Comprehensive characterization of the <Emphasis Type="Italic">cis</Emphasis>-regulatory code responsible for the spatio-temporal expression of <Emphasis Type="Italic">olSix3.2</Emphasis>in the developing medaka forebrain

Background  

Embryonic development is coordinated by sets of cis-regulatory elements that are collectively responsible for the precise spatio-temporal organization of regulatory gene networks. There is little information on how these elements, which are often associated with highly conserved noncoding sequences, are combined to generate precise gene expression patterns in vertebrates. To address this issue, we have focused on Six3, an important regulator of vertebrate forebrain development.     

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA.     

A new approach to quantify the adaptive potential of gene expression variation in gymnosperms

Variation in patterns of gene expression contributes to phenotypic diversity and can ultimately predict adaptive responses. However, in many cases, the consequences of regulatory mutations on patterns of gene expression and ultimately phenotypic differences remain elusive. A standard way to study the genetic architecture of expression variation in model systems has been to map gene expression variation to genetic loci (Fig. 1a). At the same time, in many nonmodel species, especially for long‐lived organisms, controlled crosses are not feasible. If we are to expand our understanding of the role of regulatory mutations on phenotypes, we need to develop new methodologies to study species under ecologically relevant conditions. In this issue of Molecular Ecology, Verta et al. ( 2013 ) present a new approach to analyse gene expression variation and regulatory networks in gymnosperms (Fig. 1b). They capitalized on the fact that gymnosperm seeds contain an energy storage tissue (the megagametophyte) that is directly derived from a single haploid cell (the megaspore). The authors identified over 800 genes for which expression segregated in this maternally inherited haploid tissue. Based on the observed segregation patterns, these genes (Mendelian Expression Traits) are most probably controlled by biallelic variants at a single locus. Most of these genes also belonged to different regulatory networks, except for one large group of 180 genes under the control of a putative trans‐acting factor. In addition, the approach developed here may also help to uncover the effect of rare recessive mutations, which usually remain hidden in a heterozygous state in diploid individuals. The appeal of the work by Verta et al. ( 2013 ) to study gene expression variation is in its simplicity, which circumvents several of the hurdles behind traditional expression quantitative trait locus (eQTL) studies, and could potentially be applied to a large number of species.     

Butterfly wings: the evolution of development of colour patterns

The diversity in colour patterns on butterfly wings provides great potential for understanding how developmental mechanisms may be modulated in the evolution of adaptive traits. In particular, we discuss concentric eyespot patterns, which have been shown by surgical experiments to be formed in response to signals from a central focus. Seasonal polyphenism shows how alternate phenotypes can develop through environmental sensitivity mediated by ecdysteroid hormones, whereas artificial selection and single gene mutants demonstrate genetic variation influencing the number, shape, size, position, and colour composition of the eyespots. The expression patterns of the regulatory gene Distal-less reveal that these changes can arise at several different developmental stages, and the phenotypes indicate that some forms of changed pattern may occur much more readily than others. Further study of the genes, of the developmental mechanisms, and of the functions of the patterns will provide novel insights about the evolution of morphological diversity. BioEssays 21:391–401, 1999. © 1999 John Wiley & Sons, Inc.     

Rare and common regulatory variation in population-scale sequenced human genomes

Population-scale genome sequencing allows the characterization of functional effects of a broad spectrum of genetic variants underlying human phenotypic variation. Here, we investigate the influence of rare and common genetic variants on gene expression patterns, using variants identified from sequencing data from the 1000 genomes project in an African and European population sample and gene expression data from lymphoblastoid cell lines. We detect comparable numbers of expression quantitative trait loci (eQTLs) when compared to genotypes obtained from HapMap 3, but as many as 80% of the top expression quantitative trait variants (eQTVs) discovered from 1000 genomes data are novel. The properties of the newly discovered variants suggest that mapping common causal regulatory variants is challenging even with full resequencing data; however, we observe significant enrichment of regulatory effects in splice-site and nonsense variants. Using RNA sequencing data, we show that 46.2% of nonsynonymous variants are differentially expressed in at least one individual in our sample, creating widespread potential for interactions between functional protein-coding and regulatory variants. We also use allele-specific expression to identify putative rare causal regulatory variants. Furthermore, we demonstrate that outlier expression values can be due to rare variant effects, and we approximate the number of such effects harboured in an individual by effect size. Our results demonstrate that integration of genomic and RNA sequencing analyses allows for the joint assessment of genome sequence and genome function.     

