| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 281, Issue 17, 12123-12131, April 28, 2006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1
23
From the
Laboratory of Molecular Biology, Department of Pathology, University Hospital Zurich, CH-8091 Zurich, Switzerland and the Department of Biology, Institute of Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zurich, Switzerland
Received for publication, October 4, 2005 , and in revised form, February 14, 2006.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
On their route, neural crest cells pass through highly permissive extracellular matrices, which allow rapid cellular movements. These pathways are flanked by tissues that block neural crest cell immigration and thus provide the directional information. These barrier tissues, previously identified by microsurgical manipulations, include the posterior sclerotomes (5), the perinotochordal region (6), and for a short period also the subectodermal matrix prior to melanocyte precursor invasion (7). Consequently, the streams of neural crest cells, which originally emigrate in an unsegmented fashion from the dorsal neural tube, are on their ventral path canalized into the anterior sclerotome strictly avoiding the posterior somitic halves and the more ventrally localized perinotochordal zone. This particular migration behavior finally leads to the characteristic segmental pattern of the forming sensory and sympathetic ganglia.
Since the major migration promoting substrates, fibronectin and laminin (8), are uniformly expressed in both halves of the somites (9), the guidance of the migratory neural crest cells appears to depend mainly on inhibitory cues. Several extracellular matrix and cell surface components match the candidate profile for a migration blocking function as they are selectively expressed in non-permissive tissues. Molecules consistently absent from the pathways, but highly expressed within the barriers, include chondroitin 6-sulfate proteoglycans, peanut agglutinin (PNA)4-binding glycoproteins (10, 11), F-spondin (12), semaphorin3A (13), T-cadherin (14), collagen IX (15), and, except for the dorsolateral path, ephrins (16-18).
For some of these molecules, like semaphorin3A (13) and ephrins (ephrin-B1 in avian (16) and ephrin-B2 in mammalian embryos (18)), inhibitory activities on neural crest cell migration have been demonstrated in vitro using neural tube or whole trunk explant culture systems. Unexpectedly, however, neither the gene inactivation of semaphorin3A (19) nor of ephrin-B2 (20) and the corresponding Eph receptors on neural crest cells (21, 22) resulted in the mutant mice in aberrant migration patterns through the somites. These observations suggested that a concerted action of multiple inhibitory and some attractive cues are required to guide trunk neural crest cells in vivo (1, 17, 23, 24).
Prime candidates for a cooperative partnership with the cell surface contact inhibitors of neural crest motility are extracellular matrix components belonging to the chondroitin sulfate proteoglycans (CSPGs) and PNA-binding glycoproteins (10, 11, 25, 26). Especially, the chondroitin sulfate proteoglycans versican (27) and aggrecan (28) appear to be functionally involved in the inhibition. Both proteoglycans are members of the hyalectan family forming large complexes through interactions with hyaluronan and link proteins (29). They play key roles during development, as the constitutive abrogation of their expression leads in homozygous mice to early intra-uterine (versican) or perinatal death (aggrecan) (30, 31).
At least four different isoforms of versican (V0 to V3) exist as a result of alternative splicing of two exons encoding the central glycosaminoglycan carrying domains, glycosaminoglycan (GAG)-
and GAG-
(32, 33). The largest splice variants of versican, V0 and V1, are highly expressed during embryogenesis and are frequently associated with little adhesive, fast-proliferating tissues displaying a high extracellular matrix turnover rate (34, 35). Versican V2, a smaller central nervous system-specific isoform is in contrast produced late during neurogenesis (36). It is a potent inhibitor of axonal growth and hence, seems to participate in restricting the structural plasticity of myelinated fiber tracts and impede regeneration in the mature central nervous system (37).
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
or GAG- domains of bovine, human, and mouse versicans were affinity purified from rabbit antisera previously prepared in our laboratory (32, 37, 40, 41). Monoclonal antibodies CS-56 recognizing intact chondroitin sulfate chains and 
Di-6S specific for chondroitin 6-sulfate "stubs" exposed after chondroitinase ABC digestion were obtained from Sigma and Seikagaku, respectively. Polyclonal antibodies against p75NTR were purchased from Chemicon and a monoclonal antibody specific for Sox10 (42) was a kind gift of Michael Wegner (University of Erlangen, Germany). For immunostaining of fibronectin the monoclonal antibody IST-4 (Sigma) was used. Isolation of Versican Isoforms—Intact versicans V1, V2, and a mixture of V0 and V1 were isolated from calf aorta, bovine spinal cord, and the spent culture medium of the human glioma cell line U251MG, respectively. Our previously developed procedure for the isolation of brain versican V2 was adapted for this purpose (40). Fig. 1 summarizes sources and steps involved in the protein-chemical purification of the individual isoforms.
