Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1998 Apr 28;95(9):5072–5075. doi: 10.1073/pnas.95.9.5072

Rescue of Caenorhabditis elegans pharyngeal development by a vertebrate heart specification gene

Christina Haun *, Jonathan Alexander †, Didier Y Stainier †, Peter G Okkema *,‡
PMCID: PMC20215  PMID: 9560230

Abstract

Development of pharyngeal muscle in nematodes and cardiac muscle in vertebrates and insects involves the related homeobox genes ceh-22, nkx2.5, and tinman, respectively. To determine whether the nematode and vertebrate genes perform similar functions, we examined activity of the zebrafish nkx2.5 gene in transgenic Caenorhabditis elegans. Here, we report that ectopic expression of nkx2.5 in C. elegans body wall muscle can directly activate expression of both the endogenous myo-2 gene, a ceh-22 target normally expressed only in pharyngeal muscle, and a synthetic reporter construct controlled by a multimerized CEH-22 binding site. nkx2.5 also efficiently rescues a ceh-22 mutant when expressed in pharyngeal muscle. Together, these results indicate that nkx2.5 and ceh-22 provide a single conserved molecular function. Further, they suggest that an evolutionarily conserved mechanism underlies heart development in vertebrates and insects and pharyngeal development in nematodes.

Heart development in animals as distantly related as insects and vertebrates involves related NK-2 family homeobox genes (reviewed in ref. 1). In Drosophila, the tinman homeobox gene is expressed in cardiac precursors, and tinman mutants completely lack a heart (2–4). Likewise in vertebrates, the nkx2.5 homeobox gene is expressed in myocardial precursors (5–9), and mouse Nkx2–5 mutants exhibit defects in cardiac morphogenesis and gene expression (10). This remarkable molecular conservation suggests a common mechanism controls heart development in a wide variety of species.

Nematodes have no heart or defined circulatory system; however, existing evidence suggests the nematode pharynx, a rhythmically contracting organ involved in feeding, shares functional and molecular similarities with the heart in other species. Pharyngeal muscle function, like that of vertebrate cardiac muscle, does not require nervous system input (11). Likewise at the molecular level, pharyngeal muscle development does not involve the MyoD family of myogenic regulatory factors (12, 13). Instead, pharyngeal muscle development requires the tinman/nkx2.5-related homeobox gene ceh-22. ceh-22 is expressed exclusively in pharyngeal muscle, where it binds the enhancer of the pharyngeal muscle-specific myo-2 gene, and a ceh-22 mutant displays defects in pharyngeal morphology and function (13, 14).

Is there a relationship between heart development in insects and vertebrates and pharyngeal development in nematodes? To address this question, we have examined the ability of the zebrafish nkx2.5 gene (8, 9) to substitute for ceh-22 in transgenic C. elegans. We have found that, like ceh-22 (15), nkx2.5 can activate expression of myo-2 when expressed ectopically in body wall muscle and that nkx2.5 can rescue a ceh-22 mutant when expressed in pharyngeal muscle. These results indicate that ceh-22 and nkx2.5 share a common function and suggest that development of the nematode pharynx and the vertebrate heart may involve a conserved regulatory mechanism.

MATERIALS AND METHODS

Plasmids and C. elegans Transformation Techniques.

To produce unc-54∷nkx2.5, an nkx2.5 cDNA, PCR amplified from adult zebrafish heart using primers derived from the published sequence (8, 9) was inserted downstream of the unc-54 promoter in pPD30.38 (16) using an NcoI site within the nkx2.5 5′-untranslated region. To produce ceh-22∷nkx2.5 (designated plasmid pOK102.01) or ceh-22∷ceh-22 (designated plasmid pOK102.05), cDNAs were inserted downstream of the ceh-22 promoter from pOK29.02 (14).

Two transgenic lines containing unc-54∷nkx2.5 were isolated by microinjection into wild-type N2 C. elegans, using the cotransformation marker pRF4 (17). To produce ceh-22(cc8266) mutant lines bearing either ceh-22∷nkx2.5 or ceh-22∷ceh-22 expression vectors, the balanced heterozygous strain OK 0060 [ceh-22(cc8266)/unc-42(e270) sma-1(e30)] was transformed with each construct using the pRF4 marker; two transformed homozygous ceh-22(cc8266) lines segregating from each of these initial heterozygous transformants were identified and characterized.

