Matrix Metalloprotease 1 (Mmp1) promotes cell fate change for epithelial-to-epithelial transition during regeneration after radiation damage in Drosophila

Michael Shiferaw; Lauren Orr; Korneel Hens; Tin Tin Su

doi:10.1371/journal.pgen.1012257

Abstract

Ionizing radiation (IR) is used to treat cancer, but therapeutic failure occurs when surviving cancer cells change fate and regenerate tumors through acquired stem cell-like properties. While transcriptional mechanisms underlying cell fate plasticity have been characterized, the cellular processes enabling cell movement during tissue regeneration remain unclear. We reported previously that hinge cells of the Drosophila larval wing disc convert to pouch fate and translocate to help regenerate the pouch that suffers from more IR-induced apoptosis. We report here that IR increases the expression of extracellular proteins in the hinge, including secreted proteases and cell adhesion modulators. Functional validation using RNA interference revealed that secreted Matrix Metalloprotease 1 (Mmp1) and the related secreted protease homolog Scarface (Scaf) are required in hinge cells for IR-induced cell fate conversion and translocation. IR, we found, induces Mmp1 and scaf transcripts in hinge cells via cell-autonomous JNK signaling. Overexpression of Mmp1 specifically in the hinge was sufficient to elevate Mmp1 protein levels throughout the disc and induced cell fate change of both hinge and non-hinge cells but only in the context of irradiation. Confocal imaging in a time course demonstrated that cells undergoing fate conversion remain within the epithelial layer with little evidence for delamination or epithelial-mesenchymal transition (EMT). We propose that remodeling of the extracellular environment is a critical mechanism that enables cellular reorganization during tissue regeneration. Mmp enzymes are important for cancer biology because of their role in ECM remodeling, extracellular signaling, and EMT. Our findings demonstrate for the first time that Mmp1 is necessary and sufficient for one epithelial cell type to switch to another epithelial cell type after radiation damage. These results provide a mechanistic basis for radiation therapy-induced cell fate plasticity.

Author summary

Ionizing radiation is used to treat cancer because it can kill cells. But treatment failure can occur when surviving cancer cells change fate to become more like stem cells and re-grow the tumor. More than 80% of cancers arise from epithelial cells, making it critical to understand how epithelial cells respond to radiation. We are using the Drosophila model to study how epithelial cells change fate after exposure to ionizing radiation (X-rays). We report here that radiation increased the level of two secreted enzymes in a population of radiation-resistant epithelial cells: Matrix Metalloprotease 1 (Mmp1) and Scarface (Scaf), a serine protease homolog. Both enzymes are induced through JNK signaling and are needed for radiation-resistant cells to change their identity and replace neighboring cells killed by radiation. To test if Mmp1 was sufficient for fate change, we overexpressed it in the radiation-resistant cell population. Without radiation, this had little effect. But when combined with radiation, Mmp1 overexpression produced extensive fate changes. These findings suggest that extracellular proteases may be potential targets for drugs that could be administered alongside radiation therapy to limit the ability of surviving cancer cells to regenerate tumors.

Citation: Shiferaw M, Orr L, Hens K, Su TT (2026) Matrix Metalloprotease 1 (Mmp1) promotes cell fate change for epithelial-to-epithelial transition during regeneration after radiation damage in Drosophila. PLoS Genet 22(7): e1012257. https://doi.org/10.1371/journal.pgen.1012257

Editor: Lolitika Mandal, Indian Institute of Science Education and Research Mohali, INDIA

Received: November 20, 2025; Accepted: July 16, 2026; Published: July 30, 2026

Copyright: © 2026 Shiferaw et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data that support the findings of this study are publicly available under accession no. GSE182782 in Gene Expression Omnibus (GEO) database, hosted by the National Center for Biotechnology Information (NCBI). (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE182782).

Funding: TTS received funding from the National Institutes of Health (R35 GM130374). https://www.nih.gov/ KH received funding from Biotechnology and Biological Sciences Research Council (BB/W018780/1). https://www.ukri.org/councils/bbsrc/ MS was supported in part by the National Institutes of Health (T32 GM008759). https://www.nih.gov/ Funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

More than half of all cancer patients receive ionizing radiation (IR) as part of their treatment regimen [1]. While IR effectively kills cancer cells through DNA damage, therapeutic failure frequently occurs when surviving cells regenerate tumors. This regenerative capacity is attributed to the ability of non-stem cancer cells to acquire stem cell-like properties following radiation exposure [2–4], a phenomenon that has emerged as a major obstacle to successful cancer therapy (reviewed in [5,6]). Understanding the molecular and cellular mechanisms underlying radiation-induced cell fate plasticity is therefore critical for improving therapeutic outcomes and preventing treatment resistance.

Cell fate plasticity during tissue regeneration is a conserved biological response observed across diverse organisms and tissue types. In mammalian systems, radiation damage can induce dramatic cellular reprogramming: Paneth cells in the irradiated mouse intestine, for example, dedifferentiate to replenish the stem cell compartment [7], while acinar and duct cells in damaged salivary glands adopt plasticity to regenerate functional tissue [8]. These examples demonstrate that IR can trigger normally committed cells to abandon their differentiated state and acquire regenerative capabilities. However, the molecular mechanisms that orchestrate this cellular reprogramming, particularly the physical processes that enable cells to relocate and establish new tissue domains, remain incompletely understood.

The Drosophila larval wing imaginal disc provides an exceptional model system for dissecting the mechanisms of radiation-induced regeneration. The wing disc is composed of one layer each of columnar and squamous epithelia. In 3^rd instar larvae, cells of the wing disc columnar epithelium are already specified into their future fate; the notum that will become the body wall, the pouch that will become the wing blade, and the hinge that will connect the two (Fig 1A). Of these, the hinge cells are more resistant to IR-induced apoptosis than others due to protection by Wingless (Wg, Drosophila Wnt1) and STAT92E (sole Drosophila signal transducer and activator of transcription [STAT] homolog) [9]. Remarkably, they display plasticity, changing fate from hinge to pouch identity while translocating across tissue boundaries to participate in regenerating the latter [9–11]. Recent genomic analyses have revealed that this process requires coordinated downregulation of hinge-specific transcription factors such as Zfh2, coupled with upregulation of ribosome biogenesis machinery [12]. These transcriptional changes establish the molecular framework for cell fate conversion, but the cellular and mechanical processes that enable physical cell movement and tissue reorganization during regeneration have remained largely unexplored.

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Fig 1. Fate-changing hinge cells stay within the epithelial layer after IR.

