A Reporter Assay in Lamprey Embryos Reveals Both Functional Conservation and Elaboration of Vertebrate Enhancers

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.     

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.     

后生动物非编码保守元件

生物体基因组中除了编码序列之外, 还存在大量的非编码调控序列。比较基因组学研究发现：脊椎动物、尾索动物、头索动物、果蝇、线虫等基因组中存在保守的非编码调控序列。这些非编码保守元件通常分布在与转录调控发育相关的基因上下游区域, 作为基因调控网络核心的一部分, 常常在基因表达过程中扮演转录增强子的角色。文章总结了近年来有关后生动物非编码保守元件的发现和主要特点, 并进一步就非编码保守元件在大规模基因组倍增之后的演化及其在生物躯体图式进化过程中的影响进行了综述。     

Minor change, major difference: divergent functions of highly conserved cis-regulatory elements subsequent to whole genome duplication events

Within the vertebrate lineage, a high proportion of duplicate genes have been retained after whole genome duplication (WGD) events. It has been proposed that many of these duplicate genes became indispensable because the ancestral gene function was divided between them. In addition, novel functions may have evolved, owing to changes in cis-regulatory elements. Functional analysis of the PAX2/5/8 gene subfamily appears to support at least the first part of this hypothesis. The collective role of these genes has been widely retained, but sub-functions have been differentially partitioned between the genes in different vertebrates. Conserved non-coding elements (CNEs) represent an interesting and readily identifiable class of putative cis-regulatory elements that have been conserved from fish to mammals, an evolutionary distance of 450 million years. Within the PAX2/5/8 gene subfamily, PAX2 is associated with the highest number of CNEs. An additional WGD experienced in the teleost lineage led to two copies of pax2, each of which retained a large proportion of these CNEs. Using a reporter gene assay in zebrafish embryos, we have exploited this rich collection of regulatory elements in order to determine whether duplicate CNEs have evolved different functions. Remarkably, we find that even highly conserved sequences exhibit more functional differences than similarities. We also discover that short flanking sequences can have a profound impact on CNE function. Therefore, if CNEs are to be used as candidate enhancers for transgenic studies or for multi-species comparative analyses, it is paramount that the CNEs are accurately delineated.     

Lens development depends on a pair of highly conserved Sox21 regulatory elements

Highly conserved non-coding elements (CNEs) linked to genes involved in embryonic development have been hypothesised to correspond to cis-regulatory modules due to their ability to induce tissue-specific expression patterns. However, attempts to prove their requirement for normal development or for the correct expression of the genes they are associated with have yielded conflicting results. Here, we show that CNEs at the vertebrate Sox21 locus are crucial for Sox21 expression in the embryonic lens and that loss of Sox21 function interferes with normal lens development. Using different expression assays in zebrafish we find that two CNEs linked to Sox21 in all vertebrates contain lens enhancers and that their removal from a reporter BAC abolishes lens expression. Furthermore inhibition of Sox21 function after the injection of a sox21b morpholino into zebrafish leads to defects in lens development. These findings identify a direct link between sequence conservation and genomic function of regulatory sequences. In addition to this we provide evidence that putative Sox binding sites in one of the CNEs are essential for induction of lens expression as well as enhancer function in the CNS. Our results show that CNEs identified in pufferfish-mammal whole-genome comparisons are crucial developmental enhancers and hence essential components of gene regulatory networks underlying vertebrate embryogenesis.     

Cis-regulation and conserved non-coding elements in amphioxus

The origin of the vertebrates was a major event in the evolution of morphological diversity and the genetic mechanisms responsible for this diversity, once purely theoretical, can now be approached experimentally in the genome era. With a prototypical chordate genome, vertebrate-like development and simple morphology, amphioxus provides the appropriate model for investigating the origin of the vertebrates. Comparative genomics is revealing that both conservation and divergence of genes and cis-regulatory elements involved in developmental regulatory networks are required to shape different animal body plans. This article reviews the cis-regulatory studies performed in amphioxus, the discovery of conserved non-coding elements (CNEs) across the metazoans and the examination of amphioxus CNEs. Emerging ideas on the evolution of CNEs after large-scale genome duplication events and the state of cephalochordate genomics are also discussed.     

Ancient vertebrate conserved noncoding elements have been evolving rapidly in teleost fishes

Vertebrate genomes contain thousands of conserved noncoding elements (CNEs) that often function as tissue-specific enhancers. In this study, we have identified CNEs in human, dog, chicken, Xenopus, and four teleost fishes (zebrafish, stickleback, medaka, and fugu) using elephant shark, a cartilaginous vertebrate, as the base genome and investigated the evolution of these ancient vertebrate CNEs (aCNEs) in bony vertebrate lineages. Our analysis shows that aCNEs have been evolving at different rates in different bony vertebrate lineages. Although 78-83% of CNEs have diverged beyond recognition ("lost") in different teleost fishes, only 24% and 40% have been lost in the chicken and mammalian lineages, respectively. Relative rate tests of substitution rates in CNEs revealed that the teleost fish CNEs have been evolving at a significantly higher rate than those in other bony vertebrates. In the ray-finned fish lineage, 68% of aCNEs were lost before the divergence of the four teleosts. This implicates the "fish-specific" whole-genome duplication in the accelerated evolution and the loss of a large number of both copies of duplicated CNEs in teleost fishes. The aCNEs are rich in tissue-specific enhancers and thus many of them are likely to be evolutionarily constrained cis-regulatory elements. The rapid evolution of aCNEs might have affected the expression patterns driven by them. Transgenic zebrafish assay of some human CNE enhancers that have been lost in teleosts has indicated instances of conservation or changes in trans-acting factors between mammals and fishes.     

