17.1 Introduction
The origin and diversification of novel traits is one of the most exciting topics in evolutionary developmental biology (evo-devo). The genetic and developmental mechanisms that underlie morphological innovations are, however, poorly understood, and the fact that no unambiguous definition of novelty exists does not make things easier (see Moczek, 2008; Pigliucci, 2008). While some authors suggest that only a structure for which no homologue can be found in the ancestral species or in the same organism can be considered a morphological novelty (e.g. Müller and Wagner,
1991), others emphasize its ecological importance and define novelty as a new trait that enables new functions and opens up new adaptive zones (Mayr, 1960). The animal kingdom has numerous examples of such adaptive morphological innova- tions. Feathers of birds, spinnerets of spiders, and carapaces of turtles are unique traits that have played a crucial role in the diversification of these lineages. Analysing the genetic and developmen- tal underpinnings of novel traits, however, can be a challenge when they are not represented in model organisms and the comparative method, so successful in evo-devo, is harder to apply. The co- option of existing genes, pathways, or organs in the evolution of novel traits offers the opportunity to overcome this limitation.
17.1.1 Co-option in the evolution of novelties
Among the different mechanisms that have been proposed to explain the origin of morphological novelties, the co-option, or recruitment, of pre- existing features into performing novel functions has received a great deal of attention (e.g. True and Carroll, 2002; Sanetra et al., 2005). This phe- nomenon seems to be prevalent and includes the co-option of tissues and organs as well as of single genes and whole developmental pathways, often with modification of components therein. Avian feathers, for example, have evolved from primitive feather-like epithelial outgrowths used for thermo- regulation and/or camouflage in non-avian dino- saurs (Prum, 1999), and insect wings and spider spinnerets are both derived from the respiratory organs of the common arthropod ancestor (Damen et al., 2002). The development of horns in a num- ber of beetle species seems to rely on redeployment of the arthropod limb patterning genes Distal-less and aristaless (Moczek and Nagy, 2005). The Wnt signalling pathway, involved in various develop- mental processes in vertebrates (Logan and Nusse,
2004), has been implicated in the evolution of the turtle shell (Kuraku et al., 2005).
Studies in butterflies and moths provide some spectacular examples of pathways that are shared across insects and have been co-opted in the evo- lution of colour patterns. The remarkably diverse
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lepidopteran wing patterns are built up from a mosaic of thousands of flattened pigmented scales produced by wing epidermal cells (Figure 17.1a and Plate 11). It has been proposed that these scales are homologous to insect sensory bristles, and have evolved through the recruitment of bristle-pattern- ing genes of the Achaete-Scute complex, followed by acquisition of target genes responsible for typical scale morphology (Galant et al., 1998). The variable colour patterns generated by those wing scales have themselves evolved through the co-option of a number of genetic pathways. The pigments that colour butterfly scales, for instance, derive from the redeployment of several genes from the ommochrome synthesis pathway (e.g. vermilion, cinnabar), known to function in insect eye pigmen- tation (Reed and Nagy, 2005; Reed et al., 2008). The patterns made by the spatial arrangement of col- oured scales, on the other hand, rely on genetic pathways (e.g. the Hedgehog and Wingless path- ways), involved in embryonic and wing develop- ment in butterflies and other insects (Carroll et al.,
1994; Monteiro et al., 2006). Such co-option of gen- etic pathways offers the potential to use extensive knowledge gathered from work on classical model organisms to dissect the formation of butterfly- specific colour patterns.
17.1.2 Butterfly eyespots as an example of an evolutionary novelty
The study of butterfly eyespots, characteristic pat- tern elements composed of concentric rings of dif- ferent colours (Figure 17.1b), has started to shed light on how novel patterns have arisen and diversi- fied in the Lepidoptera. Eyespots probably evolved from primitive, uniformly coloured spots through the recruitment and modification of conserved developmental genes and pathways, acquisition of signalling activity, and further diversification of colour schemes under the influence of natural selection (Brunetti et al., 2001; Monteiro, 2008). Their ecological significance in predator avoidance and sexual selection is well documented (Stevens,
2005; Costanzo and Monteiro, 2007), as is the spec- tacular diversity in eyespot morphology. Variation
in eyespot number, position, shape, size, or colour composition is found not only across species and among individuals of the same species, but often also between different wing surfaces of the same individual butterfly (Nijhout, 1991). Eyespot devel- opment is amenable to detailed characterizat- ion, ranging from the genetic pathways involved in establishing the pattern, to the molecular and cellular interactions underlying pattern specifica- tion, to the biochemical networks involved in pig- ment production (Beldade and Brakefield, 2002; McMillan et al., 2002).
