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Address correspondence to C.W. Lo, Biology Dept., Goddard Laboratory, University of Pennsylvania, Philadelphia, PA 19104-6017. Tel.: (215) 898-3693/8394. Fax: (215) 898-8780. E-mail:clo{at}sas.upenn.edu
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Abstract |
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Key Words: cadherin; connexin; wnt; neural crest; catenin
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Introduction |
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Recent studies have indicated an essential role for gap junctions in modulating the migratory behavior of neural crest cells. Gap junctions contain membrane channels that allow the movement of ions, second messengers, and small metabolites between cells (for review see Bruzzone et al., 1996). They are encoded by a multigene family known as the connexins. The gap junction protein, connexin 43 or 1 connexin (Cx43
1)* is expressed abundantly in migrating neural crest cells, and these cells are also functionally coupled via gap junction channels (Lo et al., 1999). These observations are consistent with the fact that neural crest cells migrate not as individual cells but as cohorts of cells that are organized in sheets or streams (Bancroft and Bellairs, 1976; Davis and Trinkaus, 1981). When the Cx43
1 gene is deleted in the Cx43
1 knockout mice, conotruncal heart malformations and outflow obstruction were elicited (Reaume et al., 1995). Using transgenic mouse models, we showed that the requirement for Cx43
1 in conotruncal heart development involves the cardiac neural crest cells (Ewart et al., 1997; Huang et al., 1998a; Sullivan et al., 1998). Cardiac neural crest cells, a neural crest subpopulation derived from the postotic hindbrain neural fold, play an essential role in outflow tract septation and tissue remodeling in the conotruncal region of the heart. Transgenic mice exhibiting an elevation or inhibition of Cx43
1 function targeted to the cardiac neural crest cells show outflow tract obstruction and conotruncal heart malformations (Ewart et al., 1997; Huang et al., 1998b; Sullivan et al., 1998). These heart malformations are likely due to alterations in the abundance of neural crest cells reaching the heart. Thus an elevation of gap junction communication was associated with an enhanced rate of neural crest cell migration and an increase in the abundance of neural crest cells in the outflow tract (Huang et al., 1998a). In contrast, a reduction or inhibition of gap junction communication was associated with a reduced rate of neural crest cell migration, and concomitantly, a decrease in the abundance of crest cells in the outflow tract (Huang et al., 1998a). Based on these observations, we proposed previously that gap junctions may mediate the cell-to-cell movement of second messengers and other cell-signaling molecules involved in regulating cell locomotion and in this manner help coordinate the deployment of neural crest cells to the heart (Huang et al., 1998a; Lo and Wessels, 1998).
The present study was initiated to examine the role of cadherin-based adherens junction in modulating gap junction communication and the migration of mouse cardiac neural crest cells. Studies in tissue culture cells have shown previously that gap junction formation is dependent on cadherin-mediated cellcell adhesion (Keane et al., 1988; Mege et al., 1988; Matsuzaki et al., 1990; Musil et al., 1990; Jongen et al., 1991; Meyer et al., 1992; Frenzel and Johnson, 1996). In chick embryos, N-cadherin is expressed in the neural tube and emerging neural crest cells (Hatta and Takeichi, 1986). A specific requirement for N-cadherin in neural crest cell migration was shown by the finding that the inhibition of N-cadherin with function-blocking antibody perturbed the deployment of neural crest cells (Bronner-Fraser et al., 1992). In such antibody-treated embryos, neural crest cells were found inside the lumen of the neural tube or in the extraembryonic space dorsal to the neural tube but not in the expected dorsolateral neural crest migratory pathway. In addition, studies using adenoviral vectors to ectopically express various N-cadherin constructs have shown that overexpression of N-cadherin can disrupt the normal deployment of neural crest cells (Nakagawa and Takeichi, 1998). In the present study, we sought to elucidate the role of N-cadherin in the deployment of mouse cardiac neural crest cells and whether this involves the modulation of Cx431-mediated gap junction communication.
