Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray

Abstract

Background: Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits.

Results: Here we present a microarray constructed with approximately 4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes). Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species. We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes). Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (approximately 65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time).

Conclusion: This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available. Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms.

Figure 1

The core reference set for brain up-regulated gene expression. Venn diagram of spots up regulated in brain for two individual A. burtoni. Statistical analysis (see methods) of four replicates, including two dye-swaps for each animal predicts significant regulation (p < 0.05). The intersection of these results represents the core reference set.

Figure 2

Detection of hybridization and regulation across phylogenetic distance. Heterologous hybridization of RNA from A. burtoni and seven other teleost species. The Y-axis defines the number of spots that hybridized above background (circles) for each hybridization experiment as well as the number of spots that showed significant (p < 0.05) up-regulation (bars) in brain RNA compared to mixed muscle RNA for each species. Colour coding of species is consistent throughout the manuscript

Figure 3

The detected magnitude of fold change decreases with phylogenetic distance. For all spots on the array, the fold change value from a combined analysis of all A. burtoni hybridizations was correlated independently with the fold change determined for each of the other species. The Y-axis denotes the slope of each regression, with circles colour coded as in previous figures.

Figure 4

Detection of biologically meaningful gene regulation. (3A) Core reference spots showing significant up-regulation in the brain decreases with phylogenetic distance. All 804 spots were examined in each species. The Y-axis depicts the percentage of the core reference set spots that were identified as significantly up-regulated in the brain of each species. Venn diagrams depict the relationship of identified spots from the core reference set as re-identified in each species. The number of A. burtoni reference set spots that are also significantly up-regulated in the brains of (3B) other cichlids and (3C) distantly related fish are shown in the appropriately represented area. 108 and 302 spots are not re-identified in the cichlids or distantly related fish respectively (not shown).

Figure 5

Concordant detection of regulation. All clones (vertical columns) of genes (outlined by brackets) that show > 75% concordant regulation in A. burtoni are represented. Each row represents the species used in this study (colour-coded as in Figure 1). Filled boxes represent ESTs significantly up-regulated (p < 0.05) in the brain. Numbers indicate the number of clones in each EST cluster.

Figure 6

Spots of low fold change are under represented in heterologous hybridization.The 804 reference set spots are divided into 6 classes according to fold change (> 6.0 n = 41; 5.0 – 6.0 n = 24; 4.0 – 5.0 n = 85; 3.0 – 4.0 n = 128; 2.0 – 3.0 n = 305 and 1.0 – 2.0 n = 221). For each fold change class (x-axis) the percentage of spots in that class (y-axis) that are also identified (p < 0.05) in other species are depicted by colour coded symbols. The under representation of low fold change classes is most dramatic for more distantly related fish (note salmon, green and zebrafish, yellow).