Conservation and Flexibility in the Gene Regulatory Landscape Of
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Hanly et al. EvoDevo (2019) 10:15 https://doi.org/10.1186/s13227-019-0127-4 EvoDevo RESEARCH Open Access Conservation and fexibility in the gene regulatory landscape of heliconiine butterfy wings Joseph J. Hanly1,2,3* , Richard W. R. Wallbank1,2, W. Owen McMillan2 and Chris D. Jiggins1,2 Abstract Background: Many traits evolve by cis-regulatory modifcation, by which changes to noncoding sequences afect the binding afnity for available transcription factors and thus modify the expression profle of genes. Multiple exam- ples of cis-regulatory evolution have been described at pattern switch genes responsible for butterfy wing pattern polymorphism, including in the diverse neotropical genus Heliconius, but the identities of the factors that can regulate these switch genes have not been identifed. Results: We investigated the spatial transcriptomic landscape across the wings of three closely related butterfy species, two of which have a convergently evolved co-mimetic pattern and the other having a divergent pattern. We identifed candidate factors for regulating the expression of wing patterning genes, including transcription factors with a conserved expression profle in all three species, and others, including both transcription factors and Wnt pathway genes, with markedly diferent profles in each of the three species. We verifed the conserved expression profle of the transcription factor homothorax by immunofuorescence and showed that its expression profle strongly correlates with that of the selector gene optix in butterfies with the Amazonian forewing pattern element ‘dennis.’ Conclusion: Here we show that, in addition to factors with conserved expression profles like homothorax, there are also a variety of transcription factors and signaling pathway components that appear to vary in their expression profles between closely related butterfy species, highlighting the importance of genome-wide regulatory evolution between species. Keywords: Cis-regulation, Heliconius, Butterfy, Transcription factor, Homothorax, Gene expression, Wnt signaling, Transcriptomics Background Tese elements must function by diferential binding of A major challenge in evolutionary developmental biology regulatory factors, and so to understand the evolution of is to understand how modifcations to gene expression gene regulatory networks, we must frst identify which can lead to biological diversity. In particular, variation in regulatory factors are present and able to perform this cis-regulatory elements has been repeatedly identifed as function in a given spatial and temporal context. the material source of polymorphism and divergence in Te Lepidoptera make up ~ 18% of described animal physiology, behavior, pigmentation patterns and morpho- diversity and have a vast array of wing patterns, both logical structures; Gephebase, a database of genotype– within and between species. Recent advances in genet- phenotype relationships, identifes 323 such examples of ics, genomics and experimental methods have begun to cis-regulatory evolution in diverse eukaryote clades [37]. uncover the underlying genetic and developmental basis of lepidopteran wing pattern variation [22, 33]. One of *Correspondence: [email protected] the most diverse and well-studied groups are the Helico- 1 Department of Zoology, University of Cambridge, Downing St., nius butterfies, and it is now understood that much of Cambridge CB2 3EJ, UK the variation in wing pattern in this group results from Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hanly et al. EvoDevo (2019) 10:15 Page 2 of 14 regulatory evolution at just three genes, optix, WntA and wing development. Consistent with this idea, earlier can- cortex [38, 43, 50]. CRISPR/Cas9 mutagenesis has shown didate gene studies have shown that many patterning that optix and WntA are also involved in the patterning factors previously identifed in Drosophila wing devel- of wings in multiple butterfy lineages [41, 68]. At each opment show similar patterns of expression on butterfy of these loci, there are a large diversity of complex regu- wings (Table 1). In some cases, interesting novel expres- latory alleles, controlling expression patterns across the sion domains have already been identifed in butterfy wing surface during development [13, 60, 61]. Tis regu- wings, mainly in association with eyespot elements [9, latory diversity that generates the extraordinary