Bovine tissues obtained from the local abattoir were homogenized in 6 volumes of ice-cold extraction buffer containing 4 M guanidine hydrochloride (GdnHCl), 50 mM sodium acetate, pH 5.8, 2 mM EDTA, 10 mM N-ethylmaleimide, 1 mM Pefabloc, 25 mM 6-aminocaproic acid, and 5 mM benzamidine and extracted overnight. After filtration through cheesecloth, the extract was cleared by centrifuging at 100,000 x g for 45 min. The supernatant was dialyzed extensively against 50 mM Tris, 10 mM EDTA, pH 7.0. The precipitate formed during dialysis was removed by centrifugation at 27,000 x g for 30 min. Subsequently, ammonium sulfate was added to the supernatant to reach 20% saturation. Proteins were allowed to precipitate overnight at 4 °C. Following centrifugation at 27,000 x g for 45 min the ammonium sulfate concentration was raised to 60% saturation. After an additional overnight incubation at 4 °C the precipitate was collected and resuspended in urea buffer (6 M urea, 0.25 M NaCl, 50 mM Tris, 10 mM EDTA, pH 6) followed by dialysis against 10 volumes of the same buffer. This crude extract was batch absorbed on Q-Sepharose FF (Amersham Biosciences) overnight and then packed into a column. The bound proteins were first washed with urea buffer containing 0.25 M NaCl and then eluted with a linear NaCl gradient to 1 M. 10-µl aliquots of each fraction were tested for the presence of versicans by slot blotting using antibodies against bovine GAG- (V2) or GAG- (V1) epitopes (40). Samples positive on the blot were pooled, dialyzed against 0.5 M NaCl, 20 mM Tris, 10 mM EDTA, pH 8, and batch-absorbed overnight to hyaluronan-Sepharose, which had previously been prepared according to the method of Tengblad (43) by coupling hyaluronan from bovine trachea (Sigma) to EAH-Sepharose 4B (Amersham Biosciences). Separate hyaluronan-Sepharose batches were used for each versican isoform preparation to avoid cross-contamination. The hyaluronan-Sepharose affinity column was subsequently packed and washed with a gradient of 0.5 to 3 M NaCl. Finally, hyalectans were eluted with 4 M guanidinium buffer (4 M GdnHCl, 20 mM Tris, 10 mM EDTA, pH 8). After analysis with slot blotting, the versican-containing fractions were pooled and concentrated to a volume of 500 µl using Biomax 100/Ultrafree-15 filters (Millipore).
For the V1 preparation from aorta, an additional gel filtration step on Sepharose CL-4B was required to remove partially degraded versican. For this purpose, the column was equilibrated with 3 bed volumes of the GdnHCl buffer before loading of the sample. The column was run at a slow flow rate of 0.4 ml/min. Every second fraction was tested for the presence of versican V1 on a slot blot. Antibody-reactive samples were pooled, dialyzed against PBS, and concentrated.
For the isolation of a mixture of versicans V0 and V1 from U251MG cell supernatants the proteoglycans were first precipitated from conditioned medium by adding ammonium sulfate to 70% saturation followed by purification with anion exchange and hyaluronan affinity chromatography as described above. Domain-specific antibodies recognizing the human versican homologues were used for the slot blot detection instead.
To obtain core glycoproteins, 1 to 5-µg aliquots of the versican preparations were digested with 10 milliunits of chondroitinase ABC (Seikagaku) in chondroitinase buffer (40 mM Tris, 40 mM sodium acetate, 10 mM EDTA, 1 mM Pefabloc, 3 µM pepstatin, 4 µM leupeptin, pH 8.0). Reactions were allowed to proceed overnight at 37 °C. Protein concentrations were determined with a BCA protein assay kit (Pierce).