Our studies show that N-cadherin has an essential role in mouse neural cell migration. Mouse neural crest cells express N-cadherin in regions of cellcell contact found between extended cell processes. N-cadherin is often colocalized or closely juxtaposed with Cx431 gap junctions. Interestingly, in N-cadherindeficient neural crest cells, although Cx43
1 gap junction contacts remained abundant, gap junction communication was reduced to levels like that of the Cx43
1 knockout mice. Motion analysis revealed that the speed and directionality of neural crest cell locomotion are affected differently by N-cadherin versus Cx43
1 deficiencies. These results suggest that N-cadherin and Cx43
1 gap junctions may have separable roles in neural crest cell locomotion. As ß-catenin expression was reduced in the N-cadherin but not Cx43
1-deficient neural crest cells, we further examined the possible involvement of wnt signaling in neural crest cell motility. Neural crest cells from the wnt1 knockout mouse embryos exhibited normal cell motility, even though gap junction communication was reduced to levels like that of Cx43
1-deficient neural crest cells. As with the N-cadherindeficient neural crest cells, Cx43
1 gap junction contacts remained abundant at the cell surface in wnt1-deficient neural crest cells. These observations suggest that N-cadherin and wnt1 may regulate gating of Cx43
1 gap junction channels in neural crest cells. As cadherin is known to bind p120 catenin (p120ctn), an Armadillo protein also involved in modulating cell motility (Anastasiadis and Reynolds, 2000; Grosheva et al., 2000; Noren et al., 2000), we examined p120ctn expression in neural crest cells. p120ctn was observed to be expressed abundantly in neural crest cells. It was found to be colocalized with N-cadherin and Cx43
1. Significantly, the distribution of p120ctn was altered in the Cx43
1- and N-cadherindeficient neural crest cells. Based on these findings, we propose that neural crest cell motility may be modulated by the dynamic interactions of N-cadherin and Cx43
1 with the cell's locomotory apparatus through p120ctn signaling.
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Results |
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These results suggest the possibility that N-cadherin deficiency could alter ß-cateninmediated wnt signaling in neural crest cells. Previous studies have indicated a role for wnt signaling in neural crest cell proliferation and differentiation, but whether there is also a role in cell migration is not known (Ikeya et al., 1997; Dunn et al., 2000). To examine this question, we analyzed the motility of neural crest cells from the wnt1 knockout mouse embryos. Using time-lapse videomicroscopy and motion analysis, we found no change in any cell motility parameter with the loss of wnt1 function (Table VI). We also used lithium treatment to mimic wnt signaling in neural tube explant cultures and at 10 mM concentration (the highest compatible with viability) found no significant effect on neural crest cell motility (data not shown). These results indicate that wnt1 does not have an essential role in neural crest cell motility.
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In the N-cadherin and Cx431-deficient neural crest cells, we observed distinct changes in the pattern of p120ctn distribution. In the N-cadherindeficient neural crest cells, most of the cell surface expression of p120ctn was lost; although infrequently, some p120ctn can still be found along cell processes (Fig. 6 D). Very striking was the strong localization of p120ctn in the nuclei of the N-cadherindeficient crest cells (Fig. 6 D). The increase in p120ctn immunostaining in the nuclei cannot be fully appreciated by the image in Fig. 6 D, since the immunofluorescence intensity was beyond the saturation limit of the camera. A parallel analysis of the Cx43
1-deficient neural crest cells also revealed an apparent increase in p120ctn localization in the nucleus but not as striking as that observed in the N-cadherin knockout crest cells (Fig. 6 C). In addition, there was a reduction in cell surface expression of p120ctn (Fig. 6 C compare with A and B). Quantitative analysis showed that this is due to a reduction in both the length and area of cell surface p120ctn (Table VIII). Overall, these observations suggest that the loss of N-cadherin and Cx43
1 could potentially perturb p120ctn-mediated signaling in neural crest cells.
