A Screen for Modifiers of Hedgehog Signaling inDrosophila melanogasterIdentifiesswmandmts

Abstract

Signaling by Hedgehog (Hh) proteins shapes most tissues and organs in both vertebrates and invertebrates, and its misregulation has been implicated in many human diseases. Although components of the signaling pathway have been identified, key aspects of the signaling mechanism and downstream targets remain to be elucidated. We performed an enhancer/suppressor screen in Drosophila to identify novel components of the pathway and identified 26 autosomal regions that modify a phenotypic readout of Hh signaling. Three of the regions include genes that contribute constituents to the pathway-patched, engrailed, and hh. One of the other regions includes the gene microtubule star (mts) that encodes a subunit of protein phosphatase 2A. We show that mts is necessary for full activation of Hh signaling. A second region includes the gene second mitotic wave missing (swm). swm is recessive lethal and is predicted to encode an evolutionarily conserved protein with RNA binding and Zn 1 finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity.

A Novel Interaction Between hedgehog and Notch Promotes Proliferation at the Anterior–Posterior Organizer of the Drosophila Wing

“…Note that smo RNAi not only negatively regulates Hh signaling, but also reduces output from the Hhdependent ptcGAL4 driver that drives expression of smo RNAi (Casso et al 2008). Although the consequent negative feedback may have dampened the phenotypic responses that we obtained, this effect may have been advantageous for the screen, both by masking the effects of weak modifiers and by decreasing phenotypic variability.…”

“…E(spl) codes for 13 genes, including seven basic helix-loop-helix transcriptional repressors; its genes are clustered in close proximity to one another in region 96F9-10. Df(3R)Espl3 was identified in our original screen as an enhancer of the smo RNAi wing phenotype (Casso et al 2008). While we were not able to identify a single gene in this region that enhanced smo RNAi [RNAi directed against the E(spl) complex genes HLH-mb, HLH-mD, or HLHm8 did not modify the phenotype (data not shown)], we did identify four smaller chromosomal deletions in this region that did [Df(3R)Exel6204, Df(3R)BSC751, Df(3R)BSC495, Df(3R)Espl1 ( Figure 1M and data not shown)].…”

“…Drosophila lines: smo RNAi is described in Casso et al (2008). Su(H) del47 [a 1881-base deletion that removes portions of Su(H) and l(2)35Bg] is a null allele.…”

“…Flies with smo function reduced in this way have reproducible abnormalities in both the state and the size of the 3-4 intervein regions. By making portions of the genome hemizygous in this sensitized background, we previously identified 26 autosomal segments that show haplo-insufficiency, and we characterized the roles of two genes that these deficiencies uncovered (Casso et al 2008). Here, we extend this screen to the X chromosome and describe two haploinsufficient segments on the X, as well as a new role for N signaling in the AP organizer region of the wing disc.…”

PP4 and PP2A regulate Hedgehog signaling by controlling Smo and Ci phosphorylation

“…In the Hh signaling cascade, multiple Ser/Thr kinases are involved, including PKA, GSK3 and CK1 family members. Even though PP2A has been implicated as a positive regulator in Hh signaling (Casso et al, 2008;Nybakken et al, 2005), its relevant substrates remained undetermined. Thus, it is not clear whether phosphatases are involved in regulating Smo and Ci phosphorylation, and if so which phosphatases are responsible.…”