BACKGROUND
the cytochrome p <dig>  enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes.
mammalian cyps can be separated into two major groups: those with generally narrow substrate specificity that are involved primarily in synthesis, activation or inactivation of endogenous regulatory molecules, and those involved most heavily in the metabolism of xenobiotics, but which may act as well in the metabolism of endogenous compounds.
thus, cyp enzymes can determine the persistence and action of endogenous regulatory molecules as well as many drugs and other toxicants and carcinogens.
an area growing in importance is the role of cyps in development, and in developmental toxicity of chemicals.
absolute embryo lethality upon knockout of the murine cyp oxidoreductase  demonstrates the essential role of cyp enzymes in murine development  <cit> .
many cyps potentially have roles in developmental specification.
for example, it is well known that cyp <dig> enzymes regulate levels of retinoids governing pattern formation during development  <cit> .
cyp isoforms that play essential roles in bile acid homeostasis, steroidogenesis, the vitamin d pathway, and the catabolism of many hormones may also have important roles in development  <cit> .
many chemicals that cause developmental abnormalities, including cardiovascular, neural, and connective tissue defects, are substrates for cyps, and the oxidative biotransformation of such xenobiotics may determine the cellular and organ targets of those chemicals  <cit> .
cyp enzymes that are prominent in xenobiotic metabolism could also function in producing morphogenic molecules or keeping regions free of them, creating temporal and spatial regions of morphogen action and supporting region-specific changes essential for successful development  <cit> .
the roles and regulation of most xenobiotic metabolizing cyps during development are unknown, impeding understanding of mechanisms of developmental toxicity.
many xenobiotic metabolizing cyp can be induced via transcription factors .
all cyps can be targets of exogenous chemicals that disrupt or enhance their function, whether or not they are directly involved in xenobiotic metabolism.
the zebrafish has emerged as one of the most important vertebrate model species in embryology and developmental biology  <cit> , due largely to rapid development coupled with the possibility for genetic analysis and manipulation.
multiple draft assemblies of the zebrafish genome have been produced by the wellcome trust sanger institute  <cit> .
the near completion of the zebrafish genome has made it possible to address questions of cyp gene identity and expression during zebrafish development with substantial clarity and completeness.
our objective was to identify and annotate the full suite of zebrafish cyp genes and examine their phylogenetic and shared syntenic relationships to human cyps.
we also examined cyp expression during early development in zebrafish.
cyp expression in vertebrate development has not been completely mapped for any species; prior studies in mice examined developmental expression of  <dig> cyp genes  <cit> , less than half of the mouse cyp complement  <cit> .
our analyses identify novel genes and clusters of cyp genes in zebrafish, and unanticipated waves of cyp expression during development.
RESULTS
repeated and exhaustive searching of zebrafish genome assemblies uncovered a total of  <dig> cyp genes.
based on inferred amino acid sequences, these genes fell into  <dig> cyp gene families that are also found as well in humans and other mammals.
molecular phylogenetic analysis  shows relationships among cyp genes and gene families in zebrafish and human.
as with the human cyp genes, the cyp genes in zebrafish occur in two major functional groups: those families that include enzymes involved primarily in endogenous functions ; and those that include enzymes involved heavily in oxidation of xenobiotics .
zebrafish genes in these two major groups are described in detail below, considering relationship to human cyps based on sequence identity, syntenic analysis and, where known, functional properties .
the term cyp is fully capitalized in all cases except for mouse, rat, and drosophila , and italicized only when referring to a specific gene or cdna, consistent with the nomenclature committee recommendations  <cit> .
a incorrect predicted transcript; b incomplete best blast hit chr <dig> 58640777-58651921; c two different genes concatenated into one prediction; d originally published as cyp26d1; e zv <dig> ensdarg <dig>  chromosome 20
a genewise reprediction; b no ensembl gene prediction; c identical dna; d identical dna; e incorrect n-term exon; f chr <dig>  <dig> is probably best location; g there are non-mammalian genes that share synteny but not the same subfamily name with mammalian cyp2j <dig> s. at present, annotation of fish cyp <dig> s by blast can result in misclassifying fish cyp <dig> s as cyp2js , as strictly interpreted according to nomenclature rules, there is not a cyp2j subfamily in fish.
cyp families 5-51
cyp <dig> .
zebrafish cyp5a <dig> retains 48% sequence identity with human cyp5a <dig>  and is located in a region of shared synteny with cyp5a <dig> in humans, indicating orthology.
cyp <dig>
zebrafish possess one six-exon cyp7a <dig> and one six-exon cyp7b <dig> gene, both located on chromosome  <dig>  the sequence identity between zebrafish cyp7b <dig> and mammalian cyp7bs technically precludes membership in the same subfamily, yet shared synteny  indicates orthology.
zebrafish also have a third cyp <dig> gene, cyp7c <dig>  a cyp7a-like gene located on chromosome  <dig>  possibly an ohnolog resulting from the fish-specific whole genome duplication .
a pseudogene
cyp <dig>
a 10-exon zebrafish cyp8a <dig> gene is located on chromosome  <dig>  human cyp8a <dig> catalyzes the rearrangement of pgh <dig> to prostaglandin i <dig>   <cit> .
a crystal structure of zebrafish cyp8a <dig> published in  <dig> as part of a comparative structural analysis of prostacyclin synthases, shows that zebrafish and human cyp8a <dig> exhibit nearly identical ligand-bound and ligand-free three-dimensional structures, despite exhibiting only 45% identity  <cit> .
zebrafish also possess three cyp8bs , which are single-exon genes located on chromosome  <dig> that share synteny with the single-exon human cyp8b <dig>
cyp <dig>
zebrafish have  <dig> cyp11a genes , cyp11a <dig> and cyp11a <dig>  located adjacent to one another on chromosome  <dig>  and one cyp11b-like gene, termed cyp11c <dig> .
vertebrate cyp11a <dig> synthesizes pregnenolone from cholesterol, and a maternally-derived cyp11a <dig> mrna has been suggested to form pregnenolone important in cell migration in the zebrafish zygote  <cit> .
following the onset of embryonic transcription, cyp11a <dig> is expressed in the yolk syncytial layer.
it is then expressed in the embryonic interrenal primordia, and in the interrenal glands into adulthood  <cit> .
in adult zebrafish it is expressed in the gonads, brain, and interrenal glands.
knockdown of zebrafish cyp11a <dig> leads to a shortened axis and epiboly defects .
zebrafish cyp11a <dig> is 80% identical to cyp11a <dig> at the amino acid level, but little is known about functional similarities between the two forms, or about cyp11a <dig> expression, although based on our microarray results  it appears to be expressed in the early embryo, and database est evidence indicates that it is also expressed in adult gonads.
zebrafish cyp11c <dig> exhibits 42% amino acid identity to human cyp11b <dig> , but does not share synteny with any tetrapod cyp11b genes.
cyp <dig>
cyp <dig> enzymes catalyze dual functions of steroid-17α-hydroxylase and steroid- <dig>  20-lyase.
the zebrafish genome has two cyp17a genes that are 49% identical at the amino acid level, and the two genes are located on different chromosomes .
one of these  was cloned from zebrafish ovary  <cit> .
cyp17a <dig> is expressed in gonadal tissue, but also in brain, gill, liver, and intestine.
zebrafish cyp17a <dig> is upregulated by fluorotelomer alcohols  <cit> , but not by benzopyrene  <cit> .
the functions of the two cyp <dig> s may differ   <cit> .
cyp <dig>
zebrafish have two distinct cyp <dig>  genes, cyp19a <dig> and cyp19b <dig> reported initially by kishida and callard  <cit> .
the two aromatase forms are expressed in different tissues; cyp19a <dig> is expressed principally in the ovary, but also at much lower levels in the testis, while cyp19b <dig> is expressed in brain and neural tissue, including in the olfactory bulb, ventral telencephalon, preoptic area, and ventral/caudal hypothalamic zone, and in the anterior and posterior lobes of the pituitary  <cit> .
estrogen-mediated upregulation has been observed only for the neural form, cyp19b <dig>  in adults and in embryos after  <dig> hpf  <cit> .
cyp20a <dig>
zebrafish have one cyp <dig> gene, cyp20a <dig>  61% identical to the cyp20a <dig> in humans, and they share synteny , both being adjacent to abi <dig>  at present, the substrate specificity and biological function of cyp20a <dig> are unknown, and it remains an "orphan" cyp  <cit> .
however, there is evidence for high levels of mrna expression in the hippocampus and substantia nigra of mouse  <cit> , suggesting functions in these parts of the brain implicated in important neuropathies.
cyp <dig>
zebrafish cyp21a <dig> shares 39% identity with human cyp21a <dig>  and shares synteny with the human cyp21a gene pair .
mammalian cyp21a <dig> and cyp21a <dig> convert progesterone to deoxycorticosterone via 21-hydroxylation, prominently in the adrenal gland.
the zebrafish cyp21a <dig> presumably serves a similar function, in the interrenal.
