Single locus affects embryonic segment polarity and multiple aspects of an adult evolutionary novelty

Abstract

Background: The characterization of the molecular changes that underlie the origin and diversification of morphological novelties is a key challenge in evolutionary developmental biology. The evolution of such traits is thought to rely largely on co-option of a toolkit of conserved developmental genes that typically perform multiple functions. Mutations that affect both a universal developmental process and the formation of a novelty might shed light onto the genetics of traits not represented in model systems. Here we describe three pleiotropic mutations with large effects on a novel trait, butterfly eyespots, and on a conserved stage of embryogenesis, segment polarity.

Results: We show that three mutations affecting eyespot size and/or colour composition in Bicyclus anynana butterflies occurred in the same locus, and that two of them are embryonic recessive lethal. Using surgical manipulations and analysis of gene expression patterns in developing wings, we demonstrate that the effects on eyespot morphology are due to changes in the epidermal response component of eyespot induction. Our analysis of morphology and of gene expression in mutant embryos shows that they have a typical segment polarity phenotype, consistent with the mutant locus encoding a negative regulator of Wingless signalling.

Conclusions: This study characterizes the segregation and developmental effects of alleles at a single locus that controls the morphology of a lineage-specific trait (butterfly eyespots) and a conserved process (embryonic segment polarity and, specifically, the regulation of Wingless signalling). Because no gene with such function was found in the orthologous, highly syntenic genomic regions of two other lepidopterans, we hypothesize that our locus is a yet undescribed, possibly lineage-specific, negative regulator of the conserved Wnt/Wg pathway. Moreover, the fact that this locus interferes with multiple aspects of eyespot morphology and maps to a genomic region containing key wing pattern loci in different other butterfly species suggests it might correspond to a 'hotspot' locus in the diversification of this novel trait.

Figure 1

Mutants with altered eyespots and disturbed embryogenesis. (a) Representative images of the ventral surface of wings of WT and mutant B. anynana females. (b) Representative scanning electron microscopy images of WT and mutant embryos at ~60% DT (lateral view, anterior is up and dorsal is to the left; scale bar, 100 μm). At this developmental stage, all embryos are still alive (Spr embryos die at 70% DT, while BE and Fr die at about 90% DT) and do not differ consistently in morphology, although Spr embryos seem more compacted (multiple individuals were observed for each mutant). Arrows point to thoracic legs, and arrowheads indicate mouthparts.

Figure 2

Model for the embryonic and eyespot effects of the BE/Fr/Spr locus. For each mutant phenotype, possible genotypes are shown with lines representing the locus, dots corresponding to different sites therein, mutations indicated with stars (the order and the distance between sites is arbitrary), and labels +, a, B and C representing the wild-type and three mutant alleles. Mutations at these three sites, isolated or in combination, define different alleles which can explain all our data. A single copy of the BFS^a allele has no obvious effect on eyespot morphology (the WT phenotype), but two copies produce enlarged eyespots which have 'normal' colour composition (the BE³ phenotype; phenotypically indistinguishable from BE/BE²). Mutations at sites 1 and 2 together make up the BFS^B allele; it has a dominant effect on eyespot size (the BE/BE² phenotype), and, in combination with the BFS^a allele, affects colour composition (the Spr phenotype). This explains the recessive colour composition aspect of Spr inheritance and the presence of BE³ individuals (BFS^a homozygotes) in crosses between two Spr individuals (Table 1), and is consistent with Spr having been isolated from the BE stock (see Materials and Methods). A mutation at a third site in this locus (corresponding to the BFS^C allele) affects eyespot ring boundaries (the Fr phenotype). The alleles BFS^B and BFS^C are embryonic recessive lethal and display a segment polarity phenotype.

Figure 3

Expression of segment polarity genes in embryos. (a) Ventral view of an embryo from the BE stock (including both BE and WT embryos) at 12% DT; en is expressed in the posterior compartment of each segment, which shows that the establishment of its expression is not affected. (b) Ventral view of a WT and a BE embryo at 15% DT; En protein is also detected in some anterior cells of the segments (arrow) in BE embryos. (c) At 25% DT, BE embryos appear shorter than WT, with En present in the anterior and posterior cells of each segment (arrows indicate segment borders). (d) Lateral view of a WT and a BE embryo at 40% DT. Arrows point to posterior cells of the second abdominal segment, expressing en. In BE embryos, En is also detected in anterior cells of each segment (arrowhead). (e) wg expression in embryos at 25% DT (arrows indicate segment borders). In WT, wg mRNA is detected in a single stripe per segment, while an extra wg stripe is present in each segment in a BE embryo. Segment borders allow a clear assessment of the relative position of en and wg expression stripes. Anterior is to the right; scale bar, 100 μm. Mutant embryos from Spr or Fr stocks show identical patterns of en and wg expression.

Figure 4

Ectopic eyespots and expression of eyespot patterning genes. Eyespots of WT (top) and Spr individuals (bottom) differ in colour scheme in adults, and in gene expression patterns in developing pupal wings. (a) Anterior distal part of dorsal adult forewings showing native and damage-induced eyespots formed around wound sites (stars). The colour composition of the ectopic eyespots resembles that of the native eyespots and reveals differences in the response properties of wing epidermal tissue between WT and Spr butterflies (solid and dashed rings outline the Spr native and the ectopic eyespots, respectively). These differences are reflected in the expression patterns of en (b), Dll (c) and sal (d) in 16- to 18-hr old pupal wings. Spr mutants were chosen for these experiments because they exhibit the most severe, and thus most noticeable, effects on eyespot phenotype, including damage-induced ectopic eyespots.

Figure 5

Variation in eyespot size and colour composition in Bicyclus. The different phenotypes produced by allelic variation in our study locus resemble intra- and interspecific variation in the genus (compare to Figure 1a). The wing pattern of B. anynana lines artificially selected for large eyespots [69] resembles that of BE individuals. The B. taenias eyespots with broad golden rings resemble those of Fr butterflies. Further work is necessary to determine whether the same alleles or even loci underlie these different phenotypes.