Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina1,2,3. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors4. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. We show that the Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (SpinelessON) or an ultraviolet (UV)-sensitive (SpinelessOFF) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 (refs 5, 6) leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors7,8. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.
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We thank members of the Desplan and Arikawa laboratories for discussion, and especially M. Wernet and J. Rister for suggestions. We thank M. Friedrich for clarifying insect eye homologies, A. Stolfi for discussing CRISPR/Cas9 protocols, C. Merlin for discussing butterfly injection technique, and J. Bothma for discussion. We thank A. Monteiro for providing anti-Sal and K. Shi at Genscript for help with antibody design. This work was supported by NIH grant EY13010 and the Center for Genomics and Systems Biology of NYU Abu Dhabi to C.D., and the JSPS Kakenhi grant numbers 26251036 and 20167232 to K.A. M.P. was supported by an NIH Ruth L. Kirschstein NRSA, a JSPS Short Term Fellowship award, and the Revson Biomedical Research Foundation Postdoctoral Fellowship.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Additional expression data and CRISPR/Cas9 spineless knock out results for V. cardui.
a, Antibodies to P. xuthus Pros and Dve (PxPros and PxDve) cross-react in V. cardui. As in P. xuthus, Pros labels two R7-like photoreceptors per ommatidium (green) while Dve labels bR3–8, the SVF photoreceptors equivalent to the outer photoreceptors of D. melanogaster (red), along with a stochastic subset of Pros-expressing R7-like PRs (yellow co-expression). b, Co-expression of Dve in Pros-positive R7-like photoreceptors is lost in spineless CRISPR/Cas9 knockout tissue; compare to yellow co-expression in wild-type in a. c, Sal antibodies label three photoreceptors per ommatidium in V. cardui (red). d, As in P. xuthus, Spineless antibodies (magenta) label a stochastic subset of R7-like Pros-expressing photoreceptors (green).
Extended Data Figure 2 Pleiotropic effects of the spineless mutation and sequencing of CRISPR/Cas9 generated mutations.
a, b, The spineless mutation affects antennal development in P. xuthus (a) and V. cardui (b), as shown previously for D. melanogaster. This effect is first visible at pupal stages, where missing/shortened antennae are absent in their normal channels (red arrow) and shortened structures protrude in their place (blue arrow). Strongly affected individuals have little to no remaining antennae in adult stages (at right). c, The spineless mutation affects bristle development in V. cardui, as shown previously for D. melanogaster, where some bristles are missing or reduced. in the left image, the regular comb of bristles on the tibia is interrupted in mosaic spineless mutant adults, or almost completely missing (right image). d, Mutation of spineless produced an unexpected effect on wing colour pattern. The wing of an animal injected with guide RNAs targeting both spineless and yellow during the late blastoderm stage (7–9 h after egg lay) is shown on the left. A brown-coloured yellow mutant patch of tissue is visible (yellow arrowhead), as shown in the yellow mutation in Fig. 3, but lighter patches of wing scales lacking both melanin (black) and ommachromes (oranges) are also visible, example marked with a white arrow. Similar clones were observed when only spineless sgRNAs were injected (middle and right). This effect was observed for two independent sgRNAs targeting Spineless. e, Cloning and sequencing of target regions from mutant tissues reveals a mixture of CRISPR/Cas9 generated mutations. Unmodified cDNA sequences are shown on the top line for comparison.
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Perry, M., Kinoshita, M., Saldi, G. et al. Molecular logic behind the three-way stochastic choices that expand butterfly colour vision. Nature 535, 280–284 (2016). https://doi.org/10.1038/nature18616
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