Models of eyespot formation involve the pro- duction and diffusion of one or more signalling molecules from a central organizer, the focus, and the response of the surrounding epithelial cells to the signal(s) in a threshold-like fashion, eventu- ally leading to the production of rings of differ- ent pigments (Nijhout, 1980; Dilão and Sainhas,
2004). The organizer properties of the focus are supported by experiments in which transplant- ation of the focal cells into a different position on the early pupal wing induces the formation of an ectopic eyespot around the transplanted tissue (Nijhout, 1980; French and Brakefield, 1995). The molecular identity of the signal is not known, but both Wingless and Decapentaplegic have been proposed as candidate morphogens (Monteiro et al., 2006). A number of genes have also been implicated in the determination of eyespot cen- tres and colour rings, including members of the Hedgehog pathway (Keys et al.,1999), the recep- tor gene Notch (Reed and Serfas, 2004), and the transcription factor-encoding genes Distal-less, engrailed, and spalt (Carroll et al., 1994; Brakefield et al., 1996; Brunetti et al., 2001). The latter three, for example, are expressed in scale-building cells in association with the different colour rings of the eyespots of several butterfly species (Figure
17.1h). Despite the fact that these and other genes have been implicated in eyespot development, we still know little about the interactions between them, how they regulate pigment synthesis, or the extent to which they contribute to phenotypic variation in eyespot morphology (see Beldade and Saenko, 2009).
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(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j) (k) (l)
Figure 17.1 Conserved developmental processes implicated in butterfly eyespot formation. Coloured scales covering butterfly wings
(a), and eyespot patterns formed by these scales (b), are key innovations in the lepidopteran lineage and are represented in the laboratory- tractable system, Bicyclus anynana (c, ventral view of female at rest). The formation of eyespots in B. anynana shares genetic commonalities with different conserved developmental processes such as embryonic development (d–f), wound healing (g), and wing vein patterning (j–l). Embryonic development in B. anynana has been characterized in wild-type and pleiotropic eyespot mutants (Saenko et al., 2008): wild-type embryo after completion of blastokinesis (d), the characteristic expression of the segment polarity gene engrailed at that stage (e), and a homozygous Goldeneye embryo of the same age that has failed to undergo blastokinesis (f). Expression pattern of engrailed in pupal wings in association with the gold ring of the presumptive adult eyespot (h). This expression is altered in Goldeneye eyespots (Brunetti et al., 2001; Saenko et al., 2008) in a manner that matches the change in adult eyespot colour-composition (i). Damage with a fine needle applied to the
distal part of the developing pupal wing (arrows in left panel) results in formation of ectopic eyespots around the wound site (right panel) (g). Wing venation mutants often affect eyespot patterns (all photos show the ventral surface of the hindwing): the additional vein in extra veins mutants can lead to the formation of an extra eyespot (j), while partial vein loss in Cyclops (k) and vestigial venation in veinless (l) mutants typically result in changes in eyespot size, number, and/or shape. (See also Plate 11.)
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17.1.3 Bicyclus anynana as an emerging
‘eyespot evo-devo’ model
The tropical Nymphalid B. anynana (Figure 17.1c) has been established as a laboratory system for studying the reciprocal interactions between evo- lutionary and developmental processes underlying ecologically relevant phenotypic variation, with emphasis on wing patterns (Brakefield et al., 2009). This system combines knowledge of ecology, often minimal for classical genetic model species, with experimental tractability, including recently devel- oped genomic resources (Beldade et al., 2008) and transgenic techniques (Ramos and Monteiro, 2007), and allows an integrated analysis, from the devel- opmental and genetic basis of eyespot formation to the molecular underpinnings of variation in nat- ural populations.
Here we focus on butterfly eyespots as an example of a morphological novelty and review recent findings relating to the co-option of con- served developmental processes in the evolution of eyespot patterns. First, we discuss experimental evidence for the similarities between eyespot pat- terning and wound repair. We then give examples of pleiotropic mutations isolated in B. anynana laboratory populations which affect not only eyespots but also either embryonic or wing vein development, and discuss how studies of such mutations may help elucidate the genetic path- ways involved in eyespot formation and variation. A detailed analysis of such conserved genetic net- works, extensively studied in the model organism Drosophila melanogaster, in the context of eyespot formation will be invaluable for our understand- ing of the evolutionary origin and diversification of butterfly eyespots.
