Cloning and analysis of fgfgenes

Since sword development in X. helleri requires the growth of caudal fin rays, genes that act to regulate growth in the regenerating zebrafish caudal fin appeared to be suitable candidate genes that may also be involved in sword development. Up-regulation of fgfr1 and msx gene expression has originally been observed in the blastema during zebrafish fin regeneration [25], and it had subsequently been shown that inhibition of Fgf signaling during ongoing fin regeneration prevents further outgrowth and down-regulates the established expression of blastemal msx genes [25, 26, 29, 30]. fgf24 and fgf20a encode putative Fgfr1 ligands that are expressed or fulfill important functions in zebrafish fin regeneration [25, 28]. To clone the Xiphophorus orthologs from the green swordtail, Xiphophorus helleri, we used a RT-PCR strategy and caudal fin blastemata as source for mRNA. The amplified fragment of the fibroblast growth factor receptor 1 (EU340805) covers 1248 bp of the protein's open reading frame, including parts of the IG Domain II, the complete IG Domain III and parts of the tyrosine kinase domain (see Figure A and B in Additional file 1), found in vertebrate Fgfr1 [31]. Phylogenetic reconstruction of the fgf receptor family, using coding sequence, confirmed that we cloned a partial sequence of the X. helleri Fgfr1 ortholog (Figure 2A).

The two cloned cDNA fragments of fgf24 include the complete coding sequence of fgf24, parts of the 5'UTR and the whole 3'UTR sequence. The 633 bp ORF (EU340806) of X. helleri fgf24 codes for a 210 amino acid protein with a heparin-binding growth factors/fibroblast growth factor (HBGF/FGF) family signature (see Figure A and B in Additional file 2). Phylogenetic analysis of the 633 bp cDNA sequence verified the sequence to be the X. helleri fgf24 ortholog (Figure 2B).

In addition we cloned two cDNA fragments of fgf20a, that together cover most of the coding and the complete 3'UTR sequence. The partial protein sequence, coded by 663 bp (EU340807), shows a conserved HBGF/FGF motif (see Figure A and B in Additional file 2). Interestingly, there is a QH-rich (aa 22–55) motif close the N-terminus of the sequence (see Figure A in Additional file 2). This motif could not be found in Fgf20a sequences of other vertebrate species. The phylogenetic analysis of the coding sequence confirmed it to be the X. helleri fgf20a ortholog (Figure 2C).

fgfr1 and msxCare differently regulated in caudal fins of maturing swordails and platyfish

In order to test whether Fgf-signalling is involved in sword development of the green swordtail, X. helleri (Figure 1A), we treated 4–5 month old juvenile fish with 17-α-methyltestosterone to artificially induce this process. To allow both for the simultaneous generation of large numbers of experimental animals and for timed induction of sword development we prematurely induced swords in juvenile fish. Importantly, hormonally induced swords in immature juveniles do not show any sex-related morphological differences [18, 21]. Even adult females develop a sword under testosterone treatment that is indistinguishable from the male sword both in length and pigmentation [18], therefore the sex of the individual should not bias the downstream analysis.

In developing swords, fgfr1 expression was first observed after 4 days of hormone treatment (dt), when black pigmentation along the dorsal border of the sword also becomes visible (data not shown). After 5 dt, when the outgrowth of sword rays had started, fgfr1 was mainly up-regulated in the distal tip of the main ventral sword-forming fin rays V7–V10 (Figure 1B and [21]) compared to median or dorsal rays (Figure 3A). However, fgfr1 was expressed much more strongly in V7–V9 than in V10 (Figure 3A). A slight up-regulation of fgfr1 was also detected in ray V6 (Figure 3A). Importantly, this pattern is comparable to that of msxC a gene that is strongly up-regulated in developing swords (Figure 3B and [23]). Up-regulation of fgfr1 was not observed in control fins (Figure 3C). This overlap in expression pattern of fgfr1 and msxC persists during later stages of sword outgrowth (compare Figures 3D–F and 3G–I). These finding suggest that high levels of fgfr1 expression correlate with the development of ventral caudal fin rays into swords. Furthermore, the spatio-temporal overlap of both fgfr1 and msxC expression patterns indicate a likely interaction of these genes during sword development.

