BACKGROUND
in mammals the family of s <dig> calcium binding proteins has been a target of intensive study for over  <dig> years, since the first two family members were purified from bovine brain.
until today at least  <dig> members were described in humans and a growing number in several other mammalian species.
they share an average identity of 40–60% on the amino acid level, but key features are more highly conserved.
the s <dig> genes code for small, cytoplasmic or secreted proteins that exhibit a common structure consisting of two calcium-binding loops each flanked by two alpha-helices  <cit> .
the n-terminal domain is a low affinity s100-specific domain, whereas the other one is a classical ef-hand.
a variable hinge region connects both domains.
structural examination revealed a hydrophobic region being exposed upon calcium binding which is thought to interact with hydrophobic regions of the target proteins.
the calcium-binding domains also exhibit affinity for other divalent cations such as copper and zinc  <cit> .
some family members possess dysfunctional calcium-binding domains, but there is also evidence of calcium-independent interactions in this family .
for human s <dig> family members more than  <dig> potential target proteins, both intra- and extracellular ones, have been reported.
among the interacting partners are other calcium-binding proteins , enzymes , cytoskeletal components , cell cycle regulator genes , second messenger-synthesizing enzymes  as well as kinases.
accordingly, s <dig> proteins take part in many cellular processes which may be divided into five major groups: a) modulation of the activity of some protein kinases b) modulation of other enzymatic activities c) maintenance of cell shape and motility d) modulation of signal transduction pathways and e) regulation of calcium homoeostasis  <cit> .
the active forms of s <dig> proteins often consist of homo- or heterodimers and even tetramers  <cit>  or higher aggregates  <cit> .
the expression pattern of the s <dig> family exhibits a remarkable degree of cell and tissue-specificity, so that many tissues and cell types have their unique protein composition and expression level  <cit> .
there is no evidence for s <dig> proteins in invertebrates, however, the evolutionary origin of the family has not been studied much  <cit> .
by far most publications deal with the mammalian family members, and in fact no systematic study has been performed in teleosts.
with an intent to infer properties of the family in the most recent common ancestor of teleosts and tetrapods we have characterized the s <dig> family both in shark and lamprey, and in teleosts.
we identified in each teleost species thirteen to fourteen s <dig> genes, which are under strong negative selection.
several of these genes appear to be considerably more ancient than the teleost speciation events taken into account here.
several mammalian s <dig> genes were found to have counterparts in teleosts, but eight novel genes restricted to teleosts were also detected.
furthermore we have investigated the expression of ten danio rerio s <dig> genes by rt-pcr and in situ hybridization and report highly distinctive and specialized expression patterns, suggestive of similarly specialized functions of the different family members.
RESULTS
the repertoire size of the s <dig> family of thirteen to fourteen genes is remarkably conserved across six divergent teleost species
the s <dig> proteins constitute a small family of calcium-binding proteins with highly specific expression patterns in mammals.
so far the evolution of this family has not been studied in teleosts.
up to date only two s <dig> genes were reported in fish: a homologue of the mammalian s100a was found in loach and trout  <cit>  and a new family member, ictacalcin, is present in several fish species , but absent in mammals  <cit> .
these observations are consistent with the notion that the s <dig> family may be already well established in teleost fish, but may also diverge considerably from the mammalian repertoire.
we therefore performed extended searches in the genomes of five different teleost species to establish the complete s <dig> repertoire in fish.
a recursive search strategy in the ncbi and ensembl databases using the complete repertoire of human s <dig> genes as reference set, in combination with retrieval of automated ortholog predictions  uncovered a total of  <dig> fish s <dig> genes.
fourteen s <dig> genes each were identified for zebrafish and takifugu rubripes, and thirteen genes each for three-spined stickleback, medaka, and tetraodon nigroviridis.
in addition, fourteen s <dig> genes were found in est libraries of another teleost, salmo salar.
all of these genes are novel, with the exception of one zebrafish and one takifugu gene .
we believe that the result of the data mining approach used reflects the total repertoire of the s <dig> genes in the cases of danio rerio, gasterosteus aculeatus, oryzias latipes, tetraodon nigroviridis and takifugu rubripes.
in the case of salmo salar several transcripts were found for each gene predicted, so that the repertoire reported here may approximate the complete s <dig> family present in this species.
because novel fish-specific s <dig> subclades were found, it is necessary to expand the existing s <dig> nomenclature to include these new genes .
we suggest the following criteria :
<dig>  if any ortholog of the gene was described previously, that name is kept.
this is the case for a <dig>  a <dig>  a <dig>  b, p and z members of the s <dig> family, which have mammalian orthologs, and for the fish-specific s100i.
<dig>  whenever new closely related genes correspond to a single named ortholog, that name is used, together with an additional numeral or letter to distinguish the fish genes from each other.
this is the case e.g.
for a10a and a10b of the s <dig> family.
<dig>  novel fish-specific genes and subfamilies are named consecutively by so far unused letters, starting with the letter q onwards.
s <dig> family size in cartilaginous and even jawless fish may be similar to that of bony fish
we have searched the available databases for s <dig> family members in cartilaginous fish  and a jawless fish, petromyzon marinus.
we report ten different s <dig> family members in petromyzon marinus and five s <dig> genes in callorhinchus milii plus one gene in squalus acanthias .
all genes are novel, except three genes from petromyzon <cit> , see also additional file  <dig>  the reported repertoire is expected to be incomplete, especially in the case of callorhinchus milii, for which only  <dig>  fold genomic coverage is currently available, and of course squalus acanthias.
thus the total size of the cartilaginous and jawless fish s <dig> repertoires may be rather similar to that of teleost fish, despite marked sequence divergence.
the latter necessitated further rules for nomenclature:
<dig>  if a gene from a basal taxon is clearly related to two named genes of more derived species, we suggest to always use the preceding letter.
<dig>  if no clear orthologs to any named gene can be delineated, the genes are named provisionally with x <dig>  x <dig> etc.
not to foreclose a coherent nomenclature for s <dig> genes from basal taxa, which will only be possible after completion of the respective genome projects.
s <dig> is an ancient family with a complex pattern of gene gains and losses
in order to better understand the relationships and evolutionary history of the s <dig> family members we performed a phylogenetic analysis, using the neighbor-joining method  <cit> , as well as maximum parsimony and maximum likelihood analyses using the phylip package  <cit>  .
full length coding sequences of all teleost s <dig> genes were analyzed together with human s <dig> genes, using troponins, calmodulins and other related calcium binding protein with ef-hand motifs  <cit>  as outgroup .
this phylogenetic analysis shows twenty-seven different groups of orthologs, i.e.
