A screen for modifiers of hedgehog signaling in Drosophila melanogaster identifies swm and mts

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(+) finger domains. Characterization of newly isolated alleles indicates that swm is a negative regulator of Hh signaling and is essential for cell polarity.

Figure 1.—

smo RNAi in the adult wing. (A) wild type, (B) ptcGAL4 WIZ-smo/+, (C) ptcGAL4 WIZ-smo/UAS-smo, (D) ptcGAL4 WIZ-smo/smo³, (E) ptcGAL4 WIZ-smo/+, hh¹⁰/+, (F) ptcGAL4 WIZ-smo/en^E, (G) ptcGAL4 WIZ-smo/ptc¹⁰, (H) ptcGAL4 WIZ-smo/Df(2L)BSC41, (I) ptcGAL4 WIZ-smo/mts^XE-2258, (J) ptcGAL4 WIZ-smo/Df(2L)Exel8041, (K) ptcGAL4 WIZ-smo/swm^Dh-1, (L) ptcGAL4 WIZ-smo/swm^F11, (M) ptcGAL4/10084R1 (swm RNAi), and (N) ptcGAL4 WIZ-smo/10084R1. Enhancement and suppression of the smo RNAi phenotype was scored as the distance between veins 3 and 4 in the proximal part of the wing near the anterior crossvein (see arrow in B). Ectopic vein tissue between veins 3 and 4 was seen in many genetic interactions with smo RNAi (e.g., D, G, and J), but it did not correlate with enhancement or suppression.

Figure 2.—

Results of the smo RNAi enhancer-suppressor screen. Chromosomes 2 and 3 are depicted graphically as black lines. Below, deletions and mutant chromosomes that enhance (red) or suppress (blue) this phenotype are indicated with bars. Cytological locations are marked, and candidate enhancer and suppressor genes are indicated.

Figure 3.—

The swm locus and sequence. (A) The swm locus has six exons in its swm-RA transcript (thick bars) and five introns (thin lines). The swm-RB transcript is predicted (FlyBase) to have only five exons and an alternative transcriptional start site within intron one (not shown). Both transcripts have the same predicted protein coding region (1062 amino acids, green). Noncoding regions of exons one, two, and six are in black. Two putative functional domains are shown: a CCCH-type Zn⁺ finger (yellow) and an RNA recognition motif (blue). Mutations in five swm alleles are indicated. (B) Swm protein aligned with human RBM-26 (GenBank, EAW80593) and RBM-27 (GenBank, Q9P2N5). Identity (red), strong similarity (orange), weak similarity (green), no similarity (black), Zn⁺ finger domain (yellow box), RRM (blue box). Mutations in swm alleles, in boldface type: swm^F4 (after D175 GT to AT, the last residues before the splice site); swm^Dh-1, 157-bp deletion at S249; swm^F14, Q314 to stop (CAA to TAA); swm^F11, W418 to stop (TAG to TGG); swm^F15, D923N.

Figure 4.—

swm expression in embryos and larva. In situ hybridization reveals swm expression in (A) precellular embryo, (B) germ-band extension embryo, (C) germ-band retraction embryo, (D) third instar wing disc (posterior is on the right), (E) eye/antennal disc (mf, morphogenetic furrow; st, stalk), (F) salivary gland (s, secretory cells; d, duct cells), (G) gut (gc, gastric caecae; wc, wreath cells), (H) larval brain (o, optic lobe), and (I) larval fatbody.

Figure 5.—

hh^Mrt is enhanced by swm. Wings: (A) wild type, (B) hh^Mrt/+, (C) and (D) swm^Dh-1/+; hh^Mrt/+. Third instar wing imaginal discs were stained for Ptc (E, F, and G) or Ci (H, I, and J) protein. Genotypes are as follows: (E and H) wild type; (F and I) hh^Mrt/+; and swm^Dh-1/+; (G and J) hh^Mrt/+. The arrows indicate ectopic expression of Ptc and Ci in the anterior compartment along the dorsal ventral compartment boundary.

Figure 6.—

Swm localization and function. (A–C) S2 cells expressing GFP–Swm localize GFP fluorescence to nuclei (A, phase contrast; B, fluorescence; C, merge). (D) GFP–Swm localized to nuclei in third instar salivary glands. (E and F) Wing from a swm^F15/Df(2L)Exel8041 escaper (F) has intervein regions 3–4 expanded and misoriented hairs (H), in contrast to wild-type control (E and G).