Larvae expressing G-trace under the control of 30A-GAL4 (‘30A-GAL4>G-trace’) were irradiated with 0 or 4000 R of X-rays. G-trace is P{w[+mC]=UAS-RedStinger}⁴, P{w[+mC]=UAS-FLP.D}^JD1, P{w[+mC]=Ubi-p63E(FRT.STOP)Stinger}^9F6. The discs were dissected at various times after irradiation as shown, fixed, stained for DNA and imaged for DNA, RFP, and GFP. The larvae were of the genotype w¹¹¹⁸/+ (female) or Y/+ (male); 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/+ and were produced by a cross between w¹¹¹⁸ and 30A-GAL4 > UAS-G-trace/CyO-GFP; tub-GAL80^ts/tub-GAL80^ts and sorted against CyO-GFP. Scale bar = 50 microns in G and 100 microns in other panels. (A) A diagram of a wing disc showing the notum, the hinge and the pouch regions. (B) The experimental protocol. Larvae were cultured for 2 days at 25°C before temperature shift to minimize any effect of GAL4-driven transgene expression on disc development as published [9–11]. ‘X’ denotes the duration at that temperature (e.g., 29°C X 24h = 29°C for 24 hours). (C) Representative discs from indicated time points to illustrate the increase in GFP + RFP- former hinge cells in the pouch over time (arrows). (D-E) Quantification of cell fate changes over a time course. Normalization to the hinge area (D) or the total disc area (E) gave similar results. The horizontal bars show the mean±1SEM. The numbers in the brackets are the numbers of discs examined in at least two biological replicate experiments per time point. p-values were calculated by 2-tailed t-test relative to -IR controls. (F) Confocal slices of discs with dashed lines to indicate the location of sagittal views in G. (G) Sagittal views of discs without irradiation or at 36 and 48 h after irradiation. Arrow heads show apoptotic cells with cleaved caspase Dcp1. Arrows show GFP + RFP- former hinge cells that have translocated into the pouch. The images are aligned at the hinge/hinge (H/H) fold (vertical yellow line), which is the fold within the RFP+ hinge area in C and F. H/N = hinge/notum fold. H/P = hinge/pouch fold. amp = adult muscle precursor cells that are found associated with the wing disc.

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Extracellular matrix (ECM) remodeling represents a fundamental mechanism by which cells navigate tissue boundaries and establish new positional identities during development and repair [13]. Matrix metalloproteinases and associated proteolytic enzymes facilitate cell movements by degrading structural barriers, while secreted signaling molecules coordinate cellular responses to tissue damage. Despite the recognized importance of ECM dynamics in regenerative processes, the role of matrix remodeling in radiation-induced cell fate plasticity has not been systematically investigated. This represents a significant gap in our understanding, as the physical mechanisms that allow fate-changing cells to traverse established tissue boundaries and integrate into new domains are likely to be as critical as the transcriptional programs that specify new cellular identities.

Here we demonstrate that radiation-induced cell fate plasticity in the Drosophila wing disc requires upregulation of extracellular proteins, specifically Matrix Metalloprotease 1 (Mmp1) and protease homolog Scarface (Scaf). Through a combination of transcriptomic analysis, functional genetics, and confocal imaging, we show that IR triggers JNK-dependent expression of these proteolytic enzymes specifically in fate-changing hinge cells. Importantly, we find that cells undergoing fate conversion remain within the epithelial layer during the regenerative process, suggesting that remodeling of the extracellular environment facilitates movement through existing tissue architecture rather than requiring delamination and re-integration. These findings reveal proteolysis of the extracellular environment as an essential component of radiation-induced epithelial-to-epithelial cell plasticity and provide new insights into tissue regeneration following radiation exposure.

Results

Fate-changing hinge cells stay within the epithelial layer during regeneration after IR damage

We reported previously that following irradiation with 4000R of X-rays in 3^rd instar larvae, some wing disc hinge cells do not undergo apoptosis but adopt a pouch fate over a 72-hour (h) period [9–12,14]. In those studies, G-TRACE (GAL4 Technique for Real-time And Clonal Expression), a genetically encoded lineage tracer consisting of UAS-RedStinger (real-time RFP), UAS-FLP, and Ubi-p63E(FRT.STOP)Stinger (lineage-traced GFP) ([15]; S1 Table), was expressed in the hinge using the 30A-GAL4 driver (‘30A-GAL4>G-trace’ hereafter). 30A-GAL4 maps to an intron of the dachsous (ds) gene that encodes a cadherin family member [12]. Using a temperature-sensitive GAL80^ts repressor expressed ubiquitously from a tubulin promoter, we controlled GAL4 activity temporally to initiate lineage tracing with a shift to 29°C 24 h prior to irradiation (Fig 1B).

Using these methods, we quantified the extent of fate change across a time course of regeneration (Fig 1C, quantified in D and E). We used 4000 R (40 Gy) of X-rays in all experiments; 4000R is typically used in Drosophila studies (e.g., [16]) because it kills more than half of the cells in a wing disc but is still compatible with regeneration and survival as viable adults can emerge [17]. Without IR, GFP (lineage tracer) and RFP (real-time marker) mostly overlap (Fig 1C ‘-IR’), indicating stable cell fates. After IR, we observed a gradual increase in GFP + RFP- former hinge cells that have translocated into the pouch (Fig 1C arrows). We showed previously that these cells express the pouch marker VgQ-lacZ [11], indicating fate conversion. As in previous studies, cell fate change was quantified by measuring the GFP + RFP- area in the pouch and normalizing it to the hinge area or the total disc area to compare across discs of different sizes [9–12,14]. The use of area is justified because the wing disc is composed of just two single-cell layers and our analyses focus on one of these, the columnar epithelium, that is the precursor of adult structures.

Increased RFP was not observed in the pouch during the time course. This helps us exclude the possibility that GFP+ cells in the pouch results from transient activation of 30A-GAL4 in the pouch after IR; if this were the case, we would expect to see RFP + GFP+ cells in the pouch as RFP remains detectable for at least 3 days after transient expression in control experiments (S1 Fig).

We used 36 and 48 h time points after IR as intermediate stages in fate change to ask whether former hinge cells delaminate from the columnar epithelial layer during the time course of fate change into the pouch. Sagittal views of discs at indicated locations (Fig 1F) were reconstructed in ImageJ from confocal z-sections (Fig 1G). IR-induced apoptosis results in cells that stain for cleaved caspase Dcp1 and delaminate basally (arrowheads in Fig 1G). Basal extrusion of apoptotic cells is also seen after genetic ablation of the pouch [18,19]. In contrast, GFP + RFP- former hinge cells remain within the epithelial layer at both time points with no evidence of delamination during the time course (arrows in Fig 1G); only GFP+ cells found to leave the epithelial layer were Dcp1-positive dead/dying cells (arrowheads) in 3D analysis of ten irradiated discs. However, we cannot exclude the possibility of transient delamination and re-integration.

