Screening of the genomic sequence database of Fugu rubripes with the so far known zebrafish "Hairy and Enhancer of split"-related (Her) proteins by the TBLASTN program 23 revealed 20 h/E(spl)/hey-related genes in Fugu (table 1, see additional file 1, additional file 2 and additional file 3. A BLASTN search with the compiled genes against the available cDNA database ftp://ftp.hgmp.mrc.ac.uk/pub/fugu/fugu_cdna.zip gave no matches, indicating that the dataset (3142 entries) is too small. All assembled sequences showed conserved exon-intron boundaries and no unexpected stop-codons, suggesting that the genes are functional. In general, the Fugu proteins were named according to their overall similarity to the zebrafish Her proteins. To reveal the relationships within the bHLH proteins of Fugu rubripes the sequences were aligned (fig. 1) and a corresponding tree was calculated (fig. 2A). The Fugu proteins could be clearly divided into the three classes Enhancer of split type (3 members, fig. 2A: orange box), Hey type (5 members, fig. 2A: light red box) and Hairy type (12 members, fig. 2A: bluegreen, light green, dark green and violet boxes).

In more detail: Deduced from the intronless genomic structure, which is indicative for E(spl) genes in Drosophila melanogaster24, FrHer2 (T002603/1) and both Her4 proteins (T002603/2, T002289) were identified as Enhancer of split type proteins. Fugu her2 and one of the her4 genes are located close to each other in a head to tail orientation on contig T002603 (see additional file 2). However, FrHer2 (T002603/1) does not cluster with the other 2 Enhancer of split type proteins in the tree.

Five Fugu proteins were classified as Hey (4) or Hey-related (1) compared to only one currently known zebrafish member of that class, called Gridlock 13. Homologues for each of the hey or hey-related genes in Fugu have already been isolated in higher vertebrates 52526 or were previously annotated in the genbank database (accession number: XM_068025 for "bHLH protein similar to hhesr1/hey1"). The C-terminal consensus sequence of the Hey proteins is KPYRPWGTE(I^Hey1/V^Hey2)GAF. Focussing on this consensus, FrHey1.1 (contig T004546), FrHey1.2 (contig T000531) and FrHey2 (contig T014948) could be determined for the pufferfish (table 1 and fig. 1). Interestingly, all 3 proteins show at least one exchange to the C-terminal consensus sequence. According to the similarity in the bHLH-domain the two other proteins in contig T003476 and T005657 belong also to the Hey type proteins (fig. 1). However, like mouse HRT3 (27, accession number: AF172288) FrHey3 (contig T005657) possesses the TEIGAF motif but deviates to a higher degree from the YRPW-motif. The other protein (in contig T003476) does not possess a KPYRPWGTE(I^Hey1/V^Hey2)GAF motif at the C-terminal end. Instead a completely diverged amino acid sequence is present. Furthermore, the basic domain is somehow truncated (fig. 1, see additional file 4). Interestingly, this protein consists of 4 exons. This is in contrast to the other identified Hey-genes, which have 5 exons in human and Fugu (see table 1). Nevertheless, the human orthologue, defined as "bHLH protein similar to Hesr-1/Hey1" (accession number: XM_068025, table 1, additional file 4) shows equal aberrant features from the Hey-type genes. Therefore, we will refer to the hey-related gene in Fugu as Frhhesr1-related.

12 identified pufferfish proteins belong to the Hairy class of bHLH transcription factors that can be further subdivided into 3 branches. 5 proteins belong to the c-hairy type class compared to two genes known so far from zebrafish (fig. 2: bluegreen box). At a first glance, in the second branch at least two of three proteins seem to be the zebrafish orthologues Her1 and Her7, which are involved in somitogenesis (fig. 2: violet box). A detailed analysis of the c-Hairy type class as well as of the Her1/5/7 is given in the following sections. The third branch consists of three proteins that are related to the zebrafish Her8 (table 1, fig. 2: light green box).

