miR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings.

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Dev Biol. Author manuscript; available in PMC 2017 April 01. Published in final edited form as: Dev Biol. 2016 April 1; 412(1): 83–98. doi:10.1016/j.ydbio.2016.01.041.

miR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings Kelsey Bolin1, Nicholas Rachmaninoff1, Kea Moncada2, Katharine Pula2, Jennifer Kennell2, and Laura Buttitta1,* 1University

of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor,

MI 48109

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2Vassar

College, Department of Biology, Poughkeepsie, NY 12604

Summary

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The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate.

Keywords miRNA; actin cytoskeleton; Drosophila; Imaginal wing disc

Introduction Author Manuscript

The miR-200 family plays a critical tumor suppressive role in several types of cancers (reviewed in Brabletz and Brabletz, 2010; Hill et al., 2013). In mammals the miR-200 family is made up of two genomic loci encoding microRNAs including miR-200a, miR-200b,

*

Corresponding author: [email protected]. Author contributions: K.B. and L.B. conceived of the project. K.B., N.R., K.M., K.P., J.K. and L.B. performed the experiments. L.B. and J.K wrote the manuscript with assistance from N.R. and K.B. The authors have no conflicts of interest. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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miR-200c, miR-141, and miR-429. They are most strongly expressed in epithelial tissues, have similar seed regions with only one nucleotide difference generating two classes of target sites termed miR200a sites and miR200b sites, and can regulate overlapping targets involved in multiple signaling pathways (reviewed in Bracken et al., 2015). A key role for the miR-200 family in promoting the epithelial state has been shown, and high levels of miR-200 expression in mesenchymal cells can even promote a mesenchymal to epithelial transition (Gregory et al., 2008a). In this role, the Zeb family of transcription factors are important targets, as they transcriptionally repress E-cadherin expression (Comijn et al., 2001; Eger et al., 2005). There is also a miR-200/Zeb1/2 feedback loop, where Zeb1/2 represses miR-200 expression (Bracken et al., 2008). This creates a simple model for robustly establishing and maintaining the epithelial or mesenchymal state depending upon the levels of miR-200 vs. Zeb1/2. However additional studies have also suggested Zeb1/2independent roles for miR-200 in promoting the epithelial state at least in part through targets that regulate the actin cytoskeleton such as moesin, the Formin Homology Domain Containing 1 protein (FHOD1) and WAVE3 a member of the WASP (Wiskott-Aldrich syndrome protein)/WAVE actin cytoskeleton remodeling family of proteins (Jurmeister et al., 2012; Li et al., 2014; Sossey-Alaoui et al., 2009). More recent work using an approach to identify miR-200 targets at the transcriptome-wide level, identified hundreds of potential miR-200a and miR-200b targets and revealed a predominant effect of miR-200 targets in widespread control of actin cytoskeletal regulators (Bracken et al., 2014). The current model for Zeb1/2-independent miR-200 functions in inhibition of the mesenchymal state posits that the down-regulation of miR-200 targets involved in modulating the actin cytoskeleton limits the formation of invadopodia and cell migration (Bracken et al., 2014). However this has largely been investigated in cell culture, and the effects of modulating miR-200 on the actin cytoskeleton in epithelial tissues in vivo needs to be further explored.

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Importantly, the miR-200 family can also alter cell survival and metabolism (Belgardt et al., 2015; Guo et al., 2015; Howe et al., 2011; Jin et al., 2012; Jing et al., 2015), although it also remains largely unclear exactly how these roles intersect with the function of miR-200 in establishing and maintaining the epithelial state. Several studies have shown that reduced miR-200 can lead to chemotherapy and radiotherapy resistance in cancer, which can be rescued by re-introducing miR-200 expression (Adam et al., 2009; Cortez et al., 2014; Knezevic et al., 2015; Siebzehnrubl et al., 2013). A predominant model suggests that the sensitivity of cancer cells to cytotoxic treatments is somehow coupled with the transition to a mesenchymal state (Fischer et al., 2015), but whether miR-200 can reduce cancer cell survival independent of its roles in maintaining the epithelial state remains unknown.