Protein Electrophoresis and Immunoblotting—Versican samples were separated on 4-15% PHAST SDS-polyacrylamide gels (Amersham Biosciences) under reducing conditions and stained with either Coomassie Blue (NOVEX colloidal blue staining kit, Invitrogen) or with silver stain (PHAST GEL Silver Kit; Amersham Biosciences). For immunoblotting, chondroitinase ABC-digested samples were resolved on 4-15% PHAST polyacrylamide gels followed by diffusion transfer onto Immobilon-P membranes (Millipore) at 70 °C for 30 min. The blots were blocked with 3% dry milk in PBS for 30 min followed by incubation with the primary antibodies (all diluted 1:1000 in the blocking buffer) overnight. After washing, alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse Ig secondary antibodies (BIOSOURCE International, CA; diluted 1:15000 in PBS) were allowed to bind for 1 h. The color reaction was performed with Western blue substrate solution (Promega).
|
Stripe Choice Assays—Stripe choice assays were done as described previously (37) (modified protocol from Vielmetter et al. (47)). 20 x 20-mm glass coverslips were coated in alternating stripes of test and control substrates for migratory eNCSCs. The coating solutions of the test substrate contained variable concentrations of versican isoforms (0 to 100 µg/ml) admixed to 100 µg/ml human fibronectin, whereas the control lanes were treated with 20 µg/ml fibronectin (Roche Diagnostics) alone. To exclude major differences in the coating efficiency of fibronectin upon addition of versican to the test substrate, ratios of immunofluorescence staining intensities of control and substrate lanes were determined using the image software analySIS (Soft Imaging System/Olympus; region of interest: 480 µm2, n = 10 each) (Fig. 2).
For each assay, three to four neural tubes were placed perpendicular to the stripe pattern, covered with little SN1 culture medium, and first incubated for 45 to 50 min at 37 °C in a 5% CO2 atmosphere allowing the attachment of the explants. Once the tubes adhered to the substrate, the dishes were gently flooded with medium and kept at reduced oxygen levels. For this purpose, the dishes were placed in a gas-tight modular incubator chamber, which was flushed for 3 to 5 min with a gas mixture containing 1% O2,6%CO2 and balanced N2 to generate 3 to 6% actual levels of O2. eNCSCs were allowed to migrate out of the neural tube for 20 h and were then fixed with 3.7% formaldehyde in PBS for 10 min. They were either directly visualized by phase-contrast microscopy or processed for immunofluorescence staining. Alternatively, embryonic fibroblasts were allowed to emigrate onto the stripe pattern from the surface of small uncoated circular coverslips.
Neural Crest Cell Adhesion Assay—For the adhesion assays, eNCSCs were isolated from E10.5 rat embryos as described (48) and re-plated after 15 h in culture on plastic dishes coated with fibronectin alone (100 µg/ml), versican (75 µg/ml) alone, or a mixture of versican and fibronectin (75 and 100 µg/ml, respectively). The time course and saturation of the attachment was monitored by fixing the cells after 5, 15, 30, or 45 min of incubation. The number of cells that attached after 45 min on fibronectin alone was taken as 100%.
Immunofluorescence Staining of Cells and Tissue Sections—eNCSCs were visualized by double immunofluorescence staining of fixed cultures with polyclonal antibodies against p75NTR (1:300 dilution; Chemicon) and with a monoclonal antibody against Sox10 (1:3 dilution (42)). For this purpose, cells were first blocked and permeabilized with PBS containing 10% goat serum and 0.3% Triton X-100 for 10 min. Subsequently they were incubated with the primary antibodies for 2 h at room temperature and then labeled for fluorescence detection with Cy3-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibodies (both 1:200 dilution; Jackson Laboratories). Alternatively, the substrate coats were immunostained in some experiments with polyclonal antibodies against the GAG- domain of versican (dilution: 1:100) and with the monoclonal antibody IST-4 recognizing human fibronectin.
For immunostaining of tissue sections, mouse embryos were formalin fixed, dehydrated, and embedded in paraffin. 2-µm sections of the tissue were deparaffinized in xylene and re-hydrated by a dilution series of ethanol in water. Heat unmasking of the antigens was done in 10 mM Tris, 1.7 mM EDTA, 1 mM sodium citrate, pH 7.8, in a steam cooker for 2 min at 100 °C. The sections were two times washed in PBS for 5 min and subsequently blocked, first in 0.2% gelatin and 0.5% bovine serum albumin in PBS for 30 min and then in blocking buffer from a M.O.M. kit (mouse-over-mouse, Vector Laboratories). The incubation with the monoclonal antibody against Sox10 (1:6) and the polyclonal antibodies specific for versican GAG- (1:1000) was allowed to proceed overnight at 4 °C. All antibodies were diluted in 0.5% bovine serum albumin, 0.2% gelatin, 0.02% NaN3 in PBS. After washing in PBS, the sections were incubated with Alexa 488 goat anti-rabbit IgG and Alexa 594 goat anti-mouse IgG secondary antibodies (both Molecular Probes) diluted 1:200 in PBS. Counterstaining was done with Hoechst H33258
[GenBank]
bis-benzimide stain (Invitrogen) for 2 min. Sections were finally washed in PBS and mounted in fluorescence mounting medium (Dako). Images were taken with an Olympus BX61 microscope equipped with a F-view II camera using the analySIS 3.2 software (Soft Imaging System). Red, green, and blue fluorescence were stored separately in the corresponding color channels in RGB-format. Brightness and contrast were adjusted with Photoshop 7.0.
