Download Two Cyp19 (P450 Aromatase) Genes on Duplicated Zebrafish

Two Cyp19 (P450 Aromatase) Genes on Duplicated Zebrafish Chromosomes
Are Expressed in Ovary or Brain
Evelyn Feng-Lin Chiang,* Yi-Lin Yan,† Yann Guiguen,‡ John Postlethwait,† and
Bon-chu Chung*
*Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China; †Institut National de la
Recherche Agronomique, Station SCRIBE, Campus de Beaulieu, Rennes, France; ‡Institute of Neuroscience, University of
Oregon
Cytochrome P450 aromatase (Cyp19) is an enzyme catalyzing the synthesis of estrogens, thereby controlling various
physiological functions of estrogens. We isolated two cyp19 cDNAs, termed cyp19a and cyp19b, respectively, from
zebrafish. These genes are located in linkage groups 18 and 25, respectively. Detailed gene mapping indicated that
zebrafish linkage groups 18 and 25 may have arisen from the same ancestral chromosome by a chromosome
duplication event. Cyp19a is expressed mainly in the follicular cells lining the vitellogenic oocytes in the ovary
during vitellogenesis. Cyp19b is expressed abundantly in the brain, at the hypothalamus and ventral telencephalon,
extending to the olfactory bulbs. The expression of duplicated cyp19 genes at two different tissues highlights the
evolutionary significance of maintaining two active genes on duplicated zebrafish chromosomes for specific functions in the ovary and the brain.
Introduction
Cytochrome P450 aromatase (P450aro, CYP19) is
a member of the cytochrome P450 superfamily. The
CYP19 gene encodes an enzyme controlling the synthesis of estrogens and has been identified in many different animal phyla. In vertebrates, CYP19 expression occurs in the gonads and the brain (Simpson et al. 1994).
In humans, CYP19 transcript is extensively distributed
in tissues including ovaries, placenta, adipose, and brain
(Simpson et al. 1994).
The human CYP19 gene contains 10 exons (Means
et al. 1989). Human CYP19 transcripts from different
tissues have different first exons due to tissue-specific
utilization of promoters located upstream of the first
exon (Mendelson, Evans, and Simpson 1987; Simpson
et al. 1993). Despite differences in the 59 untranslated
regions of the CYP19 transcript, CYP19 proteins from
different tissues have the same sequence, because the
first exon is noncoding.
Mice and humans have a single active Cyp19 gene,
located on murine chromosome 9 and human chromosome 15, respectively (Chen et al. 1988; Youngblood,
Nesbitt, and Payne 1989). Pigs have three active Cyp19
genes, clustered on chromosome 1q16–17, a region homologous to human chromosome 15 (Choi et al. 1997).
Cattle have one active and one transcribed-but-nonfunctional Cyp19 gene arranged in tandem on bovine chromosome 10q26 (Brunner et al. 1998). The presence of
mammalian Cyp19 genes on homologous chromosomes
but in different copy numbers indicates that these Cyp19
gene duplication and selective inactivation events occurred after mammalian speciation.
The functions of P450 aromatase from nonmammalian vertebrates have received wide attention. In fish,
Key words: zebrafish, gene duplication, cyp19, P450 aromatase,
phylogeny, steroid.
Address for correspondence and reprints: Bon-chu Chung, Institute of Molecular Biology, 48, Academia Sinica, Nankang, Taipei, Taiwan, 115, Republic of China. E-mail: [email protected].
Mol. Biol. Evol. 18(4):542–550. 2001
q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
542
cyp19 is expressed in vitellogenic follicles during oogenesis, consistent with the function of estrogen in fish
ovarian development (Tanaka et al. 1995; Fukada et al.
1996; Chang et al. 1997). Goldfish have at least two
forms of cyp19, one expressed in ovaries and the other
found in the brain (Tchoudakova and Callard 1998).
In the brain, aromatization of androgens into estrogens is an essential step in regulating a variety of physiological and behavioral processes. In vitro assays have
demonstrated the presence of aromatase activity in many
regions of the brain from rats (Roselli 1985), birds, and
fish (Gelinas and Callard 1997).
The zebrafish (Danio rerio) has become increasingly popular as a system with which to investigate vertebrate genetics and development because of the many
advantages it confers, such as its small size, rapid development, and short ovarian cycles. We report here the
isolation of two zebrafish cyp19 cDNAs, termed cyp19a
and cyp19b. The cyp19a gene is located in linkage group
18 and expressed predominantly in the ovary. The
cyp19b gene is located in linkage group 25 and expressed strictly in the brain. The presence of two cyp19
genes in zebrafish may have functional and evolutionary
significance.
Materials and Methods
Plasmid Construction
The procedure for cloning zebrafish cyp19a and
cyp19b cDNAs was described elsewhere (Chiang et al.
2001). The full-length cDNAs (GenBank accession
numbers AF183906 and AF183908) were subcloned
into the mammalian expression vector pcDNA3. The
1.3-kb 59 cyp19a or cyp19b fragment was subcloned
into pBluescriptKS1 at the EcoRI site for probe generation. Digoxigenin-11-UTP-labeled RNA probe was
synthesized from BamHI- or XhoI-linearized cyp19a
plasmid by T3 or T7 RNA polymerase to generate sense
or antisense probe. For cyp19b, transcription using T7
or T3 RNA polymerase was performed using ClaI- or
BamHI-digested plasmid as a template to generate sense
or antisense probe.