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The sole Drosophila miR-200 family homolog, miR-8 plays important roles in Insulin, Notch, and Wingless signaling as well as signaling via the fly steroid hormone ecdysone (Hyun et al., 2009; Jin et al., 2012; Kennell et al., 2008; Vallejo et al., 2011). More recently, miR-8 has been shown to also promote the epithelial state in the Drosophila intestine, where the transcription factors Escargot and the fly homolog of Zeb1, Zfh1 are critical targets (Antonello et al., 2015). Yet surprisingly, despite all of these conserved functions homozygous null miR-8 mutants are often properly patterned and viable, leaving unclear the full physiological significance of miR-8 function in these pathways during development (Hyun et al., 2009; Karres et al., 2007; Kennell et al., 2012). It also remains unclear whether Dev Biol. Author manuscript; available in PMC 2017 April 01.

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there are Zfh1-independent roles for miR-8 in promoting the epithelial state in Drosophila. One target for miR-8 previously identified is the actin regulator Enabled (Ena), which is responsible for miR-8 phenotypes observed at the neuromuscular junction in Drosophila (Loya et al., 2009; Loya et al., 2014). The targeting of Ena provides a hint that miR-8 may also play a larger role in regulating the actin cytoskeleton in Drosophila, in a manner similar to that described for miR-200.

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We previously identified miR-8 as a negative regulator of Wingless signaling in the Drosophila eye and wing (Kennell et al., 2008). We therefore examined whether mosaic analysis of miR-8 function in the wing epithelial tissue might reveal novel roles and targets for miR-8. Here we demonstrate that cell survival and the F-actin cytoskeleton is compromised by high levels of miR-8, in part through several novel direct targets of miR-8 that regulate actin dynamics via the SCAR/WAVE complex (Kunda et al., 2003) and F-actin meshwork formation (Isaji et al., 2011; Mavrakis et al., 2014). Conversely, we show that loss of miR-8 increases cell survival in the wing and resistance to DNA damage-induced apoptosis in a manner apparently unlinked to a mesenchymal transition, as epithelial organization and wing patterning remains intact in miR-8 mutants.

Results miR-8 impacts cell survival in the wing


miR-8 and its mammalian homologs have been suggested to promote and maintain the epithelial state (Antonello et al., 2015; Brabletz and Brabletz, 2010; Mongroo and Rustgi, 2010). The developing Drosophila wing has been used extensively as a model to decipher mechanisms controlling epithelial organization and morphogenesis during development. We therefore examined the role of miR-8 in the wing epithelium in more detail. miR-8 was previously suggested to be expressed in the wing pouch and notum, based upon the expression of a Gal4 enhancer trap line (NP5247) (Karres et al., 2007; Vallejo et al., 2011). To ensure the enhancer trap reflects endogenous miR-8 expression and activity in wings, we examined the expression of a miR-8 EGFP sensor transgene in the wing (Kennell et al., 2012). The sensor transgene contains EGFP with two binding sites for miR-8 in its 3′UTR, driven by the α-tubulin promoter. Targeting by endogenous miR-8 leads to decreased expression of the EGFP sensor. Consistent with the expression pattern reported for the miR-8-Gal4 enhancer trap, the miR-8 sensor revealed higher endogenous miR-8 activity in the wing pouch and notum than in the hinge (Fig. 1A). The targeting of the sensor is completely lost in miR-8 null mutants (Fig. 1B, miR-8jk22/miR-8 jk22), confirming the specificity of the sensor for endogenous miR-8. Based upon these results, we focused on examining functions of miR-8 in the developing wing pouch. We previously demonstrated that miR-8 inhibits Wg signaling via multiple direct targets (Kennell et al., 2008). Wg signaling acts as an important cellular survival factor in the Drosophila wing (Giraldez and Cohen, 2003; Johnston and Sanders, 2003). Consistent with this, manipulating levels of miR-8 expression in the wing alters cell survival. We generated clones of cells overexpressing miR-8 via Gal4/UAS transgene induction (Duffy, 2002), in otherwise normal wing tissue. We found that miR-8 expressing clones were small, exhibited apoptotic morphologies and were rapidly eliminated from the wing epithelium by 72h after Dev Biol. Author manuscript; available in PMC 2017 April 01.