17.2 Wounds and eyespots
The ability to repair wounded tissue is a funda- mental property of all multicellular organisms, and a key topic of current research (see Gurtner et al., 2008). Here we review evidence suggesting that some components of this process are involved in eyespot formation.
17.2.1 Damage-induced eyespot formation in B. anynana
Local damage to pupal wing tissue has long been known to disturb colour patterns in many lepidop- terans, and has been used to study the mechanisms of pattern formation in butterflies and moths (e.g. Kühn and Von Englehardt, 1933). In eyespot-bear- ing butterfly species, for example, damage with a very fine needle applied to the presumptive eyespot in early pupae can completely eliminate eyespots in the adults (Nijhout, 1980; French and Brakefield,
1992). Also, damage to other locations on the pupal wing epidermis can result in the formation of an ectopic eyespot around the wound site (French and Brakefield, 1995). In B. anynana, for example, rings of black and/or gold, typically poorly defined and less symmetrical than those of the native eye- spots, are found around the healed wound (Figure
17.1g). Interestingly, this type of damage produces eyespots only on the distal area of the wing, and only if applied during a very narrow time win- dow (Brakefield and French, 1995), which more or less corresponds to the period when ‘colour-ring genes’, Distal-less, engrailed, and spalt, are upregu- lated in the presumptive eyespot area (Monteiro et al., 2006). The mechanisms by which the genes and pathways of the damage response machin- ery might contribute to the formation of ectopic eyespots are as yet unclear, but insights from studies in model organisms might provide some clues.
17.2.2 Genetic mechanisms of wound repair
Comparative studies on the genetic and cellular mechanisms of wound repair and regeneration in representatives of various animal phyla have suggested their evolutionary conservation (see Woolley and Martin, 2000). For example, some steps in the wound healing process are regulated by the same transcription factor, Grainyhead, in flies and mice (Mace et al., 2005; Ting et al., 2005). Also, wound healing seems to recapitulate some aspects of embryonic morphogenesis, such as dorsal closure in flies and eyelid fusion in mice, raising the possibility of co-option of genetic path-
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ways active during embryogenesis into wound repair processes during adult life (see Martin and Parkhurst, 2004).
The mechanism by which damage results in the formation of ectopic eyespots is an intriguing question. It is known that candidate genes for eye- spot signalling also perform functions related to wound healing. Studies in fly larvae, for example, have shown that wounds act as sources of short- and long-range signalling molecules and activate downstream pathways in a gradient-like man- ner, as has been proposed for eyespot develop- ment. In Drosophila, the Jun N-terminal kinase (JNK) pathway is activated in a gradient centred around the wound (Galko and Krasnow, 2004) and up-regulates the transcription factor AP-1 which, in turn, leads to induction of decapentaple- gic, a transforming growth factor (TGF)-E family member and one of the candidate eyespot-induc- ing signals (Monteiro et al., 2006). Some evidence also exists for the involvement of the Wnt proteins in wound repair in mammals (e.g. Okuse et al.,
2005), whereas their insect homologue, Wingless, is a candidate morphogen in eyespot formation (Monteiro et al., 2006).
To investigate whether the same genetic mechan- isms that underlie wound healing are implicated in damage-induced eyespot formation in butter- flies, Monteiro et al. (2006) used immunostaining to detect the expression of known ‘eyespot genes’ around damage sites in developing pupal wings. They showed that these genes were upregulated in the epidermal cells surrounding the wound (Monteiro et al., 2006) and proposed that the for- mation of both native and ectopic eyespots relies on at least some identical molecules. Their data, however, do not distinguish between the possibil- ity that the rings of expression of the tested eyespot genes around wound sites are induced by known candidate eyespot morphogens (Monteiro et al.,
2006), or whether they result from some other, as yet unidentified, signals produced by wounded cells. Future work will focus on the identification of genes and pathways that are upregulated upon wing damage and lead to the formation of ectopic eyespots.
17.3 Embryos and eyespots
Among the spontaneous mutants maintained in laboratory populations of B. anynana (Brakefield et al., 2009), some have been characterized that are embryonic lethal in the homozygous state and have a dramatic effect on eyespot morphology in het- erozygotes (Saenko et al., 2008). Comparative ana- lysis of disturbed embryonic development in these mutants with similar phenotypes described in model insects might help identify genes involved in eyespot formation. The mechanisms of early embry- onic development are well studied in the dipteran D. melanogaster (Peel et al., 2005), and are becoming increasingly well understood in representatives of other insect orders, such as the coleopteran Tribolium castaneum and the hemipteran Oncopeltus fasciatus (reviewed in Liu and Kaufman, 2005), the hymenop- teran Nasonia vitripennis (e.g. Pultz et al., 2005; Lynch et al., 2006a), and the lepidopterans Bombyx mori (e.g. Nagy, 1995) and Manduca sexta (e.g. Kraft and Jackle,
1994). To the extent that the genetic mechanisms of embryogenesis are conserved across insects (see Peel et al., 2005; Damen, 2007; see also Chapter 16), a comparison of disturbed embryonic develop- ment in B. anynana eyespot mutants with mutants in insect model species can help identify signalling pathways and/or specific genes involved in eyespot formation and variation.
