Final Report Summary - TISSUE PATTERNING (The role of mitotic tissues in controlling the developmental clock)
How is the growth of different body parts coordinated and scaled with the overall body size to give rise to adults of correct proportions? It is well established that different organs follow autonomous growth programs and therefore grow at different speeds and during distinct stages of development1. It is therefore likely that mechanisms operate to ensure that each organ has reached an appropriate size before proceeding through developmental transitions. If not, organs would be forced to terminate growth and differentiate prematurely, giving rise to disproportionate adults. How organ growth is monitored at the organismal level and how it is coupled with developmental transitions is not well understood. In flies, the imaginal tissues (also called discs) of the larva are the equivalent of vertebrate limb buds and are the precursors of most of the visible organs in the adult fly. Perturbation of disc growth during early larval development delays larva-to-pupa transition2,3. This allows perturbed tissues to complete their growth programs and synchronise with other larval tissues before the steroid-induced transition to the pupal stage. Interestingly, larvae with little or no disc structures pupariate with normal timing4. This suggests a model whereby discs that have not completed a certain amount of growth release an inhibitory signal that delays pupariation. The molecular identification of this signal has been of longstanding interest to developmental biologists.
The main objective of this project was to screen for signals that couple patterning of mitotic tissue with the developmental clock using the power of Drosophila genetics. For this purpose, we first generated two different conditions in which manipulation of disc growth leads to a delay in larva to pupa transition. In one condition, imaginal discs undergo unlimited growth, leading to the accumulation of neoplastic tissue and the prevention of metamorphosis. In the other condition, imaginal disc growth is slowed down, leading to a systemic growth inhibition and an extension of the larval growth period. These conditions were used sequentially to screen 10.100 transgenic lines allowing Gal4-dependent targeted expression of specific RNAis (the KK collection from the Vienna Drosophila Research Center, VDRC) for candidate RNAis that could rescue the developmental delay. We identified one candidate that strongly rescued both conditions. This RNAi line targets a previously uncharacterized gene, CG14059, which we called Drosophila insulin-like peptide 8 (DILP8). DILP8 encodes a small peptide of about 150 amino acids presenting similarities with the family of insulin-related peptides. The DILP8 gene is highly induced in conditions where growth impairment produces a developmental delay. Dilp8 expression and secretion by imaginal tissues is sufficient to delay pupariation without affecting tissue integrity. In insects, the timing of the developmental transitions is triggered by a sharp increase in the production of the Prothoracicotropic hormone (PTTH), a peptidic hormone expressed in a subset of brain neurons that induces the production of the molting hormone ecdysone in the ring gland. To test if these tissues could be direct targets of DILP8, we co-cultured wt brain and ring gland complexes with discs expressing DILP8 or a non-secreted form of DILP8 (DILP8). These experiments showed that disc-secreted DILP8 acts directly on the brain complex to suppress ecdysone production. Although direct sequence comparisons have not identified an obvious ortholog for DILP8 in vertebrate genomes, in-depth analysis reveals that DILP8 shares some features with a distant insulin-like peptide family member 5, raising the possibility that peptides with similar roles may exist in vertebrates. This work was recently published (Colombani J1, Andersen DS1,2, and Leopold P2. Science 2012, 1= equal contribution, 2= corresponding authors) 6.
The condition used for the genome-wide screen described above (rn-avl RNAi) corresponded to the knockdown of avalanche (avl), a syntaxin that function in the early step of endocytosis, specifically in the discs7. The rn-avl RNAi condition results in neoplastic overgrowth of discs7 and a delay in larva-to-pupa transition of about 2 days. We reasoned that RNAi lines able to suppress the neoplastic growth in this condition would also rescue the delay in pupariation. Hence we expected to identify molecules that are crucial for driving neoplastic growth. Indeed this screen identified 8 RNAi lines that completely suppressed the neoplastic growth of the rn-avl RNAi condition as well as the associated delay. Of these, 5 candidate lines target the expression of core components of the JNK pathway including, the E2 ubiquitin-conjugating enzyme Bendless (Ubc13 in humans), the adaptor protein dTAB2, the JNKK kinase dTAK1, the JNK kinase Hemipterous, and the c-jun kinase Basket. One of the remaining lines targets the expression of a previously uncharacterized gene that we named grindelwald (grnd). Grnd encodes a transmembrane protein with some predicted structural resemblance to members of the TNFR superfamily. We have furthermore been able to show that Grnd co-localises with the apico-basal cell polarity determinant Crumbs (Crb) at the sub-apical membrane domain and is required for coupling Crb-induced loss of polarity with JNK activation and neoplastic growth. In addition, Grnd is required downstream of Eiger (Egr), the only Drosophila TNF homolog8-9, to promote MMP1 expression and invasive behaviour of RasV12/scrib-/- tumors. Finally, Grnd is also required for the physiological pro-apoptotic functions of Egr, and over-expression of Grnd lacking the extracellular domain (Grnd-intra) is sufficient to induce JNK signalling and apoptosis in flies. We propose that Grnd is a TNFR-like protein that integrates signals from Egr and apical polarity determinants to induce JNK-dependent cell death or proliferation (Andersen DS, Colombani J, and Leopold P, manuscript in preparation).
1. Cole TJ. Adv Exp Med Biol 2009; 639:1-13.
2. Halme A, et al. Curr Biol 2010; 20:458-63.
3. Stieper BC, et al. Dev Biol 2008; 321:18-26.
4. Simpson P, et al. J Embryol Exp Morphol 1980; 57:155-65.
5. Garelli A, et al., Science 2012; 336:579-82
6. Colombani J, et al. Science 2012; 336:582-5.
7. Lu and Bilder, Nat Cell Biol; 2005: 7:1232-9.
8. Moreno, E, et al. Curr Biol 2002; 12:1263-1268.
9. Kauppila, S. et al., Oncogene 2003; 22:4860-4867.