IR increases the transcript levels for extracellular proteins

To better understand the genetic basis for cell movement during regeneration, we re-analyzed the published differential gene expression data [12]. In those experiments, wing discs at 0 (-IR), 24 and 48h after irradiation were dissociated into single cells and sorted into GFP + RFP+ hinge and GFP-RFP- non-hinge populations that were subjected to genome-wide RNAseq analysis. The latter includes the pouch, the notum, and adult muscle precursor cells that are found associated with the wing disc (amp, Fig 1G). The ensuing analysis and functional tests were designed to find changes that made hinge cells more like the pouch. This work identified down-regulation of hinge-specific gene expression and up-regulation of ribosome biogenesis as key requirements [12].

Here, we mined the same RNAseq dataset but with different criteria. Starting with 8,940 transcripts, we first identified genes whose transcripts increased after irradiation within the RFP + GFP+ hinge cell population (-IR vs. + IR hinge, log₂FC ≥ 0.5 at 24 h and/or 48 h), isolating the hinge’s direct transcriptional response to radiation. Next, from this set, we selected genes with higher transcript level in the irradiated RFP + GFP+ hinge compared to irradiated GFP^-RFP^-non-hinge cells (+IR non-hinge vs. + IR hinge, log₂FC ≥ 1.0), enriching for hinge-specific responses over general tissue-wide changes. Finally, we applied a minimum expression threshold (raw mRNA count > 50) to remove low-abundance transcripts that are more susceptible to noise and less likely to be biologically meaningful. These sequential filters reduced the list from 8,940 to 1,767 irradiation-responsive genes, 136 hinge-biased candidates (S2 Table), and ultimately 34 high-confidence transcripts (S3 Table) for downstream analysis (Fig 2A).

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Fig 2. IR increases the transcript levels for extracellular proteins.

(A) Pipeline illustrating the stepwise re-analysis of the RNA-seq dataset from [12]. Sequential filters were used to select for (1) IR-induced genes in the hinge, (2) hinge-biased expression relative to non-hinge cells, and (3) a minimum expression threshold. (B) Gene Ontology (cellular-component) enrichment of the 34 hinge-biased, IR-responsive genes, showing overrepresentation of extracellular-region and ECM-related terms. (C) Heatmap showing log₂(CPM) expression values for 9 extracellular genes in hinge and non-hinge cells across three conditions: − IR, 24h after IR, and 48h after IR. (D) Line plots of RNA-seq expression for Mmp1 and scaf in hinge (GFP + RFP+ cells, blue lines) and non-hinge (GFP-RFP- cells, red lines) populations, showing induction at 24 h after IR. (E–F) Representative wing discs showing HCR-FISH for Mmp1 (E) and scaf (F) mRNAs. Larvae were treated as in Fig 1B. Wing discs were fixed at 24 or 48 h after irradiation, processed for HCR-FISH, and stained for DNA. Brackets mark the hinge region (cyan) and hinge/pouch fold (yellow). Arrows indicate hinge-enriched signal after IR. Asterisks mark trachea. Larvae were of the genotype w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + . Scale bar = 100 µm. (G-H) Mean fluorescence signal from RFP+ hinge areas were quantified as described in the Methods with disc identities blinded and normalized to the average signal from -IR samples. The numbers in the brackets are the total number of discs examined in two biological replicate experiments. p-values were calculated by 2-tailed t-test.

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Gene Ontology analysis of the hinge-biased, IR-responsive set of 34 genes revealed striking enrichment for cellular-component terms associated with the extracellular region, cell periphery, and collagen-containing extracellular matrix (Fig 2B), including 9 genes known or predicted to encode extracellular proteases/protease homologs and cell adhesion molecules (Fig 2C, graphed for two of the genes in Fig 2D). We chose 4 of these for validation by Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH): Matrix metalloproteinase 1 (Mmp1), scarface (scaf), Tiggrin (Tig) and Serpin 47C (Spn47C). In unirradiated discs, Mmp1 showed little or no expression in the hinge, with signal restricted to the hinge/pouch fold (yellow brackets in Fig 2E -IR) and the trachea that occasionally associates with the wing disc (*). At 24 and 48 h after irradiation, timepoints chosen to match those used in RNAseq analysis, Mmp1 expression increased with noticeable enrichment in the hinge (marked with RFP, white brackets and arrows in Fig 2E + IR panels; hinge/pouch fold is indicated with yellow brackets for reference). Similarly, scaf signal was minimal under basal conditions but increased markedly in the hinge following irradiation (marked with RFP in Fig 2F, cyan brackets and arrows; * marks the trachea). In contrast, Tig and Spn47C did not show clear hinge-enriched upregulation by HCR-FISH. Together, these data identify Mmp1 and scaf as genes encoding extracellular proteins with the highest expression specifically in the hinge after IR exposure. Therefore, we chose Mmp1 and scaf for further study.

IR increases the level of Mmp1 and Scaf proteins

We used antibodies against Mmp1 to ask whether IR also induces Mmp1 protein (Fig 3). Mmp1 is a secreted endopeptidase that breaks down cell adhesion during normal development and tissue repair in diverse species [20]. Mmp1 expression correlates with invasive potential and metastasis of malignant tumors [21,22]. The Drosophila Mmp1 gene encodes both predicted secreted and predicted membrane-anchored isoforms [23]. We found that Mmp1 protein levels increased after IR throughout the disc (Fig 3B’ vs. 3A’), with a more pronounced increase in the hinge (brackets and arrows in Fig 3B’ and B”’). Furthermore, the signal appeared punctate in the pouch region (Fig 3B”’ and S3 Fig). We note that while IR exposure increases Mmp1 transcripts predominantly in the hinge (Fig 2), Mmp1 protein levels increased both within and outside of the hinge. Re-analysis of the RNA-seq data by Mmp1 isoforms shows that IR increased the transcripts encoding all isoforms including secreted (RF and RJ; R for RNA) and anchored (RC) isoforms (S4 Table; [23]). Induction of a secreted isoform in the hinge by IR can explain why Mmp1 protein level increased throughout the disc. In support, experimental induction of Mmp1 in the hinge from a UAS-transgene encoding the secreted isoform, Mmp1.PF (P for protein; [23]), increased the mRNA only in the hinge but protein throughout the disc (Fig 4).

