Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies.

Eyespot patterns of nymphalid butterflies are an example of a novel trait yet, the developmental origin of eyespots is still not well understood. Several genes have been associated with eyespot development but few have been tested for function. One of these genes is the signaling ligand, wingless, which is expressed in the eyespot centers during early pupation and may function in eyespot signaling and color ring differentiation. Here we tested the function of wingless in wing and eyespot development by down-regulating it in transgenic Bicyclus anynana butterflies via RNAi driven by an inducible heat-shock promoter. Heat-shocks applied during larval and early pupal development led to significant decreases in wingless mRNA levels and to decreases in eyespot size and wing size in adult butterflies. We conclude that wingless is a positive regulator of eyespot and wing development in B. anynana butterflies.

Özsu N ，Chan QY ，Chen B ，Gupta MD ，Monteiro A ... -  《-》

Differential involvement of Hedgehog signaling in butterfly wing and eyespot development.

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.

Tong X ，Lindemann A ，Monteiro A 《PLoS One》

Wound healing, calcium signaling, and other novel pathways are associated with the formation of butterfly eyespots.

One hypothesis surrounding the origin of novel traits is that they originate from the co-option of pre-existing genes or larger gene regulatory networks into novel developmental contexts. Insights into a trait's evolutionary origins can, thus, be gained via identification of the genes underlying trait development, and exploring whether those genes also function in other developmental contexts. Here we investigate the set of genes associated with the development of eyespot color patterns, a trait that originated once within the Nymphalid family of butterflies. Although several genes associated with eyespot development have been identified, the eyespot gene regulatory network remains largely unknown. In this study, next-generation sequencing and transcriptome analyses were used to identify a large set of genes associated with eyespot development of Bicyclus anynana butterflies, at 3-6 h after pupation, prior to the differentiation of the color rings. Eyespot-associated genes were identified by comparing the transcriptomes of homologous micro-dissected wing tissues that either develop or do not develop eyespots in wild-type and a mutant line of butterflies, Spotty, with extra eyespots. Overall, 186 genes were significantly up and down-regulated in wing tissues that develop eyespots compared to wing tissues that do not. Many of the differentially expressed genes have yet to be annotated. New signaling pathways, including the Toll, Fibroblast Growth Factor (FGF), extracellular signal-regulated kinase (ERK) and/or Jun N-terminal kinase (JNK) signaling pathways are associated for the first time with eyespot development. In addition, several genes involved in wound healing and calcium signaling were also found to be associated with eyespots. Overall, this study provides the identity of many new genes and signaling pathways associated with eyespots, and suggests that the ancient wound healing gene regulatory network may have been co-opted to cells at the center of the pattern to aid in eyespot origins. New transcription factors that may be providing different identities to distinct wing sectors, and genes with sexually dimorphic expression in the eyespots were also identified.

Özsu N ，Monteiro A 《BMC GENOMICS》

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana.

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain - in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly׳s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.

Tong X ，Hrycaj S ，Podlaha O ，Popadic A ，Monteiro A ... -  《-》

Hox genes are essential for the development of eyespots in Bicyclus anynana butterflies.

The eyespot patterns found on the wings of nymphalid butterflies are novel traits that originated first in hindwings and subsequently in forewings, suggesting that eyespot development might be dependent on Hox genes. Hindwings differ from forewings in the expression of Ultrabithorax (Ubx), but the function of this Hox gene in eyespot development as well as that of another Hox gene Antennapedia (Antp), expressed specifically in eyespots centers on both wings, are still unclear. We used CRISPR-Cas9 to target both genes in Bicyclus anynana butterflies. We show that Antp is essential for eyespot development on the forewings and for the differentiation of white centers and larger eyespots on hindwings, whereas Ubx is essential not only for the development of at least some hindwing eyespots but also for repressing the size of other eyespots. Additionally, Antp is essential for the development of silver scales in male wings. In summary, Antp and Ubx, in addition to their conserved roles in modifying serially homologous segments along the anterior-posterior axis of insects, have acquired a novel role in promoting the development of a new set of serial homologs, the eyespot patterns, in both forewings (Antp) and hindwings (Antp and Ubx) of B. anynana butterflies. We propose that the peculiar pattern of eyespot origins on hindwings first, followed by forewings, could be due to an initial co-option of Ubx into eyespot development followed by a later, partially redundant, co-option of Antp into the same network.

Matsuoka Y ，Monteiro A 《-》