The molecular basis of development of the sword, asexual selected trait in the genus Xiphophorus

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The molecular basis of development of the sword, a sexual selected trait in the genus Xiphophorus

Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der

Universität Konstanz, Mathematisch-naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von Dipl. Biol. Nils Offen

Tag der mündlichen Prüfung: 19.12.2008 Referenten: Prof. Dr. Axel Meyer

Prof. Dr. Michael K. Richardson

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Acknowledgements

First, I would like to thank my Ph. D. advisor Prof. Axel Meyer who gave me the opportunity to work on this fascinating organism in his lab. I’m grateful to my supervisor PD. Dr. Gerrit Begemann for his advice, various discussion on the the projects and other stuff, inspiration and for the chance to develop and try out own ideas and to improve my scientific and personal skills. I’m thankfull for a quite fascinating time as his PhD student.

I want to thank my current and past collegues of the Meyerlab for various help in the lab and interesting discussions. I’m specially gratefull to (in no particular order) Silke Pittlik, Sebastién Wielgoss, Kai Stölting, Simone Högg, Dominique Leo, Dave Gerrad, Dirk Steincke, Elke Hespeler, Katharina Mebus, Nicola Blum, Matthias Sanetra, Cornelius Eibner, Ylenia Chiari, Maria Buske and Milena Quentin for joining me for a while on my road to Ph. D. and for a memorable time. I also want to thank Helen Gunter and Kathryn Elmer for proof-reading parts of the thesis. And last but not least I’m grateful to Janine Sieling for excellent animal care that made my work so much easier.

Some of those people become more than just colleagues to me and I’m very thankful for several activities and happy hours in and outside the lab. In this context I’d like to mention the Badminton group, which I joined for more than two years.

Especially, I want to thank my parents, my grandparents, my brother and sister and friends outside the university, in particular Martin Eggert, who gave me great support and fresh motivation to continue this work to the end.

I’m also grateful to my better half, Alexandra Schuh, who brightened up my life and supported and motivated me continuously in the last ~two years of my thesis. Last, I like to thank her mother and her stepfather for a place to relax and for delicious food.

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General Introduction

Xiphophorus fishes (Fam. Poeciliidae) are small live bearing toothcarps (Cyprinodontiformes) that are endemic to parts of Mexico and Central America [1-3].

The genus itself can be subdivided into four different groups, the northern and southern swordtails and the northern and southern platyfish, depending on their distribution relative to the Trans-Mexican Volcanic Belt in central Veracruz [3]. This classification into northern and southern swordtails, but not that of southern and northern platyfish, is further supported by several molecular phylogenies [4, 5]. However, the platyfish were originally combined in an independent genus (Platypoecilus), before Myron Gordon classified the platyfish as a subgroup within the genus Xiphophorus in 1951 [6]. To this day 26 Xiphophorus species have been described [3]. Some species such as X. andersi show a very restricted distribution pattern, whereas others such as the swordtail X. helleri and the two platyfish X. maculatus and X. variatus are the most widely distributed species (Figure 1).

The relationship among these 26 species is rather incompletely resolved, since solid phylogenies using both mitochondrial and nuclear markers comprise only 22 of the 26 described species [4, 5]. Interestingly, several Xiphophorus species can be interbred within captivity [7] and hybridization occurs also under natural conditions [5, 8].

Multiple natural hybrid zones were found between X. birchmanni and X. malinche, due to a disruption in chemical communication [8, 9]. In addition, a recent study showed that the swordtail species X. clemenciae resulted from an ancient hybridization event between a swordtail and a platy species [5]. Different populations of Xiphophorus species such as X.

helleri or X. maculatus can be amazingly variable in color pattern [10]. Several of these pigment pattern variants showed independent mendelian segregation [11, 12]. The ability to make interspecies crosses and the availability of genetic markers turned a popular aquarium fish into a model for early genetic studies in fish in the first half of the 20th century [11]. Since these days Xiphophorus fishes have become an established model organism for early genetic, as well as for behavioural studies and melanoma formation (reviewed in [13, 14]).

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Figure 1: Distribution of Xiphophorus species in Mexico and Central America

A: Distribution of: (A) X. meyeri, (B) X. gordoni, (C) X. couchianus, (D) X. xiphidium, (E) X. variatus, (F) X. birchmanni, X. continens, X. cortezi, X. malinche, X. montezumae, X. multilineatus, X.

nezahualcoyotl, X. nigrensis and X. pygmaeus, (G) X. evelynae, (H) X. andersi, (I) X. milleri and X.

kallmani, (J) three species of X. clemenciae, (K) X. helleri and X. maculatus, (K) X. alvarezi, (M) X.

signum, (N) X. mayae, (O) “PMH” type of Xiphophorus [15]

The satellite map was obtained from google maps. The distribution of Xiphophorus species was adapted from Kallman and Kazianis [3].

Swords, sexy males and choosy females

In the second half of the 20^th century Xiphophorus become a valuable organism for behavioural biologists (reviewed in [14]). Male swordtails perform a complex courtship behaviour that consists of several behavioural sequences such as lateral or sexual display [16, 17]. Interestingly, in several Xiphophorus species not all males perform a complex courtship behaviour, but show an alternative mating strategy (reviewed in [14]). In X.

nigrensis, only large males court in front of the female and perform sexual display,

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whereas small males perform a sneak and chase behaviour instead [18, 19]. The difference in body length of mature males that determine the mating strategy of the respective individual seems to result from an allelic variation in the pituitary locus (P). X.