To test if changes in the regulation of fgfr1 and msxC are linked to the absence of the sword in platyfish, we assayed the expression of both genes in the caudal fin of the platyfish X. maculatus (Figure 1C) after 5 and 10 days of testosterone treatment. The expression of both genes in caudal fins at 5 dt differs clearly between X. helleri and X. maculatus. In X. maculatus both genes are uniformly expressed with no differences between sword and non-sword rays (Figures 3J and 3K). In addition, the expression patterns of both genes in testosterone treated fins are quite similar to those of control fins (Figure 3L). At 10 dt however, both genes are up-regulated in a subset of ventral fin rays (Figures 3M–O and not shown). This expression pattern is likely to mark the fin rays that will form a small caudal extension under high exogenous levels of testosterone [20, 22]. Based on the expression data, we conclude that loss of sword ray specific regulation of fgfr1 or msxC could have been involved in the secondary loss of this trait in platyfish.

fgfr1 and msxCregulation in maturing anal fins is conserved between swordtails and platyfish

Males of both platyfish and swordtails as juveniles possess typical anal fins, that during sexual maturation transform into a gonopodium, an intromittent organ for internal fertilisation [32]. Despite some differences in gonopodium morphology between species, all gonopodia are formed by anal fin rays 3–5 that develop into a structure that can deliver sperm into the females genital tract as well as scrape out sperm of other males through hooks that are formed by modification of fin ray elements (Figure 1B and [32, 33]). We expected that gonopodia of both swordtails and platyfish show a similar spatio-temporal pattern of fgfr1 and msxC expression. Because it was impractical to identify sufficient numbers of normally developing male juvenile fish at the desired stages, we analysed the expression patterns of both genes in artificially induced gonopodia of the swordtail X. helleri and the platyfish X. maculatus.

At 5 days of testosterone treatment, strong expression of fgfr1 was found in the distal part of the main gonopodial rays 3, 4 and 5, the so-called 3–4–5 complex [34] of X. helleri (Figure 4A). Because gene expression in deeper layers of fin rays may be shielded from detection during whole mount in situ hybridisation [35], we performed in situ hybridisation on longitudinal sections which reveal strongest expression of fgfr1 in mesenchymal cells at the tip of growing gonopodial rays (Figure 4B). This pattern persists during later stages of gonopodium development (Figure 4C). In addition, fgfr1 is up-regulated in the interray tissue (Figures 4A and 4C).

As in developing swords, the spatio-temporal expression pattern of fgfr1 is similar to that of msxC, which is up-regulated in the mesenchyme of gonopodial rays 3 to 5 and in interray tissue (Figures 4D–F). Both genes are not up-regulated in untreated fins (Figure 4G).

The spatio-temporal expression pattern of fgfr1 and msxC in developing gonopodia of the platyfish X. maculatus approximately resembles the pattern found in X. helleri. Both genes are up-regulated in the distal part of the gonopodial rays 3, 4 and 5 and in the interray tissue (Figures 4H, I, J, K) compared to control fins (Figure 4L). The different shapes of the distal fin ray tips in X. maculatus and X. helleri are due to species-specific differences between the gonopodia [33].

fgfr1 and msxCshow similar expression profiles in regenerating swords

High levels of msxC transcription are also associated with regenerating sword rays after amputation [23]. It is assumed that the general mechanisms of growth control that act during early development are re-established during regeneration [36, 37]. To test whether fgfr1 is similarly regulated in regenerating and in developing sword rays, we assayed gene expression in caudal fin blastemata. The regeneration kinetics of X. helleri roughly equals that of zebrafish at 25°C, where the regenerative outgrowth starts at ~4 dpa [38]. fgfr1 is expressed in the basal layer of the epidermis and in a proximal region, which are likely to be scleroblasts (Figure 5A). msxC and fgfr1 expression overlap in these cells. Furthermore, msxC is not expressed in the basal epidermal layer, but transcription is high in the distal blastema (Figure 5B). Sword rays and non-sword rays show similar levels of fgfr1 and msxC at different stages of regenerative outgrowth (Figures 5C–F and Figures 5G, H). Both genes stay highly up-regulated in growing blastemata until 7 dpa (Figures 5E–H).