A systematic approach to identify functional motifs within vertebrate developmental enhancers

Uncovering the cis-regulatory logic of developmental enhancers is critical to understanding the role of non-coding DNA in development. However, it is cumbersome to identify functional motifs within enhancers, and thus few vertebrate enhancers have their core functional motifs revealed. Here we report a combined experimental and computational approach for discovering regulatory motifs in developmental enhancers. Making use of the zebrafish gene expression database, we computationally identified conserved non-coding elements (CNEs) likely to have a desired tissue-specificity based on the expression of nearby genes. Through a high throughput and robust enhancer assay, we tested the activity of ∼ 100 such CNEs and efficiently uncovered developmental enhancers with desired spatial and temporal expression patterns in the zebrafish brain. Application of de novo motif prediction algorithms on a group of forebrain enhancers identified five top-ranked motifs, all of which were experimentally validated as critical for forebrain enhancer activity. These results demonstrate a systematic approach to discover important regulatory motifs in vertebrate developmental enhancers. Moreover, this dataset provides a useful resource for further dissection of vertebrate brain development and function.     

Evolutionary growth process of highly conserved sequences in vertebrate genomes

Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.     

Impact of asymmetric gene repertoire between cyclostomes and gnathostomes

Extant vertebrates are divided into the two major groups, cyclostomes and gnathostomes (jawed vertebrates). The former includes jawless fishes, hagfishes and lampreys, and the latter includes all extant jawed vertebrates. In many research fields, the phenotypic traits of the cyclostomes have been considered crucial in understanding the evolutionary process from invertebrates to vertebrates. Recent studies have suggested that the common ancestor of the extant vertebrates including hagfishes and lampreys underwent two-round of whole genome duplications, and thus the genome expansion solely does not account for phenotypic differences between cyclostomes and gnathostomes. Emerging evidence from molecular phylogeny of individual gene families indicates that the gene repertoire expanded at the common ancestor of vertebrates were later reshaped asymmetrically between the two lineages, resulting in the retention of differential gene sets. This also confuses interpretation of conserved synteny which often serves as indicator of orthology and the ploidy level. In this review, current controversy and future perspectives of cyclostome genomics are discussed with reference to evolutionary developmental biology.     

De novo genesis of enhancers in vertebrates

Evolutionary innovation relies partially on changes in gene regulation. While a growing body of evidence demonstrates that such innovation is generated by functional changes or translocation of regulatory elements via mobile genetic elements, the de novo generation of enhancers from non-regulatory/non-mobile sequences has, to our knowledge, not previously been demonstrated. Here we show evidence for the de novo genesis of enhancers in vertebrates. For this, we took advantage of the massive gene loss following the last whole genome duplication in teleosts to systematically identify regions that have lost their coding capacity but retain sequence conservation with mammals. We found that these regions show enhancer activity while the orthologous coding regions have no regulatory activity. These results demonstrate that these enhancers have been de novo generated in fish. By revealing that minor changes in non-regulatory sequences are sufficient to generate new enhancers, our study highlights an important playground for creating new regulatory variability and evolutionary innovation.     

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.     

Regulatory genes in the ancestral chordate genomes

Changes or innovations in gene regulatory networks for the developmental program in the ancestral chordate genome appear to be a major component in the evolutionary process in which tadpole-type larvae, a unique characteristic of chordates, arose. These alterations may include new genetic interactions as well as the acquisition of new regulatory genes. Previous analyses of the Ciona genome revealed that many genes may have emerged after the divergence of the tunicate and vertebrate lineages. In this paper, we examined this possibility by examining a second non-vertebrate chordate genome. We conclude from this analysis that the ancient chordate included almost the same repertory of regulatory genes, but less redundancy than extant vertebrates, and that approximately 10% of vertebrate regulatory genes were innovated after the emergence of vertebrates. Thus, refined regulatory networks arose during vertebrate evolution mainly as preexisting regulatory genes multiplied rather than by generating new regulatory genes. The inferred regulatory gene sets of the ancestral chordate would be an important foundation for understanding how tadpole-type larvae, a unique characteristic of chordates, evolved.     

Developmental Genetics of Fishes: Isozymic Analyses of Differential Gene Expression

SYNOPSIS. The fundamental evolutionary position of the fishesand the plasticity of their gene expression render them wellsuited for developmental genetic analyses. Isozymes, encodedin related loci, have proven to be effective probes of differentialgene expression in the fishes. The advanced bony fishes exhibita higher temporal and spatial specificity of isozyme locus expressionthan the primitive fishes. Some isozyme loci are limited intheir expression to specific developmental periods such as hatching.The more phylogenetically recently derived loci have a morerestricted tissue expression, and tend to be expressed laterin development than more ancient loci exhibiting a more generalizedtissue expression. More closely related isozyme loci tend tooverlap more in their cellular expressions than do more distantlyrelated loci. The role of gene and genome relatedness in regulatinggene expression also has been studied by the investigation ofpreferential allele expression in interspecific hybrids. Theschedules of gene expression are often perturbed in interspecificfish hybrids. A progressive increase in the severity of developmentalabnormalities, at the morphological and enzymatic levels, occursduring embryogenesis of hybrids formed from progressively moredistantly related species. In most instances of allelic repression,the paternal allele is preferentially inhibited but occasionallythe maternal allele is selectively repressed. A non-reciprocaldevelopmental success is often observed for hybrid embryos derivedfrom reciprocal crosses. These aberrant nucleocytoplasmic interactionshave been related to models of gene regulation and the evolutionarydivergence of gene regulatory mechanisms.