cyp <dig>
cyp24a <dig> is a mitochondrial enzyme catalyzing 1α, 25-dihydroxyvitamin-d3-24-hydroxylase.
there is one cyp24a <dig> gene in zebrafish, with 63% percent amino acid identity to human cyp24a <dig>  the zebrafish enzyme is presumed to function similarly to mammalian cyp <dig> s, as vitamin d <dig> and various metabolites circulate in fish serum  <cit>  and zebrafish have a functional vitamin d receptor  with ligand specificity similar to other vertebrate vdrs  <cit> .
cyp <dig>
there are three cyp <dig> s in zebrafish.
cyp <dig> was first discovered in zebrafish  <cit>  and the retinoic acid metabolizing function appears to be conserved throughout the vertebrates  <cit> .
each zebrafish cyp <dig> is located on a separate chromosome, suggesting early establishment of these duplicated genes.
the cyp <dig> s are categorized into three separate subfamilies, with some confusion as to the proper classification, i.e., zebrafish cyp26c <dig> has previously been referred to as cyp26d <dig>  <cit> .
cyp26a <dig> and cyp26b <dig> share synteny with human cyp26a <dig> and 26b <dig> genes, respectively, while the cyp26c <dig> syntenic relationship is less distinct .
the three zebrafish cyp <dig> s are all induced by and metabolize retinoic acid, and play essential roles in hindbrain patterning  <cit> .
zebrafish cyp <dig> s also play roles in osteogenesis  <cit> , in pancreatic development  <cit> , and presumably other processes.
cyp <dig>
zebrafish have seven distinct cyp <dig> genes, distributed into three subfamilies, while in most mammals there are two or three cyp <dig> genes.
human cyp27a <dig> and cyp27b <dig> catalyze vitamin d <dig> 25-hydroxylase and 25-hydroxyvitamin d <dig> 1α-hydroxylase, respectively  <cit> .
the five zebrafish cyp27a genes are all tandemly located on chromosome  <dig> and share synteny with human cyp27a <dig> .
zebrafish cyp27b <dig> and cyp27c <dig> are located on different chromosomes .
functional properties of the various zebrafish cyp <dig> s are unknown.
cyp <dig>
a cyp39a <dig> is present in the zebrafish genome, located on chromosome  <dig>  in mammals cyp39a <dig> functions as an oxysterol-7α-hydroxylase, involved in the conversion of cholesterol to bile acids.
cyp39a <dig> has not been found in other published fish genomes, but is present in tetrapod genomes, with conserved synteny to human cyp39a <dig>  zebrafish cyp39a <dig> is approximately 4mb from a cluster of genes that share synteny with human cyp39a <dig>
cyp <dig>
zebrafish have four cyp46a genes.
one  is on chromosome  <dig>  two  are arranged in tandem on chromosome  <dig>  a fourth  is supported by est data but missing from the current assembly .
in contrast, humans have one cyp <dig>  cyp46a <dig>  human cyp46a <dig> functions both as a cholesterol 24-hydroxylase and as a 24-hydroxycholesterol-hydroxylase  <cit> .
cyp46a <dig> in mammals is a brain-specific cholesterol-metabolizing enzyme, important for maintaining brain cholesterol homeostasis and membrane function, but also with potential regulatory roles as a neurosteroid-metabolizing enzyme  <cit> .
cyp <dig>
zebrafish possess one cyp <dig> gene, cyp51a <dig>  in other animals cyp <dig> is a lanosterol-demethylase enzyme, the only cyp involved in the post-squalene portion of the cholesterol biosynthesis pathway.
cyp <dig> is an essential enzyme in the de novo synthesis of cholesterol  <cit> ).
cyp <dig> is often considered to be a "housekeeping gene" based on its ubiquitous expression and conserved function  <cit> .
zebrafish cyp <dig> has been cloned and heterologously expressed in e. coli  <cit> .
microarray analysis and in situ hybridization of embryonic zebrafish showed that cyp <dig> is strongly expressed in the eye, in the brain, and in epidermal cells of the forehead and the tail fin  <cit> , and suggesting a role for cyp <dig> and other isoprenoid synthesis enzymes in hematopoietic and vascular development.
cyp families 1- <dig>
cyp families  <dig>   <dig>   <dig>  and  <dig> include enzymes involved in drug, xenobiotic and fatty acid metabolism.
in contrast to the cyps that have principally endogenous functions, these cyp genes are much more diverse, with much less conservation of sequence between zebrafish and human.
table  <dig> provides specific information on the cyp 1- <dig> family genes.
a molecular phylogeny of clan  <dig> genes, which includes families  <dig> and  <dig> as well as cyp <dig> and cyp <dig>  is shown in figure  <dig>  molecular phylogeny of the clan  <dig> genes, including the cyp <dig> s and cyp <dig> s  and clan  <dig> genes  can be seen in figure  <dig>  some relationships between zebrafish and human cyps in families 1- <dig> suggested by molecular phylogeny have been substantiated by synteny analysis.
clan 2- cyp1s
zebrafish have five cyp <dig> genes in four subfamilies, cyp1a, cyp1b <dig>  cyp1c <dig>  cyp1c <dig>  and cyp1d <dig>  all of which have been cloned and sequenced  <cit> .
zebrafish cyp1a has exon structures similar to the human cyp1a <dig> and cyp1a <dig>  and the single cyp1b <dig> gene has a gene structure very similar to human cyp1b <dig>  the cyp1cs are closely linked single-exon genes  <cit> .
the recently identified zebrafish cyp1d <dig> has a gene structure identical to zebrafish cyp1a, and quite different from the cyp1b <dig> and cyp1cs  <cit> .
members of all four cyp <dig> subfamilies have been identified in other fishes and non-mammalian tetrapods, and the evolution of the cyp <dig> family has been discussed elsewhere in some detail  <cit> .
notably, the cyp1cs are absent and cyp1d <dig> is a pseudogene in human and some other mammals  <cit> .
cyp1a, cyp1b <dig> and the cyp1cs are inducible by ahr agonists  <cit> .
vertebrate cyp1as and cyp1b <dig> generally are involved in the metabolism of various drugs, many hydrocarbons, steroids and fatty acids.
with the exception of benzopyrene and estradiol  <cit> , little is known of the possible xenobiotic and endogenous substrates of the cyp1cs and of cyp1d <dig>
clan  <dig> - cyp2s
as in mammals, the cyp <dig> s constitute the largest cyp gene family in zebrafish, with  <dig> cyp <dig> genes, in contrast to  <dig> in humans.
the subfamilies are considered below, in order according to suggested relationships:  orthology indicated by sequence,  homologs suggested by sequence and orthology confirmed by shared synteny, and  no evident homologous relationship to human cyps.
cyp2r and cyp2u
there are single genes in each of these two subfamilies in zebrafish, cyp2r <dig> and cyp2u <dig>  these can be classified as orthologs of human cyp2r <dig> and cyp2u <dig>  based on sequence identity.
they also exhibit shared synteny with their respective human counterparts .
cyp2r <dig> is a microsomal vitamin d 25-hydroxylase in humans  <cit> .
human cyp2u <dig> is expressed in the brain and the thymus  <cit> , and catalyzes ω and ω- <dig> hydroxylation of fatty acids, including arachidonic acid  <cit> .
cyp2u <dig> appears to have a pre-vertebrate origin, and may be the oldest identifiable vertebrate cyp subfamily  <cit> .
cyp2n, cyp2p, cyp2v, cyp2ad
the  <dig> zebrafish genes in these five subfamilies occur in a clade together with human cyp2j <dig> .
analysis of gene location showed that the six cyp2ps and cyp2n <dig>  cyp2v <dig>  cyp2ad <dig>  cyp2ad <dig>  and cyp2ad <dig> all are arranged in tandem on chromosome  <dig>  this cluster of cyp genes shares synteny with human cyp2j <dig>  indicating that all  <dig> zebrafish genes at this locus share an ancestral origin with cyp2j <dig> .
cyp2j <dig> is an arachidonic acid epoxygenase with roles in the cardiovascular system.
functions of the members of the zebrafish cluster are unknown, although related genes in killifish  are arachidonic acid hydroxylases and cyp2p <dig> in particular is similar in function to human cyp2j <dig>  <cit> .
cyp2k
eight cyp2k genes are present in a tandemly duplicated array on chromosome  <dig>  this cluster of cyp2ks shares synteny with human cyp2w <dig> .
human cyp2w <dig> is a tumor-specific cyp  <cit>  that oxidizes indole and chlorzoxazone, but not fatty acids  <cit> .
cyp2k <dig>  which is not highly expressed until  <dig> dpf, has been heterologously expressed, and demonstrated to catalyze the activation of the mycotoxin aflatoxin b <dig>  to the carcinogenic exo- <dig> -afb <dig> epoxide  <cit> .
the low level cyp2k <dig> expression during zebrafish embryo and larval stages may help to explain the lack of afb <dig> toxicity in zebrafish embryos  <cit> .
