David A. Garfield

Ultrasensitive proteome analysis using paramagnetic bead technology.

In order to obtain a systems‐level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity‐limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single‐Pot Solid‐Phase‐enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub‐microgram amounts of material. These data present a first view of developmental stage‐specific proteome dynamics in Drosophila at a single‐embryo resolution, permitting characterization of inter‐individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.

Divergence Between the Drosophila pseudoobscura and D. persimilis Genome Sequences in Relation to Chromosomal Inversions

As whole-genome sequence assemblies accumulate, a challenge is to determine how these can be used to address fundamental evolutionary questions, such as inferring the process of speciation. Here, we use the sequence assemblies of Drosophila pseudoobscura and D. persimilis to test hypotheses regarding divergence with gene flow. We observe low differentiation between the two genome sequences in pericentromeric and peritelomeric regions. We interpret this result as primarily a remnant of the correlation between levels of variation and local recombination rate observed within populations. However, we also observe lower differentiation far from the fixed chromosomal inversions distinguishing these species and greater differentiation within and near these inversions. This finding is consistent with models suggesting that chromosomal inversions facilitate species divergence despite interspecies gene flow. We also document heterogeneity among the inverted regions in their degree of differentiation, suggesting temporal differences in the origin of each inverted region consistent with the inversions arising during a process of divergence with gene flow. While this study provides insights into the speciation process using two single-genome sequences, it was informed by lower throughput but more rigorous examinations of polymorphism and divergence. This reliance highlights the need for complementary genomic and population genetic approaches for tackling fundamental evolutionary questions such as speciation.

Genetic evidence reveals temporal change in hybridization patterns in a wild baboon population

The process and consequences of hybridization are of interest to evolutionary biologists because of the importance of hybridization in understanding reproductive isolation, speciation, and the influence of introgression on population genetic structure. Recent studies of hybridization have been enhanced by the advent of sensitive, genetic marker‐based techniques for inferring the degree of admixture occurring within individuals. Here we present a genetic marker‐based analysis of hybridization in a large‐bodied, long‐lived mammal over multiple generations. We analysed patterns of hybridization between yellow baboons (Papio cynocephalus) and anubis baboons (Papio anubis) in a well‐studied natural population in Amboseli National Park, Kenya, using genetic samples from 450 individuals born over the last 36 years. We assigned genetic hybrid scores based on genotypes at 14 microsatellite loci using the clustering algorithm implemented in structure 2.0, and assessed the robustness of these scores by comparison to pedigree information and through simulation. The genetic hybrid scores showed generally good agreement with previous morphological assessments of hybridity, but suggest that genetic methods may be more sensitive for identification of low levels of hybridity. The results of our analysis indicate that the proportion of hybrids in the Amboseli population has grown over time, but that the average proportion of anubis ancestry within hybrids is gradually decreasing. We argue that these patterns are probably a result of both selective and nonselective processes, including differences in the timing of life‐history events for hybrid males relative to yellow baboon males, and stochasticity in long‐distance dispersal from the source anubis population into Amboseli.

Shadow Enhancers Are Pervasive Features of Developmental Regulatory Networks

Summary Embryogenesis is remarkably robust to segregating mutations and environmental variation; under a range of conditions, embryos of a given species develop into stereotypically patterned organisms. Such robustness is thought to be conferred, in part, through elements within regulatory networks that perform similar, redundant tasks. Redundant enhancers (or “shadow” enhancers), for example, can confer precision and robustness to gene expression, at least at individual, well-studied loci. However, the extent to which enhancer redundancy exists and can thereby have a major impact on developmental robustness remains unknown. Here, we systematically assessed this, identifying over 1,000 predicted shadow enhancers during Drosophila mesoderm development. The activity of 23 elements, associated with five genes, was examined in transgenic embryos, while natural structural variation among individuals was used to assess their ability to buffer against genetic variation. Our results reveal three clear properties of enhancer redundancy within developmental systems. First, it is much more pervasive than previously anticipated, with 64% of loci examined having shadow enhancers. Their spatial redundancy is often partial in nature, while the non-overlapping function may explain why these enhancers are maintained within a population. Second, over 70% of loci do not follow the simple situation of having only two shadow enhancers—often there are three (rols), four (CadN and ade5), or five (Traf1), at least one of which can be deleted with no obvious phenotypic effects. Third, although shadow enhancers can buffer variation, patterns of segregating variation suggest that they play a more complex role in development than generally considered.

Genetics of gene expression responses to temperature stress in a sea urchin gene network

Stress responses play an important role in shaping species distributions and robustness to climate change. We investigated how stress responses alter the contribution of additive genetic variation to gene expression during development of the purple sea urchin, Strongylocentrotus purpuratus, under increased temperatures that model realistic climate change scenarios. We first measured gene expression responses in the embryos by RNA‐seq to characterize molecular signatures of mild, chronic temperature stress in an unbiased manner. We found that an increase from 12 to 18 °C caused widespread alterations in gene expression including in genes involved in protein folding, RNA processing and development. To understand the quantitative genetic architecture of this response, we then focused on a well‐characterized gene network involved in endomesoderm and ectoderm specification. Using a breeding design with wild‐caught individuals, we measured genetic and gene–environment interaction effects on 72 genes within this network. We found genetic or maternal effects in 33 of these genes and that the genetic effects were correlated in the network. Fourteen network genes also responded to higher temperatures, but we found no significant genotype–environment interactions in any of the genes. This absence may be owing to an effective buffering of the temperature perturbations within the network. In support of this hypothesis, perturbations to regulatory genes did not affect the expression of the genes that they regulate. Together, these results provide novel insights into the relationship between environmental change and developmental evolution and suggest that climate change may not expose large amounts of cryptic genetic variation to selection in this species.

