Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role. Animal development is highly stereotypic in the face of changing environmental conditions and individual genetic differences. At the same time, developmental processes can evolve rapidly, suggesting that selectable genetic variation is hidden beneath this apparent stability. To better understand the relationship between these seemingly opposed properties of robustness and evolvability, we measured how natural variation in gene expression propagates across a network of interacting genes underlying early development in sea urchins. We found that gene interactions are not equal across development: expression variation is well buffered during early development by the use of on/off switch-like (rather than continuously tunable) regulatory mechanisms, while during later development it has a greater impact on the expression of downstream target genes and on morphology. Using a breeding design, we were able to detect a substantial genetic component to the observed variation in gene expression. Interestingly, the degree of genetic contribution was greatest during early development and specifically at points of switch-like regulation, suggesting that the properties of developmental gene regulatory networks that underlie robustness also promote the accumulation of genetic variation that could seed adaptation.

The ecologically significant shift in developmental strategy from planktotrophic (feeding) to lecithotrophic (nonfeeding) development in the sea urchin genus Heliocidaris is one of the most comprehensively studied life history transitions in any animal. Although the evolution of lecithotrophy involved substantial changes to larval development and morphology, it is not known to what extent changes in gene expression underlie the developmental differences between species, nor do we understand how these changes evolved within the context of the well-defined gene regulatory network (GRN) underlying sea urchin development. To address these questions, we used RNA-seq to measure expression dynamics across development in three species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph H. tuberculata, and an outgroup planktotroph Lytechinus variegatus. Using well-established statistical methods, we developed a novel framework for identifying, quantifying, and polarizing evolutionary changes in gene expression profiles across the transcriptome and within the GRN. We found that major changes in gene expression profiles were more numerous during the evolution of lecithotrophy than during the persistence of planktotrophy, and that genes with derived expression profiles in the lecithotroph displayed specific characteristics as a group that are consistent with the dramatically altered developmental program in this species. Compared to the transcriptome, changes in gene expression profiles within the GRN were even more pronounced in the lecithotroph. We found evidence for conservation and likely divergence of particular GRN regulatory interactions in the lecithotroph, as well as significant changes in the expression of genes with known roles in larval skeletogenesis. We further use coexpression analysis to identify genes of unknown function that may contribute to both conserved and derived developmental traits between species. Collectively, our results indicate that distinct evolutionary processes operate on gene expression during periods of life history conservation and periods of life history divergence, and that this contrast is even more pronounced within the GRN than across the transcriptome as a whole. An important challenge in both evolutionary and developmental biology is understanding the genetic and molecular basis for the differences in phenotype that we observe between species. Although changes to regulatory interactions during development often play a critical role in this process, we lack detailed examples of how these differences evolve within the context of developmental gene regulatory networks (GRNs) and across the broader transcriptome as a whole. To better understand how evolutionary changes in gene regulation contribute to phenotypic differences between species, we developed a novel analytical framework to compare gene expression profiles between sea urchin species that exhibit either planktotrophic (feeding) or lecithotrophic (nonfeeding, yolk-dependent) development. We found that the shift from planktotrophy to lecithotrophy was accompanied by substantial changes to underlying gene expression profiles, and that this contrast was even more pronounced within the well-defined developmental GRN in sea urchins than across the transcriptome as a whole. Our analyses also identify cases of expression change in key regulatory genes with known developmental roles and further highlight novel candidates that may underlie derived traits in the lecithotrophic species. The comparative framework that we develop and model here is a valuable tool for identifying evolutionary changes in gene expression between species and can be readily implemented in other systems.

Background. Parrots belong to a group of behaviorally advanced vertebrates and have an advanced ability of vocal learning relative to other vocal-learni