<dig> different genes as the combined repertoire analyzed here .
six of these genes are organized in closely related gene pairs .
the delineation of the s <dig> gene family, the identification of ortholog groups and the association in gene pairs were supported by all three tree-building algorithms used .
nearly a third of the family members are present only in fish – s100i and seven novel proteins, s100q, s100r, s100s, s100t, s100u, s100v, and s100w.
remarkably, very few of these fish-specific genes have the full set of five orthologs out of five fish genomes analyzed.
when salmon is taken into account, only a single gene, s100w, is found in all  <dig> teleost species.
in four other genes one ortholog is missing, the tetraodon nigroviridis gene in s100i, the oryzias latipes ortholog in s100u, and the s100s, s100t genes in salmo salar.
only four and three species have representatives of the s100v and s100q genes, respectively.
finally, subfamily s100r is only found in pufferfish .
on the other hand, restricted duplication events have occurred for s100i, s100v  and s <dig> u .
all teleost-restricted genes that contain both a zebrafish and another teleost representative  should have been present at least in the most recent common ancestor  of neoteleostei and ostariophysii .
thus, the partial absence of several genes, e.g.
s100i in pufferfish  suggests a partial loss of these genes in the pufferfish family  although it cannot be ruled out with certainty that some apparent gene losses are actually caused by inadequacies of the currently available databases.
other examples for restricted loss in some teleost species concern s100q, u, v and the gene pair s100s/t .
two of these teleost-specific s <dig> genes  were not detected in the zebrafish genome despite extensive searches.
the most parsimonious explanation therefore is an absence of these genes in the mrca of neoteleostei and ostariophysii, i.e.
a genesis of these genes in the neoteleostei lineage .
six other genes are shared by teleost fish and mammalian species .
thus, all six genes should be more ancient than the split in actinopterygii and sarcopterygii, the separate teleost and tetrapod lineages.
four genes  are highly conserved among all six teleost species, with at least one ortholog per species being present.
however, other genes, particularly s100p, exhibit the same partial representation in teleost species as described above for some fish-specific s <dig> genes.
thus these genes appear to have been lost in some teleost sublineages.
a graphical representation of all these phylogenetic inferences is given in figure  <dig>
four of the six s <dig> genes found in the shark genome also belong to this group of genes common to teleosts and mammals .
one is an s100z ortholog, two are s100b orthologs, and one  is situated at the root of the s100a1-s100z clade .
thus, genes s100a <dig>  s100b, and s100z appear to have emerged before the divergence of cartilaginous fishes from bony fish.
since the currently available shark genome is bound to be incomplete, it may be expected that about half of the extant s <dig> genes already existed in the mrca of cartilaginous and bony fish.
moreover, the phylogenetic position of all cartilaginous fish s <dig> genes deep within the teleost phylogenetic tree demonstrates that the s <dig> family was already well established before the segregation of bony and cartilaginous fish .
in fact, the s <dig> family even precedes the earlier split in jawed/jawless vertebrates, because already ten family members were found in lamprey .
however, with possible exception of two genes related to s100b and one related to s100v, no ortholog relationship is apparent between any of the petromyzon marinus s <dig> genes and those from more derived species.
thus the phylogenetic tree reflects a complex evolutionary pattern characterized by frequent gene losses as well as extended gains of either single genes or whole gene subfamilies.
similar changes in the gene repertoire caused by complex genomic reorganization and translocation events are reported for ef hand proteins in general  <cit> .
moderate overall similarity but high degree of conservation of motifs that are characteristic for the s <dig> family
the evolutionary distances between ortholog amino acid sequences range between  <dig>  and  <dig>  , whereas paralog distances range between  <dig>  and  <dig>  .
because of this high heterogeneity among the s <dig> family and to obtain a second line of evidence in order to classify these genes reliably as members of the s <dig> family, we next examined the degree of conservation of the fish and mammalian genes.
for that purpose, sequence logos from the amino acid alignments of either the fish or mammalian s <dig> proteins alone and of a combination of all the s <dig> proteins were created and used for a comparative analysis .
all three sequence logos were very similar and showed the same general conservation pattern.
the previously described typical structure of the mammalian s <dig> proteins formed by four helices  separated by a s <dig> ef hand calcium-binding domain, a hinge and a canonical ef hand calcium-binding domain  <cit>  is conserved among the fish s <dig> proteins.
a high number of conserved residues and signature motifs in defined positions of the sequence were identified in the teleost s <dig> proteins  and found to be conserved also among fish and tetrapods, consistent with the origin of the s <dig> family well before the actinopterygian/sarcopterygian split .
these residues are located mainly in the helix domains and the calcium-binding regions, especially in the canonical ef hand.
this result is in accordance with a recent study that classifies this region as phylogenetically older than the s <dig> ef hand domains  <cit> .
in contrast, the hinge region and the c-terminus seem to be regions where there is more space for sequence variability .
this is in accordance with the observation in mammalian s <dig> proteins that the selectivity in target binding is mainly assured by the hinge region and the c-terminal tail  <cit> , i.e.
in these regions a larger divergence is expected at least between paralogs.
among the highly conserved amino acids are, as expected, the calcium-coordinating residues of the ef hands, cf.
<cit>  and several hydrophobic residues involved in dimerization  and binding to target proteins   <cit> .
a conserved cysteine close to the end of helix iv  has been shown in mammalian s100a <dig> and a <dig> to stabilize the dimer by intermolecular disulfide bridge formation  <cit> .
this cysteine is part of a conserved hydrophobic pattern f--l---l---c---f of helix iv , which has the proper spacing to form the contact surface for dimerization.
similar conserved patterns of  <dig> to  <dig> amino acids are observed for the other three helices .
most ef hands found in the teleost genes appear to be functional as judged by sequence comparison, with exception of three proteins, s100r, u, v, and some neoteleost members of the s100b subfamily.
the s100r protein has lost key residues of the canonical calcium-binding motif in the second ef hand loop, despite a generally good conservation in both domains, similar to the human s100a <dig> and a <dig> proteins  <cit> .
the s100v protein has lost most key residues of the canonical motif  <cit> .
in four of the five neoteleost s100b proteins a common three amino acid deletion has removed a key residue of the s100-specific calcium-binding loop.
nearly the same deletion also occurs in the s100v and the s100u proteins, as well as in the human s100a <dig>  a <dig>  the medaka s100a10a, and the shark cms100b <dig>  x <dig> .
the frequency of this deletion suggests a specialized function for such genes, which might act either as competitive antagonists, or alternatively, may adopt the calcium-bound conformation constitutively.
indeed, the latter has been demonstrated for the human a <dig> protein  <cit> .
s <dig> genes with such deletions do not segregate together in the phylogenetic tree , and the deletions vary slightly in length  and position .
thus, this kind of mutation has arisen several times independently.
some of these deletion events must have occurred in the mrca of cartilaginous and bony fish , others appear to have originated in the mrca of teleosts , and some are evolutionary late events restricted to a single species .
in other words, this particular deletion may be considered a local optimum in the state space of s <dig> sequences, consistent with a particular function for these genes.
strong negative selection for s <dig> genes, but virtual absence of positive selection
as previously mentioned, paralog evolutionary distance at the amino acid level is very high, with an average value of  <dig> , and a range from  <dig> – <dig>  .
such high values pinpoint the high variability in the amino acid sequences of these genes found at the intraspecies level.
in contrast, the average ortholog evolutionary distance value of  <dig>   is about  <dig>  fold lower than the one observed for the paralog comparisons .
prompted by both the high degree of intraspecies variability and the high degree of interspecies conservation of s <dig> genes we decided to investigate the evolutionary constraints that are acting on this gene family.
a strong constraint is equivalent to strong negative selection.
the degree of selective pressure is measured by investigating the relative frequency of non-synonymous  vs synonymous  substitutions  <cit> .
when the number of dn equals the number of ds, the dn/ds ratio equals  <dig>  which corresponds to neutral selection.
if the number of non-synonymous changes is higher than the number of synonymous changes, then dn/ds > <dig>  which indicates positive selection.
on the other hand, if the number of synonymous  changes is higher than the number of nonsynonymous changes, then dn/ds < <dig> and we are in the presence of negative selection  <cit> .
whenever possible, the overall dn/ds ratio within each ortholog group was determined.
all the groups show very small values, ranging from  <dig>  to  <dig> , with an average global dn/ds value of  <dig>  , which is evidence for strong negative selection.