Antibody Staining and Expression of lacZ Reporters.

Expression of MYO-2 protein was examined by staining with the monoclonal antibody 9.2.1 [kindly provided by D. M. Miller (18)] following whole mount fixation of mixed-stage populations of nematodes (19, 20). Primary antibodies were detected by immunofluoresence microscopy using Texas red-conjugated goat-anti mouse IgG (Jackson ImmunoResearch) secondary antibodies. To assay function of lacZ reporters, F1 expression assays were carried out as previously described (21); plasmids pOK21.43 (multimerized B sub-element reporter) and pOK29.02 (ceh-22∷lacZ) were microinjected at 100 μg/ml into the germ line of adult hermaphrodites and F1 progeny stained for β-galactosidase activity as late larvae or adults.

RESULTS

Zebrafish nkx2.5 Can Activate myo-2 Expression When Expressed in C. elegans Body Wall Muscle.

In wild-type C. elegans, ceh-22 is expressed exclusively in pharyngeal muscle where it activates expression of the pharyngeal muscle-specific myosin heavy chain gene myo-2 (14). Ectopic expression of ceh-22 in body wall muscle can activate expression of myo-2 (15); because myo-2 is normally never expressed in body wall muscle, this ectopic expression assay provides a sensitive test for ceh-22 function. To determine whether zebrafish nkx2.5 can function similarly to ceh-22, we expressed nkx2.5 in C. elegans body wall muscle and examined expression of the endogenous myo-2 gene by antibody staining. Two transgenic lines expressing an nkx2.5 cDNA under the control of the unc-54 body wall muscle-specific promoter were generated. In both lines, we detected myo-2 expression in the body wall muscles (Fig. 1 A and B). These results show that nkx2.5 can function like ceh-22 to induce myo-2 expression.

Figure 1.

Figure 1

Open in a new tab

Expression of zebrafish nkx2.5 in C. elegans body wall muscle activates expression of pharyngeal muscle-specific genes. (A and B) Adult animals bearing the unc-54∷nkx2.5 expression vector stained with the anti-MYO-2 antibody, 9.2.1. MYO-2 protein accumulates in pharyngeal muscles (ph) and body wall muscles (bw). Animals ectopically expressing either ceh-22 or nkx2.5 generally express MYO-2 in only a few (approximately 1–5) body wall muscle cells (15); the numbers of MYO-2 positive body wall muscles are similar in animals expressing either ceh-22 or nkx2.5. (C) An adult animal bearing the unc-54∷nkx2.5 transgene and expressing a B sub-element∷lacZ reporter in body wall muscles stained with X-Gal. β-Galactosidase staining accumulates in body wall muscle nuclei (marked by black bars).

We next asked whether Nkx2.5 directly interacts with the same sequences recognized by CEH-22 by examining expression of a reporter gene under the control of multimerized CEH-22 binding sites. CEH-22 binds a region within the myo-2 enhancer termed the B sub-element (14). In wild-type animals, a lacZ reporter under control of a synthetic enhancer consisting of four copies of a 28-bp B sub-element oligonucleotide is expressed specifically in pharyngeal muscle; only occasional expression is observed outside the pharynx (Table 1; ref. 14). In a transgenic strain bearing the unc-54∷nkx2.5 expression construct, we found a significant increase in the number of animals expressing β-galactosidase in body wall muscle (from 2.5 to 16.5%) (Table 1; Fig. 1C). To rule out the possibility that Nkx2.5 was indirectly enhancing expression of myo-2 or the B sub-element reporter by activating ectopic expression of the ceh-22 gene, we examined expression of a ceh-22∷lacZ fusion in animals bearing the unc-54∷nkx2.5 transgene. Expression of β-galactosidase was limited to pharyngeal muscle (Table 1), a pattern identical to that observed in wild-type animals (14). Thus, these data together indicate that Nkx2.5, like CEH-22, activates transcription by interacting directly with the B sub-element of the myo-2 enhancer.

Table 1.

