Drosophila matrix metalloproteinase 2 (MMP2) is specifically expressed in posterior follicle cells of stage-14 egg chambers (mature follicles) and is crucial for the breakdown of the follicular wall during ovulation, a process that is highly conserved from flies to mammals. The factors that regulate spatiotemporal expression of MMP2 in follicle cells remain unknown. Here, we demonstrate crucial roles for the ETS-family transcriptional activator Pointed (Pnt) and its endogenous repressor Yan in the regulation of MMP2 expression. We found that Pnt is expressed in posterior follicle cells and overlaps with MMP2 expression in mature follicles. Genetic analysis demonstrated that pnt is both required and sufficient for MMP2 expression in follicle cells. In addition, Yan was temporally upregulated in stage-13 follicle cells to fine-tune Pnt activity and MMP2 expression. Furthermore, we identified a 1.1 kb core enhancer that is responsible for the spatiotemporal expression of MMP2 and contains multiple pnt/yan binding motifs. Mutation of pnt/yan binding sites significantly impaired the Mmp2 enhancer activity. Our data reveal a mechanism of transcriptional regulation of Mmp2 expression in Drosophila ovulation, which could be conserved in other biological systems.

Matrix metalloproteinases (MMPs) constitute a family of enzymes involved in the degradation and remodeling of the extracellular matrix, playing crucial roles in various physiological and pathological processes, including tissue remodeling, ovulation, wound repair, immune responses, and cancer progression (Murphy, 2016; Page-McCaw et al., 2003, 2007). MMPs have been found to cleave a variety of substrates, including extracellular matrix components such as collagen, gelatin, non-collagen connective tissue components, as well as regulators of extracellular signaling (Curry and Osteen, 2003; Page-McCaw et al., 2007; Wang and Page-McCaw, 2014). Dysregulation of MMP activity occurs in various diseases and pathological conditions, leading to the progression of cancer metastasis, cardiovascular diseases, and chronic wounds (Chen et al., 2017). Consequently, precise regulation of MMP expression and activity is crucial for normal development and physiology; however, our understanding of such regulation remains limited.

The fruit fly, Drosophila melanogaster, possesses two MMP genes (Mmp1 and Mmp2) in comparison with over 20 MMP genes found in mammals (Page-McCaw et al., 2003). The highly conserved nature of Drosophila MMPs, coupled with the availability of advanced genetic techniques, makes Drosophila an ideal and straightforward model for studying MMP regulation in great detail (Page-McCaw et al., 2003). Both Drosophila MMP1 and MMP2 exist in secreted and membrane-anchored forms, each having distinct substrates (LaFever et al., 2017). Consistent with these reports, our previous work demonstrated that MMP2, but not MMP1, plays a crucial role in Drosophila ovulation (Deady et al., 2015). MMP2 is detected specifically in posterior follicle cells (∼30 cells) of stage-14 egg chambers, but not in younger-stage egg chambers. During ovulation, octopamine (OA) and Octopamine receptor in mushroom body (OAMB) signaling elicits a rise of intracellular Ca2+ in both main-body and posterior follicle cells. The Ca2+ rise in the main-body follicle cells leads to the production of reactive oxygen species (ROS), which are required for ovulation in both Drosophila and mammals (Li et al., 2018; Shkolnik et al., 2011). In addition, the Ca2+ rise in the posterior follicle cells induces MMP2 activity. This activation mediates the breakdown of the posterior follicle wall and the release of the encapsulated oocyte (Deady and Sun, 2015). However, the precise regulation of the spatiotemporal expression of MMP2 in posterior follicle cells remains unknown.

The E26 transformation-specific (ETS) transcription factors are highly conserved and play diverse roles in development, including the regulation of processes such as cell proliferation, differentiation, apoptosis, angiogenesis, migration, and pattern formation (Gutierrez-Hartmann et al., 2007; Sharrocks, 2001). In Drosophila, the ETS family of transcription factors consists of eight proteins: Ecdysone-induced protein 74EF (Eip74EF), ETS21c, ETS65A, ETS96B, ETS97D, ETS98B, Pointed (Pnt) and Yan (also referred to as Anterior open, Aop; Vivekanand, 2018). Among them, Pnt and Yan are the most extensively studied ETS factors. The pnt gene encodes three isoforms (Pnt.P1, Pnt.P2 and Pnt.P3), and all of them have an ETS domain and exhibit similar DNA-binding activity (Klambt, 1993; Scholz et al., 1993; Wu et al., 2020). Whereas Pnt.P1 is constitutively active, Pnt.P2 and Pnt.P3 transcriptional activity is stimulated by mitogen-activated protein kinases (MAPK) signaling via phosphorylation of the unique Pointed domain (Shwartz et al., 2013; Wu et al., 2020). The activity of Pnt is also regulated by Yan, which serves as an endogenous transcriptional repressor that competes with Pnt for ETS-binding motifs of their target genes (Boisclair Lachance et al., 2018; Halfon et al., 2000; Webber et al., 2018). Early studies have suggested that phosphorylation of Yan by MAPK signaling leads to nuclear export and degradation of Yan, which alleviates the transcriptional repression of Yan (O'Neill et al., 1994; Rebay and Rubin, 1995). However, recent studies have proposed a new model stating that the activator–repressor pair Pnt/Yan co-occupies target genes in order to prevent their inappropriate activation under sub-threshold signaling conditions and prime the locus for rapid transcriptional activation following the onset of upstream signaling (Webber et al., 2018). Pnt plays essential roles in various biological processes, including glial cell differentiation, heart development, longevity, embryonic ventral midline patterning, intestinal stem cell proliferation, and oogenesis (Alvarez et al., 2003; Gabay et al., 1996; Jin et al., 2015; Klaes et al., 1994; Morimoto et al., 1996; Slack et al., 2015). During Drosophila oogenesis, Pnt acts downstream of the EGFR-Ras-MAPK pathway for the establishment of the posterior and dorsal midline follicle cell fate (Lachance et al., 2009; Morimoto et al., 1996; Stevens et al., 2020; Zartman et al., 2009). Yan has also been observed in follicle cells during mid-oogenesis, where its role in border cell migration has been studied (Schober et al., 2005). Nonetheless, the role of Pnt and Yan in the final stages of oogenesis has not been studied, and the role, if any, of ETS factors in Drosophila ovulation remains unknown.

In this study, we discovered that Pnt is detected in posterior follicle cells throughout late oogenesis and overlaps with MMP2 expression in stage-14 follicle cells. Our genetic manipulation demonstrated that Pnt is both required and sufficient for MMP2 expression in posterior follicle cells, and thus for the proper breakdown of the follicular wall and follicle rupture. In addition, we observed a transient upregulation of Yan in stage-13 follicle cells, which provides a temporal regulation of Pnt activity to fine-tune MMP2 expression. Furthermore, we identified a core enhancer element of Mmp2 that is essential for the spatiotemporal expression of MMP2 in late oogenesis. We identified at least two Pnt/Yan-binding sites in the Mmp2 core enhancer element that are required for Mmp2 enhancer activity, indicating that Pnt/Yan directly bind to the Mmp2 enhancer to regulate its expression. Overall, our findings unveil a role for the ETS transcription factors Pnt and Yan in MMP2 regulation during late oogenesis and follicle rupture. Considering the highly conserved nature of ETS transcription factors, we speculate that such regulation is conserved in other species.

