Comparative genomics using fugu: a tool for the identification of conserved vertebrate cis-regulatory elements

With the imminent completion of the whole genome sequence of humans, increasing attention is being focused on the annotation of cis-regulatory elements in the human genome. Comparative genomics approaches based on evolutionary conservation have proved useful in the detection of conserved cis-regulatory elements. The pufferfish, Fugu rubripes, is an attractive vertebrate model for comparative genomics, by virtue of its compact genome and maximal phylogenetic distance from mammals. Fugu has lost a large proportion of nonessential DNA, and retained single orthologs for many duplicate genes that arose in the fish lineage. Non-coding sequences conserved between fugu and mammals have been shown to be functional cis-regulatory elements. Thus, fugu is a model fish genome of choice for discovering evolutionarily conserved regulatory elements in the human genome. Such evolutionarily conserved elements are likely to be shared by all vertebrates, and related to regulatory interactions fundamental to all vertebrates. The functions of these conserved vertebrate elements can be rapidly assayed in mammalian cell lines or in transgenic systems such as zebrafish/medaka and Xenopus, followed by validation of crucial elements in transgenic rodents.     

Fugu and human sequence comparison identifies novel human genes and conserved non-coding sequences

The compact genome of the pufferfish, Fugu rubripes, has been proposed as a 'reference' genome to aid in annotating and analysing the human genome. We have annotated and compared 85 kb of Fugu sequence containing 17 genes with its homologous loci in the human draft genome and identified three 'novel' human genes that were missed or incompletely predicted by the previous gene prediction methods. Two of the novel genes contain zinc finger domains and are designated ZNF366 and ZNF367. They map to human chromosomes 5q13.2 and 9q22.32, respectively. The third novel gene, designated C9orf21, maps to chromosome 9q22.32. This gene is unique to vertebrates, and the protein encoded by it does not contain any known domains. We could not find human homologs for two Fugu genes, a novel chemokine gene and a kinase gene. These genes are either specific to teleosts or lost in the human lineage. The Fugu-human comparison identified several conserved non-coding sequences in the promoter and intronic regions. These sequences, conserved during 450 million years of vertebrate evolution, are likely to be involved in gene regulation. The 85 kb Fugu locus is dispersed over four human loci, occupying about 1.5 Mb. Contiguity is conserved in the human genome between six out of 16 Fugu gene pairs. These contiguous chromosomal segments should share a common evolutionary history dating back to the common ancestor of mammals and teleosts. We propose contiguity as strong evidence to identify orthologous genes in distant organisms. This study confirms the utility of the Fugu as a supplementary tool to uncover and confirm novel genes and putative gene regulatory regions in the human genome.     

Genomic organization of the Hoxa4-Hoxa10 region from Morone saxatilis: implications for Hox gene evolution among vertebrates.

The physical mapping of Hox gene clusters from a limited number of vertebrates has shown an overall conservation in gene organization in which major evolutionary changes appear to be primarily restricted to the deletion of one or more genes, with the exception of the amplification of additional clusters as postulated from zebrafish. We have sequenced a 31 kb region of the HoxA cluster from the teleost Morone saxatilis (striped bass), both to provide a detailed physical map of this region and to better understand the nature of Hox cluster evolution among vertebrate taxa. We identified five linked Hox genes: Hoxa4, Hoxa5, Hoxa7, Hoxa9, and Hoxa10, which are organized similarly to those of other vertebrates. Furthermore, we have documented the absence of the Hoxa6 and Hoxa8 genes within the 31 kb contig. Comparison of our results to those published for other vertebrates suggests that the absence of Hoxa6 is a common characteristic of teleosts, whereas the absence of Hoxa8 is common to vertebrates in general, with the possible exception of zebrafish. Further comparisons between the HoxA genes from Morone with those from the pufferfish, Fugu rubripes, revealed the likely presence of a previously unreported Hoxa7 gene, or gene fragment, in the Fugu genome, which suggests that the Hoxa7 gene, unlike Hoxa6 or Hoxa8, is present in teleosts. In addition to these differences in vertebrate Hox cluster structure, we also observed a marked reduction in the length of the Hoxa4--a10 region between vertebrate lineages representative of teleosts and mammals. Comparative analysis of HoxA cluster organization among teleosts and mammals suggests that cluster length reduction and lineage-specific gene loss events are hallmarks of Hox cluster evolution.     

