miR-137 targets Myc to regulate growth during eye development

Abstract

During development, regulation of gene expression is key to cellular homeostasis. Gene expression regulation by non-coding RNAs involves the prevention of mRNA accumulation or the inhibition of translation of their target gene. In a forward-genetic screen to identify the microRNA involved in the growth and patterning of the Drosophila eye, we identified the highly conserved miR-137. Gain of function of miR-137 results in a reduced-eye phenotype by downregulating retinal determination and differentiation markers, and by upregulating negative regulators of eye development, such as Wingless (Wg) and Homothorax (Hth). Loss of function of miR-137 results in an enlarged-eye phenotype. Using bioinformatics and genetic approaches, we identified the oncogene Myc as the target of miR-137. Gain of function of Myc can rescue the reduced-eye phenotype of miR-137 gain of function, and vice versa. We tested the role of miR-137 in regulating Myc levels in the RasV12;scribRNAi, a tumor model of oncogenic cooperation that results in neoplastic tumors. Gain of function of miR-137 in the RasV12;scribRNAi background significantly reduced tumor phenotype as well as Myc levels in the eye. Our studies highlight miR-137 as a post-transcriptional regulator of Myc and a promising therapeutic target for diseases associated with Myc accumulation.

Keywords: Drosophila; Myc; Cell death; Cell proliferation; Eye development; Retina; Retinal determination; miR-137; miRNA.

Fig. 1.

Screening for miRNA involved in eye development results in the identification of miR-137. (A) The screening strategy to identify miRNA(s) involved in eye development. In the developing eye, individual miRNA(s) are misexpressed using the Gal4/UAS Target system, and their effect on eye phenotype is assayed. (B) The adult eye of an ey-Gal4 control, to which misexpressed miRNA(s) phenotypes were compared. (C) The domain of expression (UAS GFP, green) of ey-GAL4 in the third-instar larval eye-antennal imaginal disc stained for Discs large (Dlg, blue), to mark cellular outlines, and Elav (red), to mark photoreceptor neurons. (D,E) Gain of function of miR-137 in the developing eye using ey-Gal4 driver (ey>miR-137) results in the reduced-eye phenotype, as seen in (D) adults and (E) larval eye imaginal disc. (F,L,P) Quantification of adult eye surface area (μm²) using Fiji/ImageJ software with statistical analysis of normalized adult eye surface area (150 pixels/inch) carried out using an unpaired Student's t-test. The analyzed area is indicated with white dotted lines for denoted genotypes (B,D,H,J) (n=10). (G,M) Adult eye phenotype frequency of indicated genotypes (n=200, three replicates). Analysis was carried out using a two-way ANOVA with Sidak's multiple comparison test. (H) Adult eye of the eyg-Gal4 driver serves as a control. (I) The third-instar larval eye-antennal imaginal disc showing eyg-Gal4 expression (UAS-GFP, green). (J,K) eyg>miR-137 exhibits the reduced-eye phenotype in (J) adult and (K) larval eye imaginal disc. (N,O) Adult eyes of Canton-S control (N) and miR-137^KO homozygous (O) flies, which exhibit an enlarged eye phenotype. (Q) The log2 fold-change values of miR-137 normalized to 2S-rRNA in ey-Gal4, ey>miR-137 and miR-137^KO. Pairwise comparisons were made with an unpaired t-test. miR-137 is expressed in the developing eye. All graphs were plotted using GraphPad Prism 8.3.1. Data are mean±s.e.m. Statistical significance is indicated in each graph: ****P<0.0001; **P<0.01; *P<0.05; ns, non-significant. Orientation of all the imaginal discs is posterior to the left and dorsal upwards. All eye-antennal imaginal discs were captured at 20× magnification and adult eyes at 10× magnification, unless specified otherwise. Scale bars: 100 µm.

Fig. 2.

