Control of Antagonistic Components of the Hedgehog ... · CNRS UMR 6543, Centre de Biochimie, Parc...

Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.083733

Control of Antagonistic Components of the Hedgehog Signaling Pathway bymicroRNAs in Drosophila

Florence Friggi-Grelin, Laurence Lavenant-Staccini and Pascal Therond1

Institut de Recherches Signalisation, Biologie du Developpement et Cancer, Universite de Nice-Sophia Antipolis,CNRS UMR 6543, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 2, France

Manuscript received October 24, 2007Accepted for publication March 10, 2008

ABSTRACT

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diversecancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to genetranscription output. We previously showed that stability of antagonistic regulatory proteins, including thecoreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected byHh signaling activation. Here, we show that the level of these three proteins is also regulated by amicroRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 39-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway.Further, the loss of the microRNA cluster or of Dicer function modifies the 39-UTR regulation of smo andcos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targetsof microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates anhh-like phenotype, possibly due to dose compensation between the different antagonistic targets. Thisstudy reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs.

THE Hedgehog (Hh) pathway controls multiple de-velopmental processes such as pattern formation,

differentiation, and proliferation in diverse animal phylaranging from Drosophila to humans (McMahon et al.2003). Aberrant Hh pathway activation in humans hasalso been implicated in the growth and maintenance ofa variety of cancers (Ruiz i Altaba 1999; Watkins et al.2003; Fan et al. 2004; Kayed et al. 2004). A betterknowledge of Hh signaling would thus provide a greaterunderstanding of the genesis of these cancers.

Hh signaling controls the activity of the Gli/Ci tran-scription factors. In Drosophila, the activity of Ci, orig-inally called Cubitus Interruptus, is controlled by a largeintracellular multiprotein complex that includes thekinesin-like protein Costal-2 (Cos2), the serine-threonineprotein kinase Fused (Fu), and the transmembraneprotein Smoothened (Smo). Nuclear translocation ofCi is also controled by the Su(fu) protein, another cyto-plasmic anchor. In the absence of Hh, Ci associates withthis complex (Ruel et al. 2003). When secreted Hh bindsto its receptor, the transmembrane protein Patched(Ptc), Ci dissociates from the protein complex, entersthe nucleus, and induces the expression of target genessuch as decapentaplegic (dpp), the homolog of the mito-genic factor TGFb.

To identify novel regulators of the Hh pathway, amisregulation screen was previously carried out in ourlaboratory using the Drosophila wing imaginal disc as amodel (Rorth et al. 1998). In this tissue, Hh is pro-duced and released by posterior cells and then diffusesinto the anterior compartment, where it induces varioustarget genes. The correct expression of these targetgenes is necessary for the wild-type patterning of theadult wing. The study described here of one particularline, named PUAS-29B3, showed that the hh target genemisregulation resulted from the overexpression of asingle cluster of three microRNAs.

MicroRNAs are small, endogenous, noncoding RNAs,typically 21–23 nucleotides in length, that direct nega-tive posttranscriptional regulation of gene expression inplants and animals. MicroRNAs are transcribed as long,primary, mono- or polycistronic precursor transcriptsthat are processed in a two-step process involving thenuclear and cytosolic RNase III-type endonucleasesDrosha and Dicer (Dcr), respectively. MicroRNAs bindto specific complementary sequences in the 39-untrans-lated regions (39-UTRs) of messenger RNAs (mRNAs),inducing either mRNA degradation or the inhibition ofprotein synthesis, depending on the degree of miRNA:mRNA complementarity (Bartel 2004). MicroRNAshave been implicated in a variety of developmental andphysiological processes, including the control of de-velopmental timing, cell proliferation, cell fate specifi-cation, apoptosis, morphogenesis, fat metabolism, andinsulin secretion (reviewed in Di Leva et al. 2006). In

1Corresponding author: Institute of Signaling, Developmental Biology,and Cancer Research, Universite de Nice-Sophia Antipolis, CNRS,UMR 6543, Centre de Biochimie, Parc Valrose, 6108 Nice Cedex 2,France. E-mail: [email protected]

Genetics 179: 429–439 (May 2008)

addition, links between cancer development and micro-RNA expression have been described (reviewed inZhang et al. 2007).

In this study, we provide evidence that the overexpres-sion of a microRNA cluster induces a specific modifica-tion of the Hh pathway in the wing imaginal disc.Indeed two microRNAs, miR-12 and miR-283, are ableto regulate the 39-UTRs of the mRNAs encoding Cos2,Fu, and Smo and decrease the levels of the encodedproteins, resulting in ectopic expression of dpp. Flieslacking the microRNA cluster were produced by ho-mologous recombination. Suprisingly, an absence ofneither the microRNA cluster nor of Dcr induced anymodifications in the Hh signaling pathway. Neverthe-less, analysis of cells mutant for either the microRNAcluster or for Dicer function shows that both the cos2and smo 39-UTR sequences are regulated by microRNAs,strongly suggesting that antagonistic components of theHh signaling pathway are regulated by microRNAs inthe wing disc. One possible role for miR-12 and miR-283might be to dampen down the levels of Hh pathwaycomponents, particularly Cos2 and Smo, to prevent theaccidental activation or downregulation of the pathway.

MATERIALS AND METHODS

Drosophila stocks and genetic experiments: PUAS-29B3was isolated using Gal4-directed overexpression of EPg ele-ments as described (Teleman et al. 2005). Scalloped-GAL4(Sd-GAL4), MS1096-GAL4, engrailed-GAL4 (en-GAL4), andpatched-GAL4 (ptc-GAL4) lines are described in FlyBase (http://flystocks.bio.indiana.edu/). Overexpression of clones in thewing imaginal discs was produced using the ‘‘flip-out’’ technique(Basler and Struhl 1994) on heat-shocked early third instarlarvae hatched from dpp-lacZ; act,CD2,Gal4, UAS-GFPnls malescrossed to yw hs-flp122; PUAS-miR12 or yw hs-flp122, PUAS-miR283or yw hs-flp122; PUAS-miR304 or PUAS-29B3; hs-flpSb/TM6bfemales. To generate D3miR and dcr-1 mutant clones, 24- to 36-hr-old larvae with the following genotype were heat-shocked at37� for 1 hr: [hs-pM] FRT19A/D3miR FRT19A; hs-flpSb/1 andflp122; FRT82B arm-lacZ/FRT82B dcr-1Q1147X (Lee et al. 2004).