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induction. To generate larger clones for more detailed analysis, we turned to the temperature-sensitive Gal80 repressor (Gal80TS) system (McGuire et al., 2004). With Gal80TS, we can induce clone formation early, allow clones to grow in the absence of transgene induction at low temperature where the Gal80 repressor is intact (18°C), and then switch to a non-permissive Gal80 temperature (29°C) to induce the Gal4/UAS driven transgene for a short time before analysis. Using this system, we induced clones to express miR-8 in the wing epithelium for 24–72h. We stained clones expressing miR-8 with cleavedCaspase 3 (c-Casp3) antibody to detect apoptotic cells (Fan and Bergmann, 2010). Cells overexpressing miR-8 had high levels of c-Casp3 staining (Fig. 1C), indicating that miR-8 plays a role in cell survival. Similar results were also obtained with staining for a second Drosophila Caspase 1, Dcp-1 (Song et al., 1997), with Dcp-1 positivity increasing dramatically within 24h of miR-8 expression in the wing (Fig. 1D, Supplement to Fig. 1). Finally, TUNEL labeling confirmed DNA fragmentation consistent with late apoptosis in miR-8 expressing cells (Fig. 1E).

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Since high miR-8 reduced cell survival, we next examined whether the loss of miR-8 could improve cell survival. Under normal rearing conditions, the amount of apoptosis in the larval wing pouch and hinge at any given point in time is very low (gal80TS hs-

flp; act>CD2>gal4/UAS-P35; UAS-miR-81F34/+hs-flp; act>CD2>gal4/+; UAS-miR-81F34/+ tie-dye assay: hs-flp; act>CD2>lacZ, ubi>CD2>GFPNLS/+; act>CD2>gal4,UAS-RFP/UASmiR-81F34 Control: hs-flp; act>CD2>lacZ, ubi>CD2>GFPNLS/+; act>CD2>gal4,UAS-

RFP/+

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miR-8JK22 mitotic recombination clones: hs-flp; FRT 42D Ubi-GFP/FRT 42D miR-8jk22 Parental FRT42D control: hs-flp; FRT 42D Ubi-GFP/FRT 42D MARCM miR-8 mutant for FACS: hs-flp, UAS-GFPNLS, tub-Gal4; FRT42D tub-

Gal80/FRT 42D miR-8jk22 The following RNAi lines were crossed to w; tub>CD2>gal4, UAS-GFP; tub>gal80TS or w; en-gal4, UAS-GFP; tub-gal80TS: Sra-1RNAi (Trip RNAi BL#38294), JarRNAi (Trip RNAi BL#28064), EnaRNAi(Trip RNAi BL#31582), SqhRNAi(Trip RNAi BL#31542) Sra-1RNAi + JarRNAi experiments: hsflp/+; tub>CD2>gal4, UAS-GFP/+; UAS-jarRNAi/

Sra-1RNAi Sra-1RNAi + EnaRNAi experiments: hsflp/+; tub>CD2>gal4, UAS-GFP/+; UAS-enaRNAi/

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Sra-1RNAi miR-8 sensor: +/+; tubEGFP-2xmiR-8/+ and miR-8JK22/miR-8JK22; tubEGFP-2xmiR-8/+ miR-8 sponge experiment: w/+; miR-8-sponge/ap-Gal4,UAS-GFP; miR-8 sponge/tub-

Gal80TS, UAS-DIAP BskDN clone rescue: w/hs-flp; tub>CD2>gal4, UAS-GFP/+; UAS-BskDN/UAS-RFP (BL#9311) P35 clone rescue: hs-flp/UAS-P35;+; act>CD2>gal4/UAS-miR-81F34

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Experiments with Diap: w;apterous-Gal4,UAS-GFP/+; miR-81F34/UAS-DIAP, tub-gal80TS from (Buttitta et al., 2007), w;ptc-Gal4/UAS-miR-81F20; tub-gal80TS/Diap-LacZ Diap-LacZ on III (BL# 12093). All flystocks listed were generated with publicly available lines from the Bloomington Stock center, or are described in (Buttitta et al., 2007; Kennell et al., 2012; Kennell et al., 2008) Immunofluorescence