At 11 dpa, when the sword region has begun to overgrow the rest of the regenerate, fgfr1 and msxC become differently regulated in sword rays compared to other rays. Both fgfr1 (Figure 5I) and msxC (Figure 5J) are more strongly expressed in sword rays than in non-sword rays, even though this difference in expression was more clearly observed for msxC. Judging from these data, it is apparent that both genes are similarly regulated in developing as well as regenerating swords. Furthermore, it is likely that due to the lack of fgfr1 expression in the distal blastema, msxC expression in this domain is regulated by factors other than Fgfr1.

fgf24 and fgf20aare expressed in regenerating, but not developing swords

To further analyse the regulation of sword development and regeneration upstream of fgfr1, we cloned two putative ligands of Fgfr1, fgf24 and fgf20a, which are known to be involved in fin regeneration and development [27, 28, 39]. To this end we examined the expression patterns of both genes in developing and regenerating swords. We detected strong expression of fgf24 and fgf20a in caudal fin regenerates up to 3 dpa and ~1 dpa, respectively, before the transcription rate of both genes decreased (Figures 6A–D and data not shown). Therefore both genes are unlikely to play a role in the regulation of Fgf-signalling or msxC in later stages of sword regeneration, when gene regulation becomes different between sword and non-sword rays. In addition, as neither fgf24 nor fgf20a were expressed in developing swords or gonopodia (data not shown), it is unlikely that they act as ligands for Fgfr1 during these processes.

Expression levels of fgfr1 and msxCare correlated with growth rates of regenerating fin rays

To address the question whether enhanced fgfr1 and msxC expression are generally associated with extended growth of fin rays, we analysed gene expression in regenerating caudal fins of X. maculatus brushtail mutants (Figure 1D). Individuals carrying the dominant brushtail mutation are characterized by a life-long overgrowth of medial fin rays in the caudal fin (compare Figures 7A and 7B), which is independent of sex or sexual maturity [40]. The mutation causing this phenotype is not known. Mature male brushtail mutants also grow a swordlet, a small ventral fin extension (Figure 7B), similar to the ventral caudal fin extension that naturally occurs in two species of platyfish, X. andersi and X. xiphidium, and similar to that which can be artificially produced by high levels of exogenous testosterone in some species of platyfish such as X. maculatus [20, 22]. However, it lacks the pigmentation pattern typical of swords in swordtails. Since brushtail mutants are already born with a brush [40] and developing embryos are not viable when extracted from their mothers, we asked whether fgfr1 and msxC are differently expressed in regenerating brush rays, compared to more dorsal or ventral caudal fin rays. Expression of fgfr1 and msxC is strongest in the median fin rays (Figures 7C–J), which becomes particularly obvious after 4 dpa. Both genes show a graded expression pattern with a decrease of expression levels towards the dorsal and ventral fin margins (Figures 7D–F, H–J). At later stages of regeneration fgfr1 and msxC are also stronger expressed in the ventral-most caudal fin rays of males that form the swordlet, but was absent in females (compare Figure 7F to 7J, and not shown).