Transgenic lines: PUAS-microRNA lines: EcoRI–XhoI PCR frag-ments were obtained from genomic DNA using oligos 59-CGGAATTCTCCGTTGTCGTATGCCCTTGTTCTCTCTand 59-CCGCTCGAGTGCTTTGCTGCTTATTGAGTCCCACCTTAT, 59-CGGAATTCCCAACCACGTGTAGCTCCCCAAAACTGTAT and59-GCTCTAGATGAGGTGAGGTGAGCAGTTAACAGCCAATA,or 59-CGGAATTCACCCCTTACGCTGCCCCACACTTCTATTand 59-GCTCTAGAGGGCATACGACAACGGAAACAGATCAGA,and the fragments were then cloned into the pUAS vector tocreate the PUAS-miR12, PUAS-miR283, and PUAS-miR304 vec-tors, respectively. Two transgenic lines were tested for eachmicroRNA and gave similar results.

Sensor lines: Two copies of the 30 nt containing perfect miR-12, miR-283, or miR-304 target sites were cloned into the 39-UTR of the tub-EGFP vector (Brennecke et al. 2003) to obtainthe miR12-sensor, miR283-sensor, and miR304-sensor lines,respectively. Two to three transgenic lines were tested for eachsensor and gave similar results.

The cos2, smo, and fu 39-UTRs were amplified from genomicDNA with the following primers: 59-tgtgaagcaatagctcagatcctgand 59-tcacacgctgatattgagggaac, 59-CGCCTAGGtagcaagactaaa

taagcaattgatg and 59-CCGCTCGAGcgaggatttaaaatcgtttattagtt,or 59-CGCCTAGGccggcactttcttttattgcgctcag and 59-CCGCTCGAGaggtacccaacattatatcagacgctag, respectively. The 39-UTRswere cloned into the tub-EGFP plasmid (Brennecke et al.2003). Two independent transgenic strains were assayed foreach construct.

Targeted homologous recombination: Ends-out homologousrecombination was performed essentially as described (Gong

and Golic 2003). The left and right arms of the targeting pw25vector were prepared using PCR fragments generated fromgenomic DNA with the following oligos: 59-AGCAGGCGCGCCTCGCACTAACCGCATGACCAACAACcaacg and 59-TACCCGTACGgcggggttttttggggtcggattatttcgg for the downstreamflank, and 59-AGCAGCGGCCGCGCTGGTCAGGTGGCGAGTCTCAAACAATTA and 59-AGCAGCGGCCGCAACAGTTGGTCAGCGAGCACGAGAAGAAGA for the upstream flank of themicroRNA cluster, respectively. Flies carrying the targetingconstruct were crossed to flies with heat-inducible FLP recom-binase and I-SceI endonuclease (70FLP and 70I-SceI, Bloo-mington Stock Center), and the resulting progeny wereheat-shocked for 90 min at 37� on day 3 of development.Mosaic-eyed females were crossed to FM7i males, and targetingevents were mapped to the sex chromosome in the nextgeneration. The absence of the microRNA locus was verifiedby genomic PCR.

In situ hybridization and immunostaining: Embryo im-munostaining and in situ hybridization were performed asdescribed previously (Friggi-Grelin et al. 2006). Disc immu-nolabeling was performed as described previously (Gallet

et al. 2003). Antibodies were used at the following dilutions:rabbit anti-dGMAP (homemade) 1:1000; mouse anti-Smo1:100; purified rabbit anti-Cos2 (homemade) 1:1000; purifiedrabbit anti-Fu (homemade) 1:1000; monoclonal 2A1 rat anti-Ci (gift from R. Holmgren) 1:20; monoclonal 4D9 mouse anti-En (DSHB) 1:1000; monoclonal 5E10 mouse anti-Ptc (giftfrom P. Ingham) 1:400; monoclonal mouse anti-Myc (SantaCruz) 1:200; monoclonal mouse anti-b-Gal (Promega, Madi-son, WI) 1:1000; rabbit anti-b-Gal (Capel) 1:1000. Secondaryantibodies labeled with fluorescent Cy5 or Cy3 ( Jackson, WestGrove, PA) or alexa488 (Molecular Probes, Eugene, OR) wereused at a dilution of 1:200. Images were obtained by confocalmicroscopy (Leica DMR TCS_NT).

RESULTS

Identification of a new locus, overexpression of whichinduces a modification of the Hh pathway: During thecourse of a misregulation screen to search for new reg-ulators of the Hedgehog pathway in Drosophila weidentified a new EP line, named PUAS-29B3, that inducesthe outgrowth of anterior structures in the wing costaregion when induced in the wing imaginal disc (Figure1B). Several lines of evidence indicated that the defectsobserved in this line involved the misregulation of the Hhpathway. First, a similar outgrowth phenotype was ob-tained upon overexpression of Hh (data not shown).Second, the PUAS-29B3 line led to ectopic expression ofthe Hh target gene, dpp (Figure 2, A and B) (Capdevila

and Guerrero 1994; Felsenfeld and Kennison 1995).Finally, the PUAS-29B3-induced outgrowth is sensitiveto the Hh signaling level. Specifically, the outgrowthphenotype was enhanced when PUAS-29B3 was inducedin a background heterozygous for mutations in cos2 and

430 F. Friggi-Grelin, L. Lavenant-Staccini and P. Therond

suppressor of fused ½Su(fu)�, two negative components of theHh pathway (Figure 1, C and D, respectively). Moreover,the outgrowth was absent in animals in which both PUAS-29B3 and Cos2 expressions were induced (data notshown). These data encouraged us to further examinethe relationship between PUAS-29B3 and the Hhpathway.

