Figures and data
Proposed AP domains of insect wings.
(A) Comparative morphology has identified boundaries associated with color pattern variation demarcated by the M1, M3, Cu2, and 2A veins, as summarized in McKenna et al.5 (B) Examples of the 2A vein marking a clear posterior color pattern boundary across multiple butterfly families. (C) Proximal posterior features of the archetypal dipteran wing include the calypters, which are only found in Calypterata clade, and the alula, a lobe at the base of the wing blade.1 (D) mirror RNAi knockdowns result in highly specific loss of the alula in D. melanogaster.2 (E) Snodgrass’ model of the archetypal insect wing specifies three major domains along the anteroposterior axis: the remigium, the vannus, and the jugum. Homologies of these domains with the features of the dipteran wing (C), or butterfly wing pattern boundaries (A), has remained unclear.
mirror determines the identity of the far posterior vannus domain in J. coenia.
(A) In J. coenia mirror (red) is expressed posterior to the 2A vein in the late last-instar hindwing imaginal discs, marking the region that will develop into the vannus. See Figure S1 for forewing expression and controls. (B) In adult butterfly wings the vannus is the wing field posterior to the 2A vein, as shown here in J. coenia. (C) Targeted mosaic knockouts (mKOs) of mirror results in loss of vannal identity in J. coenia, including (D) reduction of the anal fold, and (E) continuation of margin color patterns along the posterior wing margin. Some mirror mKOs (C-E) also show mutation in the 2A vein along with loss (D) or reduction (E) of the 3A vein, likely associated with mosaic clone boundaries. mKOs result in asymmetric effects observed in the wings where (D,E) one wing resembles a wild-type wing (WT*) more than the other wing from the respective individual. For more mirror mKOs see Figures S2 and S3.
HCR in situ hybridization of mirror highlight posterior domain expression in J. coenia wing imaginal discs. Related to Figure 2A.
(A) mirror is expressed posterior to the 2A vein in the last-instar forewing imaginal disc of J. coenia. (B) Double in situ of mirror (Channel 1: 647nm) and wingless (Channel 2: 546 nm) in last instar J. coenia hindwing imaginal discs. Expression of wingless observed by HCR precisely matches expression in wing margin peripheral tissue and discal bands (white arrow) as previously described from chromogenic in situs.5 Bottom panel: Negative controls, lacking HCR probes, showed no signal in the 647nm and 546nm channels. Dashed lines show position of posterior veins.
mKOs of mirror result in partial or entire loss of vannus identity in J. coenia hindwings. Related to Figure 2B-E.
Arrows highlight vannal reduction-related phenotypes in the (A) dorsal side and (B) ventral side of the hindwings. Dashed lines show positions of veins. Next to vein labels, asterisks denote vein anomalies, and “-” denote missing veins. (C) Comparison of left and right dorsal hindwings from the same individual, with vannus reduction phenotype highlighted with arrows.
mirror mKOs show post-2A morphological anomalies J. coenia forewings. Related to Figure 2B-E.
mKOs of mirror result in anomalies along the forewing 2A vein, including vein bifurcation and ectopic vein fragments, which are often associated with color pattern disruption. Dashed lines show the position of 2A vein; asterisks denote 2A vein anomalies.
Identification of mirror ortholog in J. coenia.
A maximum likelihood phylogeny of J. coenia, H. erato lativitta, Tribolium castaneum, Apis mellifera, and D. melanogaster Iroquois complex genes confirms that JC_02269-RA is the ortholog of mirror.
Sanger sequencing confirms mutations at CRISPR sgRNA site in mirror mKOs.
Sanger sequencing results from three different mirror mutant individuals. Each line denotes a different wildtype (WT) or mutant allele with indels indicated in red, and the PAM site in green.