The graded expression patterns suggest that fgfr1 and msxC correlate with different growth rates of median fin rays compared to more ventral or dorsal rays. In order to test this hypothesis, we amputated the caudal fins of adult brushtail mutants and compared the growth rates of regenerating fin rays at different positions within the caudal fin. We did this by calculating the average length difference between the regenerate of the median fin ray 1 and more dorsal fin rays 4, 6 and 8 at 4 dpa and 8 dpa (Figure 8A). We found that the individual fin rays show significantly different regeneration rates (as determined by a t-test), depending on their position within the caudal fin, with the median-most ray 1 showing the fastest regeneration rate (Figure 8B). The regeneration rate decreases the more closely a fin ray is located to the dorsal edge of the fin, with regenerating ray 8 showing the slowest regeneration rate (Figure 8B). Differences in regeneration rates between fin rays according to their position in the caudal fin are more pronounced at 8 dpa (Figure 8B). The correlation between higher fgfr1 and msxC expression levels and enhanced regenerative outgrowth suggests that both genes are involved in modulating the growth rate of individual fin rays.

fgfr1expression in developing and regenerating swords

Our results show that fgfr1 is specifically up-regulated in growing fin rays of the sword and the gonopodial rays of the 3–4–5 complex as a response to the induction of male sexual trait formation by exogenously supplied testosterone. Several studies suggest that Fgfr1 plays essential roles during appendage formation and regeneration: expression of fgfr1 is re-established during regeneration of limbs and caudal fins [25, 41], and loss of fgfr1 function in these tissues blocks blastema formation and regenerative outgrowth [25, 26, 42]. Moreover, Fgfr1 is involved in supporting outgrowth of the mouse limb bud by maintaining mesenchymal cell survival and influencing the development and identity of digits [43, 44]. Interestingly, the spatio-temporal expression pattern of fgfr1 generally overlaps with that of the transcription factor msxC in developing swords and gonopodia [23]. msx genes are known to keep cells in an undifferentiated state by promoting cell proliferation [45, 46]. In zebrafish, msxB has been shown to regulate cell proliferation in the distal blastema of regenerating caudal fins [26, 47]. Accordingly, knock-down and chemical inhibition of Fgfr1-signalling abolishes the expression of msxB and msxC [25, 26]. Therefore, as judged by the up-regulation in growing sword and gonopodial rays, we propose that an increase in fgfr1-mediated signalling regulates cell-proliferation, similar to the processes in fin regeneration, through the activation of target genes like msxC.

Are there positional values that bias the ventral caudal fin for sword development? In a set of transplantation experiments it has been shown that the two major sword rays, V8 and V9, act to organize flanking rays into the developing sword [19]. This is achieved through a non-autonomous signalling process in which fin rays are induced to grow more strongly the closer they develop to V8 and V9. The development of sword rays is initiated through androgen-signalling in maturing juvenile fish, which leads to mainly autonomous growth of rays V8 and V9 and inductive signalling to flanking rays V7 and V10, which together comprise the mature sword. Moreover, the potential to develop organizing activity in V8 and V9 is established during embryonic or larval development [19]. In zebrafish ventral tail fin tissue has been fate-mapped to late gastrula stage embryos [48, 49]. It is therefore likely that positional information within the Xiphophorus caudal fin is also attained during embryonic stages. Genes of the TGFβ gene family, such as alk8/lost-a-fin or tolloid/mini fin, whose mutant phenotypes include defects of the ventral caudal fin [50, 51], could therefore provide all or part of the positional information that is used to specify sword ray fate.

To test whether fgfr1 and msxC are also differently regulated in regenerating swords, and to further characterize these interactions at the cellular level, we compared expression in regenerating sword and non-sword rays. An overlap of fgfr1 and msxC expression was detected in scleroblasts. In addition, fgfr1 is expressed in the basal layer of the epidermis, where msxC was not found, suggesting that signalling through fgfr1 alone is not sufficient to activate msxC expression in the basal epidermal layer. Likewise, msxC is temporally activated in the distal blastema, although fgfr1 is absent from this domain, suggesting that msxC is regulated by signals other than fgfr1 in the distal blastema. Interestingly, such a distal expression domain of fgfr1 is described in zebrafish [25], indicating species-specific differences in fgfr1- and, probably, in msxC-regulation between X. helleri and D. rerio. This may not be all that surprising since these two fish species are evolutionary rather distantly related [52, 53]. In addition, functional differences between zebrafish and medaka fgfr1 have recently been demonstrated [54]. We also noted that, although expression levels of fgfr1 are clearly elevated in sword rays, when compared to non-sword rays, the difference was never as strongly as that observed for msxC. A disproportionally higher activation, like for msxC, might be expected in a scenario where msxC expression is up-regulated as part of the downstream effects of signalling through Fgfr1.