The Impact of Gene Expression Variation on the Robustness and Evolvability of a Developmental Gene Regulatory Network

Changes in the nature of gene interactions during development help explain the robustness of early development and the basis for developmental evolution.

Whole-Genome Positive Selection and Habitat-Driven Evolution in a Shallow and a Deep-Sea Urchin

Comparisons of genomic sequence between divergent species can provide insight into the action of natural selection across many distinct classes of proteins. Here, we examine the extent of positive selection as a function of tissue-specific and stage-specific gene expression in two closely-related sea urchins, the shallow-water Strongylocentrotus purpuratus and the deep-sea Allocentrotus fragilis, which have diverged greatly in their adult but not larval habitats. Genes that are expressed specifically in adult somatic tissue have significantly higher dN/dS ratios than the genome-wide average, whereas those in larvae are indistinguishable from the genome-wide average. Testis-specific genes have the highest dN/dS values, whereas ovary-specific have the lowest. Branch-site models involving the outgroup S. franciscanus indicate greater selection (ωFG) along the A. fragilis branch than along the S. purpuratus branch. The A. fragilis branch also shows a higher proportion of genes under positive selection, including those involved in skeletal development, endocytosis, and sulfur metabolism. Both lineages are approximately equal in enrichment for positive selection of genes involved in immunity, development, and cell–cell communication. The branch-site models further suggest that adult-specific genes have experienced greater positive selection than those expressed in larvae and that ovary-specific genes are more conserved (i.e., experienced greater negative selection) than those expressed specifically in adult somatic tissues and testis. Our results chart the patterns of protein change that have occurred after habitat divergence in these two species and show that the developmental or functional context in which a gene acts can play an important role in how divergent species adapt to new environments.

Genetic variants regulating expression levels and isoform diversity during embryogenesis

Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown. Here we assessed the effect of genetic variation on transcriptional (expression levels) and post-transcriptional (3′ RNA processing) regulation across multiple stages of metazoan development, using 80 inbred Drosophila wild isolates, identifying thousands of developmental-stage-specific and shared QTL. Given the small blocks of linkage disequilibrium in Drosophila, we obtain near base-pair resolution, resolving causal mutations in developmental enhancers, validated transcription-factor-binding sites and RNA motifs. This fine-grain mapping uncovered extensive allelic interactions within enhancers that have opposite effects, thereby buffering their impact on enhancer activity. QTL affecting 3′ RNA processing identify new functional motifs leading to transcript isoform diversity and changes in the lengths of 3′ untranslated regions. These results highlight how developmental stage influences the effects of genetic variation and uncover multiple mechanisms that regulate and buffer expression variation during embryogenesis.

Population genetics of cis-regulatory sequences that operate during embryonic development in the sea urchin Strongylocentrotus purpuratus.

Despite the fact that noncoding sequences comprise a substantial fraction of functional sites within all genomes, the evolutionary mechanisms that operate on genetic variation within regulatory elements remain poorly understood. In this study, we examine the population genetics of the core, upstream cis‐regulatory regions of eight genes (AN, CyIIa, CyIIIa, Endo16, FoxB, HE, SM30 a, and SM50) that function during the early development of the purple sea urchin, Strongylocentrotus purpuratus. Quantitative and qualitative measures of segregating variation are not conspicuously different between cis‐regulatory and closely linked “proxy neutral” noncoding regions containing no known functional sites. Length and compound mutations are common in noncoding sequences; conventional descriptive statistics ignore such mutations, under‐representing true genetic variation by approximately 28% for these loci in this population. Patterns of variation in the cis‐regulatory regions of six of the genes examined (CyIIa, CyIIIa, Endo16, FoxB, AN, and HE) are consistent with directional selection. Genetic variation within annotated transcription factor binding sites is comparable to, and frequently greater than, that of surrounding sequences. Comparisons of two paralog pairs (CyIIa/CyIIIa and AN/HE) suggest that distinct evolutionary processes have operated on their cis‐regulatory regions following gene duplication. Together, these analyses provide a detailed view of the evolutionary mechanisms operating on noncoding sequences within a natural population, and underscore how little is known about how these processes operate on cis‐regulatory sequences.

The Evolution of Gene Regulatory Interactions

Changes in the timing and level at which genes are expressed are known to play an important role in evolution, but the mechanisms underlying changes in gene expression remain relatively obscure. Until quite recently, evolutionary biologists, like most biologists, tended to study single genes as isolated entities. These studies have added enormously to our understanding of biological evolution. But because gene regulation by its very nature involves interactions between two (or more) genes, researchers have missed a range of evolutionary phenomena that can be observed only at the level of networks of interacting genes. In this article, we consider the change in perspective that genomic technologies—particularly the advent of large-scale platforms for DNA sequencing, genotyping, and measuring gene expression—are bringing to evolutionary biology. We focus specifically on how these technologies can and are being used to increase our understanding of how and why gene expression evolves.