nigrensis males with the PL (large) allele show a higher growth rate and mature later than males with the Ps (small) allele [19]. The term P locus was originally termed by Kallman and Schreibman, who proposed that the P locus controls when the pituitary-gonadal axis is activated and sexual maturation is induced [20, 21]. They hypothesised that the P locus acts on the maturation of gonadotropic hormone producing cells. The gonadotropic hormones that are released by the pituitary gland stimulate Leydig-like cells in the testis to secrete testosterone [22, 23]. Both gonadotropic and sex hormones promote spermatogenesis and sexual maturation (reviewed [24]). Interestingly, the PS allele is maintained in populations of X. nigrensis, even though females prefer large, courting males [25]. This might be due to the fact that both P_L and P_s males can have equal fitness [26]. Firstly, Ps males mature and start reproducing earlier that PL males [19]. Secondly, the large PL males need a longer time to reach sexual maturity and are more attractive to predators and therefore likely have a higher mortality rate [19, 27]. However, the genetic nature of the P locus, even though the term was coined by Kallman and Schreibman in 1973 [20] is still unknown, as well as the mechanism how the locus actually times sexual maturation.

Besides the attempts to uncover the genetics of behaviour, Xiphophorus is more widely known as a model for sexual selection (reviewed in [14]). Many studies contribute to the understanding of female mate choice and the traits that are involved. The contribution of several traits such as chemical cues [28], colouration [29, 30], vertical bar pattern [31], body size [32] or courtship behaviour itself [33] have been studied by several groups. A first attempt to understand the genetic basis of mate choice was made by M. Cummings and co-workers in a recent study [34]. They identified a couple of genes that are differentially expressed in the brains of X. nigrensis females, when they interact with attractive males.

Another sexually selected trait, a colourful extension of the caudal fin called the sword, has been first described by Darwin himself [35]. According to a definition given by Basolo in 1991, the sword is a coloured ventral extension with 0.7-6.0 times the length of

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the caudal fin that also exhibits a black ventral margin [36]. However, Meyer and co- workers provided an alternative definition that did not include colouration and a black margin [4, 37]. As a consequence, the colourless ventral extension of X. andersi is considered a sword by this definition [4, 37], whereas after Basolo’s definition it is considered to be a protrusion [38]. In fact, Basolo’s sword definition is supported by several studies that showed the biological relevance of both length and a distinct colour pattern [39-42]. The male sword (Figure 2A) is a morphologically simple trait that is mainly formed by four bony fin rays [43, 44]. The unbranched ventral ray 9 (V9) and the branched ventral ray 8 (V8) are the two major sword rays (Figure 2B and [43]). The melanised ventral rays 10 (V10) and 7 (V7) also contribute to the sword, but grow only to half the length of the sword (Figure 3B and [43]). Besides the length, width and coloration, those rays are not further modified [43, 44]. The fin rays or lepidotrichia are also quite simple structures. A lepidotrichia consists of several segments each made up of two concave, bony units, the hemisegments (Figure 2C and [45, 46]). The different segments are linked to each other by intersegmental joints. Each hemisegment is formed by a calcified matrix, that contains collagen fibrils and glycosamino-glycans such as chondroitin sulfate and is secreted by a monolayer of scleroblasts (Figure 2C and [45- 47]). The two hemisegments enclose a core of soft tissue, such as neurons, blood vessels and fibroblasts (Figure 2C and [45-47]).

Behavioural experiments in X. helleri have shown that (1) the total length of the sword is an important criterion during mate choice which is thought to reflect a bias for large body size [33, 39] and (2) the females have a preference for a specific pattern of differently coloured stripes [40, 41]. In the green swordtail this colour pattern consists of a greenish to yellowish pigmentation that is enclosed by a ventral and a dorsal black stripe [40]. In contrast, the sword could also be a negative signal in terms of sexual selection such as in the swordtail species X. birchmanni [48]. X. birchmanni males are swordless according to Basolo’s definition and females evolved a preference against sworded males. The disdain for swords might reflect a general preference against long fins, since females also discriminate males with long dorsal fins against individuals with short ones [49]. Interestingly, the sword could also influence the maturation time of juvenile males and females in a population of green swordtails [50]. If males with either

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long or short swords are presented to a group of juvenile fish, maturation of male individuals starts later in response to a long sworded male. Since body size and sword length are positively correlated [51], a long sword might signal to the juveniles that large males are around that will outcompete them in terms of male-male competition and female mate choice, if they mature too early at a small size [32, 51]. This study by Walling and colleagues [50] proposes a more plastic control of sexual maturation than the P locus model of Kallman and Schreibman [20, 21]. In juvenile females the long sword stimulus has the opposite effect and induces earlier maturation [50].

Figure 2: The sword of X. helleri and caudal fin morphology.

(A) Male green swordtails (X. helleri) develop a brightly ornamented sword, whereas females are swordless. (B) The sword is formed by four caudal fin rays, the ventral rays (V) 7, 8, 9, 10, that are covered either with melanophores or xanthophores.

(C) Reconstruction of a teleost caudal fin. The fin rays or lepidotrichia consist of two bony hemirays that are connected by several ligaments. Both hemirays enclose a core of soft tissue containing blood vessels, fibroblasts and nerve bundles. Figures A and B taken from Eibner et al. [52] and Figure C from Becerra et al. [45].

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Sword evolution and the Sensory Exploitation-Preexisting Bias Hypothesis

The sword not only plays a prominent role in sexual selection, but also has an interesting evolutionary history. One scenario, based on a molecular phylogeny, suggests a sworded common ancestor of all extant Xiphophorus species [4, 37]. However, the ancestral presence of colouration and a black margin could neither be confirmed, nor rejected with confidence [37]. Therefore it remains unclear if the common ancestor had a Basolo-type sword [36] or only a protrusion. Only males of extant swordtail species possess a Basolo-type sword [36], whereas platyfish secondarily lost this trait ([4, 5, 37]

and Figure 3). In addition, males of three northern swordtail species, X. pygmaeus, X.

continens and X. birchmanni, do not develop a Basolo-type sword and also likely lost it secondarily ([53] and Figure 3). How the sword evolved in the first place and how it got secondary lost in some species is still under debate.

