Department of Biochemistry and Molecular Pharmacology, and Program in Neuroscience, University of Massachusetts Medical School, Worcester, MA 01655, USA
* These authors contributed equally to this work
Author for correspondence (e-mail: charles.sagerstrom{at}umassmed.edu)
Accepted 31 October 2001
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SUMMARY |
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Key words: Hox, Pbx, Meis, Prep, Hindbrain, Homeodomain, Rhombomere, Segmentation, Zebrafish
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INTRODUCTION |
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An in vivo role for Hox cofactors was first shown by analyzing mutations in the Drosophila homothorax (hth, the Meis ortholog) (Kurant et al., 1998; Pai et al., 1998
; Rieckhof et al., 1997
) and extradenticle (exd, the Pbx ortholog) (Rauskolb et al., 1993
) genes. Mutations in either gene lead to posterior transformations of embryonic segments, without affecting the expression of Hox genes, showing that both Exd and Hth are required for Hox protein function during fly development. Loss-of-function analyses in vertebrates have also revealed a requirement for pbx genes in segmentation processes during development. This is seen particularly clearly in the segmented hindbrain where disruption of the pbx4 gene in the zebrafish lazarus mutant (Pöpperl et al., 2000
) leads to abnormal segmentation. The lazarus phenotype is similar to that observed upon targeted deletion of Hox genes from paralog groups 1 and 2 in the mouse (e.g. Davenne et al., 1999
; Gendron-Maguire et al., 1993
; Goddard et al., 1996
; Lufkin et al., 1991
; Rijli et al., 1993
; Studer et al., 1996
), consistent with a role for Pbx proteins in regulating Hox function in the vertebrate hindbrain. By contrast, although several meis genes are expressed in the developing hindbrain (Sagerström et al., 2001
; Salzberg et al., 1999
; Zerucha and Prince, 2001
), no loss-of-function analyses have been reported for meis genes to date. Instead, support for meis genes acting in hindbrain development come from ectopic expression analyses showing that Meis proteins posteriorize the rostral CNS in Xenopus (Salzberg et al., 1999
) and cooperate with Pbx and Hox proteins to promote hindbrain fates in zebrafish (Vlachakis et al., 2001
). Because vertebrates have several closely related, and perhaps functionally redundant, meis genes, loss-of-function analyses for meis may best be performed by using dominant negative constructs that interfere with all Meis family members. A basis for dominant negative strategies presents itself by the fact that Meis proteins act as part of larger complexes. These complexes are probably the functional units in vivo, as evidenced by dimers and trimers being detected by co-immunopreciptation from cell extracts (Chang et al., 1997
; Ferretti et al., 2000
; Knoepfler et al., 1997
; Shen et al., 1999
). Thus, Meis sites are found adjacent to Pbx and Hox sites in several Hox-dependent promoters (Ferretti et al., 2000
; Jacobs et al., 1999
; Ryoo et al., 1999
) and the Pbx interaction domain of Meis is required for Meis function in vivo (Vlachakis et al., 2001
). Therefore, expressing a Meis protein that retains its ability to bind Pbx, but lacks other essential functions, might interfere with endogenous Meis activity. However, attempts at accomplishing this by introducing point mutations into the homeodomain (thereby preventing DNA binding) of zebrafish Meis3 and Drosophila Hth (Ryoo et al., 1999
; Vlachakis et al., 2001
) did not generate a dominant negative protein. Similarly, expressing the Meinox domain of Xenopus Meis3 in vivo did not have a dominant negative effect (Salzberg et al., 1999
), whereas expressing the Meinox domain of Hth only partially interfered with Hox function in Drosophila embryos (Ryoo et al., 1999
).