Pnt is expressed throughout late oogenesis and overlaps with MMP2 in posterior follicle cells

Previously, we reported that MMP2 is detected in posterior follicle cells of stage-14 egg chambers and is required for proper follicle rupture and ovulation (Deady et al., 2015). Using the Mmp2::GFP protein trap line (Deady et al., 2015), we confirmed that posterior MMP2 expression begins at early stage 14, but not at stage 13 or younger (Fig. S1; n>20 egg chambers). In addition, we found that MMP2 is detected in anterior follicle cells, including stretch follicle cells and centripetal migrating cells from stages 10 to 14 (Fig. S1; n>30 egg chambers). To identify the transcription factors that determine the spatiotemporal pattern of MMP2, we focused on transcription factors that are expressed specifically in posterior follicle cells. Previous work has shown that Pnt is expressed in posterior follicle cells during mid-oogenesis and is essential for posterior cell fate specification downstream of EGFR signaling (Boisclair Lachance et al., 2014; Morimoto et al., 1996; Stevens et al., 2020). If Pnt continues to be expressed in posterior follicle cells in late oogenesis (stages 11-14), it will make it a good candidate for regulating MMP2 expression. Therefore, we first analyzed the expression of Pnt in late oogenesis using a functional C-terminal GFP-tagged pnt transgene (pnt-GFP), which reports all three isoforms (Boisclair Lachance et al., 2014; Wu et al., 2020), as well as a Pnt.P1-specific antibody (Alvarez et al., 2003). Consistent with previous findings, Pnt-GFP was observed in posterior-most follicle cells from stage 6 to stage 11 (Fig. 1A-C,A′-C′). In addition, Pnt-GFP was localized in the cytoplasm at the beginning (stages 6-8) and enriched in the nuclei subsequently (stages 9-11). It is interesting to note that Pnt.P1 was only weakly detected in posterior follicle cells at stages 6-8 and was upregulated at stages 9 and 10 (Fig. 1A-B″). In addition, both Pnt-GFP and Pnt.P1 were continuously detected in posterior follicle cells throughout late oogenesis, including stage 14 (Fig. 1D-F″). We also noticed that the Pnt-GFP-expressing domain was significantly wider than the Pnt.P1-expressing domain at stage 14, but not at stages 10-13 (Fig. 1B-F″). This could result from a differential detection limit of GFP and Pnt.P1 antibodies or additional Pnt isoforms in addition to Pnt.P1 that are turned on in stage-14 follicle cells. Furthermore, co-staining of MMP2::GFP and Pnt.P1 demonstrated that MMP2 and Pnt are co-expressed in posterior follicle cells of stage-14 egg chambers (Fig. 1G-H″), suggesting a potential role of Pnt in the regulation of MMP2 expression.

Fig. 1.

Pnt overlaps with MMP2 in posterior follicle cells of stage-14 egg chambers. (A-F″) Expression of Pnt-GFP (green in A-F and white in A′-F′) and Pnt.P1 (red in A-F and white in A″-F″) in egg chambers during mid to late oogenesis. The specific stage of each egg chamber is labeled. The arrows indicate the dorsal-anterior signal of Pnt-GFP at stages 10B (B′) and 11 (C′). (G-H″) Expression of MMP2::GFP (green in G-H and white in G′-H′) and Pnt.P1 (red in G-H and white in G″-H″) in egg chambers of stage 13 (G) and 14 (H). All cell nuclei were marked with DAPI (shown in blue). Each panel represents >30 egg chambers. Scale bars: 25 µm.

Fig. 1.

Pnt overlaps with MMP2 in posterior follicle cells of stage-14 egg chambers. (A-F″) Expression of Pnt-GFP (green in A-F and white in A′-F′) and Pnt.P1 (red in A-F and white in A″-F″) in egg chambers during mid to late oogenesis. The specific stage of each egg chamber is labeled. The arrows indicate the dorsal-anterior signal of Pnt-GFP at stages 10B (B′) and 11 (C′). (G-H″) Expression of MMP2::GFP (green in G-H and white in G′-H′) and Pnt.P1 (red in G-H and white in G″-H″) in egg chambers of stage 13 (G) and 14 (H). All cell nuclei were marked with DAPI (shown in blue). Each panel represents >30 egg chambers. Scale bars: 25 µm.

Pnt is required and sufficient for MMP2 expression in late oogenesis

To determine whether Pnt regulates MMP2 expression in posterior follicle cells at stage 14, we knocked down pnt in stage-14 follicle cells with two different pnt-RNAi lines driven by 44E10-Gal4, a Gal4 driver expressed in all follicle cells at stage 14 (Deady et al., 2017). Both RNAi lines showed efficient knockdown of pnt in posterior follicle cells at stage 14 (Fig. 2A-C). We then quantified MMP2::GFP expression in control and pnt-knockdown mature follicles at stage 14 and observed that the majority of control mature follicles had either a high or medium level of MMP2::GFP expression in posterior follicle cells, whereas the majority of pnt-knockdown mature follicles showed low levels of or no (‘low/no’) MMP2::GFP expression (Fig. 2D-H, Table S1). In addition, pnt knockdown with Vm26Aa-Gal4, which is specifically expressed in follicle cells at stages 10-14 and not detected in any other tissues or developmental stages (Peters et al., 2013), also led to a significant reduction of MMP2::GFP expression (Fig. S2A-D). Altogether, these results indicate that Pnt is required for MMP2 expression in posterior follicle cells at stage 14.

Fig. 2.

Pnt is required and sufficient for MMP2 expression. (A-C) Representative images showing Pnt.P1 expression (green) in control (A), pntRNAi1 (B) and pntRNAi2 (C) mature follicles with 44E10-Gal4 driving UAS-RFP expression (44E10>RFP; red). The insets show higher magnifications of Pnt.P1 expression (white) in the boxed area. Oocytes are outlined in cyan, and arrowheads point to posterior follicle cells. (D) Representative images showing high, medium and low/no levels of posterior MMP2::GFP (white). (E-G) Representative images showing posterior MMP2::GFP expression (green) in control (E), pntRNAi1 (F) and pntRNAi2 (G) mature follicles with 44E10>RFP (red) at stage 14. The insets show higher magnifications of MMP2::GFP expression (white) in the boxed area. Oocytes are outlined in cyan, and arrowheads point to posterior follicle cells. (H) Quantification of MMP2::GFP expression in stage-14 egg chambers of control and pntRNAi driven by 44E10-Gal4. The number of mature follicles analyzed is noted above each bar. (I-K) Representative images showing ectopic MMP2::GFP expression (green) in the main-body follicle cells of stage-14 egg chambers of control (I), UAS-pnt.P1 (J) and UAS-pnt.P2 (K) with 44E10>RFP (red). The insets show higher magnifications of MMP2::GFP expression (white) in the boxed area. (L) Quantification of ectopic MMP2::GFP expression in the main-body follicle cells of stage-14 egg chambers of control, UAS-pnt.P1 and UAS-pnt.P2 flies with 44E10>RFP (n=10 egg chambers and three regions/egg chamber). Error bars represent s.d. ***P<0.001 (one-way ANOVA with post-hoc Tukey test). A.U., arbitrary units. In image panels, nuclei are labeled with DAPI and shown in blue. Scale bars: 25 µm (A-G); 50 µm (I-K).

Fig. 2.

Pnt is required and sufficient for MMP2 expression. (A-C) Representative images showing Pnt.P1 expression (green) in control (A), pntRNAi1 (B) and pntRNAi2 (C) mature follicles with 44E10-Gal4 driving UAS-RFP expression (44E10>RFP; red). The insets show higher magnifications of Pnt.P1 expression (white) in the boxed area. Oocytes are outlined in cyan, and arrowheads point to posterior follicle cells. (D) Representative images showing high, medium and low/no levels of posterior MMP2::GFP (white). (E-G) Representative images showing posterior MMP2::GFP expression (green) in control (E), pntRNAi1 (F) and pntRNAi2 (G) mature follicles with 44E10>RFP (red) at stage 14. The insets show higher magnifications of MMP2::GFP expression (white) in the boxed area. Oocytes are outlined in cyan, and arrowheads point to posterior follicle cells. (H) Quantification of MMP2::GFP expression in stage-14 egg chambers of control and pntRNAi driven by 44E10-Gal4. The number of mature follicles analyzed is noted above each bar. (I-K) Representative images showing ectopic MMP2::GFP expression (green) in the main-body follicle cells of stage-14 egg chambers of control (I), UAS-pnt.P1 (J) and UAS-pnt.P2 (K) with 44E10>RFP (red). The insets show higher magnifications of MMP2::GFP expression (white) in the boxed area. (L) Quantification of ectopic MMP2::GFP expression in the main-body follicle cells of stage-14 egg chambers of control, UAS-pnt.P1 and UAS-pnt.P2 flies with 44E10>RFP (n=10 egg chambers and three regions/egg chamber). Error bars represent s.d. ***P<0.001 (one-way ANOVA with post-hoc Tukey test). A.U., arbitrary units. In image panels, nuclei are labeled with DAPI and shown in blue. Scale bars: 25 µm (A-G); 50 µm (I-K).