One theory that is often employed to explain the evolution of the sword is the Sensory Exploitation-Preexisting Bias hypothesis [38, 42, 54, 55]. Under this theory the (female) preference and the (male) trait evolve out of concert (reviewed in [56]). Due to selection or properties of the neural and cognitive system, the receiver (e.g. the female) shows some bias to a specific signal. If a trait evolves in the male that will accidentally stimulate this pre-existing mechanism in the female, this male will have a reproductive advantage and the trait will spread in the population. Due to the receiver bias theory the female preference for swords was already present before the male sword evolved (Figure 3). This scenario is supported by the fact that the sword is only present in the Xiphophorus lineage, but females of Priapella olmecae and Poecillia latipinna show a preference for sworded males ([38, 54, 55] and Figure 3). This would imply that the preference for swords might already have been present in the last common ancestor of Xiphophorus, Priapella and Poecilia, before the sword evolved (Figure 3). Besides the sword, this receiver bias theory is also used to explain the evolution of the hair tufts on the forelegs of male wolf spiders [57] or call suffixes in Physalaemus frogs [58].

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Figure 3: Consensus taxonomy of platyfish and swordtails

The consensus taxonomy from Zauner et al. [59] was modified using phylogenetic data from Meyer et al.

and Hrbek et al. [5, 60]. For all species mentioned, it is indicated if males exhibit a sword, a protrusion or are swordless based on the definition given by Basolo [36]. The traits sword (S) and female preference for swords (P) are mapped on the cladogram. Swordless species where females show a preference for sworded males [38, 42, 55] have been indicated with a green dot. In addition, the putative origin of the poeciliinae gonopodium is indicated. The gonopodium is not found in other subfamilies within the Poeciliidae such as Aplocheilichthys [61]. The classification of Xiphophorus species in northern and southern swordtails/platyfish was adapted from Kallman and Kazianis [3].

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How the sword was lost secondarily also remains elusive. A long sword increases the metabolic cost of swimming and long, ornamented swords make a male individual more conspicuous to predators [27, 62]. It could therefore be that high predation pressure drove the loss of the sword, if its cost was actually higher than its benefit in terms of sexual selection. In fact, several studies in guppies showed that predators can indeed drive the evolution of sexually selected traits, such as coloured spots [63-65]. Interestingly, predators can also modulate the female preference for swords. If green swordtail females are exposed to a video that shows a predation event between a long-sworded male and a cichlid, the females alter their preference for males with long towards males without swords, after the video stimulus [66]. Therefore, changes in female preference could have supported a predator-driven loss of the sword. However, changes in female preference alone might not explain the loss of the sword in some species, since X. maculatus and X.

variatus females retained a preference for sworded males, even though males of these two species do not develop a sword [38, 42].

Sword development

The fascinating evolutionary history of the sword makes it a valuable system to dissect the molecular events that (1) give raise to the sword in the first place and (2) precede the loss of the sword. To answer these questions, a profound knowledge of the genetic network that promotes sword development is required. Unfortunately, the molecular mechanism of sword development is poorly understood. Zander and Dzwillo performed interspecies crosses between X. helleri and X. cortezi and could show that sword development is controlled by multiple loci [44]. They coined the term “sword genes”

(“Schwertgene”), for those genes that confer an ability to produce a sword. Interestingly, sword development can be artificially induced by exogenous testosterone, which implies that androgen signalling might activate different signalling pathways that act together during sword development ([43, 67] and Figure 4). These experiments also showed that the formation of a small ventral protrusion can be induced in some platy species by exogenous testosterone ([44] and Figure 4). Protrusions also occur naturally in males of the platy species X. andersi and X. xiphidium [38, 44]. This implies that the genetic network underlying sword development is not completely lost in those species.

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Figure 4: Schematic representations of the caudal fin of several Xiphophorus species

The first line shows the silhouettes of the caudal fin of mature males, whereas the second line shows the silhouettes of the caudal fins of methyltestosterone treated individuals of different Xiphophorus species.

The platyfish species X. maculatus and X. milleri, swordless under natural conditions, develop a small ventral protrusion under long-time methyltestosterone treatment [43, 44]. Note that in some individuals of X. helleri prolonged methyltestosterone can induce the outgrowth of the dorsal-most fin rays, which results in a so-called “Dorsalschwert” (dorsal sword; [43]). Figure taken from Zander and Dzwillo [44], modified.

In a first attempt to identify genes that are involved in sword development, Zauner and colleagues showed that the homeobox transcriptional repressor msxC (muscle segment homeobox gene C) is up-regulated in growing sword rays [59]. In a recent study, Eibner and colleagues showed that the major sword rays V9 and V8 acts as local organizer during sword development [52]. Both rays showed the capacity to induce the formation of a second, coloured sword when transplanted dorsally. However, the molecular mechanisms by which V8 and V9 promote sword development remain elusive.

