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Recruitment of a hedgehog Regulatory Circuit in Butterfly Eyespot Evolution

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Science  22 Jan 1999:
Vol. 283, Issue 5401, pp. 532-534
DOI: 10.1126/science.283.5401.532

Abstract

The origin of new morphological characters is a long-standing problem in evolutionary biology. Novelties arise through changes in development, but the nature of these changes is largely unknown. In butterflies, eyespots have evolved as new pattern elements that develop from special organizers called foci. Formation of these foci is associated with novel expression patterns of the Hedgehog signaling protein, its receptor Patched, the transcription factor Cubitus interruptus, and the engrailed target gene that break the conserved compartmental restrictions on this regulatory circuit in insect wings. Redeployment of preexisting regulatory circuits may be a general mechanism underlying the evolution of novelties.

To understand the origin of evolutionary novelties, the developmental and genetic mechanisms that produce new structures and patterns must be elucidated (1). One fundamental issue to be addressed is the extent to which novelty arises from either the redeployment of preexisting developmental programs or the separate recruitment of selected individual genes into new programs. The eyespots on butterfly wings are a recently derived evolutionary novelty that arose in a subset of the Lepidoptera and play an important role in predator avoidance (2–4). The production of the eyespot pattern is controlled by a developmental organizer called the focus, which induces the surrounding cells to synthesize specific pigments (5–7). The evolution of the developmental mechanisms that establish the focus was therefore key to the origin of butterfly eyespots. Here, we identify changes in development and gene expression associated with the evolution of eyespots.

Our screen for genes involved in eyespot evolution was based on the hypothesis that one or more of the signaling molecules that pattern the insect wing blade might also be involved in eyespot development. In Drosophila, two orthogonal systems of short-range [Hedgehog (Hh) and Serrate/Delta] and long-range [Decapentaplegic (Dpp) and Wingless (Wg)] signaling proteins organize wing imaginal disc growth, patterning, and gene expression (8). If any of these signals were involved in eyespot development, then modulation of their expression patterns near the position of eyespot foci should be observed. To test this hypothesis, we cloned the Precis coenia orthologs of the dpp,wg, and hh genes (9) and examined their expression patterns by in situ hybridization in fifth (last) larval instar wing imaginal discs, when establishment of the foci occurs (5). Only hh expression is modulated near developing foci. In mid-fifth instar imaginal discs, hh is expressed in all cells of the posterior compartment (Fig. 1B), as in Drosophila(10), but hh transcript levels are increased in a striking pattern in cells just outside of the subdivision midlines at specific positions along the proximodistal axis of the wing. These domains of increased hh transcription flank cells that have the potential to form foci. Later in the fifth instar, higher levels ofhh transcripts accumulate specifically in cells that flank the developing foci (Fig. 1C).

Figure 1

Hh signal transduction is associated with the establishment of eyespot foci. (A) Dorsal surface of the hindwing. The A/P border is indicated (Ant/Post). (B andC) In situ hybridization with a probe for hh RNA. (B) In the mid-fifth instar, hh transcripts are detected throughout the posterior compartment. However, hhtranscription is decreased in the midline (arrow) between the veins (arrowheads) and increased in regions flanking the positions of each potential focus. The heavily stained distal region (damaged in this specimen) is fated for cell death. (C) In the late fifth instar,hh transcription is sharply increased in cells flanking those destined to form an active focus (center). No increase is seen in positions that will not form active foci (top and bottom). (D and E) In situ hybridization with a probe forptc RNA. (D) In the mid-fifth instar, ptctranscripts are detected in cells just anterior to the A/P boundary. (E) In the late fifth instar, ptc transcripts are also detected in the posterior compartment in eyespot foci (arrows). (F) Immunofluorescent detection of Ci protein reveals expression throughout the anterior compartment as well as in the posterior compartment in both eyespot foci (arrows). The anterior part of the wing is toward the top and distal is to the right in all panels.

To test whether this increased level of hh transcription around developing foci is indicative of active Hh signaling, we examined the expression patterns of genes known to be modulated in response to the Hh ligand, namely, the Hh receptor encoded bypatched (ptc) and the signal transducer encoded by cubitus interruptus (ci) (11,12). In the presence of high levels of Hh, Ptc function is inhibited, resulting in the accumulation of the activator form of Ci. Because ptc is a direct target of Ci (13), cells that receive and transduce the Hh signal have increased levels ofptc transcription.

In early and mid-fifth instar P. coenia wing discs, high levels of ptc transcripts are present along the anteroposterior (A/P) boundary of the wing (Fig. 1D), and citranscripts are limited to the anterior compartment of the disc (14). These patterns are homologous to those observed inDrosophila wing discs (15–17). However, later in development, activation of ptc (Fig. 1E) and citranscription (14), accompanied by accumulation of Ci protein (Fig. 1F) (18), occurs in the posterior compartment of the butterfly wing disc in cells that are flanked by the domains of highest hh transcription and are destined to become eyespot foci. These results indicate that the Hh signal is received and transduced by cells that will differentiate as foci.

The expression of ptc and ci in eyespot foci within the posterior compartment of the butterfly wing breaks the A/P compartmental restrictions on gene expression known fromDrosophila. In Drosophila, Engrailed (En) acts in the posterior compartment to restrict Ptc and Ci to the anterior compartment through direct repression of ci transcription (15, 17), which in turn prevents ptcexpression (19, 20). ptc andci expression in the posterior compartment of P. coenia wing discs indicates that during the evolution of eyespots, either the repression of ci by En or the expression pattern of the en gene has diverged from that observed inDrosophila wings.

