The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth, e.g., a gill) on the leg of an ancestral crustacean. As a Ph.D. student in Nipam Patel’s lab at the University of California, Berkeley, I investigated this question by performing CRISPR-Cas9 knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compared my knockout phenotypes to those previously published in insects (Fig. 1). This led to an alignment of insect and crustacean legs that suggested that two leg segments that were present in the common ancestor of insects and crustaceans became incorporated into the insect body wall, moving the exite of the proximal-most leg segment dorsally, up onto the back to later form insect wings. Thus, insect wings are not novel structures, as argued by many previous authors, but instead evolved from existing, ancestral structures (Bruce and Patel, 2020).
Fig. 1. Knockout phenotypes of leg patterning genes. (a-f) Parhyale CRISPR-Cas9 phenotypes in dissected third thoracic legs (T3). Graded cyan in f indicates deletion/fusion of proximal leg segment 3. (g) Summary of leg patterning gene functional phenotypes and leg segment alignment between crustacean and insect. Colour bars correspond to remaining leg segments following knockout, semi-transparent bars indicate deleted leg segments. Open bar in dac indicates extension of dac function into tarsus of insects. Coxal plate (Cp), gill (G), tergal plate (Tp). Scale bar 50µm. Insect leg adapted from Snodgrass 1927.
Arthropods (insects, crustaceans, myriapods, and chelicerates) display a fascinating diversity of decorations, including all manner of plates, horns, helmets, gills, mimicry outgrowths, and wings. The origins and relationships of these structures has tantalized researchers for over a century, however the different numbers of leg segments in the four groups of arthropods made it difficult to determine how leg segments and other outgrowths relate to each other. To determine the origins and relationships of these structures, I compared the expression and loss-of-function phenotypes of leg patterning genes in crustaceans, insects, and arachnids using original and previously published data. Surprisingly, this comparison revealed a simple, one-to-one correspondence between the leg segments of all arthropods, with the difference in the numbers of apparent leg segments being due to the incorporation of one or two leg segments into the body wall in many arthropods. I had essentially discovered the molecular coordinate system for arthropod proximal-distal identity, much like the Hox genes are used for understanding the anterior-posterior axis. My system is therefore a powerful framework for deciphering the origins and relationships of any arthropod ectodermal structure. A coordinate system for understanding the homologies of any ectodermal structure in any arthropod unlocks a powerful system for studying the origins of novel structures, the evolvability of morphogenetic fields across vast phylogenetic distances, and the convergent evolution of shared ancestral developmental fields. This breakthrough research has led to several national and international collaborations (Bruce, 2022).
Fig. 2. Elucidating proximal leg segments in arthropods. Dissected right half and legs of embryos of a crustacean Parhyale, an insect Tribolium, and a chelicerate spider Acanthoscurria. pnr (red), ara (green), Dll (pink), DAPI (grey). In all three arthropods, leg segments 1 through 5 are identified by Dll expression, pnr marks the most dorsal domain, and the two ara armband domains bracket a region proximal to leg segment 7. In Acanthoscurria, leg segment 7 is easily identified by the coxal endite (ce) that bulges medially. (G – H) Acanthoscurria odd-r3 (yellow) is expressed in the distal region of each leg segment where the joint will later form. At this stage, proximal to leg segment 7, there is a leg-segment-like bulge (white curly brace), which expresses odd-r3 in its distal region (most visible in G’). (H) dissected walking leg 1 from Stage 11.5 embryo where morphological bulges and subdivisions of the leg segments have not yet begun. odd-r3 encircles the distal region of each leg segment, including the hypothesized proximal 8th leg segment.
Understanding how novel structures arise is a central question in both evolutionary and developmental biology. Novel structures are often defined as structures that are not derived from (homologous to) any structure in the ancestor (Muller and Wagner 1991). Previous authors working in pancrustaceans (insects and "crustaceans") had found that the same genes used to pattern insect wings were also expressed in a surprising variety of insect body wall outgrowths that looked nothing like wings. In 2017, Shiga and colleagues similarly found that this insect wing gene network was also expressed in the crustacean Daphnia magna in the carapace, a bivalved ‘‘shell’’ of exoskeleton that emerges from the back of the head and envelops the animal like a cape (Fig. 3). Shiga 2017 therefore proposed that the carapace, as well as the other outgrowths in arthropods that express the wing gene network, are novel structures that arose through the repeated co-option of this gene network.