To indentify genes that are involved in sword development one can apply a so-called candidate gene approach. The strategy behind this approach is to select genes that are functioning in a related process and to test, if these candidates are also required for sword development. Genes that are known to function either in fin ray growth or colour pattern formation are therefore putative candidate genes. Several studies focussing on fin regeneration in zebrafish revealed multiple genetic pathways, such as Hh, Wnt or Fgf signalling, to control regenerative outgrowth (reviewed in [68, 69]). In Chapter 1 we therefore focussed on Fgf signalling (Fibroblast growth factor) and studied the expression of the fgf receptor 1 and two putative ligands, fgf24 and fgf20a, during sword

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development. fgfr1 have been shown to be essential for fin ray growth during fin regeneration [70, 71]. Furthermore, Fgfr1 is an upstream regulator of msxC expression that is up-regulated during sword development [59, 70, 71]. fgf24 and fgf20a are also associated with fin development and/or regeneration and are therefore putative ligands of fgfr1[70, 72, 73]. fgf24 was shown to be expressed in fin regeneration and is essential for pectoral fin development [70, 72]. fgf20a is involved in early steps of fin regeneration and might therefore also function during sword growth initiation [73]. To test if Fgf signalling is also involved in sword development we analysed the expression pattern of fgfr1, fgf24 and fgf20a in testosterone induced swords. Induction of sword development allows one to produce a sufficient amount of individuals with swords in the same stage of development that can be used for comparative analysis. To obtain a sufficient number of naturally developing swords in comparable stages would be hardly possible, since the age when male swordtails mature can be quite variable.

We showed that fgfr1, but not fgf24 and fgf20a, is specifically up-regulated in developing swords. A similar pattern was also observed in the developing gonopodium, the modified male anal fin that is also induced by exogenous testosterone [74, 75]. fgfr1 is spatial-temporally co-expressed with msxC both in the sword and the gonopodium.

Interestingly, in testosterone treated caudal fins of platyfish, fgfr1 and msxC are differently regulated compared to swordtail fins. Both genes are only up-regulated in the ventral caudal fin after prolonged hormone treatment. Finally, we found a strong correlation of fgfr1 and msxC expression levels and fin ray growth rate, by employing the X. maculatus brushtail mutant that exhibits excessive growth of the median caudal fin rays.

Candidate gene approaches are useful to identify genes that are involved in sword development. However, this approach has one major limitation. Candidate genes are selected or not selected depending on the knowledge available on the particular gene. If no information is available on the gene or it was not shown to function in a related process it will not be selected. Therefore, most likely not all genes that function in sword development will be analysed. To perturb this problem one can apply alternative techniques where previous knowledge on gene function is not required. This could be techniques that detect differential expression of genes between different tissues or

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treatment groups. Microarray technology has proven to be useful in fin regeneration research to identify new genes involved in this process [76, 77]. However, microarrays are not available for any Xiphophorus species. Another approach would be to sequence a large quantity of different clones to analyze the transcriptome of a specific tissue or process, as it was done for fin regeneration in medaka [78]. However, large scale sequencing requires adequate technical and bioinformatical resources. A less resource- intensive method to identify putative “sword genes” is suppression subtractive hybridisation (SSH; [79]). Transcripts from two different tissues or treatment groups are pooled and subtracted against each other. The result is one pool enriched for transcripts that are differentially expressed between the two tissues. In Chapter 2 we took advantage of this method to identify genes that are differently expressed in developing swords and also gonopodia compared to juvenile fins.

We constructed a SSH library from induced swords and gonopodia and sequenced 407 clones. After eliminating redundant sequences 128 out of 201 sequences showed significant similarity. For further analysis we focussed on transcription factors and components of signalling pathways and identified four clones with similarity to rack1, dusp1, klf2 and tmsb a-like that are specifically up-regulated in developing swords and/or gonopodial rays during outgrowth. Not surprisingly, these genes are also up-regulated in regenerating caudal fins during regenerative outgrowth.

The male gonopodium

Not only the sword, but also the male anal fin is modified during sexual maturation ([80, 81] and Figure 5A). The so-called gonopodium is a specialized intromittant organ that enables the male to fertilize the female [82]. Like the sword, the Xiphophorus gonopodium is mainly formed by a small subset of fin rays, the anal fin rays 3, 4 and 5, the so called 3-4-5 complex ([81, 82] and Figure 5B, C). In contrast to the sword, these fin rays are not only modified in terms of length, but also develop complicated terminal structures [81]. The gonopodium of the green swordtail, X. helleri, exhibits several distinct morphological structures [83]. Ray 3 develops a set of segments that carry spines and a terminal hook (Figure 5C). The anterior branch of ray 4 exhibits a terminal ramus, whereas the posterior branch develops spines and serrae (Figure 5C). The terminus of the

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anterior branch of ray 5 is formed by a claw-like structure (Figure 5C). The function of the terminal structures remains elusive. It has been suggested that these structures are necessary for successful copulation [83]. This was based on the observation that females were not fertilized by males, where the distal tip of the gonopodium has been surgically removed. Another hypothesis suggests a “lock and key” mechanism for the gonopodium- genital opening-interaction that promotes prezygotic isolation [61]. In fact, the morphology of the gonopodium, in particular the morphology of the terminal structures, varies between species and was used to distinguish the different Xiphophorus species and for morphology-based phylogenetic analysis [84, 85]. However, such a mechanism should prevent hybridization, which occurs in natural population and under laboratory conditions [5, 8, 12].

Figure 5: The male gonopodium of X. helleri

The anal fin of male poeciliids like X. helleri is modified during sexual maturation (A, B). The anal fin rays 3, 4 and 5 elongate and develop several modified segments at the distal tip (B, V). These segments exhibit spines or serraes or forming either a terminal claw or hook (C). Figure A taken from Eibner et al.