Integrated gene expression profiling and linkage analysis in the rat

The combined application of genome-wide expression profiling from microarray experiments with genetic linkage analysis enables the mapping of expression quantitative trait loci (eQTLs) which are primary control points for gene expression across the genome. This approach allows for the dissection of primary and secondary genetic determinants of gene expression. The cis-acting eQTLs in practice are easier to investigate than the trans-regulated eQTLs because they are under simpler genetic control and are likely to be due to sequence variants within the gene itself or its neighboring regulatory elements. These genes are therefore candidates both for variation in gene expression and for contributions to whole-body phenotypes, particularly when these are located within known and relevant physiologic QTLs. Multiple trans-acting eQTLs tend to cluster to the same genetic location, implying shared regulatory control mechanisms that may be amenable to network analysis to identify gene clusters within the same metabolic pathway. Such clusters may ultimately underlie development of individual complex, whole-body phenotypes. The combined expression and linkage approach has been applied successfully in several mammalian species, including the rat which has specific features that demonstrate its value as a model for studying complex traits.     

Identification of regulatory variants of carboxylesterase 1 (CES1): A proof-of-concept study for the application of the Allele-Specific Protein Expression (ASPE) assay in identifying cis-acting regulatory genetic polymorphisms

It is challenging to study regulatory genetic variants as gene expression is affected by both genetic polymorphisms and non-genetic regulators. The mRNA allele-specific expression (ASE) assay has been increasingly used for the study of cis-acting regulatory variants because cis-acting variants affect gene expression in an allele-specific manner. However, poor correlations between mRNA and protein expressions were observed for many genes, highlighting the importance of studying gene expression regulation at the protein level. In the present study, we conducted a proof-of-concept study to utilize a recently developed allele-specific protein expression (ASPE) assay to identify the cis-acting regulatory variants of CES1 using a large set of human liver samples. The CES1 gene encodes for carboxylesterase 1 (CES1), the most abundant hepatic hydrolase in humans. Two cis-acting regulatory variants were found to be significantly associated with CES1 ASPE, CES1 protein expression, and its catalytic activity on enalapril hydrolysis in human livers. Compared to conventional gene expression-based approaches, ASPE demonstrated an improved statistical power to detect regulatory variants with small effect sizes since allelic protein expression ratios are less prone to the influence of non-genetic regulators (e.g., diseases and inducers). This study suggests that the ASPE approach is a powerful tool for identifying cis-regulatory variants.     

Knowing left from right: the molecular basis of laterality defects

The apparent symmetry of the vertebrate body conceals profound asymmetries in the development and placement of internal organs. Asymmetric organ development is controlled in part by genes expressed asymmetrically in the early embryo, and alterations in the activities of these genes can result in severe defects during organogenesis. Recently, data from different vertebrates have allowed researchers to put forward a model of genetic interactions that explains how asymmetric patterns of gene expression in the early embryo are translated into spatial patterns of asymmetric organ development. This model helps us to understand the molecular basis of a number of congenital malformations in humans.     

Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays

cDNA microarrays containing 1443 Arabidopsis thaliana genes were analyzed for expression profiles in major organs of Arabidopsis plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.     

Molecular anatomy of the developing limb in the coquí frog,Eleutherodactylus coqui

The vertebrate limb demonstrates remarkable similarity in basic organization across phylogenetically disparate groups. To gain further insight into how this morphological similarity is maintained in different developmental contexts, we explored the molecular anatomy of size‐reduced embryos of the Puerto Rican coquí frog, Eleutherodactylus coqui. This animal demonstrates direct development, a life‐history strategy marked by rapid progression from egg to adult and absence of a free‐living, aquatic larva. Nonetheless, coquí exhibits a basal anuran limb structure, with four toes on the forelimb and five toes on the hind limb. We investigated the extent to which coquí limb bud development conforms to the model of limb development derived from amniote studies. Toward this end, we characterized dynamic patterns of expression for 13 critical patterning genes across three principle stages of limb development. As expected, most genes demonstrate expression patterns that are essentially unchanged compared to amniote species. For example, we identified an EcFgf8‐expression domain within the apical ectodermal ridge (AER). This expression pattern defines a putatively functional AER signaling domain, despite the absence of a morphological ridge in coquí embryos. However, two genes, EcMeis2 and EcAlx4, demonstrate altered domains of expression, which imply a potential shift in gene function between coquí frogs and amniote model systems. Unexpectedly, several genes thought to be critical for limb patterning in other systems, including EcFgf4, EcWnt3a, EcWnt7a, and EcGremlin, demonstrated no evident expression pattern in the limb at the three stages we analyzed. The absence of EcFgf4 and EcWnt3a expression during limb patterning is perhaps not surprising, given that neither gene is critical for proper limb development in the mouse, based on knockout and expression analyses. In contrast, absence of EcWnt7a and EcGremlin is surprising, given that expression of these molecules appears to be absolutely essential in all other model systems so far examined. Although this analysis substantiates the existence of a core set of ancient limb‐patterning molecules, which likely mediate identical functions across highly diverse vertebrate forms, it also reveals remarkable evolutionary flexibility in the genetic control of a conserved morphological pattern across evolutionary time.