Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2404–2409, March 1997 Developmental Biology Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-specific DNA binding protein TONIA VON OHLEN†,DEREK LESSING‡,ROEL NUSSE‡, AND JOAN E. HOOPER†§ †Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, CO 80262; and ‡Developmental Biology, Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, CA 94305 Communicated by William Wood, University of Colorado, Boulder, CO, December 31, 1996 (received for review September 19, 1996) ABSTRACT Hedgehog (Hh) is a member of a family of organizing the highly ordered structure of the compound eye secreted proteins that direct patterning at multiple stages in (reviewed in refs. 5 and 15–18). both Drosophila and vertebrate development. During Drosoph- Several components of the Hh signaling pathway have been ila embryogenesis, Hh protein is secreted by the cells of the identified in Drosophila. ptc is a negative regulator of Hh posterior compartment of each segment. hh activates tran- signaling (19). Genetic analysis suggests that Hh signaling scription of wingless (wg), gooseberry (gsb), and patched (ptc)in either inactivates Ptc or overrides the ability of Ptc to repress the cells immediately adjacent to Hh-secreting cells. Hh transcription of Hh-responsive target genes. Because Ptc is a signaling is thought to involve the segment polarity gene multiple membrane spanning cell surface protein (20, 21), it cubitus interruptus (ci). ci encodes a zinc finger protein of the could be a Hh receptor (19). However, it cannot be the only Hh Gli family of sequence-specific DNA binding proteins. ci receptor, since Hh has effects in ptc null embryos (22). Two mRNA is expressed in all non-Hh expressing cells. Here we protein kinases are implicated in Hh signal transduction, fused demonstrate ci activity is both necessary and sufficient to as a positive regulator, and cAMP-dependent protein kinase A drive expression of Hh-responsive genes in the Drosophila as a negative regulator (reviewed in ref. 23). How either of embryos. We show that Ci is a sequence-specific DNA binding these fit into a signal transduction pathway remains to be protein that drives transcription from the wg promoter in determined. transiently transfected cells. We demonstrate that Ci binding In cubitus interruptus (ci) mutant embryos, transcription of sites in the wg promoter are necessary for this transcriptional Hh-responsive genes is lost (24, 25). Ci overexpression or activation. These data taken together provide strong evidence misexpression drives transcription of Hh responsive target that Ci is a transcriptional effector of Hh signaling. genes (26, 27). Ci is a member of the Gli family of sequence- specific DNA binding proteins (28, 29), a family characterized In Drosophila embryogenesis Hedgehog (Hh) plays an early by five tandem zinc fingers with highly similar sequence. Based role in segmentation and a later role in generating cell type on the presence of these zinc fingers, Orenic et al. (28) diversity within each segment (reviewed in ref. 1). The Dro- proposed that Ci acts as a transcription factor. Alexandre et al. sophila body is divided into parasegments, which are the basic (27) showed that Ci can activate transcription in yeast, through units for patterning in the developing organism. As the embryo a putative Ci binding site. These data suggest that Ci is a cellularizes, the parasegment borders form. A parasegment transcriptional effector of Hh signaling. However, direct Ci– border is delineated by expression of two secreted proteins DNA interaction has not been shown. Here we present genetic (reviewed in ref. 1). Wingless (Wg), a Wnt family member, is evidence confirming that Hh acts through Ci to activate wg and expressed in cells anterior to the parasegment border (2), and gsb transcription. We demonstrate that Ci has sequence- Hh is expressed by cells posterior to the parasegment border specific DNA binding activity and that direct interaction (3–5). During the first2haftergastrulation, secreted Hh between Ci and sequences in wg promoter is required for protein activates a signaling pathway that results in transcrip- transcriptional activation from this promoter. We conclude tion of wingless (wg), gooseberry (gsb), and patched (ptc) in cells that Ci functions as a transcriptional activator in the Hh signal that are competent to respond (6). Between 3 and6hafter transduction pathway. gastrulation, Hh takes on a new role. It participates in auto- regulatory engrailed (en) expression (7) and it organizes the MATERIALS AND METHODS pattern of dorsal cuticle hairs (8). In its latter role, Hh has been Drosophila Stocks, Crosses, and Analysis. Df(4)M62f flies proposed to act as a morphogen (8), though this remains to be were obtained from Robert Holmgren (Northwestern Univer- demonstrated (9). sity), yw; Hs-hh M3yTM3 y1 Ser from Philip Ingham (Imperial hh Dro- also specifies critical aspects of adult morphology in Cancer Research Fund), and hhGS1,rueyTM3 from Jym sophila. The cuticular structures of adult appendages derive from Mohler (Barnard College). internal epithelial sacs within the larvae, the imaginal discs. The Hs-hh M3y1; Df(4)M62fyDf(4)M62f embryos were gener- posterior compartment of each disc expresses Hh, and immedi- ated by mating Hs-hh M3y1; Df(4)M62fy1 males to ately adjacent anterior compartment cells respond to this Hh Df(4)M62fyeyD females. Embryos from this cross were col- signal by induction of the secondary signals Wg or the transform- lected and heat-shock treated as in ref. 30. ing growth factor b family member, Decapentaplegic (Dpp). hhGS1yhhGS1;ciDyciD embryos were obtained from heterozy- These signals then organize growth and patterning of both gous hhGS1yTM3;ciDy1 parents. The stage 10yearly stage 11 compartments (10–14). While the developing eye lacks distinct embryos were fixed, processed by in situ hybridization, and anterior and posterior compartments, Hh signaling is critical for scored for wild type; ciD, hh,orhh; ciD patterns; and un- scorable. Using the x2 test, the observed distribution of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: GST-Ci, glutathione-S-transferaseyCi fusion protein; b-gal, b-galactosidase; EMSA, electrophoretic mobility-shift assay. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Data deposition: The sequence reported in this paper has been 0027-8424y97y942404-6$2.00y0 deposited in the GenBank database (accession no. U84292). PNAS is available online at http:yywww.pnas.org. §To whom reprint requests should be addressed. 2404 Downloaded by guest on September 27, 2021 Developmental Biology: Von Ohlen et al. Proc. Natl. Acad. Sci. USA 94 (1997) 2405 phenotypes fit the expected 9:3:3:1 ratio with P 5 0.8 for ptc, Fragments containing mutant and nonmutant wg promoter P 5 0.44 for wg, and P . 0.9 for gsb. sequences (fragments 1 and 2) were isolated by PCR. PCR The smo ciD phenotype was determined by collecting em- fragments were end labeled with [g-32P]ATP. Quantitation bryos from smo1 cn bw spyCyo; ciDyM63a parents overnight at used a scanning densitomiter, Alpha Innotech model IS1000 188C. The embryos were fixed and assayed for wg, gsb, and ptc digital imaging system (Alpha Innotech, San Leandro, CA). RNA patterns. The stage 10yearly stage 11 embryos were Site-Directed Mutagenesis. Mutagenesis of putative Ci scored for wild type; ciD, smo,orsmo; ciD patterns; and binding sites in wg promoteryenhancer DNA used the altered unscorable. Using the x2 test, the observed distribution of sites in vitro mutagenesis kit according to manufacturers phenotypes fit the expected 9:3:3:1 ratio for wg, gsb, and ptc. instructions (Promega). A 2-kb BamHI fragment of wg pro- The fused (fu); ciD phenotype was determined by collecting moter was inserted into pAlter-1 phagemid vector. Single- embryos from fu513yfu513; ciDyM63a females crossed to fu513; stranded DNA was prepared using helper phage M13K07 or ciDyM63a males. Stage 10 and early stage 11 embryos were R408. Mutagenic primers were synthesized by Life Technol- scored for fu or fu; ciD phenotypes. Approximately 25% of the ogies. Primers used were Alt-1, TCGATGGCTACTCG- populations had a ciD like expression pattern for wg, gsb, and TCGG; Alt-2, CGCCAGCGTAGACGATGAT; Alt-3, AA- ptc. AGTGCGTAAACCATGTC; and Alt-4, AAAATGGACTT- In situ hybridizations utilized antisense-strand riboprobes, TCCCAGCGT. Each mutates one of four putative Ci binding according to standard procedures (31, 32). sites in the distal 1 kb of wg promoter. Successful oligonucle- Recombinant DNAs. To build wg-Luc, a 5-kb EcoRV fragment otide mutagenesis was confirmed by sequencing of candidate of wg promoter ligated into the SmaI site of pXP2Luc (33). To clones using Sequenase kit (United States Biochemical). construct Dwg-Luc and Dwg*-Luc, a 1-kb XhoI fragment from pAlter (Promega) containing a 2-kb BamHI fragment of wg RESULTS promoter was blunt-ended with Klenow and ligated into tk-Luc hh Acts Through ci. In the Drosophila embryo, for2hafter (33) at the SmaI site. wg-LucDS was constructed by deletion of a gastrulation, Hh signaling maintains the expression of wg, gsb, and 1-kb SalI fragment from the full-length wg-Luc construct. wg- ptc (6, 42, 43). To demonstrate ci is required for transduction of LucDB was constructed by removal of a 2-kb BamHI fragment the Hh signal, we examined expression of wg in ci null mutant from wg-Luc. mt-Ci was constructed by ligating a BglIIyNotI embryos when Hh is ubiquitously expressed under control of a fragment containing full-length Ci cDNA from NB40 (34) into heat-shock promoter (Hs-hh). In Hs-hh embryos, wg is expressed pRmHa-1 (35) at the BamHI and HincII sites.