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Discussion |
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Our studies also revealed an essential role for N-cadherin in mouse neural crest cell migration. Analysis of neural crest outgrowth area in neural tube explant cultures indicated a significant reduction in the apparent rate of neural crest cell migration in the absence of N-cadherin. These results are similar to that seen previously in Cx431-deficient neural crest cells (Huang et al., 1998a). However, time-lapse videomicroscopy and an analysis of the migratory movement of individual neural crest cells revealed distinct differences in the motility changes elicited by the loss of N-cadherin versus Cx43
1. N-cadherindeficient neural crest cells exhibited an increase in the speed of cell locomotion but a significant reduction in the directionality of cell movement. In contrast, Cx43
1 deficiency elicited only a reduction in the directionality of cell movement but no change in the speed of cell locomotion. Whereas the directionality of cell movement was reduced only in the homozygous N-cadherin and Cx43
1 knockout neural crest cells, the speed of cell locomotion was elevated in both heterozygous and homozygous N-cadherin knockout neural crest cells, thus indicating the latter cell motility parameter is subject to N-cadherin haploin sufficiency.
Modulation of neural crest cell motility by N-cadherin
Overall, our studies indicated that N-cadherin has an essential role in neural crest cell motility and that this cannot simply involve facilitating Cx431-mediated gap junction communication. We note that although the formation of cadherin-based adhesion contacts are generally associated with the cessation of cell locomotion, N-cadherin can stimulate the motility of breast cancer cells and also has been shown to facilitate tumor cell metastasis (Nieman et al., 1999; Hazan et al., 2000). In addition, ectopic expression of N-cadherin can cause cells to lose their tight cellcell contact and convert into a spindle or fibroblastic morphology (Kintner, 1992; Islam et al., 1996). Although the precise mechanism by which N-cadherin facilitates cell migration is not known, previous studies have indicated that cadherin's role in cell motility is separable from its role in cell adhesion. Thus, the ß-cateninbinding domain of cadherin required for cell adhesion is not essential for cell motility (Chen et al., 1997). Studies with retinal ganglion cells also indicated that the ß-cateninbinding domain of N-cadherin is not required for cadherin's role in axonal outgrowth (Riehl et al., 1996). Rather, recent studies suggest that cadherin may modulate cell motility by mediating signaling via the p120ctn family of Armadillo proteins. p120ctn is bound to cadherin's juxtamembrane region, a domain distinct from that mediating ß-catenin binding (Yap et al., 1998; Anastasiadis et al., 2000). Studies in various cultured cell lines show that p120ctn is also found in the cytoplasm, where it can modulate the activity of the RhoGTPases and thus affect cell motility by changing the organization of the actin cytoskeleton (Anastasiadis et al., 2000; Grosheva et al., 2000; Noren et al., 2000). In addition, p120ctn can signal to the nucleus, since it has been shown to interact with the transcription factor Kaiso and translocate into the nucleus (Daniel and Reynolds, 1999).
We observed that p120ctn is expressed abundantly in migrating mouse cardiac neural crest cells. Its distribution is similar to that described for other cell types: being localized at the membrane, diffusely in the cytoplasm, and at a low level in the nucleus. The latter was most evident when the heavy cytoplasmic p120ctn immunostaining was reduced by including Triton in the fixative. In the N-cadherindeficient neural crest cells, membrane-bound p120ctn was decreased, whereas p120ctn localization in nuclei was markedly increased. These results are consistent with a previous study showing that the exogenous expression of E-cadherin can downregulate the nuclear localization of p120ctn (Van Hengel et al., 1999). Since the Rho GTPases also have been shown to have a role in cell cycle regulation (Welsh and Assoian, 2000; Assoian and Schwartz, 2001), alterations in p120ctn signaling could potentially affect cell proliferation in the N-cadherindeficient neural crest cells. We note that ectopic N-cadherin expression in breast tumor cells was reported to slow the growth of individual tumors, a result that is consistent with our finding of an elevation of cell proliferation in the N-cadherindeficient neural crest cells (Hazan et al., 2000).
