Annotation of the Ubx genomic interval in four butterflies of the Nymphalinae sub-family.

(A) Genomic intervals spanning Antp, Ubx, and abd-A, featuring published transcript annotations from NCBI Reference Genomes of V. cardui and A. io, and manual re-annotations of the J. coenia and K. inachus genomes using published RNAseq dataset (see Methods). Exons are shown with coding (thick) and non-coding (thin) sections. No lincRNA:Ubx-AS5’ transcripts were detected in K. inachus. (B) Expression profiling of transcripts of the Ubx region in K. inachus, based on a reanalysis of published wing RNA-seq transcriptomes (Wang et al. 2022). Expression levels are plotted as DESeq2 normalized counts plots. Pairwise Wald tests adjusted for multiple test correction each assess differential expression between forewings and hindwings. ns : non-significant ; * : p < 0.05; ** : p < 0.01 ; *** : p < 0.001.

A region of hindwing-specific chromatin-opening is bordered by a TAD BE in the last intron of Ubx.

(A) Hi-C contact heatmap in fifth instar forewings of J. coenia and TAD separation scores around Ubx. A TAD boundary element (Antp-Ubx_BE) is inferred in the last intron of Ubx (vertical dotted line). (B) Differential ATAC-seq profiles, re-analyzed from a previous dataset (Mazo-Vargas et al. 2022). Top : open-chromatin profiles of forewings (FW, magenta), and hindwings (HW, green), respectively subtracted from larval head signal (purple, negative when wing signals at background-level). Bottom : subtractive ATAC-seq profile (HW-FW) revealing hindwing-enriched chromatin in the Ubx locus. Antp-Ubx_BE is in the vicinity of an isolated region of forewing-enriched opening (blue arrowhead). (C) PhastCons genomic alignment scores, with overall alignability suggesting minimal structural variation across this interval in Lepidoptera and Trichoptera.

Hindwing-enriched chromatin-opening around Ubx, and the Antp-Ubx_BE boundary, are both maintained in mid-pupal hindwings.

(A) Hi-C heatmap in J. coenia fifth instar larval forewings, and subtractive ATAC-seq profiles at this stage (hindwing-forewing), as expanded from Fig. 2 across the Hox cluster. (B) Hi-C heatmap in J. coenia mid-pupal hindwings, and subtractive ATAC-seq profiles at this stage (forewing-hindwing). Inferred TAD boundaries are shown as vertical dotted lines. Blue arrowhead : position of the Antp-Ubx_BE sgRNA.

CRISPR perturbation of Antp-Ubx_BE results in FW➞HW homeoses.

(A) Antp-Ubx_BE sgRNA targeting (cyan triangle) of a FW-enriched ATAC-peak (magenta) within the Ubx last intron. (B-C) Two examples of J. coenia Antp-Ubx_BE crispants showing mosaic FW➞HW homeoses, shown in dorsal views. CL-WT : contralateral, horizontally flipped images of forewings from the same individuals. WT HW : wild type hindwings from the same individual and mutant forewing side. Both individuals show disruption of their Radial veins (R1-R5 area). The specimen shown in C displays a partial, ectopic eyespot (asterisk). (D-E) Immunofluorescent detection of the UbdA epitope (green) in fifth instar wings disks of Antp-Ubx_BE crispants, revealing ectopic antigenicity in forewings. WT forewings of similar stage, and HW from the same crispant individuals, are shown for comparison as insets. Green autofluorescence was observed in tracheal tissues.

Rare, dual homeoses obtained from CRISPR mutagenesis of the lncRNA_Ubx-IT1 5’ region.

(A) Genomic context of the sgRNA targets (here shown in J. coenia), in the promoter and first exon of the non-coding Ubx-IT1 transcript. (B-C) Dorsal and ventral views of a J. coenia crispant displaying dual homeoses, i.e. with both FW➞HW (presumably due to Ubx gain-of-expression), and HW➞FW clones (akin to Ubx null mutations). Insets on the right describe forewing mutant clones (IT1 mKO), in apposition to CL-WT (contralateral forewings from the same individual), and WT HW (wild type hindwings from the same individual and mutant forewing side). (D) Examples of dual homeoses obtained when targeting orthologous sites in V. cardui.

Homeoses obtained from CRISPR mutagenesis of the lncRNA Ubx-AS5’ first exon.

(A) CRISPR sgRNA targets (here shown in J. coenia), in the first exon of the non-coding Ubx-AS5’ transcript. (B) A single J. coenia crispant showed a FW➞HW transformation. Insets on the right describe forewing mutant clones (AS5’ mKO), in apposition to CL-WT (contralateral forewings from the same individual), and WT HW (wild-type hindwings from the same individual and mutant forewing side). (C-D) Examples of HW➞FW homeoses obtained in J. coenia or when targeting orthologous sites in V. cardui. Scale bars: 500 μm.

CRISPR perturbation of Ubx CRM11 generates occasional dual homeotic phenotypes.

(A) Overview of ATAC-seq differential chromatin accessibility profiles (hindwing - head tissues, green ; forewing - head tissue, magenta) across the Ubx first exon. Several regions show differential opening between wings, one of which (CRM11), was targeted here for CRISPR perturbation (sites a2 and c5 indicate sgRNA targets). (B) Dual homeosis phenotypes obtained in V. cardui following dual-targeting of UbxCRE11a2c5, including homeoses in color patterns and scale morphology. (D) Additional example of a V. cardui UbxCRE11a2c5 crispant with a forewing phenotype (gain of hindwing hair patches, arrowheads). (E) Example of mild hindwing homeoses showing a white eyespot focus on the dorsal and ventral sides. These effects were previously shown to occur in coding Ubx CRISPR knock-out experiments (Tendolkar et al, 2021). Contralateral (CL) WT wings are shown for comparison with mutant wings (B-E). Colored dashed lines: wing veins. Scale bars: 500 μm.

Mosaic forewing homeoses in Heliconius butterfly spontaneous mutants.

Wild-type and mutant sides from the same individuals are shown in each panel, with one side digitally flipped to match left-to-right orientation. A. Heliconius melpomene rosina, ventral view. Wild-caught in the Osa Peninsula (Costa Rica), October 1989. B. Heliconius cydno galanthus, ventral view (magnified inset in B’). Stock culture from Organisation for Tropical Studies station, La Selva (Costa Rica), June 1990 C. Heliconius himera, dorsal view (magnified inset in C’). Stock Culture in the butterfly farm Heliconius Butterfly Works in Mindo (Ecuador), March 2008.

Summary of wing homeosis phenotypes obtained from mutational interrogation.

(A) CRISPR targets at non-coding regions across the Ubx region, here visualized in J. coenia. (B) Summary of injection and adult phenotype data obtained across CRISPR experiments. FW/HW crispants : total number of individuals with forewing or hindwing homeotic clones, regardless of the injected species. Individuals with dual homeosis are counted in both categories. Nmut/Ninj : number of crispants obtained (Nmut), over the number of injected embryos for each species. Bold: experiments with consistent effects in only one segment. See Table 1 for details.

CRISPR mutational interrogation experiments at putative Ubx regulatory regions