Control of antagonistic components of the hedgehog signaling pathway by microRNAs in Drosophila

Abstract

Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. We previously showed that stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. Here, we show that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3'-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 3'-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs.

Figure 1.—

The PUAS-29B3 line controls the expression of a microRNA cluster and induces anterior wing outgrowth. (A) Wild-type adult wing and (B) PUAS-29B3/Sd-GAL4 wing obtained at 27°. Note that when flies were grown at 18°, a temperature at which GAL4 activity is much lower, this phenotype is not observed (data not shown). (C) PUAS-29B3/Sd-GAL4; cos2^w1/+ and (D) PUAS-29B3/Sd-GAL4; Su(Fu)^LP/+ wings obtained at 18°. Arrows indicate outgrowth of anterior wing tissue in the costa region. (E) Localization of the PUAS-29B3 element in gene CG33206, which encodes dGMAP. The microRNA cluster, composed of miR-283, miR-304, and miR-12, is present in the fifth intronic sequence of dGMAP. (F) In situ hybridization for dGMAP mRNA (red), followed by dGMAP immunostaining (green) in PUAS-29B3; en-GAL4 embryos. No increase in dGMAP protein was observed in the en domain. Note that endogenous dGMAP mRNAs are present in all cells but levels are low compared to ectopic RNA expression (middle). Note also that dGMAP proteins are present in all ectodermal cells but their level is artifactually decreased in the en cells where the dGMAP in situ detection is very high (right). (G–L) GFP expression in tub-EGFP (G), miR12-sensor (H and J), miR283-sensor (I and K), miR304-sensor (L), wt discs (G–I), or in PUAS-29B3; ptc-GAL4 discs (J–L). The pictures shown in G, H, and I were obtained with a similar laser setting on the confocal microscope. Note that the laser setting is different for H and J (in which laser intensity has been increased) and thus sensor level is not comparable for these two panels. G–I are illustrative of three independent lines for each sensor. Dotted lines indicate the A/P border. (G–L) Magnification, 200×.

Figure 2.—

Ectopic dpp expression is due to overexpression of two microRNAs, miR-12 and miR-283. dpp expression is visualized with the dpp-lacZ reporter gene (red) in wing imaginal discs. Clones of cells driving expression of 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 anterior clones following overexpression of PUAS-29B3, two copies of miR-12, or one copy each of miR-12 and miR-283. Note that ectopic dpp expression is mostly observed at the anterior 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, likely due to tissue deformation in the outgrowth.

Figure 3.—

Overexpression of miR-12 or miR-283 modifies the levels of Cos2, Fu, Smo, and Ci proteins. Clones of cells driving expression 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 decreases 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 a higher 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 (shaded bars), or PUAS-miR283 (open bars) clones compared to surrounding wild-type cells. In PUAS-29B3 clones, the decrease in Cos2 and Fu levels is more pronounced than in PUAS-miR12 or PUAS-miR283 clones. **P < 0.001; *P < 0.04.

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 3′-UTR binding sites for miR-12 and miR-283, respectively. Each site is presented below, showing the conservation between Drosophila pseudoobscura and 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 3′-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-miR12 imaginal discs (D–G) or in ptc-GAL4/UAS-miR283 (H–K) wing imaginal discs. Note that no modifications in EGFP expression were observed when miR-12 or miR-283 were overexpressed in the tub-EGFP control line (D and H).

Figure 5.—

An absence of the microRNA cluster does not lead to Hh phenotype but changes the level of the sensor constructs. (A–H) Clones of cells homozygous mutant for Δ3miR are marked by the absence of Myc (red). Cos2 (A–A′), Fu (B–B′), Smo (C–C′), Ci (D–D′) and dGMAP (H–H′) proteins are visualized by immunofluorescence (green). GFP expression of Cos2- (E–E′), Fu- (F–F′) and Smo- (G–G′) sensor constructs is in green. No modifications in the levels of the proteins (A–D) were observed except for dGMAP (H). In contrast GFP level of the sensor constructs modestly increased (E–G, arrows) in Δ3miR mutant clones.

Figure 6.—

Absence of Dcr-1 function changes Cos2- and Smo-sensor level without affecting their protein levels. (A–E) Clones of cells homozygous mutant for dcr-1 are marked by the absence of β-Gal (red). Cos2 (A–A″), Fu (B–B″), and Smo (C–C″) proteins are visualized by immunofluorescence (green). No modifications in the levels of the proteins were observed. GFP expression of miR12 (D–D″), Cos2- (E–E″), and Smo- (F–F″) sensor lines is in green. Note that GFP level strongly increases in dcr-1 mutant clones. Note also that, although dcr-1 mutant clones have been induced in L1, as attested by the big size of the wt twin spots (bright red), they reach a small size only likely due to cell lethality. Due to technical difficulties we could not analyze Fu-sensor constructs in dcr-1 mutant clones.