Gain of function of miR-137 downregulates retinal determination and retinal differentiation genes. (A-F″) Expression of the retinal determination genes (A-C″) Eya (green) and (D-F″) Dac in eye-antennal imaginal disc of (A,A′,D,D′) ey-Gal4, (B,B′,E,E′) ey>miR-137 and (C-C″,F-F″) hsFLP-out clones of miR-137. (A′,B′,C′,D′,E′,F′) Single channel images for (A′,B′,C′) Eya and (D′,E′,F′) Dac. Elav (red) marks the retinal neuron. (B,B′) Eya and (E,E′) Dac expression is downregulated posterior to the MF in the ey>miR-137 background, which exhibits a reduced-eye phenotype. FLP-out clones showed abnormal spacing between neighboring photoreceptors and exhibited downregulation of (C,C′) Eya and (F,F′) Dac. (C″,F″) Elav indicates where retinal neurons are affected. (G-L″) Expression of a retinal differentiation genes (G-I″) Ato (green) and (J-L″) Sca (green) in eye-antennal imaginal disc of (G,G′,J,J′) ey-Gal4 control, (H,H′,K,K′) ey>miR-137 and (I,I′,I″,L,L′,L″) FLP-out clones of miR-137. (G′,H′,I′,J′,K′,L′) Single channel images for (G′,H′,I′) Ato and (J′,K′,L′) Sca. (H,H′) Ato and (K,K′) Sca expression is downregulated in MF in the ey>miR-137 background and a similar observation was seen in (I,I′,L,L′) hsFLP-out clones. All the imaginal discs are oriented posterior to the left and dorsal upwards. All eye-antennal imaginal discs were captured at 20× magnification, unless specified otherwise. Scale bars: 100 µm.

Fig. 3.

Gain of function of miR-137 blocks morphogenetic furrow progression and ectopically induces the negative regulators of eye fate. (A-C′) Eye-antennal imaginal discs stained for the dpp-LacZ reporter (green), which marks the morphogenetic furrow (MF) in the (A,A′) ey-Gal4 (control) and (B,B′) ey>miR-137 background, and in (C,C′) heat shock FLP-out clones of miR-137 in the developing eye imaginal disc. Elav (red) marks the retinal neuron. (A′,B′,C′) Single channel images for dpp-lacZ expression. dpp-lacZ is significantly downregulated in ey>miR-137 background (marked by arrow in C′) when compared to the control and in FLP-out clones. dpp-LacZ is downregulated in the border of the clone and does not extend to the dorsal margin (marked by arrows in B,D,H). (D-I″) Eye-antennal imaginal disc of third-instar larvae stained for (D-F″) Wg and (G-I″) Hth. (D-F″) Wg (green), a negative regulator of MF progression, is ectopically induced in (E,E′) the ey>miR-137 background and in (F,F′) the FLP-out clone of miR-137 (outlined in yellow) in comparison to the control (D,D′) ey-Gal4. (G-I″) Hth (green), a head fate marker and negative regulator of eye fate, is ectopically induced in (H,H′) the ey>miR-137 background and in (I-I″) the FLP-out clone of miR-137 (outlined in yellow) in comparison to the control (G,G′) ey-Gal4. (D′,E′,F′,G′,H′,I′) Single channel images for (D′,E′,F′) Wg and (G′,H′,I′) Hth. All the imaginal discs are oriented posterior to the left and dorsal upwards. All eye-antennal imaginal discs were captured at 20× magnification, unless specified otherwise. Scale bars: 100 µm.

Fig. 4.

Gain of function of miR-137 triggers cell death in the developing eye. (A-B′,D-E′,G-H′) Eye-antennal imaginal discs of third-instar larvae stained for Elav (red), (A-B′) Dcp1 (green, a marker for apoptotic cells), (D-E′) hid5′-WT-GFP (green, a proapoptotic gene, hid reporter) and (G-H′) TUNEL (green, a marker for dying cells nuclei) in (A,A′,D,D′,G,G′) ey-Gal4 and (B,B′,E,E′,H,H′) ey>miR-137 background. Single channel images for (A′,B′) Dcp-1, (D′,E′) hid5′-WT-GFP and (G′,H′) TUNEL alone. (C,F,I) Statistical analysis showed that ectopic expression of miR-137 (ey>miR-137) exhibits a significant increase in (C) Dcp-1, (F) hid5′-WT-GFP and (I) TUNEL-positive nuclei as compared to the ey-Gal4 controls as shown in the graphs. The signal intensity was calculated in three regions of interest per disc within a sample of n=5 for each genotype. (J-M) Ectopic expression of baculovirus P35, which blocks cell death, in (J,K) ey-Gal4 (ey>P35, control) and (L,M) ey>miR-137 (ey>miR-137+P35) background. P35 rescues the ey>miR-137 reduced-eye phenotype, as seen in (L) eye imaginal disc and (M) adult eye. (N) Statistical analysis showed a decrease in Dcp1-positive cells. Dcp1-positive cells were calculated in a sample of n=5 for each genotype. (C,F,I,N) The statistical analysis was carried out using an unpaired Student's t-test. Data are mean±s.e.m. Statistical significance is indicated in each graph: ***P<0.001; **P<0.01; *P<0.05. All graphs were plotted using GraphPad Prism 8.3.1. All the imaginal discs are oriented posterior to the left and dorsal upwards. All eye-antennal imaginal discs were captured at 20× magnification and adult eyes at 10× magnification, unless specified otherwise. Scale bars: 100 µm.