Next, we tested whether overexpression of specific Pnt isoforms was able to induce MMP2 expression ectopically. Overexpression of either Pnt.P1 or Pnt.P2 using 44E10-Gal4 in stage-14 follicle cells was able to induce ectopic MMP2::GFP expression outside the posterior domain, with Pnt.P1 doing so more efficiently than Pnt.P2 (Fig. 2I-L). In contrast, overexpression of Pnt.P1, but not Pnt.P2, using Vm26Aa-Gal4 was able to induce ectopic MMP2::GFP expression before stage 14 (Fig. S2E-G′), even though both could induce ectopic MMP2::GFP at stage 14 (Fig. S2H-J′). Therefore, Pnt.P1 is sufficient to induce MMP2 expression outside the posterior domains in earlier stages, whereas Pnt.P2 is only sufficient to induce MMP2 at stage 14 outside posterior domains. We also noticed that Pnt.P1, not Pnt.P2, was able to induce precocious expression of Hindsight (Hnt; also known as Pebbled, Peb) in main-body follicle cells (Fig. S2E-G′), a transcription factor that is upregulated at stage 14 and required for MMP2 expression, but is not normally detected from stages 10B to 13 (Deady et al., 2017). This ectopic expression of Hnt may explain the difference between Pnt.P1 and Pnt.P2 in inducing MMP2 expression. Overall, these findings demonstrate that Pnt is both required and sufficient for MMP2 expression in follicle cells.

Pnt in mature follicle cells is required for MMP2-mediated follicle rupture

Our previous work demonstrated that posterior MMP2 in mature follicles is activated by OA/OAMB-mediated intracellular Ca2+ rise and is essential for proper follicle rupture and ovulation (Deady and Sun, 2015; Deady et al., 2015). Here, we examined whether Pnt is essential for OA/OAMB-induced MMP2 activity and follicle rupture. We first applied our previously developed in situ zymography assay (see Materials and Methods; Deady and Sun, 2015) to evaluate MMP2 activity after OA stimulation. We found that OA stimulation led to posterior gelatinase activity in 69% of control mature follicles (Fig. 3A,D). In contrast, the percentage of mature follicles with pnt knockdown using 44E10-Gal4 that had posterior gelatinase activity was only 12.8% and 22.6% for each RNAi line (Fig. 3B-D). We further tested whether these mature follicles were competent for OA-induced follicle rupture with our ex vivo follicle rupture assay (see Materials and Methods; Deady et al., 2015; Knapp et al., 2018). In contrast to the ∼45% rupture rate in control mature follicles, pnt-knockdown mature follicles showed a significant reduction in rupture rate (7.7% and 14.9% for pntRNAi1 and pntRNAi2, respectively) in response to OA stimulation (Fig. 3E-H). In addition, pnt-knockdown mature follicles did not respond to stimulation with ionomycin (Fig. 3I-L), a potent Ca2+ ionophore that can bypass OA/OAMB signaling to induce follicle rupture (Deady et al., 2017). Similar results were obtained when pnt was knocked down using Vm26Aa-Gal4 (Fig. S2K-M). Altogether, these findings support the conclusion that Pnt is required for MMP2 expression and thus for OA-induced MMP2 activation and follicle rupture.

Fig. 3.

Pnt is required for OA-induced MMP2 activation and follicle rupture. (A-C) Representative images showing gelatinase activity (green) in mature follicles after a 3-h culture with 20 μM OA. Control (A), pntRNAi1 (B) and pntRNAi2 (C) mature follicles with 44E10>RFP (red) were used. Mature follicles with posterior gelatinase activity are marked by arrowheads. (D) Quantification of mature follicles with posterior gelatinase activity. (E-G) Representative images showing control (E), pntRNAi1 (F) and pntRNAi2 (G) mature follicles with 44E10>RFP (red) after a 3-h culture with 20 μM OA. The ruptured mature follicles are marked with arrowheads, and the brightfield images are pseudocolored blue. (H) Quantification of OA-induced follicle rupture. (I-K) Representative images showing control (I), pntRNAi1 (J) and pntRNAi2 (K) mature follicles with 44E10>RFP (red) after a 3-h culture with 2 μM ionomycin. The ruptured mature follicles are marked with arrowheads, and the brightfield images are pseudocolored blue. (L) Quantification of ionomycin-induced follicle rupture. The number of mature follicles analyzed is noted above each bar. ***P<0.001 (one-way ANOVA with post-hoc Tukey test). Error bars represent s.d. Scale bars: 700 µm.

Fig. 3.

Pnt is required for OA-induced MMP2 activation and follicle rupture. (A-C) Representative images showing gelatinase activity (green) in mature follicles after a 3-h culture with 20 μM OA. Control (A), pntRNAi1 (B) and pntRNAi2 (C) mature follicles with 44E10>RFP (red) were used. Mature follicles with posterior gelatinase activity are marked by arrowheads. (D) Quantification of mature follicles with posterior gelatinase activity. (E-G) Representative images showing control (E), pntRNAi1 (F) and pntRNAi2 (G) mature follicles with 44E10>RFP (red) after a 3-h culture with 20 μM OA. The ruptured mature follicles are marked with arrowheads, and the brightfield images are pseudocolored blue. (H) Quantification of OA-induced follicle rupture. (I-K) Representative images showing control (I), pntRNAi1 (J) and pntRNAi2 (K) mature follicles with 44E10>RFP (red) after a 3-h culture with 2 μM ionomycin. The ruptured mature follicles are marked with arrowheads, and the brightfield images are pseudocolored blue. (L) Quantification of ionomycin-induced follicle rupture. The number of mature follicles analyzed is noted above each bar. ***P<0.001 (one-way ANOVA with post-hoc Tukey test). Error bars represent s.d. Scale bars: 700 µm.

Downregulation of Yan at stage 14 is required for MMP2 expression and follicle rupture

Although Pnt is required and sufficient to induce MMP2 expression, it is still unclear why MMP2 is only induced at stage 14, given that Pnt is expressed throughout late oogenesis (Fig. 1D-F). Because the transcriptional activity of Pnt can be suppressed by Yan (Boisclair Lachance et al., 2014, 2018; Webber et al., 2018), we hypothesized that Yan regulates Pnt activity and MMP2 expression in late oogenesis. We first analyzed the expression of Yan in late oogenesis using an anti-Yan antibody. Consistent with a previous report (Boisclair Lachance et al., 2014), Yan was not detected in main-body follicle cells from stages 10A to 12 (Fig. 4A-B′). However, Yan was rapidly upregulated in all follicle cells at stage 13 (Fig. 4C-D′; n>30 egg chambers) and slowly downregulated first in main-body follicle cells at early stage 14 and then in posterior and anterior follicle cells at late stage 14 (Fig. 4E-H′). By late stage 14, there was no detectable Yan in any follicle cells (Fig. 4G-H′). The dynamic expression of Yan in late oogenesis suggests that Yan may fine-tune Pnt activity to control MMP2 expression.

Fig. 4.

Downregulation of Yan at stage 14 is required for MMP2 expression and follicle rupture. (A-H′) Yan (green in A-H and white in A′-H′) is dynamically expressed in follicle cells during late oogenesis. The specific stage of each egg chamber is labeled; the ‘a’ refers to the anterior portion, and the ‘p’ refers to the posterior portion. The arrows in E′ and F′ indicate the anterior and posterior expression of Yan at early stage 14. Nuclei are labeled with DAPI and shown in blue. (I-K) Representative images showing MMP2::GFP expression (green) in control (I), UAS-yanAct (J) or UAS-yanWT (K) mature follicles with 44E10>RFP (red). We noticed that the RFP signal (red arrows) is located more toward the oocyte membrane in UAS-yanAct and UAS-yanWT mature follicles. The insets show higher magnifications of MMP2::GFP expression (white) in boxed areas. Oocytes are outlined in cyan, nuclei are labeled with DAPI in blue, and arrowheads point to posterior follicle cells. (L) Quantification of MMP2::GFP expression in control, UAS-yanAct and UAS-yanWT mature follicles. The number of egg chambers analyzed is noted above each bar. (M-O) Quantification of OA-induced posterior gelatinase activity (M), OA-induced follicle rupture (N), and ionomycin-induced follicle rupture (O) in control, UAS-yanAct and UAS-yanWT mature follicles. All quantifications were performed after a 3-h culture. Mature follicles were isolated according to 44E10>RFP expression. The number of mature follicles analyzed is noted above each bar. ***P<0.001 (one-way ANOVA with post-hoc Tukey test). NS, not significant. Error bars represent s.d. Scale bars: 50 µm.