Two Zebrafish cyp19 Genes on Duplicate Chromosomes
RT-PCR
Zebrafish (D. rerio) were maintained at 28.58C
as described (Westerfield 1995). Total RNA was isolated from zebrafish tissues and RT-PCR was performed according to established procedures at 30 cycles of 948C for 40 s, 558C for 1 min, and 728C for
1 min (Hwang et al. 1997; Lee et al. 1998). The
cyp19a primers (59-ATGGTGAGGAAACTCTCATC-39 and 59-ACTTTCTTCTGCCAGGTGTC-39)
and the cyp19b primers (59-AGAGCAATAATTACACAGGG-39 and 59-CTGTTGCGAAGTCCATTTCA-39) amplified cDNA fragments between nucleotides 336 and 731 and between nucleotides 392 and
907, respectively. The actin primers (Hwang et al.
1997) amplified a 340-bp fragment as a control for
RNA amounts in each tissue sample. PCR products
were analyzed on 1.2% agarose gels, followed by hybridization with cyp19a or cyp19b probes to confirm
their identities.
Phylogenetic Analysis
DNA sequence alignment and homology analysis
were performed using the GCG sequence analysis program
(GCG, Madison, Wis.). Phylogenetic relationships of aromatase genes were derived by aligning sequences by
CLUSTAL X (http://www-igbmc.u-strasbg.fr/BioInfo/
ClustalX/Top.html). Zebrafish aromatase amino acid sequences were compared against GenBank sequences using
blastp, and then the matching sequences were imported
into CLUSTAL X. Sequences were trimmed until they
were unambiguously alignable, and trees were generated
by the neighbor-joining method using NJplot (http://
pbil.univ-lyon1.fr/software/njplot.html) (Saitou and Nei
1987; Perriere and Gouy 1996). A P450 gene from Drosophila melanogaster served as an outgroup to root the
tree. As a measure of the statistical validity of each node
in the phylogenetic analysis, we utilized the bootstrapping method (Efron and Gong 1983; Felsenstein 1985;
Swofford et al. 1996). Bootstrapping involves resampling the sequence data with replacement to create a
series of samples with the same size as the original data
and constructing new phylogenetic trees. The alignment
is available on request.
Gene Mapping
For single-strand conformation polymorphism
(SSCP) analysis, genomic DNA from C32 and SJD parental strains was amplified using primers specific to the
sequence of the 39 untranslated region of the cyp19a and
cyp19b genes. Primers for cyp19a (GCGTGCAGCTTATCCTCAGA and CAATAAAAAAGGTTACAGAAATTAAGTCAC) amplified a 228-bp fragment (nucleotides 1530–1757). Primers for cyp19b (ATTATTCGCCCTCCTGTCATTTT and AGCCACCTGTATACTTTCCCTCAA) amplified a 474-bp fragment
(nucleotides 1743–2216). The SSCPs were genotyped
on DNA from 96 F2 progeny from the cross that had
previously been genotyped for over 800 PCR-based
markers (Goff et al. 1992; Postlethwait et al. 1994,
543
1998; Johnson et al. 1996; Knapik et al. 1996, 1998).
Additional loci were previously mapped (Gates et al. 1999;
Geisler et al. 1999; Hukriede et al. 1999) (http://
zfish.wustl.edu/, http://www.map.tuebingen.mpg.de/,http://
mgchd1.nichd.nih.gov:8000/zfrh/current.html). The strain
distribution patterns were analyzed using MapManager
(http://mcbio.med.buffalo.edu/mapmgr.html), and maps
were constructed with MapMaker (Lander et al. 1987).
Conserved Syntenies
The sequences of zebrafish loci, including SSLPs
(Knapik et al. 1996, 1998) (http://zebrafish.mgh.harvard.
edu/mapping/ssrpmappindex.html), expressed sequence
tags available in GenBank, and cloned genes were compared with the sequences of human and mouse genes in
GenBank using the blastx algorithm (http://www.ncbi.
nlm.nih.gov/blast/blast.cgi). The map locations of human
and mouse genes with very high similarities were found
in On Line Mendelian Inheritance in Man (http://www.
ncbi.nlm.nih.gov/Omim/searchomim.html), GeneMap’99
(http://www.ncbi.nlm.nih.gov/genemap/), LocusLink (http:
//www.ncbi.nlm.nih.gov/genome/guide/), and Mouse Genome databases (http://www.informatics.jax.org/searches/
markerpform.shtml). Human/mouse comparative mapping
was accomplished at http://www.ncbi.nlm.nih.gov/
Homology/.
In Situ Hybridization
In situ hybridization was performed according to
previously described protocols with some modifications
(Westerfield 1995). Tissues were removed from zebrafish under a dissecting microscope and fixed in 4% paraformaldehyde at 48C overnight. Following successive
proteinase K and acetic anhydride treatment, hybridization was carried out with digoxigenin-labeled antisense
or sense RNA probes (100 ng/200 ml solution) at 658C
overnight. Tissues were then washed and residual probes
removed by RNase A digestion. The hybridization signal was detected after incubation with anti-digoxigeninalkaline phosphatase Fab fragments (Boehringer Mannheim), followed by color reaction as previously described (Hu et al. 1999). For more precise signal localization, some hybridized samples were embedded in
CRYOMATRIX (Shandon, Pittsburgh, Pa.), followed by
cryosection. Cryosections were mounted on slides and
observed under a microscope.