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Fig 3. IR induces Mmp1 and Scaf proteins.

(A-B”’) Larvae were treated as in Fig 1B. Wing discs were fixed at 24 h after irradiation and stained for DNA and with antibodies against Mmp1. The genotype was w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/+ (abbreviated as 30A-GAL4). The disc in B-B’ was imaged at higher magnification and shown in B”-B”’. Brackets and arrow in B’ and B”’ indicate the elevated Mmp1 expression in the hinge that is marked with RFP in B and B.” (C-D”’) 4-5-day old larvae of the genotype Scaf-GFP/Scaf-GFP were irradiated with 0 or 4000R of X-rays. The animals were cultured at 25°C throughout the experiment. Wing discs were fixed at 24 h after irradiation and stained for DNA. The discs in C-C’ and D-D’ are imaged at higher magnification and shown in C”-C”’ and D”-D”’, respectively. (E-F) Mean fluorescence signal from whole discs were quantified as described in the Methods and normalized to the average signal from -IR samples. The numbers in the brackets are the total number of discs examined in two biological replicate experiments. Scale bar = 100 microns in A-D’ and 50 microns in B”-D”’. p-values were calculated by 2-tailed t-test.

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Fig 4. Overexpression of Mmp1 mRNA in the hinge increases Mmp1 protein throughout the wing disc.

Larvae were treated as in Fig 1B. Wing discs were fixed at 72 h after the shift to 29°C and processed to detect Mmp1 mRNA by HCR-FISH (A-B”’) or fixed at 48 h after temperature shift and stained with anti-Mmp1 antibodies (C-D”’). The discs were also stained for DNA. The genotypes were w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/+ (A, C) and w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Mmp1 (B, D). The discs in B-B’ and D-D’ were imaged at higher magnification and shown in B’‘-B’“ and D’‘-D’“, respectively. Brackets and arrow in D”’ indicate the elevated Mmp1 expression in the hinge that is marked with RFP in D.” Scale bar = 100 microns in A-D’ and 50 microns in B”-D”’. (E-F) Mean fluorescence signal from RFP+ hinge areas (E) or the whole disc (F) were quantified as described in the Methods and normalized to the average signal from -IR samples. The numbers in the brackets are the total number of discs examined in two biological replicate experiments. p-values were calculated by 2-tailed t-test.

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Scaf is a predicted secreted serine protease with a defective catalytic site that is expected to render it inactive [24]. It is required for dorsal closure of Drosophila embryo and for adult male terminalia rotation [24,25], and for the closure of the pupal thorax [26], all processes in which two epithelial cell layers migrate towards each other and fuse. To monitor Scaf protein levels, we generated larvae expressing a C-terminal in-frame fusion of GFP to full-length Scaf (to be called Scaf-GFP). Scaf-GFP expression in unirradiated discs, we found, matches the published expression pattern of a protein trap version wherein part of Scaf was replaced with GFP [26], with signal noted in the hinge (Fig 3C’, brackets) and the stalk (Fig 3C’, arrow). The Scaf protein trap is a loss-of-function mutant whereas Scaf-GFP is homozygous viable and, therefore, likely to retain Scaf function. Like Mmp1, scaf mRNA was induced by IR predominantly in the hinge (Fig 2F, 2H), but Scaf-GFP increased throughout the disc (Fig 3C’ vs. 3D’, magnified in C”’ and D”’). Similarly, like Mmp1, experimental induction of a Scaf transgene in half of the wing disc was shown before to increase signal throughout the disc [24]. These results are consistent with Scaf being a secreted protein.

JNK activity is needed to induce Mmp1 and Scaf after IR

Mmp1 and Scaf are transcriptional targets of JNK signaling in other contexts. For example, JNK is needed to increase Mmp1 transcripts [27] or protein [28] in Drosophila tumors. Likewise, transcriptional induction of scaf in the leading-edge cells during embryonic dorsal closure requires JNK signaling [24,25] and JNK regulates Scaf expression in the stalk (arrow in Fig 3C’) of wing discs [26]. In addition, Scaf antagonizes JNK signaling during dorsal closure [24,25,29], much like Puckered (Puc) phosphatase that is induced by JNK and inhibits JNK signaling in a negative feedback loop. UAS-puc has been used to inhibit JNK signaling in many contexts including in the wing disc in Drosophila [30]. To investigate whether JNK activity is needed for the induction of Mmp1 and Scaf after IR in the wing discs, we expressed UAS-puc using the protocol in Fig 1B. Thus, Puc expression was initiated concurrently with G-trace at 24h before IR and was limited to the 30A-GAL4 expression domain in the hinge. Puc expression, we find, eliminated the IR-induced increase in Mmp1 and scaf transcripts in the hinge (Fig 5A-5D’, quantified in G). Puc expression in the hinge also reduced the IR-induced elevation of Mmp1 protein in the hinge (Fig 5 compare E”’ to F”’, quantified in H). The effect of Puc on Mmp1 protein in the rest of the disc, however, was less pronounced and IR-induced Mmp1 puncta in the pouch remained.

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Fig 5. JNK activity is needed to induce Mmp1 and Scaf after IR.

Larvae were treated as in Fig 1B. Wing discs were fixed at 24 h after irradiation and processed to detect scaf and Mmp1 mRNA by HCR-FISH (A-D’) or stained with antibodies to Mmp1 (E-F). The genotypes were w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/+ (A, C, E) or w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-puc (B, D, F). The boxed regions in E” and F” were imaged at higher magnification and further amplified 2-fold digitally and shown in E”’- and F”’. Scale bar = 100 microns in A-D’, 50 microns in E-E” and F-F” and 10 microns in E”’ and F”’. (G-H) Mean fluorescence signal from RFP+ hinge areas were quantified as described in the Methods with disc identities blinded and normalized to the average signal from 30A > GAL4 controls. The numbers in the brackets are the total number of discs examined in two biological replicate experiments. p-values were calculated by 2-tailed t-test.