these low dn/ds values combined with the high divergence between the s <dig> genes suggest not only an ancient origin of these genes but also indicate that individual genes of this family are slowly evolving.
however, as the strong negative selection could be masking some positive selection events at the individual codon level, we also determined the dn/ds ratio separately for each individual sequence position.
results are shown for two of the ortholog groups, the s100i and s100z.
these two clades represent two different evolutionary scenarios, the first showing evidence for recent gene duplications in zebrafish and lacking one ortholog in takifugu rubripes, while the second is a very well conserved group with orthologs in all six fish species.
also they show two different values for their evolutionary distances, of  <dig>  and  <dig>  respectively .
as predicted by the global dn/ds analysis, a considerable portion of the total sequence – 25% for the s100i and 35% for s100z – is under negative selection, with the second value being closer to the average value of  <dig> sites per ortholog group .
no evidence for positive selection was found, suggesting that positive selection does not appear to play a significant role in the evolution of the s <dig> genes.
a comparison between s100i and s100z shows rough similarity in the pattern of negative selection , although no specific motifs could be identified between genes .
gene structure of s <dig> genes is highly conserved between fish and mammalian species
the mammalian s <dig> genes are found to have a common structure consisting of usually three exons  <cit> .
the first exon contains only 5'utr, part of the second and third exons code for the orf and a 3'utr is located in the last portion of the third exon  <cit> .
for the fish s <dig> genes we restricted analysis to the coding exons, since prediction of noncoding utrs is currently not very reliable.
we found that  <dig> of the analyzed  <dig> teleost s <dig> genes conform to the pattern observed in ortholog genes in humans.
however, another seven teleost fish genes have their orf split in three exons, one of them in most cases very small and always located at one end of the orf .
for zebrafish several, but not all, of these predicted exons have been confirmed by rt-pcr .
each major exon contains one complete ef-hand domain.
without exception these two exons are joined at the same position in the sequence .
thus these exons may be as old as the s <dig> family itself and the first such gene may have arisen from a duplication of this domain.
in contrast, the intron/exon borders of the small exons  show a variable position in the n-terminal and c-terminal regions and occur in few species only, consistent with a late origin in the teleost lineage.
s <dig> fish genes are distributed both as singletons and small clusters in the genome
all mammalian s100a genes are clustered in the genome, whereas s100b, g, p, and z are located as singletons on different chromosomes  <cit> .
in zebrafish the scenario is somewhat similar, with two smaller clusters of  <dig>  and  <dig>  mb each located on the chromosomes  <dig> and  <dig>  respectively, and composed of seven  and four  genes, respectively.
similarly, in the other teleost fish species analyzed small clusters of maximally five genes are found .
these clusters always contain phylogenetically less related genes and, in contrast to the situation in mammals, cluster gene associations differ between species.
however, and remarkably so, there are two s <dig> gene pairs, which are stably linked in the teleost genome, whenever present in the same species, albeit not closely related phylogenetically .
conversely, a closely related gene pair  consists of a singleton gene  and a member of a cluster .
thus, among teleost s <dig> genes, genomic linkage is not at all correlated with degree of phylogenetic relationship, consistent with an ancient evolutionary origin of these clusters.
in total, three zebrafish genes, s100b, s100v <dig> and s100z, are present as singletons in the chromosomes  <dig>   <dig>  and  <dig>  respectively .
this feature is conserved across all five teleost species studied; additionally s100p, which is absent in zebrafish, is always a singleton, whenever it occurs .
the isolated genomic location of these family members appears to be an evolutionary ancient feature of the s <dig> family, since all members of this group also found in mammals  share the singleton location.
the two clusters present in zebrafish are situated on chromosomes  <dig> and  <dig>  which are largely syntenic  <cit>  and result from the teleost-specific whole genome duplication, which occurred after the actinopterygian/sarcopterygian split  <cit> .
two of the three gene pairs observed in the teleost s <dig> repertoire  are distributed between these two clusters , consistent with a duplication of their ancestral genes by this whole genome duplication nearly  <dig> million years ago.
this interpretation is supported by the observation that the gene pair a10a/b has a single mammalian ortholog.
moreover, very similar clusters are present in chromosomes  <dig> and  <dig> from tetraodon nigroviridis and chromosomes  <dig> and  <dig> from oryzias latipes .
both chromosome pairs are syntenic to each other, and to zebrafish chromosomes  <dig> and  <dig>  <cit> .
the third recognizable gene pair in teleosts  is shared both with mammals and with cartilaginous fish, but a <dig> is always a member of a cluster , whereas z never is, consistent with a very ancient origin of this gene duplication, before the emergence of the cluster itself.
in total, three genes from the human s100a cluster on chromosome  <dig>  a <dig>  a <dig> and a <dig> are found in the teleost clusters.
together with the synteny of this region of human chromosome  <dig> with the corresponding teleost cluster regions  <cit>  these data are consistent with a small ancestral cluster of s <dig> genes in the mrca of teleosts and tetrapods plus some isolated ancestral s <dig> genes.
in contrast, other gene pairs occur closely linked in the chromosome , indicating a local tandem duplication event as cause of such gene pairs.
expression patterns of s <dig> genes range from highly specific to fairly broad distributions
to obtain an overview of the tissue specificity of expression for the whole family we performed rt-pcr with twelve different tissues from adult zebrafish.
for a higher spatial resolution of the expression patterns at the cellular level we performed whole mount in situ hybridization of zebrafish larvae  <dig> days post fertilization.
at this stage zebrafish have completed organogenesis and major behavioral patterns are already functional.
eight of ten genes analyzed show expression in the larval stage, with expression patterns ranging from highly restricted to spatially broad distributions .
the results of the in situ hybridizations are often consistent with those from the rt-pcr .
a general tendency of the rt-pcr to show broader expression may be related to the higher sensitivity of that method compared to in situ hybridization.
additionally, a developmental regulation, i.e.
a late onset of expression, may explain some of the differences in the results, especially the absence of two s <dig> transcripts  in larval tissues .
for s100b a late onset of expression was confirmed by the detection of strong signals in adult brain .
highly restricted expression of s <dig> genes within the sensory nervous system
three zebrafish s <dig> genes show a remarkably restricted expression in just one or two different larval neuronal populations.
zebrafish s100s is expressed in the neuromasts of the lateral line and the inner ear .
labeled structures in the inner ear may include hair cells .
in sections of the tail region the central portion of the neuromasts is labeled , consistent with an expression in sensory hair cells.
thus in the larvae particular sensory structures and possibly some neurons innervating them are labeled.
the rt-pcr results  show a general expression in all sensory systems examined which are in detail barbels and lips, brain, eyes, olfactory system and additionally the genitourinary system.
the in situ expression pattern of s100s is quite similar to that of the highly related zebrafish s100t, which is also expressed in the neuromasts of the lateral line and the inner ear , the latter in lower levels compared to s100s.
zebrafish develop approximately 9– <dig> neuromasts along one side  <cit> , which have their counterparts on the opposite side and all of them are stained by either probe.
in rt-pcr s100t expression is additionally observed in non-sensory tissue such as bone, gills, heart, liver and muscles.
zebrafish s100z  is the gene with the spatially most restricted expression pattern in the larvae.
only a single tissue expresses s100z, the olfactory placode, and within that tissue expression is restricted to a few large cells , by morphology presumably neurons.
a far broader expression pattern in adult fish is suggested by the rt-pcr results and may suggest a late ontogenetic onset of expression in those tissues.
the related s100a <dig> was not detected at all in larval tissues, but shows an expression in the adult genitourinary system, skin, olfactory epithelium and brain according to rt-pcr-results.
