Ed Yong, The Atlantic wrote:Scientists Can Now Repaint Butterfly Wings

Thanks to CRISPR, scientists are studying animal evolution in ways that were previously thought to be impossible.

By Ed Yong, The Atlantic, 18th September 2017

When the butterfly emerged from its pupa, Robert Reed was stunned. It was a Gulf fritillary—a bright-orange species with a few tigerlike stripes. But this butterfly had no trace of orange anywhere. It was entirely black and silver. “It was the most heavy-metal butterfly I’ve ever seen,” Reed says. “It was amazing to see that thing crawl out of the pupa.”

Reed’s team at Cornell University had created the metal butterfly by deleting just one of its genes, using the revolutionary gene-editing technique known as CRISPR. And by performing the same feat across several butterfly species, the team showed that this one gene, known as optix, controls all kinds of butterfly patterns. Red becomes black. Matte becomes shiny. Another gene, known as WntA, produces even wilder variations when it’s deleted. Eyespots disappear. Boundaries shift. Stripes blur.

These experiments prove what earlier studies had suggested—that optix and WntA are “paintbrush genes,” says Anyi Mazo-Vargas, one of Reed’s students. “Wherever you put them, you’ll have a pattern.”

Ed Yong, The Atlantic wrote:Biologists have long been smitten by butterflies, and not just for their pretty colors. These insects are perfect subjects for addressing two of the most fundamental questions in the study of evolution. First, where do new things come from? Butterflies all evolved from a moth ancestor, so how did a presumably dull-winged insect give rise to a kaleidoscopic dynasty of some 18,000 species, each with a distinctive pattern of colors and shapes plastered on its wings? Also, what are the genes behind these patterns? How did a limited set of DNA come to produce patterns of such astonishing diversity and often-baffling complexity?

Many scientists, Reed included, have addressed that second question. By carrying out painstaking cross-breeding experiments, and by working out where in the wings various genes are active, they identified a handful of pattern-defining genes, with colorful names like optix, doublesex, and cortex. “It was convincing but we didn’t know exactly what these genes were doing,” says Reed. Without the ability to delete the genes, and see if their absence changed the butterfly wings, “we didn’t have the final proof. There’s been this frustrating wall that I’ve banged my head against.”

CRISPR changed everything. This technique, used by bacteria for billions of years and harnessed by scientists in the last five, allows researchers to cut and edit DNA far more easily and precisely than ever before. As I’ve argued before, the oft-cited concerns that CRISPR will usher in a dystopic era of designer babies are overblown. But scientists are already exploiting it, to do experiments that would have been impossible a decade ago. They’ve used CRISPR to probe the weaknesses of cancer cells, study how bodies are built, and to learn how our feet evolved from fishy fins. And Reed has used it to finally do the gene-deleting experiments that had long eluded him.

By deleting the optix gene in a wide variety of butterflies, team member Linlin Zhang showed that red parts of the wing consistently turn black. The Gulf fritillary transforms from a vivid orange insect into a dark inky one. The small postman loses the vivid red streaks on its hind wings. And the painted lady loses its complex psychedelic patterns and becomes almost monochrome. “They just turn grayscale,” says Reed. “It makes these butterflies look like moths, which is pathetically embarrassing for them.”

These results reveal another side to CRISPR’s power: It’s so versatile that scientists can quickly manipulate the same genes in many species, including those that aren’t standard parts of laboratory life. For years, scientists have relied on a few handfuls of “model systems”—species that they can easily breed, study, and manipulate in laboratories. But CRISPR “fully unlocks butterflies as a model system,” says Wei Zhang from the University of Chicago, who published the first study that used the technique on butterflies.

Mazo-Vargas et al, 2017 wrote:Abstract

Butterfly wing patterns provide a rich comparative framework to study how morphological complexity develops and evolves. Here we used CRISPR/Cas9 somatic mutagenesis to test a patterning role for WntA, a signaling ligand gene previously identified as a hotspot of shape-tuning alleles involved in wing mimicry. We show that WntA loss-of-function causes multiple modifications of pattern elements in seven nymphalid butterfly species. In three butterflies with a conserved wing-pattern arrangement, WntA is necessary for the induction of stripe-like patterns known as symmetry systems and acquired a novel eyespot activator role specific to Vanessa forewings. In two Heliconius species, WntA specifies the boundaries between melanic fields and the light-color patterns that they contour. In the passionvine butterfly Agraulis, WntA removal shows opposite effects on adjacent pattern elements, revealing a dual role across the wing field. Finally, WntA acquired a divergent role in the patterning of interveinous patterns in the monarch, a basal nymphalid butterfly that lacks stripe-like symmetry systems. These results identify WntA as an instructive signal for the prepatterning of a biological system of exuberant diversity and illustrate how shifts in the deployment and effects of a single developmental gene underlie morphological change.