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JNK, Mmp1 and Scaf are needed for IR-induced fate plasticity

To address the role of JNK, Mmp1 and Scaf in IR-induced fate change and translocation of hinge cells, we used UAS-transgenes to express Puc, dominant negative Basket (Bsk, Drosophila JNK) and dsRNA against Mmp1 and scaf specifically in the hinge. 30A-GAL4 > G-trace and the protocol in Fig 1B was used so transgene expression initiated 24h before IR and continued for 3d after IR when fate change was assayed as in Fig 1C. Fate change was quantified at 72 h after IR because this is the time of maximal fate change before larvae are lost to pupariation, consistent with our previous studies [9–12]. We find that Puc, Bsk^DN, Mmp1 RNAi and Scaf RNAi reduced IR-induced fate plasticity significantly compared to w¹¹¹⁸ or w RNAi controls (Fig 6A-6H, quantified in K-L). We conclude that JNK activity, Mmp1 and Scaf are needed cell-autonomously in the hinge for IR-induced cell fate plasticity and translocation into the pouch.

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Fig 6. JNK, Mmp1, and Scaf are needed for IR-induced cell fate plasticity and translocation.

Larvae were treated as in Fig 1B. Wing discs were fixed at 72h after irradiation and stained for DNA. (K-L) show quantification of data from discs such as those in (A-J). The numbers in the brackets are the total number of discs examined in at least two biological replicate experiments. p-values were calculated by 2-tailed t-test. (M) shows a graphical summary of how IR induces Mmp1 and Scaf expression and fate plasticity after IR. Scale bar = 100 microns. The genotypes were: A-B: w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + . C-D: UAS-bsk^DN/+ or UAS-bsk^DN/Y; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + . E-F: w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Mmp1 RNAi. G-H: w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Scaf RNAi. I-J: UAS-bsk^DN/+ or UAS-bsk^DN/Y; 30A-GAL4 > UAS-G-trace/UAS-Mmp1; tub-GAL80^ts/ + . K-L: GAL4 control = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + . UAS-puc = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-puc, bsk^DN = UAS-bsk^DN/+ or UAS-bsk^DN/Y; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + , bsk^DN;UAS-scaf = UAS-bsk^DN/+ or UAS-bsk^DN/Y; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-scaf, bsk^DN;UAS-Mmp1 = UAS-bsk^DN/+ or UAS-bsk^DN/Y; 30A-GAL4 > UAS-G-trace/UAS-Mmp1; tub-GAL80^ts/ + , w RNAi = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-w RNAi, Mmp1 RNAi = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Mmp1 RNAi, Scaf RNAi = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Scaf RNAi.

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Overexpression of Mmp1 synergizes with IR to induce excessive fate change

To address sufficiency, we examined the effect of overexpressing Mmp1 or Scaf from UAS-transgenes in the context of 30A-GAL4 > G-trace. The protocol in Fig 1B was used so transgene expression initiated 24h before IR and continued for 3d after IR when fate change was assayed as in Fig 1C. Mmp1 overexpression had little effect on its own without IR (Fig 7A-A”). In irradiated discs, however, Mmp1 overexpression resulted in increased fate change both in and outside the hinge. Examples include the loss of RFP expression in the hinge (brackets in Fig 7B-B”), indicative of loss of hinge-specific gene expression without translocation to the pouch; and gain of RFP in the pouch (arrows in Fig 7 C-C”). Antibody staining for the hinge marker Zfh2 shows that loss of RFP (driven by hinge-specific 30A/Ds-GAL4) in the hinge can occur without loss of Zfh2 (arrows in Fig 7 E”-E’”’). Likewise, gain of RFP in the pouch can occur without gaining Zfh2 expression (brackets in D”-D’”’ and E” to E’”’). Such findings suggest that fate change under these conditions is incomplete. Analysis of additional hinge markers may be needed to understand the extent of this phenomenon.

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Fig 7. Overexpression of Mmp1 induces excessive fate change but only after IR exposure.

Larvae were treated as in Fig 1B. Wing discs were fixed at 72 h after irradiation and stained for DNA. (A-C) The discs were imaged on a widefield microscope. Brackets in B’-B” indicate areas of the hinge that have lost RFP expression. Arrows in C’-C” indicate RFP signal in the pouch. (D-E) The discs were stained also with an antibody against Zfh2 and imaged on a confocal microscope. D-D”” show a single confocal slice. Arrows show that GFP + RFP+ overgrowth in the notum shows Zfh2 expression. Bracket shows GFP+ cells in the pouch that show faint RFP signal but no Zfh2 expression. E-E”” show a maximum projection z-stack of confocal slices. Arrows indicate part of the hinge that lost GFP and RFP expression but still show Zfh2 expression. Brackets indicate GFP + RFP+ cells in the pouch that lack Zfh2 expression. (F) The fraction of discs that show RFP + GFP+ growth in the notum like in panels B and D. The numbers in the brackets are the total number of discs examined in three biological replicate experiments. p-values were calculated by 2-tailed t-test. Scale bar = 100 microns. The genotypes were: A-E: w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Mmp1. F: GAL4 control = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + . UAS-Mmp1 = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-Mmp1, UAS-Mmp1^Δcat = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/UAS-Mmp1^Δcat; tub-GAL80^ts/ + .

https://doi.org/10.1371/journal.pgen.1012257.g007

The most penetrant phenotype observed was the outgrowth from the notum of an ectopic RFP+ hinge replete with hinge folds (arrows in Fig 7D-D”’, quantified in Fig 7F). Such ectopic hinges express Zfh2 and enclose a pouch-like area (arrow in Fig 7’”’). In contrast, ectopic discs were not observed in irradiated discs without Mmp1 overexpression, and Mmp1 without the catalytic domain (Mmp1^Δcat [31]) induced ectopic hinges to a significantly lower level (Fig 7F), suggesting that catalytic activity of Mmp1 is needed for full effect. We have published previously that knock down of nucleosome remodeler Nurf-38 induces ectopic hinge and pouch structures from the notum in about 40% of irradiated wing discs [10]. Thus, the ability of Mmp1 overexpression to induce ectopic structures in the notum reported here surpasses the highest levels we have seen before. We note that some cells with ectopic RFP, for example those in the pouch and the notum in Fig 7C’, are in locations with endogenous expression of Wg, which we have shown before to be required for IR-induced hinge-to-pouch fate change [9]. But other areas with ectopic RFP, like those in the ectopic outgrowths, are by definition not in locations with endogenous Wg expression.

Disruption of the hinge and outgrowth from the notum makes quantification and normalization of hinge-to-pouch fate change difficult in these discs but these results allow us to conclude that Mmp1 overexpression in the hinge promotes fate plasticity throughout the wing disc after irradiation. In contrast, overexpression of scaf with the same protocol had no statistically significant effect on fate change (S4 Fig). Mmp1^Δcat also did not interfere with IR-induced hinge-to-pouch fate change (S4 Fig), consistent with the published data that its ability to act as a dominant negative is phenotype-dependent; producing adult bristle phenotypes but with little effect in the larval stages [31].