Expression of lacZ reporters in body wall muscle

Construct Percent F1 transformants expressing β-galactosidase in body wall muscle*
Wild type (N2) OK0027†
B sub-element∷lacZ 2.5% (n = 118) 16.5% (n = 151)
ceh-22∷lacZ ND 0% (n = 69)
Open in a new tab
*

lacZ reporters were injected into the germ line of adult hermaphrodites, and the entire F1 brood was stained to detect β-galactosidase expression (21). Using this “F1 expression assay,” both reporters are strongly expressed in pharyngeal muscle (14). The total number of F1 transformants (n), recognized by pharyngeal expression of β-galactosidase, was counted, and they were then examined for ectopic β-galactosidase expression in body wall muscle. 

†

The unc-54∷nkx2.5 fusion in strain OK 0027 is maintained as an extrachromosomal array that is segregated to 55% of F1 progeny; therefore, approximately 30% of the F1 transformants bearing the unc-54∷nkx2.5 fusion express B sub-element∷lacZ in body wall muscle. 

nkx2.5 Can Substitute for ceh-22 During Normal Pharyngeal Development.

In addition to its role in myo-2 activation, CEH-22 likely regulates other genes required for pharyngeal development. Indeed, a ceh-22 mutant exhibits profound contractile and morphological defects in the pharynx, despite expressing myo-2 nearly as well as wild type (15). To examine the extent to which Nkx2.5 and CEH-22 are functionally equivalent, we asked if expression of nkx2.5 in pharyngeal muscle could rescue a ceh-22 mutant. The loss-of-function mutation ceh-22(cc8266) causes pharyngeal muscle defects that interfere with feeding, resulting in a lethal/slow growth phenotype (15). Approximately one-quarter of ceh-22(cc8266) mutants arrest shortly after hatching, whereas the remaining mutants grow slowly to adulthood. These mutant adults exhibit characteristic morphological and contractile defects in the pharynx and have a starved appearance typical of feeding defective mutants. We placed the nkx2.5 cDNA under control of the ceh-22 promoter and introduced this fusion (designated ceh-22∷nkx2.5) into ceh-22(cc8266) mutants. For comparison, we also introduced a similar fusion containing the ceh-22 cDNA under the control of the ceh-22 promoter (designated ceh-22∷ceh-22) into ceh-22(cc8266) mutants.

We tested whether expression of nkx2.5 or ceh-22 could rescue the larval arrest/slow growth phenotype by picking synchronized embryos and counting the number of animals reaching adulthood after 4 days at 20°C (Table 2). Ninety-eight percent of the wild-type embryos reached adulthood under these conditions. In comparison, only 36% of the untransformed ceh-22(cc8266) mutants reached adult stage, and these adults had a thin, pale appearance indicative of poor feeding (22). For ceh-22(cc8266) mutants transformed with ceh-22∷nkx2.5 or ceh-22∷ceh-22, 68% and 77% of the animals reached adulthood within 4 days, respectively, and all of these transformed adults had a well fed appearance. The animals that did not reach adulthood likely did not contain the transforming DNA, which in C. elegans is maintained as a semi-stable extrachromosomal array (17); however, we cannot rule out the possibility that neither nkx2.5 nor ceh-22 completely rescued the mutant phenotype. Nonetheless, the ability of the ceh-22∷nkx2.5 transgene to rescue ceh-22(cc8266) growth defects nearly as well as ceh-22∷ceh-22 was quite striking.

Table 2.

nkx2.5 and ceh-22 rescue the growth defects in ceh-22(cc8266)

Genotype Percent animals reaching adult after 4 days at 20°C* n
Wild type (N2) 98 305
ceh-22(cc8266) 36 230
ceh-22(cc8266); cuEx(ceh-22∷nkx2.5 cDNA) 68 104
ceh-22(cc8266); cuEx(ceh-22∷ceh-22 cDNA) 77 203
Open in a new tab
*

Embryos segregating from the indicated strains were picked to fresh plates and incubated at 20°C. After 4 days, the number of animals reaching adulthood was determined. Neither ceh-22∷ceh-22 nor ceh-22∷nkx2.5 rescued a partially penetrant sterile phenotype exhibited by ceh-22(cc8266) (ref. 15; data not shown). This phenotype is rescued by transformation with a ceh-22 genomic DNA fragment; thus, we believe the ceh-22∷ceh-22 and ceh-22∷nkx2.5 may be missing sequences necessary for rescue of sterility. 

