Gene duplication, gene loss and evolution of expression domains in the vertebrate nuclear receptor NR5A (Ftz-F1) family

Abstract

Fushi tarazu factor 1 (Ftz-F1, NR5A) is a zinc-finger transcription factor that belongs to the nuclear receptor superfamily and regulates genes that are involved in sterol and steroid metabolism in gonads, adrenals, liver and other tissues. To understand the evolutionary origins and developmental genetic relationships of the Ftz-F1 genes, we have cloned four homologous Ftz-f1 genes in zebrafish, called ff1a, ff1b, ff1c and ff1d. These four genes have different temporal and spatial expression patterns during development, indicating that they have distinct mechanisms of genetic regulation. Among them, the ff1a expression pattern is similar to mammalian Nr5a2, while the ff1b pattern is similar to that of mammalian Nr5a1. Genetic mapping experiments show that these four ff1 genes are located on chromosome segments conserved between the zebrafish and human genomes, indicating a common ancestral origin. Phylogenetic and conserved synteny analysis show that ff1a is the orthologue of NR5A2, and that ff1b and ff1d genes are co-orthologues of NR5A1 that arose by a gene-duplication event, probably a whole-genome duplication, in the ray-fin lineage, and each gene is located next to an NR6A1 co-orthologue as in humans, showing that the tandem duplication occurred before the divergence of human and zebrafish lineages. ff1c does not have a mammalian counterpart. Thus we have characterized the phylogenetic relationships, expression patterns and chromosomal locations of these Ftz-F1 genes, and have demonstrated their identities as NR5A genes in relation to the orthologous genes in other species.

Figure 1. Phylogenetic tree of Ftz-F1 (ff1) sequences

Numbers indicate the number of times the branching was obtained from 1000 bootstrap runs. The marker of 0.1 is the length that corresponds to a 10% sequence difference. Zebrafish ff1b and ff1d can be grouped into the NR5A1 group, and ff1a into the NR5A2 group. Both zebrafish and pufferfish ff1c are segregated as an outgroup. Species abbreviations: Cau, Carassius auratus, goldfish; Cin, Ciona intestinalis, an ascidian; Cga, Clarias gariepinus, North African catfish; Dme, Drosophila melanogaster, fruitfly; Dre, Danio rerio, zebrafish; Gga, Gallus gallus, chicken; Hsa, Homo sapiens, human; Mmu, Mus musculus, mouse; Obo, Odontesthes bonariensis, a percomorph fish; Oke, Oncorhynchus keta, chum salmon; Ola, Oryzias latipes, medaka; One, Oncorhynchus nerka, sockeye salmon; Oni, Oreochromis niloticus, nile tilapia; Sal, Salvelinus alpinus, charr; Tni, Tetraodon nigroviridis, pufferfish; Tru, Takifugu rubripes, pufferfish; Xla, Xenopus laevis, frog. Species_gene name, accession numbers: Cau_FTZF1, AF526537; Cin_FTZF, ci0100139008; Cga_FTZF1A, AY014862; Dme_CG8676PA, NP_476932; Dre_ff1a, NM_131463; Dre_ff1b, AF198086; Dre_ff1c, AF327373; Dre_ff1d, AY212920; Gga_SF1, BAA76713; Hsa_NR5A1, NP_004950; Hsa_NR5A2, NP_003813; Mmu_Nr5a1, NP_620639; Mmu_Nr5a2, NP_109601; Obo_sf1, AY323199; Oke_FF1I, AF242223; Sal_ftf1b, AF468977; Oke_FF1II, AF242224; Ola_FTZF1, AB016834; One_FF1I, AF242225; One_FF1II, AF242226; Oni_SF1, AB060814; Tni_ff1c, CAG12178; Tru_ff1a, scaffold_5225.2457.4; Tru_ff1c, SINFRUP00000152795; Tru_ff1b, SINFRUP00000132871; Sal_ftf1a, AF468978; Xla_FtzF1roRA, AAA18356; Xla_FtzF1roRB, AAA18357.

Figure 2. Comparison of human chromosome Hsa9 and zebrafish chromosomes DreLG8 and DreLG21

The gene segments are shown as thick lines and gene orientations are indicated by arrows. Zebrafish ff1b and ff1d, and human NR5A1 are all located next to NR6A1. Besides, ff1d and NR5A1 are in the same gene orientation syntenic with several similar genes.

Figure 3. Comparison of human, zebrafish and fugu chromosome segments surrounding the ff1c gene

The zebrafish chromosome segment DreLG3 is shown at the top, and the human chromosome segments homologous with the DreLG3 are shown below. A fugu gene segment corresponding to the region is shown at the bottom. NOTCH3 and/or ff1c, RDH8, XAB2 and STXBP2 can be found in linked loci in zebrafish, fugu and human.

Figure 4. Expression patterns of (a) ff1 genes during early development, and (b) ff1c and (c) ff1d in different adult tissues detected by RT-PCR

RNA was isolated from different stages or from adult tissues, and primers for specific ff1 genes were used for RT-PCR. The abbreviations for the developmental stages are as follows: C, cleavage; B, blastula, G, gastrula, S, segmentation, P, pharyngula. 1.5, 2, 4 and 6 represent the number of days post-fertilization. N, negative control. Expressions of ff1a and ff1b genes begin during segmentation and become more evident during pharyngula. The ff1c gene is expressed starting at 4 days post-fertilization. Transcripts of ff1d start maternally at the cleavage stage and persist throughout all examined time points. Actin expression was used as a control.

Figure 5. Expression patterns of ftz-f1 homologues in zebrafish head (A–D) and trunk (E–H) regions 26 h post-fertilization

In the head, both ff1a (A) and ff1b (B) are detected in the hypothalamus (hy), but the sizes of expression domains are different. In addition, ff1a is expressed in trigeminal ganglion (tg). In contrast with ff1a and ff1b, ff1c (C) expression is not detected at this stage, and ff1d (D) is ubiquitously expressed. In the trunk, ff1a (E) is detected in spinal neurons (sn), somites (sm) and endoderm. Different from wide distribution of ff1a, ff1b (F) is detected only in head kidney primordia (hk). The ff1c gene is not expressed in the trunk (G), and ff1d is ubiquitous expressed in the dorsal part of the trunk (H).

Figure 6. Model of NR5A/NR6A gene evolution

The ancestral gene segment contains one NR5 gene. After tandem duplication, this gene became the ancestor of NR5 and NR6 in vertebrates. Two more rounds of gene amplification ensue, resulting in four sets of the gene segment. Some of the genes were subsequently lost in the lobe-fin lineage, leading to the current human genome with one NR6A1 and three NR5A genes. In the ray-fin lineage, the genome was duplicated further, followed by loss of chromosomal segments, forming zebrafish chromosomes with two nr6a1 and four ff1 genes.