Fig. 4.

Downregulation of Yan at stage 14 is required for MMP2 expression and follicle rupture. (A-H′) Yan (green in A-H and white in A′-H′) is dynamically expressed in follicle cells during late oogenesis. The specific stage of each egg chamber is labeled; the ‘a’ refers to the anterior portion, and the ‘p’ refers to the posterior portion. The arrows in E′ and F′ indicate the anterior and posterior expression of Yan at early stage 14. Nuclei are labeled with DAPI and shown in blue. (I-K) Representative images showing MMP2::GFP expression (green) in control (I), UAS-yanAct (J) or UAS-yanWT (K) mature follicles with 44E10>RFP (red). We noticed that the RFP signal (red arrows) is located more toward the oocyte membrane in UAS-yanAct and UAS-yanWT mature follicles. The insets show higher magnifications of MMP2::GFP expression (white) in boxed areas. Oocytes are outlined in cyan, nuclei are labeled with DAPI in blue, and arrowheads point to posterior follicle cells. (L) Quantification of MMP2::GFP expression in control, UAS-yanAct and UAS-yanWT mature follicles. The number of egg chambers analyzed is noted above each bar. (M-O) Quantification of OA-induced posterior gelatinase activity (M), OA-induced follicle rupture (N), and ionomycin-induced follicle rupture (O) in control, UAS-yanAct and UAS-yanWT mature follicles. All quantifications were performed after a 3-h culture. Mature follicles were isolated according to 44E10>RFP expression. The number of mature follicles analyzed is noted above each bar. ***P<0.001 (one-way ANOVA with post-hoc Tukey test). NS, not significant. Error bars represent s.d. Scale bars: 50 µm.

The downregulation of Yan at stage 14 prompted us to investigate whether this downregulation is required for Pnt-regulated MMP2 expression. We utilized 44E10-Gal4 to overexpress either a wild-type Yan (yanWT) or a constitutively active Yan (yanAct), in which the phosphoacceptor residues of all eight consensus sites were replaced with a non-phosphorylatable alanine (Rebay and Rubin, 1995), in stage-14 follicle cells and analyzed MMP2 expression. MMP2::GFP expression was significantly reduced in stage-14 posterior follicle cells with overexpression of yanAct but not yanWT (Fig. 4I-L, Table S1). Antibody staining showed that Yan protein was detected at high levels in early stage-14 follicle cells with overexpression of either yanAct or yanWT (Fig. S3A-B′,E-F′); however, YanWT was readily degraded in late stage-14 follicle cells, whereas YanAct persisted robustly in the anterior and posterior regions of the late stage-14 egg chambers (Fig. S3C-D′,G-H′). This difference in the extended expression of posterior Yan between yanAct and yanWT may explain why only overexpression of yanAct is sufficient to disrupt MMP2::GFP expression. In addition, we used the Flip-out/Gal4 system (Pignoni and Zipursky, 1997) to randomly generate clones that have Gal4 drive yanAct overexpression. We found that yanAct-overexpressing clone cells at the posterior end of mature follicles showed no MMP2::GFP expression (Fig. S4A-A″; 10/10 clones). Altogether, our data indicate that downregulation of Yan at stage 14 is required for the upregulation of MMP2.

Consistent with this conclusion, we also observed that mature follicles with overexpression of yanAct showed a significant reduction of posterior gelatinase activity after OA stimulation (Fig. 4M), OA-induced follicle rupture (Fig. 4N) and ionomycin-induced follicle rupture (Fig. 4O). Surprisingly, we found that mature follicles with overexpression of yanWT also showed similar defects to yanAct, although less severe (Fig. 4M-O).

Next, we investigated whether Yan expression at stage 13 is required for the prevention of precocious MMP2 expression. We knocked down yan at stage 13 with Vm26Aa-Gal4 driving yan-RNAi expression. Owing to difficulty in precisely determining the stage of the egg chambers, we were unable to conclude whether precocious MMP2 expression occurred in yan-knockdown egg chambers. Therefore, we used the Flip-out/Gal4 system again to knock down yan expression; this approach provides internal wild-type controls and is much more sensitive. We observed that MMP2::GFP was precociously upregulated in yan-knockdown clone cells, but not in adjacent wild-type posterior follicle cells at stage 13 (Fig. S4B-B″; 5/5 clones). This result suggests that Yan at stage 13 likely plays a role in preventing Pnt activity and precocious MMP2 upregulation. Altogether, our data indicate that Pnt and Yan work together to regulate MMP2 expression precisely in stage-14 posterior follicle cells.

A 1.1 kb enhancer is responsible for precise MMP2 expression in late oogenesis

Next, we tested whether Pnt and Yan regulate Mmp2 transcription directly. It is difficult to use genomic assays, such as chromatin immunoprecipitation or CUT&RUN (Meers et al., 2019), to determine whether Pnt and Yan bind to Mmp2 enhancers, because there are fewer than 30 follicle cells expressing Pnt in each mature follicle. Therefore, we decided to take the traditional approach of dissecting the enhancer region of the Mmp2 gene to find the transcription factor-binding sites.

We cloned the upstream 5.1 kb intergenic region of Mmp2 (Fig. 5A), which possibly contains Mmp2 enhancers for MMP2 expression, and inserted it into the pRed-SA reporter vector (Le Poul et al., 2020) to detect the enhancer activity by nuclear dsRed reporter expression. Consistent with our prediction, dsRed was consistently detected in anterior follicle cells from stages 10A to 14 (Fig. 5B,D,F,H; n>50 egg chambers). In contrast, dsRed began to be detected in posterior tip follicle cells at early stage 14 and reached peak expression with a posterior-to-anterior concentration gradient pattern at late stage 14 (Fig. 5C,E,G,I). The observed expression pattern entirely mirrored that of MMP2::GFP expression (Fig. S1), suggesting that the 5.1 kb fragment, denoted as ME.full, encompasses all the enhancers necessary for Mmp2 expression during late oogenesis.

Fig. 5.

Identification of the core enhancer for MMP2 expression in late oogenesis. (A) Schematic illustrating the Mmp2 genomic region and different enhancer reporters. The genomic locus of Mmp2 and its adjacent gene Uba1 are depicted. A 5.1 kb fragment upstream of Mmp2 (ME.full), as well as truncated fragments from ME.full (ME. T-1, ME. T-2, ME. T-3 and ME.core) is integrated into upstream of dsRed (red), nuclear localization signal (NLS, cyan) and SV40 terminator (gray) in the pRedSA vector. The expression patterns of these enhancer reporters are depicted at the right. (B-I) Expression of ME.full-dsRed reporter (red) in both anterior (a) and posterior (p) ends of egg chambers during late oogenesis. The insets show higher magnifications of ME.full-dsRed expression (white) in boxed areas. The specific stage of each egg chamber is labeled, and early, mid and late stage 14 were determined according to Hnt expression, nurse cell nuclei, and dorsal appendage morphology, as in our previous study (Deady et al., 2017). (J-Q) Expression of ME. T-1-dsRed (red in J,K), ME. T-2-dsRed (red in L,M), ME. T-3-dsRed (red in N,O) and ME.core-dsRed (red in P,Q) in both anterior and posterior end of egg chambers at late stage 14. Insets show higher magnifications of dsRed expression (white) in boxed areas. Nuclei are labeled with DAPI and shown in blue. Each panel represents >30 egg chambers. Scale bars: 50 μm.