We also examined the pharyngeal morphology of representative adult ceh-22(cc8266) mutants and ceh-22(cc8266) animals bearing either the ceh-22∷nkx2.5 or ceh-22∷ceh-22 fusion by Nomarski microscopy (Fig. 2). Nine of nine untransformed ceh-22(cc8266) mutants displayed an abnormal pharyngeal morphology (Fig. 2B). The pharynx appeared thicker than wild type, particularly in the region where the isthmus joins the terminal bulb; in addition, the grinder, a cuticular structure located in the lumen of the pharynx, appeared in an abnormal orientation and did not invert completely during pharyngeal pumping. In contrast, nearly all of the ceh-22(cc8266) animals bearing ceh-22∷nkx2.5 or ceh-22∷ceh-22 displayed a normal pharyngeal morphology (Fig. 2 C and D); only 1/17 and 1/19 of these adults, respectively, appeared abnormal, whereas the pharyngeal contractions and inversion of the grinder appeared normal in all transgenic animals.

Figure 2.

Figure 2

Open in a new tab

Expression of nkx2.5 or ceh-22 in pharyngeal muscle rescues a ceh-22 mutant. Nomarski micrographs of the adult pharynx in wild type (A), a ceh-22(cc8266) mutant (B), and a ceh-22(cc8266) mutant rescued with either the ceh-22∷nkx2.5 (C) or the ceh-22∷ceh-22 (D) expression vectors. The poorly formed grinder (g) and thickened junction of the pharyngeal isthmus and terminal bulb (i-tb) characteristic of ceh-22(cc8266) are marked in B.

DISCUSSION

Our results demonstrate that zebrafish nkx2.5 and ceh-22 are functionally interchangeable in C. elegans. Nkx2.5 can bind sites within the chromosome normally targeted by CEH-22 and interact productively and specifically with the C. elegans transcriptional machinery. Mutational analysis of the B sub-element of myo-2 indicates that CEH-22 interacts with additional C. elegans factors to regulate gene expression (14). Whatever the nature of the interactions with these other as yet unidentified proteins, Nkx2.5 seems capable of reproducing them. Amino acid sequence identity between Nkx2.5 and CEH-22 is greatest within the homeodomain (68% identity), although two short regions of similarity are also found in conserved positions upstream and downstream of the homeodomain (Fig. 3). The region of conservation downstream of the homeodomain is part of an NK2-specific domain like that found in Nkx2.5 (1, 8, 9); however, deletion of this divergent NK2 domain from CEH-22 has no detectable effect on its ability to activate myo-2 when expressed in body wall muscle (R. Reardon and P.G.O., unpublished work). It therefore seems likely that the homeodomain and perhaps the conserved upstream peptide are the primary determinants mediating function of CEH-22 and Nkx2.5, although other regions of low primary sequence identity but high functional homology may also contribute to activity.

Figure 3.

Figure 3

Open in a new tab

Regions of sequence identity between CEH-22 and Nkx2.5. (A) Schematic diagram of CEH-22 and Nkx2.5 indicating the positions of conserved sequences (numbered according to the predicted protein sequence). The overall levels of amino acid sequence identity are reported for the homeodomains and for the portions of the proteins outside the homeodomains. (B) Sequence alignments between conserved regions of CEH-22 and Nkx2.5. Amino acid identities are indicated by a colon (:) and similarities by a period (.); numbering indicates the location of these sequences within the primary amino acid sequence. A portion of the NK2-specific domain consensus reported in ref. 1 is shown with residues present in both CEH-22 and Nkx2.5 underlined.

Similar experiments examining function of murine Nkx2–5 in Drosophila indicate that derivatives of Nkx2–5 can also partially rescue the tinman mutant, suggesting a conservation of function also exists between tinman and Nkx2–5 (R. Bodmer, personal communication; G. Ranganayakulu, R. Harvey, and E. Olson, personal communication). Together these results and ours suggest that a key step in the development of cardiac muscle in vertebrates and insects and pharyngeal muscle in nematodes is controlled by a common factor. Expression of tinman and nkx2.5 in the hearts of Drosophila (23, 24) and the chicken, respectively (25), involve upstream signals mediated by transforming growth factor-β superfamily and wingless signaling pathways; it will be important to determine whether similar upstream signals regulate ceh-22 expression in the C. elegans pharynx to understand the extent to which the pathways specifying heart and pharyngeal development are conserved.