functions of the other cyp2ks are not known.
cyp2y
cyp2y <dig> and cyp2y <dig> are tandemly arranged on chromosome  <dig>  they share synteny with a cluster of cyp <dig> genes for human drug-metabolizing enzymes, including cyp2a <dig>  cyp2a <dig>  cyp2b <dig>  cyp2f <dig>  and cyp2s <dig> located on human chromosome  <dig> .
the human genes at this locus include genes induced by pxr agonists  and genes induced by ahr agonists .
whether cyp2y <dig> and cyp2y <dig> have functional or regulatory properties in common with any of these human cyps is not known.
cyp2x
there are nine cyp2x genes present in two separate arrays of tandemly duplicated genes, on chromosomes  <dig> and  <dig>  two of the cyp2x genes  , have two exact copies in the current genome assembly  likely as an artifact of the assembly process, so there is some uncertainty as to the exact number of cyp2xs.
the cyp2x genes do not appear to share synteny with any mammalian cyp.
catalytic activities of cyp2xs in zebrafish are unknown, although cyp2x <dig> from channel catfish has been cloned  <cit>  and heterologously expressed  <cit> .
aminopyrine and benzphetamine demethylase activities were observed, but no biological function has as yet been ascribed.
cyp2aa
due to overlapping gene predictions, the current genome assemblies and gene counts are not accurate in the region on chromosome  <dig> where the cyp2aa genes are located.
ten cyp2aa genes are present in a tandemly duplicated array on chromosome  <dig>  genewise  <cit>  predictions indicate that there are  <dig> cyp2aas, with high identity to each other , but no clear orthology to other cyp <dig> s. at least three have been cloned .
some cyp2aa genes appear to be induced by pxr agonists .
the cyp <dig> s most closely related to the cyp2aas are the cyp2xs, and cyp2r <dig> .
cyp2ae
the two cyp2ae genes are present as tandem duplicates on chromosome  <dig>  approximately  <dig> kb downstream from the cyp2aa cluster.
no shared synteny is evident with mammalian genomes, and is unclear even for other published fish genomes.
however, the cyp2aes cluster phylogenetically with the cyp2j orthologs cyp2n/p/v/ae , suggesting a possible insertion of a set of duplicated cyp2j orthologs.
clan  <dig> - cyp3s
the zebrafish genome includes five cyp <dig> genes, cyp3a <dig>  and four cyp3cs, cyp3c1-3c <dig> .cyp3a <dig> and cyp3c <dig> were previously cloned and heterologously expressed  <cit> .
cyp3a <dig> is a <dig> exon gene located in chromosome  <dig>  and is 54% identical to human cyp3a <dig>  zebrafish cyp3c1-cyp3c <dig> are all 13-exon genes located in tandem on zebrafish chromosome  <dig> .
other fish species also have multiple cyp <dig> genes located in this region , and chickens appear to have two homologs at that locus.
it is likely that the previously named human pseudogenes cyp3a-se <dig> and cyp3a-se <dig>  <cit>  are the orthologous remnants of these cyp3s, as they are located in the same region  as the immediately adjacent foxk1gene .
qiu et al.
found that in most vertebrates apparently intact  cyp <dig> genes are located within one of two genomic regions, labeled cyp3hr <dig> and cyp3hr <dig>  <cit> .
zebrafish cyp3cs are within cyp3hr <dig>  while the human cyp3as are in cyp3hr <dig>  zebrafish cyp3a <dig> does not share synteny with cyp <dig> s in other fish for which genomic information is available .
cyp3a <dig> is inducible by some pxr agonists  <cit> .
other fish cyp3as are prominent in catalyzing testosterone 6β-hydroxylase  <cit> , and this is likely true for zebrafish cyp3a <dig> as well.
activities are not known for the other zebrafish cyp <dig> s.
clan  <dig> - cyp4s
there are four cyp <dig> genes in zebrafish, cyp4f <dig>  cyp4t <dig>  cyp4v <dig>  and cyp4v <dig> , in contrast to humans, which have  <dig> cyp <dig> s. mammalian cyp4f enzymes function as omega-hydroxylases of c16-c <dig> fatty acids, including eicosanoids such as leukotriene b <dig>  <cit> .
the full range of substrates of cyp4v enzymes is not yet known, although human cyp4v <dig> is a selective omega-hydroxylase of saturated, medium-chain fatty acids  <cit> .
the cyp <dig> s appear to have less involvement in xenobiotic metabolism than many cyp <dig> s, cyp <dig> s or cyp <dig> s, although some xenobiotics  induce cyp4ts possibly via peroxisome proliferator-activated receptor alpha  and gamma   <cit> .
zebrafish cyp4f <dig> and the cyp4vs share synteny with their human counterparts, but zebrafish cyp4t <dig> does not occupy a shared syntenic position with other cyp <dig> s.
developmental expression
using single-color agilent custom whole-transcriptome microarrays, we analyzed the expression of  <dig> cyp genes over the course of early development in zebrafish .
zebrafish embryos were staged and sampled from  <dig> hours post-fertilization  through  <dig> hpf, and four biological replicates were sampled and analyzed .
after normalization, probes exhibiting saturation or signals that were not above background were removed , leaving  <dig> probes for examination of expression patterns.
none of the excluded probes were for cyps, indicating that all  <dig> cyps examined were expressed during development.
bayesian estimation of temporal regulation  showed  <dig> significant differentially expressed probes , similar to the results of a naïve anova analysis.
of the  <dig> cyp genes examined,  <dig> were found not to have differential expression during development.
all others had one or more probes that indicated significant differential expression between sampling times.
expression patterns of a subset of  <dig> cyps were assessed using qpcr.
molecule counts were calculated by using plasmid-derived standard curves, and expression levels were normalized to arnt <dig>  we previously observed  <cit>  that arnt <dig> has significantly less variability during development than β-actin, a conclusion supported here by the single-color microarray data .
nine of the eleven cyps examined with qpcr exhibited linear relationships between the arnt2-normalized microarray data and the arnt2-normalized qpcr molecule counts .
patterns of expression
several zebrafish cyps exhibited expression levels that varied by  <dig> fold or greater  during development.
among these are cyp2p <dig>  cyp11a <dig>  cyp26a <dig>  cyp26b <dig>  cyp2aa7-cyp2aa <dig>  cyp2aa <dig>  cyp2y <dig>  cyp2y <dig>  and cyp <dig> .
some had highest levels of expression at the earliest time point .
others showed a peak at intermediate times, and still others showed elevated expression at the latest time sampled .
previous investigators have observed similar variation in expression levels for some of these cyps, including cyp2p <dig>  <cit> , cyp11a <dig>  <cit> , and the cyp <dig> s  <cit> .
an alternate way of depicting developmental expression is a within-gene normalized heat-map, which shows the relative expression of all cyps over the course of development .
while the absolute expression levels of different genes may be low, especially in comparison to "housekeeping" genes such as β-actin or arnt <dig>  within-gene normalization can reveal the relative changes in gene expression throughout the time series, and allow more ready comparison of profile clusters.
this approach revealed a striking picture of waves of cyp gene expression as development progresses.
the waves of cyp expression occurred with different clusters expressed most strongly usually only at one or two sequential time points  during development.
cyp2k <dig>  cyp2n <dig>  cyp3a <dig>  and cyp8b <dig> appear to have bimodal expression, with elevated levels both at  <dig> and  <dig> hpf .
a similar bimodal expression pattern was seen with cyp5a <dig>  cyp21a <dig>  cyhp2k <dig>  cyp8b <dig>  and cyp4t <dig>  which had the highest relative expression at 3hpf and  <dig> hpf.
clustering of gene expression patterns  revealed co-expression of tandemly duplicated cyps, evident in the expression clustering of subsets of the cyp2aas, the cyp2ks, the cyp2ys, the cyp46as, and the cyp27as .
however, other clusters, such as the set of cyp <dig> subfamilies that share synteny with human cyp2j <dig> did not exhibit complete co-expression: cyp2p <dig>  cyp2p <dig>  cyp2ad <dig>  and cyp2ad <dig> were mostly highly expressed at  <dig> hpf, while cyp2p <dig>  cyp2p <dig>  and cyp2v <dig> were most highly expressed at 6- <dig> hpf.
the observation that some cyps showed high levels of transcript at  <dig> hpf, implies that these could involve maternally-derived transcript in oocytes.
a number of cyp genes identified by cluster affinity search analysis using mev  <cit>  appear to have a maternal signal in the microarray data, based on the decreasing expression levels from  <dig> hpf .
to begin assessing maternal transcript we examined selected cyp in unfertilized eggs.
qpcr analysis of unfertilized eggs showed that cyp1a, cyp2v <dig>  cyp2aa <dig>  and cyp <dig> had significant arnt2-normalized expression levels in unfertilized eggs .
analysis of the larger complement of cyp genes for maternal transcript in unfertilized eggs requires microarray analysis, and is ongoing.