Here we first demonstrate that highly divergent members of the Meis family display the same activity in promoting hindbrain fates, suggesting that conserved regions within Meis family members carry out this function. We proceed to define this essential region and find that it resides within the Meinox domain, a region previously implicated in Pbx binding. The activity of this region, M1, is independent of Pbx binding, suggesting that Meis proteins contribute a distinct activity to the complex. The M1 region does not encode a known motif and we hypothesize that it may interact with an auxiliary protein. This data predicts that, to inhibit Meis function the M1 domain must be removed from the Hox-cofactor complex, and we took advantage of the fact that nuclear localization of zebrafish Meis proteins is mediated by Pbx proteins (Vlachakis et al., 2001). We find that expressing the Pbx4/Lzr N-terminus in zebrafish embryos sequesters Meis proteins in the cytoplasm, thereby keeping them out of transcription complexes in the nucleus. Embryos without nuclear Meis displayed severe defects in hindbrain development. In particular, gene expression specific to rhombomere (r) 3 and r4 was largely lost and rhombomere boundaries do not form properly in this region. Neuronal differentiation in this region was also affected, e.g. nV branchiomotor neurons in r3 and Mauthner neurons in r4 were lost. Our results suggest that the Meis Meinox domain contributes an activity in addition to Pbx binding and show that Meis proteins are required for proper specification of r3 and r4 during hindbrain development.
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MATERIALS AND METHODS |
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RNA injections, western blots, immunoprecipitations, in situ hybridization and immunostaining was performed as described previously (Vlachakis et al., 2001).
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RESULTS |
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A search of the zebrafish EST database revealed several ESTs with sequence homology to murine Prep1. One of these, fc13f10, was obtained and sequenced. Sequence analysis revealed that zebrafish Prep1 has a similar domain structure to other Meis proteins (Fig. 1A; Prep1 Accession Number, AY052752). Prep1 is most similar to Meis3 in the homeodomain (71% identical at the amino acid level) and in the M1 and M2 domains (55% and 86% identical, respectively) that have been implicated in Pbx binding (Knoepfler et al., 1997). Other regions of Prep1, i.e. the N-terminus, the region between the M1 and M2 domains, the C-terminus and the region between the M2 domain and the homeodomain, were less than 26% identical. The fc13f10 Prep1 EST has been mapped to between 52.2 and 52.3 cM from the top of LG9 by the zebrafish mapping consortium.
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Our sequence comparison (Fig. 1A) revealed that the M1 and M2 domains, which have been implicated in binding to Pbx, are well conserved between Meis3 and Prep1, suggesting that Prep1 may interact with Pbx proteins in a manner similar to Meis3. To determine whether Prep1 interacts with Pbx4/Lzr, the most prevalent Pbx protein during early zebrafish development (Pöpperl et al., 2000), we used an in vitro co-immunoprecipitation assay. Pbx4/Lzr was expressed alone or together with MYCMeis3 or MYCPrep1 and precipitated with anti-MYC antibody. We find that both MYCMeis3 (Fig. 1J, lane 2) and MYCPrep1 (lane 4) interact with Pbx4/Lzr. The anti-MYC antibody did not crossreact with Pbx4/Lzr (lane 6). We have previously shown that zebrafish Meis3 depends on Pbx proteins for its nuclear localization (Vlachakis et al., 2001
), and that this requires an intact Meinox motif in Meis3, consistent with Meis3 interacting with Pbx proteins to access the nucleus in vivo. To determine if Prep1 behaves the same way, we tested its subcellular localization in the presence or absence of co-expressed Pbx4/Lzr. We find that at 5 hpf MYCPrep1 is primarily cytoplasmic in the absence of Pbx4/Lzr (Fig. 1K), but localizes to the nucleus when Pbx4/Lzr is co-expressed (Fig. 1L).
We have previously shown that, although Hoxb1b can interact with Pbx4/Lzr to induce ectopic expression of hoxb1a in r2 of the hindbrain, co-expression of Meis3 with Pbx4/Lzr and Hoxb1b leads to ectopic expression of both hoxb1a and hoxb2 in a broad domain, resulting in transformation of the rostral CNS to a hindbrain fate (Vlachakis et al., 2001). To determine whether Prep1 can function to induce hindbrain fates in a manner similar to Meis3, we co-expressed Prep1 with Pbx4/Lzr and Hoxb1b in developing zebrafish embryos and scored for ectopic expression of the hoxb1a and hoxb2 hindbrain genes. Western blot analysis showed that MYCMeis3 and MYCPrep1 were expressed at similar levels (Fig. 1P). Expression of MYCPrep1 or MYCMeis3 by themselves had no effect on hoxb1a or hoxb2 expression (not shown). By contrast, expressing MYCMeis3 or MYCPrep1 together with Pbx4/Lzr and Hoxb1b resulted in massive ectopic expression of both hoxb1a (not shown) and hoxb2 (Fig. 1M-O) anterior to their normal expression domains, leading to anterior truncations. Because Prep1 represents the most divergent Meis family member known, these results suggest that all known members of the zebrafish Meis family, despite differences in sequence and expression pattern, share the ability to promote hindbrain fates.