The role of connexins in neural crest cell motility
The present study together with our earlier work showed that Cx431 gap junctions play an essential role in neural crest cell motility. We proposed previously that Cx43
1-mediated gap junction communication might regulate neural crest cell migration by facilitating the cell-to-cell movement of second messengers and other cell-signaling molecules involved in cell locomotion (Huang et al., 1998a; Lo and Wessels, 1998). This model was based on our finding of an apparent correlation between changes in the level of dye coupling and alterations in the apparent rate of neural crest cell migration elicited by the gain or loss of Cx43
1 function. However, in the present study we showed that neural crest cells from the wnt1 knockout mouse embryos have normal cell motility even though they exhibited only a low level of functional coupling via gap junction channels. In addition, our studies showed no consistent correlation between cell motility changes and the stepwise decrease in dye coupling in neural crest cells derived from the heterozygous and homozygous N-cadherin or Cx43
1 knockout mouse embryos. This divergence between changes in neural crest cell motility and alterations in the level of dye coupling would suggest that carboxyfluorescein injection is not an appropriate method for examining the role of Cx43
1 in cell motility. Thus, carboxyfluorescein may not be a suitable gauge of the movement of molecules that are important in cell motility. Since wnt1-deficient neural crest cells continued to exhibit a low level of residual dye coupling, in principle this may be sufficient to mediate the cell-to-cell movement of molecules involved in modulating cell motility. However, this explanation is somewhat problematic, given that Cx43
1- and N-cadherindeficient neural crest cells have dye coupling levels comparable to that of the wnt1-deficient neural crest cells.
Another possibility to consider is that the role of Cx431 in motility may involve a novel function. Since Cx43
1 is extensively colocalized with p120ctn, perhaps connexins like cadherins may have a role in modulating p120ctn signaling. It is of significance to note that the distribution of p120ctn is altered in the Cx43
1-deficient neural crest cells. In the wnt1-deficient neural crest cells, Cx43
1 gap junctions continued to be expressed in abundance at the cell surface. Since Cx43
1 gap junctions are also maintained in N-cadherindeficient neural crest cells, presumably it is not merely whether Cx43
1 gap junctions are present but perhaps the modulation of Cx43
1 interaction with other proteins that is of critical importance in cell motility.
The integration of Cx431 and N-cadherin in cell motility
In light of the binding of p120ctn by Cx431 and N-cadherin and the colocalization or juxtapositioning of Cx43
1 and N-cadherin at the cell surface, we hypothesize that Cx43
1 and N-cadherin may be integrated within a cell surface complex that can modulate cell motility by transducing signals to the cytoskeleton and the nucleus. This would allow cell adhesion contacts and gap junctions to be regulated dynamically in conjunction with cell locomotion. Since p120ctn family members are subject to differential splicing and are typically expressed in multiple isoforms (Reynolds, et al., 1994; Staddon et al., 1995; Mo and Reynolds, 1996; Keirsebilck et al., 1998), it is possible that different p120ctn isoforms may bind Cx43
1 versus N-cadherin and thus differentiate the roles of cadherin versus connexin in cell motility. In preliminary studies, we observed that Cx43
1 coimmunoprecipitated with only one of two p120ctn bands expressed in NIH3T3 cells (unpublished data). In future studies, it may be interesting to examine another p120ctn family member, Armadillo repeat protein deleted in velocardiofacial syndrome (ARVCF; Mariner et al., 2000), a gene situated in a chromosome region deleted frequently in patients with velocardiofacial syndrome, a congenital condition involving cranial and cardiac neural crest defects (Young et al., 1980; Shprintzen et al., 1981; Sirotkin et al., 1997). Studies in polarized epithelial cells have indicated that scaffold proteins play an important role in integrating components of the apical junctional complexes with signal transduction pathways (Yeaman et al., 1999). In this regard, it is interesting to note that the scaffold protein ZO-1 has been shown to bind Cx43
1 (Toyofuki et al., 1998); ZO-1 also has been observed to have a role in cadherin-based cell adhesion (Itoh et al., 1997). In future studies, it will be important to examine, on a biochemical level, the interactions of Cx43
1 and N-cadherin and their interactions with ZO-1, p120ctn, and other cytoplasmic proteins. However, these studies will likely necessitate the use of model tissue culture systems, since at best only a few thousand neural crest cells can be obtained from each postotic neural tube explant culture.