non-neuronal expression of two s <dig> genes, restricted to a single cell type or tissue
zebrafish s100a10a shows one of the most restricted patterns .
expression is restricted to the intestinal bulb and the intestine with intestinal loops being clearly visible.
expression slightly decreases from intestinal bulb to comparatively low levels in the anal region.
rt-pcr was negative, consistent with the described specificity .
the expression is limited to the epithelial cells of the intestine and gut .
zebrafish s100a <dig> exhibits an extremely specific in situ hybridization pattern .
s100a <dig> seems to be restricted to isolated large cells located on the yolk sac and the pericardial cavity of the fish.
there are about  <dig> cells labeled in the whole larva.
rt-pcr with adult tissues suggests expression in gills and olfactory epithelium.
the cellular morphology, larval staining pattern and the adult expression in gills suggest expression in mr-cells, also known as ionocytes  <cit> .
these cells control ionic composition of body fluids, especially calcium levels.
results obtained by rt-pcr indicate a much broader distribution, possibly at levels below the threshold for in situ hybridization.
three s <dig> genes exhibit broad expression in several tissues and cell types
zebrafish s100i.
<dig>  expression  was previously described by  <cit>  and  <cit> .
our data are consistent with their findings of expression in the epithelial cells of barbels, gills, olfactory rosettes and skin.
s100i.
<dig> is expressed throughout the whole skin with high levels of expression in the rim of the dorsal  and pectoral  fin.
high expression levels were observed in the urogenital opening .
moreover, s100i.
<dig> exhibits also strong expression in cells of the olfactory placode .
additionally expression can be observed throughout the pharyngeal arches  and the lateral line excluding the neuromasts , which appear as light spots in the middle of the larvae.
in this respect s100i.
<dig> shows the complementary pattern to s100s, t, which label the neuromasts, but not the surrounding tissue.
the in situ hybridization signals may represent the combined expression pattern of s100i.
<dig> and s100i.
<dig>  since their probes share over 90% homology at the nucleotide level, and thus cross-reactivity cannot be excluded.
however, rt-pcr results for s100i.
<dig> are expected to be specific, since a primer from its unique, short exon was used.
rt-pcr results for s100i.
<dig> probably represent a mix of both s100i transcripts, since no completely specific primers were possible there.
the results show expression of both genes in all tissues tested, with exception of muscle, where only s100i.
<dig> could be detected.
zebrafish s100a10b  exhibits high expression in the lower jaw  in particular in the branchial arches.
epithelial cells are labeled, but cartilaginous tissue is devoid of staining.
olfactory placode and the whole larval trunk do not express s100a10b.
thus this expression pattern is a subset of that observed for s100i.
<dig>  unexpectedly, rt-pcr reveals expression of s100a10b in every tissue examined.
partial correlation of expression pattern similarity with coding sequence similarity
the highly related genes s100s and s100t show a nearly identical larval expression pattern, and the duplicate s100i genes appear to exhibit a very similar expression pattern as well.
however, expression patterns for the duplicate a10a genes are very different, with the a10b pattern a subset of the unrelated s100i.
<dig>  i.
<dig>  and a completely different pattern being observed for a10a.
this partial correlation is consistent with the interpretation that in some cases  regulatory elements have been conserved alongside the coding regions of these genes, which are spread far apart in the genome.
additionally, for the two s100i genes, which are clustered, regulatory elements shared within the cluster might synchronize the expression pattern.
in this context it is noteworthy that the close genomic linkage of s100i to s100a10b is conserved in the genome of all fish species that possess both genes, and indeed, a10b expression is quite similar to that of s100i , consistent with partially shared regulatory elements.
on the other hand, a <dig> and s are also linked closely, and their expression patterns are completely different.
thus regulation of s <dig> gene expression follows a complex pattern presumably involving several different mechanisms as suggested for humans, cf.
<cit> .
discussion and
CONCLUSIONS
the s <dig> family represents the largest family of calcium-binding proteins with ef hands.
s <dig> proteins have multiple and essential functions in many different tissues, mainly in cells of epithelial origin.
however, most studies have been performed in mammals, and the evolutionary emergence of the family is not clear.
we have performed an extensive search in six teleost genomes and identified in total fourteen distinct subfamilies, which constitute the fish s <dig> gene repertoire.
individual species exhibit thirteen to fourteen different s <dig> genes.
upon closer investigation a complex pattern of gene gain and gene loss emerges, which results in a roughly stable total repertoire size for each species.
a similar family size is observed in amphibians , whereas a preponderance of gene gains leads to an enlarged s <dig> repertoire size in mammals .
the presence of several s <dig> family members from cartilaginous fish  deep inside the phylogenetic tree implies that the s <dig> family predates at least the segregation of bony from cartilaginous fish.
moreover, already ten members of the s <dig> family have been identified in a jawless primitive fish .
thus the origin of the family may lie with very early vertebrate stages although individual family members only become reliably recognizable in the time period between the jawed/jawless segregation and the divergence of cartilaginous from bony fish around  <dig> to  <dig> million years ago  <cit> .
the somewhat variable composition of each species repertoire suggests that gene function may be variable between species.
in fact, an a11-like gene from xenopus  <cit>  is expressed in olfactory receptor neurons, i.e.
may have acquired the function served in zebrafish by s100z.
such flexibility is observed irrespective of a generally strong negative selection within the individual s <dig> genes, i.e.
the evolutionary changes within the regulatory regions may be much faster than those within the coding regions.
on the other hand, some functional specializations appear quite stable, which may be inferred from the expression of human s100a <dig> in polarized epithelia  <cit>  parallel to the expression of the zebrafish s100a10a in the intestinal epithelium.
however, it should be noted that human a <dig> is constitutionally in the, calcium-bound' configuration due to a deletion in the s <dig> ef hand, in contrast to the zebrafish a <dig> genes.
moreover, the other member of this gene pair  shows a radically different distribution.
another example is s100b, which is expressed in brain in zebrafish as well as in mammalian species  <cit> .
the genomic structure of teleost s <dig> genes is markedly similar to that of mammalian genes, with two to three coding exons in similar distances, and a conserved domain structure of one ef hand per each main exon.
some similarities are also observed for the genomic location.
most mammalian s <dig> genes lie together with some other genes in one large cluster, the so-called epidermal differentiation complex   <cit>  for which common regulatory elements have been discussed .
two smaller clusters of four to seven members in teleosts are syntenic to the human cluster and thus suggest the presence of an ancestral small cluster of s <dig> genes.
teleost cluster configuration does not correlate with sequence similarity, in contrast to the mammalian edc.
thus the teleost clusters may represent the ancestral cluster composition more closely, while extended local gene duplications appear to have enlarged the mammalian cluster.
while the predominantly epithelial and epithelial-derived cellular expression of mammalian s <dig> genes is maintained in zebrafish, the actual regulatory mechanisms responsible for this similar expression may be different.
no evidence for shared regulatory elements is observed in teleost fish, as judged from both sequence divergence and expression pattern similarity of neighboring genes .
zebrafish s <dig> genes exhibit highly specific and characteristically different patterns of expression like their mammalian counterparts .
duplicated genes show both redundancy and divergence in their expression pattern.
larval expression is limited to different types of epithelial cells and neural cells of epithelial origin .
our findings provide a firm basis for unraveling the molecular nature of the s100-like immunoreactivity observed in teleost fish tissues in several earlier publications, e.g.
