Department of Biological Sciences, Louisiana State University
Correspondence: E-mail: mnoor{at}lsu.edu.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: gene expression hybrid sterility reproductive isolation DNA microarrays
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both theoretical and empirical studies suggest that failures in the regulation of gene expression may contribute to hybrid dysfunctions (see review in Orr and Presgraves 2000). For example, Johnson and Porter (2000; see also Porter and Johnson [2002]) modeled the evolution of regulatory genetic pathways and found that binding strength of proteins to promoter regions can provide biologically plausible hybrid sterility. An empirical example is provided by the overexpression of Xmrk-2 oncogene (Xiphophorus melanoma receptor kinase) that is associated with tumor formation and hybrid lethality in crosses between swordtails (Xiphophorus helleri) and platyfish (X. maculatus) (Schartl 1995; Schartl et al. 1999). To evaluate the role of gene expression in hybrid sterility, technological advances can now enable researchers to examine expression patterns of hundreds or thousands of genes in hybrids relative to nonhybrids simultaneously.
Genome-wide expression profiling can rapidly identify whether qualitative failures in gene expression are associated with hybrid male sterility, and if so, what genes or genetic pathways are responsible. Two hypotheses may predict the identity and the location of genes that are misexpressed in hybrids, and hence possibly associated with sterility. First, genes expressed predominantly in males and the male germ line, and thus often subject to accelerated microevolutionary divergence (see review in Singh and Kulathinal 2000), may be most likely to be deregulated in hybrids. In Drosophila, it has been shown that male reproductive proteins, such as accessory gland proteins (Acps), can be twice as divergent between species as nonreproductive proteins (e.g., Civetta and Singh 1995). The coding regions of their corresponding genes exhibit a significant excess of nonsynonymous over synonymous substitutions, suggesting that positive Darwinian selection accelerated their divergence (e.g., Swanson et al. 2001). Second, X-chromosomal genes may be misexpressed in hybrids more than autosomal genes, corresponding with suggestions of a disproportionate X-effect in hybrid sterility (Charlesworth, Coyne, and Barton 1987; Coyne and Orr 1989; True et al. 1996).
To evaluate the potential for gene misexpression to cause sterility in hybrids, we must first ascertain whether and how much gene misexpression is detectable in hybrids and the nature of the misexpressed transcripts. In a previous study, Reiland and Noor (2002) documented a handful of transcripts misexpressed in hybrids of Drosophila pseudoobscura and D. persimilis relative to pure species, but their differential display technique had very low resolution for detecting quantitative differences in gene expression. Here, we apply much higher resolution techniques, microarrays and real-time fluorescent quantitative PCR, to examine differences in gene expression between two Drosophila species (D. simulans and D. mauritiana) and their sterile F1 hybrids. Infertility in male hybrids between these species is associated with spermiogenic, or postmeiotic, failure at the cytological level (Wu et al. 1992). We evaluate whether genes with male-specific patterns of expression, or those located on the X chromosome, are disproportionately disrupted in hybrids, testing the hypotheses laid out above, and we discuss the implications of our findings with regard to Haldane's rule (Haldane 1922).
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oligonucleotide Microarray Assays
For each replicate, 5 µg of total RNA were obtained from 23 seven-day post-eclosion adult Drosophila simulans, D. mauritiana, and F1 hybrid males with the QIAGEN RNeasy kit. A total of 14 Affymetrix GeneChip Drosophila Genome array chips were used: 5 x D. simulans, 4 x D. mauritiana, 5 x F1 hybrids. The GeneChip Sample Cleanup Module (QIAGEN) was used according to the Affymetrix standard hybridization protocol. Double-stranded cDNA was synthesized with a T7-(dT)24 primer; cRNA was synthesized and biotin-labeled in an in vitro transcription reaction using the ENZO BioArray HighYield RNA Transcript Labeling Kit. The final concentration of cRNA in the fragmentation mixture was 0.5 µg/µL. The target cRNA was hybridized to arrays that allow one to monitor the relative abundance of more than 14,000 mRNA transcripts. Patterns of hybridization were detected in an Affymetrix scanner, and the results were analyzed using Affymetrix Microarray Suite (MAS) 5.0 software. The software Detection algorithm calculates the Discrimination Score from each probe pair (Perfect Match vs. Mismatch), assesses probe saturation, calculates a Detection P value from the one-sided Wilcoxon Signed Rank test, and assigns a Present, Marginal, or Absent Call in relation to a threshold value Tau = 0.015. All transcripts were scaled to a target intensity value of 10,000.