Modulation of gap junction communication by N-cadherin and wnt1
Our finding that Cx431 gap junctions persisted even as dye coupling was virtually eliminated in the N-cadherin and wnt1-deficient neural crest cells would suggest that wnt1 signaling and N-cadherin can modulate gating of the Cx43
1 gap junction channel. These results contrast with a previous study of mammalian tissue culture cells, indicating that Cx43
1 is transcriptionally regulated by the wnt1/ß-cateninsignaling pathway (van der Heyden et al., 1998). Studies in the Xenopus embryo have shown that wnt signaling can modulate gap junction communication (Olson et al., 1991; Olson and Moon, 1992; Heasman et al., 1994), but this likely is posttranscriptionally regulated, since the effects of wnt on coupling occurs before the activation of zygotic transcription. It is interesting to note that more recent work suggests that the wnt modulation of gap junction communication in Xenopus embryos involves ß-catenin but is independent of cadherin's role in cell adhesion (Krufka et al., 1998). As ß-catenin is extensively colocalized with Cx43
1 in neural crest cells, an interesting possibility is that the inhibition of dye coupling in the wnt1- and N-cadherindeficient neural crest cells may arise from the perturbation of ß-catenin and Cx43
1 interactions. For example, ß-catenin interactions could modulate conformation of the cytoplasmic tail of Cx43
1 and thus affect gating of the gap junction channel as proposed in the particlereceptor model (Homma et al., 1998). We note that the cell surface localization of ß-catenin is greatly reduced in both wnt1- and N-cadherindeficient neural crest cells. A possible role for p120ctn in gating the gap junction channel also may be worth considering. In addition, as the cytoplasmic tail of Cx43
1 contains a consensus phosphorylation site for GSK3ß (Lau et al., 1996), alterations in wnt signaling could alter phosphorylation of the cytoplasmic tail of Cx43
1 and thus bring about conformational changes that may perturb channel activity directly or indirectly. In future studies, it will be important to define the cytoplasmic domain of Cx43
1 to which ß-catenin and p120ctn binds and determine whether mutations that disrupt such interactions may affect gating of the gap junction channel.
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Materials and methods |
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Neural tube explant cultures
To obtain embryos for neural crest outgrowth studies, mice were mated, and the day the vaginal plug was found was considered E0.5. Embryos were collected on E8.5, and the yolk sac from each embryo was harvested for genotyping by PCR analysis. The method used to establish the neural crest outgrowth cultures was described previously (Huang et al., 1998a) and was adapted from those described by Murphy et al. (1991) and Moiseiwitsch and Lauder (1994). In brief, the hindbrain neural folds of E8.5 embryos were collagenase/dispase treated, and the dorsal ridge of the neuroepithelium spanning the postotic region of the hindbrain neural fold was surgically removed from the surrounding tissue and cultured on a fibronectin-coated Petri dish in DME with high glucose and 10% FBS. The cultures were maintained for 2448 h at 37°C with 5% CO2. Lineage studies in mouse embryos have confirmed that neural crest cells from the postotic hindbrain neural fold give rise to the cardiac crest cells, as has been found in chick embryos (Fukiishi and Morriss-Kay, 1992; for review see Kirby and Waldo, 1995).
Neural crest dye coupling analyses in vitro and in vivo
To quantitate gap junction communication, dye coupling experiments were carried out in vitro and in vivo as described previously. For the in vitro studies, microelectrode impalements were made into neural crest cells in 24-h explant cultures (Huang et al., 1998a). Microelectrodes were filled with 5% carboxyfluorescein and dye was iontophoretically injected for 2 min. The number of dye-filled cells found at the termination of dye injection was recorded. To examine dye coupling in vivo, impalements and dye injections were carried out into the dorsolateral region of the postotic hindbrain neural fold, the region where presumptive cardiac crest cells are found, and the number of dye filled cells recorded after 2 min as described above.
