Frizzled2 receives the WntA morphogen during butterfly wing pattern formation

Abstract

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes that occur in a developing epithelium. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its modes of reception and signal transduction remain elusive. Butterfly genomes encode four homologues of the Frizzled-family of Wnt receptors. Here we show that CRISPR mosaic knock-outs of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss-of-function in multiple nymphalids. While WntA mosaic clones result in intermediate patterns of reduced size, consistently with a morphogen function, fz2 clones are cell-autonomous. Shifts in pupal expression in WntA crispants show that WntA and fz2 are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity (PCP) in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning, and shed light on the functional diversity of insect Frizzled receptors.

Lepidopteran scale cells derive from sensory organ precursors through a canonical lineage

“…These results suggest that Notch-driven lateral inhibition is coupled with the differential expression of CAMs to drive SOP spacing and specification within the butterfly pupal wing epithelium. Other genes showing expression restricted to cluster SOP include those previously described in early scale development Hr38 and fz2, and the asymmetric cell division factor inscuteable (insc; Banerjee et al, 2023;Hanly et al, 2023;Le Borgne et al, 2002;Nolo et al, 2000;Prakash et al, 2024). HCR confirmed SOP-specific expression of fz2 and a POU gene pdm3, with no described roles in SOP specification (Figure 3F-G).…”

“…The identities of clusters for wing margin and trachea could be inferred based on known expression profiles. The cluster margin contains the genes fz3, Wnt6, wg, and cut; expression of all these genes has previously been localised to the wing margin region in multiple species of butterfly (Banerjee et al, 2023;Dohrmann et al, 1989;Hanly et al, 2023;Macdonald et al, 2010). Additional markers of this cluster included the transmembrane transporter CG15406, a S1A non-peptidase homolog CG31326, the extracellular matrix gene M-spondin (mspo) and the transcription factors Beadex (Bx) and hairy (hry).…”

“…C. Dotplot of putative marker genes with top expression in identified cell identities. Expression and/or functional data is present for genes with * (Banerjee et al, 2023;Hanly et al, 2023;Perry et al, 2016;Pomerantz, 2021;Prakash et al, 2024). Genes in bold were tested for expression in D-E''' and genes with black triangles were tested for function in Figures 5-6.…”

“…Each subcluster shows semi-independent gene expression profiles with non-overlapping marker genes. Genes in bold were tested for in situ expression in Figure 3 and genes with * have known expression data either from this paper or from previous publications (Ficarrotta et al, 2022;Hanly et al, 2023;Livraghi et al, 2024;Pomerantz, 2021;Prakash et al, 2024).…”

“…Early competence in the undifferentiated SOP modulates scale colour fate Of note, the snRNAseq dataset revealed novel insights into the development of butterfly wing scales. We identified pattern-related genes restricted to the early SOP cells like WntA and fz2, and Ivory in the later scale-building cells, the latter specifically implicated in melanic patterns across Lepidoptera (Banerjee et al, 2023;Fandino et al, 2024;Hanly et al, 2023;Livraghi et al, 2024;Tian et al, 2024). Additionally, we identified a gene, pdm3, that contributes to scale cell development and differentiation, which is not found in analogous contexts in Drosophila.…”