Comparing both fish species, the number of h/E(spl)/hey-related genes in pufferfish is twice the number of these genes currently known from zebrafish. Evidence from Hox cluster analysis suggests that the extent of duplication in Fugu, if any, is far less extensive than in zebrafish 28. Therefore, it is likely that more h/E(spl)/hey-related genes in zebrafish exist. Deduced from the comparative tree it can be estimated that at least for hey type genes and in particular for genes of the c-hairy type class further zebrafish homologues have to be expected.

A screen with the 20 conceptually translated Fugu proteins against the human genomic sequence database yielded 10 h/E(spl)/hey-related genes (for accession numbers see Materials and Methods). These 10 genes can be further divided into 6 h/E(spl) genes, 3 hey genes (hey1, hey2, heyL, 26) and 1 hey-related gene (hhesr1, accession number: XM_068025). In Fugu, 15 h/E(spl) genes, 4 hey-genes and 1 hey-related gene were identified. Deduced from the sequence alignment of the Hey type proteins (see additional file 4), FrHey1.1 and FrHey1.2 are similar to human Hey1, FrHey2 is similar to human Hey2 and FrHey3 is similar to human HeyL. One copy of the hey-related gene exists in both genomes (hhesr1, accession number: XM_068025, and Frhhesr1). While the set of Hey- and Hey-related genes is quite comparable in both organisms, the number of h/E(spl) genes in Fugu (15) is 2.5 times higher than in the human genome (6). This indicates a duplication of those genes in Fugu or loss of members of the h/E(spl) genes in human. It is not known how these genes are expressed in Fugu but interestingly the majority of the zebrafish her genes is expressed in subsets of differentiating neuroblasts during nervous system development (101229; her8 unpublished). Since fish possess a unique set of neurons and sensory organs, compared to higher vertebrates, it might be that selective duplication of the hairy genes was one source that led to neuronal/sensory cell diversification.

The 5 c-Hairy type proteins of the Hairy class of bHLH transcription factors in Fugu rubripes can be divided into three distinct c-Hairy subclasses, namely the c-Hairy1 class and c-Hairy2 class 4 and a third novel c-Hairy class, which we will refer to as the c-Hairy-related class since no apparent orthologue from chicken is known so far. Members of the three subclasses can be distinguished by length of the amino acid sequence between the orange domain and the WRPW motif (fig. 3). FrHer9 (in contig T000078) possesses the longest amino acid sequence stretch between the two domains and therefore belongs to the c-Hairy1 class, like zebrafish Her9 12.

In FrHer6.1 (in contig T007628) and FrHer6.2 (in contig T000396) a sequence stretch of around 30–40 amino acids just N-terminal of the WRPW-motif is missing compared to c-Hairy1 class members (fig. 3). This feature is indicative for c-Hairy2 class proteins 4. Thus, FrHer6.1 and FrHer6.2 belong to the c-Hairy2 class and can be considered as Her6 homologues.

Deduced from the high conservation in the bHLH domain to the other c-Hairy proteins, FrHer10.1 (in contig T000956) and FrHer10.2 (in contig T008912) constitute a third to our knowledge novel c-Hairy class. In both proteins a sequence stretch of around 40 amino acids is missing, like in the c-Hairy2 class. In addition, a second stretch just C-terminal to the orange domain is absent compared to the already known two classes (fig. 3). Proteins of this c-Hairy type are so far not known in other vertebrates. However, these two Fugu proteins show similarity to the Hes2 proteins from mouse and human (fig. 3). But in the Hes2 proteins the number of amino acids between the orange domain and the WRPW motif is even more reduced in comparison to the c-Hairy-related proteins. To elucidate whether the class of c-Hairy-related proteins is unique in Fugu, the shotgun sequences of Tetraodon nigroviridis were screened with the 5 Fugu c-hairy type nucleotide sequences. Like in Fugu, the two c-Hairy-related class members (TnHer10.1 and TnHer10.2, TnHer10_1 and TnHer10_2 in fig. 3) and also the second member of the c-Hairy2 class were found (TnHer6.2, TnHer6_2 in fig. 3) supporting that respective proteins have to be expected in zebrafish.