discussion
in this study we identified the total cyp complement in zebrafish, and assessed patterns of expression of these genes during normal embryonic development.
zebrafish are an increasingly important model in developmental toxicology, pharmacology and chemical effects on disease.
knowing the identity and regulation of cyp genes is essential to strong inference regarding chemical effects in this model, and to assess pathways of metabolism of xenobiotics and endobiotics, and the relationship to cyp roles in these processes in humans.
the  <dig> zebrafish cyps occur in the same  <dig> gene families that are found in humans and other mammals, but with differences in numbers of genes, and often with uncertain function.
cyp families 5-51
many of the cyp gene families in this group have single genes in zebrafish, as they do in humans, and show a high degree of conservation of sequence with their human counterparts.
these genes also exhibit similar syntenic relationships as found in human.
together, the sequence data and gene location data indicate that in many of these families the genes are direct orthologs of their human  counterparts.
where there is 1: <dig> correspondence in these gene families, i.e., with cyp5a <dig>  7a <dig>  7b <dig>  8a <dig>  20a <dig>  21a <dig>  24a <dig>  26a <dig>  26b <dig>  39a <dig>  46a <dig> and 51a <dig>  it most likely indicates conservation of enzyme activities and physiological function.
in other gene families in the "endogenous set", defining relationships between human cyps with important endogenous functions and the zebrafish homologs is complicated by the presence of multiple closely related paralogs in zebrafish, not found in human.
thus, zebrafish have two cyp17as, two cyp19as, and four cyp46as, while humans have only half that number in each case.
such doubling of the numbers of genes in several cyp gene families could be the result of individual gene duplication, or could be remnants of the third round of whole genome duplication , with the retention of duplicated genes in zebrafish.
zebrafish cyp paralogs that are co-orthologs of the human cyps could have distinct functions, as a result of function partitioning  or differential regulation .
this has been observed for cyp <dig>  with cyp19a <dig>  and cyp19a <dig>  aromatases displaying distinct expression patterns and inducibility.
the neural form  exhibits sensitivity to induction by estrogen, while the ovarian form appears to be mostly recalcitrant to induction by estrogen receptor agonists  <cit> .
while functions have not been confirmed for zebrafish, the two cyp17a genes in other fish also appear to represent enzyme sub-functionalization following gene duplication.
tilapia and medaka cyp17a <dig> possess both steroid-17α-hydroxylase and  <dig>  20-lyase activities, as does mammalian cyp17a <dig>  but both tilapia and medaka cyp17a <dig> possess only 17α-hydroxylase activity, as they only convert pregnenolone or progesterone to 17α- hydroxy products, but do not perform the subsequent conversion to androstenedione or dhea  <cit> .
there also are significant differences in spatial expression patterns for the duplicated cyp17a genes during development, and during the spawning cycle in other fish  <cit> .
we did not see any substantial temporal separation of cyp17a <dig> and cyp17a <dig> expression during development, although cyp17a <dig> is more strongly expressed .
in some families of "endogenous" genes, zebrafish have more than twice the number of genes than occur in humans, and some have novel subfamilies as well.
this is evident in the cyp27s, and the cyp46s, where the numbers of genes are greater than would be expected to have resulted from wgd- <dig>  it is likely that the ancestral condition would be one of fewer genes, with expansion in zebrafish rather than loss in humans.
that expansion in zebrafish could involve wgd, but is most evident in tandem duplication as well as translocation.
this is clearly suggested in cyp <dig> by the four genes that share synteny with the single human cyp27a <dig>  and the presence of cyp27b <dig> and 27c <dig>  which do not share synteny with human cyps.
the biological significance of some duplicated genes also could involve distinctions in temporal or organ- and cell-specific regulation, but determining this can be complicated by the strong possibility of substrate overlap.
cyp families 1-4
zebrafish cyp genes in families  <dig>   <dig>  and  <dig> are more diverse than in humans, and sequence identities often are too low to discern orthology between zebrafish and mammalian genes in these families.
however, analysis of the additional character of shared synteny clarifies evolutionary relationships between human and zebrafish genes in these families.
cyp family  <dig> differs from families 1- <dig>  as there are fewer cyp <dig> genes in zebrafish than there are in mammals.
however, like the cyp <dig> s, cyp <dig> s and cyp <dig> s, the cyp <dig> s also are involved with  xenobiotics, while this is seldom the case with cyp5-cyp <dig> genes.
consistent with phylogeny, the fish cyp <dig> and cyp <dig> clades appear as sister groups to the mammalian clades for these genes.
however, as discussed earlier  <cit> , both mammals and fish share the cyp <dig> subfamilies cyp1a and cyp1b.
zebrafish also express cyp1cs, which do not occur in humans, and cyp1d <dig>  a pseudogene in human.
all mammalian cyp <dig> s are in a single subfamily, cyp3a, which occurs in zebrafish as well.
however, zebrafish cyp3a <dig> shares synteny with single exon pseudogene cyp3as in human , while the novel cyp3c subfamily  <cit>  shares synteny with the functional human cyp3a <dig>  3a <dig> and 3a <dig>  <cit> .
fugu and possibly other fishes also have a second cyp <dig> subfamily, cyp3b, about which nothing is known  <cit> .
the functional similarities in different taxa, suggest similar biological roles for the homologous cyp <dig> s and cyp <dig> s. thus, cyp3as are the primary catalysts of testosterone 6β-hydroxylase in fish and mammals  <cit> , and mammalian and fish cyp1as and likely cyp1bs are prominent in the metabolism of some pah pro-carcinogens.
the roles of orthologous cyps in metabolism of particular compounds can differ between taxa, however.
thus, the regio-specific oxidation of bap and the rates of metabolism of planar hah appear to differ in degree between fish and mammalian cyp1as  <cit> , apparently reflecting species differences in cyp1a structures  <cit> .
the functions of the novel cyp <dig> s and cyp <dig> s are less well defined, although zebrafish cyp1cs and cyp1d <dig> have been expressed and functions have been determined with bap  <cit> , estradiol  <cit> , and a number of other exogenous and endogenous substrates .
little is known of the function of the cyp3cs, but cyp3c <dig> appears not to be responsive to chemicals that induce cyp3a <dig>  <cit> .
identifying zebrafish-human orthologs is most difficult in the cyp <dig> family, where the differences in cyp <dig> divergence between zebrafish and humans obscure many homologous relationships.
thus as noted, of the  <dig> zebrafish and  <dig> human cyp <dig> subfamilies, only two  warrant the same designation in zebrafish as in humans based on sequence identity.
while the disparity between zebrafish, or other fish, and mammals is exaggerated by evolutionary distance, our analysis of shared synteny indicates that members of distinct subfamilies in mammals and fish still may bear co-orthology.
the cyp2j-related genes are a key example.
the single clade of  <dig> genes in the zebrafish cyp2n, 2p, 2v, and 2ad subfamilies and human cyp2j <dig> , implies relationship between the fish and human genes.
our analysis shows the zebrafish genes occur in tandem in a cluster that shares synteny with cyp2j <dig>  indicating co-orthology .
this suggests that there are catalytic functions among these zebrafish cyps that are similar to the human cyp2j <dig>  a similar hypothesis was borne out in functional characterization of previously identified killifish cyp2p <dig>  which also clusters with the cyp2js on phylogenetic analysis.
heterologously expressed cyp2p <dig> exhibited nearly identical regio- and stereoselectivity for oxidation of arachidonic acid as human cyp2j <dig>  consistent with molecular phylogeny indicating a shared ancestral origin  <cit> .
notably, at present there is little or nothing known about the catalytic or biological functions or the chemical regulation of the majority of zebrafish cyp <dig> s.
zebrafish cyps in development
in addition to annotating the full complement of cyp genes, we analyzed the expression of  <dig> cyp genes over the course of development in zebrafish .
several of these zebrafish cyps exhibited markedly elevated expression levels at some time during development, but a large number, including more than three-quarters of the total genomic complement, showed distinct temporal expression patterns .
importantly, gene expression profiling performed on whole embryos often under-estimates the importance of tissue- or cell-specific gene expression due to dilution effects.
our array results are similar to developmental expression that has been determined for some individual cyp genes .
developmental roles have been established principally for some of the "endogenous" cyps.
such genes include cyp11a <dig>  which is essential for the synthesis of pregnenolone, critical for cell migration  <cit> ; and the three cyp <dig> s, which contribute to retinoic acid gradients that regulate hindbrain and neural crest patterning  <cit>  and osteogenesis  <cit> .
we and others have seen complex cyp expression patterns in development.
developmental roles of cyps in families 1- <dig> are unknown, although morpholino knockdown of cyp1cs appears to protect from developmental toxicity of dioxin  and over-expression of cyp2p <dig> caused developmental abnormalities, including cardiovascular abnormalities  <cit> , suggesting developmental significance of these genes.
the roles for many cyps, including roles in development, cannot necessarily be inferred from sequence identities.
thus, cyp20a <dig> is an 'orphan' cyp that does not have a defined function in zebrafish or in humans  <cit> , and some large subfamilies  do not have any homologs in mammals.