Mazo-Vargas et al, 2017 wrote:The multitude of patterns found in developing organisms is achieved by a small number of conserved signaling pathways, which raises an important question. How does biodiversity arise from the sharing of constituents across a single tree of life? One explanation for this apparent paradox is that conserved regulatory genes evolve new “tricks” or roles during development (1). Assessing this phenomenon requires comparing the function of candidate genes across a dense phylogenetic sampling of divergent phenotypes. Here, the patterns on butterfly wings provide an ideal test case. The development of scale-covered wings, their structural and pigment complexity, and an elaborate patterning system are key features of the Lepidoptera (moths and butterflies), which form about 10% of all species known to humankind (2). Wing patterns across the group are fantastically diverse and are often shaped by natural and sexual selection (3). Studies in fruit flies, butterflies, and moths have implicated secreted Wnt-signaling ligands as color pattern inducers (4–8). In butterfly wings, two lines of evidence suggest a prominent patterning role for the Wnt ligand gene WntA in particular. First, WntA was repeatedly mapped as a locus driving pattern-shape adaptations involved in mimicry, and a total of 18 WntA causative alleles have been identified across a wide phylogenetic spectrum (9–13). Second, WntA expression marks developing wing domains that prefigure the position and shape of pattern elements of various color compositions (10, 14).

The nymphalid groundplan provides a conceptual framework to understand pattern variation in butterflies (3). Under this framework, patterns are organized into parallel subdivisions of autonomous color pattern complexes known as “symmetry systems,” which are arranged across the dorsal and ventral surfaces of both the fore- and hindwing (14–19) (Fig. 1 A–C). This arrangement is thought to represent a putative archetype of a butterfly wing pattern, and diversity is created by modifying elements within and among these symmetry systems (3). WntA is typically expressed in three of the four symmetry systems (14): the small proximal pattern called Basalis (B), the large median pattern called the Central Symmetry System (CSS), and the Marginal Band System (MBS), which features laminar stripes bordering the wing. Here we used CRISPR/Cas9 mutagenesis to impair WntA function and assess its patterning roles in Nymphalidae, the largest butterfly family that radiated around 90 Mya (20). We characterize the developmental function of WntA in species representative of the nymphalid groundplan and then show that WntA has acquired divergent patterning roles in several lineages.

Mazo-Vargas et al, 2017 wrote:Significance

Our study assesses the long-held hypothesis that evolution of new gene functions underlies the diversification of animal forms. To do this, we systematically compared the patterning roles of a single gene across seven butterfly species. Under a null hypothesis of gene stasis, each knockout experiment should yield directly comparable phenotypes. We instead observed a varied repertoire of lineage-specific effects in different wing regions, demonstrating that the repeated modification of a key instructive signal was instrumental in the complex evolution of wing color patterns. These comparative data confirm the heuristic potential of CRISPR mutagenesis in nontraditional model organisms and illustrate the principle that biodiversity can emerge from the tinkering of homologous genetic factors.

Mazo-Vargas et al, 2017 wrote:Results and Discussion

We injected Cas9/sgRNA duplexes into 1–6 h butterfly embryos at a syncytial stage (n = 5,794 eggs). As only a fraction of the dividing nuclei are edited, the resulting mosaicism can bypass the deleterious effects of developmental mutations and yields G0 escapers that survive until the adult stage for phenotypic analysis (21–23). We performed CRISPR injections in seven nymphalid species to induce frameshift mutations in WntA-coding exons. About 10% of hatchlings (240 of 2,293 survivors) yielded adult butterflies with mosaic knockout (mKO) pattern defects on their wings (SI Appendix, Figs. S1–S9 and Tables S1 and S2).

WntA Induces Central Symmetry Systems. First we used CRISPR to test the effects of WntA loss-of-function on the wing patterns of the Common Buckeye Junonia coenia (tribe: Junoniini). WntA mKOs resulted in a complete loss of the CSS, consistent with WntA expression that prefigures its shape and position in the wing imaginal disks (Fig. 1 D and E and SI Appendix, Fig. S1). The WntA-positive forewing B element was lost while the wg-positive D1-D2 elements (8, 24) were unaffected (Fig. 1F). The B-D1-D[/sup]2[/sup] patterns have a similar color composition, indicating that WntA and wg play interchangeable roles in their induction. In contrast, the double loss of the distinct B and CSS patterns also illustrates the regional specificity of WntA-signaling color outputs across the wing surface. In the marginal section of the wing (Fig. 1G), WntA mKOs resulted in a contraction of the MBS and in a shift of chevron patterns known as the distal parafocal elements (dPF) (17, 19). WntA may impact these distal elements by participating in complex patterning dynamics in the marginal section of the wing (25).

