The miR-310/13 cluster antagonizes β-catenin function in the regulation of germ and somatic cell differentiation in the Drosophila testis

Abstract

MicroRNAs (miRNAs) are regulators of global gene expression and function in a broad range of biological processes. Recent studies have suggested that miRNAs can function as tumor suppressors or oncogenes by modulating the activities of evolutionarily conserved signaling pathways that are commonly dysregulated in cancer. We report the identification of the miR-310 to miR-313 (miR-310/13) cluster as a novel antagonist of Wingless (Drosophila Wnt) pathway activity in a functional screen for Drosophila miRNAs. We demonstrate that miR-310/13 can modulate Armadillo (Arm; Drosophila β-catenin) expression and activity by directly targeting the 3'-UTRs of arm and pangolin (Drosophila TCF) in vivo. Notably, the miR-310/13-deficient flies exhibit abnormal germ and somatic cell differentiation in the male gonad, which can be rescued by reducing Arm protein levels or activity. Our results implicate a previously unrecognized function for miR-310/13 in dampening the activity of Arm in early somatic and germline progenitor cells, whereby inappropriate/sustained activation of Arm-mediated signaling or cell adhesion may impact normal differentiation in the Drosophila male gonad.

Keywords: Armadillo; Beta-catenin; MicroRNA; Stem cells; Wingless.

Fig. 1.

Identification of miR-310/13 in an RNAi-based targeted screen for miRNAs that suppress Wg pathway activity downstream of Axin. (A) The primary screen. miRNAs were tested for their ability to modulate Wg reporter (dTF12) activity in Clone 8 and S2R+ cells, where the pathway was ectopically activated by Axin dsRNA. (B) Unbiased cluster analysis of averaged log normalized scores for each screened miRNA in Clone 8 and S2R+ cells. (C) Highlighted clusters of strong inhibitors of the Wg reporter (top panel; orange box in B), including the previously reported Wg antagonist miR-8. Note the functional clustering of members of the miR-310/13 cluster. Also highlighted are potent activators of the Wg reporter (bottom panel; red box in B), including the previously published Wg agonist miR-315. (D,D′) Epistasis analyses. miR-310/13 strongly inhibits the dTF12-luciferase reporter when the Wg pathway is activated by dsRNA-mediated knockdown of Axin or by cDNA expression of ΔNLRP6 or Dsh. No significant inhibition by miR-310/13 was observed upon pathway activation with the constitutively active S37Aβ-cat. (E) Alignment of mature regions of Drosophila miR-310, miR-311, miR-312 and the human ortholog hsa-miR-25. d, Drosophila; a, Anopheles. (F,G) Alignments of multiple arm (F) and pan (G) 3′-UTR transcripts in various Drosophila and insect species revealing a conservation of miR-310/13 binding sites in arm and pan mRNA. (H) Predicted binding sites of miR-310/13 components in the 3′-UTR of arm and pan. (E-H) Gray shading highlights the conserved regions. (I-J′) pan 3′-UTR (I) and arm 3′-UTR (J) containing luciferase sensor reporters are significantly downregulated in the presence of miR-310/13, compared with control sensor containing full-length cDNA of arm lacking the 3′-UTR (Δ3′UTR, J′). (K) Western blot (WB) showing the ability of the miR-310/13 cluster and its individual components to significantly downregulate levels of Arm protein in S2R+ cells treated with Axin dsRNA. (L) hsa-miR-25 inhibits Wnt3a-induced activation of the SuperTOPFlash/STF16 reporter in human embryonic kidney (HEK293T) cells. Error bars indicate s.d. (n=4). P-values by Student’s t-test. RLU, relative luciferase units.

Fig. 2.

miR-310/13 flip-out clones mimic loss of Arm activity by downregulating expression of endogenous Arm. (A-C) Flip-out (FO) clones ectopically expressing miR-310/13 and GFP are difficult to recover in the wing imaginal disc (B; see range of clone recovery in the inset), compared with control GFP WT clones generated under identical conditions (A). The viability of miR-310/13-expressing GFP⁺ clones is robustly restored upon co-expression of Arm^S10 lacking the 3′-UTR (C). (D) Quantification of data generated in A-C as percentage clone recovery with respect to total disc area. The error bars for minima and maxima for percentage of disc area are indicated. (E-E′′) FO clones co-expressing miR-310/13 and p35. Note the recovery of these clones compared with the miR-310/13 FO clones shown in Fig. 1B. (F,G) x-z sections of the UAS-miR-310/13, UAS-p35 FO clones reveal that the recovered clones are small, composed of just one to three cells (red and yellow arrowheads, F), suggesting a failure in proliferation. Cells within the clones also appeared to be delaminating from the epithelial plane (G); red arrowhead indicates a cell at an early stage of delamination, whereas the white arrowhead marks a cell that has completely delaminated from the epithelial plane. This is likely to be due to loss of adhesion upon downregulation of Arm by miR-310/13. (H-H′) High-magnification image of an early p35, miR-310/13 FO clone reveals significant reduction of endogenous Arm expression at the cell junctions within the clone (red arrowheads in H′,H′).