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Wing discs from wandering larvae at the third larval instar were fixed with 4% paraformaldehyde/1X PBS and processed as described (Buttitta et al., 2007), except for staining with anti-Sparc and TUNEL labeling, which required modifications described below. EdU incorporation was performed for 15 min in Ringer’s solution and detected using Click-iT EdU Alexa Fluor 555 Imaging Kit from Life Technologies. For UV treatment L3 larvae were placed in uncovered plastic 6cm dishes and exposed to 240mJ of UV using a Stratalinker 2400 with a UVC bulb as described (Kang and Bashirullah, 2014). Animals were allowed to recover for the indicated time and fixed and stained for DCP-1 immunofluorescence. The antibodies and reagents used are as follows Anti-Casp3: rabbit 1:100 (Cell Signaling)


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TUNEL: see protocol Anti-Sparc: see protocol; mouse-anti Sparc 4 uL in 40 uL PAT + 0.3% Triton-X (gift of Eduardo Moreno, Bern, Switzerland) or rabbit anti-Sparc 1:100 (gift of M. Ringuette, Toronto, Canada). Anti-pJNK: 1:100 rabbit (Promega) F- actin staining: 1:100 in PBS rhodamine-labelled phalloidin (Invitrogen) Anti-LacZ: 1:5,000 Rabbit (Cappel) or 1:500 mouse (Promega) Anti-Sra-1: 1:1000 rabbit (provided by Alexis Gautreau, Laboratoire d’Enzymologie et Biochimie Structurales CNRS, FRANCE) Anti-Jaguar: 1:20 mouse (3C7 provided by Kathryn Miller, St. Louis, MO, USA)

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Anti-Enabled: 1:50 mouse (Developmental Studies Hybridoma Bank, DSHB, USA) Anti-Sqh: 1:300 mouse (provided by Robert Edwin Ward IV, University of Kansas, USA) Anti-Dcp1: 1:100 rabbit (Cell signaling) Anti-Pnut 1:100 mouse (DSHB, USA) Anti-Abp1: 1:250 Rabbit (provided by Michael Kessels Jena University Hospital, Germany) Anti-Diap 1:100 goat (Santa Cruz Biotechnology) Anti-Arm 1:100 mouse (DSHB, USA) Anti-DE-Cad 1:20 rat (DSHB, USA)

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Anti-βPS1 (Integrin) 1:100 mouse (DSHB, USA) Anti-MMP1 1:100 mouse (DSHB, USA) Anti-PH3 1:2,000 Rabbit (Upstate Biotechnology) Clone size measurements

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Crosses to compare miR-8 expressing clone sizes with and without P35 were performed with a UAS-P35 on the X, such that only females expressed P35 while males served as a non-P35 control from the exact same vial. Similarly, crosses to compare miR-8 expressing clone sizes with and without UAS-BskDN were performed with males carrying UAS-BskDN on III over a UAS-RFP transgene, such that only RFP negative animals expressed BskDN while RFP positive animals served as non- BskDN control from the exact same vials. This was done to ensure that the level of heat-shock, efficiency of clone formation and rearing conditions were identical between the two genotypes compared in Fig. 3. Crosses to collect animals to make miR-8 clones and control FRT42D clones were performed in parallel. Animals were roughly synchronized by collecting eggs for 12 h on grape-agar plates. Hatched larvae were transferred to vials uncrowded vials (50/vial) containing cornmeal-agar food and reared at 25°C for 24h. Clones were induced by 18 min. heat-shock in a 37°C water bath and animals were reared for 75h at 25°C prior to dissection and fixation at the


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wandering L3 stage. For clone size measurements, images were analyzed using Nikon NIS Elements D software. The “polygon area” tool was used to outline the wing pouch as defined by the location of the hinge wrinkles. Next, individual clones were outlined with the “polygon area tool” labeling each one for area measurement export to an Excel spreadsheet. The area of all clones of the same genotype: all GFP+ and all GFP- were summed. This was used to calculate the percentage of the total wing pouch area taken up by GFP+ and GFPclones respectively. The average percentage of the wing pouch taken up by each clone type was then averaged and the standard deviation of the percentages, and the p-value were calculated using an unpaired, two-tailed t-test. In vitro 3′UTR reporter assays