[52], Figure C from Clark et al. [83], modified. (scalebar B: 500 µM)

As for the sword, less is known about the developmental pathways that shape the gonopodium. The poeciliid gonopodium is thought to have evolved only once and therefore all extant species are likely to share a similar developmental program to shape

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the gonopodium ([61] and Figure 4). Based on morphological studies of naturally developing and testosterone-induced gonopodia of Gambusia affinis, Turner proposed a two step model for gonopodium development [75, 80]. During the first phase when the testis starts to mature and releases low levels of testosterone, accelerated growth of the 3- 4-5 complex is promoted. The segments that are formed during the first phase are the same size or larger than the “juvenile” segments [80]. When the testis develops further, the testosterone secretion increases and phase 2 is initiated [75, 80]. During this phase so- called differentiation areas arise in a specific temporal sequence at a specific location within the developing gonopodium. These areas add specific structures (e.g. hooks, spines) or segments of smaller size to the rays. The difference in segment length is thought to be a result of local changes in the growth and/or segmentation rate. However, these differentiation areas are a descriptive term and do not describe a kind of molecular organizer. Therefore, the molecular mechanisms that organize the formation of terminal structures are unknown. The two step model is somehow supported by testosterone induction experiments in Gambusia affinis and X. maculatus [74, 75]. Juvenile fish treated with exogenous testosterone develop a gonopodium where outgrowth and formation of terminal segments start almost simultaneously. These gonopodia are much shorter than the natural ones and lack the segments that are formed in phase 1.

First molecular studies on induced gonopodia of Gambusia affinis showed that two androgen receptors are expressed during gonopodium development [86]. Inhibition of androgen signalling results in reduced gonopodium growth, which supports a function of androgen signalling in growth control of the 3-4-5 complex. This study also revealed shh and ptc1 to be essential for gonopodium outgrowth [86]. Interestingly, the expression of both genes is controlled by androgen receptors, supporting the role of androgen receptor as a molecular switch that controls gonopodium development. As in developing swords fgfr1 and msxC are up-regulated in developing gonopodia ([59] and Chapter 1), which suggests a role of Fgf signalling in gonopodial growth control.

In Chapter 3 we focussed on the role of retinoic acid (RA) signalling during gonopodium development, for two reasons. RA signalling is essential for appendage development in vertebrates [87-89] and it provides positional information along the proximo-distal axis in developing and regenerating limbs [89-92]. Therefore, RA

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signalling might either play a general role in gonopodium development or a specific role in establishing the proximo-distal polarity within the gonopodium. RA, a small lipophilic, diffusible molecule is synthesised by retinaldehyde dehydrogenases (Aldh1as) and stimulates gene expression through binding to two types of receptors, retinoic acid receptors (RARs) and retinoic X receptors (RXRs) [93]. In this study we showed that aldh1a2, a RA synthesising enzyme, and two RA receptors, rarγ-a and rarγ-b, are expressed in developing gonopodia. Inhibiting RA synthesis with DEAB increases the length of newly formed terminal segments, whereas the segment length decreases when RA signalling is over-activated by exogenous RA. In addition, aldh1a2 is co-expressed with one of the androgen receptors (arβ)in developing gonopodia. Interestingly, this expression domain of both genes in the distal tip of the gononopodial rays is not found in developing swords, whereas both rarγ receptors are similarly expressed in developing swords and gonopodia.

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Chapter I

Fgfr1 signalling in the development of a sexually selected trait in vertebrates, the sword of swordtail fish

Nils Offen, Nicola Blum, Axel Meyer and Gerrit Begemann BMC Developmental Biology (2008: 8)

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1.1 Abstract

Background: One of Darwin’s chosen examples for his idea of sexual selection through female choice was the “sword”, a colourful extension of the caudal fin of male swordtails of the genus Xiphophorus. Platyfish, also members of the genus Xiphophorus, are thought to have arisen from within the swordtails, but have secondarily lost the ability to develop a sword. The sustained increase of testosterone during sexual maturation initiates sword development in male swordtails. Addition of testosterone also induces sword-like fin extensions in some platyfish species, suggesting that the genetic interactions required for sword development may be dormant, rather than lost, within platyfish. Despite considerable interest in the evolution of the sword from a behavioural or evolutionary view point, little is known about the developmental changes that resulted in the gain and secondary loss of the sword. Up-regulation of msxC had been shown to characterize the development of both swords and the gonopodium, a modified anal fin that serves as an intromittent organ, and prompted investigations of the regulatory mechanisms that control msxC and sword growth.

Results: By comparing both development and regeneration of caudal fins in swordtails and platyfish, we show that fgfr1 is strongly up-regulated in developing and regenerating sword and gonopodial rays. Characterization of the fin overgrowth mutant brushtail in a platyfish background confirmed that fin regeneration rates are correlated with the expression levels of fgfr1 and msxC. Moreover, brushtail re-awakens the dormant mechanisms of sword development in platyfish and activates fgfr1/msxC-signalling.

Although both genes are co-expressed in scleroblasts, expression of msxC in the distal blastema may be independent of fgfr1. Known regulators of Fgf-signalling in teleost fins, fgf20a and fgf24, are transiently expressed only during regeneration and thus not likely to be required in developing swords.

Conclusion: Our data suggest that Fgf-signalling is involved upstream of msxC in the development of the sword and gonopodium in male swordtails. Activation of a gene regulatory network that includes fgfr1 and msxC is positively correlated with fin ray

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growth rates and can be re-activated in platyfish to form small sword-like fin extensions.

These findings point towards a disruption between the fgfr1/msxC network and its regulation by testosterone as a likely developmental cause for sword-loss in platyfish.