Results
Characterization of Zebrafish Cyp19a and Cyp19b
We isolated from a zebrafish cDNA library two different forms of cyp19 cDNAs, termed cyp19a and
cyp19b, encoding proteins of 517 and 511 amino acids,
respectively. Two other zebrafish cyp19a and cyp19b sequences had previously been deposited in GenBank.
One sequence (accession number AF004521) differs
from our cyp19a sequence (accession number
AF183906) by 15 bases, presumably due to sequencing
or PCR errors. These differences led to conservative
544
Chiang et al.
changes of four amino acids and a premature termination resulting in the loss of eight amino acids at the Cterminus of cyp19a in the sequence of accession
AF004521. The other sequence in GenBank (accession
number AF120031) is a partial sequence. It differs from
our cyp19b (accession number AF183908) in the overlapped region by three bases, resulting in a change of
one amino acid.
There is an overall 60% sequence identity between
zebrafish Cyp19a and Cyp19b, indicating that they are
only distantly related. Sequence alignment shows clustered sequence identities along the entire length of the
proteins, showing that they belong to the same protein
family (fig. 1). Cyp19’s from all species contain five
conserved regions, namely, the amino-terminal transmembrane domain (I), the substrate-binding loop (II),
the distal a-helix (III), the steroid-binding domain (IV),
and the heme-binding region (V) (Chen and Zhou 1992).
The distal helix I (region II) and the proximal hemebinding helix L (region V) are the two most conserved
regions. These two helices together form the heme-binding pocket allowing electron transport to take place
(Chen and Zhou 1992).
To understand the tissue distribution of cyp19 gene
expression, RNA was extracted from various adult zebrafish tissues for RT-PCR analysis using either cyp19aor cyp19b-specific primers, followed by hybridization
with gene-specific probes (fig. 2). The cyp19a gene was
expressed abundantly in the ovary and moderately in the
testis, intestine, and brain. We found that cyp19b was
expressed only in the brain. To check that the primers
were indeed gene-specific, we used cyp19a and cyp19b
plasmids in control experiments. The amplification of
actin transcripts in these experiments indicated that the
quality of RNA preparation from each tissue was similar. While cyp19a distribution in the ovary and brain is
similar to the distribution observed for mammalian
CYP19, the presence of a strong cyp19b expression in
the brain is unique for zebrafish and goldfish (Tchoudakova and Callard 1998).
Gene Phylogenies
To study the evolution of Cyp19 genes, we conducted a phylogenetic analysis (fig. 3). A P450 gene
from D. melanogaster served as an outgroup to root the
tree. The results showed that the vertebrate Cyp19 gene
branched as expected from the known evolutionary relationships of the species. All fish Cyp19 genes clustered
together on the same branch, suggesting that they were
all orthologs of the single mammalian Cyp19 gene. The
fish branch, however, bifurcated into two subbranches
with a high bootstrap value (944/1,000). One of these
branches contained the brain form of Cyp19, while the
other branch contained ovarian Cyp19.
This phylogenetic tree provides evidence that there
was a Cyp19 gene duplication in the teleost lineage after
its divergence from the tetrapod lineage. In zebrafish and
tilapia, there are two forms of Cyp19. The ovarian forms
are more closely related to each other than they are to
the brain form of the same species. This suggests that
there was a gene duplication event before the divergence
of the Ostariophysi (including Cypriniform fish, such as
zebrafish) and the Acanthopterygii (including the Perciform fish, such as tilapia) (Nelson 1994). The goldfish
appears to have four forms of Cyp19, consistent with its
known recent tetraploidization event about 20 MYA
(Ohno, Wolf, and Atkin 1968; Wolf et al. 1969; Risinger
and Larhammar 1993).
Comparative Mapping
Scoring polymorphisms in our MOP haploid mapping panel, we mapped zebrafish cyp19a and cyp19b to
the proximal portion of the lower arm of LG18 and the
upper arm of LG25, respectively (fig. 4). The human
aromatase gene CYP19 is located on chromosome 15
(cytogenetic location 15q21.1 and physical location
15p165.55 cR). After integrating these data with existing
gene maps (Johnson et al. 1996; Postlethwait et al. 1998;
Gates et al. 1999; Geisler et al. 1999; Hukriede et al.
1999) (http://zfish.wustl.edu/), we found that on LG18,
marker Z10264 is highly similar (10210, blastx) to human fibrillin1 (FBN1), located at 15q21.1 along with
CYP19. On the radiation hybrid map, marker Z10264 is
similarly physically very close to CYP19 (15p167.92cR
vs. 15p165.55cR). Zebrafish cyp19a is also syntenic with
expressed sequence tag (EST) fa08a06, which is highly
similar to human glycine amidinotransferase (GATM).
All three loci are tightly linked in Hsa15q21.1.
Likewise, on LG25, cyp19b is syntenic with ESTs
fb37h03 and fb61a08, which show very high similarity
to ubiquitous mitochondrial creatine kinase (CKMT1)
and very long-chain fatty-acid-Coenzyme A ligase-1
(FACVL1), respectively, on human chromosome Hsa15.
Similarly, on LG25, mariposa (Moens et al. 1996) (also
called fkd3; Odenthal and Nusslein-Volhard 1998) is
syntenic with cyp19b. The two mouse orthologs of these
genes, Cyp19 and Mf3 (or Fkh5), are closely linked on
mouse chromosome 9 at 31.0 and 41.0 cM, respectively,
and the human ortholog, FKH5 (accession number
AF055080) is in human 15q21–q26. Altogether, at least
10 human loci, CKMT1, CYP19, FBN1, GATM,
FACVL1, CYPB, CYP11A1, KIAA0735, FKH5, and
MEF2Ai, are all in a small region of Hsa15 that shares
extensive syntenies with both LG25 and LG18 in the
zebrafish genome (fig. 5).