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All 3′UTR reporter genes were generated by cloning a stop codon, followed by the entire 3′UTR of the predicted miR-8 target, downstream of lacZ in pAclacZ (Invitrogen). Kc167 Drosophila cells were transiently transfected with the indicated 3′UTR reporter gene along with pAc control or pAc-miR-8, and pAc-luciferase, to control for transfection efficiency. βgalactosidase and luciferase activities were measured as described previously (Blauwkamp et al., 2008). TUNEL labeling detailed protocol

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For Rhodamine-TUNEL labeling the Apoptag Detection Kit was used (Millipore) with the following modifications. Tissues were fixed with 4% paraformaldehyde (PFA)/PBS. Staining for clonal markers (ie. GFP) was performed via standard procedures before TUNEL labeling. Before TUNEL reaction, tissues were fixed in 100% methanol for 6 min. followed by washes with PBS + 0.1% Triton-X. Tissues were then exposed to Equilibration Buffer for 2 min. prior to addition of the TdT enzyme. Tissues were incubated with TdT enzyme for 1hr at 37C and stopped by addition of diluted Stop/Wash Buffer Digoxygenin (DIG)-labeled dT was detected with anti-DIG-Rhodamine antibody and samples were mounted on slides using standard procedures. Sparc labeling detailed protocol For anti-Sparc staining, tissues were fixed for 1 hour in 4% PFA/PBS and blocked in PBS + 1% BSA + 0.3% Triton-X overnight. Clone 30A/B4 anti-Sparc antibody (provided by Dr. E Moreno) or rabbit anti-dSparc (provided by Dr. M. Ringuette) was used at 4 uL in 40 uL of blocking solution or 1:100 respectively and incubated with tissues for 1–2 days at room temperature. Tissues were washed and secondary antibody labeling was performed overnight as previously described.

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Microscopy All images were obtained using a Zeiss LSM 510 confocal or a Leica SP5 confocal except for Fig. 6D and Supplement to Fig. 5G, which was obtained using a Leica DMI6000 epifluorescence system with de-convolution (ImageQuant). All images were cropped, rotated and processed using Adobe Photoshop. For brightness/contrast the Auto Contrast function was used. All brightness/contrast adjustments were applied equally on the entire image. All x/z optical sections were obtained on a Leica SP5 confocal. Images for x/z sections are


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maximum projections of 1–3 y-sections of 0.5 micron intervals or less. All image quantifications were performed using Image J. Flow cytometry Flow cytometry on larval wings was performed as described (Flegel et al., 2013). Western blots

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Wandering third instar wild type (w; +/+) and miR-8 mutant (w; miR-8JK22/miR-8JK22 or w; miR-8JK22/miR-8Δ1 (Karres et al., 2007)) larvae were lysed in RIPA buffer or for Sra-1 detection directly in Laemmli buffer. The primary antibodies used were mouse anti-Sqh (1:5000), mouse anti-Jar (1:20), rabbit anti-Abp1 (1:1000), rabbit anti-Sra-1 (1:1,000), mouse anti-Pnut (1:500, 4C9H4 concentrate from DSHB) and mouse anti-tubulin (1:500 for E7 or 1:1,000 for 12A10, DSHB). HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were visualized using ECL kits and digitally imaged to avoid band saturation. Bands were analyzed using NIH ImageJ. Due to band interference, the tubulin control for the blots of Abp1 and Sqh are from a separate blot which was loaded identically and processed in parallel.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

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We thank Dr. A. Miller, Dr. K. Cadigan, the Buttitta and Kennell lab members for helpful discussions. We thank Dr. E. Moreno, Dr. M. Ringuette, Dr. A. Gautreau, Dr. M. Kesells, Dr. R. Ward, IV and Dr. K. Miller for kindly providing antibodies. Additional stocks and antibodies were obtained from the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center and the Developmental Studies Hybridoma Bank (DSHB). Work in the Buttitta Lab was supported by NIH R00 GM086517 and startup funding from the University of Michigan, J.K. was supported by NIH F32GM074465 and R15 GM101598.

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Highlights 1.

miR-8 impacts cell survival and targets cytoskeletal regulators in Drosophila

2.

miR-8 alters apoptosis resistance in Drosophila independent of an epithelial-tomesenchymal transition

3.

Persistence of cells with supra-physiological miR-8 disrupts epithelial organization.

Author Manuscript Author Manuscript Author Manuscript Dev Biol. Author manuscript; available in PMC 2017 April 01.