<cit> .
taken together we have identified and characterized the teleost s <dig> gene family with respect to gene structure and expression pattern.
these genes exhibit characteristics similar to the mammalian gene family in several respects.
considering the importance of s <dig> genes in the pathogenesis of epidermal disease – particular s <dig> proteins are markedly over expressed in psoriasis, wound healing, skin cancer, inflammation, cellular stress, and other epidermal pathological states  <cit>  – it appears advantageous to study these processes in the zebrafish model system .

 in mammals the family of s calcium binding proteins has been a target of intensive study for over years, since the first two family members were purified from bovine brain. until today at least members were described in humans and a growing number in several other mammalian species. they share an average identity of 40–60% on the amino acid level, but key features are more highly conserved. the s genes code for small, cytoplasmic or secreted proteins that exhibit a common structure consisting of two calcium-binding loops each flanked by two alpha-helices. the n-terminal domain is a low affinity s100-specific domain, whereas the other one is a classical ef-hand. a variable hinge region connects both domains. structural examination revealed a hydrophobic region being exposed upon calcium binding which is thought to interact with hydrophobic regions of the target proteins. the calcium-binding domains also exhibit affinity for other divalent cations such as copper and zinc. some family members possess dysfunctional calcium-binding domains, but there is also evidence of calcium-independent interactions in this family. for human s family members more than potential target proteins, both intra- and extracellular ones, have been reported. among the interacting partners are other calcium-binding proteins, enzymes, cytoskeletal components, cell cycle regulator genes, second messenger-synthesizing enzymes as well as kinases. accordingly, s proteins take part in many cellular processes which may be divided into five major groups: a) modulation of the activity of some protein kinases b) modulation of other enzymatic activities c) maintenance of cell shape and motility d) modulation of signal transduction pathways and e) regulation of calcium homoeostasis. the active forms of s proteins often consist of homo- or heterodimers and even tetramers or higher aggregates. the expression pattern of the s family exhibits a remarkable degree of cell and tissue-specificity, so that many tissues and cell types have their unique protein composition and expression level. there is no evidence for s proteins in invertebrates, however, the evolutionary origin of the family has not been studied much. by far most publications deal with the mammalian family members, and in fact no systematic study has been performed in teleosts. with an intent to infer properties of the family in the most recent common ancestor of teleosts and tetrapods we have characterized the s family both in shark and lamprey, and in teleosts. we identified in each teleost species thirteen to fourteen s genes, which are under strong negative selection. several of these genes appear to be considerably more ancient than the teleost speciation events taken into account here. several mammalian s genes were found to have counterparts in teleosts, but eight novel genes restricted to teleosts were also detected. furthermore we have investigated the expression of ten danio rerio s genes by rt-pcr and in situ hybridization and report highly distinctive and specialized expression patterns, suggestive of similarly specialized functions of the different family members.  the repertoire size of the s family of thirteen to fourteen genes is remarkably conserved across six divergent teleost species the s proteins constitute a small family of calcium-binding proteins with highly specific expression patterns in mammals. so far the evolution of this family has not been studied in teleosts. up to date only two s genes were reported in fish: a homologue of the mammalian s100a was found in loach and trout and a new family member, ictacalcin, is present in several fish species, but absent in mammals. these observations are consistent with the notion that the s family may be already well established in teleost fish, but may also diverge considerably from the mammalian repertoire. we therefore performed extended searches in the genomes of five different teleost species to establish the complete s repertoire in fish. a recursive search strategy in the ncbi and ensembl databases using the complete repertoire of human s genes as reference set, in combination with retrieval of automated ortholog predictions uncovered a total of fish s genes. fourteen s genes each were identified for zebrafish and takifugu rubripes, and thirteen genes each for three-spined stickleback, medaka, and tetraodon nigroviridis. in addition, fourteen s genes were found in est libraries of another teleost, salmo salar. all of these genes are novel, with the exception of one zebrafish and one takifugu gene. we believe that the result of the data mining approach used reflects the total repertoire of the s genes in the cases of danio rerio, gasterosteus aculeatus, oryzias latipes, tetraodon nigroviridis and takifugu rubripes. in the case of salmo salar several transcripts were found for each gene predicted, so that the repertoire reported here may approximate the complete s family present in this species. because novel fish-specific s subclades were found, it is necessary to expand the existing s nomenclature to include these new genes. we suggest the following criteria: if any ortholog of the gene was described previously, that name is kept. this is the case for a a a b, p and z members of the s family, which have mammalian orthologs, and for the fish-specific s100i. whenever new closely related genes correspond to a single named ortholog, that name is used, together with an additional numeral or letter to distinguish the fish genes from each other. this is the case e.g. for a10a and a10b of the s family. novel fish-specific genes and subfamilies are named consecutively by so far unused letters, starting with the letter q onwards. s family size in cartilaginous and even jawless fish may be similar to that of bony fish we have searched the available databases for s family members in cartilaginous fish and a jawless fish, petromyzon marinus. we report ten different s family members in petromyzon marinus and five s genes in callorhinchus milii plus one gene in squalus acanthias. all genes are novel, except three genes from petromyzon, see also additional file the reported repertoire is expected to be incomplete, especially in the case of callorhinchus milii, for which only fold genomic coverage is currently available, and of course squalus acanthias. thus the total size of the cartilaginous and jawless fish s repertoires may be rather similar to that of teleost fish, despite marked sequence divergence. the latter necessitated further rules for nomenclature: if a gene from a basal taxon is clearly related to two named genes of more derived species, we suggest to always use the preceding letter. if no clear orthologs to any named gene can be delineated, the genes are named provisionally with x x etc. not to foreclose a coherent nomenclature for s genes from basal taxa, which will only be possible after completion of the respective genome projects. s is an ancient family with a complex pattern of gene gains and losses in order to better understand the relationships and evolutionary history of the s family members we performed a phylogenetic analysis, using the neighbor-joining method, as well as maximum parsimony and maximum likelihood analyses using the phylip package. full length coding sequences of all teleost s genes were analyzed together with human s genes, using troponins, calmodulins and other related calcium binding protein with ef-hand motifs as outgroup. this phylogenetic analysis shows twenty-seven different groups of orthologs, i.e. different genes as the combined repertoire analyzed here. six of these genes are organized in closely related gene pairs. the delineation of the s gene family, the identification of ortholog groups and the association in gene pairs were supported by all three tree-building algorithms used. nearly a third of the family members are present only in fish – s100i and seven novel proteins, s100q, s100r, s100s, s100t, s100u, s100v, and s100w. remarkably, very few of these fish-specific genes have the full set of five orthologs out of five fish genomes analyzed. when salmon is taken into account, only a single gene, s100w, is found in all teleost species. in four other genes one ortholog is missing, the tetraodon nigroviridis gene in s100i, the oryzias latipes ortholog in s100u, and the s100s, s100t genes in salmo salar. only four and three species have representatives of the s100v and s100q genes, respectively. finally, subfamily s100r is only found in pufferfish. on the other hand, restricted duplication events have occurred for s100i, s100v and s u. all teleost-restricted genes that contain both a zebrafish and another teleost representative should have been present at least in the most recent common ancestor of neoteleostei and ostariophysii. thus, the partial absence of several genes, e.g. s100i in pufferfish suggests a partial loss of these genes in the pufferfish family although it cannot be ruled out with certainty that some apparent gene losses are actually caused by inadequacies of the currently available databases. other examples for restricted loss in some teleost species concern s100q, u, v and the gene pair s100s/t. two of these teleost-specific s genes were not detected in the zebrafish genome despite extensive searches. the most parsimonious explanation therefore is an absence of these genes in the mrca of neoteleostei