The Meinox domain is sufficient to mediate the activity of Meis family proteins
Because Prep1 and Meis3 can both promote hindbrain fates, the sequences responsible for this activity must be shared between the two proteins. Meis3 and Prep1 have highest sequence identity in the Meinox domain (consisting of the M1, I and M2 regions) and in the homeodomain. Although this is consistent with Meis proteins mediating their in vivo effects solely by binding Pbx and DNA, thereby perhaps stabilizing Pbx/Hox complexes, it remains possible that other domains in Meis proteins are essential for function, or that the Meinox and homeodomain have activities in addition to Pbx and DNA binding. To determine which domains are necessary for Meis protein function, we generated a series of Meis3 deletion constructs (Fig. 2A) and tested whether they could promote hindbrain fates upon co-expression with Pbx4/Lzr and Hoxb1b in zebrafish embryos.
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To test this possibility, we set out to determine if a Meinox domain lacking the ability to bind Pbx still retains activity. To carry out this experiment it became necessary to devise a means for the Meinox domain to participate in Pbx/Hox complexes without being able to interact with Pbx (Fig. 2B). We replaced the N-terminus of Pbx4/Lzr (containing the PBC-A and PBC-B domains required for Meis binding) with the Meis N-terminus (containing the Meinox domain). This eliminates the normal interaction between the Meinox domain and Pbx4/Lzr, but as the chimeric protein retains the Hox interaction motif in Pbx4/Lzr, it still ensures that the Meinox domain is part of the Pbx/Hox transcription complex bound to DNA. Notably, as this construct lacks the PBC-A and PBC-B domains, it can not bind endogenous Meis proteins. To also eliminate the ability of this construct to bind endogenous Pbx proteins, we used a Meinox domain that contains multiple amino acid substitutions in the M1 (aa 64-67 KCELNNSQ) and M2 (L141
A; E142
A) motifs. We have previously shown that this mutated Meinox domain can not bind to Pbx4/Lzr in vivo (Vlachakis et al., 2001
) and we confirmed that the resulting fusion protein, BMNPbx4, does not bind endogenous Pbx by performing co-immunoprecipitations on lysates from embryos expressing BMNPbx4 (Fig. 3V). To ensure that BMNPbx4 localizes to the nucleus, we also introduced a nuclear localization signal (NLS) at its N-terminus.
BMNPbx4 is expressed at similar levels to Meis3 following microinjection (Fig. 3A, compare lanes 2 and 11) and localizes to the nucleus (Fig. 3L), as expected. Expression of BMNPbx4 alone resulted in embryos with normal expression of hoxb1a and hoxb2 (not shown), whereas co-injection with Hoxb1b resulted in embryos exhibiting ectopic hoxb1a (Fig. 3Q) and hoxb2 (Fig. 3U). This phenotype was qualitatively and quantitatively similar to the phenotype produced by expressing the Meinox domain together with Pbx4/Lzr and Hoxb1b (Fig. 3P,T; Table 1). This result indicates that the BMNPbx4 chimera now contains the combined activities of Pbx4/Lzr and Meis3.