|
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
650 kDa; V1, 520 kDa; and V2, 400 kDa). Degradation products were nearly absent. Only two minute bands migrating in the size range of the V2 isoform became weakly apparent on immunoblots of the U251MG and aorta preparations (Fig. 3C). Their immunoreactivity with GAG--specific polyclonal antibodies clearly excluded an identity with the V2 variant, which lacks this domain. Except for some low molecular mass components later introduced by the treatment with chondroitinase ABC, no other contaminating proteins were detectable in Coomassie Blue and silver-stained gels. The yields of these preparations were 0.25 mg of versican V0/V1 per liter of culture supernatant, 0.2 mg of versican V1 per 100 g of aortic tissue, and 0.3 mg of versican V2 per 100 g of spinal cord, respectively (referred to the protein content). Versicans V0/V1 and V1 Specifically Restrict the Migration of Early Neural Crest Stem Cells in Vitro—Having sufficient amounts of intact versicans at hand, we could now study their effect on the migration behavior of eNCSCs in stripe-choice assays. These in vitro assays simulate the metameric expression pattern of versican V0 and V1 within the posterior sclerotome during the active neural crest cell migration period occurring around stage 20 in chick (35) and around E9.5 in mouse embryos (Fig. 4A). Consequently, neural tubes from E9.5 mouse embryos were used as the stem cell source for the in vitro experiments. The explants were cultured on coverslips coated with alternating stripes of the migration-promoting extracellular matrix protein fibronectin and the versican test substrate admixed to fibronectin. Like in vivo, the eNCSCs emigrated from the neural tube as sheets and moved onto the substrate-coated surface. This migration remained uniform in assays, in which the coverslips were exclusively coated with 20 and 100 µg/ml fibronectin in a stripe pattern (Fig. 4C). The picture changed, however, dramatically, when eNCSCs were confronted with lanes of fibronectin alternating with stripes treated with a mixture of versican V0 and V1 plus fibronectin (Fig. 4B). In these experiments, the continuous sheets of the migratory neural crest cells divided into separate streams shortly after leaving the neural tube subsequently advancing only on the versican-free surfaces. This selective movement greatly differed from the behavior of embryonic trunk fibroblasts, which did not display a substrate preference. In a comparable stripe choice assay, the fibroblasts migrated uniformly, even at a high versican coating concentration (Fig. 5).
In contrast to the fibroblasts, early embryonic neural crest stem cells avoided versican V0/V1 containing substrates already at coating concentrations of versican as low as 25 µg/ml (Fig. 6, B and F), clearly overriding the migration promoting effect of fibronectin (100 µg/ml) also present in these lanes. Whereas the inhibition was not yet complete at this low versican V0/V1 level, no crossing of the cells could be observed anymore at higher coating concentrations (Fig. 6, C and G and D and H). Of note, parallel experiments with intact versican V1 from bovine aorta gave very similar results in this experimental setting (data not shown).
|
Versican Core Glycoprotein Retains the Inhibitory Capacity after Removal of the Chondroitin Sulfate Side Chains—To investigate, whether the inhibitory function of versican V0/V1 originates from the GAG moiety or from the core glycoproteins, we digested our preparations with chondroitinase ABC and tested them again in stripe choice assays (Fig. 7). Prior to these experiments, the efficient removal of the chondroitin sulfate side chains had been confirmed by slot and Western blot analysis with the monoclonal antibodies CS-56 against intact chondroitin sulfate and Di-6S recognizing the stubs of chondroitin 6-sulfate exposed next to the core protein linker region after GAG cleavage (Fig. 7, A and B). Because intact versican V0/V1 was in the CS-56 slot blot still detected at a 1:100 dilution, but no staining was observed in a 1:2-diluted sample after chondroitinase ABC treatment, we concluded that the digestion was more than 98% complete. The stripe choice experiments demonstrated that the versican core glycoproteins were still able to restrict the migration of eNCSCs. Similar to the results with the intact proteoglycans, the effect was directly proportional to the increasing concentration of the GAG-free versican core glycoprotein, although some reduction of the inhibitory capacity was observed. At low coating concentrations (25 µg/ml) of digested V0/V1, cells migrating out of the neural tube showed only a marginal stripe restriction (Fig. 7C). At concentrations of 50µg/ml (Fig. 7D) and higher, almost all the cells followed the stripe pattern migrating again on areas coated with fibronectin alone.
