Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair

  1. Jingqiang Wang
  2. Chunye Liu
  3. Lingli He
  4. Zhiyao Xie
  5. Lanyue Bai
  6. Wentao Yu
  7. Zuoyun Wang
  8. Yi Lu
  9. Dong Gao
  10. Junfen Fu  Is a corresponding author
  11. Lei Zhang  Is a corresponding author
  12. Yi Arial Zeng  Is a corresponding author
  1. Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, National Children’s Regional Medical Center, China
  2. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China
  3. Human Anatomy & Histoembryology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, China
  4. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, China
  5. School of Life Science and Technology, ShanghaiTech University, China

Abstract

Ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and OSE stem cells rapidly generate new cells for the repair. How the stem cell activation is triggered by the rupture and promptly turns on proliferation is unclear. Our previous study has identified that Protein C Receptor (Procr) marks OSE progenitors. In this study, we observed decreased adherent junction and selective activation of YAP signaling in Procr progenitors at OSE rupture site. OSE repair is impeded upon deletion of Yap1 in these progenitors. Interestingly, Procr+ progenitors show lower expression of Vgll4, an antagonist of YAP signaling. Overexpression of Vgll4 in Procr+ cells hampers OSE repair and progenitor proliferation, indicating that selective low Vgll4 expression in Procr+ progenitors is critical for OSE repair. In addition, YAP activation promotes transcription of the OSE stemness gene Procr. The combination of increased cell division and Procr expression leads to expansion of Procr+ progenitors surrounding the rupture site. These results illustrate a YAP-dependent mechanism by which the stem/progenitor cells recognize the murine ovulatory rupture, and rapidly multiply their numbers, highlighting a YAP-induced stem cell expansion strategy.

Editor's evaluation

This is a well conducted and interesting study that increases our knowledge of mechanisms governing healing after ovarian rupture. This study specifically demonstrates the importance of Procr+ progenitors that are positively regulated by YAP signalling.

https://doi.org/10.7554/eLife.75449.sa0

Introduction

During the adult reproductive cycles, the ovarian surface epithelium (OSE) undergoes recurring ovulatory rupture and repair (Gaytán et al., 2005; Auersperg et al., 2001). After ovulation, to maintain the physiological function and morphology of the ovary, the wound is completely closed within 12 hr to 3 days following rupture (Burdette et al., 2006; Ng and Barker, 2015; Tan and Fleming, 2004). Cells surrounding the damaged sites are required to respond to the wound by turning on cell proliferation to supply sufficient cells as building blocks for regeneration (Wang et al., 2017). Our previous study has identified that Procr+ OSE stem/progenitor cells are the major contributor for ovulatory rupture repair. Targeted ablation of these cells hampers the repair (Wang et al., 2019). Interestingly, we observed that Procr+ cells expand instantly upon ovulation, reminiscent of a result of symmetric division (Wang et al., 2019). It remains unknown how the stem cell is triggered by the ovulation event, and what is the signal that links the rupture to the instant stem cell expansion.

The cue for this stem/progenitor cell amplification likely comes from a particular extracellular signal occurring upon ovulation. One possibility is that the follicular fluid expelled during ovulation consists of Wnts and other potential niche signals (Ahmed et al., 2006; Boyer et al., 2010; Nilsson et al., 2001; Parrott et al., 2000), which may regulate Procr+ stem/progenitor cell expansion. Another possibility is the involvement of mechanical force-induced signals, either through the change of adherent junctions or via a directed mechanism during ovulation, resulting in Procr+ stem/progenitor cell expansion.

YAP (Yes-associated protein, also known as YAP1) signaling is an evolutionarily conserved pathway and a master regulator of organ size and tissue growth during animal development (Moya and Halder, 2019). As a downstream effector, YAP is critical for regeneration in different organs, through triggering cell proliferation, cell survival, or expansion of stem and progenitor cell compartments (Johnson and Halder, 2014; Lin et al., 2017b; Moya and Halder, 2016; Panciera et al., 2016; Patel et al., 2017; Xiao et al., 2016; Zhang and Del Re, 2017). YAP is a transcriptional coactivator protein that shuttles between the cytoplasm and nucleus, and regulates the expression of target genes, such as Ccn1 and Birc5, through binding with TEAD transcription factors (Chen et al., 2001; Lin et al., 2017a; Piccolo et al., 2014; Totaro et al., 2018; Zhao et al., 2008). Vgll4, a member of Vestigial-like proteins, serves as a transcriptional repressor of YAP through direct interactions with TEADs (Chen et al., 2004). Previous studies from us and others have demonstrated the important roles of Vgll4 plays during development and regeneration in various tissues (Feng et al., 2019; Suo et al., 2020; Lin et al., 2016; Yu et al., 2019). Cell–cell junctions links cells to each other in epithelial tissues, and is an upstream negative regulator of YAP (Ramos and Camargo, 2012; Yang et al., 2015). Mechanical forces regulate cell–cell adhesion stability, and cell–cell adhesion junctions may be intrinsically weak at high forces (Pinheiro and Bellaϊche, 2018). It has been shown that disruption of adherent junctions turns on YAP nuclear activities in lung stem/progenitor cells (Zhou et al., 2018). However, whether YAP signaling is implicated in ovulatory rupture repair is unknown.

