The complexity of alternative splicing of hagoromo mRNAs is increased in an explosively speciated lineage in East African cichlids Yohey Terai*, Naoko Morikawa*, Koichi Kawakami†, and Norihiro Okada*‡§ *Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan; †Department of Developmental Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan; and ‡Division of Cell Fusion, National Institute of Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585 Aichi, Japan Edited by Tomoko Ohta, National Institute of Genetics, Mishima, Japan, and approved August 29, 2003 (received for review May 12, 2003) The adaptive radiation of cichlid fishes in the lakes of East Africa other fish, including cichlids. With insertional mutagenesis, the is a prime example of speciation. The choice of cichlid mates on the zebrafish gene hagoromo (hag) was shown to be responsible for basis of a variety of coloration represents a potential basis for pigment pattern formation (17). Recently, we cloned a cichlid speciation that led to adaptive radiation. Here, we characterize the homolog of hag and showed a correlation between the morpho- cichlid homolog of the zebrafish hagoromo (hag) gene that was logical diversity of the Great Lakes lineage and the evolutionary recently cloned and characterized from a pigmentation mutant. rate of amino acid sequence changes in the hag gene (18). Here Although only one hag mRNA was reported in zebrafish, cichlids we show a correlation between the explosive speciation of express nine different hag mRNAs resulting from alternative splic- cichlids and the complexity of alternative splicing variants of this ing. The hag mRNAs are expressed between the myotome and the putative pigmentation gene. Diversification of the function of epidermis where pigment cells are located, suggesting the cichlid the gene in cichlids has been achieved by alternative splicing that hag gene is involved in pigmentation. The hag mRNA splicing evolved over a very short evolutionary period. Together, our pattern does not fluctuate among individuals from each of two results suggest that pigmentation diversity was attained through species, suggesting that alternative splice site choice is fixed within both amino acid substitutions and posttranscriptional regulatory species. Furthermore, cichlids in lineages that underwent explosive mechanisms. speciation expressed a greater variety of hag mRNAs than those in Materials and Methods lineages that did not undergo such a degree of speciation, sug- gesting that species in the explosively speciated lineage acquired Determination of the Structure of the Cichlid hag Gene. Isolation of a complex regulatory mechanism of alternative splicing over a very the cichlid hag cDNA was described in our previous work (18). short evolutionary period. Here, we provide an example in which Briefly, the cichlid hag gene was mapped by using inter-exon alternative splicing may play a role in mate choice, leading to PCR with genomic DNA of Labidochromis caeruleus as a cichlid speciation through diversification of gene function by template. We used pairs of forward (F) and reverse (R) primers production of multiple mRNAs from a single gene. to amplify introns 1 (e1F, e2R); 2 (e2F, e3R); 3 (e3F, e4R); 4 (e4F, e5R); 5 (e5F, e6R); 6 (e6F, e7R); 7 (e7F, e8R); and 8 (e8F, e9R). akes Victoria, Malawi, and Tanganyika in the East African Ͼ Ͼ LRift Valley harbor roughly 500, 500, and 200 endemic Detection of Each Type of hag mRNA. Total RNA (5 ␮g) extracted species of cichlid fishes, respectively (1–5). These fishes provide from whole bodies of several cichlid species by using TRIzol a spectacular example of adaptive radiation of living vertebrates reagent (Invitrogen) was used for synthesis of cDNA with an (1, 2). The fishes exploit almost all resources available to oligo(dT) primer. The first round of PCR was performed to freshwater fishes (1), and they are ecologically and morpholog- amplify all cDNAs by using 1% of the synthesized cDNAs as a ically highly diverse despite having evolved during a very short template. Primers for the e9terminal type were e1F and e9R2, evolutionary period (6, 7). whereas those for the i5terminal type were e1F and XgoodR1. The formation of color patterns in these fishes is important for The PCR conditions were 30 s at 94°C, 30 s at 60°C, and 2 min mate choice and is therefore of particular interest from an at 72°C for 35 cycles. By using 1 ␮l of 1% of the product of the evolutionary standpoint (8, 9). Cichlids in Lake Victoria can first PCR as template, a second PCR was performed with the potentially interbreed without loss of fertility (10), but species following conditions: 30 s at 94°C, 30 s at 60°C, and 2 min at 72°C are sexually isolated as