Fig. 5.

The gain-of-function phenotype of miR-137 does not exhibit domain constraint. (A-D) Gain of function of miR-137 in the dorsal half of the adult eye (A,C, marked by white dotted lines) using the DE-Gal4 driver (domain marked by GFP reporter) marked by expression of mini-white reporter (red, white dotted line outlines the domain). (C,D) The gain of function of miR-137 in the background of DE-Gal4 (DE>miR-137) reduces the dorsal domain of the eye field in (C) the adult eye and (D) the eye-imaginal disc. (B,D) Eye-antennal imaginal disc of third-instar larvae stained for Elav (red), Dlg (blue) and GFP (green) reporter. (E) Dorsal half expression of DE>miR-137 (n=10) normalized to the control DE-Gal4 (n=10). (E) The bar graph shows the length of the dorsal half of eye in DE>miR-137 (n=10) normalized to the control, DE-Gal4 (n10). (F,H) Adult wings of (F) nub-Gal4 and (H) nub>miR-137. The wings of nub>miR-137 (H) are reduced when compared to the control (F) nub-Gal4. (G,I) Wing imaginal discs of (G) nub-Gal4 and (I) nub>miR-137 stained for Engrailed (En) (red, a posterior compartment fate marker) and GFP (green, reporter marks nub-Gal4 expression domain). (J) A nub-Gal4 expression domain in the wing disc (n=5) of nub>miR-137 normalized to control nub-Gal4 (n=5). The nub-Gal4 domain is significantly reduced in the nub>miR-137 background. Data are mean±s.e.m. Statistical significance is indicated in each graph: ****P<0.0001; **P<0.01. All graphs were plotted using GraphPad Prism 8.3.1. All eye-antennal imaginal discs were captured at 20× magnification and adult eyes at 10× magnification, unless specified otherwise. Scale bars: 100 µm.

Fig. 6.

Screening for miR-137 gene target(s) identified Myc. (A) The miR-137 binding site on its target(s) predicted by the bioinformatic tools TargetScan and DIANA. (B-H) Loss-of-function phenotypes in the adult eye of the putative targets identified using bioinformatics approaches. Adult eye phenotypes of (B) dcr2; ey-Gal4 (control), (C) dcr; ey>Myc^RNAi, (D) dcr; ey>Inx2^RNAi, (E) dcr; ey>bi^RNAi, (F) dcr; ey>msi^RNAi, (G) dcr; ey>mei-P26^RNAi and (H) dcr; ey>scrt^RNAi. In these experiments, dcr2 is co-expressed in the ey expression domain for a stronger RNA interference effect. (C) dcr; ey>Myc^RNAi and (D) dcr; ey>Inx2^RNAi exhibit a strong reduction in eye size when compared to the control (B). (I) The normalized eye-surface area of miR-137 targets (n=10 per genotype) compared to the control dcr; ey-Gal4. The statistical analysis was performed using a one-way ANOVA with Dunnett's multiple comparison test. (J) Relative transcriptional (expression) levels of Myc (from triplicate sets) obtained using quantitative PCR (qPCR) in ey-Gal4 and ey>miR-137 backgrounds. Statistical analysis was carried out using an unpaired Student's t-test for independent samples. (K) Eye-antennal imaginal disc of third-instar larvae stained for Elav (red), Dlg (blue) a membrane-specific marker and GFP (green) reporter expressing in ey-Gal4 domain. (L,M) The Tubulin–GFP–miR-137 sensor shows ubiquitous expression of GFP under the control of tubulin promoter in the eye discs (L) and the ey>miR-137+miR-137-sensor (ey>miR-137+sensor) shows reduced GFP fluorescence in ey-producing cells the eye discs (M). (N) Quantification of GFP shows a significant reduction in GFP intensity in ey>miR-137+miR-137-sensor eye discs when compared to the control. The statistical analysis was carried out using an unpaired Student's t-test. Data are mean±s.e.m. Statistical significance is indicated in each graph: ****P<0.0001; ***P<0.001; **P<0.01. All graphs were plotted using GraphPad Prism 8.3.1. All eye-antennal imaginal discs were captured at 20× magnification and adult eyes at 10× magnification unless specified otherwise. Scale bars: 100 µm.