and ostariophysii, i.e. a genesis of these genes in the neoteleostei lineage. six other genes are shared by teleost fish and mammalian species. thus, all six genes should be more ancient than the split in actinopterygii and sarcopterygii, the separate teleost and tetrapod lineages. four genes are highly conserved among all six teleost species, with at least one ortholog per species being present. however, other genes, particularly s100p, exhibit the same partial representation in teleost species as described above for some fish-specific s genes. thus these genes appear to have been lost in some teleost sublineages. a graphical representation of all these phylogenetic inferences is given in figure four of the six s genes found in the shark genome also belong to this group of genes common to teleosts and mammals. one is an s100z ortholog, two are s100b orthologs, and one is situated at the root of the s100a1-s100z clade. thus, genes s100a s100b, and s100z appear to have emerged before the divergence of cartilaginous fishes from bony fish. since the currently available shark genome is bound to be incomplete, it may be expected that about half of the extant s genes already existed in the mrca of cartilaginous and bony fish. moreover, the phylogenetic position of all cartilaginous fish s genes deep within the teleost phylogenetic tree demonstrates that the s family was already well established before the segregation of bony and cartilaginous fish. in fact, the s family even precedes the earlier split in jawed/jawless vertebrates, because already ten family members were found in lamprey. however, with possible exception of two genes related to s100b and one related to s100v, no ortholog relationship is apparent between any of the petromyzon marinus s genes and those from more derived species. thus the phylogenetic tree reflects a complex evolutionary pattern characterized by frequent gene losses as well as extended gains of either single genes or whole gene subfamilies. similar changes in the gene repertoire caused by complex genomic reorganization and translocation events are reported for ef hand proteins in general. moderate overall similarity but high degree of conservation of motifs that are characteristic for the s family the evolutionary distances between ortholog amino acid sequences range between and, whereas paralog distances range between and. because of this high heterogeneity among the s family and to obtain a second line of evidence in order to classify these genes reliably as members of the s family, we next examined the degree of conservation of the fish and mammalian genes. for that purpose, sequence logos from the amino acid alignments of either the fish or mammalian s proteins alone and of a combination of all the s proteins were created and used for a comparative analysis. all three sequence logos were very similar and showed the same general conservation pattern. the previously described typical structure of the mammalian s proteins formed by four helices separated by a s ef hand calcium-binding domain, a hinge and a canonical ef hand calcium-binding domain is conserved among the fish s proteins. a high number of conserved residues and signature motifs in defined positions of the sequence were identified in the teleost s proteins and found to be conserved also among fish and tetrapods, consistent with the origin of the s family well before the actinopterygian/sarcopterygian split. these residues are located mainly in the helix domains and the calcium-binding regions, especially in the canonical ef hand. this result is in accordance with a recent study that classifies this region as phylogenetically older than the s ef hand domains. in contrast, the hinge region and the c-terminus seem to be regions where there is more space for sequence variability. this is in accordance with the observation in mammalian s proteins that the selectivity in target binding is mainly assured by the hinge region and the c-terminal tail, i.e. in these regions a larger divergence is expected at least between paralogs. among the highly conserved amino acids are, as expected, the calcium-coordinating residues of the ef hands, cf. and several hydrophobic residues involved in dimerization and binding to target proteins. a conserved cysteine close to the end of helix iv has been shown in mammalian s100a and a to stabilize the dimer by intermolecular disulfide bridge formation. this cysteine is part of a conserved hydrophobic pattern f--l---l---c---f of helix iv, which has the proper spacing to form the contact surface for dimerization. similar conserved patterns of to amino acids are observed for the other three helices. most ef hands found in the teleost genes appear to be functional as judged by sequence comparison, with exception of three proteins, s100r, u, v, and some neoteleost members of the s100b subfamily. the s100r protein has lost key residues of the canonical calcium-binding motif in the second ef hand loop, despite a generally good conservation in both domains, similar to the human s100a and a proteins. the s100v protein has lost most key residues of the canonical motif. in four of the five neoteleost s100b proteins a common three amino acid deletion has removed a key residue of the s100-specific calcium-binding loop. nearly the same deletion also occurs in the s100v and the s100u proteins, as well as in the human s100a a the medaka s100a10a, and the shark cms100b x. the frequency of this deletion suggests a specialized function for such genes, which might act either as competitive antagonists, or alternatively, may adopt the calcium-bound conformation constitutively. indeed, the latter has been demonstrated for the human a protein. s genes with such deletions do not segregate together in the phylogenetic tree, and the deletions vary slightly in length and position. thus, this kind of mutation has arisen several times independently. some of these deletion events must have occurred in the mrca of cartilaginous and bony fish, others appear to have originated in the mrca of teleosts, and some are evolutionary late events restricted to a single species. in other words, this particular deletion may be considered a local optimum in the state space of s sequences, consistent with a particular function for these genes. strong negative selection for s genes, but virtual absence of positive selection as previously mentioned, paralog evolutionary distance at the amino acid level is very high, with an average value of, and a range from –. such high values pinpoint the high variability in the amino acid sequences of these genes found at the intraspecies level. in contrast, the average ortholog evolutionary distance value of is about fold lower than the one observed for the paralog comparisons. prompted by both the high degree of intraspecies variability and the high degree of interspecies conservation of s genes we decided to investigate the evolutionary constraints that are acting on this gene family. a strong constraint is equivalent to strong negative selection. the degree of selective pressure is measured by investigating the relative frequency of non-synonymous vs synonymous substitutions. when the number of dn equals the number of ds, the dn/ds ratio equals which corresponds to neutral selection. if the number of non-synonymous changes is higher than the number of synonymous changes, then dn/ds > which indicates positive selection. on the other hand, if the number of synonymous changes is higher than the number of nonsynonymous changes, then dn/ds < and we are in the presence of negative selection. whenever possible, the overall dn/ds ratio within each ortholog group was determined. all the groups show very small values, ranging from to, with an average global dn/ds value of, which is evidence for strong negative selection. these low dn/ds values combined with the high divergence between the s genes suggest not only an ancient origin of these genes but also indicate that individual genes of this family are slowly evolving. however, as the strong negative selection could be masking some positive selection events at the individual codon level, we also determined the dn/ds ratio separately for each individual sequence position. results are shown for two of the ortholog groups, the s100i and s100z. these two clades represent two different evolutionary scenarios, the first showing evidence for recent gene duplications in zebrafish and lacking one ortholog in takifugu rubripes, while the second is a very well conserved group with orthologs in all six fish species. also they show two different values for their evolutionary distances, of and respectively. as predicted by the global dn/ds analysis, a considerable portion of the total sequence – 25% for the s100i and 35% for s100z – is under negative selection, with the second value being closer to the average value of sites per ortholog group. no evidence for positive selection was found, suggesting that positive selection does not appear to play a significant role in the evolution of the s genes. a comparison between s100i and s100z shows rough similarity in the pattern of negative selection, although no specific motifs could be identified between genes. gene structure of s genes is highly conserved between fish and mammalian species the mammalian s genes are found to have a common structure consisting of usually three exons. the first exon contains only 5'utr, part of the second and third exons code for the orf and a 3'utr is located in the last portion of the third exon. for the fish s genes we restricted analysis to the coding exons, since prediction of noncoding utrs is currently not very reliable. we found that of the analyzed teleost s genes conform to the pattern observed in ortholog genes in humans. however, another seven teleost fish genes have their orf split in three exons, one of them in most cases very small and always located at one end of the orf. for zebrafish