In this study, we investigated how OSE stem/progenitor cells are triggered by the rupture postovulation and divide subsequently. We found that, in the proximity of rupture site, decreased adherent junction is associated with increased incidence of Yap1 nuclear localization in OSE cells. Interestingly, only Procr+ OSE cells displayed a low level of Vgll4, allowing YAP signaling activation, and conditional deletion of Yap1 in Procr+ cells hampers OSE repair. We generated a new TetO-Vgll4 mouse. Ectopic expression of Vgll4 in the stem/progenitor cells using Procr-rtTA;TetO-Vgll4 mice blocked OSE ovulatory repair. Moreover, we found that YAP signaling activation resulted in Procr+ cells expansion at the rupture site, through the combination of inducing cell division, and directly activating Protein C Receptor (Procr) transcription. The activation of Procr is essential, as when Procr was deleted, stemness property was lost and OSE repair was hindered.

Results

Decreased E-cadherin expression at the rupture site and selective activation of YAP signaling in Procr+ cells

To investigate what could be the potential extracellular stimuli at the rupture site, we performed immunostaining of various adherent or tight junction components on ovarian sections. To increase rupture incidences, superovulation was induced by injection of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG), and the ovaries were harvested at 0.5 days after HCG injection when ovulation just occurred (Figure 1—figure supplement 1A). Interestingly, we found that E-cadherin, α-catenin, and ZO-1 staining is markedly decreased at the proximal region of rupture (defined as within 20 cells on one side of the rupture in section) compared to other regions, that is, rupture distal region (Figure 1A and Figure 1—figure supplement 1C) and nonrupture region (Figure 1—figure supplement 1B,D). As adherent junction has been implicated as a modulator of YAP signaling (Kim et al., 2011; Schlegelmilch et al., 2011; Varelas et al., 2010; Yang et al., 2015), we examined YAP activities at the rupture area by immunostaining. We observed an increased incidence of nuclear Yap1 at the proximal region of rupture compared to other regions (Figure 1B–D). These results suggest that compromised adherent junctions resulting from ovulatory rupture are associated with Yap1 nuclear localization in OSE cells surrounding the wound.

Figure 1 with 2 supplements see all
Rupture-induced YAP signaling activation is preferentially activated in Procr+ progenitors at the rupture sites.

(A) Sections from wild-type ovaries at ovulation stage were stained with Krt8 (K8) and E-cadherin (E-cad). Confocal images showed less E-cad in the ovarian surface epithelium (OSE) of proximal regions surrounding the rupture sites (views #1, #3 in A) compared with distal regions (views #2, #4 in A). Scale bar, 100 μm for zoom out and 10 μm for zoom in. n = 3 mice and 15 images. (B–D) Sections from wild-type ovaries at ovulation stage were stained with K8 and Yap1. Confocal images (B, C) and quantification (D) showed Yap1 nuclear localization in the OSE was only observed in the proximal regions surrounding the rupture sites (B, D), but not in the distal regions (B, D) or the nonrupture sites (C, D). Scale bar, 100 μm for zoom out and 20 μm for zoom in. n = 3 mice and 15 images. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. (E–G) Procr-rtTA;TetO-H2B-GFP+/− mice were fed with doxycycline for 3 days and harvested at ovulation stage. Confocal images of ovarian sections with K8 and Yap1 staining (E, F) and quantification (G) were showed. Nuclear Yap1 staining is preferentially detected in Procr+ (histone 2B-GFP+) cells in rupture proximal region (arrowheads in E), whereas at the nonrupture site, Yap1 staining was cytoplasmic regardless in Procr+ (arrows in F) or Procr− cells (F). Scale bar, 100 μm for zoom out and 10 μm for zoom in. n = 3 mice and 15 images. One-way analysis of variance (ANOVA) with Tukey test is used for comparison of multiple groups. ***p < 0.001. (H–J) Combination of Procr and Ccn1 double fluorescent in situ with K8 antibody immunohistochemistry staining (H–I). Confocal images showed colocalization of Procr and Ccn1 in the OSE at the rupture sites (arrowhead in H), while at nonrupture regions, both Procr+ and Procr− cells had low incidence of Ccn1 expression (I). Quantification showed increased Ccn1 expression in Procr+ cells at rupture sites compared with Procr− cells at rupture sites or Procr+ cells at nonrupture regions (J). Scale bar, 100 μm for zoom out and 1 μm for zoom in. n = 3 mice and 15 images. One-way ANOVA with Tukey test is used for comparison of multiple groups. ***p < 0.001.