One branch of the tree consists of the Fugu homologues of zebrafish her1, her5 and her7 (see fig. 2B violet box and table 1). The her1 and her7 genes in zebrafish have been shown to be cyclically expressed and are thought to play an important role in somitogenesis. Besides, they are arranged in a head to head orientation (accession number: AF292032). In contrast, Frher1 (in contig T002307) is orientated in the same manner to Frher5. Frher7 was identified on another contig (T014589). But sequence similarity does not strictly mean functional similarity. It has been shown that the specificity of the biological action in vivo of proteins of the H/E(spl) family is mediated by the orange domain 30. Sequence comparison of the orange domain of FrHer5 with zebrafish Her7 and Her5 shows that the domain from the Fugu protein is equally similar to that of the zebrafish proteins (fig. 4). Besides, FrHer5 possesses a prolin residue C-terminal of the WRPW motif, which is indicative for Her7/Hes7 proteins of zebrafish, mouse and human. Moreover, in the FrHer7 basic domain the prolin residue, which is necessary for the repressive function by binding to N-boxes 31, is missing. Instead, a histidin residue is present at this position (fig. 4). Additionally, the loop region of FrHer7 is shorter in comparison to zebrafish Her7. Length differences in the loop region are known to be responsible for functional specificity 32. All observed differences indicate that FrHer7 is not the functional equivalent of zebrafish Her7. Furthermore, the Her1, Her5 and Her7 proteins in Tetraodon show identical characteristics as the Fugu proteins (fig. 4). Conclusively, evidence from sequence analysis implies that the Fugu her1/5 gene complex is equivalent to the zebrafish her1/7 gene complex.

In the upstream sequences of the c-hairy1 and c-hairy2 class genes Frher6.1 (T007628) and Frher9 (T000078) a sequence stretch of approximately 100 nucleotides matches with the proximal part in the promoters of x-hairy2a and hhes1. The corresponding promoter parts of the orthologous genes in Tetraodon and zebrafish show identical composition, organisation and localization of the regulatory elements with respect to the 5' end of the corresponding gene (fig. 5). The fish her6 and her9 upstream sequences consist of a CCAAT-box, a TATA box and a SPS motif. It has been shown that this site is a crucial element in the regulation of the x-hairy2a gene, which is responsible for the (striped) expression in the PSM in Xenopus33. The SPS motif is a bipartite binding site for the Suppressor of hairless protein. The binding sites are separated by 30 or 29 nucleotides in the promoters of E(spl) genes of Drosophila melanogaster and higher vertebrates, respectively 3334. One of the binding sites occurs in a reverse orientation to the other. Furthermore, a hexamer motif, which lies between or within the motifs, has a functional aspect.

Although the promoters consist of identical regulatory elements, on the level of nucleotide sequence they can be divided into two groups that correspond to the c-Hairy1 and c-Hairy2 protein classes (fig. 5). In particular, this classification is even reflected in the intervening regions of the two Su(h) sites of the identified SPS motifs. Thus, the promoters of the c-hairy class genes show more similarities within their classes in different species than among themselves in the same species.

The zebrafish her1/7 gene complex was compared with the Fugu her1/5 gene complex. The intergenic region of zebrafish is 11.4 kb large (accession number: AF292032), the region of Fugu consists of 2.4 kb. Alignment of the intergenic regions of the two clusters did not show large sequence stretches of significant similarity. Since conservation of promoter sequences most of the time show functional regions the reverse must not be true 35. Therefore, we comparatively analysed the zebrafish her1/7 promoter with the upstream sequences of the Fugu her1/5 gene complex and Tetraodon her1 by MatInspector 36. First, we focused on potential Su(h) binding sites in the pufferfish promoters since Fugu and Tetraodon are closely related. Re-examination focussing on Su(h) sites that are able to constitute a SPS motif yielded one site in the intergenic region of the Fugu her1/5 gene complex and in the upstream sequence of Tetraodon her1 (table 2). The SPS sequences in the her1 promoters of both pufferfish deviate from the other sites identified so far. In both Su(h) sites of Tetraodon her1 one nucleotide exchange is present at the same position. Both SPS sequences in Fugu her1/5 and Tetraodon her1 show a distance of 32 nucleotides between the two Su(h) binding motifs (table 2). Deduced from this data we suggest that: 1. The distance between the two Su(h) sites has to be expected in the range of 29 – 32 nucleotides for fish. 2. Single exchanges to the GTGGGAA consensus might exist. Taking these parameters into account examination of the zebrafish her1/7 promoter yielded one SPS candidate sequence (table 2). The two potential Su(h) sites have a distance of 31 nucleotides and a hexamer motif. Both sites deviate from the GTGGGAA consensus. Nevertheless, for the Su(h) protein of Drosophila it has been shown, that it binds to oligonucleotide sequences with the consensus RTGRGAR 34. At least one binding site matches to this consensus. The second Su(h) site deviates in one position from the identified motifs in pufferfish. However, it has to be experimentally verified, whether the identified SPS sequences in this study are targets for transcriptional regulation. Screening with a model according to these findings gave no further SPS motifs in the upstream sequences of the other Fugu genes than already identified (see Materials and Methods).