Expression of cyp19a in the Ovary and cyp19b in the
Brain
To examine cyp19a and cyp19b expression in more
detail, we performed whole-mount in situ hybridization
with various zebrafish tissues. Neither cyp19a nor
cyp19b transcripts were detected by in situ hybridization
in the testis, heart, or liver (data not shown). The cyp19a
signal was detected mainly in the vitellogenic follicles
of the adult ovary (fig. 6A). It was not present in the
previtellogenic follicles (fig. 6A). This finding shows
that the cyp19a expression level varies during follicular
development, in agreement with previous studies of
Two Zebrafish cyp19 Genes on Duplicate Chromosomes
545
FIG. 1.—Sequence alignment of Cyp19 proteins from different species. Cyp19 from humans, mice, chickens, the ovarian (ov) and brain
(Br) forms of goldfish (gdfish), and zebrafish (zfish) are aligned. Gaps introduced during the alignment are shown by dots. The five conserved
regions are underlined and marked with Roman numerals I–V.
cyp19 expression in rainbow trout, tilapia, and medaka
(Tanaka et al. 1992; Fukada et al. 1996; Chang et al.
1997).
To determine the regional and cellular expression
of cyp19a, the ovary was further sectioned for histolog-
ical study. Cells at the outer edges of vitellogenic follicles surrounding the zona pellucida expressed cyp19a
(fig. 6B and C), suggesting that these follicular cells
were theca/granulosa layers. There was no signal in the
ovarian tissues when the sense riboprobe was used (data
546
Chiang et al.
FIG. 2.—Expression pattern of cyp19 in different adult tissues
detected by RT-PCR. RNA was isolated from different tissues, and
primers specific for cyp19a or cyp19b were used for RT-PCR, followed
by hybridization with their specific cDNA probes. The cyp19a and
cyp19b lanes show plasmid templates used in PCR reactions as a specificity control. Primers for the actin gene were also used as a control
for the quality of the RNA preparation.
FIG. 4.—The locations of cyp19a and cyp19b on zebrafish linkage
groups. Loci of the form Z928 are simple sequence length polymorphisms (Knapik et al. 1996, 1998; Shimoda et al. 1999) (http://
zebrafish.mgh.harvard.edu/mapping/ssrpmappindex.html). Loci of the
form 14V550 are random amplified polymorphic DNAs (Johnson et
al. 1996; Postlethwait et al. 1998), and loci given in bold are cloned
genes.
not shown), indicating that the hybridization signal detected was specific for the cyp19a transcript.
RT-PCR analysis showed cyp19b expression only
in zebrafish brain (fig. 2). In situ hybridization revealed
that the distribution of cyp19b transcript was mainly in
the hypothalamus and ventral telencephalon, extending
to the olfactory bulb (fig. 7B). No signal was detected
in the brain hybridized with a sense cyp19b probe (fig.
7A).
From RT-PCR analysis, cyp19a expression was
also detected in zebrafish brain, although less abun-
FIG. 3.—Phylogenetic tree of vertebrate aromatase genes. The
common names of the organism, followed by the expression domains
of the genes and the accession numbers, are shown. O 5 ovary; B 5
brain. Numbers indicate the hits supporting the branching pattern from
1,000 bootstraps. The marker of 0.1 is the length that corresponds to
a 10% sequence difference.
FIG. 5.—Comparative syntenies around aromatase genes. Loci
shown on LG18 and LG25 are compiled from sources listed in Materials and Methods. Syntenies are shown, but loci are listed in the
order in which they appear on the human chromosome. FKH5 is localized somewhere in the cytogenetic region indicated. The results
show that these two linkage groups have a number of loci found on
human chromosome 15. These results suggest that zebrafish linkage
groups 18 and 25 are duplicates of a portion of human chromosome
15q. A hypothesis for the evolution of chromosomes, aromatase genes,
and expression domains is presented below the synteny maps.
Two Zebrafish cyp19 Genes on Duplicate Chromosomes
547
FIG. 7.—Expression of cyp19b in adult brain. Ventral views. A,
Sense probe. There is no signal when the sense probe is used. B,
Antisense probe. The cyp19b transcripts are found at the hypothalamus
and ventral telencephalon, extending to the olfactory bulb. HT 5 hypothalamus; OB 5 olfactory bulb; VTel 5 ventral telencephalon.
FIG. 6.—Expression of cyp19a in the adult ovary. A, Wholemount in situ hybridization. Only the large vitellogenic follicles gave
a positive stain. Staining was absent in the smaller previtellogenic follicles. B, Sectioning of the ovary. A blue hybridization signal was
observed in the layer of cells surrounding the yolk-laden stage III
oocytes. C, Higher magnification of the follicle, showing that the staining is in the follicular cell layer surrounding the oocyte. Abbreviations:
y, yolk; z, zona pellucida; t/g, theca/granulosa layers.
dantly than the cyp19b transcript. Hybridization of the
brain with antisense cyp19a riboprobe confirmed a lower expression of the gene, which, like that of cyp19b, is
mainly concentrated in the hypothalamic area (data not
shown). These results show that the cyp19b transcript is
the major aromatase transcript in zebrafish brain.