PUAS-29B3 induces overexpression of a microRNAcluster: To identify the gene responsible for the out-growth phenotype, we first localized the PUAS elementof the 29B3 line. This element was present in an intronof the dGMAP gene (Friggi-Grelin et al. 2006) (Figure1E). When the PUAS-29B3 insertion was expressed inthe engrailed (en) expression domain, dGMAP transcripts

Figure 1.—The PUAS-29B3 linecontrols the expression of a microRNAcluster and induces anterior wing out-growth. (A) Wild-type adult wing and(B) PUAS-29B3/Sd-GAL4 wing ob-tained at 27�. Note that when flies weregrown at 18�, a temperature at whichGAL4 activity is much lower, this pheno-type is not observed (data not shown).(C) PUAS-29B3/Sd-GAL4; cos2w1/1 and(D) PUAS-29B3/Sd-GAL4; Su(Fu)LP/1wings obtained at 18�. Arrows indicateoutgrowth of anterior wing tissue inthe costa region. (E) Localization ofthe PUAS-29B3 element in geneCG33206, which encodes dGMAP. ThemicroRNA cluster, composed of miR-283, miR-304, and miR-12, is presentin the fifth intronic sequence of dGMAP.(F) In situ hybridization for dGMAPmRNA (red), followed by dGMAP im-munostaining (green) in PUAS-29B3;en-GAL4 embryos. No increase in dGMAPprotein was observed in the en domain.Note that endogenous dGMAP mRNAsare present in all cells but levels are lowcompared to ectopic RNA expression(middle). Note also that dGMAP proteinsare present in all ectodermal cells buttheir level is artifactually decreased inthe en cells where the dGMAP in situ de-tection is very high (right). (G–L) GFPexpression in tub-EGFP (G), miR12-sensor(H and J), miR283-sensor (I and K),miR304-sensor (L), wt discs (G–I), or inPUAS-29B3; ptc-GAL4 discs ( J–L). Thepictures shown in G, H, and I were ob-tained with a similar laser setting on theconfocal microscope. Note that the lasersetting is different for H and J (in whichlaser intensity has been increased) andthus sensor level is not comparable forthese two panels. G–I are illustrative ofthree independent lines for each sensor.Dotted lines indicate the A/P border.(G–L) Magnification, 2003.

miRNAs Regulate Hedgehog Pathway 431

were observed in the characteristic en embryonic stripes,but no dGMAP protein was produced likely due to theabsence of the normal initiation codon in the inducedtranscripts (Figure 1F). Further, the overexpression ofthe corresponding cDNA did not affect the Hh pathway(data not shown). Interestingly, a microRNA cluster(Lagos-Quintana et al. 2001; Aravin et al. 2003; Lai

et al. 2003) was identified in the intronic sequence ofthe gene (Figure 1E). The PUAS-29B3 locus contains acluster of three previously uncharacterized microRNAs,miR-12, miR-283, and miR-304 (Lagos-Quintana et al.2001; Aravin et al. 2003; Lai et al. 2003). Because thiscluster is embedded within the dGMAP gene and isoriented in the same direction as the gene, we reasonedthat it would be coexpressed with the dGMAP transcript.To verify this and to follow the expression of themicroRNAs, sensor lines (Brennecke et al. 2003) forthe three microRNAs were generated. Two complemen-tary sites with perfect matches for each microRNA wereplaced downstream of an EGFP gene controlled by theubiquitously expressed tubulin (tub) promoter. Thebinding of the microRNAs to their complementary 39-UTR sequences was expected to block the translation ofEGFP mRNA and thereby decrease the level of EGFP. Inthe three sensor lines, we observed a uniform decreasein the EGFP level (Figure 1, H and I, and data notshown) compared to the tub-EGFP control line (Figure1G), the control line lacked microRNA sites down-stream of the EGFP gene and was analyzed using asimilar laser setting. This indicated that, like dGMAP,the intronic microRNAs are uniformly expressed in thewing imaginal disc, suggesting that they are subjected tothe same regulation as the host gene, as previously pro-posed (Friggi-Grelin et al. 2006). The miR12-sensorline, however, showed a stronger decrease in the level ofEGFP (Figure 1H) than did the miR-283 and miR-304lines (Figure 1I and data not shown), suggesting that itwas more highly expressed in the disc. These data are in

agreement with the finding that miR-12 is one of themost abundant Drosophila microRNAs (Lagos-Quin-

tana et al. 2001) and are also consistent with a Northernanalysis of the three microRNAs reported in anotherstudy (Leaman et al. 2005). It is therefore possible thatmiR-12, which is processed from the same transcript asmiR-283 and miR-304, is more stable than the two othermicroRNAs or might just be more efficient.

To verify that PUAS-29B3 induces the expression ofthis microRNA cluster, we tested the sensitivity of eachsensor line to PUAS-29B3 activation. When PUAS-29B3was induced in the patched (ptc) expression domainalong the A/P border of the wing imaginal disc (usingthe ptc-GAL4 driver), we observed a decrease in thelevel of GFP at the A/P border in the three sensor lines(Figures 1, J–L).

miR-12 and miR-283 overexpression affects the Hhpathway: In view of the above results, it seemed possiblethat the induced PUAS-29B3 phenotype resulted fromthe combinatorial expression of one or more of thethree microRNAs. To test this possibility, we constructednew PUAS transgenic lines to be able to independentlyexpress miR-12, miR-283, or miR-304. We inducedclones of cells overexpressing 29B3, miR-12, miR-283,or miR-304 in the wing imaginal discs. In PUAS-29B3clones located in the A compartment, ectopic dppexpression was observed (100% of the clones, Figure2, A and B), consistent with the outgrowth observed inadult wings (Figure 1B). When miR-12 or miR-283 wereinduced separately, however, no ectopic dpp expressionwas observed (Figure 2, E and F, and data not shown).But ectopic dpp expression was observed when the miR-12 level was increased by expressing two copies of thetransgene at the anterior edge of the wing pouch (100%of the clones, Figure 2, G and H) or when miR-12 andmiR-283 were overexpressed together (100% of theclones located at the edge, Figure 2, C and D). No mod-ifications in Hh target gene expression were observed in