Fig. 3.

Overexpression of the miR-310/13 cluster in Drosophila imaginal discs phenocopies Wg loss of function. (A-A′) Dpp-lacZ expression is restricted by Wg to the dorsal compartment of WT Drosophila leg discs (yellow arrowhead in A′). ns^*, non-specific staining. (B-B′) Misexpression of miR-310/13-dsRED using ptc-GAL4 leads to Dpp-lacZ derepression and expansion to the ventral compartment of the leg disc (yellow arrowhead in B′; quantified in supplementary material Fig. S2). Note the expression of miR-310/13 in the ptc expression domain, visualized by dsRED expression in B′. Red arrowheads (A′,B′) indicate mild expansion of the endogenous expression of Dpp-lacZ evident in the dorsal compartment. (C-I′) Effect of misexpression of miR-310/13 on sens, a Wg target gene, and wing patterning/growth of the wing imaginal disc. Sens is expressed in two rows at the dorsoventral boundary of the wing disc and is used as a readout for Wg signaling activity (C). C96-GAL4-driven expression of Axin leads to downregulation of Sens (D, arrowhead), adult wing notching, and depletion of sensory bristles (H,H′, black arrowhead), compared with the WT control (C,G,G′). Misexpression of miR-310/13-dsRED in the C96 domain (note dsRED expression in E) phenocopies Axin overexpression, both in its ability to repress Sens (F, arrowheads) and by causing wing notching in the adult (I,I′, black arrowheads). (J,K) Co-expression of arm lacking a 3′-UTR together with miR-310/13 leads to significant rescue of wing notching and bristle loss, in addition to the formation of ectopic sensory bristles (J) similar to those observed in flies overexpressing Arm alone under C96-GAL4 (K). (G′,J′,K′) Magnification of dorsal wing margins from WT flies (G′), flies that co-express an active form of Arm (Arm^*) and miR-310/13 (J′), and those expressing Arm^* alone (K′). Note that the increase in dorsal sensory wing margin bristles in flies expressing Arm^* alone (K′) is rescued by the co-expression of miR-310/13 (J′). Boxes indicate the regions magnified.

Fig. 4.

miR-310/13 null flies (d59/d59) exhibit a male-specific fertility defect. (A,B) d59/d59 males exhibit severe sterility. Hatching rate of eggs laid by d59/d59 males (n=200) crossed to WT females is significantly decreased (gray bars) compared with a WT control cross (black bars) (A). d59/d59 females exhibit no significant fertility defect (B). (C) Model of WT testes (see text for details). (E,E′) GFP sensor containing multimerized miR-312 binding sites displays a marked reduction of the GFP signal in the apical and medial domain of the testis, but not in the nuclei of the muscle sheath cells (arrowheads in E; see quantification of GFP sensor fluorescence intensity in supplementary material Fig. S2I-K). (D,D′) Note that there is increased cytoplasmic GFP expression in both soma and germ in the control testis. However, we consistently see much weaker GFP expression in germ cells compared with soma. (F-F′′) WT testis showing the germline marker Vasa in green, DAPI in blue, and the CySCs/early somatic cyst cell marker Tj in red. (G-G′′) Testes of d59/d59 flies exhibit abnormal accumulations of early germ cells and somatic cyst cells. The germ cell clusters (outlined by green dashed line in G′-G′′) accumulate further away from the hub compared with WT control. The cells within these clusters display condensed nuclear morphology, as shown by DAPI staining (arrowheads in G′) and appear to be poorly differentiated. The germ cell clusters are almost always associated with ectopic Tj⁺ cells (G,G′′), which show a marked increase in number compared with WT (compare G′′ with F′′). (H,I) Ectopic cyst somatic cells are positive for the CySC/early cyst cell marker Zfh1 in d59 mutant testes (I), compared with WT (H). Note that the bright Zfh1⁺ cells (arrowheads in I) are associated with germ cell clusters (shown in the merge in supplementary material Fig. S2M). (J,K) Synchronous cell division in abnormal germ cell clusters in d59/d59 testes (K), compared with WT (J), as revealed by EdU staining. (L-M′) Abnormal germ cell accumulations in d59 homozygous testes harbor branched fusomes (M′). 1B1 marks fusomes (red) (L,M). GSCs contain ‘dot’ fusomes, and the degree of fusome branching in germ cells correlates with the extent of differentiation (L,L′). miR-310/13-deleted testes display extensive branched fusomes within abnormal germ cell clusters (yellow arrows in M,M′; higher magnification in M′ inset). (N-N′′) Rescue of the germ cell accumulation phenotype in d59/d59 males expressing a genomic construct spanning the mir-310-313 locus as well as a region upstream of the transcription start site (miR-310/13L). Asterisks mark the hub.