Expression differences obtained from this assay may be confounded by sequence differences between these species and D. melanogaster. Hence, we focus on those transcripts that show little difference in hybridization between the two pure species assayed but a large difference between the pure species and the F1 hybrid males. As the F1 hybrids were produced from the same strains as the pure species evaluated, they would possess no sequence difference from them, and differences in hybridization between these pure species and these F1 hybrids must reflect differences in transcript abundance.
Real-Time Polymerase Chain Reaction
For the real-time polymerase chain reaction (RT-PCR), 250 ng of total RNA prepared as described above were reverse transcribed in a reaction bearing 5 mM MgCl2, 50 mM KCl, 10 mM TrisHCl, 1 mM dNTPs, 20 U RNasin, 20 U reverse transcriptase (SuperScript II), 2.5 µM of the target primer, and 50 nM of 18SrRNA reverse primer (Applied Biosystems [ABI]). All real-time PCR primers and fluorescent probes were designed using Primer Express 2.0 software (ABI), and their sequences are available by request. All target probes were FAM-labeled (Biosearch Technologies), and the normalizer probe (18S rRNA) was VIC-labeled (ABI). Pre-developed ABI TaqMan Assay Reagents and ABI standard protocols were used to prepare the RT-PCR reaction mixture. The probes contain a reporter dye at the 5' end of the probe and a quencher dye at the 3' end of the probe. During the reaction, the reporter dye and quencher dye are separated, resulting in increased fluorescence of the reporter. All PCRs consisted of an initial 2 min at 50°C and 10 min at 95°C, 40 cycles of 95°C for 15 s and at 60°C for 1 min. Applied Biosystems Prism 7000 SDS software was used for visualization and quantification of the amplification products.
Statistical Analysis
Standard one-way analyses of variance (ANOVAs) were used for detecting differences in expression between each pure species individually and the F1 hybrids. In addition, we used Bayesian regularized t-tests (Baldi and Long 2001; Long et al. 2001) to compare microarray expression signals between pure species and F1 hybrids. In this analysis, a prior estimate of variance within groups is estimated by the weighted average of a prior estimate of the variance for that gene (obtained from the local weighted average of the variance of other genes) and the experimental estimate of the variance for that gene. This leads to the desirable property of the Bayesian approach converging toward the t-test as the number of replicates increases and prior variance tends to zero. The mean difference in threshold cycle number (CT) was tested with a post hoc Duncan test after the 2-way repeated measures ANOVA analysis, in which genotype (D. simulans, D. mauritiana, and hybrids) effect was significant (P < 0.05). There were two levels of replications: two independent reverse transcriptase reactions and two PCR replicates of each sample within the RT reaction. All results were repeatable when different cycle thresholds were tested and were largely independent of whether or not normalization for 18SrRNA was applied.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Real-Time PCR Analysis
We validated the putative underexpression of Mst84Dc with quantitative fluorescent real-time PCR (RT-PCR, TaqMan Applied Biosystems). In this assay, we used a repeated measures ANOVA in which 12 bulk-RNA samples from males (4 each from D. simulans, D. mauritiana, and F1 hybrids) were analyzed, and each sample was replicated in two reverse-transcription reactions followed by independent RT-PCR assays. The cDNA from all F1 samples consistently amplified exponentially after 4 more PCR cycles than pure species samples, indicated as higher threshold cycle (CT) values for hybrid samples than for nonhybrid samples (fig. 4a). This delayed amplification suggests a lower concentration of the transcript in hybrids than in pure species (table 1).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the transcripts identified, Mst98Cb, has been described in some detail previously (Schäfer et al. 1993, White-Cooper et al. 1998). Schäfer et al. (1993) hypothesized it to function as a structural protein of the sperm tail. Mst98Cb is transcribed prior to spermiogenesis but not translated until after meiosis. As the spermatogenetic failure of these hybrids is postmeiotic, but transcription of Mst98Cb and the other genes identified is premeiotic (as is most transcription involved in Drosophila spermatogenesis; see Fuller [1998]), the misexpression in hybrids may contribute to sterility rather than being a mere by-product of possessing incompletely developed sperm.
Although we confirmed quantitative misexpression of Mst98Cb and CG5762 in hybrids, this misexpression was not statistically significant from the microarray analysis alone. Hence, the RT-PCR assay succeeded in detecting underexpression suggested by microarray analysis but not confirmed with our statistical tests, suggesting that the latter (especially the Bayesian approach) might increase type II statistical error. This exemplifies the need for an independent validation of microarray-based studies with more sensitive techniques such as RT-PCR (Rajeevan et al. 2001) and northern blots (Taniguchi et al. 2001).
Implications for Haldane's Rule
If one sex of interspecies hybrids is sterile or inviable, it is typically the heterogametic sex; a pattern referred to as Haldane's rule (see review in Orr 1997). In the past decade, three theories have emerged that may partially or completely explain Haldane's rule in a wide variety of taxa. First, if alleles conferring deleterious interactions in hybrids are typically recessive, heterogametic hybrids, which bear only a single X chromosome, will exhibit sterility earlier in evolutionary divergence than homogametic hybrids (Muller 1942; Turelli and Orr 1995). This theory has been supported by several recent observations (see review in Orr 1997), although it does not appear to completely explain hybrid sterility in Drosophila and mammals (see Wu, Johnson, and Palopoli 1996). A second, but not mutually exclusive, theory suggests that hybrid male sterility in particular may evolve rapidly, through either sexual selection or unique physiological properties of spermatogenesis (sometimes called "faster male": Wu and Davis 1993; Wu, Johnson, and Palopoli. 1996). This theory is supported by the higher number of factors conferring male sterility over female sterility in genetic mapping studies of D. simulans and D. mauritiana (e.g., True, Weir, and Laurie 1996) and the observation of greater male sterility in mosquito species lacking a degenerate Y chromosome (Presgraves and Orr 1998). However, it predicts that Haldane's rule should be weaker in Lepidoptera, where males are homogametic, than in Drosophila, and this prediction is not supported (Presgraves 2002). The final theory that may contribute to Haldane's rule is that the relative rate of evolution of sex-chromosomal genes may exceed that of autosomal genes if new advantageous mutations are typically recessive (sometimes called "faster X": Charlesworth, Coyne, and Barton 1987). This theory predicts an excess of hybrid sterility factors on the sex chromosomes, which is observed weakly (True et al. 1996), and generally higher rates of evolution of sex-chromosomal genes, something which is not detected in Drosophila (Betancourt, Presgraves, and Swanson 2002).
In our analyses, transcripts known to be predominantly male-specific in their expression were significantly overrepresented among underexpressed genes in hybrids. The male-specificity of misexpression is consistent with the faster-male theory and the repeated observations of rapid evolution of male reproductive proteins. The results demonstrate that genes with male-specific patterns of expression are more prone to disruption in hybrids of these species than genes without sex-specific expression. Indeed, our results may be conservative because male-specific genes are often among the most rapidly evolving (e.g., Singh and Kulathinal 2000; Swanson et al. 2001), so some of the most divergent male-specific genes may not have been surveyed here because of sequence divergence from D. melanogaster.
The lack of a disproportionate number of X-chromosome genes being misexpressed in hybrids may incorrectly appear to militate against the faster-X theory. The transcripts misexpressed in hybrids are likely to be downstream genetic targets of the changes that cause hybrid dysfunctions. Although genes involved in spermatogenesis are likely to regulate other genes involved in spermatogenesis or male secondary sexual traits, most X-linked genes probably do not regulate exclusively other X-linked genes (but see Boutanaev et al. 2002). As such, our results do not refute the faster-X theory.
Prospective
Our study is one of very few that have attempted to determine why genes might fail to interact properly in hybrids, resulting in sterility (e.g., Reiland and Noor 2002). Although this study has not directly shown that the observed quantitative disruptions in gene expression in hybrids cause sterility, we have shown that many genes do have disruptions in their expression in hybrids, including several that contribute to spermatogenesis. This study has further illustrated the utility of microarrays and other technologies for the global analysis of gene expression, to evolutionary studies. In addition, it has established a panel of genes on which to focus further investigations of the genetics of hybrid sterility. This panel constitutes a dramatic advance given that the past 70 years of study has identified one only gene (Ting et al. 1998). Given that we have now documented that gene misexpression does occur in sterile species hybrids, clear next steps will include (1) documenting transcriptional variation among various life cycle stages and tissues and (2) comparing gene expression in sterile and fertile males among multigenerational backcross lines to determine whether the disruptions in gene expression are directly associated with sterility.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Affymetrix Microarray Suite., 2002. Version 5.0. Affymetrix, Santa Clara, Calif.
Andrews, J., G. G. Bouffard, C. Cheadle, J. Lu, K. G. Becker, and B. Oliver. 2000. Gene discovery using computational and microarray analysis of transcription in the Drosophila melanogaster testis. Genome Res. 10:1841-1842.
Baldi, P., and A. D. Long. 2001. A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes. Bioinformatics 17:509-519.
Betancourt, A. J., D. C. Presgraves, and W. J. Swanson. 2002. A test for faster X evolution in Drosophila. Mol. Biol. Evol. 19:1816-1819.
Boutanaev, A. M., A. I. Kalmykova, Y. Y. Shevelyov, and D. I. Nurminsky. 2002. Large clusters of co-expressed genes in the Drosophila genome. Nature 420:666-669.[CrossRef][ISI][Medline]
Braidotti, G., and D. P. Barlow. 1997. Identification of a male meiosis-specific gene, Tcte2, which is differentially spliced in species that form sterile hybrids with laboratory mice and deleted in t chromosomes showing meiotic drive. Dev. Biol. 86:85-99.
Charlesworth, B., J. A. Coyne, and N. H. Barton. 1987. The relative rates of evolution of sex chromosomes and autosomes. Am. Nat. 130:113-146.[CrossRef][ISI]
Civetta, A., and R. S. Singh. 1995. High divergence of reproductive tract proteins and their association with postzygotic reproductive isolation in Drosophila melanogaster and Drosophila virilis group species. J. Mol. Evol. 41:1085-1095.[ISI][Medline]
Coyne, J. A., and H. A. Orr. 1989. Patterns of speciation in Drosophila. Evolution 43:362-381.[ISI]
Coyne, J. A., and H. A. Orr. 1997. "Patterns of speciation in Drosophila" revisited. Evolution 51:295-303.[ISI]
Dobzhansky, T. 1936. Studies on hybrid sterility: II. Localization of sterility factors in Drosophila virilis Sturt.x lummei Hackman hybrids. Genetics 21:113-135.
Dobzhansky, T. 1937. Genetics and the origin of species. Columbia University Press, New York.
Fuller, M. T. 1998. Genetic control of cell proliferation and differentiation in Drosophila spermatogenesis. Semin. Cell. Dev. Biol. 9:433-444.[CrossRef][ISI][Medline]
Haldane, J. B. S. 1922. Sex ratio and unisexual sterility in hybrid animals. J. Genet. 12:101-109.
Jin, W., R. M. Riley, R. D. Wolfinger, K. P. White, G. Passador-Gurgel, and G. G. Gibson. 2001. The contributions of sex, genotype and age to transcriptional variance in Drosophila melanogaster. Nat. Genet. 29:389-395.[CrossRef][ISI][Medline]
Johnson, N. A. 2000. Gene interactions and the origin of species. Pp. 197212 in J. B. Wolf, E. D. Brodie III, and M. J. Wade, eds. Epistasis and the evolutionary process. Oxford University Press, New York.
Johnson, N. A., and A. H. Porter. 2000. Rapid speciation via parallel, directional selection on regulatory genetic pathways. J. Theor. Biol. 205:527-542.[CrossRef][ISI][Medline]
Leemans, R., B. Egger, T. Loop, L. Kammermeier, H. He, B. Hartmann, U. Certa, F. Hirth, and H. Reichert. 2000. Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 97:12138-12143.
Long, A. D., H. J. Mangalam, B. Y. P. Chan, L. Tolleri, G. W. Hatfield, and P. Baldi. 2001. Global gene expression profiling in Escherichia coli K12: improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. J. Biol. Chem. 276:19937-19944.
Muller, H. J. 1940. Bearing of the Drosophila work on systematics. Pp. 185268 in J. Huxley, ed. The new systematics. Clarendon Press, Oxford.
Muller, H. J. 1942. Isolating mechanisms, evolution and temperature. Biol. Symp. 6:71-125.
Orr, H. A. 1997. Haldane's rule. Annu. Rev. Genet. 28:195-218.
Orr, H. A., and D. C. Presgraves. 2000. Speciation by postzygotic isolation: forces, genes and molecules. BioEssays 22:1085-1094.[CrossRef][ISI][Medline]
Parisi, M., R. Nuttal, D. Naiman, G. Bouffard, J. Malley, J. Andrews, S. Eastman, and B. Oliver. 2003. Paucity of genes on the Drosophila X chromosome showing male-biased expression. Science 299:697-700.
Porter, A. H., and N. A. Johnson. 2002. Speciation despite gene flow when developmental pathways evolve. Evolution 56:2103-2111.[ISI][Medline]
Presgraves, D. C. 2002. Patterns of postzygotic isolation in Lepidoptera. Evolution 56:1168-1183.[ISI][Medline]
Presgraves, D. C., and H. A. Orr. 1998. Haldane's rule in taxa lacking hemizygous X. Science 282:952-954.
Price, T. D., and M. B. Bouvier. 2002. The evolution of F1 postzygotic incompatibilities in birds. Evolution 56:2083-2089.[ISI][Medline]
Rajeevan, M. S., S. D. Vernon, N. Tayasavang, and E. R. Unger. 2001. Validation of array-based gene expression profiles by real-time (kinetic) RT-PCR. J. Mol. Diagn. 3:26-31.
Rawson, P. D., and R. S. Burton. 2002. Functional coadaptation between cytochrome c and cytochrome c oxidase within allopatric populations of a marine copepod. Proc. Natl. Acad. Sci. USA 99:12955-12958.
Reiland, J., and M. A. F. Noor. 2002. Little qualitative RNA misexpression in sterile male F1 hybrids of Drosophila pseudoobscura and D. persimilis. BMC Evolutionary Biology 2:16.[CrossRef][Medline]
Sasa, M. M., P. T. Chippindale, and N. A. Johnson. 1998. Patterns of postzygotic isolation in frogs. Evolution 52:1811-1820.[ISI]
Schäfer, M., D. Borsch, A. Hulster, and U. Schäfer. 1993. Expression of a gene duplication encoding conserved sperm tail proteins is translationally regulated in Drosophila melanogaster. Mol. Cell. Biol. 13:1708-1718.[Abstract]
Schartl, M. 1995. Platyfish and swordtails: a genetic system for the analysis of molecular mechanisms in tumor formation. Trends Genet. 11:185-189.[CrossRef][ISI][Medline]
Schartl, M., U. Hornung, H. Gutbrod, J.-N. Volff, and J. Wittbrodt. 1999. Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element. Genetics 153:1385-1394.
Singh, R. S., and R. J. Kulathinal. 2000. Sex gene pool evolution and speciation: a new paradigm. Genes Genet Syst. 75:119-130.[CrossRef][ISI][Medline]
Swanson, W. J., A. G. Clark, H. M. Waldrip-Dail, M. F. Wolfner, and C. F. Aquadro. 2001. Evolutionary EST analysis identifies rapidly evolving male reproductive proteins in Drosophila. Proc. Natl. Acad. Sci. USA 19:7375-7379.[CrossRef]
Taniguchi, M., K. Miura, H. Iwao, and S. Yamanaka. 2001. Quantitative assessment of DNA microarrayscomparison with Northern blot analyses. Genomics 71:34-39.[CrossRef][ISI][Medline]
Ting, C.-T., S.-C. Tsaur, M.-L. Wu, and C.-I. Wu. 1998. A rapidly evolving homeobox at the site of a hybrid sterility gene. Science 282:1501-1504.
True, J. R., B. S. Weir, and C. C. Laurie. 1996. A genome-wide survey of hybrid incompatibility factors by the introgression of marked segments of Drosophila mauritiana chromosomes into Drosophila simulans. Genetics 142:819-837.
Turelli, M., and H. A. Orr. 1995. The dominance theory of Haldane's rule. Genetics 140:389-402.
Turelli, M., and H. A. Orr. 2000. Dominance, epistasis and the genetics of postzygotic isolation. Genetics 154:1663-1679.
White-Cooper, H., M. A. Schäfer, L. S. Alphey, and M. T. Fuller. 1998. Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila. Development 125:125-134.
Wu, C.-I., and A. W. Davis. 1993. Evolution of postmating reproductive isolation: the composite nature of Haldane's rule and its genetic bases. Am. Nat. 142:187-212.[CrossRef][ISI]
Wu, C.-I., N. A. Johnson, and M. F. Palopoli. 1996. Haldane's rule and its legacy: why are there so many sterile males? Trends Ecol. Evol. 11:281-284.[CrossRef][ISI]
Wu, C.-I., D. E. Perez, A. W. Davis, N. A. Johnson, E. L. Cabot, M. F. Palopoli, and M.-L. Wu. 1992. Molecular genetic studies of postmating reproductive isolation in Drosophila. Pp. 191212 in N. Takahata and A. G. Clark, eds. Molecular paleo-population biology. Springer-Verlag, Berlin.