The nicotinic acetylcholine receptor gene family of the pufferfish,Fugu rubripes

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission at nerve-muscle junctions and in the brain. However, the complete gene family of nAChRs has not so far been reported for any vertebrate organism. We have identified the complete nAChR gene family from the reference genome of the pufferfish, Fugu rubripes. It consists of 16 alpha and 12 non-alpha candidate subunits, making it the largest vertebrate nAChR gene family known to date. The gene family includes an unusual set of muscle-like nAChR subunits comprising two alpha1s, two beta1s, one delta, one epsilon, and one gamma. One of the beta1 subunits possesses an aspartate residue and N-glycosylation sites hitherto shown to be necessary for delta-subunit function. Potential Fugu orthologs of neuronal nAChR subunits alpha2-4, alpha6, and beta2-4 have been identified. Interestingly, the Fugu alpha5 counterpart appears to be a non-alpha subunit. Fugu possesses an expanded set of alpha7-9-like subunits and no alpha10 ortholog has been found. Two new candidate beta subtypes, designated beta5 and beta6, may represent subunits yet to be found in the human genome. The Fugu nAChR gene structures are considerably more diverse than those of higher vertebrates, with evidence of "intron gain" in many cases. We show, using RT-PCR, that the Fugu nAChR subunits are expressed in a variety of tissues.     

Characterization of the pufferfish Otx2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification

The Otx2 gene, containing a highly conserved paired-type homeobox, plays a pivotal role in the development of the rostral head throughout vertebrates. Precise regulation of the temporal and spatial expression of Otx2 is likely to be crucial for proper head specification. However, regulatory mechanisms of Otx2 expression remain largely unknown. In this study, the Otx2 genome of the puffer fish Fugu rubripes, which has been proposed as a model vertebrate owing to its highly compact genome, was cloned. Consistently, Fugu Otx2 possesses introns threefold smaller in size than those of the mouse Otx2 gene. Otx2 mRNA was transcribed after MBT, and expressed in the rostral head region throughout the segmentation and pharyngula periods of wild-type Fugu embryos. To elucidate regulatory mechanisms of Otx2 expression, the expression of Otx2-lacZ reporter genes nearly covering the Fugu Otx2 locus, from -30.5 to +38.5 kb, was analyzed, by generating transgenic mice. Subsequently, seven independent cis-regulators were identified over an expanse of 60 kb; these regulators are involved in the mediation of spatiotemporally distinct subdomains of Otx2 expression. Additionally, these expression domains appear to coincide with local signaling centers and developing sense organs. Interestingly, most domains do not overlap with one another, which implies that cis-regulators for redundant expression may be abolished exclusively in the pufferfish so as to reduce its genome size. Moreover, these cis-regions were also able to direct expression in zebrafish embryos equivalent to that observed in transgenic mice. Further comparative sequence analysis of mouse and pufferfish intergenic regions revealed eight highly conserved elements within these cis-regulators. Therefore, we propose that, in vertebrate evolution, the Otx2 promoter acquires multiple, spatiotemporally specific cis-regulators in order to precisely control highly coordinated processes in head development.     

The G protein-coupled receptors in the pufferfish <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

Background

Guanine protein-coupled receptors (GPCRs) constitute a eukaryotic transmembrane protein family and function as “molecular switches” in the second messenger cascades and are found in all organisms between yeast and humans. They form the single, biggest drug-target family due to their versatility of action and their role in several physiological functions, being active players in detecting the presence of light, a variety of smells and tastes, amino acids, nucleotides, lipids, chemicals etc. in the environment of the cell. Comparative genomic studies on model organisms provide information on target receptors in humans and their function. The Japanese teleost Fugu has been identified as one of the smallest vertebrate genomes and a compact model to study the human genome, owing to the great similarity in its gene repertoire with that of human and other vertebrates. Thus the characterization of the GPCRs of Fugu would provide insights to the evolution of the vertebrate genome.