fgfr1 and msxCare part of a network that regulates sword development

We previously proposed that the gene regulatory network underlying sword development did not evolve anew, but was co-opted from the gonopodium [23]. The 3–4–5 complex of the gonopodium is likely to have evolved only once in the common ancestors of all poeciliid fish and is thus evolutionary older than the sword, which originated in its fully developed form in the lineage leading to the genus Xiphophorus [5, 32, 34]. In support of this view, the regulatory dynamics of fgfr1 and msxC show very similar expression patterns during gonopodium growth and caudal fin regeneration that are conserved between swordtails and platyfish, whereas differences in later stages of gonopodium development are likely to reflect differences in species-specific morphology [33]. The similarities in gene expression between swords and gonopodia supports the hypothesis that sword development evolved by co-option of an androgen-regulated genetic network from the evolutionary older gonopodium. fgfr1 is most likely one of a number of regulatory nodes within an intricate network of signalling pathways that converge on the activation of msx genes. The observation that there is only partial co-expression between fgfr1 and msxC during regeneration supports this notion.

There is compelling evidence that msx genes are also regulated by Bmp signalling during development and regeneration of vertebrate appendages [55–57]. For example, inhibition of BMP signalling in the developing mouse limb leads to down-regulation of Msx2 [57], while over-activation of the pathway results in up-regulation of Msx1 and Msx2 [55]. Activation may be direct, as Bmp4 induces the interaction of Smads with Msx1 regulatory sequences [58]. Bmp-regulation of Msx genes seems to be widely conserved, since inhibition of Bmp signalling also leads to down-regulation of msxb in regenerating fins [56] and to a loss of Msx1 expression in the regenerating tail of Xenopus tadpoles [59]. Wnt signalling is a third component of this regulatory network. Manipulation of its activity in regenerating limbs, fins and tadpole tails showed that Wnt signalling regulates the expression of msx and fgf genes and is important for regenerative outgrowth [60–62]. Recent work in the poeciliid fish Gambusia affinis had shown that induction of sonic hedgehog (shh) expression by androgens is required for gonopodium formation [63]. Given that Shh controls dermal bone development in the zebrafish caudal fin [47], it is likely that also Shh signalling may be required for sword development. Functional tests will be required to dissect the molecular network that controls msxC expression to better understand how these different signalling pathways act together to shape the male sword. The similarities in gene expression between swords and gonopodia supports the hypothesis that sword development evolved by co-option of a modular androgen-regulated fgfr1/msxC network from the evolutionary older gonopodium.

Juveniles of X. maculatus fail to up-regulate fgfr1 and msxC in the ventral caudal fin rays at testosterone-levels that otherwise cause sword development in swordtails after 4 days of treatment. It is only after prolonged exposure that both genes start to be expressed more strongly in the ventral-most rays. This expression marks the development of a "swordlet", a small colourless caudal fin extension, that develops in some platy species under excessive exposure to testosterone [20, 22]. A swordlet is also found naturally in the species X. andersi and X. xiphidium and outside the genus Xiphophorus in other species of poeciliid fishes, e.g. in Poecilia petenensis. The failure to up-regulate the fgfr1/msxC network in response to endogenous androgens may have caused the loss of swords in the lineage leading to the platyfish. Since artificially high levels of testosterone can overcome this inhibition ([20, 22] and Figure 3), the evolutionary changes that led to sword-loss may have been quantitative ones that could have altered the strength of genetic interactions within the fgfr1/msxC network or its upstream regulation. One attractive possibility for the evolutionary loss of swords therefore would be an acquired loss in the sensitivity of the fgfr1/msxC network to be activated by androgen-signalling (Figure 9), possibly at the level of fgf regulation.

