*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
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Abstract |
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Introduction |
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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 5' 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 1q1617, 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, 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.
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Materials and Methods |
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RT-PCR
Zebrafish (D. rerio) were maintained at 28.5°C as described (Westerfield 1995
). Total RNA was isolated from zebrafish tissues and RT-PCR was performed according to established procedures at 30 cycles of 94°C for 40 s, 55°C for 1 min, and 72°C for 1 min (Hwang et al. 1997
; Lee et al. 1998
). The cyp19a primers (5'-ATGGTGAGGAAACTCTCATC-3' and 5'-ACTTTCTTCTGCCAGGTGTC-3') and the cyp19b primers (5'-AGAGCAATAATTACACAGGG-3' and 5'-CTGTTGCGAAGTCCATTTCA-3') 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 3' untranslated region of the cyp19a and cyp19b genes. Primers for cyp19a (GCGTGCAGCTTATCCTCAGA and CAATAAAAAAGGTTACAGAAATTAAGTCAC) amplified a 228-bp fragment (nucleotides 15301757). Primers for cyp19b (ATTATTCGCCCTCCTGTCATTTT and AGCCACCTGTATACTTTCCCTCAA) amplified a 474-bp fragment (nucleotides 17432216). 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, 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/ssr_map_index.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/marker_form.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 4°C overnight. Following successive proteinase K and acetic anhydride treatment, hybridization was carried out with digoxigenin-labeled antisense or sense RNA probes (100 ng/200 µl solution) at 65°C overnight. Tissues were then washed and residual probes removed by RNase A digestion. The hybridization signal was detected after incubation with anti-digoxigenin- alkaline 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.
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Results |
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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 -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 heme- binding 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
).
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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 15_165.55 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 (10-10, 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 (15_167.92cR vs. 15_165.55cR). 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.
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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 ).
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Discussion |
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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 17ß-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 functions, 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 tissue- specific 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.
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Acknowledgements |
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Footnotes |
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1 Keywords: zebrafish
gene duplication
cyp19
P450 aromatase
phylogeny
steroid
2 Address for correspondence and reprints: Bon-chu Chung, Institute of Molecular Biology, 48, Academia Sinica, Nankang, Taipei, Taiwan, 115, Republic of China. mbchung{at}sinica.edu.tw
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