Figure 2.—Ectopic dpp expres-sion is due to overexpression oftwo microRNAs, miR-12 and miR-283. dpp expression is visualizedwith the dpp-lacZ reporter gene(red) in wing imaginal discs.Clones of cells driving expressionof PUAS-29B3 (A and B), PUAS-miR12 alone (E and F), or PUAS-miR12 together with PUAS-miR283(C and D), are labeled with GFP(green). Ectopic dppZ expression(arrows) is observed in more ante-riorclones following overexpressionofPUAS-29B3, twocopiesof miR-12,or one copy each of miR-12 andmiR-283. Note that ectopic dpp ex-pression ismostlyobservedat thean-terior edge of the wing pouch, as if

this region is more sensitive to the activation of the Hh pathway. We also note that the expression of Dpp in the clones is variable, likelydue to tissue deformation in the outgrowth.


clones overexpressing miR-304 (data not shown). To-gether, these results suggest that the PUAS-29B3 phe-notype is caused by the overexpression of both miR-12and miR-283. The limited activation of dpp by individualmiR expression might be due to a decreased stability ofmiR transcripts compared to the precursor mRNAsinduced by PUAS-29B3.

Several components of the Hh pathway are potentialtargets of the microRNA cluster: To determine whichcomponents of the Hh pathway are targeted by miR-12and miR-283, we analyzed the levels of several proteinsin the pathway in cells overexpressing either of the

two microRNAs. In the Hh pathway, a multicomponentcytoplasmic complex including the Smo, Fu, and Cos2proteins controls the stability and activity of the tran-scriptional factor Ci, which in turn regulates the ex-pression of Hh target genes. In cells overexpressingmiR-304, no modification was observed, however, incells overexpressing either miR-12 (Figure 3, A–F) ormiR-283 (Figure 3, I–N), all three proteins—Cos2(Figure 3, A, B, I, and J), Fu (Figure 3, C, D, K, andL), and Smo (Figure 3, E, F, M, and N)—were destabi-lized. In addition, Ci was stabilized (Figure 3, G and H,and data not shown), likely due to the decreased level of

Figure 3.—Overexpression of miR-12 or miR-283 modifies the levels of Cos2, Fu, Smo, and Ci proteins. Clones of cells drivingexpression of PUAS-miR12 (A–H) or PUAS-miR283 (I–L) are labeled with GFP. Cos2 (A, B, I, and J), Fu (C, D, K, and L), Smo (E,F, M, and N), and Ci (G and H) proteins are visualized by immunofluorescence (red). Induction of microRNA expression de-creases the level of Fu, Cos2, and Smo proteins in both anterior and posterior clones, whereas Ci is stabilized in anterior clones.Dotted lines indicate the A/P border. Arrows indicate the most significant clones. Note that the antibody against Cos2 gives ahigher background than the one against Fu, which likely accounts for the seemingly stronger affects on Fu than on Cos2.(O) Percentage of the decrease in the levels of Cos2, Fu, and Smo proteins in PUAS-29B3 (solid bars), PUAS-miR12 (shadedbars), or PUAS-miR283 (open bars) clones compared to surrounding wild-type cells. In PUAS-29B3 clones, the decrease inCos2 and Fu levels is more pronounced than in PUAS-miR12 or PUAS-miR283 clones. **P , 0.001; *P , 0.04.


Cos2 (Wang and Holmgren 1999). The decrease in thelevels of these three proteins in 29B3 clones was greaterthan in miR-12 or miR-283 clones (Figure 3O). Thus,ectopic dpp expression was probably observed in thePUAS-29B3 clones (Figure 2, A and B), but not in themiR-12 clones, because of the higher destabilization ofCos2 protein in the former context.

smo, cos2, and fu are regulated by the microRNAscluster: In view of this data we hypothesized that thesegenes might represent direct targets of the microRNAs.Binding sites of microRNAs are possible to identifybecause a 6- to 8-base ‘‘seed’’ of contiguous pairing ispresent between the 39 end of the binding site and the 59

end of the microRNA (Brennecke et al. 2005; Rajewsky

2006) and is conserved among the 39-UTRs of homol-ogous genes in related species. Using these criteria, weidentified potential miR-12 and miR-283 binding siteson both the cos2 and smo 39-UTRs (Figure 4, A and B,respectively), as well as two potential miR-12 sites on thefu 39-UTR (Figure 4C).

To determine whether the microRNAs can regulatethe 39-UTRs of cos2, smo, and fu, we established newsensor lines in which the EGFP gene was fused to the 39-UTRs of these genes. In the absence of microRNAinduction, these sensor lines displayed uniform EGFPexpression in wing imaginal discs (data not shown).However, when miR-12 was overexpressed in the ptcdomain, EGFP expression decreased in all three sensorlines at the A/P boundary (Figure 4, E–G), stronglysuggesting that miR-12 can regulate, likely directly, the39-UTRs of cos2, smo, and fu and thereby diminish theexpression of the encoded proteins. When the sameexperiments were performed with miR-283, we ob-tained similar results in the Cos2 and Smo-sensor lines(Figure 4, I and J, respectively), but not in the Fu-sensorlines (Figure 4K). We suspect that the decreased levelsof Fu observed upon miR-283 overexpression (Figure 3,I and J) were likely a consequence of the decreased levelof Cos2, consistent with results from a previous study(Ruel et al. 2003) and not due to the direct regulation offu mRNA by the microRNA. No modifications in thelevels of the three sensor lines were observed upon miR-304 overexpression.