In Xenopus, it has been shown that the SPS regulatory motif and a specific 3'UTR element are sufficient to drive mesoderm expression 33. Since the zebrafish her1 gene is exclusively expressed in the PSM, we assume that SPS sequences are specific mesoderm regulator elements in fish and presumably in vertebrates.

To detect conserved regions, the upstream sequences of the Fugu genes, for which we could identify the first exon, were compared with the "genbank" database at NCBI by using the BLASTN algorithm under low stringency conditions 23. Only in the case of the upstream sequence of Frhhesr1-related (in contig T003476) and the corresponding human, mouse and zebrafish promoters (fig. 6; see Materials and Methods for retrieval of the sequences from the different organisms) an extraordinary high degree of similarity was observed. The sequences are not conserved over the entire length. Highly conserved blocks are disrupted by diverged sequence stretches between the different species. The conserved blocks do not seem to contain any regulatory motif characterized so far. It is unlikely that the conserved regions do code for a protein. By using GENSCAN http://genes.mit.edu/GENSCAN.html one potential exon 500 bp upstream of the Start-Methionine in Fugu was identified, which is not conserved in mouse and human. However, a BLASTP search for the deduced 31 amino acid long peptide gave no significant similarities. A sorted six-frame translation with the Fugu sequence with a minimum ORF size of 50 amino acids and any Start Codon gave 18 ORFs that showed no remarkable similarities in a BLASTP search. Furthermore, a BLASTX search over the entire sequence gave no positive results. However, it cannot be excluded that so far uncharacterized miRNAs (reviewed in 37) are encoded within the conserved regions. Nevertheless, it would be worthwhile to investigate if this sequence truly represents a promoter and what function the respective protein carries out.

By using the TBLASTN program 23 the Fugu database (first release from 25.10.01) at the UK HGMP Resource Centre http://Fugu.hgmp.mrc.ac.uk/19 was screened with the 9 different Her proteins of Danio rerio. The accession numbers of the used zebrafish bHLH genes are: her1: X97329, her2: X97330, her3: X97331, her4: X97332, her5: X95301, her6: X97333, her7: AF240772, her8: AY007990, her9: AF301264. The TBLASTN results were screened manually for the occurrence of sequence motifs known to be conserved in H/E(spl)-related proteins. These motifs are the basic domain with the consensus sequence KPx(M/V/I)E(K/R)(R/K)R, the highly conserved second loop with the consensus sequence (L/V)EKA(D/E)(I/V)LE and the WRPW motif located at the C-terminal end of the proteins. For further analysis, at least two of these motifs had to occur in a reasonable distance within a contig. Because the N- and/or C-termini often were missing in the automatically annotated proteins http://www.jgi.doe.gov/fugu/index.html, a training set for identification of the exonic regions was created, which contains the following known hairy genes: Drosophila hairy: X15904, human hes1: L19314, mouse hes1: D16464, mouse hes7: AB050104, zebrafish her1 and her7: AF292032 (see additional file 5). The regions of coding sequences, which we have conceptually translated into the respective proteins of Fugu rubripes, are depicted in table 1. Sequence LGS99222 was used for identification of the FrHer6.1 C-terminus (PAAVSPGAPSGNTDSVWRPW). Sequence LGS286123 was used for identification of the FrHey1 C-terminus (WGLEIGAF). The BLASTX algorithm 38 was used to identify the closest relatives to the Fugu Her proteins in zebrafish and higher vertebrates (table 1).