as well, the different numbers of genes that are co-orthologs of single human cyps precludes assignment of a function to any one, which requires empirical determination.
this is true for the multiple co-orthologs in the "endogenous" cyp families, such as the cyp <dig> s, the cyp <dig> s and cyp46's, as well as for most those in the "xenobiotic" cyp families.
the issues in cyp <dig> exemplify the questions and approaches.
zebrafish cyp11a <dig> is expressed throughout development, as in the murine model, but the cyp11a <dig> knockdown is not lethal  <cit> .
as zebrafish have two cyp11a genes and a cyp11c gene, it is possible that overlapping substrate specificity and spatiotemporal expression patterns might allow one to substitute for the other in loss-of-function studies.
there is a greater dearth of information regarding cyp genes that may have maternally derived transcripts deposited in oocytes.
our analysis of transcripts of a few selected cyp genes, cyp1a, cyp2v <dig>  cyp2aa <dig>  and cyp20a <dig>  showed that transcripts for all four were present in unfertilized zebrafish eggs, and that the levels of transcript could be substantial.
cyp <dig> mrna also has been reported in oocytes  <cit> , and cyp1a transcript also was reportedly recently by others  <cit> .
the significance of maternal transcript of these cyps is not known.
whether other cyps also have maternal transcripts deposited in oocytes, and what influences that deposition, is under investigation.
CONCLUSIONS
the identification of the full complement of  <dig> zebrafish cyps, and determination that the majority of cyps have distinct developmental patterns of expression opens the door to assessing their role not only in development, but also in the response of embryos to toxic chemicals.
for many cyp genes involved in xenobiotic metabolism, inferring relationships among the genes between zebrafish and human based on sequence is difficult, complicated by remnants of whole genome duplication in the teleost line, uneven gene duplication and gene loss in different animal and gene lineages, and the possibility of gene conversion of closely linked paralogs  <cit> .
nevertheless, shared synteny indicates orthologous relationships of many zebrafish and human cyps, and also indicates which zebrafish cyps have no apparent homologs in humans.
the lineage-specific diversification makes possible the acquisition of new functions  and regulation.
in most cases, however, functions of the individual paralogous cyps in zebrafish have yet to be determined.
those functions, and the timing, location and magnitude of expression in development will determine the strength of inference from developmental toxicological studies with the zebrafish model.

 the cytochrome p enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. mammalian cyps can be separated into two major groups: those with generally narrow substrate specificity that are involved primarily in synthesis, activation or inactivation of endogenous regulatory molecules, and those involved most heavily in the metabolism of xenobiotics, but which may act as well in the metabolism of endogenous compounds. thus, cyp enzymes can determine the persistence and action of endogenous regulatory molecules as well as many drugs and other toxicants and carcinogens. an area growing in importance is the role of cyps in development, and in developmental toxicity of chemicals. absolute embryo lethality upon knockout of the murine cyp oxidoreductase demonstrates the essential role of cyp enzymes in murine development. many cyps potentially have roles in developmental specification. for example, it is well known that cyp enzymes regulate levels of retinoids governing pattern formation during development. cyp isoforms that play essential roles in bile acid homeostasis, steroidogenesis, the vitamin d pathway, and the catabolism of many hormones may also have important roles in development. many chemicals that cause developmental abnormalities, including cardiovascular, neural, and connective tissue defects, are substrates for cyps, and the oxidative biotransformation of such xenobiotics may determine the cellular and organ targets of those chemicals. cyp enzymes that are prominent in xenobiotic metabolism could also function in producing morphogenic molecules or keeping regions free of them, creating temporal and spatial regions of morphogen action and supporting region-specific changes essential for successful development. the roles and regulation of most xenobiotic metabolizing cyps during development are unknown, impeding understanding of mechanisms of developmental toxicity. many xenobiotic metabolizing cyp can be induced via transcription factors. all cyps can be targets of exogenous chemicals that disrupt or enhance their function, whether or not they are directly involved in xenobiotic metabolism. the zebrafish has emerged as one of the most important vertebrate model species in embryology and developmental biology, due largely to rapid development coupled with the possibility for genetic analysis and manipulation. multiple draft assemblies of the zebrafish genome have been produced by the wellcome trust sanger institute. the near completion of the zebrafish genome has made it possible to address questions of cyp gene identity and expression during zebrafish development with substantial clarity and completeness. our objective was to identify and annotate the full suite of zebrafish cyp genes and examine their phylogenetic and shared syntenic relationships to human cyps. we also examined cyp expression during early development in zebrafish. cyp expression in vertebrate development has not been completely mapped for any species; prior studies in mice examined developmental expression of cyp genes, less than half of the mouse cyp complement. our analyses identify novel genes and clusters of cyp genes in zebrafish, and unanticipated waves of cyp expression during development.  repeated and exhaustive searching of zebrafish genome assemblies uncovered a total of cyp genes. based on inferred amino acid sequences, these genes fell into cyp gene families that are also found as well in humans and other mammals. molecular phylogenetic analysis shows relationships among cyp genes and gene families in zebrafish and human. as with the human cyp genes, the cyp genes in zebrafish occur in two major functional groups: those families that include enzymes involved primarily in endogenous functions; and those that include enzymes involved heavily in oxidation of xenobiotics. zebrafish genes in these two major groups are described in detail below, considering relationship to human cyps based on sequence identity, syntenic analysis and, where known, functional properties. the term cyp is fully capitalized in all cases except for mouse, rat, and drosophila, and italicized only when referring to a specific gene or cdna, consistent with the nomenclature committee recommendations. a incorrect predicted transcript; b incomplete best blast hit chr 58640777-58651921; c two different genes concatenated into one prediction; d originally published as cyp26d1; e zv ensdarg chromosome 20 a genewise reprediction; b no ensembl gene prediction; c identical dna; d identical dna; e incorrect n-term exon; f chr is probably best location; g there are non-mammalian genes that share synteny but not the same subfamily name with mammalian cyp2j s. at present, annotation of fish cyp s by blast can result in misclassifying fish cyp s as cyp2js, as strictly interpreted according to nomenclature rules, there is not a cyp2j subfamily in fish. cyp families 5-51 cyp. zebrafish cyp5a retains 48% sequence identity with human cyp5a and is located in a region of shared synteny with cyp5a in humans, indicating orthology. cyp zebrafish possess one six-exon cyp7a and one six-exon cyp7b gene, both located on chromosome the sequence identity between zebrafish cyp7b and mammalian cyp7bs technically precludes membership in the same subfamily, yet shared synteny indicates orthology. zebrafish also have a third cyp gene, cyp7c a cyp7a-like gene located on chromosome possibly an ohnolog resulting from the fish-specific whole genome duplication. a pseudogene cyp a 10-exon zebrafish cyp8a gene is located on chromosome human cyp8a catalyzes the rearrangement of pgh to prostaglandin i. a crystal structure of zebrafish cyp8a published in as part of a comparative structural analysis of prostacyclin synthases, shows that zebrafish and human cyp8a exhibit nearly identical ligand-bound and ligand-free three-dimensional structures, despite exhibiting only 45% identity. zebrafish also possess three cyp8bs, which are single-exon genes located on chromosome that share synteny with the single-exon human cyp8b cyp zebrafish have cyp11a genes, cyp11a and cyp11a located adjacent to one another on chromosome and one cyp11b-like gene, termed cyp11c. vertebrate cyp11a synthesizes pregnenolone from cholesterol, and a maternally-derived cyp11a mrna has been suggested to form pregnenolone important in cell migration in the zebrafish zygote. following the onset of embryonic transcription, cyp11a is expressed in the yolk syncytial layer. it is then expressed in the embryonic interrenal primordia, and in the interrenal glands into adulthood. in adult zebrafish it is expressed in the gonads, brain, and interrenal glands. knockdown of zebrafish cyp11a leads to a shortened axis and epiboly defects. zebrafish cyp11a is 80% identical to cyp11a at the amino acid level, but little is known about functional similarities between the two forms, or about cyp11a expression, although based on our microarray results it appears to be expressed in the early embryo, and database est evidence indicates that it is also expressed in adult gonads. zebrafish cyp11c exhibits 42% amino acid identity to human cyp11b, but does not share synteny with any tetrapod cyp11b genes. cyp cyp enzymes catalyze dual functions of steroid-17α-hydroxylase and steroid- 20-lyase. the zebrafish genome has two cyp17a genes that are 49% identical at the amino acid level, and the two genes are located on different chromosomes. one of these was cloned from zebrafish ovary. cyp17a is expressed in gonadal tissue, but also in brain, gill, liver, and intestine. zebrafish cyp17a is upregulated by fluorotelomer alcohols, but not by benzopyrene. the functions of the two cyp s may differ. cyp zebrafish have two distinct cyp genes, cyp19a and cyp19b reported initially by kishida and callard. the two aromatase forms are expressed in different tissues; cyp19a is expressed principally in the