Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly eyespots

Wnt signaling is involved in the differentiation of eyespot color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here we explore the localization and function of armadillo/{beta}-catenin dependent (canonical) and armadillo/{beta}- catenin independent (non-canonical) Wnt signaling in eyespot development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all seven wnt ligand and four frizzled receptor transcripts present in the genome of this species, and testing the function of arm and frizzled4 using CRISPR-Cas9. During mid to late larval wing development, Arm protein was present in cells at the center of the future eyespots, the foci, and the wing margin, but wnts expressed on the wing, wnt1 (wingless), wnt6, and wnt10 showed expression only some distance away from the foci, along the wing margin. The receptor frizzled9 was expressed in the wing margin and in finger-like projections leading to the foci during early larval wing development, overlapping in expression with Arm. At the same time, the receptor frizzled4 showed a novel expression pattern, anti-localized with Arm, where it is likely transducing non-canonical Wnt signaling. In the early pupal stage, wnt1 was newly expressed in the foci, as previously shown, along with Arm. In addition, frizzled4 and frizzled9-mediated Wnt signaling is likely repressing the expression of frizzled2, as these receptors have anti-colocalized expression domains. Arm had a conserved expression in three other nymphalid butterflies, and functional knockouts of arm and frizzled4 in B. anynana showed that both genes are essential for the differentiation of eyespots. These results show that distinct Wnt signaling pathways are essential for eyespot development in butterflies and are likely interacting to control their active domains.