Deletion of the microRNA cluster does not lead toHh pathway deregulation: To assess the importance ofthese microRNAs for fly development, flies lacking thecluster were produced by homologous recombination(Gong and Golic 2003) and were named D3miR.The mutant line was verified by PCR and sequencing(supplemental Figure 1 and data not shown). Mutantslacking the three microRNAs presented strong lethalitybefore the L1 stage (88.3%). Because most of the mu-tant embryos died well before the end of embryogene-sis, we were unable to analyze the cuticular pattern ofthe dead embryos, although we did stain these embryoswith phalloidin, DAPI, and a Golgi marker. No pheno-types were observed that were reminiscent of Hh lack of

function (supplemental Figure 2). In D3miR dead em-bryos, nuclei are observed (supplemental Figure 2A)but cell membranes are lacking (supplemental Figure2B), suggesting that lethality in these animals takesplace before cellularization, whereas D3miR escaperembryos presented wild-type organization (supplemen-tal Figure 2G). We also analyzed Wg expression in theescaper embryos that survived until later stages (sup-plemental Figure 2I). Because Wg expression is de-pendent upon Hh signaling activity between 3 and 6–7hr after egg laying, any mutations affecting the Hhpathway should affect the Wg pattern. However, nodifferences in the Wg pattern were identified betweenthe wt and the D3miR mutant embryos. Finally, in view ofthe role of Hh in the egg chamber, homeostasis of germline and follicular cells, we also stained the egg chamberof D3miR adult escaper mutants with DAPI and phalloi-din. No defects in the egg chamber organization wereobserved (supplemental Figure 2, K–K$). Adult struc-tures of the D3miR escaper mutants, e.g., wings, eyes,legs, and abdomen, were also analyzed. No differencesfrom wild-type animals were observed (data not shown).We also sensitized the background for Hh signaling andanalyzed the D3miR escaper mutants in heterozygousmutant backgrounds for Cos2, Smo, and Su(fu). We didnot observe any Hh-like phenotypes in D3miR; cos2w1/1,in D3miR; smoIIX43/1, or in D3miR; SufuLP/1 mutantanimals (data not shown).

A likely explanation for the observed lethal pheno-type was that these three microRNAs are involved inessential processes unrelated to Hh signaling regulation(Lim et al. 2005). For example, miR-12, miR-283, andmiR-304 could have targets other than smo, cos2, and fu,and the regulation of these other targets could becritical for animal viability. Alternatively, it also seemedpossible that the lethality was due to the reduced levelsof dGMAP observed in the D3miR line (Figure 5, H–H9,and supplemental Figure 2, H and K$), possibly due tomodified splicing of the microRNA-containing intron.To discriminate between these hypotheses, we askedwhether expressing dGMAP or different microRNAscould rescue the lethality of the D3miR mutant. Wefound that ubiquitous expression of individual micro-RNAs driven by arm-Gal4 rescues the viability of theD3miR mutant by 17 to 25%, whereas dGMAP did notsignificantly increase the viability of mutant animals(supplemental Figure 3). These results demonstratedthat the lethality of the D3miR mutants is mainly theconsequence of the absence of the microRNA clusterand not of GMAP activity but it is possible that theabsence of dGMAP might still contribute to the lethalityobserved in the D3miR mutants. Moreover the microRNA-rescue animals look comparable to wild type. Indeed, wedid not observe any phenotypes reminiscent of the Hhphenotype. Taken together, these results show that thelethality of the D3miR mutant is not due to Hh signalingmisregulation.


Smo, cos2, and fu 39-UTRs are physiological targets ofmir-12 and mir-283: To confirm that the Hh pathway isnot modified in D3miR mutant animals, we inducedclones of cells mutant for the microRNA cluster in thewing discs at the L1 stage (Figure 5). We observednormal levels of Hh pathway components such as theCos2, Fu, Smo, and Ci proteins (Figure 5, A–A9, B–B9,

C–C9, and D–D9, respectively), as well as normal ex-pression of Hh target genes (data not shown). Becausethe absence of the microRNA cluster had no conse-quences for the Hh pathway, it seemed possible that,although expressed in this tissue, the cluster has nofunctional role in the wing imaginal disc. To test thishypothesis, we analyzed GFP expression from the Cos2-

Figure 4.—smo, cos2, and fu mRNAs are regulated by the 29B3-microRNA cluster. Schematics of cos2 (A), smo (B), and fu (C)mRNAs. Green squares indicate the open reading frames of the transcripts. Blue and red bars represent potential 39-UTR bindingsites for miR-12 and miR-283, respectively. Each site is presented below, showing the conservation between Drosophila pseudoobscuraand Anopheles gambiae and theoretical miR-12 or miR-283 binding sites. Conserved residues are shaded in red, with stars below.Yellow bars represent the primers used to amplify the 39-UTR sequences to establish the Cos2-, Smo-, and Fu-sensor lines. (D–K)GFP expression in tub-EGFP (D and H), Cos2- (E and I), Smo- (F and J), and Fu- (G and K) sensor lines in ptc-GAL4/UAS-miR12imaginal discs (D–G) or in ptc-GAL4/UAS-miR283 (H–K) wing imaginal discs. Note that no modifications in EGFP expressionwere observed when miR-12 or miR-283 were overexpressed in the tub-EGFP control line (D and H).


(Figure 5, E–E9), Fu- (Figure 5, F–F9), and Smo- (Figure5, G–G9) sensor constructs in the D3miR mutant clones.Clones were induced at the L1 stage and discs dissected4 days later to minimize the possibility that perduranceof the microRNAs might mask a loss-of-function phe-notype. As shown in Figure 5, E–G, a small increase inthe GFP level was observed in the mutant cells, suggest-ing that miR-12 and miR-283 do indeed regulate the 39-UTRs of the three genes in wing imaginal discs.

Absence of Dicer activity does not lead to Hhpathway deregulation: We were surprised to only ob-serve a modest increase in GFP sensor levels in theD3miR mutant cells. Redundancy with other microRNAsmight explain why no strong increase in the GFP sensorlevel was seen. This seemed to be a reasonable hypoth-esis, since the Drosophila genome is predicted to encodeas many as 100 miRNAs, and it is common to find indi-vidual mRNAs targeted by multiple miRNAs (Bren-

necke et al. 2005). It was thus possible that potentialphenotypes resulting from a lack of the microRNA clus-ter were masked by functional redundancy with othermicroRNAs that can also bind smo, cos2, and fu mRNAs.

