New Negative Feedback Regulators of Egfr Signaling inDrosophila

Abstract

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 Egfrresponsive 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 Sulf1 is required for the termination of intestinal stem cell division during regeneration

“…Loss of Sulf1 also led to elevated activities of JAK-STAT, EGFR and Hh signaling. These results are consistent with previously established roles of Sulf1 in regulating EGFR and Hh signaling in the developing wing (Butchar et al, 2012;Wojcinski et al, 2011). In addition, it has been shown that JAK-STAT signaling is regulated by HSPGs in the ovary (Hayashi et al, 2012).…”

Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration

“…Given that the Sulf1{KO; mCherry} allele retains Sulf1 endogenous regulatory sequences, it can be used as not only a molecular null mutant but also as a highly reliable Sulf1 transcriptional reporter. We examined mCherry expression in the wing imaginal disc where Sulf1 is expressed at high levels along the anteroposterior and dorsoventral boundaries (Butchar et al, 2012;Kleinschmit et al, 2010;Wojcinski et al, 2011;You et al, 2011). The mCherry signal was detected in the previously reported pattern (Fig.…”

“…Thus, it is important to understand how expression of mammalian and Drosophila Sulfs is regulated. In the Drosophila wing disc, EGFR and Wg pathways induce expression of Sulf1, which in turn negatively regulates these pathways, indicating that Sulf1 forms negative-feedback loops for EGFR and Wg signaling (Butchar et al, 2012;Kleinschmit et al, 2010). Further study is required to reveal how Sulf1 transcription is precisely regulated during midgut homeostasis and regeneration, and how Sulf1-mediated feedback systems contribute to stabilizing stem cell systems and/or preventing cancer formation.…”

“…This can positively or negatively regulate signal transduction in a context-dependent manner (Ai et al, 2003;Kleinschmit et al, 2013;Wojcinski et al, 2011). In the Drosophila wing disc, Sulf1 negatively regulates the FGF, Wg, EGFR and Hh pathways (Butchar et al, 2012;Kamimura et al, 2006;Kleinschmit et al, 2013Kleinschmit et al, , 2010Wojcinski et al, 2011;You et al, 2011). Given that some of the signaling ligands known to regulate ISC division in the midgut are heparan-sulfate-dependent factors, including Vein (Vn; an EGFR ligand), Hh, and Wg, we hypothesized that dynamic regulation of 6-O sulfation might be important for controlling ISC mitotic activity during regeneration.…”

“…Sulfs specifically remove 6-O sulfate groups from highly sulfated regions of heparan sulfate. In Drosophila, the single Sulf gene, Sulf1, modulates FGF, Wg, Hh and EGFR signaling during development (Butchar et al, 2012;Dani et al, 2012;Kamimura et al, 2006;Kleinschmit et al, 2013Kleinschmit et al, , 2010Wojcinski et al, 2011;You et al, 2011).…”