a consequence of mate choice (8, 9). Male for 20 cycles, with pairs of primers to detect e9terminal-i2ret cichlids are known for their display of bright breeding coloration, (i2F, e9R2); e9terminal-i3ret (i3F, e9R2); e9terminal-e3less and the ability of females to choose conspecific males on the (we3F, e9R2); i5terminal coding region (e1F, e5R); i5terminal- basis of their color pattern has been documented extensively by i1ret (i1F or i1FL for Lamprologini species, XgoodR1); observations in natural environments and behavioral experi- i5terminal-i2ret (i2F, XgoodR1); i5terminal-i3ret (i3F, ments (8, 9). It is therefore expected that the genes responsible XgoodR1); and i5terminal-e3less (we3F, XgoodR1) (see Fig. for formation of cichlid color pattern diversity must have either 1d). mRNAs retaining intron 4 were probed by using primers i4F evolved rapidly via mutations or undergone dynamic changes (specific to intron 4) and e9R2 under the same conditions such as alternative splicing that broadened their functional described above. potential (11, 12) during adaptive radiation in each lake. Al- though cichlid body color is based on pigment pattern formation, In Situ Hybridization. We used a 1-kb fragment of e9terminal-fl the mechanisms underlying this process remain unknown. In (full length) cDNA to generate sense and antisense RNA probes zebrafish, various mutations affecting pigmentation have been described (13, 14), and some relevant genes have been cloned and characterized (e.g., sparse, nacre, and hagoromo; refs. This paper was submitted directly (Track II) to the PNAS office. 15–17). Abbreviations: hag, hagoromo; F, forward; R, reverse. The genes and mutations involved in zebrafish pigmentation §To whom correspondence should be addressed. E-mail: [email protected]. may provide potential clues to pigmentation mechanisms in © 2003 by The National Academy of Sciences of the USA 12798–12803 ͉ PNAS ͉ October 28, 2003 ͉ vol. 100 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2132833100 Downloaded by guest on September 25, 2021 with a digoxigenin labeling kit (Roche Applied Science). In situ hybridization was performed as described (19), except that 30 min was added to each washing time, and each sample was incubated in a solution of proteinase K until just before the sample began to fall apart (Ϸ1 h, depending on the sample size). Primers. Primers were designed on the basis of cichlid hag cDNA sequences (GenBank accession nos. AB107792 and AB107793). The sequences of the primers were as follows: e1F (5Ј-AGCC- GCCTCTCTCAAGTTTGTAGAAGC-3Ј), e2R (5Ј-CATCTCA- GAGGAATTCTTCTTCTGCAGAC-3Ј), e2F (5Ј-TGCAGAAG- AAGAATTCCTCTGAGATGGA-3Ј), e3R (5Ј-CTTTGTGG- ATATGGTACACCTTTATGTCAG-3Ј), e3F (5Ј-CTGCTGAC- ATAAAGGTGTACCATATCCACA-3Ј), e4R (5Ј-TCCGTCTT- TAGCATCCACACAGTTCAC-3Ј), e4F (5Ј-TTCTCAGGTC- ATAACCAGGAGGTGAACT-3Ј), e5R (5Ј-ACTCTGTCACAC- ATGGGAATTGTATCTCT-3Ј), e5F (5Ј-CAGAGATACAAT- TCCCATGTGTGACAGAGT-3Ј), e6R (5Ј-AGTGCCTAA- GAATATCGATCGTTCAACATC-3Ј), e6F (5Ј-GAACTTCTC- CCCTCTCTGCATCTGG-3Ј), e7R2 (5Ј-ACAGTCGAAT- AAAGGTATCATAACCACAGG-3Ј), e7F (5Ј-TCTCCGTTC- CAGCTCTTCACCTGT-3Ј), e8R2 (5Ј-ATTCAGTCTGCCGTT- TGTCCCAA-3Ј), e8F (5Ј-CCAGACAGATGGAAACCACAT- GATAGC-3Ј), e9R2 (5Ј-TCTGAAGTCCAGCGAATGCACAG- 3Ј), XgoodR1 (5Ј-GTATCGCCCAGATTTCCTACAACAA-3Ј), i1F (5Ј-GATGAAAGGGTCAGATGTGCAT-3Ј), i1FL (5Ј- ATGTTGATGTGTCATCTGAACATGT-3Ј), i2F (5Ј-AACTGT- EVOLUTION TCATCATATCTCTGCCTGA-3Ј), i3F (5Ј-CTCCTGTCAG- CATAAGAGAGGTCAAGACT-3Ј), i4F (5Ј-ATCTAAAT- CATCATGTGTTCAGTTATTGGT-3Ј), and we3F (5Ј-GAAT- TCCTCTGAGATGGAAAACTAATGAC-3Ј). Results Isolation of Various Types of hag mRNA from Cichlid Fishes. The exon–intron structure of the cichlid hag cDNA was determined (Fig. 1a) by using the hag gene cDNA from L. caeruleus (18). Unlike the zebrafish hag cDNA, the cichlid hag cDNA comprised two major types terminating at different polyadenylation sites (Fig. 1a). One type, designated ‘‘e9terminal,’’ ended at exon 9, whereas the other, designated ‘‘i5terminal,’’ ended within intron 5 with the polyadenylation site located Ϸ600 bp downstream of exon 5 (Fig. 1a). RNA of the e9terminal type and the i5terminal type was amplified from the cichlids L. caeruleus (data not shown) and Melanochromis auratus by using RT-PCR with primers specific to each type. Several shorter and longer frag- ments were detected in addition to the cDNA containing the expected number of exons (e9terminal-fl and i5terminal-fl, Fig. 1b). The relative levels of the different mRNAs were presumed to be comparable, because PCR for each analysis was performed under almost identical conditions. Further cloning and sequence analyses revealed the presence of nine different hag mRNAs that could be grouped into four Fig. 1. (a) Structure of the cichlid hag.