One way to circumvent this potential problem was toblock all microRNA synthesis. In Drosophila, Dcr-1 is

essential for microRNA-directed repression as it par-ticipates in the digestion of pre-microRNAs to formmicroRNAs (Lee et al. 2004). In dcr-1 mutants, there-fore, microRNA synthesis and function are inhibited. Totest our hypothesis, we created dcr-1 mutant clones inthe wing imaginal discs (Figure 6). We also inducedmutant clones in a Minute background but we did notobtain significantly bigger clones (data not shown). Astrong increase in the GFP level of the miR12-sensor wasobserved in the dcr-1 mutant cells (Figure 6, D–D$),confirming that the microRNAs are absent in theseclones. Nevertheless these mutant clones contained nor-mal levels of Hh pathway components such as Cos2, Fu,and Smo (Figure 6, A–A$, B–B$, and C–C$, respec-tively), as well as normal expression of Hh target genes(data not shown). In contrast, GFP levels in the Cos2-(Figure 6, E–E$) and Smo- (Figure 6, F–F$) sensor lineswere strongly increased (by 40%) in the dcr-1 mutantclones. Thus, although the sensor lines for the cos2 andsmo 39-UTRs showed increased expression in the dcr-1mutant clones, confirming that a loss-of-function phe-notype was not masked by perdurance of the miRNAs,the absence of Dcr activity affected neither the Cos2 andSmo protein levels nor the Hh pathway.

Figure 5.—An absence of themicroRNA cluster does not leadto Hh phenotype but changesthe level of the sensor constructs.(A–H) Clones of cells homozygousmutant for D3miR are marked bythe absence of Myc (red). Cos2(A–A9), Fu (B–B9), Smo (C–C9),Ci (D–D9) and dGMAP (H–H9)proteinsare visualizedby immuno-fluorescence (green). GFP expres-sion of Cos2- (E–E9), Fu- (F–F9)and Smo- (G–G9) sensor con-structs is in green. No modifica-tions in the levels of the proteins(A–D) were observed except fordGMAP (H). In contrast GFP levelof the sensor constructs mod-estly increased (E–G, arrows) inD3miR mutant clones.


DISCUSSION

We found that cos2, fu, and smo mRNA can beregulated by a cluster of microRNAs, including miR-12and miR-283, in Drosophila wing disc. The overexpres-sion of this cluster decreases the levels of Smo, Cos2, and

Fu proteins and activates the Hh pathway, as evidenced

by the induction of dpp expression in the wing imaginal

discs and by the adult wing outgrowth. The experiments

presented here with the 39-UTR sensors of smo, fu, or

cos2 are in favor of a direct binding. To constitute a real

Figure 6.—Absence of Dcr-1 function changesCos2- and Smo-sensor level without affectingtheir protein levels. (A–E) Clones of cells homo-zygous mutant for dcr-1 are marked by the ab-sence of b-Gal (red). Cos2 (A–A$), Fu (B–B$),and Smo (C–C$) proteins are visualized by immu-nofluorescence (green). No modifications in thelevels of the proteins were observed. GFP expres-sion of miR12 (D–D$), Cos2- (E–E$), and Smo-(F–F$) sensor lines is in green. Note that GFPlevel strongly increases in dcr-1 mutant clones.Note also that, although dcr-1 mutant clones havebeen induced in L1, as attested by the big size ofthe wt twin spots (bright red), they reach a smallsize only likely due to cell lethality. Due to tech-nical difficulties we could not analyze Fu-sensorconstructs in dcr-1 mutant clones.


proof of a direct effect, further experiments as directbiochemical binding assay or compensatory mutationbetween the 39-UTR and the miRNAs will be necessaryto perform.

The three programs (Stark et al. 2003; Grun et al.2005; Griffiths-Jones et al. 2006) that have beencreated to genomewide predictions of DrosophilamiRNA targets provide lists of presumptive miR-12,and miR-283 regulated genes. In addition to ourin vivo validations, miR-12 binding sites are predictedon the 39-UTR of ci and no sites were found on the 39-UTR of the Su(fu) gene (Griffiths-Jones et al. 2006).We did not observe a decrease in either of these twoproteins in the microRNA cluster overexpressing clones(data not shown). It is interesting to note that Flynt

et al. (2007) recently showed that Su(fu) mRNA, encod-ing another negative regulator of Hedgehog signaling,is targeted by miR-214 in zebrafish. Absence of miR-214results in the reduction of muscle cell types, the spec-ification of which is dependent on Hh pathway activity.Nevertheless, our study shows that in Drosophila wingdiscs an absence of microRNA does not modify the Hhpathway, raising the question of what the role of micro-RNAs in Drosophila Hh pathway regulation is.

Could the microRNAs overexpression phenotype thatwe identified be artifactual and simply the result offorced overexpression of the microRNA cluster in atissue in which it should be silent? We believe the answeris no, because Northern blot analysis (Leaman et al.2005) and the increase of our miR-sensor in the dcr-1mutant clones (Figure 6) showed that the microRNAcluster is indeed expressed in this tissue. This, to us,suggests that the cluster likely has a role in this tissue inwhich it is normally present. Is the microRNA clusterregulation of the cos2 and smo 39-UTRs physiological?We think so, because an absence of either the microRNAcluster or of Dicer in the wing imaginal disc induces anincrease in the Cos2- and Smo-sensor lines. This, to us,signifies that the microRNAs expressed from the clusterregulate the cos2 and smo 39-UTRs and thus display somefunctionality in the disc during larval development.Altogether, these data clearly show that we have notcreated an artifactual situation in which the microRNAcluster is expressed in a tissue in which it should notbe present. The miRs overexpression was also testedon embryonic patterning but it did not lead to anyphenotype (data not shown), suggesting that the miRcluster regulation on the Hh pathway is specific to larvaltissues.

