TGF-α ligands can substitute for the neuregulin Vein in Drosophila development.

ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-α-type and a single neuregulin-like called vein (vn). Here, we used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, we found that the essential role of vn in development could be largely replaced by expression of any TGF-α ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. Our results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity.

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila.

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.

New negative feedback regulators of Egfr signaling in Drosophila.

The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. Here we used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We suggest Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.

Drosophila embryonic 'fibroblasts': extending mutant analysis in vitro.

The in vivo analysis of Drosophila using genetics, with almost a hundred year history, has produced an immense body of knowledge about biology. In vitro analysis, while arguably the poor cousin to its in vivo relative, has a utility--in biochemical analyses and in cell-based screening, for example, with RNAi. A major block to the development of in vitro analysis has been the lack of an efficient genetic method to derive cell lines from mutant Drosophila strains. We recently discovered that expression of activated Ras (Ras(V12)) provides cells in vitro with both a survival and a proliferative advantage and hence promotes the generation of cell lines. In this addendum, we provide new data describing the genesis of seven cell lines corresponding to a rumi mutant, which demonstrate that the method can be used to derive lines and study genetic mutants in vitro.