Analyses of neural crest cell migration and proliferation
Images of the neural tube explant cultures were acquired by videomicroscopy at 24 and 48 h, and the outgrowth area was measured as described previously (Huang et al., 1998a). In brief, this entailed using NIH Image software to measure the area and perimeter of the outgrowth to obtain the migration index, which is the outgrowth area (mm2) divided by the perimeter (mm) of the explant. This normalized for variations in the outgrowth area arising from differences in the shape of the explant. For monitoring cell proliferation, 48-h explant cultures were incubated with BrdU (10 µM/ml) for 2 h, then fixed, and processed for immunodetection using an anti-BrdU antibody as described previously (Huang et al., 1998a). To determine the total cell number in the outgrowth after BrdU immunostaining, the cultures were further stained with hematoxylin. Cell counting was carried out using NIH Image. The migration and proliferation obtained were analyzed statistically using Statview (SAS Institute, Inc.).
Time-lapse videomicroscopy and motion analysis
For time-lapse videomicroscopy, images of the explants were captured every 10 min over a 20-h interval using IPLab (Scanalytics) or Openlab (Improvision, Inc.) imaging software. For these studies, the explant cultures were maintained at 37°C on a heated microscope stage in phosphate-buffered L-15 medium containing 10% FBS. The images were then converted to a Quicktime movie for viewing and motion analysis. The motion analysis was carried out using the Dynamic Image Analysis Software (Solltech, Inc.). Using this software, we determined the speed of cell locomotion, the directionality of cell movement (the net distance achieved divided by the total distance traveled), the direction change (the change in direction of cell movement in degrees), and the persistence of cell movement (the speed of cell locomotion divided by direction change in gradients). The data obtained was statistically evaluated by ANOVA using Statview (SAS Institute, Inc.).
Immunohistochemistry
For immunohistochemical staining of neural crest cells in explant cultures, neural tube explants were plated on glass coverslips coated with 15 µg/ml of fibronectin. After 2448 h of culture, the cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. In some cases, cells were first incubated with 3.7% formaldehyde containing 0.3% Triton X-100 for 3 min followed by postfixation in 4% paraformaldehyde for an additional 15 min. For immunostaining, the coverslips were incubated for 2 h at room temperature with the primary antibody diluted in blocking buffer (1% BSA and 2% FBS with 0.3% Triton X-100 in PBS), then washed with PBS three times for 1 h or longer, and incubated with the secondary antibody for 45 min at room temperature. After washing, the coverslips were mounted with SlowFade antifade reagent (Molecular Probes) on glass slides and examined by epifluorescence illumination using a 40 or 63x oil objective on a Leica DMRE microscope. All of the images were captured using a 5 Mhz Micromax cooled CCD camera. For some samples, images were obtained as Z stacks comprised of 0.2-µm optical slices and then subjected to deconvolution analysis by volume neighbor or iterative deconvolution algorithms using the Openlab deconvolution software (Improvision, Inc.).
The antibodies used included mouse monoclonal antiN-cadherin antibody from Zymed Laboratories (33-3900; used at 1:100 dilution), mouse monoclonal antiß-catenin IgG (C19220; used at 1:100 dilution), mouse monoclonal anti-p120ctn antibody (P17920; 1:500 dilution), and mouse monoclonal antip120-FITCconjugated antibody from BD Transduction Laboratory (P17924; 1:50 dilution), and rabbit polyclonal Cx431 antibody 18-A8 made against the cytoplasmic tail of Cx43
1 (1:1500 dilution; supplied by Dr. Elliot Hertzberg, Albert Einstein College of Medicine, Bronx, NY). The secondary antibodies used were Cy3- or FITC-conjugated goat antimouse or antirabbit IgG obtained from Jackson ImmunoResearch Laboratories. For double immunostaining with the N-cadherin and p120ctn mouse antibodies, cells were first incubated with the N-cadherin antibody followed by Cy3-conjugated antimouse antibody. Then they were further incubated with the FITC-conjugated p120ctn antibody.
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Footnotes |
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* Abbreviations used in this paper: Cx43
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Acknowledgments |
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Supported by grants from the National Science Foundation IBN-31544 (to C.W. Lo), and the National Institutes of Health HD36457, HD39946, and HL36059 (to C.W. Lo), and HL57554 (to G.L.R. Radice).
Submitted: 8 May 2001
Accepted: 5 June 2001
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