Discussion
In this report, we describe the characterization of
two zebrafish cyp19 genes. These two genes are located
on different chromosomes and encode proteins of about
60% sequence identity. Our phylogenetic analysis indicated that these two genes are orthologous to each other,
arising through an ancient gene duplication event. Was
that event a tandem gene duplication involving only
cyp19, or did it involve duplication of a large chromosome region or an entire chromosome? This question is
important, because if a large chromosome region was
involved, all of the regulatory regions would be present
in duplicate just after the event, whereas if there were a
tandem duplication, the regulatory sequences may or
may not have been duplicated. If the two cyp19 genes
map very near each other, then this would provide support for a tandem duplication, but if the two genes map
on different chromosomes with evidence of chromosome duplication, then the tandem duplication hypothesis would be less likely.
A chromosome duplication event may have occurred in all ray-finned fish, accounting for the presence
of duplicated genes in zebrafish. One example is the
arrangement of the HOX-clusters, which are present as
duplicated copies for each of the four HOX-bearing
chromosomes in mammals (Amores et al. 1998; Postlethwait et al. 1998). It is, however, also possible that
there was a tandem duplication of the original cyp19
gene and that a translocation event broke the duplication-bearing chromosome in two between the two cyp19
duplicates. We favor the chromosome duplication hypothesis over the translocation hypothesis because other
parts of LG18 and LG25 also contain genes orthologous
to syntenic regions of genes on human chromosomes 11,
19, 22, and a small part of 7 (our unpublished data).
LG18 and LG25 appear to be duplicated chromosome
copies.
The zebrafish cyp19a gene is expressed mainly in
the follicular cells during vitellogenesis (fig. 6). It is
absent in the previtellogenic follicles. This observation
is consistent with the increase in P450 aromatase activity and production of 17b-estradiol in follicles during
vitellogenesis and the rapid decline before final oocyte
maturation in various fishes (Young, Kagawa, and Nagahama 1983; Kanamori, Adachi, and Nagahama 1988;
Sakai et al. 1988; Tanaka et al. 1995; Chang et al. 1997).
Therefore, the function of cyp19a could be the participation of vitellogenesis in ovarian follicular
development.
The strong expression of cyp19b in zebrafish brain
is mainly in the hypothalamus and sensory control tissues, such as the ventral telencephalon and olfactory
bulbs (fig. 7). The production of estrogens in these brain
regions has been correlated with neuroendocrine func-
548
Chiang et al.
tions, sexual behavior, and sexual differentiation during
the development of the central nervous system (Naftolin, Ryan, and Petro 1972; Honda et al. 1998; Hutchison
et al. 1999; Kellogg and Lundin 1999; Steckelbroeck et
al. 1999; Zwain and Yen 1999). The presence of large
amounts of cyp19b in zebrafish brain suggests a special
function for locally produced estrogens. With such a
large amount of cyp19b produced, the zebrafish may be
a good model with which to study the function of estrogen in the brain.
The distinct expression profiles of cyp19a and
cyp19b suggest differences in their physiological functions. We present a hypothesis for the evolution of the
chromosomal segment containing Cyp19 (fig. 5). In the
last common ancestor of zebrafish and mammals, the
Cyp19 gene was expressed strongly in the ovary and
weakly in the brain under the control of separate tissuespecific regulatory elements. This supposition was supported by the analysis of the human CYP19 promoter.
The ovary uses a proximal promoter regulated by cAMP,
and the brain and adipose tissues use other promoters
regulated by cytokines (Simpson et al. 1997). There was
apparently genome duplication after the divergence of
ray-finned and lobe-finned fishes (Amores et al. 1998;
Postlethwait et al. 1998). Usually after gene duplication,
one gene will be inactivated if there is no evolutionary
pressure to keep the function of both genes (Bailey,
Poulter, and Stockwell 1978; Takahata and Maruyama
1979; Li 1980). In the zebrafish, however, both Cyp19a
and Cyp19b retain enzymatic activities (our unpublished
data), indicating that there has been evolutionary pressure for the preservation of functions in both genes. According to the duplication-degeneration-complementation model (Force et al. 1999), the function of both duplicate genes will be retained if gene duplication is followed by reciprocal degeneration of essential
tissue-specific regulatory elements. Thus, the brain form
of Cyp19 would be unable to compensate for loss of
function of ovarian Cyp19. Females lacking the ovarian
expression of Cyp19 would probably be sterile, as has
been shown for humans and mice (Shozu et al. 1991;
Fisher et al. 1998). Similarly, the lack of cyp19b expression in the brain might have a yet unknown deleterious effect on zebrafish. This brain function might be
uncovered by a mutation in the brain-specific cyb19b
gene in zebrafish.
In the lobe-finned lineage, the single Cyp19 gene
apparently retained regulatory elements for Cyp19 expression in the ovary and brain. The chromosome segment shown in figure 5 apparently remained intact in
the human lineage and became part of Hsa15, but in the
mouse lineage it was separated by translocations so that
portions of this segment are on three mouse chromosomes (Mmu), Mmu9, Mmu2, and Mmu7 (fig. 5). It is
known that the rodent lineage has suffered substantially
more chromosome translocations and other rearrangements than the human lineage (O’Brien et al. 1999).
After the divergence of mammals, the Cyp19 gene in
cattle and pigs probably underwent additional tandem
duplication, resulting in two Cyp19 genes clustered on
bovine chromosome 10 and three Cyp19 genes on swine
chromosome 1 (Choi et al. 1997; Brunner et al. 1998).
