest cells moving from three different experiments combined together for control and Slit2-exposed neural crest cells. The lapsed time is 2.5 h.
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Acknowledgments |
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This work was supported in part by a postdoctoral fellowship to M.E. De Bellard from the National Multiple Sclerosis Society (FA 1383-A-1) and by a United States Public Health Service grant (HD-15527) to M. Bronner-Fraser.
Submitted: 13 January 2003
Revised: 11 June 2003
Accepted: 16 June 2003
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