As miR-12 and miR-283, and likely redundant miRs,are present in every cell of the wing disc, one possibilityis that their normal roles are to dampen down the levelsof Hh pathway components, particularly Cos2 and Smo,to prevent the accidental activation or downregulationof the pathway. Indeed, expressing both the microRNAcluster and its targets in the same tissue could provide ameans of ‘‘buffering stochastic fluctuations’’ in mRNA

levels or in protein translation rates within the Hh sig-naling pathway, as has been proposed for other pro-cesses (Hornstein and Shomron 2006).

Absence of the microRNA cluster does not lead toHh pathway deregulation: Our data possibly indicatethat miRNAs are able to regulate two antagonisticcomponents of the pathway, Cos2 and Smo. We havepreviously shown that the stability of these two proteinsis ‘‘interdependent’’: an increased level of Cos2 in thewing imaginal disc lowers the level of Smo, and, in theopposite direction, increased Smo decreases the level ofCos2 (Ruel et al. 2003). We propose that the interregu-lation of Cos2/Smo levels is independent of theirrelative activities because Cos2 effect on Smo levels isobserved in posterior cells in which Cos2 activity isstrongly inhibited by the constitutive activation of thepathway. Therefore, eliminating the miRNA-mediatedinhibition of Cos2 and Smo in D3miR or dcr-1 mutantcells likely initially increased the levels of both proteins,but then the resulting higher levels of each proteinpresumably downregulated the other; the net variationof Cos2 and Smo levels would therefore be null. Wefavor this hypothesis because the independent Smo- andCos2-sensor lines, which are unaffected by this Cos2/Smo interregulation, showed increased levels of GFPstaining in D3miR and dcr-1 mutant animals (Figures 5,E–G, and 6, E and F, respectively). This suggests that thelevels of both Cos2 and Smo are increased in the mutantanimals but, because of the downregulation of eachprotein by the other, no ultimate alterations in the levelsof the proteins are observed. If so, we would not neces-sarily expect to see an Hh phenotype in the miR mutant.

MicroRNAs misexpression and cancers: Our screencreated a situation in which the expression of themicroRNA cluster is deregulated, ultimately destabiliz-ing Cos2 protein levels and thereby activating Ci and Hhtarget gene expression. Importantly, a similar situationmight be encountered during tumoral development.Aberrant Hh signaling activity is known to trigger thedevelopment of diverse cancers (Ruiz i Altaba 1999;Watkins et al. 2003; Fan et al. 2004; Kayed et al. 2004).While several of these tumors have been linked tomutations in Hh signaling components, not all of themhave, leaving open the possibility that they are caused byother factors such as microRNA misexpression. Inter-estingly, more than half of the known human microRNAgenes are located near chromosomal breakpoints asso-ciated with cancer (Calin et al. 2004a,b), and in somedocumented cases the microRNAs are amplified, lead-ing to overexpression (Lu et al. 2005). Some upregu-lated microRNAs are possibly able to bind mRNAsencoding negative regulators of Hh signaling, such asSu(fu) or Ptc, and could thus induce the misactivationof the Hh pathway, as is observed in some cancers.Therefore, a fine analysis of microRNA expression levelsand the levels of known Hh components should beconsidered in studies of Hh pathway-related cancers.


What does our manuscript add to the current knowl-edge about miRNA regulation? Our study shows for thefirst time that a cluster of three microRNAs can targetseveral antagonistic components of the same pathwayin vivo. This is novel and unexpected. This raises thequestion of how to interpret the miRNA expressionsignatures observed in human tumors. Indeed, as statedabove, it has been proposed that miRNAs are differen-tially expressed in human cancers and contribute tocancer development. The working hypothesis in thecancer/miRNAs field is that key cancer genes areregulated by aberrant expression of miRNAs. The iden-tification of a specific miRNA:mRNA interactor pair isgenerally accepted as being of biological importancewhen the mRNA encodes a tumor suppressor or anoncogene whose expression is modified in the tumor.Our study shows indirectly this is an oversimplified view,because identifying an oncogene or tumor suppressoras a target of a miRNA may not provide a full explana-tion for tumor development if the same miRNA hitsother antagonistic components of the same pathwaythat nullify the effect of the identified miRNA:mRNAinteractor pair.

We thank S. Cohen for providing the tub-EGFP vector and fortransgenic lines, R. Carthew for the dicer-1 mutant line, and all mem-bers of P. Therond’s lab for discussions. F.F.G. was supported by apostdoctoral fellowship from Fondation de France. This work wassupported by grants from Fondation de France, Centre National de laRecherche Scientifique, and Ligue Nationale Contre le Cancer‘‘equipe labellisee 2008’’ to P.P.T.

LITERATURE CITED

Aravin, A. A., M. Lagos-Quintana, A. Yalcin, M. Zavolan, D.Marks et al., 2003 The small RNA profile during Drosophila mel-anogaster development. Dev. Cell 5: 337–350.

Bartel, D. P., 2004 MicroRNAs: genomics, biogenesis, mechanism,and function. Cell 116: 281–297.

Basler, K., and G. Struhl, 1994 Compartment boundaries and thecontrol of Drosophila limb pattern by hedgehog protein. Nature368: 208–214.

Brennecke, J., D. R. Hipfner, A. Stark, R. B. Russell and S. M.Cohen, 2003 bantam encodes a developmentally regulated mi-croRNA that controls cell proliferation and regulates the pro-apoptotic gene hid in Drosophila. Cell 113: 25–36.

Brennecke, J., A. Stark, R. B. Russell and S. M. Cohen, 2005 Prin-ciples of microRNA-target recognition. PLoS Biol. 3: e85.

Calin, G. A., C. G. Liu, C. Sevignani, M. Ferracin, N. Felli et al.,2004a MicroRNA profiling reveals distinct signatures in B cellchronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101:11755–11760.

Calin, G. A., C. Sevignani, C. D. Dumitru, T. Hyslop, E. Noch et al.,2004b Human microRNA genes are frequently located at frag-ile sites and genomic regions involved in cancers. Proc. Natl.Acad. Sci. USA 101: 2999–3004.