1.2 Background

Charles Darwin conceived not only the theory of natural selection, but also recognized that a theory of sexual selection is necessary to explain the presence of conspicuous traits in male animals that could not have arisen by natural selection [35]. A number of studies provided evidence that sexual selection increases taxonomic diversity, although it remains somewhat controversial if and how sexual selection alone can cause speciation (reviewed in [94, 95]). The body of theory about sexual selection has been extended through several new insights that explain the evolution of sexually selected traits and mating behaviour. Fishes of the genus Xiphophorus are a popular model in which various aspects of sexual selection have been studied extensively (reviewed in [14]). The most prominent sexually selected trait in male swordtail fish of the genus is the sword, a conspicuously pigmented elongation of the ventral caudal fin. The sword consists of several components, i.e. a ventral fin elongation and a characteristic pigmentation pattern [37, 96]. In the green swordtail X. helleri, it consists of centrally located yellow-orange or green coloured rays, that are flanked dorsally and ventrally by rays with strong melanisation (Figure 1.1A, B and [40]). Both length and colouration are important for mating success [32, 40].

The evolutionary history of the sword is of particular interest. One scenario, supported by molecular phylogenies, suggests that all extant Xiphophorus species, swordtails and sword-less platyfish, descended from a common, sworded ancestor [4, 5, 37]. Moreover, short extensions of the ventral portion of the caudal fin are also phylogenetically widespread and, for example, are found in Poecilia petenensis [97]. Platyfish (Figure 1.1C), a common name that is used to describe several swordless species that belong to a monophyletic clade within the genus Xiphophorus, however, secondarily lost their sword during evolution, possibly because the costs in terms of natural selection were higher than the gain in terms of sexual selection. Nonetheless, females of some platy species in which this has been tested still prefer sworded males over the swordless males of their own

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species [38, 42]. The preference for elongated caudal fins seems to be much older than the trait itself, since it is also present in at least one species of the sister genus Priapella [54]. Therefore, the sword is thought to have evolved in response to a pre-existing female bias, such as a general preference for the apparent size [32, 42]. Due to this interesting evolutionary history, the sword presents a valuable model to study how evolution acts at the molecular level to generate or abolish a sexually selected trait. This objective has also driven research in other animals, e.g. the colour morphs in males of the livebearing fish Poecilia parae [98], the exaggerated hypercephaly in stalk-eyed flies [99], or the horns of dung beetles [100]. All of these are examples of model systems in which the basis of change in male exaggerated traits under sexual selection is amenable to genetic dissection.

Figure 1.1: Xiphophorus species and strains used.

Adult morphologies of X. helleri (A) and X. maculatus (C) used in this study as representatives of the swordtail and platyfish lineages. Overview and nomenclature of adult fin rays (B) in the sword [43] of X. helleri and in the gonopodium [85] that is formed by swordtails and platyfish males. The caudal fin ray overgrowth mutant brushtail (D). Note that C and D show different strains of X. maculatus that exhibit dissimilar body colouration independent of the brushtail mutation.

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One way to address this question in swordtails is to dissect the genetic pathways that might be involved in the development of the sword and to characterize these within a phylogenetic framework of the entire genus that involves swordtails and platyfish. So far, only little is known about the molecular basis of sword development. Hybridisation experiments between X. helleri and X. cortezi revealed that multiple genes control sword development, which were collectively termed ‘‘sword genes’’ (“Schwertgene”), i.e.

genes or alleles that confer an ability to produce a sword in hybrids of platyfish and swordtails [44]. In addition, fin ray transplantation experiments have shown that sword rays are characterized by the possession of an organizing activity that induces neighbouring fin rays to contribute to the sword [52]. Sword-induction experiments with juvenile swordtails, treated with exogenous testosterone, revealed that testosterone is a sufficient and essential factor that induces sword development [43, 67]. Exogenous testosterone also induces the development of the gonopodium (Figure 1.1B), a modified anal fin used as a copulation organ that is common to all fish in the family Poeciliidae (the livebearing toothcarps). This might suggest that androgen signalling regulates a molecular pathway that induces both sword and gonopodium development. Interestingly, some platy species develop a small ventral extension of the caudal fin through testosterone treatment [43, 67, 101], suggesting that the genetic machinery underlying sword development is still partly intact even in normally swordless platyfishes. Most likely, this machinery has never been lost completely, even though it might have been inactive for more than a million years [4, 5].

Genes that regulate growth-dependent processes like fin regeneration are good candidates for genes involved in sword development. A candidate gene approach revealed msxC (muscle segment homeobox gene C), a gene known to act in fin regeneration, to be specifically up-regulated in developing swords and gonopodia [59].

By combining available genetic and phylogenetic data, it was hypothesized that genes and pathways that shape the evolutionarily older gonopodium have been partly adapted for sword development [59].

Other putative candidate genes for sword development are upstream regulators of msxC, such as components of the Fgf (Fibroblast growth factor) signalling pathway. Fgf signalling controls epithelial-mesenchymal interactions in the external genital anlagen of

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mammalian embryos [102]. Fibroblast growth factor receptor 1 (Fgfr1) appears to regulate msxC and msxB expression during caudal fin regeneration in zebrafish and is required for regenerative outgrowth of fin rays [70, 71]. Furthermore, Fgf ligands such as those encoded by the fgf24 and fgf20a genes have been shown to play a role in caudal fin regeneration or pectoral fin development [72, 73]. To test a putative role of Fgf-signalling in sword development we cloned the fgf receptor 1 and two fgf orthologs, fgf24 and fgf20a, from the swordtail X. helleri and analysed their expression pattern in developing swords and gonopodia as well as regenerating swords. From a developmental point of view, we asked whether regulation of fgf genes expression is associated with growth of the sword and gonopodium during development and sword regeneration. From an evolutionary standpoint, we were interested in evaluating whether potential differences in fgf gene expression between swordtails and platy species contribute to the understanding of the molecular changes that led to the loss of the sword during evolution. Furthermore, we analysed the expression of fgfr1 and msxC in regenerating caudal fins in the platyfish X. maculatus fin overgrowth mutant brushtail, where medial rays of the caudal fin continue to grow throughout the entire life of the animal (Figure 1.1D). We show that genes are regulated similarly in regenerating sword rays and elongated brush rays, although sword regeneration proceeds differently from regeneration in brushtail.