Fig. 7.

miR-137 targets Myc during eye development. (A-B′) Gain of function of Myc (ey>Myc) (A,A′) and miR-137 (ey>miR-137) (B,B′) in (A,B) adult eye and (A′,B′) eye imaginal disc. (C′,D′) Eye-antennal imaginal discs stained for Elav, a pan-neuronal marker (red), and Dlg, a membrane-specific marker (green). (C-D′) Modulation of Myc levels in ey>miR-137 background using (C,C′) gain of function of Myc (ey>miR-137+Myc) and (D,D′) loss of function of Myc (ey>miR-137+Myc^RNAi), as seen in (C,D) adult eye and (C′,D′) eye imaginal disc. (E) The normalized eye-surface area of (A) ey>Myc, (B) ey>miR-137, (C) ey>miR-137+Myc and (D) ey>miR-137+Myc^RNAi showed a significant increase in eye size in ey>miR-137 +Myc compared to controls (ey>miR-137, n=10 per genotype). (F) Statistical analysis of adult eye phenotype frequency between (B) ey>miR-137 and (C) ey>miR-137+ Myc. There was significant rescue seen in ey>miR-137+ Myc when compared to ey>miR-137. (G-H′) Loss of function of miR-137 in (G,G′) ey-Gal4 (miR-137^KO, ey-Gal4) and (H,H′) ey>Myc (miR-137^KO, ey>Myc) background produces a wild-type phenotype in (G,H) adult eye and (G′,H′) eye imaginal disc. (I-J′) Loss of function of Myc in (I,I′) miR-137^KO (ey>miR-137^KO+Myc^RNAi) and (J,J′) ey-Gal4 (ey>Myc^RNAi) background, as seen in (I,J) adult eye and (I′,J′) eye imaginal disc, produces a reduced-eye phenotype. (K) Statistical analysis of adult eye phenotype frequency in (J) ey>Myc^RNAi and (D) ey>miR-137+Myc^RNAi. The reduced-eye phenotype is classified into: (1) 1° reduced eye, representing reduced eye in one or both of the eyes (blue); (2) 2° reduced eye, representing highly reduced to no eye in one of the eyes (purple); and (3) 3° reduced eye, representing highly reduced to no eye in both the eyes (pink). (L) The normalized eye-surface area of ey-Gal4 (Fig. 1B,C), miR-137^KO/+, ey-Gal4 (G,G′), miR-137^KO/+, ey>Myc (H,H′), miR-137^KO/+, ey>Myc^RNAi (I,I′) and dcr; ey>Myc^RNAi (J,J′). (M-O) Eye-antennal imaginal disc of (M) Canton-S, (N) miR-137^KO and (O) miR-137^CR stained for Elav (red) and Myc (green). (P) Mean integrated density of Myc levels from Canton-S (M), miR-137^KO (N) and miR-137^CR (O). The signal intensity of Myc was calculated in five regions of interest per disc within a sample of n=5 for each genotype. Myc intensity was significantly increased in miR-137^KO and miR-137^CR. (Q-Q″) Eye-antennal imaginal disc of (Q) random flp out clones of miR-137sp (hsFLP; Act>miR-137sp+ GFP) stained for Elav (red) and Myc (blue). (Q′) Single channel image of GFP marking the clones of miR-137sp. (Q′′) Single channel image of Myc. Myc intensity was dramatically increased with flp out clones of miR-137sp. (R) Mean integrated density of Myc levels between control and flp out clones of miR-137sp. The signal intensity of Myc was calculated at the ROI (50×50) of flp out clones (n=5) and non-flp out (n=5) of the same disc. Myc intensity was significantly increased in hsFLP-out clones of miR-137sp. One-way ANOVA with Tukey's multiple comparison test was used for comparing more than two groups, and an unpaired Student's t-test was used to compare two groups. For adult frequency analysis, three replicates of 200 flies (200×3=600) were used to calculate the frequency of each genotype. Data are mean±s.e.m. Statistical significance is indicated in each graph: ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05. All eye-antennal imaginal discs were captured at 20× magnification and adult eyes at 10× magnification unless specified otherwise. Scale bars: 100 µm.