|
|
Versican Interferes with the Substrate Adhesion of eNCSCs to Fibronectin—To test, whether intact versican interferes with fibronectin-mediated cell adhesion, uniform Sox10 and p75-positive eNCSC populations were re-plated on culture dishes coated with versican V0/V1 alone (75 µg/ml), fibronectin alone (100 µg/ml), and a mixture of versican and fibronectin (75 and 100 µg/ml, respectively). These experiments demonstrated that in relation to the number of cells binding to fibronectin, less then 10% adhered to the surface coated with versican V0/V1 plus fibronectin, whereas on pure versican V0/V1 (75 µg/ml) no cell adhesion was observed, even long after the cell attachment had reached saturation in the control dishes (Fig. 8). Hence, these comparative in vitro assays provided strong evidence that versicans inhibit neural crest cell migration by negatively controlling the binding of neural crest cells to adhesion-promoting extracellular matrix substrates such as fibronectin.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Prerequisite for our functional studies has been the identification of suitable sources for the isolation of the intact versicans. This has in the past been hampered by the fact that the largest versican splice variants V0 and V1 are predominantly expressed during early phases of embryonic development (34, 35), where they are subjected to a highly dynamic turnover most likely being controlled by the action of specific ADAMTS proteinases and matrix metalloproteinases (51, 52). Consequently, the yields of intact versican are minimal, when embryonic tissues are used as a source (e.g. 30 µg of core protein from the limb buds of 750 chick embryos (49)). During maturation, the expression of the V0 and V1 isoforms is greatly reduced and the core proteins left in the adult organism are largely fragmented as demonstrated by protein-chemical analysis of tissue extracts and by immunological stainings of neo-epitopes exposed after physiological cleavage with ADAMTS proteinases (51, 52). Despite this partial degradation, V0 and V1 fragments stay incorporated in various elastic tissues such as blood vessels (50, 53) and skin (41) and can therefore be detected by immunohistochemical techniques. Whereas these fragments may still contribute to the mechanical properties of mature extracellular matrices, it appears likely that the limited proteolysis of versicans V0 and V1 is required for the abrogation of their functions in cell proliferation and migration during embryonic development.
|
|
domain. This is concluded from the fact that they are, unlike versican V2, reactive with both, GAG- and GAG- domain-specific antibodies. Hence, from our tissue expression studies (32) and protein chemical analysis, we have currently no evidence that versican V2 is expressed outside of the central nervous system as has been indicated in a recent report (54).
Whereas the aorta extract contained predominantly intact versican V1 with only trace amounts of versican V0, a roughly equal proportion of these isoforms could be isolated from the glioma preparation. Both versican isoforms were after chondroitinase ABC digestion strongly reactive with the monoclonal antibody Di-6S (3B3) used in various immunohistological studies (10, 11, 25, 26). Because all proteoglycan isoforms of versican also bind the peanut agglutinin,5 it appears that at least parts of the CS-6 epitopes and PNA-binding carbohydrates are directly linked to the core proteins of versican V0 and V1 in tissues forming barriers to neural crest cell migration and axonal growth. The relationship between PNA-binding fragments of versican V0 and V1 and the previously described axon growth inhibitory glycoproteins isolated from the chick somites (55) is currently unknown.
Because primary cell cultures of dissociated embryonic trunks of chicken and mouse express the large versicans, V0 and V1, we have performed most of our functional studies with preparations containing a mixture of these isoforms. Nonetheless, versican V1 alone proved to be similarly effective in analogous experiments (data not shown). In all stripe choice assays, fibronectin was added to the versican coating solutions. This way, we have excluded that versicans not simply act as non-permissive substrates for migratory neural crest stem cells, but rather function as active inhibitors, which suppress the migration-promoting properties of fibronectin. As fibronectin is in the embryonic trunk present in both pathways and barrier tissues (9), the stripe choice assays are closely reflecting the in vivo situation. In comparison to other inhibitory molecules that have previously been tested in a similar experimental set-up (13, 15, 18), only relatively low versican concentrations had to be applied to observe maximal inhibition. Considering molar relationships, versican V0 and V1 may even be the most potent inhibitors of neural crest cell migration.