1.3 Results

Cloning and analysis of fgf genes

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 [70], 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 [70, 71, 103, 104]. fgf24 and fgf20a encode putative Fgfr1 ligands that are expressed or fulfill important functions in zebrafish fin regeneration [70, 73]. To clone the Xiphophorus orthologs from the green swordtail, Xiphophorus helleri, we used a RT-PCR strategy and caudal fin blastemata as source for

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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 (Figures S1.1A,B), found in vertebrate Fgfr1 [105]. Phylogenetic reconstruction of the fgf receptor family, using coding sequence, confirmed that we cloned a partial sequence of the X. helleri Fgfr1 ortholog (Figure 1.2A).

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 (Figures S1.2A, B).

Phylogenetic analysis of the 633 bp cDNA sequence verified the sequence to be the X.

helleri fgf24 ortholog (Figure 1.2B).

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 (Figures S1.2A, B). Interestingly, there is a QH-rich (aa 22-55) motif close the N-terminus of the sequence (Figure S1.2A).

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 1.2C).

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Figure 1.2: Phylogeny of fgf genes.

Phylogenetic analysis of vertebrate fgf receptors (A), fgf8/17/18/24 (B) and fgf9/16/20 (C) families using PhyML (upper values) and Mr. Bayes (lower values). For analysis the coding regions of fgf genes cDNAs were used. The position of the X. helleri orthologs of fgfr1, fgf24 and fgf20a within the three phylogenies is highlighted (red box).

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fgfr1 and msxC are 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 1.1A), 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 [43, 44]. Even adult females develop a sword under testosterone treatment that is indistinguishable from the male sword both in length and pigmentation [44], 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 1.1B and [43]) compared to median or dorsal rays (Figure 1.3A).

However, fgfr1 was expressed much more strongly in V7-V9 than in V10 (Figure 1.3A).

A slight up-regulation of fgfr1 was also detected in ray V6 (Figure 1.3A). Importantly, this pattern is comparable to that of msxC a gene that is strongly up-regulated in developing swords (Figure 1.3B and [59]). Up-regulation of fgfr1 was not observed in control fins (Figure 1.3C). This overlap in expression pattern of fgfr1 and msxC persists during later stages of sword outgrowth (compare Figures 1.3D-F and 1.3G-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.

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Figure 1.3: Expression of fgfr1 and msxC in the developing sword.

X. helleri fgfr1 is up-regulated during sword development. When maturation is induced by exogenous testosterone, fgfr1 is up-regulated in the ventral-most caudal fin rays in developing swords at 5 and 10 days of treatment (dt)(A, F). Weaker expression of fgfr1 can also be detected in non-sword rays (A, D, E). fgfr1 is not up-regulated in untreated control fins (C). fgfr1 expression overlaps with msxC, which is also up- regulated in developing sword rays ([59], B and I). In later stages of treatment, up-regulation of both genes in the distal part of the dorsal-most rays is observed in some individuals (G), which may develop a small

“upper sword” ([67]). Like fgfr1, msxC expression is also detected in non-sword rays (G, H). When maturation is induced by exogenous testosterone in the platyfish, X. maculatus fgfr1 and msxC are similarly expressed in all caudal fin rays after 5 dt (J and K). The expression levels are comparable to untreated fish (L). After 10 dt fgfr1 is more strongly expressed in the ventral-most fin-rays (O) compared to other rays (M, N), which may correspond to the formation of a small ventral swordlet [67, 101]. White arrowheads indicate gene expression. (X. helleri: n= 10 for every stage and probe; X. maculatus: 5 dt: n= 5; 10 dt and controls: n= 3; scale bars: 200 µm)

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 1.1C) 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

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between sword and non-sword rays (Figures 1.3J and 1.3K). In addition, the expression patterns of both genes in testosterone treated fins are quite similar to those of control fins (Figure 1.3L). At 10 dt however, both genes are up-regulated in a subset of ventral fin rays (Figures 1.3M-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 [67, 101]. 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 msxC regulation 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 [61]. 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 1.1B and [61, 85]). We expected that gonopodia of both swordtails and platyfish show a similar spatio-temporal pattern of fgfr1and 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 [82] of X. helleri (Figure 1.4A). Because gene expression in deeper layers of fin rays may be shielded from detection during whole mount in situ hybridisation [106], 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 1.4B). This pattern persists during later stages of gonopodium development (Figure 1.4C). In addition, fgfr1 is up-regulated in the interray tissue (Figures 1.4A and C).

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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 1.4D-F). Both genes are not up-regulated in untreated fins (Figure 1.4G).

Figure 1.4: Expression of fgfr1 and msxC in the developing gonopodia of X. helleri and X. maculatus.

fgfr1 and msxC are both expressed in developing gonopodia of X. helleri and X. maculatus. In X. helleri fgfr1 is up- regulated at 5 days (A) and 10 days (C) of treatment in mesenchymal cells (B) of the main gonopodium-forming rays 3-5 compared to control fins (G). In addition fgfr1 is strongly expressed in the interray tissue of those rays (A, C). As in developing swords, fgfr1 expression overlaps with msxC expression (D-F). In early stages of gonopodium development (5 dt) of the platyfish X. maculatus, the expression patterns of fgfr1 (H) and msxC (J) resemble that of X. helleri.

Both genes are up-regulated in the same set of fin rays compared to untreated controls (L). Expression of both genes (I, K) at 10 dt is comparable to that of X.

helleri with species-specific differences in the shape of growing rays. Black arrowheads indicate the expression in the distal part of the fin rays, white arrowheads indicate inter-ray expression.