Results

We classified the GPCRs in the Fugu genome and our analysis of its 316 membrane-bound receptors, available on the public databases as well as from literature, detected 298 GPCRs that were grouped into five main families according to the GRAFS classification system (namely, Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin). We also identified 18 other GPCRs that could not be grouped under the GRAFS family and hence were classified as ‘Other 7TM’ receptors. On comparison of the GPCR information from the Fugu genome with those in the human and chicken genomes, we detected 96.83% (306/316) and 96.51% (305/316) orthology in GPCRs among the Fugu-human genomes and Fugu-chicken genomes, respectively.

Conclusions

This study reveals the position of pisces in vertebrate evolution from the GPCR perspective. Fugu can act as a reference model for the human genome for other protein families as well, going by the high orthology observed for GPCRs between Fugu and human. The evolutionary comparison of GPCR sequences between key vertebrate classes of mammals, birds and fish will help in identifying key functional residues and motifs so as to fill in the blanks in the evolution of GPCRs in vertebrates.

     

The G protein-coupled receptors in the pufferfish Takifugu rubripes

Background  

Guanine protein-coupled receptors (GPCRs) constitute a eukaryotic transmembrane protein family and function as “molecular switches” in the second messenger cascades and are found in all organisms between yeast and humans. They form the single, biggest drug-target family due to their versatility of action and their role in several physiological functions, being active players in detecting the presence of light, a variety of smells and tastes, amino acids, nucleotides, lipids, chemicals etc. in the environment of the cell. Comparative genomic studies on model organisms provide information on target receptors in humans and their function. The Japanese teleost Fugu has been identified as one of the smallest vertebrate genomes and a compact model to study the human genome, owing to the great similarity in its gene repertoire with that of human and other vertebrates. Thus the characterization of the GPCRs of Fugu would provide insights to the evolution of the vertebrate genome.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

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.     

Comparative analysis of a 229-kb medaka genomic region, containing the zic1 and zic4 genes, with Fugu, human, and mouse

Medaka is one of the prominent model animals, which also include other fishes such as Fugu and zebrafish. Its genome is relatively compact but has not been well characterized. Here we have sequenced a 229-kb region of medaka, containing the Double anal fin (Da) locus, and compared its structure to those in Fugu, human, and mouse. This region, representing a gene-poor region, contains no major rearrangements and can be readily compared among different species. Comparison of G+C contents and repeats suggested that medaka and Fugu are highly related as expected and that medaka is more similar to mammals than Fugu is. Sequence comparisons of developmental genes zic1 and zic4, identified within this region, revealed that zic1, but not zic4, is highly conserved among vertebrates. The 5' coding region of zic4 is, however, extremely homologous among fishes with little synonymous substitutions, implying its distinct function in fish.     

The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis

The G-protein coupled cannabinoid receptors CB(1) and CB(2) are activated by Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of cannabis, and mediate physiological effects of endogenous cannabinoids ('endocannabinoids'). CB(1) genes have been identified in mammals, birds, amphibians and fish, whilst CB(2) genes have been identified in mammals and in the puffer fish Fugu rubripes. Therefore, both CB(1) and CB(2) receptors probably occur throughout the vertebrates. However, cannabinoid receptor genes have yet to be identified in any invertebrate species and the evolutionary origin of cannabinoid receptors is unknown. Here we report the identification of CiCBR, a G-protein coupled receptor in a deuterostomian invertebrate - the urochordate Ciona intestinalis - that is orthologous to vertebrate cannabinoid receptors. The CiCBR cDNA encodes a protein with a predicted length (423 amino-acids) that is the intermediate of human CB(1) (472 amino-acids) and human CB(2) (360-amino-acid) receptors. Interestingly, the protein-coding region of the CiCBR gene is interrupted by seven introns, unlike in vertebrate cannabinoid receptor genes where the protein-coding region is typically intronless. Phylogenetic analysis revealed that CiCBR forms a clade with vertebrate cannabinoid receptors but is positioned outside the CB(1) and CB(2) clades of a phylogenetic tree, indicating that the common ancestor of CiCBR and vertebrate cannabinoid receptors predates a gene (genome) duplication event that gave rise to CB(1)- and CB(2)-type receptors in vertebrates. Importantly, the discovery of CiCBR and the absence of orthologues of CiCBR in protostomian invertebrates such as Drosophila melanogaster and Caenorhabditis elegans indicate that the ancestor of vertebrate CB(1) and CB(2) cannabinoid receptors originated in a deuterostomian invertebrate.     