brushtailenhances sword development in platyfish

We analysed fin ray regeneration in X. maculatus brushtail, a mutant that spontaneously arose in platy strains within the pet trade (Figure 1D). The brush resembles the sword with regard to size, but occurs in both sexes, consists of medial fin rays, and lacks conspicuous pigmentation [40]. In contrast to swords, brushes are formed during embryonic or larval development and show allometric growth throughout the whole life of an individual. In adult male platyfish, brushtail often also induces a swordlet on the ventral caudal fin margin (M. Schartl, personal communication). The X. maculatus brushtail fish used in our studies are not congenic and may contain alleles derived from hybrids between swordtails and X. maculatus. It could thus be argued that brushtail hybrids are more inclined to develop swords due to introgression of sword alleles. However, we note that formation of the swordlet in males is always linked to the brushtail phenotype and does not occur in male brushless siblings that derive from a cross between brushtail parents. Therefore brushtail enhances a dormant program of sword development [64], similar to prolonged exposure to testosterone in X. maculatus. We thus reasoned that the mechanisms of fin growth in swords and brushes may be similar. Our analysis of fin ray regeneration revealed that (1) regeneration rates in brushtail and swordtail caudal fins differed in that sword rays regenerate at the same rate as the rest of the fin (Figures 5 and 7 and [23]), whereas rays within the brush exhibit accelerated regeneration rates the closer they are to the middle of the fin, and (2) expression levels of fgfr1 and msxC show a positive correlation with regeneration rates. Therefore, brushtail mutants lend support to the hypothesis that the strength of fgfr1/msxC expression is correlated with the rate of regenerative growth, which in brushtail mirrors the life-long overgrowth of the brush. In contrast, sword rays initially exhibit regeneration rates and strong fgfr1/msxC expression indistinguishable from non-sword rays. Growth and gene expression ceases once rays have reconstituted the previous fin length, while fgfr1/msxC transcription remains high in the sword. The correspondence of gene expression levels in swords, gonopodia, and in brushtail suggests that fgfr1 and msxC expression are a general readout of proliferation in fin rays.

Pharmacological interference with fgfr1 signalling is not practicable in Xiphophorus fish, as treatments using the standard zebrafish assay, where many fish are treated in small volumes of water, cause a high mortality, while treatments in volumes used in our testosterone experiments would require excessive amounts of the drug. Moreover, electroporation of morpholinos has failed due to persistent problems with the initial injection, which has to be performed into growing fin rays, rather than into a regeneration blastema (not shown). Therefore we had to resolve to associative studies of gene expression in a phylogenetic framework and in mutants to study the involvement of candidate genes in sword development.

As an explanation of the development of a swordlet in adult male brushtail platyfish, our findings suggest that fgfr1/msxC-mediated signalling is constitutively activated throughout the brushtail caudal fin and already in young larvae of both sexes, with strong activation in medial rays and lower, yet elevated levels at the margins. Although endogenous levels of testosterone in platyfish are usually not sufficient to induce or maintain a sword, growth of a swordlet could nevertheless ensue if in maturing male platyfish both testosterone- and brushtail-activated signalling pathways converged on the activation of the fgfr1/msxC regulatory network. brushtail simulates the effect of high exogenous doses of testosterone on swordlet formation in platyfish, in agreement with the idea that the fgfr1/msxC network is a major downstream target of androgen signalling during sword development (Figure 9). It is interesting to note that brushtail re-awakens a dormant program for sword development that had been evolutionary lost in platyfish. Therefore, swordlets likely employ at least parts of the developmental program controlling sword growth [64]. An examination of the molecular mechanisms that activate fgfr1/msxC signalling in brushtail will be valuable for exploring the regulation of this gene network in controlling the growth of ventral caudal fin rays.