Although ceh-22, nkx2.5, and tinman seem to share common function, it is not clear that they are orthologs. These genes are members of the larger family of NK2 homeobox genes and other family members function in a variety of tissues (1). Indeed the CEH-22 homeodomain shares highest sequence identity with mouse Nkx2–2 and Drosophila vnd (14), which are primarily expressed in the developing nervous system (26, 27). It will be interesting to determine whether these or other NK2 family members will also function like nkx2.5 to activate myo-2. It is noteworthy that another C. elegans NK2 homeobox gene, ceh-24, is expressed outside the pharynx in some neurons and the vulval muscles (28), suggesting certain NK2 family members may be incapable of activating myo-2.

The nematode pharynx, vertebrate heart, and insect heart are all muscular tubes that pump liquids, although the specific functions of these organs are very different. What is the significance of the fact that these different organs utilize a common myogenic mechanism? One interesting observation is that in all vertebrates thus far examined, nkx2.5 (or the related genes nkx2.3 and nkx2.7) is expressed not only in the anterior lateral plate mesoderm, which will give rise to the heart, but also in the anterior endoderm, which will give rise to parts of the anterior digestive tract (5–9). Likewise, Drosophila tinman is transiently expressed in an anterior region of the embryo surrounding the stomodeum (2), which gives rise to the mouth, pharynx, and esophagus (29). Perhaps the ancestral function of ceh-22/tinman/nkx2.5 was to demarcate a “module” (as defined in ref. 30) that gave rise to an anteriorly located contractile tube used for feeding, circulation, or both. The mesodermal component of this field may have been co-opted during evolution to form the very distinct structures found in modern phyla.

Acknowledgments

We are grateful to Andrew Fire for encouragement and critical reading of this manuscript. This work was funded by a grant from National Institutes of Health (to P.G.O.) and grants from National Institutes of Health, American Heart Association, and the March of Dimes and Packard Foundations (to D.Y.R.S.). J.A. is currently an American Heart Association predoctoral fellow and a trainee in the Medical Scientist Training Program at University of California, San Francisco. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health National Center for Research Resources.