Co-expression of UAS-scaf in the hinge (S2 Fig) did not rescue the fate change defects in bsk^DN discs, suggesting that additional JNK targets are needed (Fig 6K). The effect of co-expression of UAS-Mmp1 was hard to interpret because UAS-Mmp1 on its own induces excessive fate change. 25% of bsk^DN; UAS-Mmp1 discs (9/36 in two biological replicates) were deformed with RFP + GFP+ cells in the pouch area, precluding quantification of hinge-to-pouch fate change (Fig 6I). Fate change was quantified in the remainder of the discs (Fig 6J) and found to be not significantly different from bsk^DN (Fig 6K).

Discussion

We report here that hinge cells of the Drosophila larval wing disc remain within the epithelial layer as they change fate into pouch cells during regeneration after IR damage. Two extracellular proteins, Mmp1 and Scaf, are induced by IR through JNK signaling in the hinge and are needed cell-autonomously in the hinge for fate change and translocation. JNK is activated in Drosophila wing discs at 24 h after exposure to 3000-4000R of X-rays [32]. Therefore, our results provide a molecular pathway that links IR to fate plasticity through JNK and Mmp1/Scaf (Fig 6M). Radiation did not induce Mmp2 reproducibly (S4 Table), the only other Mmp family protease in Drosophila [12], suggesting functional specificity between Mmp proteases. Previous studies showed that Mmp1 and Mmp2 have different substrates in vitro [23], and that Mmp1 preferentially cleaves cell-cell junctions while Mmp2 preferentially cleaves the basement membrane [33,34]. Our finding that Mmp1 but not Mmp2 is induced specifically by radiation and allows the hinge cells to translocate to the pouch is consistent with these known characteristics of the two Mmp proteases. We propose that Mmp1 helps to remodel cell-cell interactions to allow fate-changing hinge cells to translocate to the pouch while remaining in the epithelial layer. This is the first report we are aware of wherein an Mmp protease is required for one epithelial cell type to switch into another. Epithelial-to-mesenchymal transition (EMT) is a critical component of cell movement and rearrangement both during development and in cancer metastasis. The reverse, mesenchymal-to-epithelial transition (MET), is equally important for development and for cancer cells to invade the target organ but is less completely understood than EMT [35]. What we describe here is different from these processes in that we observed epithelial-to-epithelial transition without an obvious mesenchymal intermediate; hinge cells appear to remain in the epithelial layer (this report) and transcriptome analysis did not show upregulation of mesenchymal markers such as twist and snail [12]. The closest parallel may be the regeneration of intestinal stem cells in mice wherein columnar epithelial cells in the upper intestinal crypt change fate and repopulate crypt base columnar stem cells after the latter have been depleted [36–40]. While gene expression changes and regulators of cell fate plasticity have been identified for this process, mechanisms for physical translocation of cells have not been studied. It would be interesting to test whether homologs of conserved ECM regulators we identified have a role in cell fate plasticity in the mouse intestine.

Scaf was the other ECM protein we found with a role in hinge-to-pouch fate change and translocation. Like Mmp1, depletion of Scaf inhibited fate plasticity. Unlike Mmp1, however, overexpression of Scaf had little effect. Scaf like Mmp1 is a JNK transcriptional target ([25] and this report). The relationship between Scaf and JNK is complex; depletion of Scaf phenocopies over-active JNK signaling in the embryo and adult [24,25,29] but defective JNK signaling in the larva/pupa [26]. Our findings are consistent with the view that both low (endogenous) and high (overexpression) levels of Scaf can promote JNK-dependent fate plasticity. How exactly Scaf, a secreted protease homolog with a predicted inactive catalytic site, plays this role remains to be investigated.

Overexpression of Mmp1 transcripts from a UAS-Mmp1 construct specifically in the hinge was sufficient to elevate Mmp1 protein levels throughout the disc and induced cell fate change of both hinge and non-hinge cells in irradiated discs. Irradiation also induced Mmp1 transcripts in the hinge and elevated Mmp1 protein levels throughout the disc. Increased Mmp1 protein levels resulting from localized Mmp1 transcription is not surprising given that Mmp1 is a secreted protein. In irradiated discs, however, fate change was confined to the hinge. One possible reason is that higher Mmp1 protein levels were induced by overexpression than by IR (compare Fig 4D’ to Fig 3B’). If so, it follows that hinge cells can change fate and translocate at lower levels of Mmp1 than the rest of the disc. This would be consistent with inherent features of the hinge that distinguish it from the rest of the wing disc, including differential cytoskeletal arrangements and higher propensity for oncogenic transformation upon depletion of tumor suppressor function [41].

Transcriptional induction of Mmp1 is one mechanism by which radiation promotes fate plasticity. But that cannot be the whole picture because overexpressing Mmp1 on its own had little effect, promoting fate plasticity only when combined with radiation. What other consequences of radiation besides Mmp1 induction are needed for fate plasticity? Our published studies identified the requirement for epigenetic regulators [10], apoptotic caspases providing non-apoptotic function [11], mitotic division [10], cytoskeletal components [14], down regulation of hinge determinants [12] and upregulation of ribosome biogenesis genes [12], with radiation exposure bringing about some of these. We propose that not one single change, but a plethora of molecular changes cooperate to allow hinge-to-pouch plasticity after radiation damage in the wing disc.

Cell fate plasticity after irradiation [6], particularly the acquisition of progenitor-like status by cancer cells, is implicated in treatment failure [3,5]. Mmp1 has long been recognized as an important factor in cancer cell metastasis [42,43]. Mmp1 is also needed for brain tumors to metastasize to the ovary in Drosophila [44]. As such, Mmp inhibitors have been studied extensively for the treatment of cancer but with little or no success [45,46]. Our results suggest that application of such inhibitors in the context of radiation therapy, specifically by administering them post IR, may be beneficial.