Embryonic development in B. anynana (described in Saenko et al., 2008) is characterized by a long- germ mode of segmentation, as in most other lepi- dopterans and dipterans (Kraft and Jackle, 1994), but differs from that of D. melanogaster in some important aspects. For example, at about half way through embryonic development, lepidopterans go through a characteristic movement within the egg that results in the reversal from a ventral to a dorsal flexion, called blastokinesis (Broadie et al., 1991; Figure 17.1d). Other aspects of embry- onic development, such as segment patterning by segment polarity and Hox genes (Figure 17.1e) and limb patterning by Distal-less, are similar in B. anynana and other insects (Saenko et al., 2008). This suggests that the direct comparison of dis- rupted embryonic development in pleiotropic B. anynana eyespot mutants with described mutants
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in model insects could be a useful approach for identifying new pathways and candidate genes for eyespot development (Saenko et al., 2008).
17.3.1 The homozygous lethal mutation
Goldeneye
Goldeneye is one such pleiotropic mutation that is embryonic lethal in homozygotes (Saenko et al.,
2008) and affects eyespot colour composition in het- erozygous adults (Brunetti et al., 2001). In Goldeneye butterflies, the scales that typically form the black inner ring of B. anynana eyespots are replaced with gold-coloured scales characteristic of the outer ring (Figure 17.1i), and the expression of genes associ- ated with the different colour rings changes accord- ingly (Brunetti et al., 2001). Analysis of embryonic development in Goldeneye homozygotes has shown that these embryos do not undergo blastokinesis, subsequently become shorter and thicker than wild-type embryos of the same age, and end up dying shortly after the time at which blastokinesis would normally have occurred (Saenko et al., 2008; Figure 17.1f).
The study of disturbed embryogenesis in Goldeneye has suggested that genes involved in blastokinesis may play a role in eyespot formation (Saenko et al., 2008). Unfortunately, the specific gen- etic regulation of embryonic movements in insects, including blastokinesis in the Lepidoptera, is poorly understood, despite the fact that several mutations have been identified that disturb these processes (e.g. Ueno et al., 1995; Schock and Perrimon, 2002; Van der Zee et al., 2005; Panfilio et al., 2006). Even though the Goldeneye embryonic phenotype did not show resemblance to described mutants in other species, and despite the fact that we do not know whether embryonic movements are regulated by similar mechanisms across insect lineages, analysis of candidate genes affecting this process seems like a valuable starting point for further genetic dissec- tion of variation in eyespot morphology.
17.3.2 Conservation versus divergence in insect embryonic development
The strategy of comparing disturbed embryonic phenotypes between B. anynana eyespot mutants
and mutants in genetic model systems will be useful only to the extent that the genetic mechan- isms of embryonic development are conserved across insects. Recent studies extending the ana- lysis of insect embryonic development outside D. melanogaster (see Peel et al., 2005, Damen, 2007; Peel, 2008) have shown that while some aspects of embryonic development are indeed highly conserved (e.g. the functions of segment polarity and Hox genes), others appear to be unexpectedly diverged (e.g. the functions of gap and pair-rule genes; see Chapter 16).
The analysis of embryonic lethality in three other pleiotropic B. anynana mutants, in which development appears to be disturbed during the segmented germband stage, is more prom- ising for identifying mutated genes (P. Beldade and S. V. Saenko, unpublished data). Unlike blas- tokinesis, this stage of embryogenesis is highly conserved among arthropods (e.g. Farzana and Brown, 2008), and the genes and developmental pathways that regulate it have been studied in greater detail in model organisms (see Galis et al.,
2002). Still, whatever the embryonic stage affected in any pleiotropic eyespot mutant, because direct comparison of disturbed eyespot phenotypes with
‘eyespot mutants’ in model species is impossible (model insects do not have eyespots!), comparative analysis of embryonic phenotypes in such mutants remains a valuable first approach.