For identification of the c-hairy homologues as well as her1, her5 and her7 in Tetraodon nigroviridis the corresponding Fugu sequences were used to screen the shotgun sequence database at genoscope by BLASTN http://www.genoscope.cns.fr/externe/tetraodon/Ressource.html and the trace archive http://www.ncbi.nlm.nih.gov/Traces/trace.cgi? at NCBI using megaBLAST. The Sequences, which are described here, can be retrieved at the Ensembl Trace Server http://trace.ensembl.org or at the NCBI Trace archive http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?. Sequences with the accession numbers 89970374, 89981571, 90016949, 90052606, 90066642, 90222617, 90222618, 90275465, 90275466, 90351844, 90421676, 90430225, 95723915, 95738908, 95996399, 97329806, 97457947, 97625306, 99082743, 99085687, 99124118, 99344199, 99379652, 99390932, 99545674 and 101019329 were assembled for 1 kb upstream of the first exon up to 5 kb downstream of the third exon of Tetraodon her1. The fourth exon of Tetraodon her1 was identified in sequence COAG1029AA11SP1 (reverse complement) at position 97–614. For Tetraodon her5 the sequences with the following accession numbers were assembled: C0AH037AD02SP1, C0AG530DE07SP1, C0AG873DH06LP1, C0AG1038AA11SP1, C0AG1120DA10SP1 and 90098825, 90098826, 90176607, 90411020, 90411021, 95688576, 95703158, 96052770, 97373175, 97658773, 99123371, 99159494, 99248382, 99288485, 101130421. For Tetraodon her6.1 the sequences with the following accession numbers were assembled: 89972279, 90058082, 90064995, 90119043, 90135880, 90238776, 90289277, 90444073, 90476831 and C0AG202CG02SP1, C0AG336DF02SP1, C0AG465AB08LP1, C0AG467BE05LP1, C0AG600DE09LP1, C0AG635CC05LP1, C0AG1333AE06SP1. Tetraodon her6.2 was assembled with sequences of the following accession numbers: 97704800, C0AG515DB08SP1 and C0AG1340BB02SP1. For Tetraodon her7 the sequences with the accession numbers C0BG101CF02LP1, C0AG256AH01SP1, C0AG325AF02LP1, C0AG542AG07SP1, C0AG575CF01SP1, C0AG735BC08SP1, C0AG793CF06SP1, C0AG799AD01LP1, C0AG900AF01SP1, C0AG1022CB06LP1, C0AG1034AH05SP1, C0AG1243AA03SP1, C0AG1342BE07LP1, C0AG1541DH04LP1, C0AH109BE02LP1 and C0BG045CF02SP1 were used. For Tetraodon her9 the sequences with the accession numbers C0AA019AG08C1, C0AG201DA11SP1, C0AG302BF10LP1, C0AG622DB06LP1, C0AG997DB09LP1, C0AG1070BF12SP1, C0AG1174AH07SP1, C0AG1255CB09SP1 and C0AG1350CB05LP1 were utilised. For Tetraodon her10.1 the sequences with the accession numbers C0AG132BG03LP1, C0AG214BF08LP1, C0AG471CC07SP1, C0AG852AF01LP1, C0AG1268AC03SP1, C0BG067CF10LP1, 97442563 and 100725254 were assembled. For Tetraodon her10.2 the following sequences were assembled and the reverse complement of it was analysed: 101123589, C0AG438BH03LP1, C0AG859BF07LP1, C0AH077AD07SP1, C0BG092BE02LP1 (see additional file 6).

The human genome database was screened with the 20 Fugu proteins by TBLASTN http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs. The accession numbers (contig id, protein id) in which the human H/E(spl)/Hey-related proteins were identified, are: Hes1/Hry (NT_005571, gi: 20536141, XP_039925), Hes2 (AL031848, gi: 4914512, CAB46198), Hes4 (NT_032954, gi: 20472908, NP_066993), Hes5 (NT_004350, gi: 20535853, not annotated yet, 1. exon 161111–161164 and 2. exon 161264–161803), Hes6 (NT_005120, gi: 20535565, NP_061115), Hes7 (NT_010823, gi: 20560291, NP_115969). Hey1 (NT_008209, gi: 20544348, XP_113553), Hey2 (NT_030710, gi: 20550030, NP_036391), HeyL (NT_004511, gi: 20537057, NP_055386), protein similar to Hesr-1 (NT_006169, gi: 20535686, XP_068025).