Additional constructs were generated to better delineate the region of the Meis3 N-terminus required for this activity. We first generated a construct containing only the I domain fused to Pbx4/Lzr. This construct (IPbx4; Fig. 2B) is expressed at the same level as Meis3 following injection (Fig. 3A, lane 12) and localizes to the nucleus (not shown). IPbx4 lacks in vivo activity (Table 1), confirming that the I domain is not required for function and also showing that simply fusing sequences to the Pbx4/Lzr C-terminus is not sufficient for activity. We then added the M1 domain (containing the same amino acid substitutions as in BMNPbx4) onto the IPbx4 construct to generate BM1IPbx4 (Fig. 2B). This construct is expressed at the same level as other constructs (Fig. 3, lane 13) and localizes to the nucleus (Fig. 3M). BM1IPbx4 has no effect when expressed by itself (not shown), but leads to ectopic hoxb1a and hoxb2, as well as anterior truncation similar to those seen with the BMNPbx4 construct, when co-expressed with Hoxb1b (Table 1). On the basis of the data from the deletion analysis and the chimeric constructs, we conclude that the Meinox domain has a function in addition to Pbx binding and that the M1 domain is sufficient for this function, at least in our ectopic expression system. We do not think that the M1 domain acts by stabilizing the fusion protein, because a fusion protein lacking the M1 domain (IPbx4) does not appear to be less stable over time in vivo than one that retains the M1 domain (BMNPbx4; Fig. 3W). Instead, we speculate that the M1 domain may serve as a binding site for an auxiliary protein.
Expression of the Pbx4/Lzr N-terminus sequesters Meis proteins in the cytoplasm
Our finding that the M1 domain is sufficient for Meis activity provides a rationale for a dominant negative strategy. In particular, it might not be sufficient to eliminate the DNA binding capacity of Meis to generate a dominant negative construct because such a construct will retain the M1 domain. Instead, we set out to devise a strategy where the M1 domain is kept out of Pbx/Hox complexes. Specifically, as the M1 domain is also involved in Pbx binding, we hypothesized that expressing a construct that sequesters Meis proteins away from Pbx/Hox complexes might act in a dominant negative fashion. To test this possibility we generated a construct expressing only the N-terminus of Pbx4/Lzr, containing the PBC-A and PBC-B domains required for binding to Meis, but lacking the motifs required for binding Hox proteins and for nuclear localization (Fig. 4A). We observed that this construct (MycCPbx4) was cytoplasmically located at 12 hpf following expression in zebrafish embryos (Fig. 4B). By contrast, injected MycMeis3 is found exclusively in the nucleus at this stage of development (Fig. 4C), probably as a result of nuclear transport by endogenous Pbx, which has become highly expressed by this stage (Vlachakis et al., 2001
). Strikingly, when
CPbx4 is co-expressed with MycMeis3, MycMeis3 is found primarily in the cytoplasm (Fig. 4D). These data are consistent with
CPbx4 competing with endogenous Pbx proteins for binding to Meis3 in the cytoplasm and subsequently retaining Meis3 in the cytoplasm. This result raises the possibility that
CPbx4 might act in a dominant negative fashion by keeping Meis proteins out of nuclear Pbx/Hox complexes.
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To explore further the effect of CPbx4 on r3 and r4 development, we analyzed neuronal differentiation in this region. Both the primary reticulospinal neurons and the branchiomotor neurons display a segment-specific distribution in the hindbrain, permitting us to characterize the effect of
CPbx4 on neuronal differentiation in individual rhombomeres. We find that 73% (30/41) of
CPbx4-injected embryos lack one or both r4-specific Mauthner neurons (Fig. 4U-W). Using an anti-Islet1 antibody we also observed an effect on branchiomotor neurons in 70% (21/30)
CPbx4-injected embryos. This effect is strongest in r3, as most embryos lack nV branchiomotor neurons on at least one side of the midline in r3 (Fig. 4S,T). Because there are only a few islet-1 positive cells in r4 it is difficult to determine whether it is affected, although this region occasionally seems to be reduced in size, in agreement with the observed loss of r4 Mauthner neurons. nVII neurons in r6 and r7 are also affected, although less severely, perhaps as a result of these neurons originating in r4 before migrating to r6 and r7 (Chandrasekhar et al., 1997
). By contrast, nV neurons in r2 are largely unaffected. These results are consistent with the observed effect of
CPbx4 on gene expression and suggest that specification of r3 and r4 is particularly dependent on Meis function.