|
How the signal initiated through the contact of moving neural crest cells with versican is translated into the inhibition of cellular migration is currently still open. Versicans may either directly activate a versican-specific receptor on the surface of neural crest cells or act indirectly by sterically interfering with the interaction between migration promoting substrates and their integrin receptors (58, 59). The sterical hindrance model is supported by the observation that the complete removal of the chondroitin sulfate side chains leads only to a modest reduction, but not to the abolition of the inhibitory activity. This relatively small effect could be caused by a partial collapse of the core protein in the absence of the highly sulfated glycosaminoglycan side chains, greatly diminishing the hydrodynamic size of the versican molecule and possibly allowing the re-establishment of a few interactions between the cell surface and the permissive substrate. Along this line, it appears plausible that the extent of inhibition is in vivo controlled by the expression of specific splice forms varying in core protein size and number of chondroitin sulfate side chains covalently attached to them.
Despite the fact that no versican-specific transmembrane signaling molecule has been identified as yet, the versican-receptor hypothesis should, however, not be discarded. Only recently, we have shown that the central nervous system-derived versican V2 efficiently blocks axonal growth (37). Schweigreiter et al. (60) could subsequently demonstrate that this inhibition is mediated by a signaling cascade involving two members of the Rho family of small GTPases, RhoA and Rac1. This study has shown that the axonal contact with versican V2 activates RhoA and inactivates Rac1 in cerebellar granule cells. Consequently, the existence of an unknown versican receptor has been postulated that triggers this reaction finally leading to a growth cone collapse. An analogous signaling mechanism could also be responsible for the inhibition of neural crest cell migration by versican V0 and V1. During cellular movement, the activation of Rho is in general associated with retraction of migratory cells, whereas the activation of Rac promotes the formation of membrane protrusions at the leading edge (61). Integrin receptors are key mediators of these responses (62). For instance, the activation of RhoA and its downstream target, ROCK, directly affects the migration of leukocytes by decreasing the affinity of the 41 integrin to its extra-cellular ligands (63). Interestingly, the same fibronectin-binding integrin is known for its central role in the locomotion of neural crest cells (64, 65). Hence, a RhoA-mediated signaling process engaged upon contact with versican V0 and V1 could also negatively regulate the integrin-dependent migration of neural crest cells during embryonic development.
The suppression of cell-matrix interactions by versican V0 and V1 may not be sufficient to completely abrogate neural crest cell invasion of barrier tissues in vivo, as some neural crest cells could eventually switch from a collective chain-type movement (66) to a random amoeboid migration pattern (reviewed in Ref. 67). For a few cells, such an erratic locomotion within the caudal versican-containing portion of the sclerotome has indeed been observed in chicken trunk explants after disrupting the interaction between the EphB3 receptor on migratory neural crest cells and ephrin-B1 on the surface of posterior sclerotomal cells by addition of soluble ephrin-B1 (1, 16). Nonetheless, constitutive inactivation of the corresponding ephrin or Eph receptor genes alone has not led to an aberrant phenotype in regard to trunk neural crest cell migration (20). This indicates that the cell surface-bound ephrins and the extracellular matrix-embedded versicans V0 and V1 may together direct the migration of neural crest stem and progenitor cells through the rostral sclerotome. Thus, future investigations will possibly have to rely on complex mouse models carrying combinations of multiple constitutively and/or conditionally inactivated genes to elucidate, how versicans, ephrins, and the other putative migration inhibitors (summarized in Fig. 9) join in vivo their functions to regulate the highly precise migration patterns of the various neural crest cell subpopulations in a concerted action.
|
FOOTNOTES |
|---|
1 Present address: Dept. of Laboratory Medicine, Children's Hospital, Harvard Medical School, Boston, MA 02115.
2 Present e-mail address: maurice.kleber{at}gmx.ch.
3 To whom correspondence should be addressed. Tel.: 41-44-255-3945; Fax: 41-44-255-4440; E-mail: dieterzi{at}pathol.unizh.ch.
4 The abbreviations used are: PNA, peanut agglutinin; eNCSCs, early neural crest stem cells; GdnHCl, guanidine hydrochloride; PBS, phosphate-buffered saline; GAG, glycosaminoglycan; ADAMTS, a disintegrin and metalloproteinase with thrombospondinlike motifs.
5 M. Matasci and D. R. Zimmermann, unpublished observation.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