For examination of the corresponding promoter regions 2 kb upstream of the Start-Methionine were used in a BLASTN search 23 to detect conserved regulatory elements. Search for potential transcription factor binding sites was done using the MatInspector program 36. Due to bad sequence reads we were not able to identify the first exon in 4 genes (indicated with n.d. in table 1), which were therefore excluded from upstream sequence analysis.

For identification of the zebrafish her6 and her9 upstream sequences the corresponding cDNA sequences were used for a blastn search against the trace archive at NCBI. The sequences with the accession numbers 15682371 and 25608918 were assembled for the her6 promoter. The first 53 nucleotides of the published her6 cDNA sequence were found on a different sequence stretch. The sequences with the accession numbers 25425900, 98681634, 15611875, 98577137, 100352443 and 102690745 were assembled for around 1 kb upstream of her9 (see additional file 6).

For comparison of the promoter region of the Frhhesr1-related gene in contig T003476 (table 1) the corresponding human sequence (accession number: 16931048, nt 18662–20664) was used for a BLASTN search against the trace archive to obtain corresponding sequences of mouse and zebrafish. The mouse upstream region was assembled using trace archive files with the following accession numbers 34431248 nt 1–741 of 741, 20297240 nt 135–751 of 751 (reverse complement), 7193321 nt 1–628 of 705 (reverse complement) and 43361903 nt 272–942 of 942 (reverse complement). The zebrafish promoter was compiled by utilizing the following files (accession numbers): 83152916 nt 1–602 of 612 (reverse complement), 30330598 nt 191–612 of 612 and 42667125 nt 1–612 of 612. All sequences are immediate to the first ATG of the respective hhesr1-related coding sequence, except for zebrafish. Here, no sequence information about the 3'end of this promoter as well as the beginning of the coding sequence was available. Potential exonic regions in the upstream sequence of the Frhhesr1-related gene were investigated by GENSCAN http://genes.mit.edu/GENSCAN.html and potential ORFs were identified by BioEdit 39.

The SPS sequence in the upstream sequence of Frher9 (T000078) was identified at position 38130–38161. The SPS sequence in the promoter region of Frher6 (T007628) was localized at position 7320–7289 (reverse complement). The Frher1/5 gene complex was found in contig T002307 and a corresponding SPS sequence was identified at position 8677–8643 (reverse complement). Deduced from the created dataset of SPS sites, we used FASTM at Genomatix http://www.genomatix.de/cgi-bin/fastm2/fastm.pl to define a model for this motif for fish. The model consists of two binding site elements separated by 20 to 45 nucleotides (distance from start of element 1 to start of element 2). Both elements had to occur on one strand, the elements are defined as: GTGRRAR and WTYMCAC. With this dataset, we re-examined all available upstream sequences of the Fugu genes for the occurrence of SPS sites.

Alignments of amino acid sequences were done making use of the CLUSTALW algorithm 40 in the program BioEdit or by using the Pileup program of the GCG software package 4142. Phylogenetic trees based on these alignments were computed by the Neighbor-Joining method 43 with a bootstrap support of 100 replicates. For the tree calculations the program PHYLIP 44 was used. Trees were displayed using Treeview 45. The program GeneDoc was used for displaying the alignments 46. For the alignment of the c-hairy homologues the accession numbers of the compared genes are: c-hairy1: AF032966, c-hairy2: 4, mouse hes1: NM008235, x-hairy1: U36194, x-hairy2A: AF383159. For comparsion, the mouse hes2 (NM_008236) and human hes2 (CAB46198) were used.

For alignment of the upstream sequences the DIALIGN program 47 from Genomatix http://www.genomatix.de/cgi-bin/dialign/dialign.pl was used.