several, but not all, of these predicted exons have been confirmed by rt-pcr. each major exon contains one complete ef-hand domain. without exception these two exons are joined at the same position in the sequence. thus these exons may be as old as the s family itself and the first such gene may have arisen from a duplication of this domain. in contrast, the intron/exon borders of the small exons show a variable position in the n-terminal and c-terminal regions and occur in few species only, consistent with a late origin in the teleost lineage. s fish genes are distributed both as singletons and small clusters in the genome all mammalian s100a genes are clustered in the genome, whereas s100b, g, p, and z are located as singletons on different chromosomes. in zebrafish the scenario is somewhat similar, with two smaller clusters of and mb each located on the chromosomes and respectively, and composed of seven and four genes, respectively. similarly, in the other teleost fish species analyzed small clusters of maximally five genes are found. these clusters always contain phylogenetically less related genes and, in contrast to the situation in mammals, cluster gene associations differ between species. however, and remarkably so, there are two s gene pairs, which are stably linked in the teleost genome, whenever present in the same species, albeit not closely related phylogenetically. conversely, a closely related gene pair consists of a singleton gene and a member of a cluster. thus, among teleost s genes, genomic linkage is not at all correlated with degree of phylogenetic relationship, consistent with an ancient evolutionary origin of these clusters. in total, three zebrafish genes, s100b, s100v and s100z, are present as singletons in the chromosomes and respectively. this feature is conserved across all five teleost species studied; additionally s100p, which is absent in zebrafish, is always a singleton, whenever it occurs. the isolated genomic location of these family members appears to be an evolutionary ancient feature of the s family, since all members of this group also found in mammals share the singleton location. the two clusters present in zebrafish are situated on chromosomes and which are largely syntenic and result from the teleost-specific whole genome duplication, which occurred after the actinopterygian/sarcopterygian split. two of the three gene pairs observed in the teleost s repertoire are distributed between these two clusters, consistent with a duplication of their ancestral genes by this whole genome duplication nearly million years ago. this interpretation is supported by the observation that the gene pair a10a/b has a single mammalian ortholog. moreover, very similar clusters are present in chromosomes and from tetraodon nigroviridis and chromosomes and from oryzias latipes. both chromosome pairs are syntenic to each other, and to zebrafish chromosomes and. the third recognizable gene pair in teleosts is shared both with mammals and with cartilaginous fish, but a is always a member of a cluster, whereas z never is, consistent with a very ancient origin of this gene duplication, before the emergence of the cluster itself. in total, three genes from the human s100a cluster on chromosome a a and a are found in the teleost clusters. together with the synteny of this region of human chromosome with the corresponding teleost cluster regions these data are consistent with a small ancestral cluster of s genes in the mrca of teleosts and tetrapods plus some isolated ancestral s genes. in contrast, other gene pairs occur closely linked in the chromosome, indicating a local tandem duplication event as cause of such gene pairs. expression patterns of s genes range from highly specific to fairly broad distributions to obtain an overview of the tissue specificity of expression for the whole family we performed rt-pcr with twelve different tissues from adult zebrafish. for a higher spatial resolution of the expression patterns at the cellular level we performed whole mount in situ hybridization of zebrafish larvae days post fertilization. at this stage zebrafish have completed organogenesis and major behavioral patterns are already functional. eight of ten genes analyzed show expression in the larval stage, with expression patterns ranging from highly restricted to spatially broad distributions. the results of the in situ hybridizations are often consistent with those from the rt-pcr. a general tendency of the rt-pcr to show broader expression may be related to the higher sensitivity of that method compared to in situ hybridization. additionally, a developmental regulation, i.e. a late onset of expression, may explain some of the differences in the results, especially the absence of two s transcripts in larval tissues. for s100b a late onset of expression was confirmed by the detection of strong signals in adult brain. highly restricted expression of s genes within the sensory nervous system three zebrafish s genes show a remarkably restricted expression in just one or two different larval neuronal populations. zebrafish s100s is expressed in the neuromasts of the lateral line and the inner ear. labeled structures in the inner ear may include hair cells. in sections of the tail region the central portion of the neuromasts is labeled, consistent with an expression in sensory hair cells. thus in the larvae particular sensory structures and possibly some neurons innervating them are labeled. the rt-pcr results show a general expression in all sensory systems examined which are in detail barbels and lips, brain, eyes, olfactory system and additionally the genitourinary system. the in situ expression pattern of s100s is quite similar to that of the highly related zebrafish s100t, which is also expressed in the neuromasts of the lateral line and the inner ear, the latter in lower levels compared to s100s. zebrafish develop approximately 9– neuromasts along one side, which have their counterparts on the opposite side and all of them are stained by either probe. in rt-pcr s100t expression is additionally observed in non-sensory tissue such as bone, gills, heart, liver and muscles. zebrafish s100z is the gene with the spatially most restricted expression pattern in the larvae. only a single tissue expresses s100z, the olfactory placode, and within that tissue expression is restricted to a few large cells, by morphology presumably neurons. a far broader expression pattern in adult fish is suggested by the rt-pcr results and may suggest a late ontogenetic onset of expression in those tissues. the related s100a was not detected at all in larval tissues, but shows an expression in the adult genitourinary system, skin, olfactory epithelium and brain according to rt-pcr-results. non-neuronal expression of two s genes, restricted to a single cell type or tissue zebrafish s100a10a shows one of the most restricted patterns. expression is restricted to the intestinal bulb and the intestine with intestinal loops being clearly visible. expression slightly decreases from intestinal bulb to comparatively low levels in the anal region. rt-pcr was negative, consistent with the described specificity. the expression is limited to the epithelial cells of the intestine and gut. zebrafish s100a exhibits an extremely specific in situ hybridization pattern. s100a seems to be restricted to isolated large cells located on the yolk sac and the pericardial cavity of the fish. there are about cells labeled in the whole larva. rt-pcr with adult tissues suggests expression in gills and olfactory epithelium. the cellular morphology, larval staining pattern and the adult expression in gills suggest expression in mr-cells, also known as ionocytes. these cells control ionic composition of body fluids, especially calcium levels. results obtained by rt-pcr indicate a much broader distribution, possibly at levels below the threshold for in situ hybridization. three s genes exhibit broad expression in several tissues and cell types zebrafish s100i. expression was previously described by and. our data are consistent with their findings of expression in the epithelial cells of barbels, gills, olfactory rosettes and skin. s100i. is expressed throughout the whole skin with high levels of expression in the rim of the dorsal and pectoral fin. high expression levels were observed in the urogenital opening. moreover, s100i. exhibits also strong expression in cells of the olfactory placode. additionally expression can be observed throughout the pharyngeal arches and the lateral line excluding the neuromasts, which appear as light spots in the middle of the larvae. in this respect s100i. shows the complementary pattern to s100s, t, which label the neuromasts, but not the surrounding tissue. the in situ hybridization signals may represent the combined expression pattern of s100i. and s100i. since their probes share over 90% homology at the nucleotide level, and thus cross-reactivity cannot be excluded. however, rt-pcr results for s100i. are expected to be specific, since a primer from its unique, short exon was used. rt-pcr results for s100i. probably represent a mix of both s100i transcripts, since no completely specific primers were possible there. the results show expression of both genes in all tissues tested, with exception of muscle, where only s100i. could be detected. zebrafish s100a10b exhibits high expression in the lower jaw in particular in the branchial arches. epithelial cells are labeled, but cartilaginous tissue is devoid of staining. olfactory placode and the whole larval trunk do not express s100a10b. thus this expression pattern is a subset of that observed for s100i. unexpectedly, rt-pcr reveals expression of s100a10b in every tissue examined. partial correlation of expression pattern similarity with coding sequence similarity the highly related genes s100s and s100t show a nearly identical larval expression pattern, and the duplicate s100i genes