Materials and methods

Drosophila stocks

Drosophila stocks are listed in S1 Table and are described in published sources [15] and FlyBase [47]. To create the scaf-GFP knock-in line, a single guide RNA (gRNA) targeting the stop codon of scaf was cloned into the pCFD3 vector following the published protocol for CRISPR/Cas9-mediated genome editing in Drosophila [48]. A Drosophila-codon–optimized GFP sequence was introduced in-frame at the C-terminus of Scaf using a homology-directed repair (HDR) donor plasmid containing ~500 bp left and right homology arms flanking the scaf stop codon. The HDR construct also contained a 3xP3-dsRed fluorescent marker cassette for screening. Both the pCFD3-gRNA and HDR donor plasmids were co-injected into nanos-Cas9.attP2 embryos (BDSC 78782). Successful GFP insertions were identified by DsRed fluorescence and subsequently validated by PCR and Sanger sequencing. To generate the UAS-scaf overexpression construct, the full-length scaf coding sequence (CDS) was amplified by PCR from Drosophila melanogaster cDNA and cloned into a 5xUAS-attB expression vector. The resulting plasmid was injected into nos-phiC31; attP2 embryos (BDSC 99002) to mediate site-specific integration via ΦC31 recombination. Transgenic flies were screened for marker expression, and insertions were confirmed by PCR and sequencing (plasmid maps available upon request).

Larval culture and irradiation

After crossing, adult flies were fed on cornmeal-molasses food (Fly Food R, LabExpress) for 3 days at 25°C to boost egg production. Embryos were collected and larvae were raised on Nutri-Fly Bloomington Formula food (Genesee Scientific) at 25°C unless otherwise noted. The cultures were monitored daily for signs of crowding, typically seen as ‘dimples’ in the food surface as larvae try to increase the surface area for access to air. Cultures were split at the first sign of crowding. For temperature shifts, we used baths filled with Lab Armor beads (ThermoFisher), which we found gave more consistent and reproducible data than water baths. Larvae in food were irradiated in a Faxitron Cabinet X-ray System Model RX-650 (Lincolnshire, IL) at 115 kV and 5.33 rad/sec.

Fixation and antibody staining

Wing discs were dissected in PBS for all experiments. For visualization of RFP/GFP without antibody staining, wing discs were fixed in 4% paraformaldehyde in PBS for 30 minutes (min) and washed three times in PBT (PBS + 0.1% Tween-20).

For cleaved Dcp1 antibody staining, wing discs were fixed in 4% paraformaldehyde in PBS for 30 min, and washed three times in PBS, permeabilized in PBTx (0.5% Triton X-100) for 10 min and rinsed in PBTx. The discs were blocked in 5% Normal Goat Serum (NGS) in PBTx for at least 30 min and incubated overnight at 4°C in primary antibody in block. The discs were rinsed thrice in PBTx and incubated in secondary antibody in block for 2 h at room temperature or overnight at 4°C.

For Mmp1 and Zfh2 antibody staining and to visualize Scaf-GFP, wing discs were fixed in 10% paraformaldehyde in PBS for 30 min and washed three times in PBT. For staining with antibodies against Mmp1, the discs were blocked in 5% NGS in PBT for at least 30 min and incubated overnight at 4°C in primary antibody in block. The discs were rinsed thrice in PBT and incubated in secondary antibody in block for 2 h at room temperature or overnight at 4°C.

We used the following primary antibodies: cleaved Dcp1 (1:100, rabbit polyclonal, Cell Signaling Cat# 9578); Mmp1 (1:10 dilution of a 1:1:1 mixture of three mouse monoclonal antibodies against the catalytic domain, 5H7B11, 3A6B4 and 3B8D12, Developmental Studies Hybridoma Bank); Zfh2 (1:400, rat polyclonal [49]). Secondary antibodies were used at 1:200–500 (Jackson).

The discs were counter-stained with 10 µg/ml Hoechst33258 in PBT or PBTx for 2 min, washed 3 times, and mounted on glass slides in Fluoromount G (SouthernBiotech).

RNA Fluorescence In Situ Hybridization (FISH)

FISH to detect Mmp1 and scaf transcripts was performed using the HCR RNA-FISH v3.0 system (Molecular Instruments), following the manufacturer’s protocol for samples in solution with the following modifications: 3 pmol instead of 2 pmol for probes and 9 pmol instead of 30 pmol for hairpins. HCR probe sets for Mmp1 (FlyBase gene ID: FBgn0035049) and scaf (FlyBase gene ID: FBgn0033033) were designed against consensus sequences common to all annotated isoforms (S1 Data).

Wing discs were fixed in 4% paraformaldehyde in PBS, washed three times with PBTx, and permeabilized in PBTx (0.5% Triton-X100) for 10 minutes at room temperature, and rinsed in PBTx. For the detection stage, samples were pre-hybridized in 500 μL of probe hybridization buffer for 30 minutes at 37°C. Probe solution was prepared by adding 3 pmol each of HCR probe sets for Mmp1 and scaf (both designed for use with B3 amplifier; 3 μL of 1 μM stock each) to 500 μL of probe hybridization buffer at 37°C. Pre-hybridization solution was removed and probe solution was added to samples, which were then incubated overnight (>12 hours) at 37°C. Excess hairpins were removed by washing with 500 μL of 5 × SSCT (5 × saline-sodium citrate buffer with 0.1% Tween 20) at room temperature.

For the amplification stage, samples were pre-amplified in 500 μL of amplification buffer for 30 minutes at room temperature. 9 pmol each of B3 hairpin h1 and B3 hairpin h2 were prepared by snap-cooling 3 μL of 3 μM stock (heated at 95°C for 90 seconds, then cooled to room temperature in a dark drawer for 30 minutes), with hairpins snap cooled in separate tubes. Hairpin solution was prepared by adding snap-cooled B3 h1 and B3 h2 hairpins (3 μL each) to 500 μL of amplification buffer at room temperature. Pre-amplification solution was removed, and hairpin solution was added, followed by overnight incubation (>12 hours) in the dark at room temperature. Excess probes were removed by washing with 500 μL of 5 × SSCT at room temperature: 2 × 5 minutes, 2 × 30 minutes, and 1 × 5 minutes. Samples were stored at 4°C protected from light before microscopy. Mmp1 and scaf probe sets were designed to work with the B3 amplifier system. Each transcript was detected separately under identical experimental conditions.

Imaging

Confocal images (Fig 1F-1G) were acquired on a Nikon Ti2-E inverted microscope equipped with an AXR confocal scanner (NSPARC system) and controlled by NIS-Elements software. Images were typically collected as z-stacks, although the number and thickness of optical sections varied between experiments. Most datasets were acquired at a resolution of 1024 × 1024 pixels, corresponding to a field of view of ~442.6 × 442.6 µm. All other brightfield and fluorescent images were acquired on a Leica DMR compound microscope using a Q-Imaging R6 CCD or an ORCA-Halo sCMOS camera and either Ocular or Micro-Manager software. Image processing and analysis were performed in Fiji/ImageJ (v2.9.0) using standard tools and plugins.