The ligands required for Fgfr1 activation during fin development remain elusive

To examine the regulation of fin growth upstream of fgfr1, we isolated fgf20a and fgf24 as potential ligands in caudal fins that develop a sword under testosterone treatment. In zebrafish, fgf20a plays an essential role in blastema formation, while fgf24 is expressed in regenerating caudal fins and is required for pectoral fin bud initiation [25, 27, 28, 39]. Therefore, both genes are putative candidates to control early phases of sword and gonopodium development such as the re-initiation of fin growth under testosterone. We show that both genes are transiently expressed in regenerating fin rays and are down-regulated after 3 dpa, suggesting that fgf20a and fgf24 fulfill similar functions in blastema formation in zebrafish and X. helleri. Since neither gene was expressed at any detectable level in developing swords or gonopodia, other fgfs remain attractive candidates that might mediate the activation of sword development downstream of testosterone-activated androgen receptors. A number of other Fgfs are known with confirmed roles during appendage formation and regeneration, such as Fgf2, Fgf4, Fgf8 and Fgf10, which are able to induce regeneration in Xenopus limbs or chick limb buds [65–67], and Fgf16 as well as Fgf8, Fgf4 and Fgf10, which are involved in zebrafish pectoral fin development (reviewed in [68]). Further investigation of these candidate genes should uncover the ligand of Fgfr1 that controls development of the sword and gonopodium.

Fish stocks and maintenance

Xiphophorus helleri and X. maculatus were taken from stocks kept at the animal research facility at the University of Konstanz. The X. maculatus brushtail mutant, a commercial breed, was obtained from a local pet shop. Fish were maintained on a 12:12 h light:dark cycle at 24°C in 110-litre densely planted aquaria and were fed TetraMin flakes and Artemia.

Testosterone treatment and fin regeneration

Up to six juvenile individuals of X. helleri, aged between 3 and 6 months, were placed in a 30-litre tank. 17-α-Methyltestosterone (1 mg/ml stock solution in ethanol; Sigma-Aldrich, Munich, Germany) was added to the water twice a week to a final concentration of 10 μg/l. After 5 or 10 days of testosterone treatment approximately 1/3 of the distal part of the caudal fin and approximately 2/3 of the anal fin was amputated using a sterile razor blade. For fin amputation fish were anesthetized by incubation in a solution of 0.08 mg/ml tricaine (3-aminobenzoicacid-ethylester-methanesulfonate; Sigma-Aldrich, Munich, Germany).

For regeneration experiments adult X. helleri and X. maculatus individuals were anesthetized and 1/3 of the caudal fin was amputated. Subsequently, fins were allowed to regenerate at 24°C for variable time periods, without addition of 17-α-Methyltestosterone, depending on the experiment. Fish were anesthetized again and the blastema was removed. Fins and blastemata used for in situ hybridisation were fixed in 4% paraformaldehyde (Sigma-Aldrich, Munich, Germany) in phosphate buffered saline (PBS) overnight, transferred to methanol and stored at -20°C until use.

Cloning fgfr1, fgf24 and fgf20a

Total RNA was isolated from caudal fin blastemata 24 hpa (for fgf20a), 48 hpa (for fgf24) and 72 hpa (for fgfr1) and used for cDNA synthesis as described [23]. Degenerate Primers were designed based on an alignment of cDNAs from Danio rerio, Tetraodon nigroviridis and Takifugu rubripes to amplify cDNA fragments of the desired genes. A 1299 kb fgfr1 fragment was amplified by PCR using the Primer pair fgfr-fw1: 5'-CCIGAIAARATGGARAARAARYTGCAYGC-3' and fgfr-rev1: 5'-CIGGIACYTGGTMIGGRTTRTARCA-3'. A 605 bp fgf24 fragment was amplified by PCR using the Primers fgf24-fw1: 5'-GAKAGIGCARCRIGYIYGGAIRC-3' fgf24-rev1: 5'-GTCCICYYIKCCYTTKGGYTGGCGC-3' and fgf24-rev2: 5'-CCAGTATAAATAAMAYRACAGACAC-3'. A 497 bp fgf20a fragment was amplified by PCR using the Primer pair fgf20a-fw2: 5'-GGSTCTCATTTCGTYCTCAC-3' and fgf20-rev0: 5'-GTRTTRTACCARTTYTCYTC-3'.