In summary, we have characterized two zebrafish
Cyp19 genes, located on duplicated chromosomal segments. The cyp19a gene is expressed mainly in the ovary. The cyp19b gene is expressed only in the brain. The
retention of two active cyp19 genes on duplicated zebrafish chromosomes demonstrates the importance of
these genes in the two different tissues during evolution.
Acknowledgments
We thank Ching-Fong Chang, Sheng-Ping Hwang,
Jye-I Wang, and Jao-Lun Chen for discussion and help
in setting up the system; Graham Corley-Smith for helping with primer design; and Mary Wyatt for reading the
manuscript. This work was supported by grants NSC882611-B-001-008-B24 and NSC87-2311-B-001-032-B24
from the National Science Council and by Academia
Sinica, Republic of China, NIH grant R01RR10715, and
NSF grant IBN-9728587.
LITERATURE CITED
AMORES, A., A. FORCE, Y.-L. YAN et al. (13 co-authors). 1998.
Zebrafish hox clusters and vertebrate genome evolution.
Science 282:1711–1714.
BAILEY, G. S., R. T. POULTER, and P. A. STOCKWELL. 1978.
Gene duplication in tetraploid fish: model for gene silencing
at unlinked duplicated loci. Proc. Natl. Acad. Sci. USA 75:
5575–5579.
BRUNNER, R. M., T. GOLDAMMER, R. FUERBASS, J. VANSELOW,
and M. SCHWERIN. 1998. Genomic organization of the bovine aromatase encoding gene and a homologous pseudogene as revealed by DNA fiber FISH. Cytogenet. Cell Genet. 82:37–40.
CHANG, X. T., T. KOBAYASHI, H. KAJIURA, M. NAKAMURA, and
Y. NAGAHAMA. 1997. Isolation and characterization of the
cDNA encoding the tilapia (Oreochromis niloticus) cytochrome P450 aromatase (P450arom): changes in P450arom
mRNA, protein and enzyme activity in ovarian follicles during oogenesis. J. Mol. Endocrinol. 18:57–66.
CHEN, S., and D. ZHOU. 1992. Functional domains of aromatase cytochrome P450 inferred from comparative analyses
of amino acid sequences and substantiated by site-directed
mutagenesis. J. Biol. Chem. 267:22587–22594.
CHEN, S. A., M. J. BESMAN, R. S. SPARKES, S. ZOLLMAN, I.
KLISAK, T. MOHANDAS, P. F. HALL, and J. E. SHIVELY. 1988.
Human aromatase: cDNA cloning, Southern blot analysis,
and assignment. DNA 7:27–38.
CHIANG, E. F.-L., Y.-L. YAN, S.-K. TONG, P.-H. HSIAO, Y. GUIGUEN, J. POSTLETHWAIT, and B.-c. CHUNG. 2001. Characterization of duplicated zebrafish cyp19 genes. J. Exp. Zool.
(in press).
CHOI, I., D. L. TROYER, D. L. CORNWELL, K. R. KIRBY DOBBELS, W. R. COLLANTE, and F. A. SIMMEN. 1997. Closely
related genes encode developmental and tissue isoforms of
porcine cytochrome P450 aromatase. DNA Cell Biol. 16:
769–777.
EFRON, B., and G. GONG. 1983. A leisurely look at the bootstrap, the jacknife, and cross-validation. Am. Stat. 37:36–
48.
FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an
approach using the bootstrap. Evolution 39:783–791.
Two Zebrafish cyp19 Genes on Duplicate Chromosomes
FISHER, C. R., K. H. GRAVES, A. F. PARLOW, and E. R. SIMPSON. 1998. Characterization of mice deficient in aromatase
(ArKO) because of targeted disruption of the cyp19 gene.
Proc. Natl. Acad. Sci. USA 95:6965–6970.
FORCE, A., M. LYNCH, F. B. PICKETT, A. AMORES, Y. L. YAN,
and J. POSTLETHWAIT. 1999. Preservation of duplicate genes
by complementary, degenerative mutations. Genetics 151:
1531–1545.
FUKADA, S., M. TANAKA, M. MATSUYAMA, D. KOBAYASHI,
and Y. NAGAHAMA. 1996. Isolation, characterization, and
expression of cDNAs encoding the medaka (Oryzias latipes) ovarian follicle cytochrome P450 aromatase. Mol. Reprod. Dev. 45:285–290.
GATES, M. A., L. KIM, E. S. EGAN, T. CARDOZO, N. H. I.
SIROTKI, S. T. DOUGAN, D. LASHKARI, R. ABAGYAN, A. F.
SCHIER, and W. S. TALBOT. 1999. A genetic linkage map
for zebrafish: comparative analysis and localization of genes
and expressed sequences. Genome Res. 9:334–347.
GEISLER, R., G. J. RAUCH, H. BAIER et al. (25 co-authors).
1999. A radiation hybrid map of the zebrafish genome. Nat.
Genet. 23:86–89.
GELINAS, D., and G. V. CALLARD. 1997. Immunolocalization
of aromatase- and androgen receptor-positive neurons in the
goldfish brain. Gen. Comp. Endocrinol. 106:155–168.
GOFF, D. J., K. GALVIN, H. KATZ, M. WESTERFIELD, E. LANDER, and C. J. TABIN. 1992. Identification of polymorphic
simple sequence repeats in the genome of the zebrafish.
Genomics 14:200–202.