Quantification of fluorescence signal

Mean fluorescence signals were measured from microscope images using ImageJ, either from the whole disc or from RFP+ hinge area as indicated in the figure legends, taking care to avoid the trachea that can show bright signal. Mean fluorescence from three areas adjacent to the disc (ventral and two lateral sides of each disc) was averaged and subtracted from the mean fluorescence signal of the disc area of interest, to correct for background fluorescence. Average background-corrected signal for the control group was used to normalize within each biological replicate, to allow comparison across multiple biological replicates.

Statistical analysis

For sample size justifications, we used a simplified resource equation from [50]; E = Total number of animals − Total number of groups, where E value of 10–20 is considered adequate. When we compare two groups (e.g., -/ + IR), n = 6 per group or E = 10 would be adequate. All samples met or exceeded this criterion. A 2-tailed Student t-test with assumed equal variance was used.

Supporting information

S1 Fig. Transiently expressed RFP persists for at least 3 days.

Wing discs were dissected from larvae treated as shown in E, fixed, and stained for DNA. Panels A’-D’ are shown at identical intensity display so that RFP signal can be directly compared across panels. RFP is mostly undetectable without temperature shift to inactivate GAL80 (A’) but is significantly induced after 24h at 29°C (B’), followed by a decline after incubation for 2 (C’) or 3 (D’) days at 18°C to reactivate GAL80. Panel D” shows the image in panel D’ but with increased brightness, to illustrate the presence of detectable RFP signal even after 3 days at 18°C. Larvae were of the genotype 30A-GAL4, G-trace/ + ; tub-GAL80^ts/ + that result from a cross of 30A-GAL4, G-trace/CyO-GFP; GAL80^ts/GAL80^ts with w¹¹¹⁸ and sorted against GFP from the CyO balancer. Scale bar = 100 microns.

https://doi.org/10.1371/journal.pgen.1012257.s001

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S2 Fig. Scaf mRNA is enriched in the hinge when UAS-scaf is driven by 30A-GAL4.

Larvae of the genotype w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-scaf were treated as in Fig 1B without irradiation. Wing discs were fixed at 72 h after the shift to 29°C and processed to detect scaf mRNA by HCR-FISH. The discs were also stained for DNA. Scale bar = 100 microns.

https://doi.org/10.1371/journal.pgen.1012257.s002

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S3 Fig. Mmp1 puncta are on the basal side of the pouch epithelium.

Wing discs like the one shown in Fig 3B” were imaged on the confocal microscope. The genotype of the larvae was w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + . Larvae were treated as in Fig 1B. Wing discs were fixed at 24 h after irradiation and stained for DNA and with antibodies against Mmp1. Scale bar = 100 microns.

https://doi.org/10.1371/journal.pgen.1012257.s003

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S4 Fig. Overexpression of Scaf or Mmp1^Δcat had little effect on IR-induced fate change.

Larvae were treated as in Fig 1B. Wing discs were fixed at 72 h after irradiation and stained for DNA. Fate change was quantified as described for Fig 1. The numbers in the brackets are the total number of discs examined in at least two biological replicate experiments. p-values were calculated by 2-tailed t-test. The genotypes were: w¹¹¹⁸ = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/ + , UAS-scaf = w¹¹¹⁸/+or Y/ + ; 30A-GAL4 > UAS-G-trace/ + ; tub-GAL80^ts/UAS-scaf. UAS-Mmp1^Δcat = w¹¹¹⁸/+ or Y/ + ; 30A-GAL4 > UAS-G-trace/UAS-Mmp1^Δcat; tub-GAL80^ts/+.

https://doi.org/10.1371/journal.pgen.1012257.s004

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S1 Table. Key Reagents.

https://doi.org/10.1371/journal.pgen.1012257.s005

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S2 Table. Gene Annotation for 136 Differentially Expressed Genes.

https://doi.org/10.1371/journal.pgen.1012257.s006

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S3 Table. Gene Annotation for 34 Differentially Expressed Genes.

https://doi.org/10.1371/journal.pgen.1012257.s007

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S4 Table. Mmp1 and Mmp2 transcript levels before and after irradiation.

https://doi.org/10.1371/journal.pgen.1012257.s008

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S1 Data. Consensus sequences used in RNA Fluorescence In Situ Hybridization.

https://doi.org/10.1371/journal.pgen.1012257.s009

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Acknowledgments

Drosophila stocks from the Bloomington Drosophila Stock Center (NIH P40 OD018537) were used in this study. This work was funded by an NIH grant to TTS (R35 GM130374) and by a BBSRC grant to KH (BB/W018780/1). MS was supported in part by the NIH/CU Signaling and Cellular Regulation Training Program (T32 GM008759). Monoclonal antibodies against Drosophila Mmp1 developed by G. M. Rubin were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. We acknowledge the use of the Light Microscopy Core Facility at the University of Colorado Boulder (RRID:SCR_018993) and thank its director Dr. James Orth for help and advice.

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Figures

Abstract

Author summary

Introduction

Results

Fate-changing hinge cells stay within the epithelial layer during regeneration after IR damage

IR increases the transcript levels for extracellular proteins

IR increases the level of Mmp1 and Scaf proteins

JNK activity is needed to induce Mmp1 and Scaf after IR

JNK, Mmp1 and Scaf are needed for IR-induced fate plasticity

Overexpression of Mmp1 synergizes with IR to induce excessive fate change

Discussion

Materials and methods

Drosophila stocks

Larval culture and irradiation

Fixation and antibody staining

RNA Fluorescence In Situ Hybridization (FISH)

Imaging

Quantification of fluorescence signal

Statistical analysis

Supporting information

S1 Fig. Transiently expressed RFP persists for at least 3 days.

S2 Fig. Scaf mRNA is enriched in the hinge when UAS-scaf is driven by 30A-GAL4.

S3 Fig. Mmp1 puncta are on the basal side of the pouch epithelium.

S4 Fig. Overexpression of Scaf or Mmp1Δcat had little effect on IR-induced fate change.

S1 Table. Key Reagents.

S2 Table. Gene Annotation for 136 Differentially Expressed Genes.

S3 Table. Gene Annotation for 34 Differentially Expressed Genes.

S4 Table. Mmp1 and Mmp2 transcript levels before and after irradiation.

S1 Data. Consensus sequences used in RNA Fluorescence In Situ Hybridization.

Acknowledgments

References

S4 Fig. Overexpression of Scaf or Mmp1^Δcat had little effect on IR-induced fate change.