Variations on the WntA Groundplan Theme. Next we asked if the instructive roles of WntA were phylogenetically conserved, using two other nymphalid butterflies with a groundplan organization, the Specked Wood Pararge aegeria (tribe: Satyrini) (15) and the Painted Lady Vanessa cardui (tribe: Nymphalini) (18). WntA mKOs yielded consistent effects by eliminating the CSS and distalizing the parafocal elements in these two species (Figs. 1 H–K and 2 A–H and SI Appendix, Fig. S10). Of note, in the V. cardui hindwing, the complex wave-like patterns of the CSS were lost upon severe WntA mKO and reduced in more intermediate forms (Fig. 2 I and J). These two species also highlighted other aspects of WntA phenotypic effects. In P. aegeria hindwings, the mKO-mediated disruption of the marginal system resulted in an apparent expansion of the eyespot outer rings (SI Appendix, Fig. S10D). V. cardui WntA mKOs resulted in the reduction of each dorsal forewing eyespot (P values < 10−4; Fig. 2 K–M) and generated color composition defects in the ventral forewing eyespots (Fig. 2N and SI Appendix, Fig. S3). Only V. cardui forewings are known to express WntA in their eyespots (14). We thus infer that WntA was co-opted in the eyespot gene regulatory network of the V. cardui lineage to elaborate upon the patterning of this complex feature (26). Overall, comparisons in three species show that multifaceted modulations of WntA function have shaped variations on the basic nymphalid groundplan theme.

WntA Induces Pattern Boundaries in Heliconius. We next focused on species that departed more markedly from the nymphalid groundplan configuration, starting with the hyperdiverse Heliconius clade (tribe: Heliconiini). We performed CRISPR mKOs in Central American morphs of two species, Heliconius erato demophoon and Heliconius sara sara. WntA removal resulted in an expansion of light-color patterns in both cases (Fig. 3 A and F). In H. erato demophoon, WntA expression marked melanic patches that contour forewing red and hindwing yellow stripes (Fig. 3 B and D). Predictably, its loss-of-function resulted in the loss of the corresponding boundaries with black contours being replaced by expansions of red or yellow (Figs. 3 C and E). H. sara forewing disks showed a proximal and central WntA expression domains that each correspond to melanic fields that frame the signature yellow stripes of this butterfly (Fig. 3G). Both melanic intervals were lost following WntA mKOs (Fig. 3H), yielding an almost uniformly yellow forewing surface. Hindwings showed a similar effect with WntA deficiency resulting in melanic-to-yellow switches in the anteroproximal half of the wing (Fig. 3J). Interestingly, this treatment also revealed a cryptic stripe of red patches. A similar phenotype is observed in subspecies of H. sara, as well as in its sister species Heliconius leucadia (SI Appendix, Fig. S11), suggesting that modulations of Wnt signaling could underlie these cases of natural variation. Overall, these data support previous predictions that groundplan elements such as the CSS can be homologized to what form the apparent contours of Heliconius patterns (27–29). WntA is best thought as a prepatterning factor that determines boundaries between color fields, a view that is compatible with the replacement effects of mKOs, where WntA-deficient cells acquire the color fate of the adjacent territory. This property may explain why cis-regulatory tinkering of WntA expression seems to underlie the repeated modification of color pattern shapes across this explosive radiation (9–12), as it allows the coordinated modulation of colorfate on either side of a moving boundary.

Antagonistic Roles of WntA in Adjacent Patterns. Compared with Heliconius, the closely related Gulf Fritillary butterfly (Agraulis vanillae) has modified the nymphalid groundplan differently to produce its distinctive wing pattern (27). Rather than continuous stripes, A. vanillae shows dispersed silver spots of identical color composition, each consisting of a core of highly reflective “mirror” scales (30) and an outline of black scales. A subset of silver spots express WntA or wg (14), and accordingly, all of the WntA+[i] patterns contracted or disappeared in [i]WntA mKOs (Fig. 3 K–N and SI Appendix, Fig. S6). Among the wg+ elements (forewing D1 and D2), only D1 coexpressed WntA and was specifically reduced in WntA mKOs (SI Appendix, Fig. S12), suggesting that silver spots respond to overall Wnt dosage. WntA mKOs also resulted in a drastic expansion of WntA-free (WntA−) patterns (Fig. 3O). Importantly, butterflies treated with exogenous heparin, a ligand-binding molecule with Wnt gain-of-function effects (9, 14, 31, 32), showed the opposite outcome: expanded WntA+ and reduced WntA− patterns (14). These reverse effects of CRISPR loss-of-function vs. heparin gain-of-function suggest that WntA activates and represses two distinct sets of patterns, and the repressed domain in fact shows a secondary wave of WntA expression in late larval instar wing disks (Fig. 3P). This observation leads us to propose that the dual effect of WntA may be due to a biphasic deployment, with a first wave of WntA pattern activating expression followed by an inhibitory event in the Wnt repressed territory. Testing this working model will require the identification and expression profiling of WntA-signaling targets in A. vanillae.