References

  • 1.Harvey R. Dev Biol. 1996;178:203–216. doi: 10.1006/dbio.1996.0212. [DOI] [PubMed] [Google Scholar]
  • 2.Bodmer R, Jan L Y, Jan Y N. Development. 1990;110:661–669. doi: 10.1242/dev.110.3.661. [DOI] [PubMed] [Google Scholar]
  • 3.Bodmer R. Development. 1993;118:719–729. doi: 10.1242/dev.118.3.719. [DOI] [PubMed] [Google Scholar]
  • 4.Azpiazu N, Frasch M. Genes Dev. 1993;7:1325–1340. doi: 10.1101/gad.7.7b.1325. [DOI] [PubMed] [Google Scholar]
  • 5.Lints T J, Parsons L M, Hartley L, Lyons I, Harvey R P. Development. 1993;119:419–431. doi: 10.1242/dev.119.2.419. [DOI] [PubMed] [Google Scholar]
  • 6.Tonissen K F, Drysdale T A, Lints T J, Harvey R P, Krieg P A. Dev Biol. 1994;162:325–328. doi: 10.1006/dbio.1994.1089. [DOI] [PubMed] [Google Scholar]
  • 7.Schultheiss T, Xydas S, Lassar A B. Development. 1995;121:4203–4214. doi: 10.1242/dev.121.12.4203. [DOI] [PubMed] [Google Scholar]
  • 8.Lee K, Xu Q, Breitbart R E. Dev Biol. 1996;180:722–731. doi: 10.1006/dbio.1996.0341. [DOI] [PubMed] [Google Scholar]
  • 9.Chen J-N, Fishman M C. Development. 1996;122:3809–3816. doi: 10.1242/dev.122.12.3809. [DOI] [PubMed] [Google Scholar]
  • 10.Lyons I, Parsons L M, Hartley L, Li R, Andrews J E, Robb R, Harvey R P. Genes Dev. 1995;9:1654–1666. doi: 10.1101/gad.9.13.1654. [DOI] [PubMed] [Google Scholar]
  • 11.Avery L, Horvitz H R. Neuron. 1989;3:473–485. doi: 10.1016/0896-6273(89)90206-7. [DOI] [PubMed] [Google Scholar]
  • 12.Krause M, Fire A, White Harrison S, Priess J, Weintraub H. Cell. 1990;63:907–919. doi: 10.1016/0092-8674(90)90494-y. [DOI] [PubMed] [Google Scholar]
  • 13.Chen L, Krause M, Sepanski M, Fire A. Development. 1994;120:1631–1641. doi: 10.1242/dev.120.6.1631. [DOI] [PubMed] [Google Scholar]
  • 14.Okkema P G, Fire A. Development. 1994;120:2175–2186. doi: 10.1242/dev.120.8.2175. [DOI] [PubMed] [Google Scholar]
  • 15.Okkema P G, Ha E, Haun C, Chen W, Fire A. Development. 1997;124:3965–3973. doi: 10.1242/dev.124.20.3965. [DOI] [PubMed] [Google Scholar]
  • 16.Mello C C, Fire A. In: Methods in Cell Biology: C. elegans. Shakes D, Epstein H, editors. San Diego: Academic; 1995. pp. 452–482. [Google Scholar]
  • 17.Mello C C, Kramer J M, Stinchcomb D, Ambros V. EMBO J. 1991;10:3959–3970. doi: 10.1002/j.1460-2075.1991.tb04966.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Miller D M, Stockdale F E, Karn J. Proc Natl Acad Sci USA. 1986;83:2305–2309. doi: 10.1073/pnas.83.8.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Miller, D. M. & Shakes, D. C. in Methods in Cell Biology: C. elegans, eds. Shakes, D. & Epstein, H. (Academic, San Diego), pp. 365–394.
  • 20.Finney M, Ruvkun G B. Cell. 1990;63:895–905. doi: 10.1016/0092-8674(90)90493-x. [DOI] [PubMed] [Google Scholar]
  • 21.Okkema P G, Harrison S W, Plunger V, Aryana A, Fire A. Genetics. 1993;135:385–404. doi: 10.1093/genetics/135.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Avery L. Genetics. 1993;133:897–917. doi: 10.1093/genetics/133.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Frasch M. Nature. 1995;374:464–467. doi: 10.1038/374464a0. [DOI] [PubMed] [Google Scholar]
  • 24.Park M, Wu X, Golden K, Axelrod J D, Bodmer R. Dev Biol. 1996;177:104–116. doi: 10.1006/dbio.1996.0149. [DOI] [PubMed] [Google Scholar]
  • 25.Schultheiss T M, Burch J B, Lassar A B. Genes Dev. 1997;11:451–462. doi: 10.1101/gad.11.4.451. [DOI] [PubMed] [Google Scholar]
  • 26.Jimenez F, Martin-Morris L E, Velasco L, Chu H, Sierra J, Rosen D R, White K. EMBO J. 1995;14:3487–3495. doi: 10.1002/j.1460-2075.1995.tb07355.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Price M, Lazzaro D, Pohl T, Mattei M G, Ruther U, Olivo J C, Duboule D, Di Lauro R. Neuron. 1992;8:241–255. doi: 10.1016/0896-6273(92)90291-k. [DOI] [PubMed] [Google Scholar]
  • 28.Harfe B, Fire A. Development. 1998;125:421–429. doi: 10.1242/dev.125.3.421. [DOI] [PubMed] [Google Scholar]
  • 29.Fullilove S L, Jacobson A G. In: The Genetics and Biology of Drosophila. Ashburner M, Wright T R F, editors. 2c. New York: Academic; 1978. pp. 105–227. [Google Scholar]
  • 30.Raff R A. The Shape of Life: Genes, Development, and the Evolution of Animal Form. Chicago: University of Chicago Press; 1996. [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES