Interspecific profiling of gene expression informed by comparative genomic hybridization: A review and a novel approach in African cichlid fishes

Abstract

Modern genomic approaches have facilitated great progress in our understanding of the molecular and genetic underpinnings of ecological and evolutionary processes. Analysis of gene expression through heterologous hybridization in particular has enabled genome-scale studies in many ecologically and evolutionarily interesting species. However, these studies have been hampered by the difficulty of comparing-on a common array platform-gene-expression profiles across species due to sequence divergence altering the dynamics of hybridization. All too often, comparisons of expression profiles across species were limited to contrasting lists of gene or even of just functional categories. Here we review these issues and propose a novel solution. Exploiting the diverse cichlid lineages of East Africa as our model-system, we then present results from an experimental case study that compares the neural gene-expression profiles of males and females of two species that differ in mating system. Using a single microarray platform that contains genes from one species, Astatotilapia burtoni, we conducted a total of 16 direct comparisons for neural gene-expression level between individual males and females from a pair of sister species, the polygynous Enantiopus melanogenys and the monogamous Xenotilapia flavipinnis. Next, we conducted a meta-analysis with previously published data from two different intra-specific expression studies to determine whether sex-specific neural gene expression is more closely associated with behavioral phenotype than it is with gonadal sex. Our results indicate that the gene expression profiles are species-specific to a large extent, as relatively few genes show conserved expression patterns associated with either sex. Finally, we describe how competitive genomic DNA hybridizations between the two focal species allow us to assess the degree to which divergence of sequences biases the results. We propose a masking technique that correlates interspecific expression ratios obtained with cDNA with hybridization ratios obtained with genomic DNA for the same set of species and determines threshold sequence divergence to reduce false positives. Our approach should be applicable to a wide range of interesting questions related to the evolution and ecology of gene expression.

Comparative gene expression profiles for highly similar aggressive phenotypes in male and female cichlid fishes (Julidochromis)

“…The 45 sexbiased genes consistent between the two species are much fewer than the genes that were found to be phenotype specific (see below). Similarly, a recent study of monogamous and polygynous cichlids found that most differences in gene expression were both sex and species specific, with few genes showing sex-specific gene expression across species (Machado et al, 2009). In a previous review on the topic, Ellegren and Parsch (Ellegren and Parsch, 2007) reported that many of the genes that show sex-specific expression within species are those that are most sequence diverged between species, suggesting functional and therefore likely expression divergence.…”

“…In each hybridization, equal quantities of Cy3 and Cy5 samples (1.5-2.5g) were combined in hybridization buffer (Ambion SlideHyb buffer #1) and applied to the second-generation cDNA array (GEO platform ID: GLP6416) designed for the African cichlid A. burtoni (Renn et al, 2004;Salzburger et al, 2008), which contains 20,000 features, representing ~16,000 unique sequences (including 35% anonymous features) derived from brain, skin and mixed organ libraries from individuals of all ages, sexes and phenotypes, and has been validated for a variety of cichlid species (Renn et al, 2004;Aubin-Horth et al, 2007;Machado et al, 2009). Cross-species comparative genomic DNA hybridization for these Julidochromis species identified fewer than 10 microarray features that are substantially influenced by sequence or copy number differences between these species (data not shown).…”

“…The development of reliable methods to globally assay gene expression levels and associate these gene expression patterns with behavior has revolutionized our understanding of the relationship between gene expression and complex behaviors such as aggression (Edwards et al, 2006), addiction (Lewohl et al, 2000;Rhodes and Crabbe, 2005;Buitenhuis et al, 2009), maternal care (Gammie et al, 2007), social behavior (Whitfield et al, 2003) (reviewed by Robinson et al, 2008), and even mate choice (Cummings et al, 2008). Among African cichlids, microarray techniques have been used to examine the basis of a variety of behaviors including dominance and subordinate behavior in male Astatotilapia burtoni , various phenotypes in Neolamprologus pulcher breeding groups (AubinHorth et al, 2007) and different mating strategies in species from the Ectodini clade (Machado et al, 2009). Combined, those studies have identified a number of genes whose expression strongly correlates with aggressive behavior or with sex, thus demonstrating that certain sex-specific behaviors in cichlids are accompanied by differences in gene expression.…”

“…Microarrays can be used to ask these comparative questions but become less useful for comparing phenotypes between species as genetic divergence increases (Machado et al, 2009;Renn et al, 2004;Cohen et al, 2007). In closely related species, microarrays have been successfully used to investigate whether differences in phenotype are reflected by changes in phenotype-specific gene expression.…”

Genomics of adaptation and speciation in cichlid fishes: recent advances and analyses in African and Neotropical lineages

“…The genetic basis of this behavioural variability and plasticity has recently been a focus of research using genomic and transcriptomic methods. There may be considerable sex-specific and speciesspecific gene regulation associated with breeding systems such as monogamy or polygyny [63]. In the polygynous mating system of A. burtoni, social dominance and therefore reproductive potential is associated with differences in gonad size, growth, hormone levels and coloration.…”