Genomic structure and evolutionary conservation of the tyrosinase gene family from Fugu

The tyrosinase gene family encompasses three members, tyrosinase, tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct), which encode for proteins implicated in melanin synthesis. In human and mouse, genomic organization is known for all three genes, revealing common features of regulatory elements and of exon/intron structure. We have set out to identify the complete family from a more primitive vertebrate, the pufferfish Fugu (Takifugu rubripes), which is characterized by a compact genome. We had recently isolated and characterized the Fugu tyrosinase gene (Genesis 28 (2000) 99-105). We now report the isolation and characterization of the two other members of the family, Tyrp1 and Dct. Regulatory sequences from these genes function in mouse pigment cells and are able to mediate reporter gene expression. Our results demonstrate the existence of all three tyrosinase family members in teleosts and underline the evolutionary conservation of the pigmentary system.     

A degenerate ParaHox gene cluster in a degenerate vertebrate

The ParaHox genes consist of 3 homeobox gene families, Gsx, Xlox, and Cdx, all of which have fundamental roles in development. Xlox (known as IPF1 or PDX1 in vertebrates), for example, is crucial for development of the vertebrate pancreas and is also involved in regulation of insulin expression. The invertebrate amphioxus has a gene cluster containing one gene from each of the gene families, whereas in all vertebrates examined to date there are additional copies resultant from ParaHox gene cluster duplications at the base of the vertebrate lineage. Extant vertebrates basal to bony and cartilaginous fish are central to the question of when and how these multiple genes arose in the vertebrate genome. Here, we report the mapping of a ParaHox gene cluster in 2 species of hagfishes. Unexpectedly, these basal vertebrates have lost a functional Xlox gene from this cluster, unlike every other vertebrate examined to date. Furthermore, our phylogenetic analyses suggest that hagfishes may have diverged from the vertebrate lineage before the duplications, which created the multiple ParaHox clusters in jawed vertebrates.     

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal β-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.     

Evolution of the genetic machinery of the visual cycle: a novelty of the vertebrate eye?

The discovery in invertebrates of ciliary photoreceptor cells and ciliary (c)-opsins established that at least two of the three elements that characterize the vertebrate photoreceptor system were already present before vertebrate evolution. However, the origin of the third element, a series of biochemical reactions known as the "retinoid cycle," remained uncertain. To understand the evolution of the retinoid cycle, I have searched for the genetic machinery of the cycle in invertebrate genomes, with special emphasis on the cephalochordate amphioxus. Amphioxus is closely related to vertebrates, has a fairly prototypical genome, and possesses ciliary photoreceptor cells and c-opsins. Phylogenetic and structural analyses of the amphioxus sequences related with the vertebrate machinery do not support a function of amphioxus proteins in chromophore regeneration but suggest that the genetic machinery of the retinoid cycle arose in vertebrates due to duplications of ancestral nonvisual genes. These results favor the hypothesis that the retinoid cycle machinery was a functional innovation of the primitive vertebrate eye.     

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Identification and characterisation of five novel miniature inverted-repeat transposable elements (MITEs) in amphioxus (Branchiostoma floridae)

As the sister group to vertebrates, amphioxus is consistently used as a model of genome evolution for understanding the invertebrate/vertebrate transition. The amphioxus genome has not undergone massive duplications like those in the vertebrates or disruptive rearrangements like in the genome of Ciona, a urochordate, making it an ideal evolutionary model. Transposable elements have been linked to many genomic evolutionary changes including increased genome size, modified gene expression, massive gene rearrangements, and possibly intron evolution. Despite their importance in genome evolution, few previous examples of transposable elements have been identified in amphioxus. We report five novel Miniature Inverted-repeat Transposable Elements (MITEs) identified by an analysis of amphioxus DNA sequence, which we have named LanceleTn-1, LanceleTn-2, LanceleTn-3a, LanceleTn-3b and LanceleTn-4. Several of the LanceleTn elements were identified in the amphioxus ParaHox cluster, and we suggest these have had important implications for the evolution of this highly conserved gene cluster. The estimated high copy numbers of these elements implies that MITEs are probably the most abundant type of mobile element in amphioxus, and are thus likely to have been of fundamental importance in shaping the evolution of the amphioxus genome.