However, my work on the origin of insect wings suggested a different hypothesis. Given that the insect wing is just another kind of exite (multipotent side lobe of the leg), and that many arthropods have incorporated the leg base with its exite into the body wall, I hypothesized that the reason that all of these different outgrowths express wing genes is not due to repeated co-option, but because they are all exites inherited from a common ancestor.
To determine whether the Daphnia carapace is an exite on the proximal-most leg segment that was incorporated into the body wall or is a novel structure that arose through co-option, I compared the gene function work in Shiga 2017 with the expression of pannier and araucan, genes that I had previously shown to pattern the proximal leg region of all arthropods examined, as well as the "wing" genes vestigial and scalloped. I compared the expression of these genes in Daphnia to a more basally-branching pancrustacean, Parhyale, to represent the ancestral condition prior to the evolution of the carapace, as well as to a more derived pancrustacean, the insect Tribolium. The expression patterns of these genes in Daphnia was consistent with my hypothesis that the carapace is a greatly expanded exite on the proximal-most leg segment, rather than a co-option of "wing" genes. The leg that the carapace exite emerges from is the maxilla 2 of the head (mouthparts are modified legs) . The Daphnia carapace, therefore, appears to be homologous to (the same as) the Parhyale tergal plate and the insect wing, but emerging on the head rather than the thorax. Remarkably, the vestigial-positive tissue that gives rise to the carapace on the Daphnia head appears to also be present on the heads of Parhyale and Tribolium as a small, inconspicuous protrusion. Thus, rather than a novel structure resulting from gene co-option, the Daphnia carapace appears to have arisen from a shared, ancestral structure that persists in a cryptic state in other arthropod lineages. Cryptic persistence of unrecognized serial homologs may thus be a general solution for the origin of novel structures. (Bruce and Patel, 2022).
Fig. 3. Many arthropods have tergal plates (pink shaded) on many body segments, including maxilla 2 (Mx2) and the 2nd thoracic segment (T2). Despite differences in their shape, size, and function, these tergal plates all appear to be exites of the proximal-most leg segment that was incorporated into the body wall to varying degrees.
An iconic feature of insects is the apparent lack of legs on the abdomen, which is believed to be due to the repression of the leg-patterning gene Distalless (Dll) by abdominal Hox genes. However, in contrast to these molecular observations, it is not widely appreciated that the embryos of most insect groups do in fact form paired protrusions on most abdominal segments that appear to be homologous to the thoracic legs. However, these degenerate before hatching to form the abdominal body wall. To resolve this discordance between molecular and morphological observations, I examined the expression patterns of pannier and araucan, genes known to distinguish proximal leg segments in all arthropods, in embryos of the flour beetle Tribolium castaneum. In Tribolium embryos, all pregenital abdominal segments develop leg-like paired protrusions, and the stripes of pannier and araucan expression that delineate the proximal leg segments of the thorax are also expressed in the same configuration around these abdominal protrusions. This suggests that insect abdominal legs are homologous to only the proximal portion of the thoracic legs, which in insect adults forms the body wall (lateral tergum and pleura). Importantly, these truncated abdominal legs appear to retain the ancestral ability to form multi-functional exites (gills, plates, etc). Thus, just as insect wings on the thorax evolved from an ancestral exite on a proximal leg segment that had been incorporated into the body wall (Bruce and Patel, 2020), so too did the various insect outgrowths on the abdomen. These cryptic, truncated abdominal legs – likely inherited from their crustacean ancestors – appear to be an important wellspring for new functions in insects, such as caterpillar prolegs, gills, leaf-like structures for camouflage and colorful spines for aposematic warning (Bruce and Patel, 2023).
Fig. 3. Abdominal leg nubs in Tribolium embryos. Top: earlier embryo. Bottom: later embryo. Arrow points to 4th abdominal leg nub that later degenerates into the body wall. araucan (ara, green) is expressed in two stripes down the length of the embryo, one dorsal stripe and one lateral stripe, as well as a circular patch on leg segment 6 (coxa) of each thoracic leg. The two stripes bracket the proximal-most 8th leg segment that carries both the wing and the spiracle. vestigial (vg, pink) marks the future wing serial homologs: the wing, elytra, and tergal plates, as well as certain cells in the ventral nerve cord. Gray, DAPI, marks all cell nuclei.
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