Fig. 5.

Identification of the core enhancer for MMP2 expression in late oogenesis. (A) Schematic illustrating the Mmp2 genomic region and different enhancer reporters. The genomic locus of Mmp2 and its adjacent gene Uba1 are depicted. A 5.1 kb fragment upstream of Mmp2 (ME.full), as well as truncated fragments from ME.full (ME. T-1, ME. T-2, ME. T-3 and ME.core) is integrated into upstream of dsRed (red), nuclear localization signal (NLS, cyan) and SV40 terminator (gray) in the pRedSA vector. The expression patterns of these enhancer reporters are depicted at the right. (B-I) Expression of ME.full-dsRed reporter (red) in both anterior (a) and posterior (p) ends of egg chambers during late oogenesis. The insets show higher magnifications of ME.full-dsRed expression (white) in boxed areas. The specific stage of each egg chamber is labeled, and early, mid and late stage 14 were determined according to Hnt expression, nurse cell nuclei, and dorsal appendage morphology, as in our previous study (Deady et al., 2017). (J-Q) Expression of ME. T-1-dsRed (red in J,K), ME. T-2-dsRed (red in L,M), ME. T-3-dsRed (red in N,O) and ME.core-dsRed (red in P,Q) in both anterior and posterior end of egg chambers at late stage 14. Insets show higher magnifications of dsRed expression (white) in boxed areas. Nuclei are labeled with DAPI and shown in blue. Each panel represents >30 egg chambers. Scale bars: 50 μm.

To identify the minimal core enhancer element responsible for MMP2 expression, a series of deletion constructs were generated (Fig. 5A; see Materials and Methods). Among these constructs, the proximal 2.6 kb fragment (ME. T-1) exhibited an identical expression profile to the ME.full construct (Fig. 5J,K). However, the proximal 1.5 kb fragment (ME. T-2) did not show any expression during late oogenesis (Fig. 5L,M). Furthermore, the distal 3.6 kb fragment (ME. T-3) displayed an expression pattern comparable to the ME.full reporter (Fig. 5N,O). At least 30 egg chambers were observed in each case and showed the same expression pattern. These findings indicate that the core enhancer element lies within the 1.1 kb overlapping region between ME. T-1 and ME. T-3. Consequently, we cloned this core enhancer (designated ME.core) and confirmed its activity in late oogenesis, with a similar expression pattern to that of ME.full (Fig. 5P,Q). Collectively, these results suggest that the 1.1 kb ME.core serves as the core enhancer responsible for regulating the spatiotemporal expression of MMP2 during late oogenesis.

Pnt and Yan regulate ME.core activity in late oogenesis

Upon identification of the core enhancer responsible for regulating MMP2 expression, we set out to determine whether the ME.core activity is regulated by Pnt and Yan. When pnt was knocked down in stage-14 follicle cells with 44E10-Gal4, there was a significant reduction of the ME.core reporter expression at the posterior end of mature follicles (Fig. 6A-C′). In addition, we found that overexpression of either Pnt.P1 or Pnt.P2 was sufficient to induce ectopic ME.core-dsRed expression in main-body follicle cells at stage 14 (Fig. 6D-F), whereas only overexpression of Pnt.P1 was sufficient to induce precocious ME.core expression before stage 14 (Fig. S5). Furthermore, the expression of the ME.core-dsRed reporter was also drastically reduced in posterior follicle cells with overexpression of yanAct but not yanWT at stage 14 (Fig. 6G-I′, Fig. S4C-C″). Finally, knockdown of yan led to precocious upregulation of the ME.core-dsRed reporter at stage 13 (Fig. S4D-E″). At least 30 mature follicles and ten Flip-out/Gal4 clones were analyzed for each condition, and all showed the same phenotype. All these results are consistent with the role of Pnt and Yan in regulating MMP2::GFP expression. Therefore, we conclude that Pnt and Yan regulate the ME.core enhancer either directly or indirectly to control the precise expression of MMP2 in posterior follicle cells at stage 14.

Fig. 6.

ME.core reporter expression in mature follicles with altered expression of Pnt and Yan. (A-C′) Representative images showing the expression of ME.core-dsRed (red in A-C and white in A′-C′) and Pnt.P1 (green in A-C) in control (A), pntRNAi1 (B) and pntRNAi2 (C) mature follicles with 44E10-Gal4. (D-F) Representative images showing the expression of ME.core-dsRed (red in D-F) and Pnt.P1 (green in D-F) in control (D), UAS-Pnt.P1 (E), and UAS-Pnt.P2 (F) mature follicles with 44E10-Gal4. Insets show higher magnifications of ME.core-dsRed expression (white) in boxed areas. (G-I′) Representative images showing the expression of ME.core-dsRed (red in G-I and white in G′-I′) and Hnt (green in G-I) in control (G), UAS-yanAct (H) and UAS-yanWT (I) mature follicles with 44E10-Gal4. Nuclei are labeled with DAPI and shown in blue. Each panel represents >30 egg chambers. Scale bars: 50 μm.

Fig. 6.

ME.core reporter expression in mature follicles with altered expression of Pnt and Yan. (A-C′) Representative images showing the expression of ME.core-dsRed (red in A-C and white in A′-C′) and Pnt.P1 (green in A-C) in control (A), pntRNAi1 (B) and pntRNAi2 (C) mature follicles with 44E10-Gal4. (D-F) Representative images showing the expression of ME.core-dsRed (red in D-F) and Pnt.P1 (green in D-F) in control (D), UAS-Pnt.P1 (E), and UAS-Pnt.P2 (F) mature follicles with 44E10-Gal4. Insets show higher magnifications of ME.core-dsRed expression (white) in boxed areas. (G-I′) Representative images showing the expression of ME.core-dsRed (red in G-I and white in G′-I′) and Hnt (green in G-I) in control (G), UAS-yanAct (H) and UAS-yanWT (I) mature follicles with 44E10-Gal4. Nuclei are labeled with DAPI and shown in blue. Each panel represents >30 egg chambers. Scale bars: 50 μm.

Pnt and Yan directly regulate the expression of MMP2 in mature follicles

Lastly, we examined whether Pnt/Yan directly bind to the ME.core to regulate MMP2 expression. To address this, we took several approaches to predict the Pnt/Yan-binding sites in the ME.core. First, we used the Pnt binding motif identified through direct assays (Nitta et al., 2015; Zhu et al., 2011) to search for the potential Pnt-binding sites in the ME.core enhancer element using the Catalog of Inferred Sequence Binding Preferences (CIS-BP) database (Weirauch et al., 2014), which resulted in 11 binding sites (Fig. S6, Table S2). Among them, six binding sites (ETS1-ETS3 and ETS6-ETS8) contained the known Pnt/Yan consensus core binding motif 5′-GGAA/T-3′ (Boisclair Lachance et al., 2018; Hollenhorst et al., 2011). Because ETS1 and ETS2 had the highest binding score, we hypothesized that ETS1 and ETS2 are potential Pnt-binding sites. To test this hypothesis, we generated ME.core reporters with a mutation in ETS1, ETS2, or both, following the rationale used to study Pnt-binding sites at the eve gene locus (Boisclair Lachance et al., 2018; Halfon et al., 2000; Fig. 7A). By quantifying the dsRed intensity in follicle cells at the posterior tip (magenta circles in Fig. 7B′-E′) and slightly anterior regions (green circles in Fig. 7B′-E′), we found a significant reduction of reporter expression in mature follicles with either mutant ETS1, ETS2, or ETS1+ETS2 double mutation (Fig. 7B-G, Table S3). Notably, the ETS1 mutant ME.core reporter (ME.core-Δ1) showed a stronger reduction than the ETS2 mutant ME.core reporter (ME.core-Δ2), and there was no further decrease in ETS1/2 double mutant ME.core reporter (ME.core-Δ1+2) in comparison with the ME.core-Δ1 reporter. Altogether, our data strongly suggest that Pnt directly binds to ETS1- and ETS2-binding sites of the 1.1 kb ME.core enhancer to regulate MMP2 expression in posterior follicle cells in late oogenesis in conjunction with Yan (Fig. 7H).