To confirm that this phenotype is specific, we attempted to rescue CPbx4-injected embryos by co-expressing pbx4/lzr mRNA. We expected Pbx4/Lzr to compete with
CPbx4 for Meis binding in the cytoplasm and bring Meis proteins to the nucleus where they could interact with Hox proteins and activate transcription. We find that expressing pbx4/lzr mRNA, along with
CPbx4 mRNA, rescued hoxb1a expression to virtually normal levels in all embryos (43/43). We attribute this high frequency of rescue to
CPbx4 not entering the nucleus. Thus, once Meis proteins have entered the nucleus together with Pbx4/Lzr, they are inaccessible to the
CPbx4 dominant negative protein. We also used the BMNPbx4 construct to rescue
CPbx4-injected embryos. Because BMNPbx4 does not interact with Pbx, it should not be affected by the
CPbx4 dominant negative construct. Furthermore, as it contains the M1 domain it should be able to rescue Meis activity in
CPbx4-expressing embryos. We find that expression of BMNPbx4 together with
CPbx4 restores hoxb1a expression in all embryos (30/30), but that the rescued expression is less complete than following rescue with pbx4/lzr. We attribute this difference to BMNPbx4 being less active than wild-type Meis3 in vivo (Table 1). This result further shows that the effect of
CPbx4 is because of its interference with endogenous Meis activity.
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DISCUSSION |
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What role do Meis proteins play in the multimeric transcription complexes?
Several reports have shown that Meis, Pbx and Hox proteins can interact to form trimeric complexes (Berthelsen et al., 1998a; Ferretti et al., 2000
; Jacobs et al., 1999
; Ryoo et al., 1999
; Shen et al., 1999
; Vlachakis et al., 2000
) and that Hox and Meis need to interact with Pbx to function in vivo (Vlachakis et al., 2001
). Although these data suggest that Meis/Pbx/Hox complexes exist in vivo, the role that each protein plays within the complex remains unclear. Possible roles for Hox and Pbx proteins derive from their interaction with transcriptional coactivators (Chariot et al., 1999
; Saleh et al., 2000
) and corepressors (Asahara et al., 1999
; Saleh et al., 2000
). The absence of such interactions for Meis proteins has led to the suggestion that they stabilize Pbx/Hox complexes by binding both to DNA and to Pbx. In possible disagreement with this hypothesis, it has been found that, although Meis proteins require an intact Pbx interaction domain, they do not require an intact homeodomain to synergize with Pbx and Hox proteins (e.g. Berthelsen et al., 1998a
; Vlachakis et al., 2001
), although this has only been analyzed during conditions of Meis overexpression. In this report we identify a domain essential for function near the Pbx interaction motif of Meis3. By mutating residues required for Pbx binding and transferring the domain from Meis3 onto Pbx4/Lzr, we show that this activity is retained even when Pbx binding is abolished. We interpret our results to mean that Meis proteins contribute an activity to the multimeric complexes in addition to stabilization. Because this domain does not contain any known motifs we hypothesize that it serves as a binding site for an auxiliary protein required for transcription activity.
Furthermore, if Meis proteins serve only to stabilize Pbx/Hox complexes it should be possible to generate a dominant negative form of Meis by disrupting DNA binding while retaining Pbx binding. We did not observe reproducible dominant negative phenotypes using such constructs (Vlachakis et al., 2001) (N. V. and C. G. S., unpublished), and although a similar construct does not have an effect in Xenopus embryos (Salzberg et al., 1999
), expressing a homeodomain-less Hth construct in Drosophila has a mild dominant negative effect on Hox-dependent functions (Ryoo et al., 1999
). Our identification of a required domain adjacent to the Pbx interaction domain explains these results given that constructs lacking the homeodomain will retain the M1 domain and will not be strongly dominant negative. Our results instead support the idea that to interfere with Meis function, this essential domain must be kept out of the multimeric complexes.
For what Hox-dependent processes are Meis proteins required?