Capdevila, J., and I. Guerrero, 1994 Targeted expression of thesignaling molecule decapentaplegic induces pattern duplica-tions and growth alterations in Drosophila wings. EMBO J. 13:4459–4468.

Di Leva, G., G. A. Calin and C. M. Croce, 2006 MicroRNAs: fun-damental facts and involvement in human diseases. Birth DefectsRes. C Embryo Today 78: 180–189.

Fan, L., C. V. Pepicelli, C. C. Dibble, W. Catbagan, J. L. Zarycki

et al., 2004 Hedgehog signaling promotes prostate xenografttumor growth. Endocrinology 7: 7.

Felsenfeld, A. L., and J. A. Kennison, 1995 Positional signaling byhedgehog in Drosophila imaginal disc development. Develop-ment 121: 1–10.

Flynt, A. S., N. Li, E. J. Thatcher, L. Solnica-Krezel and J. G.Patton, 2007 Zebrafish miR-214 modulates Hedgehog signal-ing to specify muscle cell fate. Nat. Genet. 39: 259–263.

Friggi-Grelin, F., C. Rabouille and P. Therond, 2006 The cis-Golgi Drosophila GMAP has a role in anterograde transportand Golgi organization in vivo, similar to its mammalian orthologin tissue culture cells. Eur. J. Cell. Biol. 85: 1155–1166.

Gallet, A., R. Rodriguez, L. Ruel and P. P. Therond, 2003 Cho-lesterol modification of hedgehog is required for trafficking andmovement, revealing an asymmetric cellular response to hedge-hog. Dev. Cell 4: 191–204.

Gong, W. J., and K. G. Golic, 2003 Ends-out, or replacement, genetargeting in Drosophila. Proc. Natl. Acad. Sci. USA 100: 2556–2561.

Griffiths-Jones, S., R. J. Grocock, S. van Dongen, A. Bateman andA. J. Enright, 2006 miRBase: microRNA sequences, targets andgene nomenclature. Nucleic Acids Res. 34: D140–D144.

Grun, D., Y. L. Wang, D. Langenberger, K. C. Gunsalus and N.Rajewsky, 2005 microRNA target predictions across seven Dro-sophila species and comparison to mammalian targets. PLoSComput. Biol. 1: e13.

Hornstein, E., and N. Shomron, 2006 Canalization of develop-ment by microRNAs. Nat. Genet. 38: S20–S24.

Kayed, H., J. Kleeff, S. Keleg, J. Guo, K. Ketterer et al.,2004 Indian hedgehog signaling pathway: expression and reg-ulation in pancreatic cancer. Int. J. Cancer 110: 668–676.

Lagos-Quintana, M., R. Rauhut, W. Lendeckel and T. Tuschl,2001 Identification of novel genes coding for small expressedRNAs. Science 294: 853–858.

Lai, E. C., P. Tomancak, R. W. Williams and G. M. Rubin, 2003 Com-putational identification of Drosophila microRNA genes. GenomeBiol. 4: R42.

Leaman, D., P. Y. Chen, J. Fak, A. Yalcin, M. Pearce et al.,2005 Antisense-mediated depletion reveals essential and spe-cific functions of microRNAs in Drosophila development. Cell121: 1097–1108.

Lee, Y. S., K. Nakahara, J. W. Pham, K. Kim, Z. He et al., 2004 Dis-tinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117: 69–81.

Lim, L. P., N. C. Lau, P. Garrett-Engele, A. Grimson, J. M. Schelter

et al., 2005 Microarray analysis shows that some microRNAs down-regulate large numbers of target mRNAs. Nature 433: 769–773.

Lu, J., G. Getz, E. A. Miska, E. Alvarez-Saavedra, J. Lamb et al.,2005 MicroRNA expression profiles classify human cancers. Na-ture 435: 834–838.

McMahon, A. P., P. W. Ingham and C. J. Tabin, 2003 Develop-mental roles and clinical significance of hedgehog signaling.Curr. Top. Dev. Biol. 53: 1–114.

Rajewsky, N., 2006 microRNA target predictions in animals. Nat.Genet. 38: S8–S13.

Rorth, P., K. Szabo, A. Bailey, T. Laverty, J. Rehm et al., 1998 Sys-tematic gain-of-function genetics in Drosophila. Development125: 1049–1057.

Ruel, L., R. Rodriguez, A. Gallet, L. Lavenant-Staccini and P. P.Therond, 2003 Stability and association of Smoothened, Costal2and Fused with Cubitus interruptus are regulated by Hedgehog.Nat. Cell Biol. 5: 907–913.

Ruiz i Altaba, A., 1999 Gli proteins and Hedgehog signaling: de-velopment and cancer. Trends Genet. 15: 418–425.

Stark, A., J. Brennecke, R. B. Russell and S. M. Cohen, 2003 Iden-tification of Drosophila microRNA targets. PLoS Biol. 1: E60.

Teleman, A. A., Y. W. Chen and S. M. Cohen, 2005 DrosophilaMelted modulates FOXO and TOR activity. Dev. Cell 9: 271–281.

Wang, Q. T., and R. A. Holmgren, 1999 The subcellular localiza-tion and activity of Drosophila cubitus interruptus are regulatedat multiple levels. Development 126: 5097–5106.

Watkins, D. N., D. M. Berman, S. G. Burkholder, B. Wang, P. A.Beachy et al., 2003 Hedgehog signalling within airway epithelialprogenitors and in small-cell lung cancer. Nature 422: 313–317.

Zhang, B., X. Pan, G. P. Cobb and T. A. Anderson, 2007 microRNAsas oncogenes and tumor suppressors. Dev. Biol. 302: 1–12.

Communicating editor: J. A. Lopez


Control of Antagonistic Components of the Hedgehog ... · CNRS UMR 6543, Centre de Biochimie, Parc...

Documents

Transcript of Control of Antagonistic Components of the Hedgehog ... · CNRS UMR 6543, Centre de Biochimie, Parc...