To obtain fgf24/fgf20a fragments of appropriate size for RNA probe generation 3'RACE reactions were performed using a 3'RACE kit (Roche). A ~1,3 kb fgf24 fragment was amplified using the gene-specific primers fgf24R-fw1: 5'-CTACAGCAGGACCACGGGCAAAC-3' and fgf24R-fw2: 5'-CAAGAAAGGCTCACGCACCACGC-3'. A ~1,2 kb fgf20a fragment was amplified using the gene-specific primers fgf20a-fw1: 5'-CATCAGAGGAGTGGACAGCGGC-3' and fgf20a-fw2: 5'-CGCCATGAACAGCAAGGGGGAG-3'. The PCR products were gel-purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and cloned into the pCRII-TOPO vector (Invitrogen, Karlsruhe, Germany) for sequencing.

Whole-mount in situ hybridisation

Antisense and sense RNA probes were generated using a digoxigenin labelling kit (Roche, Mannheim, Germany). Probes for fgfr1, fgf24 and fgf20a were generated from the cDNA fragments listed above. msxC probes were generated from a 635 bp cDNA fragment [23]. Because of the high sequence conservation within the genus, X. helleri antisense probes could also be used in the platyfish X. maculatus.

In situ hybridisation of Xiphophorus fins and blastemata were performed as described [25] with several modifications. Prehybridisation was done 4 h at 68°C in formamide solution (50% formamide, 5× SSC, 0,1% Tween-20, pH to 6 with 1 M citric acid). Post-hybridisation washing steps were initiated at 68°C with formamide solution. To block unspecific binding sites 0,5% blocking reagent (Roche, Mannheim, Germany) in PBST (PBS/0.1% Tween-20) was used. Antibody incubation was done at 4°C overnight. After fixation of stained fins/blastemata, the tissue was washed twice for 20 min in PBST, 20 min in ethanol/PBST (70:30) and 20 min in 100% ethanol and stored at 4°C. The specificity anti-sense probes were verified with sense probe experiments.

In situhybridisation on longitudinal sections

4 day-old caudal fin blastemata and anal fins from individuals treated with 17-α-Methyltestosterone for 5 days were fixed in 4 % Paraformaldehyde (Sigma-Aldrich, Munich, Germany). Longitudinal sections of 10 μm thickness were created using a Reichert-Jung Autocut 2040 Microtome and in situ hybridisation was performed as described [69].

Microscopy and image editing

Fin explants and brushtail caudal fins were analysed using a Zeiss Stemi SV11 Apo. Pictures were taken using the AxioVision software v3.1 (Zeiss, Jena, Germany) and the digital camera Zeiss AxioCam MRc. The pictures were processed using Adobe Photoshop.

Fin ray measurement and calculation

Pictures from regenerating caudal fins of males and females were photographed at 4 and 8 days post amputation. The regenerate length of the median fin ray 1 and more dorsal fin rays 4, 6 and 8 was measured using the software ImageJ [70]. In case of bifurcation both semi-rays' regenerates were measured and the average was calculated. To eliminate variation in regeneration speed between the individuals, the difference in length between the regenerate of the median ray 1 and the three other ray regenerates were calculated for each fish. Last, the average was calculated for each difference and graphically presented using Microsoft Excel. A paired, one-tail t-test was used to determine whether the calculated average distances differ significantly from each other.

Phylogenetic analysis and motif prediction