ovary, but also at much lower levels in the testis, while cyp19b is expressed in brain and neural tissue, including in the olfactory bulb, ventral telencephalon, preoptic area, and ventral/caudal hypothalamic zone, and in the anterior and posterior lobes of the pituitary. estrogen-mediated upregulation has been observed only for the neural form, cyp19b in adults and in embryos after hpf. cyp20a zebrafish have one cyp gene, cyp20a 61% identical to the cyp20a in humans, and they share synteny, both being adjacent to abi at present, the substrate specificity and biological function of cyp20a are unknown, and it remains an "orphan" cyp. however, there is evidence for high levels of mrna expression in the hippocampus and substantia nigra of mouse, suggesting functions in these parts of the brain implicated in important neuropathies. cyp zebrafish cyp21a shares 39% identity with human cyp21a and shares synteny with the human cyp21a gene pair. mammalian cyp21a and cyp21a convert progesterone to deoxycorticosterone via 21-hydroxylation, prominently in the adrenal gland. the zebrafish cyp21a presumably serves a similar function, in the interrenal. cyp cyp24a is a mitochondrial enzyme catalyzing 1α, 25-dihydroxyvitamin-d3-24-hydroxylase. there is one cyp24a gene in zebrafish, with 63% percent amino acid identity to human cyp24a the zebrafish enzyme is presumed to function similarly to mammalian cyp s, as vitamin d and various metabolites circulate in fish serum and zebrafish have a functional vitamin d receptor with ligand specificity similar to other vertebrate vdrs. cyp there are three cyp s in zebrafish. cyp was first discovered in zebrafish and the retinoic acid metabolizing function appears to be conserved throughout the vertebrates. each zebrafish cyp is located on a separate chromosome, suggesting early establishment of these duplicated genes. the cyp s are categorized into three separate subfamilies, with some confusion as to the proper classification, i.e., zebrafish cyp26c has previously been referred to as cyp26d. cyp26a and cyp26b share synteny with human cyp26a and 26b genes, respectively, while the cyp26c syntenic relationship is less distinct. the three zebrafish cyp s are all induced by and metabolize retinoic acid, and play essential roles in hindbrain patterning. zebrafish cyp s also play roles in osteogenesis, in pancreatic development, and presumably other processes. cyp zebrafish have seven distinct cyp genes, distributed into three subfamilies, while in most mammals there are two or three cyp genes. human cyp27a and cyp27b catalyze vitamin d 25-hydroxylase and 25-hydroxyvitamin d 1α-hydroxylase, respectively. the five zebrafish cyp27a genes are all tandemly located on chromosome and share synteny with human cyp27a. zebrafish cyp27b and cyp27c are located on different chromosomes. functional properties of the various zebrafish cyp s are unknown. cyp a cyp39a is present in the zebrafish genome, located on chromosome in mammals cyp39a functions as an oxysterol-7α-hydroxylase, involved in the conversion of cholesterol to bile acids. cyp39a has not been found in other published fish genomes, but is present in tetrapod genomes, with conserved synteny to human cyp39a zebrafish cyp39a is approximately 4mb from a cluster of genes that share synteny with human cyp39a cyp zebrafish have four cyp46a genes. one is on chromosome two are arranged in tandem on chromosome a fourth is supported by est data but missing from the current assembly. in contrast, humans have one cyp cyp46a human cyp46a functions both as a cholesterol 24-hydroxylase and as a 24-hydroxycholesterol-hydroxylase. cyp46a in mammals is a brain-specific cholesterol-metabolizing enzyme, important for maintaining brain cholesterol homeostasis and membrane function, but also with potential regulatory roles as a neurosteroid-metabolizing enzyme. cyp zebrafish possess one cyp gene, cyp51a in other animals cyp is a lanosterol-demethylase enzyme, the only cyp involved in the post-squalene portion of the cholesterol biosynthesis pathway. cyp is an essential enzyme in the de novo synthesis of cholesterol ). cyp is often considered to be a "housekeeping gene" based on its ubiquitous expression and conserved function. zebrafish cyp has been cloned and heterologously expressed in e. coli. microarray analysis and in situ hybridization of embryonic zebrafish showed that cyp is strongly expressed in the eye, in the brain, and in epidermal cells of the forehead and the tail fin, and suggesting a role for cyp and other isoprenoid synthesis enzymes in hematopoietic and vascular development. cyp families 1- cyp families and include enzymes involved in drug, xenobiotic and fatty acid metabolism. in contrast to the cyps that have principally endogenous functions, these cyp genes are much more diverse, with much less conservation of sequence between zebrafish and human. table provides specific information on the cyp 1- family genes. a molecular phylogeny of clan genes, which includes families and as well as cyp and cyp is shown in figure molecular phylogeny of the clan genes, including the cyp s and cyp s and clan genes can be seen in figure some relationships between zebrafish and human cyps in families 1- suggested by molecular phylogeny have been substantiated by synteny analysis. clan 2- cyp1s zebrafish have five cyp genes in four subfamilies, cyp1a, cyp1b cyp1c cyp1c and cyp1d all of which have been cloned and sequenced. zebrafish cyp1a has exon structures similar to the human cyp1a and cyp1a and the single cyp1b gene has a gene structure very similar to human cyp1b the cyp1cs are closely linked single-exon genes. the recently identified zebrafish cyp1d has a gene structure identical to zebrafish cyp1a, and quite different from the cyp1b and cyp1cs. members of all four cyp subfamilies have been identified in other fishes and non-mammalian tetrapods, and the evolution of the cyp family has been discussed elsewhere in some detail. notably, the cyp1cs are absent and cyp1d is a pseudogene in human and some other mammals. cyp1a, cyp1b and the cyp1cs are inducible by ahr agonists. vertebrate cyp1as and cyp1b generally are involved in the metabolism of various drugs, many hydrocarbons, steroids and fatty acids. with the exception of benzopyrene and estradiol, little is known of the possible xenobiotic and endogenous substrates of the cyp1cs and of cyp1d clan - cyp2s as in mammals, the cyp s constitute the largest cyp gene family in zebrafish, with cyp genes, in contrast to in humans. the subfamilies are considered below, in order according to suggested relationships: orthology indicated by sequence, homologs suggested by sequence and orthology confirmed by shared synteny, and no evident homologous relationship to human cyps. cyp2r and cyp2u there are single genes in each of these two subfamilies in zebrafish, cyp2r and cyp2u these can be classified as orthologs of human cyp2r and cyp2u based on sequence identity. they also exhibit shared synteny with their respective human counterparts. cyp2r is a microsomal vitamin d 25-hydroxylase in humans. human cyp2u is expressed in the brain and the thymus, and catalyzes ω and ω- hydroxylation of fatty acids, including arachidonic acid. cyp2u appears to have a pre-vertebrate origin, and may be the oldest identifiable vertebrate cyp subfamily. cyp2n, cyp2p, cyp2v, cyp2ad the zebrafish genes in these five subfamilies occur in a clade together with human cyp2j. analysis of gene location showed that the six cyp2ps and cyp2n cyp2v cyp2ad cyp2ad and cyp2ad all are arranged in tandem on chromosome this cluster of cyp genes shares synteny with human cyp2j indicating that all zebrafish genes at this locus share an ancestral origin with cyp2j. cyp2j is an arachidonic acid epoxygenase with roles in the cardiovascular system. functions of the members of the zebrafish cluster are unknown, although related genes in killifish are arachidonic acid hydroxylases and cyp2p in particular is similar in function to human cyp2j. cyp2k eight cyp2k genes are present in a tandemly duplicated array on chromosome this cluster of cyp2ks shares synteny with human cyp2w. human cyp2w is a tumor-specific cyp that oxidizes indole and chlorzoxazone, but not fatty acids. cyp2k which is not highly expressed until dpf, has been heterologously expressed, and demonstrated to catalyze the activation of the mycotoxin aflatoxin b to the carcinogenic exo- -afb epoxide. the low level cyp2k expression during zebrafish embryo and larval stages may help to explain the lack of afb toxicity in zebrafish embryos. functions of the other cyp2ks are not known. cyp2y cyp2y and cyp2y are tandemly arranged on chromosome they share synteny with a cluster of cyp genes for human drug-metabolizing enzymes, including cyp2a cyp2a cyp2b cyp2f and cyp2s located on human chromosome. the human genes at this locus include genes induced by pxr agonists and genes induced by ahr agonists. whether cyp2y and cyp2y have functional or regulatory properties in common with any of these human cyps is not known. cyp2x there are nine cyp2x genes present in two separate arrays of tandemly duplicated genes, on chromosomes and two of the cyp2x genes, have two exact copies in the current genome assembly likely as an artifact of the assembly process, so there is some uncertainty as to the exact number of cyp2xs. the cyp2x genes do not appear to share synteny with any mammalian cyp. catalytic activities of cyp2xs in zebrafish are unknown, although cyp2x from channel catfish has been cloned and heterologously expressed. aminopyrine and benzphetamine demethylase activities were observed, but no biological function has as yet been ascribed. cyp2aa due to overlapping gene predictions, the current genome assemblies and gene counts are not accurate in the region on chromosome where the cyp2aa genes are located. ten cyp2aa genes are present in a tandemly duplicated array on chromosome genewise predictions indicate that there are cyp2aas, with high identity to each other, but no clear orthology to other cyp s. at least three have been cloned. some cyp2aa genes appear to be induced by pxr agonists. the cyp s most closely related to the cyp2aas are the cyp2xs, and cyp2r. cyp2ae the two cyp2ae genes are present as tandem duplicates on chromosome approximately kb downstream from the cyp2aa cluster. no shared synteny is evident with mammalian genomes, and is unclear even for other published fish genomes. however, the cyp2aes cluster phylogenetically with the cyp2j orthologs cyp2n/p/v/ae, suggesting a possible insertion of a set of duplicated cyp2j