Our experiments reveal a role for Meis proteins in the development of the hindbrain, particularly r3 and r4. Notably, this region of the hindbrain expresses Hox genes only from paralog group 1 and 2, and the phenotype we observe is similar to that of mice lacking paralog group 1 and 2 Hox genes (Barrow and Capecchi, 1996; Davenne et al., 1999
; Studer et al., 1996
). Because expression of paralog group 1 and 2 Hox genes is controlled by Hox proteins acting in an auto- and cross-regulatory fashion, we suggest that Meis proteins are essential cofactors for Hox proteins in this capacity. Although both murine hoxb1 and hoxb2 have Meis binding sites adjacent to Hox and Pbx binding sites in their enhancers (Ferretti et al., 2000
; Jacobs et al., 1999
), the Meis site in the hoxb1 enhancer is not essential for expression (Ferretti et al., 2000
). These data may indicate that, although Meis proteins are required for both hoxb1 and hoxb2 expression, binding to the Meis site is dispensable for hoxb1 expression.
Our results also indicate that hoxb1a and hoxb2 expression is dependent on Meis, whereas hoxb1b expression is not. This finding correlates with the fact that hoxb1b (the zebrafish counterpart to murine hoxA1) is the earliest Hox gene expressed in zebrafish. Because there are no other Hox proteins present to regulate initial hoxb1b expression, it is possible that its expression is regulated by a Hox-independent mechanism, and that Meis proteins are therefore not required. Once hoxb1b is expressed it may then act with meis and pbx to crossregulate the transcription of later expressed Hox genes. Indeed, we have shown that co-expression of Hoxb1b with Meis3 and Pbx4/Lzr is sufficient to induce ectopic hoxb1a and hoxb2 expression in zebrafish (Vlachakis et al., 2001) and murine hoxA1 probably regulates directly the expression of hoxB1 (the murine counterpart to zebrafish hoxb1a) (Pöpperl et al., 1995
).
Meis proteins may also be required for the proper formation of other structures. For instance, although r2 retains hoxa2 expression in CPbx4-injected embryos, it occasionally also expresses ectopic ephA4 and there may be similar subtle effects on more caudal rhombomeres, as well as on regions outside the hindbrain. Furthermore, because our dominant negative approach relies on the
CPbx4 construct binding to Meis, any Meis functions that are independent of Pbx binding would not be detected in our experiments.
The phenotype we observe as a result of interfering with Meis activity is also qualitatively similar to that of the lazarus mutant (which carries a mutation in the pbx4 gene) (Pöpperl et al., 2000). Particularly, in both cases gene expression is affected primarily in r3 and r4 and less in r1, r2 or r5-r7. This suggests that Pbx and Meis function in the same pathway during hindbrain development. This is consistent with work in Drosophila, where the phenotypes of hth and exd mutants are largely indistinguishable (Kurant et al., 1998
; Pai et al., 1998
; Rieckhof et al., 1997
) and the genes are thought to act in the same pathway. An explanation for Meis and Pbx acting in the same pathway in the hindbrain probably comes from Meis proteins not interacting directly with Hox proteins expressed in the hindbrain (primarily paralog group 1-4), whereas Pbx proteins do. Therefore, Meis proteins can only act as Hox cofactors in the hindbrain by binding to Pbx. Our finding that Meis and Pbx loss-of-function give similar hindbrain phenotypes is therefore consistent with all hindbrain Hox functions that require Pbx also requiring Meis. However, although the meis loss-of-function and lazarus phenotypes are qualitatively similar, they differ quantitatively. Surprisingly, we observe both a higher frequency and a more severe effect on hindbrain gene expression in the absence of Meis function than reported for the lazarus mutant. We speculate that this is unlikely to be a result of Pbx-independent effects of Meis proteins on Hox function, but may instead stem from the presence of maternal pbx4/lzr transcript, as well as additional pbx genes expressed in the lazarus mutant (Pöpperl et al., 2000
). If this is correct, complete removal of Pbx activity might be required to conclusively define the relative roles of Pbx and Meis in regulating Hox function.
Note added in press
While this work was under review two other manuscripts reporting Meis loss of function phenotypes were published (Dibner et al., 2001; Waskiewicz et al., 2001
).
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ACKNOWLEDGMENTS |
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