Fig. 7.

ETS-binding motifs in ME.core are critical for the enhancer activity in mature follicles. (A) Schematic depicting the mutational analysis of ETS-binding sites in ME.core. The location and sequence of two predicted ETS-binding sites (ETS1 and ETS2) are shown, and the ETS core binding sequence (GGAA/T) is highlighted in blue. The mutated sequences are labeled and underlined in red. ME.core-Δ1, ME.core- Δ2 and ME.core- Δ1+2 are ME.core with ETS1, with ETS2, and with ETS1/2 mutation, respectively. (B-E′) Representative images showing the expression of ME.core-dsRed (red in B and white in B′), ME.core-Δ1-dsRed (red in C and white in C′), ME.core-Δ2-dsRed (red in D and white in D′), and ME.core-Δ1+2-dsRed (red in E and white in E′) in mature follicles. The square and rectangular insets in B′-E′ show higher magnifications of dsRed expression in follicle cells of the corresponding boxed areas. Nuclei are labeled with DAPI and shown in blue. The nuclei outlined in magenta and green were used for dsRed quantification. These nuclei are from follicle cells at the most posterior tip and the second or third row towards the anterior, respectively. (F,G) Quantification of the dsRed intensity in the nuclei of follicle cells at the posterior tip (G) and the second or third row towards the anterior (F). ***P<0.001 (one-way ANOVA with post-hoc Tukey test). Error bars represent s.d. A.U., arbitrary units. (H) Diagram showing the regulation of MMP2 expression in late oogenesis. At late stage 13 and early stage 14, Yan inhibits MMP2 transcription by binding to the ME.core region and preventing Pnt activity in posterior follicle cells. At mid-stage 14, Yan is downregulated, and Pnt can occupy the ME.core region and induce MMP2 expression, likely with the help of Hnt. By late stage 14, MMP2 is robustly expressed and responds to OA/Oamb signaling to mediate follicle rupture. The coloured circles represent follicle cell nuclei, with the specific transcription factor indicated by the color. Each panel represents >30 egg chambers. Scale bars: 20 μm.

Fig. 7.

ETS-binding motifs in ME.core are critical for the enhancer activity in mature follicles. (A) Schematic depicting the mutational analysis of ETS-binding sites in ME.core. The location and sequence of two predicted ETS-binding sites (ETS1 and ETS2) are shown, and the ETS core binding sequence (GGAA/T) is highlighted in blue. The mutated sequences are labeled and underlined in red. ME.core-Δ1, ME.core- Δ2 and ME.core- Δ1+2 are ME.core with ETS1, with ETS2, and with ETS1/2 mutation, respectively. (B-E′) Representative images showing the expression of ME.core-dsRed (red in B and white in B′), ME.core-Δ1-dsRed (red in C and white in C′), ME.core-Δ2-dsRed (red in D and white in D′), and ME.core-Δ1+2-dsRed (red in E and white in E′) in mature follicles. The square and rectangular insets in B′-E′ show higher magnifications of dsRed expression in follicle cells of the corresponding boxed areas. Nuclei are labeled with DAPI and shown in blue. The nuclei outlined in magenta and green were used for dsRed quantification. These nuclei are from follicle cells at the most posterior tip and the second or third row towards the anterior, respectively. (F,G) Quantification of the dsRed intensity in the nuclei of follicle cells at the posterior tip (G) and the second or third row towards the anterior (F). ***P<0.001 (one-way ANOVA with post-hoc Tukey test). Error bars represent s.d. A.U., arbitrary units. (H) Diagram showing the regulation of MMP2 expression in late oogenesis. At late stage 13 and early stage 14, Yan inhibits MMP2 transcription by binding to the ME.core region and preventing Pnt activity in posterior follicle cells. At mid-stage 14, Yan is downregulated, and Pnt can occupy the ME.core region and induce MMP2 expression, likely with the help of Hnt. By late stage 14, MMP2 is robustly expressed and responds to OA/Oamb signaling to mediate follicle rupture. The coloured circles represent follicle cell nuclei, with the specific transcription factor indicated by the color. Each panel represents >30 egg chambers. Scale bars: 20 μm.

The precise regulation of MMP expression and activity is crucial for extracellular matrix homeostasis, organ health, and physiological functions. However, our understanding of the transcriptional regulation of MMP in multiple organisms is very limited. In our previous work, we demonstrated the spatiotemporal expression of MMP2 in posterior follicle cells of stage-14 egg chambers in the Drosophila ovary and uncovered its essential role in follicle rupture and ovulation. We also identified a zinc-finger transcription factor, Hnt, that is upregulated in stage-14 follicle cells and is required for MMP2 expression in posterior follicle cells (Deady et al., 2017). However, Hnt is expressed in all follicle cells, whereas MMP2 is restricted to posterior follicle cells. Determining the factors restricting MMP2 expression in posterior follicle cells remains a fundamental question in the field of reproduction. We have identified roles for the ETS-family transcriptional regulators Pnt and Yan in the precise regulation of MMP2 expression in posterior follicle cells (Fig. 7H). The restricted posterior expression of Pnt, which functions as a key activator of MMP2 expression, provides a spatial constriction of MMP2 expression in posterior follicle cells. In addition, the transient expression of Yan in stage-13 follicle cells prevents the premature expression of MMP2, which ensures that MMP2 is only turned on in stage-14 posterior follicle cells. As a twofold ratio change of Pnt/Yan can robustly affect Pnt activity in eye discs (Bernasek et al., 2023), it is highly likely that the upregulation of Yan antagonizes Pnt transcriptional activity in stage-13 egg chambers to prevent premature MMP2 expression.

It is unclear why Pnt could not induce MMP2 expression before stage 13 in posterior follicle cells given that Pnt is expressed in these cells throughout late oogenesis, whereas Yan is only turned on at stage 13. A possible explanation is that additional transcription factors are required for MMP2 expression in conjunction with Pnt. A promising candidate is Hnt, which is only turned on at stage 14 and is required for MMP2 expression (Deady et al., 2015, 2017). Consistent with this idea, the premature upregulation of MMP2 by Pnt.P1 overexpression was accompanied by the upregulation of Hnt before stage 14 (Fig. S2F). Future work will be required to determine whether Hnt directly binds to the MMP2 enhancer and/or forms a transcriptional complex with Pnt.

Through classical enhancer analysis, we identified a 1.1 kb core enhancer element for Mmp2 expression in late oogenesis in Drosophila. By introducing mutations in the potential Pnt/Yan-binding sites (ETS1 and ETS2), the core enhancer activity was significantly impaired (Fig. 7B-G). The ETS1 site seems to play major roles compared with the ETS2 site, which is consistent with the fact that the ETS1 site is highly conserved across the 14 sequenced Drosophila species (Fig. S6B). Alternatively, the mild reduction in ME.core-Δ2 reporter might be due to an insufficient block of Pnt binding as there are additional GGAA core motifs that are not disrupted by the introduced mutation. In any case, none of the mutations led to a complete blockade of core enhancer activity, as weak dsRed expression was still detected in posterior follicle cells in the mutation analysis. A possible explanation is that additional Pnt/Yan-binding sites, such as ETS3 (highly conserved and similar to ETS1), may work cooperatively with ETS1/ETS2 sites to allow Pnt/Yan binding and regulation of Mmp2 enhancer activity, similar to a previously reported mechanism of eve gene regulation (Boisclair Lachance et al., 2018). Additionally, other transcription factors may bind to the core enhancer to regulate enhancer activity. Nevertheless, our experimental data provide strong support to our conclusion that Pnt directly regulates MMP2 expression in posterior follicle cells in late oogenesis in Drosophila.

