Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly wing patterns.

Wnt signaling members are involved in the differentiation of cells associated with eyespot and band 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/ß-catenin dependent (canonical) and Armadillo/ß-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

Vermilion and cinnabar are involved in ommochrome pigment biosynthesis in eyes but not wings of Bicyclus anynana butterflies.

If the same pigment is found in different tissues in a body, it is natural to assume that the same metabolic pathways are deployed similarly in each tissue. Here we show that this is not the case for ommochromes, the red and orange pigments found in the eyes and wings of butterflies. We tested the expression and function of vermilion and cinnabar, two known fly genes in the ommochrome pathway, in the development of pigments in the eyes and in the wings of Bicyclus anynana butterflies, both traits having reddish/orange pigments. By using fluorescent in-situ hybridization (HCR3.0) we localized the expression of vermilion and cinnabar in the cytoplasm of pigment cells in the ommatidia but observed no clear expression for either gene on larval and pupal wings. We then disrupted the function of both genes, using CRISPR-Cas9, which resulted in the loss of pigment in the eyes but not in the wings. Using thin-layer chromatography and UV-vis spectroscopy we identified the presence of ommochrome and ommochrome precursors in the orange wing scales and in the hemolymph of pupae. We conclude that the wings either synthesize ommochromes locally, with yet unidentified enzymes or incorporate these pigments synthesized elsewhere from the hemolymph. Different metabolic pathways or transport mechanisms, thus, lead to the presence of ommochromes in the wings and eyes of B. anynana butterflies.

Laser Microdissection-Mediated Isolation of Butterfly Wing Tissue for Spatial Transcriptomics.

The assignment of specific patterns of gene expression to specific cells in a complex tissue facilitates the connection between genotype and phenotype. Single-cell sequencing of whole tissues produces single-cell transcript resolution but lacks the spatial information of the derivation of each cell, whereas techniques such as multiplex FISH localize transcripts to specific cells in a tissue but require a priori information of the target transcripts to examine. Laser dissection of tissues followed by transcriptome analysis is an efficient and cost-effective technique that provides both unbiased gene expression discovery together with spatial information. Here, we detail a laser dissection protocol for total RNA extraction from butterfly larval and pupal wing tissues, without the need of paraffin embedding or the use of a microtome, that could be useful to researchers interested in the transcriptome of specific areas of the wing during development. This protocol can bypass difficulties in extracting high quality RNA from thick fixed tissues for sequencing applications.