Fig. 8.

miR-137 exhibits growth regulation function in the developing eye. (A,D) Eye imaginal disc (A) and adult eye (D) of ey-Gal4 control. (B,E) ey>Ras^V12+ scrib^RNAi serves as a model for oncogenic cooperation resulting in neoplastic tumors and overgrowth. (C,F) Gain of function of miR-137: ey>miR-137+ Ras^V12+ scrib^RNAi significantly rescues the overgrowth phenotype of (B,E) ey> Ras^V12+ scrib^RNAi both at the eye imaginal disc level and in the adult eye. (A-C) All eye-imaginal discs are stained for Myc (green) and Elav (red). (G) Statistical analyses of normalized adult eye surface area (pixels/inch) were carried out using one-way ANOVA with Tukey's multiple comparison test for (A) ey-Gal4, (C) ey> Ras^V12+ scrib^RNAi and (E) ey> miR-137+Ras^V12+ scrib^RNAi (n=10 per genotype). The eye surface is reduced by gain of function of miR-137 in the ey> Ras^V12+ scrib^RNAi background. (H) Average intensity of Myc levels within the three backgrounds: (1) ey-Gal4, (2) ey> Ras^V12+ scrib^RNAi and (3) ey>miR-137+ Ras^V12+ scrib^RNAi. The signal intensity of Myc was calculated in three region of interest per disc within a sample of n=5 for each genotype. Statistical analysis was performed using an unpaired Student's t-test for independent samples. Myc intensity was significantly reduced in the ey>miR-137+ Ras^V12+ scrib^RNAi background. (I) Relative transcriptional (expression) levels of Myc using the quantitative real-time PCR (qRT-PCR) in ey-Gal4, ey>Ras^V12+scrib^RNAi and ey> Ras^V12+scrib^RNAi+miR-137 backgrounds. Statistical analysis was carried out in triplicate, using an unpaired Student's t-test for independent samples. Myc transcript levels were significantly increased in ey>Ras^V12+scrib^RNAi compared to ey-Gal4 and decreased in ey> Ras^V12+scrib^RNAi+miR-137 background compared to ey-Gal4. (J,K) Eye-antennal imaginal disc of (J) ey-Gal4 and (K) ey>miR-137 stained for phospho-histone 3 (pH3) (green, a marker for cell division) and Elav (red). (L) Statistical analysis of pH3 counts in the retinal field marked by Elav in comparable region of interest in ey-Gal4 (n=5) and ey>miR-137 (n=5) shows a significant difference in proliferating cells between control and ey>miR-137. The statistical analysis was carried out using an unpaired Student's t-test. Quantification of pH3 and eye-surface area was calculated using Fiji/ImageJ software to assay the differences. Data are mean±s.e.m. Statistical significance is indicated in each graph: ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05. All graphs were plotted using GraphPad Prism 8.3.1. All the imaginal discs are oriented posterior to the left and dorsal upwards. All eye-antennal imaginal discs were captured at 20× magnification and adult eyes at 10× magnification unless specified otherwise. Scale bars: 100 µm.

Fig. 9.

A model to demonstrate the role of miR-137 in growth regulation by targeting Myc during eye development. (A) When miRNAs are expressed at normal levels, a normal eye is produced. (B) Gain of function of miR-137 in the ey-Gal4 domain (ey>miR-137) results in a reduced-eye phenotype. Our results indicate that miR-137 targets Myc to reduce the size of eye. (C) miR-137 targets Myc to regulate growth during development. Gain of function of miR-137 can rescue overgrowth phenotypes observed in the tumor model of oncogenic cooperation in Drosophila. (D) Our results show that miR-137 targets Myc to regulate cell proliferation and cell death in the developing eye and in other tissues.