HONDA, S., N. HARADA, S. ITO, Y. TAKAGI, and S. MAEDA.
1998. Disruption of sexual behavior in male aromatase-deficient mice lacking exons 1 and 2 of the cyp19 gene.
Biochem. Biophys. Res. Commun. 252:445–449.
HU, M.-C., Y.-Y. HUANG, N.-C. HSU, H. LI, and B.-C. CHUNG.
1999. Tissue-specific, hormonal and developmental regulation of CYP11A1/LacZ expression in transgenic mice
leads to adrenocortical zone characterization. Endocrinology 140:5609–5618.
HUKRIEDE, N. A., L. JOLY, M. TSANG et al. (17 co-authors).
1999. Radiation hybrid mapping of the zebrafish genome.
Proc. Natl. Acad. Sci. USA 96:9745–9750.
HUTCHISON, J. B., A. WOZNIAK, C. BEYER, M. KAROLCZAK,
and R. E. HUTCHISON. 1999. Steroid metabolising enzymes
in the determination of brain gender. J. Steroid Biochem.
Mol. Biol. 69:85–96.
HWANG, S. P. L., M. F. TSOU, Y. C. LIN, and C. H. LIU. 1997.
The zebrafish BMP4 gene: sequence analysis and expression pattern during embryonic development. DNA Cell
Biol. 16:1003–1011.
JOHNSON, S. L., M. A. GATES, M. JOHNSON, W. S. TALBOT, S.
HORNE, K. BAIK, S. RUDE, J. R. WONG, and J. H. POSTLETHWAIT. 1996. Centromere-linkage analysis and the consolidation of the zebrafish genetic map. Genetics 142:1277–
1288.
KANAMORI, A., S. ADACHI, and Y. NAGAHAMA. 1988. Developmental changes in steroidogenic responses of ovarian follicles of amago salmon (Oncorhynchus rhodurus) to chum
salmon gonadotropin during oogenesis. Gen. Comp. Endocrinol. 72:13–24.
KELLOGG, C. K., and A. LUNDIN. 1999. Brain androgen-inducible aromatase is critical for adolescent organization of environment-specific social interaction in male rats. Horm.
Behav. 35:155–162.
KNAPIK, E. W., A. GOODMAN, O. S. ATKINSON et al. (20 coauthors). 1996. A reference cross DNA panel for zebrafish
(Danio rerio) anchored with simple sequence length polymorphisms. Development 123:451–460.
549
KNAPIK, E. W., A. GOODMAN, M. EKKER, M. CHEVRETTE, J.
DELGADO, S. NEUHAUSS, N. SHIMODA, W. DRIEVER, M. C.
FISHMAN, and H. J. JACOB. 1998. A microsatellite genetic
linkage map for zebrafish (Danio rerio). Nat. Genet. 18:
338–343.
LANDER, E. S., P. GREEN, J. ABRAHAMSON, A. BARLOW, M. J.
DALY, S. E. LINCOLN, and L. NEWBURG. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181.
LEE, H.-H., H.-T. CHAO, Y.-J. LEE, S.-G. SHU, M.-C. CHAO,
J.-M. KUO, and B.-C. CHUNG. 1998. Identification of four
novel mutations in the CYP21 gene in congenital adrenal
hyperplasia in the Chinese. Hum. Genet. 103:304–310.
LI, W. H. 1980. Rate of gene silencing at duplicate loci: a
theoretical study and interpretation of data from tetraploid
fishes. Genetics 95:237–258.
MEANS, G. D., M. S. MAHENDROO, C. J. CORBIN, J. M. MATHIS, F. E. POWELL, C. R. MENDELSON, and E. R. SIMPSON.
1989. Structural analysis of the gene encoding human aromatase cytochrome P-450, the enzyme responsible for estrogen biosynthesis. J. Biol. Chem. 264:19385–19391.
MENDELSON, C. R., C. T. EVANS, and E. R. SIMPSON. 1987.
Regulation of aromatase in estrogen-producing cells. J. Steroid Biochem. 27:753–757.
MOENS, C. B., Y. L. YAN, B. APPEL, A. G. FORCE, and C. B.
KIMMEL. 1996. Valentino: a zebrafish gene required for normal hindbrain segmentation. Development 122:3981–3990.
NAFTOLIN, F., K. J. RYAN, and Z. PETRO. 1972. Aromatization
of androstenedione by the anterior hypothalamus of adult
male and female rats. Endocrinology 90:295–298.
NELSON, J. S. 1994. Fishes of the world. John Wiley, New
York.
O’BRIEN, S. J., M. MENOTTI RAYMOND, W. J. MURPHY, W. G.
NASH, J. WIENBERG, R. STANYON, N. G. COPELAND, N. A.
JENKINS, J. E. WOMACK, and J. A. MARSHALL GRAVES.
1999. The promise of comparative genomics in mammals
[in process citation]. Science 286:458–481.
ODENTHAL, J., and C. NUSSLEIN VOLHARD. 1998. Fork head
domain genes in zebrafish. Dev. Genes Evol. 208:245–258.
OHNO, S., U. WOLF, and N. B. ATKIN. 1968. Evolution from
fish to mammals by gene duplication. Hereditas 59:169–
187.
PERRIERE, G., and M. GOUY. 1996. WWW-Query: an on-line
retrieval system for biological sequence banks. Biochimie
78:364–369.
POSTLETHWAIT, J. H., S. L. JOHNSON, C. N. MIDSON et al. (15
co-authors). 1994. A genetic linkage map for the zebrafish.
