Distal‐Less Regulates Eyespot Patterns and Melanization in Bicyclus Butterflies

Abstract

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether overexpression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies.

Hexapoda: Comparative Aspects of Later Embryogenesis and Metamorphosis

“…3.21G, H ;Warren et al 1994 ;Suzuki and Palopoli 2001 ;Tomita and Kikuchi 2009 ). After proleg outgrowth has been initiated, abd -A is subsequently required for their maintenance in A3-A6 (Tomita and Kikuchi 2009 ;Chen et al 2013 ). The posterior prolegs express Dll but do not express abd -A , and their development is initiated prior to that of the more anterior prolegs .…”

Self-Similarity, Distortion Waves, and the Essence of Morphogenesis: A Generalized View of Color Pattern Formation in Butterfly Wings

“…The induced inhibitory signals inhibit further propagation of the original calcium signals, finalizing the position and shape of the black spot. Calcium oscillations stimulate cells to undergo genome amplification and to express a set of regulatory genes such as Wnt-family genes (Monteiro et al 2006;Martin and Reed 2014), spalt and Distal-less (Monteiro et al 2013;Adhikari and Otaki 2016;Dhungel et al 2016;Zhang and Reed 2016). Alternatively, calcium oscillations may be stabilized by the Wnt/Ca 2þ transduction pathway that involves intracellular calcium release (Kühl et al 2000;Kohn and Moon 2005).…”

Nymphalid eyespot serial homologues originate as a few individualized modules

“…The flip side of this story, however, is that the presence of an interrupted network creates developmental potential for future reactivations of the network in the same wing sectors at a later stage in evolution [36]. For instance, differentiation of eyespots in the M2 and M3 sectors in B. anynana, the two 'interrupted' sectors normally without eyespots, was recently shown possible via the overexpression of Dll during the late larval stages of development [37]. Importantly, while Dll levels were overexpressed across the whole wing, additional eyespots only appeared in the wing sectors that were already pre-patterned with Sal, as well as other eyespot centre marker genes, earlier in development [37].…”

“…For instance, differentiation of eyespots in the M2 and M3 sectors in B. anynana, the two 'interrupted' sectors normally without eyespots, was recently shown possible via the overexpression of Dll during the late larval stages of development [37]. Importantly, while Dll levels were overexpressed across the whole wing, additional eyespots only appeared in the wing sectors that were already pre-patterned with Sal, as well as other eyespot centre marker genes, earlier in development [37]. This shows that gradual evolution in expression levels of a single gene may be sufficient to reactivate a previously interrupted network.…”