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Fig. 1. miR-8 is expressed in the wing pouch where it impacts cell survival

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(A). In a wild type background, miR-8-sensor expression is decreased in the wing pouch and notum. (B). Expression of the miR-8-sensor is rescued in miR-8 mutant wings (miR-8JK22/ miR-8JK22), suggesting that functional miR-8 is endogenously expressed in the wing pouch and notum. (C). GFP-labeled clones overexpressing miR-8 were generated using the tub>CD2>Gal4/UAS, Gal80TS system with heat-shock induced recombination from 38–80 hr of development and transgene induction at 28°C for 24–72hr prior to wing disc dissection at wandering third larval instar (L3). miR-8 overexpressing clones are positive for (C) cleaved-Caspase 3 (c-Casp3), (D) Dcp-1 (the full timecourse is shown in Supp. Fig. 1) and (E) TUNEL. (F) Negatively marked miR-8−/− clones with GFP-labeled twinspots were generated in larval wings using heat-shock induced FLP/FRT42D mitotic recombination and measured 75h post clone induction. miR-8 mutant tissue is overrepresented within the wing pouch compared to wild-type sibling clones (shown in additional data in Supp. Fig. 1) and FRT 42D control clones lacking the miR-8 allele. N indicates the total number of twinspots measured. (G) The DNA content and (H) cell size (via Forward scatter FSC) of miR-8 mutant clones is similar to wild-type GFP positive sibling clones. (I) Tissues were induced to express a miR-8 sponge using en-Gal4/UAS, Gal80TS with incubation at 28°C for 70hr


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prior to wing disc dissection. Wandering L3 larvae were exposed to 240mJ of UV and assayed 5–12hr later for cell death by Dcp-1 (staining shown in Supp. Fig. 1).


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Author Manuscript Author Manuscript Author Manuscript Fig. 2. miR-8 reduces Diap1 levels post-transcriptionally

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(A–B) miR-8 + GFP-expressing clones were generated using tub>CD2>Gal4/UAS, Gal80TS with heat-shock induced recombination at 38–48hr of development and transgene induction at 28°C for 24h prior to wing dissection at L3. miR-8 reduces Diap levels within 24hours (C–D) miR-8 expression driven by patched (ptc)-Gal4/UAS, Gal80TS for 24h reduces Diap. (E–F) miR-8 does not affect Diap-LacZ expression when overexpressed using the ptc-Gal4 driver. (G–H) Restoring Diap expression does not prevent apoptosis of high miR-8 expressing cells. Expression of miR-8 with UAS-Diap was driven in the dorsal wing using apterous (ap)-Gal4/UAS, Gal80TS for 24h. Apoptosis was assayed by Dcp-1 positivity and Diap expression confirmed using anti-Diap. Dev Biol. Author manuscript; available in PMC 2017 April 01.

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Fig. 3. Cells with high miR-8 activate JNK signaling and are basally extruded

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(A.B) GFP-expressing clones or (C,D) miR-8 + GFP-expressing clones were generated using tub>CD2>Gal4/UAS, Gal80TS with heat-shock induced recombination at 38–48hr of development and transgene induction at 28°C for 48h prior to wing dissection at L3. Cells expressing GFP only in the wing pouch remain in the epithelium of disc proper. (B and D show representative x/z optical sections, all optical x/z sections are oriented with the peripodial epithelium marking the apical side of the disc at top). (C, D) Cells expressing high levels of miR-8 in the pouch are located basally and often have pyknotic nuclei. The arrow in D indicates basally located pyknotic nuclei. (E) miR-8 overexpression also causes increased phospho- JNK (pJNK) within miR-8 clones and in non miR-8-expressing cells bordering the clones (indicated by arrows, arrowheads indicate small miR-8 clones not outlined). Note that the large, round pJNK positive cells visible are located in the peripodial epithelia, which has been previously described (Tamori et al., 2010). This wing is from an L3 larva just prior to wandering to minimize the peripodial signal. (F) miR-8 overexpression using tub>CD2>Gal4 leads to elimination of most cells from the wing pouch by 72h and the few remaining cells are basally located (F′ x/z optical section of F). (G) Co-expression of


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miR-8 with a dominant negative form of Basket (BskDN) partially increases clone recovery (by 3.9-fold increase in clone area) at 72h. (G′) However miR-8+BskDN expressing cells are still basally located. (H– J) Co-expression of miR-8 with the apoptosis inhibitor P35 fully prevents clone elimination from the wing pouch (clone area is increased more than 10-fold comparing clones induced in parallel in H and I). (I, J) miR-8+P35 clones exhibit a rounded morphology, enlarged nuclei and are most often basally located.