orthologs. clan - cyp3s the zebrafish genome includes five cyp genes, cyp3a and four cyp3cs, cyp3c1-3c.cyp3a and cyp3c were previously cloned and heterologously expressed. cyp3a is a exon gene located in chromosome and is 54% identical to human cyp3a zebrafish cyp3c1-cyp3c are all 13-exon genes located in tandem on zebrafish chromosome. other fish species also have multiple cyp genes located in this region, and chickens appear to have two homologs at that locus. it is likely that the previously named human pseudogenes cyp3a-se and cyp3a-se are the orthologous remnants of these cyp3s, as they are located in the same region as the immediately adjacent foxk1gene. qiu et al. found that in most vertebrates apparently intact cyp genes are located within one of two genomic regions, labeled cyp3hr and cyp3hr. zebrafish cyp3cs are within cyp3hr while the human cyp3as are in cyp3hr zebrafish cyp3a does not share synteny with cyp s in other fish for which genomic information is available. cyp3a is inducible by some pxr agonists. other fish cyp3as are prominent in catalyzing testosterone 6β-hydroxylase, and this is likely true for zebrafish cyp3a as well. activities are not known for the other zebrafish cyp s. clan - cyp4s there are four cyp genes in zebrafish, cyp4f cyp4t cyp4v and cyp4v, in contrast to humans, which have cyp s. mammalian cyp4f enzymes function as omega-hydroxylases of c16-c fatty acids, including eicosanoids such as leukotriene b. the full range of substrates of cyp4v enzymes is not yet known, although human cyp4v is a selective omega-hydroxylase of saturated, medium-chain fatty acids. the cyp s appear to have less involvement in xenobiotic metabolism than many cyp s, cyp s or cyp s, although some xenobiotics induce cyp4ts possibly via peroxisome proliferator-activated receptor alpha and gamma. zebrafish cyp4f and the cyp4vs share synteny with their human counterparts, but zebrafish cyp4t does not occupy a shared syntenic position with other cyp s. developmental expression using single-color agilent custom whole-transcriptome microarrays, we analyzed the expression of cyp genes over the course of early development in zebrafish. zebrafish embryos were staged and sampled from hours post-fertilization through hpf, and four biological replicates were sampled and analyzed. after normalization, probes exhibiting saturation or signals that were not above background were removed, leaving probes for examination of expression patterns. none of the excluded probes were for cyps, indicating that all cyps examined were expressed during development. bayesian estimation of temporal regulation showed significant differentially expressed probes, similar to the results of a naïve anova analysis. of the cyp genes examined, were found not to have differential expression during development. all others had one or more probes that indicated significant differential expression between sampling times. expression patterns of a subset of cyps were assessed using qpcr. molecule counts were calculated by using plasmid-derived standard curves, and expression levels were normalized to arnt we previously observed that arnt has significantly less variability during development than β-actin, a conclusion supported here by the single-color microarray data. nine of the eleven cyps examined with qpcr exhibited linear relationships between the arnt2-normalized microarray data and the arnt2-normalized qpcr molecule counts. patterns of expression several zebrafish cyps exhibited expression levels that varied by fold or greater during development. among these are cyp2p cyp11a cyp26a cyp26b cyp2aa7-cyp2aa cyp2aa cyp2y cyp2y and cyp. some had highest levels of expression at the earliest time point. others showed a peak at intermediate times, and still others showed elevated expression at the latest time sampled. previous investigators have observed similar variation in expression levels for some of these cyps, including cyp2p, cyp11a, and the cyp s. an alternate way of depicting developmental expression is a within-gene normalized heat-map, which shows the relative expression of all cyps over the course of development. while the absolute expression levels of different genes may be low, especially in comparison to "housekeeping" genes such as β-actin or arnt within-gene normalization can reveal the relative changes in gene expression throughout the time series, and allow more ready comparison of profile clusters. this approach revealed a striking picture of waves of cyp gene expression as development progresses. the waves of cyp expression occurred with different clusters expressed most strongly usually only at one or two sequential time points during development. cyp2k cyp2n cyp3a and cyp8b appear to have bimodal expression, with elevated levels both at and hpf. a similar bimodal expression pattern was seen with cyp5a cyp21a cyhp2k cyp8b and cyp4t which had the highest relative expression at 3hpf and hpf. clustering of gene expression patterns revealed co-expression of tandemly duplicated cyps, evident in the expression clustering of subsets of the cyp2aas, the cyp2ks, the cyp2ys, the cyp46as, and the cyp27as. however, other clusters, such as the set of cyp subfamilies that share synteny with human cyp2j did not exhibit complete co-expression: cyp2p cyp2p cyp2ad and cyp2ad were mostly highly expressed at hpf, while cyp2p cyp2p and cyp2v were most highly expressed at 6- hpf. the observation that some cyps showed high levels of transcript at hpf, implies that these could involve maternally-derived transcript in oocytes. a number of cyp genes identified by cluster affinity search analysis using mev appear to have a maternal signal in the microarray data, based on the decreasing expression levels from hpf. to begin assessing maternal transcript we examined selected cyp in unfertilized eggs. qpcr analysis of unfertilized eggs showed that cyp1a, cyp2v cyp2aa and cyp had significant arnt2-normalized expression levels in unfertilized eggs. analysis of the larger complement of cyp genes for maternal transcript in unfertilized eggs requires microarray analysis, and is ongoing. discussion in this study we identified the total cyp complement in zebrafish, and assessed patterns of expression of these genes during normal embryonic development. zebrafish are an increasingly important model in developmental toxicology, pharmacology and chemical effects on disease. knowing the identity and regulation of cyp genes is essential to strong inference regarding chemical effects in this model, and to assess pathways of metabolism of xenobiotics and endobiotics, and the relationship to cyp roles in these processes in humans. the zebrafish cyps occur in the same gene families that are found in humans and other mammals, but with differences in numbers of genes, and often with uncertain function. cyp families 5-51 many of the cyp gene families in this group have single genes in zebrafish, as they do in humans, and show a high degree of conservation of sequence with their human counterparts. these genes also exhibit similar syntenic relationships as found in human. together, the sequence data and gene location data indicate that in many of these families the genes are direct orthologs of their human counterparts. where there is 1: correspondence in these gene families, i.e., with cyp5a 7a 7b 8a 20a 21a 24a 26a 26b 39a 46a and 51a it most likely indicates conservation of enzyme activities and physiological function. in other gene families in the "endogenous set", defining relationships between human cyps with important endogenous functions and the zebrafish homologs is complicated by the presence of multiple closely related paralogs in zebrafish, not found in human. thus, zebrafish have two cyp17as, two cyp19as, and four cyp46as, while humans have only half that number in each case. such doubling of the numbers of genes in several cyp gene families could be the result of individual gene duplication, or could be remnants of the third round of whole genome duplication, with the retention of duplicated genes in zebrafish. zebrafish cyp paralogs that are co-orthologs of the human cyps could have distinct functions, as a result of function partitioning or differential regulation. this has been observed for cyp with cyp19a and cyp19a aromatases displaying distinct expression patterns and inducibility. the neural form exhibits sensitivity to induction by estrogen, while the ovarian form appears to be mostly recalcitrant to induction by estrogen receptor agonists. while functions have not been confirmed for zebrafish, the two cyp17a genes in other fish also appear to represent enzyme sub-functionalization following gene duplication. tilapia and medaka cyp17a possess both steroid-17α-hydroxylase and 20-lyase activities, as does mammalian cyp17a but both tilapia and medaka cyp17a possess only 17α-hydroxylase activity, as they only convert pregnenolone or progesterone to 17α- hydroxy products, but do not perform the subsequent conversion to androstenedione or dhea. there also are significant differences in spatial expression patterns for the duplicated cyp17a genes during development, and during the spawning cycle in other fish. we did not see any substantial temporal separation of cyp17a and cyp17a expression during development, although cyp17a is more strongly expressed. in some families of "endogenous" genes, zebrafish have more than twice the number of genes than occur in humans, and some have novel subfamilies as well. this is evident in the cyp27s, and the cyp46s, where the numbers of genes are greater than would be expected to have resulted from wgd- it is likely that the ancestral condition would be one of fewer genes, with expansion in zebrafish rather than loss in humans. that expansion in zebrafish could involve wgd, but is most evident in tandem duplication as well as translocation. this is clearly suggested in cyp by the four genes that share synteny with the single human cyp27a and the presence of cyp27b and 27c which do not share synteny with human cyps. the biological significance of some duplicated genes also could involve distinctions in temporal or organ- and cell-specific regulation, but determining this can be complicated by the strong possibility of substrate overlap. cyp families 1-4 zebrafish cyp genes in families and are more diverse than in humans, and sequence identities often are too low to discern orthology between zebrafish and mammalian genes in these families. however, analysis of the additional character of shared synteny clarifies evolutionary relationships between human