appear to exhibit a very similar expression pattern as well. however, expression patterns for the duplicate a10a genes are very different, with the a10b pattern a subset of the unrelated s100i. i. and a completely different pattern being observed for a10a. this partial correlation is consistent with the interpretation that in some cases regulatory elements have been conserved alongside the coding regions of these genes, which are spread far apart in the genome. additionally, for the two s100i genes, which are clustered, regulatory elements shared within the cluster might synchronize the expression pattern. in this context it is noteworthy that the close genomic linkage of s100i to s100a10b is conserved in the genome of all fish species that possess both genes, and indeed, a10b expression is quite similar to that of s100i, consistent with partially shared regulatory elements. on the other hand, a and s are also linked closely, and their expression patterns are completely different. thus regulation of s gene expression follows a complex pattern presumably involving several different mechanisms as suggested for humans, cf.. discussion and  the s family represents the largest family of calcium-binding proteins with ef hands. s proteins have multiple and essential functions in many different tissues, mainly in cells of epithelial origin. however, most studies have been performed in mammals, and the evolutionary emergence of the family is not clear. we have performed an extensive search in six teleost genomes and identified in total fourteen distinct subfamilies, which constitute the fish s gene repertoire. individual species exhibit thirteen to fourteen different s genes. upon closer investigation a complex pattern of gene gain and gene loss emerges, which results in a roughly stable total repertoire size for each species. a similar family size is observed in amphibians, whereas a preponderance of gene gains leads to an enlarged s repertoire size in mammals. the presence of several s family members from cartilaginous fish deep inside the phylogenetic tree implies that the s family predates at least the segregation of bony from cartilaginous fish. moreover, already ten members of the s family have been identified in a jawless primitive fish. thus the origin of the family may lie with very early vertebrate stages although individual family members only become reliably recognizable in the time period between the jawed/jawless segregation and the divergence of cartilaginous from bony fish around to million years ago. the somewhat variable composition of each species repertoire suggests that gene function may be variable between species. in fact, an a11-like gene from xenopus is expressed in olfactory receptor neurons, i.e. may have acquired the function served in zebrafish by s100z. such flexibility is observed irrespective of a generally strong negative selection within the individual s genes, i.e. the evolutionary changes within the regulatory regions may be much faster than those within the coding regions. on the other hand, some functional specializations appear quite stable, which may be inferred from the expression of human s100a in polarized epithelia parallel to the expression of the zebrafish s100a10a in the intestinal epithelium. however, it should be noted that human a is constitutionally in the, calcium-bound' configuration due to a deletion in the s ef hand, in contrast to the zebrafish a genes. moreover, the other member of this gene pair shows a radically different distribution. another example is s100b, which is expressed in brain in zebrafish as well as in mammalian species. the genomic structure of teleost s genes is markedly similar to that of mammalian genes, with two to three coding exons in similar distances, and a conserved domain structure of one ef hand per each main exon. some similarities are also observed for the genomic location. most mammalian s genes lie together with some other genes in one large cluster, the so-called epidermal differentiation complex for which common regulatory elements have been discussed. two smaller clusters of four to seven members in teleosts are syntenic to the human cluster and thus suggest the presence of an ancestral small cluster of s genes. teleost cluster configuration does not correlate with sequence similarity, in contrast to the mammalian edc. thus the teleost clusters may represent the ancestral cluster composition more closely, while extended local gene duplications appear to have enlarged the mammalian cluster. while the predominantly epithelial and epithelial-derived cellular expression of mammalian s genes is maintained in zebrafish, the actual regulatory mechanisms responsible for this similar expression may be different. no evidence for shared regulatory elements is observed in teleost fish, as judged from both sequence divergence and expression pattern similarity of neighboring genes. zebrafish s genes exhibit highly specific and characteristically different patterns of expression like their mammalian counterparts. duplicated genes show both redundancy and divergence in their expression pattern. larval expression is limited to different types of epithelial cells and neural cells of epithelial origin. our findings provide a firm basis for unraveling the molecular nature of the s100-like immunoreactivity observed in teleost fish tissues in several earlier publications, e.g.. taken together we have identified and characterized the teleost s gene family with respect to gene structure and expression pattern. these genes exhibit characteristics similar to the mammalian gene family in several respects. considering the importance of s genes in the pathogenesis of epidermal disease – particular s proteins are markedly over expressed in psoriasis, wound healing, skin cancer, inflammation, cellular stress, and other epidermal pathological states – it appears advantageous to study these processes in the zebrafish model system. 
in larval tissue expression is often restricted to specific cell types like keratinocytes, hair cells, ionocytes and olfactory receptor neurons as demonstrated by in situ hybridization. many aspects of the genomic arrangement and location of mammalian s genes are retained in the teleost s gene family, including a completely conserved intron/exon border between the two ef hands. individual species feature distinctive subsets of thirteen to fourteen genes that result from local gene duplications and gene losses. eight of the fourteen s subfamilies are unique for teleosts, while six are shared with mammalian species and three of those even with cartilaginous fish. despite a complex pattern of gene gains and losses the total repertoire size is remarkably constant between species. zebrafish s genes exhibit highly specific and characteristic expression patterns, showing both redundancy and divergence in their cellular expression. several s family members are found in jawless fish already, but none of them are clear orthologs of cartilaginous or bony fish s genes. we report that the teleost s repertoire comprises fourteen different subfamilies which show remarkable similarity across six divergent teleost species. we have performed an extensive search in several teleost genomes to establish the s gene family in fish. the evolutionary origin of this family is still uncertain and most studies have examined mammalian family members. all teleost s genes show the expected structural features and are subject to strong negative selection. the origin of the s family predates at least the segregation of jawed from jawless fish and some extant family members predate the divergence of bony from cartilaginous fish.
 among the ef-hand calcium-binding proteins the subgroup of s proteins constitute a large family with numerous and diverse functions in calcium-mediated signaling. the evolutionary origin of this family is still uncertain and most studies have examined mammalian family members.  we have performed an extensive search in several teleost genomes to establish the s gene family in fish. we report that the teleost s repertoire comprises fourteen different subfamilies which show remarkable similarity across six divergent teleost species. individual species feature distinctive subsets of thirteen to fourteen genes that result from local gene duplications and gene losses. eight of the fourteen s subfamilies are unique for teleosts, while six are shared with mammalian species and three of those even with cartilaginous fish. several s family members are found in jawless fish already, but none of them are clear orthologs of cartilaginous or bony fish s genes. all teleost s genes show the expected structural features and are subject to strong negative selection. many aspects of the genomic arrangement and location of mammalian s genes are retained in the teleost s gene family, including a completely conserved intron/exon border between the two ef hands. zebrafish s genes exhibit highly specific and characteristic expression patterns, showing both redundancy and divergence in their cellular expression. in larval tissue expression is often restricted to specific cell types like keratinocytes, hair cells, ionocytes and olfactory receptor neurons as demonstrated by in situ hybridization.  the origin of the s family predates at least the segregation of jawed from jawless fish and some extant family members predate the divergence of bony from cartilaginous fish. despite a complex pattern of gene gains and losses the total repertoire size is remarkably constant between species. on the expression level the teleost s proteins can serve as precise markers for several different cell types. at least some of their functions may be related to those of their counterparts in mammals. accordingly, our findings provide an excellent basis for future studies of the functions and interaction partners of s genes and finally their role in diseases, using the zebrafish as a model organism. 