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Author Manuscript Author Manuscript Author Manuscript Fig. 4. Apoptotic signaling in miR-8 expressing cells leads to compensatory proliferation

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(A–C) Heat-shock induced clones expressing RFP alone (A) or RFP with miR-8 (B) were generated using the “Tie-Dye” method 72–80h hours before dissection of wandering L3 wings. We observed loss of the miR-8 expressing clones (red) and increased size of nearby GFP clones. We noted that “distant” LacZ clones (blue) were produced at a much lower rate than the other clones especially in the miR-8 expressing background, as previously described (Worley et al., 2013) therefore our distant measurements consist of fewer clones. Yellow bar = 50um. Error bars are std. dev. ***pCD2>Gal4/UAS system with heat-shock induced recombination at 30hr of development. Cells were labeled for S-phase with a 20 min EdU pulse and M-phase using Phospho-histone H3 Ser10 (PH3) positivity. DNA is labeled with Hoechst 33258. (F,G) Large clones expressing miR-8+P35 exhibit reduced EdU labeling while adjacent non-expressing tissue exhibits increased EdU positive cells. (H–K) Cell cycle analysis by flow cytometry was performed on larval wings containing GFP-labeled clones (H), expressing GFP+P35 (I), GFP+miR-8 (J) and miR-8+P35+GFP (K). The black trace shows the cell cycle profile of the non-clonal cells in the tissue. P35 expression alone shifts the cell cycle distribution in the wing slightly toward G2, while cells expressing miR-8 or miR-8+P35 show a dramatic shift toward late G2.


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Author Manuscript Author Manuscript Author Manuscript Fig. 5. High miR-8 remodels the wing epithelium and reduces apical f-actin

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GFP-labeled clones overexpressing miR-8 were generated by heat-shock induced recombination using tub>CD2>Gal4/UAS, Gal80TS with miR-8 expression induced at 28°C for 48h prior to dissection at wandering L3. (A) Apical x/y sections show little effect of miR-8 expression on E-cadherin (E-cad) intensity or localization. (B) Basal x/y sections show holes of E-cad (indicated by arrows) due to extrusion of miR-8-expressing cells from the wing epithelium. The inset shows an x/z section of this wing, confirming the loss of basal E-cad in a miR-8 clone undergoing basal extrusion. (C–G) GFP-labeled clones overexpressing miR-8 +P35 were generated using heat-shock induced recombination of actin>CD2>Gal4/UAS and dissected 72h post clone induction. (C) Persistent miR-8 + P35


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leads to ectopic E-cad and integrin (β-PS). (D) miR-8 + P35 clones show concentrated foci of Arm staining in the center, (E) which in x/z optical sections correspond to locations of folded epithelium caused by basal extrusion of miR-8 cells. (F) Persistent miR-8 + P35 leads to Sparc and MMP1 upregulation, (G) with Sparc mis-localized apically (arrows). (H,I) Cells expressing miR-8 without P35 also show apical localization of Sparc. (J,K) Cells expressing miR-8 have reduced apical f-actin, (L) which can be seen by 24h of miR-8 expression in an optical x/z section. In this case miR-8 + GFP expression is driven by apterous (ap)-Gal4,Gal80TS with a shift to 28°C 24h prior to dissection. Ap-Gal4 drives in the dorsal wing, oriented to the left in this x/z section. The arrow indicates the dorso-ventral boundary.


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Author Manuscript Author Manuscript Author Manuscript Fig. 6. Additional targets of miR-8 impact the actin cytoskeleton and epithelial organization

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(A) The 3′UTR of potential targets was cloned downstream of a LacZ coding region, and cotransfected into Kc167 cells with a miR-8 expression vector. The 3′UTRs of Sra-1, jar, sqh, CalpA, Abp1, pnut, Arp3, or Vang caused a significant decrease in LacZ expression in the presence of miR-8 (p

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