and zebrafish genes in these families. cyp family differs from families 1- as there are fewer cyp genes in zebrafish than there are in mammals. however, like the cyp s, cyp s and cyp s, the cyp s also are involved with xenobiotics, while this is seldom the case with cyp5-cyp genes. consistent with phylogeny, the fish cyp and cyp clades appear as sister groups to the mammalian clades for these genes. however, as discussed earlier, both mammals and fish share the cyp subfamilies cyp1a and cyp1b. zebrafish also express cyp1cs, which do not occur in humans, and cyp1d a pseudogene in human. all mammalian cyp s are in a single subfamily, cyp3a, which occurs in zebrafish as well. however, zebrafish cyp3a shares synteny with single exon pseudogene cyp3as in human, while the novel cyp3c subfamily shares synteny with the functional human cyp3a 3a and 3a. fugu and possibly other fishes also have a second cyp subfamily, cyp3b, about which nothing is known. the functional similarities in different taxa, suggest similar biological roles for the homologous cyp s and cyp s. thus, cyp3as are the primary catalysts of testosterone 6β-hydroxylase in fish and mammals, and mammalian and fish cyp1as and likely cyp1bs are prominent in the metabolism of some pah pro-carcinogens. the roles of orthologous cyps in metabolism of particular compounds can differ between taxa, however. thus, the regio-specific oxidation of bap and the rates of metabolism of planar hah appear to differ in degree between fish and mammalian cyp1as, apparently reflecting species differences in cyp1a structures. the functions of the novel cyp s and cyp s are less well defined, although zebrafish cyp1cs and cyp1d have been expressed and functions have been determined with bap, estradiol, and a number of other exogenous and endogenous substrates. little is known of the function of the cyp3cs, but cyp3c appears not to be responsive to chemicals that induce cyp3a. identifying zebrafish-human orthologs is most difficult in the cyp family, where the differences in cyp divergence between zebrafish and humans obscure many homologous relationships. thus as noted, of the zebrafish and human cyp subfamilies, only two warrant the same designation in zebrafish as in humans based on sequence identity. while the disparity between zebrafish, or other fish, and mammals is exaggerated by evolutionary distance, our analysis of shared synteny indicates that members of distinct subfamilies in mammals and fish still may bear co-orthology. the cyp2j-related genes are a key example. the single clade of genes in the zebrafish cyp2n, 2p, 2v, and 2ad subfamilies and human cyp2j, implies relationship between the fish and human genes. our analysis shows the zebrafish genes occur in tandem in a cluster that shares synteny with cyp2j indicating co-orthology. this suggests that there are catalytic functions among these zebrafish cyps that are similar to the human cyp2j a similar hypothesis was borne out in functional characterization of previously identified killifish cyp2p which also clusters with the cyp2js on phylogenetic analysis. heterologously expressed cyp2p exhibited nearly identical regio- and stereoselectivity for oxidation of arachidonic acid as human cyp2j consistent with molecular phylogeny indicating a shared ancestral origin. notably, at present there is little or nothing known about the catalytic or biological functions or the chemical regulation of the majority of zebrafish cyp s. zebrafish cyps in development in addition to annotating the full complement of cyp genes, we analyzed the expression of cyp genes over the course of development in zebrafish. several of these zebrafish cyps exhibited markedly elevated expression levels at some time during development, but a large number, including more than three-quarters of the total genomic complement, showed distinct temporal expression patterns. importantly, gene expression profiling performed on whole embryos often under-estimates the importance of tissue- or cell-specific gene expression due to dilution effects. our array results are similar to developmental expression that has been determined for some individual cyp genes. developmental roles have been established principally for some of the "endogenous" cyps. such genes include cyp11a which is essential for the synthesis of pregnenolone, critical for cell migration; and the three cyp s, which contribute to retinoic acid gradients that regulate hindbrain and neural crest patterning and osteogenesis. we and others have seen complex cyp expression patterns in development. developmental roles of cyps in families 1- are unknown, although morpholino knockdown of cyp1cs appears to protect from developmental toxicity of dioxin and over-expression of cyp2p caused developmental abnormalities, including cardiovascular abnormalities, suggesting developmental significance of these genes. the roles for many cyps, including roles in development, cannot necessarily be inferred from sequence identities. thus, cyp20a is an'orphan' cyp that does not have a defined function in zebrafish or in humans, and some large subfamilies do not have any homologs in mammals. as well, the different numbers of genes that are co-orthologs of single human cyps precludes assignment of a function to any one, which requires empirical determination. this is true for the multiple co-orthologs in the "endogenous" cyp families, such as the cyp s, the cyp s and cyp46's, as well as for most those in the "xenobiotic" cyp families. the issues in cyp exemplify the questions and approaches. zebrafish cyp11a is expressed throughout development, as in the murine model, but the cyp11a knockdown is not lethal. as zebrafish have two cyp11a genes and a cyp11c gene, it is possible that overlapping substrate specificity and spatiotemporal expression patterns might allow one to substitute for the other in loss-of-function studies. there is a greater dearth of information regarding cyp genes that may have maternally derived transcripts deposited in oocytes. our analysis of transcripts of a few selected cyp genes, cyp1a, cyp2v cyp2aa and cyp20a showed that transcripts for all four were present in unfertilized zebrafish eggs, and that the levels of transcript could be substantial. cyp mrna also has been reported in oocytes, and cyp1a transcript also was reportedly recently by others. the significance of maternal transcript of these cyps is not known. whether other cyps also have maternal transcripts deposited in oocytes, and what influences that deposition, is under investigation.  the identification of the full complement of zebrafish cyps, and determination that the majority of cyps have distinct developmental patterns of expression opens the door to assessing their role not only in development, but also in the response of embryos to toxic chemicals. for many cyp genes involved in xenobiotic metabolism, inferring relationships among the genes between zebrafish and human based on sequence is difficult, complicated by remnants of whole genome duplication in the teleost line, uneven gene duplication and gene loss in different animal and gene lineages, and the possibility of gene conversion of closely linked paralogs. nevertheless, shared synteny indicates orthologous relationships of many zebrafish and human cyps, and also indicates which zebrafish cyps have no apparent homologs in humans. the lineage-specific diversification makes possible the acquisition of new functions and regulation. in most cases, however, functions of the individual paralogous cyps in zebrafish have yet to be determined. those functions, and the timing, location and magnitude of expression in development will determine the strength of inference from developmental toxicological studies with the zebrafish model. 
cyp enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. the results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research. there are genes in cyp families to most of which are direct orthologs of human cyps that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. many cyps potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for cyps. here we identify and annotate the full suite of cyp genes in zebrafish, compare these to the human cyp gene complement, and determine the expression of cyp genes during normal development. the high degree of sequence similarity suggests conservation of enzyme activities for these cyps, confirmed in reports for some steroidogenic enzymes, and the cyp retinoic acid hydroxylases. there are orthologous relationships for some cyp s and some cyp s between zebrafish and human. transcript profiling by microarray and quantitative pcr revealed that the majority of zebrafish cyp genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. zebrafish have a total of cyp genes, distributed among gene families found also in mammals. complexity is much greater in gene families and which include cyps prominent in metabolism of drugs and pollutants, as well as of endogenous substrates.
 increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome p gene regulation and function. cyp enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. many cyps potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for cyps. here we identify and annotate the full suite of cyp genes in zebrafish, compare these to the human cyp gene complement, and determine the expression of cyp genes during normal development.  zebrafish have a total of cyp genes, distributed among gene families found also in mammals. there are genes in cyp families to most of which are direct orthologs of human cyps that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. the high degree of sequence similarity suggests conservation of enzyme activities for these cyps, confirmed in reports for some steroidogenic enzymes, and the cyp retinoic acid hydroxylases. complexity is much greater in gene families and which include cyps prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. there are orthologous relationships for some cyp s and some cyp s between zebrafish and human. in contrast, zebrafish have cyp genes, compared to in human, with only two recognized as orthologous based on sequence. analysis of shared synteny identified cyp gene clusters evolutionarily related to mammalian cyp s, as well as unique clusters.  transcript profiling by microarray and quantitative pcr revealed that the majority of zebrafish cyp genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. transcripts of some cyp occur also in oocytes. the results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research. 