17.4 Veins and eyespots
Models for butterfly wing pattern formation pro- pose an important role for wing veins and the wing margin (see Nijhout, 1991). An association between wing venation and either patterns of col- ourful stripes and bands or eyespot formation has been supported by the phenotypic characteriza- tion of spontaneous venation mutants in Papilio and Heliconius species (Koch and Nijhout, 2002; Reed and Gilbert, 2004) and in B. anynana (Saenko et al., 2008), respectively. Models of eyespot forma- tion have suggested that wing veins act as sources of diffusible molecules involved in the determin- ation of eyespot-organizing centres, the foci (see Nijhout et al., 2003; Evans and Marcus, 2006), but this role, as well as the nature or even the existence
E V OL U T ION A R Y NO V E L T IES IN B U T T E RFL Y W ING S 189
of the proposed diffusible signals, has not yet been
shown experimentally.
17.4.1 Parallels between fruitfly and butterfly vein development
The mechanisms of vein patterning in Drosophila are fairly well understood (reviewed in Blair, 2007), as is the role of veins in the distribution of melanin precursors in newly eclosed fruitflies (True et al.,
1999). As is often the case for work in non-model systems, this knowledge is an invaluable starting point for our understanding of the mechanisms behind vein establishment and its role in pattern formation in butterfly wings.
Unsurprisingly, positional specification in butterfly wing discs seems to be achieved in a man- ner very similar to that described for Drosophila. Division of the developing wing discs into antero- posterior and dorsoventral compartments is marked by the expression of the genes engrailed and apterous, respectively, and proximal–distal patterning is regulated by Distal-less and wing- less (Carroll et al., 1994). The signalling pathways that are involved in the positioning and differenti- ation of longitudinal and cross veins in Drosophila (reviewed in Marcus, 2001, and Crozatier et al.,
2004) might also be conserved between the lin- eages of Diptera and Lepidoptera (De Celis and Diaz-Benjumea, 2003). Functional analysis of homologues of known Drosophila vein patterning genes during butterfly wing development will be instrumental in our understanding of vein estab- lishment and role in butterfly wings.
17.4.2 Wing venation and eyespot patterns in B. anynana mutants
Study of B. anynana mutants with disturbed ven- ation has started to explore the functional relation- ship between wing veins and eyespot formation, and has suggested that eyespot patterning depends on normal formation of veins and tracheae (Saenko et al., 2008). Mutations that lead to the addition of veins, such as extra veins (Figure 17.1j) can also lead to the appearance of extra eyespots, presum- ably when the ectopic vein bisects an existing
eyespot signalling centre or because the additional vein itself acts as an inducer of eyespot formation. Conversely, partial or complete loss of wing veins often leads to formation of smaller, fewer and/or misshaped eyespots, as in the Cyclops (Figure 17.1k) and veinless (Figure 17.1l) mutants of B. anynana (Saenko et al., 2008).
Surgical manipulations on developing wings of B. anynana venation mutants have provided the first insights into the mechanisms by which loss of veins can interfere with eyespot formation. The absence of eyespots on the dorsal surface of vein- less individuals, investigated by transplanting the signalling focus of a wild-type pupa into a vein- less wing, has shown that vestigial venation leads to impaired determination of the eyespot focus or lack of focal signal (Saenko et al., 2008). The molecu- lar mechanisms of such relationship are yet to be explored.
17.5 Concluding remarks
Here we have reviewed knowledge of the genetic and developmental mechanisms of eyespot forma- tion, and discussed new approaches to the study of these lineage-specific structures, based on the commonalities with conserved developmental pro- cesses such as wound healing, embryonic develop- ment, or vein patterning.
How much the study of laboratory populations can tell us about variation in natural populations remains a crucial issue in evo-devo. In particular, the extent to which the mutants of large effect identified in the laboratory are relevant for nat- ural variation within and across species is still debated (see Haag and True, 2001). Whilst it seems unlikely that pleiotropic mutations with negative effects on other traits (e.g. embryonic lethality in Goldeneye, or fragile wings in mutants with ves- tigial venation) will contribute to variation in natural populations, it is possible that the same loci harbour other, less deleterious, alleles rele- vant to variation in eyespot patterns. Future work will explore the extent to which loci identified in laboratory eyespot mutants contribute to vari- ation segregating in natural populations and to variation across species.
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17.6 Acknowledgements
We thank Max Telford and Tim Littlewood for organizing the Novartis Foundation Symposium on Animal Evolution and inviting us to contribute
to this book. We also thank Paul Brakefield, Vernon French, and Antónia Monteiro for many inspiring discussions on butterfly wing patterns.
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