(X. helleri: n= 10 for every stage and probe; X. maculatus: 5 dt: n= 5; 10 dt and controls: n= 3; scale bars: A, C, D, F-L: 200 µm; B and E: 100 µm)

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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 1.4H, I, J, K) compared to control fins (Figure 1.4L). The different shapes of the distal fin ray tips in X. maculatus and X. helleri are due to species- specific differences between the gonopodia [85].

fgfr1 and msxC show similar expression profiles in regenerating swords

High levels of msxC transcription are also associated with regenerating sword rays after amputation [59]. It is assumed that the general mechanisms of growth control that act during early development are re-established during regeneration [107, 108]. 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 [109]. fgfr1 is expressed in the basal layer of the epidermis and in a proximal region, which are likely to be scleroblasts (Figure 1.5A). 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 1.5B). Sword rays and non-sword rays show similar levels of fgfr1 and msxC at different stages of regenerative outgrowth (Figures 1.5C-F and Figures 1.5G, H). Both genes stay highly up-regulated in growing blastemata until 7 dpa (Figures 1.5E-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 1.5I) and msxC (Figure 1.5J) 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.

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fgf24 and fgf20a are 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 [72, 73, 110]. 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 1.6A-D and data not shown). Therefore both genes are unlikely to play a role in the regulation of Fgf-

Figure 1.5: Expression of fgfr1 and msxC during caudal fin regeneration.

fgfr1 is expressed in the regenerating caudal fin blastema. In situ hybridisation on longitudinal sections at 4 days post amputation (dpa) reveal fgfr1 expression in the basal layer of the epidermis and in scleroblasts (A). msxC expression overlaps with that of fgfr1 in scleroblasts (B) and shows additional expression in the distal blastema (B, G, H). There is no overall clearly visible difference in expression of fgfr1 (C-F) and msxC (G, H) between sword and non-sword regenerates until 7 dpa. At 11 dpa fgfr1 (I) and msxC (J) show higher levels of expression in regenerating sword rays than in non-sword rays, though this difference is more obvious for msxC.

White arrowheads indicate expression in scleroblasts, black arrowheads the msxC expression domain in the distal blastema and white arrows the plain of amputation.

bl= basal epidermal layer; db= distal blastema; e= epidermis; l= lepidotrichia;

m= mesenchyme (4 dpa fgfr1: n= 8; 7 dpa fgfr1: n= 5; 7 dpa msxC: n= 5; 11 dpa fgfr1:

n= 5; 11 dpa msxC: n= 4; scale bars: A and B: 100 µm, C-J: 200 µm)

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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 msxC are 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 1.1D). Individuals carrying the dominant brushtail mutation are characterized by a life-long overgrowth of medial fin rays in the caudal fin (compare Figures 1.7A and 1.7B), which is independent of sex or sexual maturity [10]. The mutation causing this phenotype is not known. Mature male brushtail mutants also grow a swordlet, a small ventral fin extension (Figure 1.7B), 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 [67, 101]. However, it lacks the pigmentation pattern typical of swords in swordtails. Since brushtail mutants are already born with a brush [10] 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 1.7C-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

Figure 1.6: Expression of X. helleri fgf24 during fin regeneration.

fgf24 is expressed in the wound epidermis at 3 dpa (A, B). Expression diminishes after 3dpa and is almost absent by 4 dpa (C, D). fgf24 is not differentially expressed in sword rays (B) compared to non-sword rays (A). White arrowheads indicate the expression in wound epidermis and white arrows the level of amputation.

(3dpa : fgf24: n= 6; 4 dpa fgf24: n= 5; scale bars: 200 µm)

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ventral fin margins (Figures 1.7D-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 1.7F to 1.7J, and not shown).

Figure 1.7: Expression of fgfr1 and msxC in regenerating caudal fins of brushtail mutants.

Compared to a wildtype platyfish (A), X. maculatus brushtail mutants possess elongated median caudal fin rays (B). Male brushtail mutants also develop a small ventral extension of the caudal fin (swordlet).

fgfr1 and msxC show a graded expression pattern in regenerating caudal fins of brushtail mutants at different stages of regeneration with strongest expression in the median fin rays (C-J). fgfr1 (C-F) and msxC (G-J) are expressed in a similar pattern as in X. helleri regenerating caudal fins. At later stages of regeneration, fgfr1 (F) and msxC show stronger expression in the ventral-most caudal fin rays of males compared to females (J). White arrowheads indicate expression in scleroblasts, black arrowheads the msxC expression domain in the distal blastema and white arrows the plain of amputation. (n= 3 for every stage and probe; scale bars: A and B: 1 mm; C-J: 200 µm)

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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 1.8A). 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 1.8B). 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 1.8B). Differences in regeneration rates between fin rays according to their position in the caudal fin are more pronounced at 8 dpa (Figure 1.8B). 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.

Figure 1.8. Different regeneration rate of brushtail fin rays, depending on their position in the caudal fin.

The regenerate’s length of four different fin rays, highlighted in the schematic drawing of an adult brushtail fin (A) were measured at 4 days post amputation (dpa) and 8 dpa. The regenerate’s length of the dorsal fin rays 4, 6 and 8 were then compared to that of the median fin ray 1.

Dorsal fin rays regenerate more slowly than the median fin ray 1, shown as average length difference between fin ray regenerates (B). The difference in regeneration rate increases the closer a fin ray is located to the dorsal edge of the fin.

The position dependence of regeneration rates is more obvious at 8dpa (n= 11;

*P<0.00001, t-test).

The molecular basis of development of the sword, asexual selected trait in the genus Xiphophorus