variation 44]. in wing patterns nonetheless acts against a background of If this is in fact the case, then the pattern variation highly conserved regulatory factors that underlie insect we observe in Heliconius and other butterfies could be Table 1 A summary of single-gene expression studies performed on developing butterfy wings, indicating whether notable diferences in domains of expression have been described in butterfies relative to D. melanogaster Gene Species Any novel domains? References Ultrabithorax J. coenia [63, 64] apterous J. coenia, B. anynana Localized reduction in expression associated [9, 47] with ventralization of pattern engrailed/invected J. coenia Eyespot associated [9] P. rapae, B. anynana, Saturnia pavonia, Anther- [42] aea polyphemus scalloped J. coenia [9] wingless V. cardui, A. vanillae, J. coenia, Battus philenor, B. Expressed in association with basal and mar- [35, 42, 44] mori, M. sexta, Z. morio, B. anynana, P. rapae ginal pattern elements cut V. cardui, A. vanillae, J. coenia, Battus philenor, B. [35] mori, M. sexta, Z. morio hedgehog J. coenia, B. anynana Eyespot associated [26] cubitus interruptus J. coenia, B. anynana Eyespot associated [26] patched J. coenia, B. anynana Eyespot associated [26] achaete-scute J. coenia [14] Notch H. erato, H. melpomene, A. vanillae P. rapae, M. [49, 51] sexta, V. cardui, J. coenia, B. anynana Optix Heliconius spp, J. coenia, V. cardui, Butterfy specifc, in association with specifc [36, 50] scale types WntA Heliconius spp, Limenitis arthemis, A. vanillae, J. Butterfy specifc, in association with specifc [15, 40, 41] coenia, Danaus plexippus pattern elements doublesex Papilio, B. anynana [5, 31] aristaless1 & aristaless2 J. coenia, Limenitis arthemis, Spodoptera orni- Wing pattern associated, including Discalis II [39] thogalli, Ephestia kuehniella elements Distal-less H. erato, H. melpomene, A. vanillae P. rapae, M. Dynamic eyespot-associated expression [6, 9, 51] sexta, V. cardui, J. coenia, B. anynana, Saturnia pavonia, Antheraea polyphemus spalt major V. cardui, P. rapae, P. oleracea, Colias philodice, C. Eyespot associated [42, 57, 67] eurytheme, B. anynana decapentaplegic J coenia, B. anynana Parallel to veins, in addition to A-P [9, 11] Ecdysone receptor J coenia Eyespot associated [28] pSmad B. anynana, P. rapae Eyespot associated [42] Antennapedia B. anynana, Heteropsis iboina, Pararge aegeria Eyespot associated [54] and Melanargia galathea, Lasiommata megera, J. coenia, M. cinxia, Inachis io, Caligo memnon cortex H. melpomene, H. numata Butterfy specifc, pattern associated [43] armadillo B. anynana [11] BarH1 Colias Butterfy specifc, pigment associated [65] Many of the genes listed, in addition to having domains of expression that are homologous to those found in D. melanogaster, are also expressed in association with eyespots Hanly et al. EvoDevo (2019) 10:15 Page 3 of 14 generated by the diferential ‘readout’ of a highly con- established that modifed the expression of yellow to gen- served set of transcription factors that efectively ‘pre- erate novel diversity. Likewise, expression of pigmenta- pattern’ the wing (Fig. 1a). Tese conserved expression tion genes in the thorax of D. melanogaster is repressed patterns could then provide input to the regulatory ele- by the expression of the transcription factor stripe, which ments of pattern switch genes like optix (Fig. 1b), and specifes fight muscle attachment sites. Te shape of this in turn, modifcations of these elements could lead to thoracic element is thus constrained by factors that spec- production of wing pattern diversity (Fig. 1c), a mecha- ify fight muscle pattern [16]. On the other hand, there nism that allows for the gain of novel phenotypes with are also examples of Drosophila pigmentation evolving the avoidance of deleterious pleiotropic efects [48]. Tis by modifcation to the trans-regulatory landscape, by hypothesis has previously been examined at the within- which conserved components of the regulatory land- species level in Heliconius through transcriptomics [20]. scape themselves change in expression profle to afect Key examples of this mode of regulatory evolution have downstream melanin synthesis domains. For example, been described in evolution of melanic patterns in Dros- Dll in D. biarmipes and D. prolongata has gained addi- ophila species. For example, the protein Engrailed is a tional expression profles to its usual peripheral pattern, deeply conserved component that specifes the posterior in correlation with