The origin of insect wings is a biological mystery that has fascinated scientists for centuries. Identification of tissues homologous to insect wings from lineages outside of Insecta will provide pivotal information to resolve this conundrum. Here, through expression and clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) functional analyses in Parhyale, we show that a gene network similar to the insect wing gene network (preWGN) operates both in the crustacean terga and in the proximal leg segments, suggesting that the evolution of a preWGN precedes the emergence of insect wings, and that from an evo-devo perspective, both of these tissues qualify as potential crustacean wing homologues. Combining these results with recent wing origin studies in insects, we discuss the possibility that both tissues are crustacean wing homologues, which supports a dual evolutionary origin of insect wings (that is, novelty through a merger of two distinct tissues). These outcomes have a crucial impact on the course of the intellectual battle between the two historically competing wing origin hypotheses.
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We thank N. Patel and H. Bruce for technical assistance and sharing their preprint, and X. Franch-Marro for the Ph-vvl clone. We also thank the Center for Bioinformatics and Functional Genomics (CBFG) and the Center for Advanced Microscopy and Imaging (CAMI) at Miami University for technical support, S. Yi for technical assistance and T. Ohde, A. Fernándes, D. Linz, N. Patel, H. Bruce, A. Martin and other members of the Tomoyasu and Patel labs for helpful discussions. This work is supported by the Miami University Faculty Research Grants Program (CFR) (to Y.T.), the National Science Foundation (NSF) (IOS1557936 to Y.T.), an NSF Graduate Research Fellowship (to C.M.C.-H.) and EDEN: Evo-Devo- Eco Network (NSF-IOS0955517 to C.M.C.-H.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Alignment for the Vg Tondu domain. b, c, POU domain (b) and homeodomain (c) alignments of Nub and Vvl. Both Nub and Vvl belong to the POU-homeodomain protein family; however, each class possesses some characteristic amino acids in the conserved domains (yellow and blue highlighted amino acids). d, Ap homeodomain alignment. Characteristic amino acids found among the two classes of Apterous (A and B) and the corresponding vertebrate homologs are highlighted. The presence of characteristic amino acids for A and B classes indicates that the emergence of these two arthropod Apterous classes preceded the divergence of crustaceans and hexapods. Amino acid sequences for Parhyale proteins were translated from the cloned cDNA sequences. In some cases, additional sequence was added from the published genome assembly (Ph-apA and Ph-apB) or additional cDNA clones (Ph-vvl)49 to obtain the longest conserved domain amino acid sequence possible. Amino acid sequences for other species were obtained from NCBI and OrthoDB except for Af-ApB homeodomain which was obtained from26. Species abbreviations: Af- Artemia franciscana, Am-Apis mellifera, Bg-Blattella germanica, Bm-Bombyx mori, Da-Daphnia magna, Dm- Drosophila melanogaster, Dr- Danio rerio, Mm- Mus musculus, Ph-Parhyale hawaiensis, Of-Oncopeltus fasciatus, Tc-Tribolium castaneum.
a, vg KO2 alignments. b, vg KO3 alignments. c, apA KO2 alignments. d, apA KO3 alignment. The top line in each alignment shows the WT sequence with the targeted site (green) and the Protospacer adjacent motif (PAM) (blue highlight). Red in brackets with “Δ” indicates the number of base pairs deleted from that region and blue in brackets with “+” indicates the number of base pairs added to that region. Yellow nucleotides indicate SNPs.
a, nub KO1. b, nub KO3. c, apA KO2. d, apA KO3. “M” refers to the sacrificed hatchling that exhibited visible mutant phenotypes, while “WT” with gene prefix indicates CRISPR/Cas9 injected individuals that lacked any visible abnormalities. “WT” with no gene prefix is the negative control, with the corresponding genomic region isolated from un-injected WT hatchlings. “+” and “-” indicate presence and absence of T7 endonuclease I. Asterisks indicate T7 endonuclease I digested bands.
Individual with curled tergal phenotype (arrowhead).
Arrow indicates strong expression of Ph-apB in the brain. Embryo is ~stage 21.
A simplified version of the wing gene network (WGN) described in Drosophila. The six genes investigated in this study are indicated in red.
Expression pattern for all genes analyzed in this study.
Supplementary Tables 1–3.
Ph-vg expression pattern (confocal).
Confocal image for WT.
Confocal stack for WT.
Confocal image for Ph-vg KO.
Confocal stack for Ph-vg KO.
Ph-nub expression pattern (confocal).
Confocal image for Ph-nub KO.
Confocal stack for WT (T4).
Confocal stack for Ph-nub KO (T4).
Ph-apA expression pattern (confocal).
Confocal image for Ph-apA KO.
Confocal stack for Ph-apA KO.
Confocal stack for Ph-apA KO (severe).
Ph-ci expression pattern.
Ph-En visualization (confocal).
Ph-omb expression pattern (confocal).
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Clark-Hachtel, C.M., Tomoyasu, Y. Two sets of candidate crustacean wing homologues and their implication for the origin of insect wings. Nat Ecol Evol 4, 1694–1702 (2020). https://doi.org/10.1038/s41559-020-1257-8
Nature Ecology & Evolution (2020)
Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments
Nature Ecology & Evolution (2020)