The ME.core enhancer element we identified reflects the expression of endogenous MMP2 in both anterior stretch follicle cells and posterior follicle cells of egg chambers. It is unlikely that Pnt regulates ME.core and MMP2 expression in anterior stretch follicle cells because Pnt is not detected in stretch follicle cells (Boisclair Lachance et al., 2014). Future work is required to further truncate the ME.core sequence to identify the minimal element and transcription factors for MMP2 expression in anterior stretch follicle cells, which would lead to improved understanding of the more complex regulatory network of MMP2 expression in Drosophila ovary and beyond. Although the ME.core completely reflects the anterior and posterior MMP2 expression in the ovaries, we noticed that there are some basal dsRed signals detected in main-body follicle cells at late stage 14 from ME.core and ME. T-3 reporters, but not in ME.full and ME. T-1 reporters (Fig. S7). This suggests that the 1.5 kb proximal sequence upstream of the Mmp2 transcriptional start site contains some DNA elements that suppress random transcriptional activity to ensure that MMP2 expression is restricted to anterior and posterior follicle cells.

Previous studies have focused on the antagonistic paradigm between the activator Pnt and the repressor Yan in various developmental processes. In the absence of EGFR-Ras-MAPK signaling, Yan outcompetes Pnt to repress the expression of target genes. Transduction of this signaling pathway leads to the phosphorylation and degradation of Yan while concomitantly inducing phosphorylation and activation of Pnt.P2 (Brunner et al., 1994; Gabay et al., 1996; O'Neill et al., 1994; Rebay and Rubin, 1995). It is interesting to note that overexpression of Pnt.P2 led to a much weaker effect on the upregulation of MMP2 than did overexpression of Pnt.P1, even in stage-14 follicle cells. This is consistent with observations in many other systems and could be because Pnt.P2 has limited transcription activity without being phosphorylated by MAPK (O'Neill et al., 1994; Qiao et al., 2006; Shwartz et al., 2013; Tootle et al., 2003). We also observed that ectopic YanWT is degraded more robustly than YanAct in stage-14 follicle cells. This implies that there is potential MAPK activity in stage-14 follicle cells to phosphorylate and degrade endogenous Yan. Future work will be required to determine whether MAPK activity is detected in stage-14 follicle cells and whether they are required for MMP2 expression and follicle rupture.

It is worth mentioning that the EGFR-Ras-MAPK signaling pathway plays crucial roles downstream of the luteinizing hormone surge to induce ovulation in mammals (Hsieh et al., 2007; Panigone et al., 2008). In addition, multiple MMPs are expressed in somatic follicle cells of mammalian (and human) preovulatory follicles (Curry and Osteen, 2003; McCord et al., 2012; Rosewell et al., 2015), and MMP activity is detected at the apex of ovulatory follicles right before follicle rupture (Curry et al., 2001), which is essential for ovulation (Curry and Osteen, 2003). However, the transcription factors downstream of MAPK required to induce ovulation remain unknown, and whether the EGFR-Ras-MAPK signaling pathway regulates MMP expression in somatic follicle cells has not yet been determined. Our work in Drosophila shows that Pnt, one of the downstream transcription factors of the EGFR-Ras-MAPK pathway, regulates Drosophila MMP2 expression in somatic follicle cells of stage-14 egg chambers and, thus, follicle rupture. As Pnt is homologous to mammalian ETS1 and ETS2, whereas Yan is homologous to mammalian ETS variant 6 (Etv6) (Roukens et al., 2008), our work in Drosophila suggests that ETS1, ETS2 and Etv6 may play a role in regulating MMP expression in mammalian preovulatory follicles downstream of the luteinizing hormone surge. In fact, it has been shown that ETS1 and ETS2 regulate MMP expression in human NIH3T3 fibroblasts, neuroblastoma cells and squamous carcinoma cells, indicating that ETS-MMP regulation is involved in cancer progression (Behrens et al., 2001; Taki et al., 2006; Westermarck et al., 1997). Therefore, the role of ETS transcription factors in regulating MMP expression may be conserved across species and in multiple organ systems, which awaits future exploration.

Drosophila genetics and clone induction

Flies were reared on standard cornmeal and molasses food at 25°C, except noted otherwise. All RNAi-knockdown experiments except those with the Flip-out/Gal4 system were performed at 29°C with UAS-dcr2 to increase the efficiency of the gene knockdown. Experiments with gene overexpression were also performed at 29°C. For pnt expression analysis, pnt-GFP [Bloomington Drosophila Stock Center (BDSC), stock #42680], a functional pnt transgene tagged with GFP-FLAG-PreScission-TEV-BLRP at the C terminus (Boisclair Lachance et al., 2014), was used. The protein trap line Mmp2::GFP/CyO (Deady et al., 2015) was generated using the recombinase-mediated cassette exchange method (Nagarkar-Jaiswal et al., 2015) and used for analyzing MMP2 expression. Vm26Aa-Gal4 (Peters et al., 2013) and 44E10-Gal4 (Deady et al., 2017) were used to drive gene or RNAi expression in follicle cells starting at stages 10 and 14, respectively. Three RNAi lines were used: UAS-pntRNAi1 (BDSC, B35038), UAS-pntRNAi2 (Vienna Drosophila Resource Center, V105390) and UAS-yanRNAi (BDSC, B34909). The following overexpression lines from the BDSC were used: UAS-Pnt.P1 (B869), UAS-Pnt.P2 (B399), UAS-yanAct (B5789) and UAS-yanWT (B5790). Isolation and identification of stage-14 mature follicles for ex vivo culture assays were performed using either Oamb-RFP (Knapp et al., 2019) or UAS-RFP driven by 44E10-Gal4. For the Mmp2 enhancer-related experiments, Vm26Aa-Gal4, UAS-dcr2; ME.core-dsRed and w; ME.core-dsRed;44E10-Gal4, UAS-dcr2 were used to cross with corresponding RNAi or overexpression stocks. Control flies for all experiments were prepared by crossing Gal4 drivers to Oregon-R wild-type flies.

For Flip-out/Gal4 clone generation (Pignoni and Zipursky, 1997), hsFLP; Mmp2::GFP/CyO; act<CD2<Gal4, UAS-RFP was crossed to UAS-yanRNAi and UAS-yanAct. For generating the ME.core reporter-related Flip-out/Gal4 clones, hsFLP; ME.core-dsRed; act<CD2<Gal4, UAS-GFP was crossed to UAS-yanRNAi and UAS-yanAct. For clone induction, 2- to 3-day-old adult female flies were heat-shocked in a 37°C water bath for 10 min (for hsFLP; Mmp2::GFP/Cyo; act<CD2<Gal4, UAS-RFP line) or 45 min (for hsFLP; ME.core-dsRed/Cyo, act<CD2<Gal4, UAS-GFP line) and raised with males in wet-yeasted vials for 2-3 days before dissection. The clone cells were marked by act-Gal4 driving UAS-RFP or UAS-GFP expression.

Immunostaining and microscopy

Immunostaining was performed following a standard procedure (Sun and Spradling, 2012). The following primary antibodies were used: mouse anti-Yan (1:10; Developmental Study Hybridoma Bank, 8B12H9; Rebay and Rubin, 1995), mouse anti-Hnt (1:75; Developmental Study Hybridoma Bank, 1G9) (Yip et al., 1997), rabbit anti-Pnt.P1 (1:400; a gift from Dr James Skeath, Washington University School of Medicine, St. Louis, MO, USA) (Alvarez et al., 2003), rabbit anti-GFP (1:4000; Invitrogen, A11122), mouse anti-GFP (1:1000; Invitrogen, 3E6) and rabbit anti-RFP (1:2000, MBL International, PM005). Alexa Fluor 488 and Alexa Fluor 568 goat secondary antibodies [1:1000; Invitrogen, 488-mouse (A11001), 488-rabbit (A11008), 568-mouse (A11004), 568-rabbit (A11011)] were used. Images were acquired using a Leica TCS SP8 confocal microscope or Leica MZ10F fluorescent stereoscope with a sCOMS camera (PCO. Edge) and assembled using Photoshop software (Adobe) and ImageJ. Confocal microscope settings for imaging were kept consistent for each set of experiment.