Science 264:699–703.
POSTLETHWAIT, J. H., Y. L. YAN, M. A. GATES et al. (25 coauthors). 1998. Vertebrate genome evolution and the zebrafish gene map. Nat. Genet. 18:345–349.
RISINGER, C., and D. LARHAMMAR. 1993. Multiple loci for synapse protein SNAP-25 in the tetraploid goldfish. Proc. Natl.
Acad. Sci. USA 90:10598–10602.
ROSELLI, C. E. 1985. Distribution and regulation of aromatase
activity in the rat hypothalamus and limbic system. Endocrinology 117:2471–2477.
SAITOU, N., and M. NEI. 1987. The neighbor-joining method:
a new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4:406–425.
SAKAI, N., T. IWAMATSU, K. YAMAUCHI, N. SUZUKI, and Y.
NAGAHAMA. 1988. Influence of follicular development on
steroid production in the medaka (Oryzias latipes) ovarian
follicle in response to exogenous substrates. Gen. Comp.
Endocrinol. 71:516–523.
550
Chiang et al.
SHIMODA, N., E. W. KNAPIK, J. ZINITI, C. SIM, E. YAMADA, S.
KAPLAN, D. JACKSON, F. DE SAUVAGE, H. JACOB, and M.
C. FISHMAN. 1999. Zebrafish genetic map with 2000 microsatellite markers. Genomics 58:219–232.
SHOZU, M., K. AKASOFU, T. HARADA, and Y. KUBOTA. 1991.
A new cause of female pseudohermaphroditism: placental
aromatase deficiency. J. Clin. Endocrinol. Metab. 72:560–
566.
SIMPSON, E. R., M. S. MAHENDROO, G. D. MEANS, M. W.
KILGORE, C. J. CORBIN, and C. R. MENDELSON. 1993. Tissue-specific promoters regulate aromatase cytochrome P450
expression. J. Steroid Biochem. Mol. Biol. 44:321–330.
SIMPSON, E. R., M. S. MAHENDROO, G. D. MEANS et al. (12
co-authors). 1994. Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrinol.
Rev. 15:342–355.
SIMPSON, E. R., M. D. MICHAEL, V. R. AGARWAL, M. M. HINSHELWOOD, S. E. BULUN, and Y. ZHAO. 1997. Expression
of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J. 11:29–36.
STECKELBROECK, S., D. D. HEIDRICH, B. STOFFEL WAGNER, V.
H. HANS, J. SCHRAMM, F. BIDLINGMAIER, and D. KLINGMULLER. 1999. Characterization of aromatase cytochrome
P450 activity in the human temporal lobe. J. Clin. Endocrinol. Metab. 84:2795–2801.
SWOFFORD, D. L., G. J. OLSEN, P. J. WADDELL, and D. M.
HILLIS. 1996. Phylogenetic inference. Pp. 407–514 in D. M.
HILLIS, C. MORITZ, and B. K. MABLE, eds. Molecular systematics. Sinauer, Sunderland, Mass.
TAKAHATA, N., and T. MARUYAMA. 1979. Polymorphism and
loss of duplicate gene expression: a theoretical study with
application of tetraploid fish. Proc. Natl. Acad. Sci. USA
76:4521–4525.
TANAKA, M., S. FUKADA, M. MATSUYAMA, and Y. NAGAHAMA.
1995. Structure and promoter analysis of the cytochrome P-
450 aromatase gene of the teleost fish, medaka (Oryzias
latipes). J. Biochem. 117:719–725.
TANAKA, M., T. M. TELECKY, S. FUKADA, S. ADACHI, S. CHEN,
and Y. NAGAHAMA. 1992. Cloning and sequence analysis
of the cDNA encoding P-450 aromatase (P450arom) from
a rainbow trout (Oncorhynchus mykiss) ovary; relationship
between the amount of P450arom mRNA and the production of oestradiol-17 beta in the ovary. J. Mol. Endocrinol.
8:53–61.
TCHOUDAKOVA, A., and G. V. CALLARD. 1998. Identification
of multiple CYP19 genes encoding different cytochrome
P450 aromatase isozymes in brain and ovary. Endocrinology 139:2179–2189.
WESTERFIELD, M. 1995. The zebrafish book: a guide for the
laboratory use of zebrafish (Danio rerio). University of
Oregon Press, Eugene.
WOLF, U., H. RITTER, N. B. ATKIN, and S. OHNO. 1969. Polyploidization in the fish family Cyprinidae, order Cypriniformes. I. DNA-content and chromosome sets in various
species of Cyprinidae. Humangenetik 7:240–244.
YOUNG, G., H. KAGAWA, and Y. NAGAHAMA. 1983. Evidence
for a decrease in aromatase activity in the ovarian granulosa
cells of amago salmon (Oncorhynchus rhodurus) associated
with final oocyte maturation. Biol. Reprod. 29:310–315.
YOUNGBLOOD, G. L., M. N. NESBITT, and A. H. PAYNE. 1989.
The structural genes encoding P450scc and P450arom are
closely linked on mouse chromosome 9. Endocrinology
125:2784–2786.
ZWAIN, I. H., and S. S. YEN. 1999. Neurosteroidogenesis in
astrocytes, oligodendrocytes, and neurons of cerebral cortex
of rat brain. Endocrinology 140:3843–3852.
DAVID IRWIN, reviewing editor
Accepted October 31, 2000