In Vivo MMP Functions Figure 7. Phenotype arising upon Dpp knock-down in last instar larvae. (A) Lateral views of wild-type imago (A) and arrested dpp (RNAi) animal at pupal-adult transformation (B) are shown. This phenotype is dominant, however, some animals exhibited a milder phenotype, in which they ecdysed to imagoes but had affected wings (D) and leg deformations (F,G) of mainly the tarsus when compared to wild-type wing (C) and leg (E). Scale bar, A–B, 500 mm; C–G, 250 mm. doi:10.1371/journal.pone.0004751.g007 Dorsal and Dpp are part of two coordinated ancestral self-organized systems in the beetle embryo [32]. In contrast to Drosophila, Tribolium has an ancestral type of development, including a short germ band embryo with gradual segmentation and continuously limbs growth starting from an appendage bud shared with most insects and other arthropods as well as with vertebrates [25]. Hence, findings from Tribolium, when compared to findings from Drosophila and other animal model organisms, provide a deeper and a more comprehen- sive understanding of conserved as well as derived molecular mechanisms. Metamorphosis Here, we show that RNAi-mediated silencing of MMP-1 leads to an arrest in the initial stage of Tribolium pupation resulting in phenotypes with altered development of antennae, compound eyes, wings, legs, and head. As a first step towards addressing molecular mechanisms behind this phenotype, we investigated the potential responsibility of the prominent morphogen Dpp because results from D. melanogaster suggested an possible role of Dpp on observed MMP-1 (RNAi) phenotype. Knock-down of Dpp leads to an arrest of pupal to adult transition during adult morphogenesis giving a phenotype with pupal characteristics and adult-like tanning of the cuticula. Since MMP-1 (RNAi) animals arrest in an earlier stage of metamorphosis, we propose that Dpp has no or only little impact on MMP-1 knock-down phenotype. Figure 8. MMP-2 knock-down in adults result in offspring with gut defects. Tribolium larvae that are exposed to UV-light, resulting in auto-fluorescence of their cuticle and intestine. (A) Auto-fluorescence of the posterior end of a wild-type first instar larva showed an intestine that is normally s-shaped in a typical way in up to three segments (arrow-heads). (B) In contrast, examination of the posterior end of a first instar MMP-2 (RNAi) larva revealed an abnormal intestine with a twisted hind gut and a half truncated ileum loop. Closer examination of dissected hind guts from wild-type (C) and MMP-2 (RNAi) (D) larvae revealed no obvious differences except a kink at the end of the colon of MMP-2 (RNAi) larvae (indicated by an arrow). Gut epithelium seems not to be affected since cell appearance by DAPI-mediated nucleus staining indicated no differences. Scale bars, A–B, 100 mm; C–D, 50 mm; E–F, 5 mm. doi:10.1371/journal.pone.0004751.g008 In Drosophila, MMPs have been co-opted for maggot specific metamorphosis processes including head evasion and notum connation [38]. Both processes have recently been demonstrated to depend on imaginal disc eversions which require basement membrane remodeling by particularly MMPs [39]. Since Tribolium has no imaginal discs and instead limbs grow continuously starting from an appendage bud, we postulate that MMP-1 (RNAi) PLoS ONE | www.plosone.org 8 March 2009 | Volume 4 | Issue 3 | e4751