Fig. 5.

Interfering with Arm activity and cell junctions in both somatic and germ cells rescues the abnormally differentiated germ cell clusters in d59 homozygous testes. (A,A′) Fz3RFP (Wg reporter) expression is restricted to cells of the somatic lineage as marked by eya. (B,B′) Wg-lacZ expression is increased in CySCs and their early progeny (arrowheads) proximal to the hub, indicating that Wg signaling may be active in these cells. Star marks non-specific staining. (C,C′) d59/CyO-Twist-GFP (CTG); Fz3RFP reporter testes exhibit normal morphology of germ cells (Vasa⁺ cells). (D,D′) d59/d59; Fz3RFP testes exhibit both a stronger RFP signal and an expansion in the domain expressing Fz3RFP reporter (D′). Yellow arrowheads indicate Fz3RFP, Tj⁺ cells. Ectopic RFP expression is seen in germ cells (white arrowheads, D′). (E-J′) Rescue of the germ and somatic cell accumulation phenotype in d59/d59 flies upon overexpression of Axin using the soma-specific C587-GAL4 driver (E,E′) or upon RNAi-mediated depletion of Arm (G,G′) and a partial rescue upon RNAi-knockdown of E-cad (I,I′) in the soma. Similar observations were made with germline expression of Axin (F,F′) or short hairpins against arm (H,H′) and E-cad (J,J′). Note the reappearance of properly differentiating germ cell clusters and normal nuclear morphology in each rescue example. (K-K′′) Overexpression of Arm in both germ (nos-GAL4) and somatic (Tj-GAL4) cells resulted in the formation of ectopic germ cell clusters (K′,K′′) associated with a distinct set of Tj⁺ cells (K′). Ectopic two-cell clusters in K′ are marked by the dashed yellow line. Asterisks mark the hub.

Fig. 6.

Model for miR-310/13 regulation of Arm function. (A) Components of the miR-310/13 cluster may directly target the mRNA for Arm/β-cat and/or Pan/TCF, modulating their levels. miRNA-mediated modulation of Arm levels can result in changes in transcription of target genes and in cell-cell adhesion. (B) Model representing the phenotype observed in miR-310/13-deficient testis (right) compared with WT testes (left). Poorly differentiated germ cell clusters (green) are enveloped by an increased number of somatic cyst cells (red). Arrows depict putative signaling interactions between germ cells and soma. We propose that high levels of Wg/Arm activity, and additional signaling interactions between the expanded soma and the associated germ cells, maintain the germ cells in early stages of differentiation. Moreover, the miR-310/13 cluster may function to diminish Arm activity/expression in order to facilitate further steps in the differentiation and maturation of germ and cyst somatic cells. (C) Model for the cellular mechanisms that might influence the miR-310/13 mutant phenotype. Deletion of miR-310/13 may lead to increased levels of cytosolic Arm, which can modulate both transcription of target genes and the strength of cell-cell contacts (see supplementary material Fig. S4). Increased transcription could potentially affect the proliferative state of either somatic or germline cells in a cell-autonomous or non-autonomous fashion. Modulation of cell adhesion could also perturb the differentiation process non-autonomously. Supporting this model is the observation that reduction in Arm or E-cad levels in either cell lineage leads to rescue of the abnormal germ cell accumulation phenotype (Fig. 5; supplementary material Fig. S3).