Quantification of MMP2::GFP and ME-dsRed expression

For the MMP2::GFP expression quantification in RNAi experiments (Figs 2H, 4L, Fig. S2D), we categorized MMP2::GFP expression into three levels: high, medium and low/no as in our previous studies (Fig. 2D) (Deady et al., 2017; Knapp et al., 2019; Oramas et al., 2023). We first identified late stage-14 mature follicles using the 44E10-Gal4/UAS-RFP or Oamb-RFP expression, DAPI signal, and egg morphology, and then scored the MMP2::GFP expression level. Researchers were unaware of the sample genotypes during scoring to prevent bias. Three biological replicates were carried out for each experiment and data were combined. The percentage of mature follicles with MMP2::GFP expression in each category was calculated and plotted.

For MMP2::GFP expression quantification in gain-of-function analysis (Fig. 2L), ten stage-14 egg chambers from each condition were randomly picked and imaged using the Leica SP8 confocal microscope with the same settings. Quantification of MMP2::GFP intensity was then carried out using ImageJ. We subtracted the background of each image using the rolling ball feature (setting the rolling ball radius to 50 pixels), and then measured the mean intensity of three randomly selected regions of main-body follicle cells for each follicle (see also Table S1).

For ME-dsRed expression quantification in ETS mutation experiments (Fig. 7F,G), ten late stage-14 egg chambers from each condition were randomly picked and imaged with the same Leica SP8 confocal scope settings. Early and late stage-14 egg chambers are equivalent to stage-14A and stage-14C egg chambers, respectively, defined in our previous study (Deady et al., 2017). In short, late stage-14 mature follicles were characterized by medium levels of Hnt in follicle cell nuclei, fully extended dorsal appendages, and the absence of nurse cell nuclei. By contrast, early stage-14 egg chambers only display anterior and posterior nuclear expression of Hnt, have a slightly shorter dorsal appendage, and have some nurse cell nuclei remnants. Quantification of the dsRed intensity of different reporters was carried out using ImageJ as described above. We selected the nuclei of five follicle cells at the posterior tip and at a slightly anterior region. In total, 50 follicle cells (five cells per mature follicle) were measured per region per genotype.

Ex vivo follicle rupture and gelatinase assay

The ex vivo follicle rupture assay was performed as previously described (Beard et al., 2023; Deady and Sun, 2015; Knapp et al., 2018). In brief, ovaries were dissected from 5- to 6-day-old virgin females fed with wet yeast for 3 days, and stage-14 mature follicles with an intact follicle cell layer were isolated in Grace's insect medium (Caisson Labs) and separated into groups of about 30. These mature follicles were cultured at 29°C for 3 h in culture medium (Grace's insect medium+10% fetal bovine serum+1% penicillin-streptomycin) supplemented with 20 μM OA (Sigma-Aldrich) or 2 μM ionomycin (Cayman Chemical Company). Each data point represents the percentage of ruptured mature follicles per group with data represented as mean±s.d.

In situ zymography for detecting gelatinase activity (MMP2 activity) was performed as previously described (Deady and Sun, 2015; Deady et al., 2017). Following the isolation of mature follicles as described above, 20 μM OA and 10 μg/ml of DQ-gelatin conjugated with fluorescein (Invitrogen) were added to the culture media and cultured in 29°C for 3 h. Fluorescein is quenched by DQ-gelatin (the substrate of gelatinase) and emits a fluorescent signal after DQ-gelatin is broken down by gelatinases. After 3 h, mature follicles with posterior fluorescein signal were counted, and data were represented as the percentage of mature follicles with posterior green fluorescein signal (Deady et al., 2015).

Mmp2 enhancer analysis and transgenic animals

A 5.1 kb genomic DNA upstream of the Mmp2 transcription starting site was amplified using PCR from genomic DNA extracted from Oregon-R flies. The PCR products with KpnI and BamHI restriction sites at the terminal were ligated into the pENTR-TOPO vector (Thermo Fisher Scientific) and confirmed by Sanger sequencing. The 5.1 kb Mmp2 enhancer (named ‘ME.full’) was then subcloned into a pRedSA vector (a kind gift from Dr Nicolas Gompel) (Le Poul et al., 2020) by utilizing KpnI and BamHI to obtain pRedSA-ME.full vector. For generating truncated Mmp2 enhancer vectors, specific fragments in pRedSA-ME.full vector were deleted with specific restriction enzyme pairs, and the remaining vector was re-ligated after blunting. PacI+KpnI, PashAI+KpnI and PashAI+BamHI were used to obtain ME. T-1 (∼2.6 kb), ME. T-2 (∼1.5 kb) and ME. T-3 (∼3.6 kb), respectively (Fig. 5A). The ME.core fragment (∼1.1 kb), ME.core-Δ1, ME.core-Δ2 and ME.core-Δ1+2 were amplified using traditional PCR or overlapping PCR and inserted into the pRedSA vector by utilizing similar strategies as for ME.full. All mutated enhancer sequences were confirmed by Sanger sequencing. All primers used are listed in Table S4.

To generate transgenic Mmp2 enhancer reporter flies (ME.full-dsRed, ME. T-1-dsRed, ME. T-2-dsRed, ME. T-3-dsRed, ME.core-dsRed, ME.core-Δ1-dsRed, ME.core-Δ2-dsRed and ME.core-Δ1+2-dsRed), ɸC31-mediated transgenesis was applied through a service provided by BestGene Inc. by following a standard protocol. All constructs were integrated at the genomic attP site VK00016 on chromosome 2 (Le Poul et al., 2020).

ETS-binding sites prediction and conservation analysis

To predict the potential binding sites of Pnt/Yan in the 1.1 kb ME.core sequence, we performed motif scanning and prediction using the CIS-BP website. We executed the motif scan of the ME.core sequence using directly determined binding motifs of Pnt (from Drosophila melanogaster, CIS-BP ID: T177272_2.00) using the default setting. Detailed information of predicted ETS-binding sites is summarized in Table S2.

Conservation analysis of predicted ETS-binding sites was performed using the UCSC genome browser. The phyloP and phastCons scores along the genomic locus (Chr2R:9686506..9685375) of ME.core were presented to investigate the conservation of each ETS-binding site at both single base and species level. The detailed sequence alignment of ETS-binding sites among 14 Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. suzukii, D. ananassae, D. eugracilis, D. elegans, D. takahashii, D. rhopaloa, D. ficusphila, D. psedoobscura, D. persimilis) with relative intact genome assembly was performed to analyze the detailed sequence conservation.

Statistical analysis

Statistical tests were performed using Prism 9 (GraphPad). Sample sizes were chosen based on previous research experience. For expression analysis, at least 30 egg chambers from more than ten flies were examined. For quantification between control and mutant, three biological repeats were used.

Quantification results are presented as mean±s.d. as indicated. For comparison of more than two means, one-way ANOVA with post-hoc Tukey's test was used. For comparison of distribution, Chi-square test was used.

We thank Drs Wu-Min Deng, Allan Spradling, James Skeath and Nicolas Gompel for sharing fly lines, vectors and other reagents; Bloomington Drosophila Stock Center and Vienna Drosophila Resource Center for fly stocks; and Developmental Studies Hybridoma Bank for antibodies. We appreciate constructive comments from anonymous reviewers. We also appreciate the excellent support from UConn Imaging Core facility. The Leica SP8 confocal microscope is supported by an NIH Award (S10OD016435) to Akiko Nishiyama.

Author contributions

Conceptualization: J.S.; Methodology: B.Z., E.M.K., E.S., J.S.; Validation: B.Z., R.O.; Formal analysis: B.Z., E.M.K., E.S.; Investigation: B.Z., E.M.K., E.S., J.S.; Resources: J.S.; Data curation: B.Z., E.M.K., E.S., R.O.; Writing - original draft: B.Z., E.M.K., J.S.; Writing - review & editing: B.Z., E.M.K., E.S., R.O., J.S.; Visualization: B.Z., E.M.K., E.S., R.O.; Supervision: J.S.; Project administration: J.S.; Funding acquisition: J.S.

Funding

J.S. is supported by the University of Connecticut Start-Up Fund, and National Institute of Child Health and Human Development grants (R01-HD086175 and R01-HD097206). Deposited in PMC for release after 12 months.

Data availability

All relevant data can be found within the article and its supplementary information.

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Competing interests

The authors declare no competing or financial interests.