EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph

Abstract

Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation mode.

Regulation of leg size and shape: Involvement of the Dachsous‐fat signaling pathway

“…1B; see Fig. 7) (Mito et al, 2002;Nakamura et al, 2008b). In the fifth instar, the tibiae, tibial spurs, tarsi, and tarsal claws are restored in a miniature form (Fig.…”

“…In Drosophila, the Egfr ligands are known to be Vein (Vn), Spitz, and Gurken, among which Vn acts as a morphogenetic signal to establish new distal fates at the tip of the developing leg that is located at the center of the leg imaginal disc (Campbell, 2002;Galindo et al, 2002Galindo et al, , 2005. In Gryllus limb bud, Egfr is also likely to be involved in establishment of the PD axis (Nakamura et al, 2008b). However, since no homolog of vn has been identified in other insect orders, an Egfr ligand might be a Tgfa-like protein (Lynch et al, 2010).…”

Lowfat, a mammalian Lix1 homologue, regulates leg size and growth under the Dachsous/Fat signaling pathway during tissue regeneration†

“…Whole-mount in situ hybridization of embryos and regenerating legs were carried out using a semiautomatic hybridization instrument (HYBRI-MASTER HS5100, ALOKA, Tokyo) and with InSitu Chip (InSitu Chip [L], ALOKA, Tokyo; Ogasawara et al, 2006) as previously described (Mito et al, 2002;Nakamura et al, 2007Nakamura et al, , 2008bBando et al, 2009). …”

“…In the fifth instar, miniaturized versions of the tibiae, tibial spurs, tarsi, and tarsal claws are restored. In the sixth instar, the missing portions of the amputated legs are nearly fully restored and exhibit a nearly normal appearance (Mito et al, 2002;Nakamura et al, 2007Nakamura et al, , 2008b. When an amputated tibia is transplanted with different level along the tibia, intercalary growth occurs between host stump and graft piece to regenerate the missing part (Nakamura et al, 2007).…”

Triggering the regeneration and tissue repair programs

“…The epithelial growth factor (EGF) pathway is influential in patterning the distally regenerated structures of the cricket limbs. Moreover, as observed during development, it appears to act downstream of the canonical Wnt pathway, suggesting that EGF plays a similar function during regeneration and development in insects (Nakamura et al, 2008b). Emili Salo (University of Barcelona, Spain) highlighted the central role of both BMP and Wnt signaling in the morphogenesis of regenerating planaria.…”