The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of beta-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

Winged insects underwent an unparalleled evolutionary radiation, but mechanisms underlying the origin and diversification of wings in basal insects are sparsely known compared with more derived holometabolous insects. In the neopteran species Oncopeltus fasciatus, we manipulated wing specification genes and used RNA-seq to obtain both functional and genomic perspectives. Combined with previous studies, our results suggest the following key steps in wing origin and diversification. First, a set of dorsally derived outgrowths evolved along a number of body segments including the first thoracic segment (T1). Homeotic genes were subsequently co-opted to suppress growth of some dorsal flaps in the thorax and abdomen. In T1 this suppression was accomplished by Sex combs reduced, that when experimentally removed, results in an ectopic T1 flap similar to prothoracic winglets present in fossil hemipteroids and other early insects. Global geneexpression differences in ectopic T1 vs. T2/T3 wings suggest that the transition from flaps to wings required ventrally originating cells, homologous with those in ancestral arthropod gill flaps/epipods, to migrate dorsally and fuse with the dorsal flap tissue thereby bringing new functional gene networks; these presumably enabled the T2/T3 wing's increased size and functionality. Third, "fused" wings became both the wing blade and surrounding regions of the dorsal thorax cuticle, providing tissue for subsequent modifications including wing folding and the fit of folded wings. Finally, Ultrabithorax was co-opted to uncouple the morphology of T2 and T3 wings and to act as a general modifier of hindwings, which in turn governed the subsequent diversification of lineage-specific wing forms.wing origins | Sex combs reduced | Ultrabithorax | RNA-seq | vestigial S ome 350 million years ago, the development of insect wings was a seminal event in the evolution of insect body design (1, 2). The ability to fly was critical to insects becoming the most diverse and abundant animal group, and the origin of such novelty has been a focus of intense scientific inquiry for more than a century (3, 4). More recently, through studies of genetic model systems such as Drosophila, the mechanisms of wing morphogenesis have been elucidated (5-12). Still lacking however is a comprehensive understanding of transitional steps connecting the morphology of structures observed in the fossil record with that of the modern-day insects, including wing origins and subsequent diversification.The initial stages of insect wing evolution are missing from the fossil record and it is therefore necessary to use indirect evidence from fossils that postdate the origin and initial radiation of pterygotes (2). Larvae of many of those taxa featured dorsally positioned outgrowths on each of the thoracic and abdominal segments (2, 13), apparently serial homologs (i.e., similar structures likely arising from a common set of developmental mechanisms). Diverse lineages independently lost those dorsal appendages on the abdomen while un...

Differential enlargement of hind (T3) legs represents one of the hallmarks of insect evolution. However, the actual mechanism(s) responsible are yet to be determined. To address this issue, we have now studied the molecular basis of T3 leg enlargement in Oncopeltus fasciatus (milkweed bug) and Acheta domesticus (house cricket). In Oncopeltus, the T3 tibia displays a moderate increase in size, whereas in Acheta, the T3 femur, tibia, and tarsus are all greatly enlarged. Here, we show that the hox gene Ultrabithorax (Ubx) is expressed in the enlarged segments of hind legs. Furthermore, we demonstrate that depletion of Ubx during embryogenesis has a primary effect in T3 legs and causes shortening of leg segments that are enlarged in a wild type. This result shows that Ubx is regulating the differential growth and enlargement of T3 legs in both Oncopeltus and Acheta. The emerging view suggests that Ubx was co-opted for a novel role in regulating leg growth and that the transcriptional modification of its expression may be a universal mechanism for the evolutionary diversification of insect hind legs.

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development.

Diversification of leg appendages is one of the hallmarks of morphological evolution in insects. In particular, insect hind (T3) legs exhibit a whole spectrum of morphological diversification, ranging from uniform to extremely modified. To elucidate the developmental basis of T3 leg evolution, we have examined the expression patterns of two homeotic genes, Ultrabithorax and abdominal-A (collectively referred to as UbdA), in a broad range of species. First, our results show that UbdA expression in hemimetabolous insects is localized only in specific T3 leg segments undergoing differential growth (compared to their foreleg counterparts). In contrast, in basal hexapod and insect lineages, the absence of the UbdA signal coincides with uniform leg morphology. The same situation exists in first instar larvae of holometabolous insects, in which absence of UbdA expression in the embryonic T3 legs is associated with the lack of larval T3 leg diversification. Second, there is a clear difference in the timing of expression between species with greatly enlarged T3 leg, such as crickets and grasshoppers, and species that exhibit more moderate enlargement of hind legs, such as mantids and cockroaches. In the former, the UbdA expression starts much earlier, coinciding with the elongation of T3 limb buds. In the latter, however, the UbdA expression starts at much later stages of development, coinciding with the establishment of distinct leg segments. These results suggest that diversification of insect hind legs was influenced by changes in both the spatial and temporal regulation of the UbdA expression.