We examined whether the expression of en, or of its paralog invected (inv), was modulated during focal establishment. In early fifth instar wing discs, these genes are expressed evenly throughout the posterior compartment (14). However, later in development, En and Inv are expressed at much higher levels in foci (Fig. 2A) (21) and are completely coincident with the expression of Ci (Fig. 2B). In pupal wing discs, when focal establishment is complete, expression of En/Inv is maintained in the focus and expands to cells around it (Fig. 2C). These results suggest that during the course of eyespot evolution, there was a relaxation of the strict En-mediated repression ofci that occurs in the posterior compartment ofDrosophila. Furthermore, the coincidence of the expression domains of en/inv and ci, rather than ofen/inv and hh, indicates that during focal establishment en and inv are targets, rather than inducers, of Hh signaling.

Figure 2

En/Inv is expressed at high levels in eyespot foci. (A) Detection of En/Inv with the monoclonal antibody 4F11 reveals expression throughout the posterior compartment with higher levels present in eyespot foci (arrows). en/inv transcripts are expressed in identical patterns (14). (B) Double immunofluorescent detection of Ci (red) and En/Inv (green) shows that focal expression of these proteins is completely coincident. (C) After pupation, En/Inv protein is present at high levels in the eyespot focus and in surrounding scale-building cells (aligned in rows).

In P. coenia, as in most species of butterflies, eyespots are found only in the posterior compartment of the wing, the normal domain of hh and en/invexpression. However, a few butterfly families contain species with eyespots in the anterior compartment. If hh signaling plays a role in focal establishment and all butterfly eyespot foci develop by the same mechanism, as is suggested by correlations in their variance (22), then expression of Hh signaling components and en/inv should be associated with foci located in the anterior as well as the posterior compartment. To test whether Hh signaling accompanies focal establishment regardless of its location in the wing, we examined hindwing imaginal discs of Bicyclus anynana, a species that forms eyespots on both sides of the A/P compartment border (Fig. 3A). Both En/Inv and Ci are coexpressed in all B. anynana eyespot foci, including the one in the anterior compartment (Fig. 3, B and C). Thus, the expression of the Hh signaling pathway and en/inv is associated with the development of all eyespot foci and has become independent of A/P compartmental restrictions.

Figure 3

Hh signal transduction is associated with the establishment of all eyespot foci, regardless of their anteroposterior location. (A) Eyespots occur in both the anterior and posterior compartments (border indicated) on the ventral surface of theB. anynana hindwing. (B) In the late fifth instar wing imaginal disc, En/Inv is expressed at high levels in all eyespot foci. En/Inv in the anteriormost focus (arrow) is well within the anterior compartment of the wing and does not contact cells in the posterior compartment. (C) High-magnification view of the region surrounding the A/P boundary reveals that En/Inv (green) and Ci (red) expression are coincident in the eyespot foci.

The novel expression patterns of hh, ptc,ci, and en/inv could result from independent or dependent changes in their regulation during eyespot evolution. Experimental evidence from Drosophila and comparative analysis of butterflies leads us to infer that some changes in the expression of hh pathway components were primary whereas others were secondary consequences. For instance, beginning in the late third instar of the developing Drosophila wing disc, the ability to express en/inv in response to Hh signaling is a general property of Ci-expressing cells. This competence is present throughout the anterior compartment (23) but is only used just anterior to the A/P boundary to pattern intervein tissue (24, 25). Expression of en/inv just to the anterior of the A/P boundary in P. coenia and B. anynana indicates that this regulatory circuit is conserved in butterflies (note overlap of En/Inv and Ci in Fig. 3C). The similarity between the induction of en/inv by Hh at the A/P boundary and in eyespot foci in late wing development suggests that during eyespot evolution, the Hh-dependent regulatory circuit that establishes foci was recruited from the circuit that acts along the A/P boundary of the wing (Fig. 4).

Figure 4

Model for the recruitment of the Hh signaling pathway in the evolution of butterfly eyespot foci. Hh induction ofen/inv expression in cells just anterior to the A/P boundary is a conserved feature of wing development. This activation is dependent on binding of the Hh ligand to the membrane receptor Ptc and the conversion of the transcription factor Ci to its activator form. In eyespot-bearing butterflies, the same Hh-regulated pathway is used to establish eyespot foci. hh expression is modified in butterflies through repression along the midline of each wing subdivision and increased transcription in cells flanking the eyespot foci. In butterflies, repression of ci in the posterior compartment is relaxed for these cells to express ptc and transduce the Hh signal.

For this Hh regulatory circuit to operate in focal development, two primary spatial regulatory changes must have evolved. First, mechanisms must have evolved that modulate levels of hhexpression along the proximodistal axis of the wing field. Second, because reception of the Hh signal depends on expression of the Ptc receptor, which in turn depends on Ci function, the restriction ofci from the posterior compartment must have been relaxed.ptc and en/inv expression would then evolve as secondary consequences of these regulatory changes. This recruitment of an entire regulatory circuit through changes in the regulation of a subset of components increases the facility with which new developmental functions can evolve and may be a general theme in the evolution of novelties within extant structures.

  • * These authors contributed equally to this work.

  • Present address: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

  • Present address: Howard Hughes Medical Institute, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.

  • § To whom correspondence should be addressed. E-mail: sbcarrol{at}facstaff.wisc.edu

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