In this report, we demonstrate the evolutionary and developmental flexibility of the regulatory networks underlying butterfly wing patterning. Virtually all the wing patterning genes thus far identified in butterflies are known to play other deeply conserved, non-wing patterning roles in insect development. This has been recognized since the first gene expression patterns were reported in butterflies , and led to wing patterns serving as a popular illustrative case study of gene co-option. Here we undertook an expanded exploration for wing patterning functions for En/Inv, Dll, Ci, and Spalt. These transcription factors have all been proposed to have been co-opted to eyespot development from various ancestral functions, including appendage development, anterior–posterior compartmentalization, and wing vein development [26,27,28,29]. In this study we asked whether these genes may play some additional roles in wing patterning beyond eyespot development, and we presented new evidence for their likely roles in wing pupal cuticle marking and, in the case of Spalt, wing margin color pattern determination as well.
Patterning the border ocelli system
One finding of the work presented here was that En/Inv, Dll, and Ci precisely mark domains on the J. coenia last-instar forewing disc that correspond to the position and shape of pupal cuticle markings. Much of the pupal cuticle is secreted by the forewing during pupation, and positional associations suggest that the black coloration on the pupal cuticle is produced by the Dll-, En/Inv-, and Ci-expressing wing cells. These findings would indicate that the border ocelli system not only determines eyespot color patterns, but also plays a role in patterning and coloration of the pupal cuticle. Some of these pupal cuticle markings occur where gene expression occurs, but there are no adult eyespots. This suggests that the presence of En/Inv, Dll, Ci, and Spalt in the last-instar wing disc is, by itself, insufficient for eyespot formation. In turn, we speculate that other genes are likely necessary to induce the production of the eyespot focal signal. Alternatively, repressors may be present in some pre-pattern spots expressing En/Inv, Dll, Ci, and Spalt, thus preventing adult eyespot formation, but allowing other patterning elements to form on the cuticle. In any case, our findings support Taira and Otaki , who proposed that eyespot foci can function in pupal cuticle patterning. It is important to recognize that gene expression studies such as this one have been extremely helpful in identifying patterning genes that are then subsequently supported by knockdown studies [21, 30,31,32,33]. Unfortunately, however, in the spalt CRISPR/Cas9 deletion experiments we did not observe effects on pupal markings or anterior white wing spots. We urge caution in overinterpreting negative mosaic results, however. It is possible that because of the variability of spalt somatic mutations, that we simply did not generate mutant cells in these regions, that there were pupal viability issues in potentially informative knockouts, or that the mutant Spalt protein may have retained some of its original function. More functional work is required to assess this.
Our work also demands a reassessment of how border ocelli system patterns are determined. From the last-instar Spalt staining (Fig. 3a), we see that seven wing cells have spots of spalt expression, while a subset of five of these cells also show En/Inv, Dll, and Ci co-expression. All this is in spite of the fact that only two of these spots of co-expression will ultimately go on to produce an adult eyespot. Defining the position of the center of the wing cell is a critical step that must precede these gene expression events. Once this position is defined, a combination of different genes can be expressed which ultimately determine whether eyespots, pupal cuticle markings, and/or simple (white) spots ultimately form on the wing. Thus, our results lead us to envision an expanded model of border ocelli system where pattern elements along the anterior–posterior axis are positioned through a shared process, likely involving Spalt, then combinatorial effects of other ligands and transcription factors determine the final characteristic of specific individual elements, i.e., inductive eyespot foci, cuticle markings, simple spots, etc.
The role of spalt in post-morphogen color pattern specification
The transcription factor Spalt appears to play multiple functions during butterfly wing patterning. For example, functional knockouts show that it plays distinct roles in both vein determination and eyespot patterning . Here we describe an additional role of Spalt in wing margin color patterning. We observed that Spalt is expressed in a discrete line of cells along the proximal boundary of the border lacuna in last-instar wing discs. These are the cells that will become the margin of the adult wing. This expression domain expands during the early pupal stage, after the morphogen induction phase, to encompass a larger domain of scale-building cells along the wing margin. The interface between Spalt-positive and Spalt-negative scale-building cells has the distinctive, species-specific shape as the EIII submarginal bands in both J. coenia and B. anynana (Fig. 5), suggesting a connection between spalt and marginal band patterning. In Drosophila, spalt is involved in positioning the wing veins . In the fly wing the transcription factor Knirps is expressed in, and defines, the L2 wing vein, and the positioning of Knirps is controlled by Spalt and Optix . Ultimately the L2 wing vein in Drosophila forms at the anterior-edge of the interface or boundary between Spalt-expressing and non-expressing cells [27, 34]. This mechanism has a striking similarity to the proposed positioning of the butterfly EIII submarginal band which appears to be patterned at the boundary between Spalt-positive and Spalt-negative cells in the pupal wing disc (Fig. 5), and in the future it would be interesting to examine Knirps expression to test whether this boundary formation system may have been co-opted for butterfly color patterning.
To test the function of spalt in wing margin color patterning, we looked at CRISPR/Cas9-generated spalt deletion mosaics in J. coenia. Using this technique, previous work demonstrated the importance of spalt in eyespot and vein formation . Here, we further show that spalt deletion results in loss of submarginal band color patterns on the adult wing (Fig. 6). These results not only confirm that spalt is necessary for submarginal band formation, but also suggest it has a highly specialized function in specifically promoting the EIII submarginal band. On both ventral and dorsal wing surfaces we observed mosaics where the EIII element is missing, but the EI and EII marginal bands appear to be undisturbed. This is especially striking in the individuals shown Fig. 6a, where a section of EIII is gone, but the EI and EII bands are unaffected. Furthermore, this loss of EIII also reveals a section of a red background pattern, implying epistasis between spalt and this red optix-induced element . The epistatic masking of an optix color pattern by a Wnt-induced pattern has also recently been shown in Heliconius butterflies [32, 35], suggesting a deep conservation of patterning system interactions in which spalt appears to play a key role.
The effects of spalt knockouts on margin color patterns are quite different than those of Dll knockouts, which result in a loss of all wing margin color patterns in both J. coenia and B. anynana . We infer that, in the context of wing margin color patterning, Dll likely plays an early role in determining the entire margin pattern system, consistent with its wing margin expression in last-instar wing discs. Then spalt likely plays a later role in specifically elaborating the EIII pattern. Extending this speculative model further, we propose spalt as a candidate for a morphogen readout factor in pupal wings, since it has a highly specific role in determining very particular subpatterns of a system likely to be induced by inductive morphogen signaling [18, 32].