Repurposing of WntA in a Reduced Groundplan. Finally, we used the lack of visible CSS in monarchs (Danaus plexippus; tribe: Danaini) as an example of extreme divergence from the nymphalid groundplan. WntA lacked a CSS median stripe expression as expected and was instead detected around the presumptive veins, indicative of a potential role in the induction of vein-dependent patterns (33). WntA mKO adults showed drastic expansions of the white interveinous patterns (Fig. 4), which are usually visible as thin outlines of the veins in WT ventral wings. In addition, white dot elements that ornate the marginal region expanded and fused following WntA mKO. Other WntA mKO monarchs showed a small dorsal patch of ectopic interveinous scales in the crossvein region, demonstrating maximal WntA expression in hindwings (SI Appendix, Fig. S13). Consistent with a Wnt loss-of-function, this mild phenotype was reproduced by injection of dextran sulfate, a drug treatment that emulates Wnt signal inhibition in other butterflies (14, 32) (SI Appendix, Fig. S14). Overall, expression and functional data suggest that WntA was again repurposed, in this case as a repressor of interveinous white scales in the monarch lineage.

Lessons from Somatic CRISPR Phenotypes. Somatic mutagenesis yielded loss-of-function data in the G0 adults of seven butterfly species, an achievement that would have been unrealistic in the pre-CRISPR era. Experimental replication using various single-guide RNA (sgRNA) targets ruled out a contribution of off-target lesions, and genotyping experiments revealed a predominance of frameshift, presumably null WntA alleles (SI Appendix, Figs. S8 and S9). Variations in clone size, allelic dosage, and the possible occurrence of hypomorphic mutations could underlie complex cases of mosaicism, explaining the range of observed effects (Fig. 2J and SI Appendix, Figs. S1–S7). Inferring the allelic composition of wing mutant clones from their genotyping is complicated by the movement of insect wing epithelial cells following adult emergence (34), as well as by the presence of cell contaminants that are unlikely to underlie the pattern phenotype (e.g., tracheal cells, neurons, hemocytes). We attempted the generation of germline mutations in V. cardui to bypass the experimental limitations of somatic heterogeneity. Following the injection of a single sgRNA targeting the WntA stop codon, we obtained an adult female bearing a modification of the forewing CSS (SI Appendix, Fig. S15). Six G1 offsprings displayed the same phenotype and were all heterozygous for a 16-bp indel mutation, resulting in a C-terminal Cys-Asn-Stop → Gly-Ser-Arg-Stop editing of the predicted WntA protein. This allele was passed to a second generation but was subsequently lost due to an episode of high mortality in our stock. Nonetheless, this preliminary result illustrates the potential of CRISPR to induce a variety of loss-of-function alleles, which could be propagated via the germline for tackling future developmental questions where mosaicism is a concern.

Mazo-Vargas et al, 2017 wrote:Conclusions.

The Nymphalidae family comprises about 6,000 butterfly species, most of which can be identified by their wing patterns. We used this system as a proxy of morphological evolution and found that a single signal articulates its underlying complexity, as shown by the variety of WntA mKO phenotypes obtained across different wing regions and species. Our data highlight three major results. First, WntA is associated with multiple pattern elements within the same individual, including within the same wing surface, e.g., both the adjacent Basalis and CSS patterns require WntA in J. coenia forewings, despite distinct color compositions, whereas CSS stripes often differ between wing surfaces (dorsal vs. ventral, forewing vs. hindwing). Wnt signaling may combine with selector genes that mark distinct wing domains to mediate these regional-specific outputs within a single individual (24, 35). Second, spatial shifts in WntA expression cause pattern-shape evolution, exemplified by the multitude of species-specific manifestations of the CSS. Cis-regulatory variants of WntA (9–12), or alternatively, modulations of the trans-regulatory landscape that controls WntA expression, may have fashioned these macroevolutionary shifts. Finally, WntA evolves new patterning functions. It was co-opted into forewing eyespot formation in the V. cardui lineage, evolved a localized pattern-inhibiting role in A. vanillae, and was repurposed for the patterning of vein-contouring markings in monarchs. In summary, WntA instructs the formation of multiple wing-pattern elements in the nymphalid radiation, demonstrating the importance of prepatterning processes in the unfolding of complex anatomy. The versatility of this signaling factor illustrates how the repeated tinkering of a developmental gene can foster boisterous evolutionary change.