Our previous study has established that Procr+ progenitor cells surrounding the wound instantly proliferate upon rupture and are responsible for OSE repair (Wang et al., 2019). We therefore investigated whether Procr+ cells close to the rupture site are associated with YAP signaling activities. We performed Yap1 immunostaining using Procr-rtTA;tetO-H2B-GFP reporter, in which H2B-GFP signal marks Procr-expressing cells. Superovulation was induced in these animals by PMSG and HCG injections, and ovaries were harvested 0.5 days after HCG injection. We found that Procr+ (H2B-GFP+) cells at the rupture proximal region (referred to as rupture site from here on) have significantly higher nuclear Yap1 staining (75.9% ± 1.7%) compared to Procr− cells (39.6% ± 1.0%) (Figure 1E, G), or compared to Procr+ cells at the nonrupture region (Figure 1F, G). This was further validated by RNA double in situ hybridization with Procr and a YAP target gene Ccn1. We found that, at the rupture site, Ccn1 is preferentially activated in Procr+ OSE cells, with 44.8% ± 2.9% of Procr+ cells being Ccn1+, which is markedly higher than that of Procr− cells (8.9% ± 0.8%) (Figure 1H, J). At the nonrupture site, both Procr+ and Procr− cells had a rather low expression of Ccn1 expression (Figure 1I, J). Furthermore, we FACS-isolated Procr+ and Procr− OSE cells from the rupture sites (Figure 1—figure supplement 2A), validated the epithelial identity by Krt8 (K8) staining (Figure 1—figure supplement 2B). qPCR analysis indicated that Procr+ cells at rupture sites have a higher level of Birc5 expression compared to Procr− cells (Figure 1—figure supplement 2C). Together, these results suggest that YAP signaling was specifically activated in Procr+ cells at the rupture site. Considering the role of YAP signaling in promoting cell proliferation, these results are in line with our previous observations that only Procr+, but not Procr−, cells at the rupture site displayed increased proliferation (Wang et al., 2019).

Deletion of Yap1 in Procr+ cells hinders rupture repair and progenitor proliferation

To investigate whether YAP signaling is important for OSE repair, we deleted Yap1 specifically in Procr+ cells using Procr-CreER;Yap1fl/fl mice (Yap1-cKO). Yap1fl/fl mice was used as control (Ctrl). Tamoxifen (TAM) was administered in 4-week-old mice, following by superovulation at 2 days after TAM injection (Figure 2A). The impact on OSE repair by Yap1 deletion was analyzed by ovary whole-mount imaging. At 4.5 dpi (ovulation), the two groups had similar ruptures (Figure 2B, E). At 6 dpi, Ctrl ovaries underwent rapid repairing (Figure 2C, E), and the OSE was completely recovered by 7.5 dpi (Figure 2D, E). In contrast, the OSE repair in Yap1-cKO ovaries was significantly delayed at both 6 and 7.5 dpi (Figure 2C–E). The efficacy of Yap1 deletion and the reduced expression of the target gene Ccn1 in OSE cells were validated by qPCR analyses (Figure 1—figure supplement 2D).

Deletion of Yap1 in Procr+ cells hinders ovarian surface epithelium (OSE) rupture repair and progenitor proliferation.

(A) Illustration of TAM induction and superovulation strategy. (B–E) Yap1 was deleted in Procr+ cells using Procr-CreER;Yap1fl/flmice (Yap1-cKO), and Yap1fl/fl mice was used as control (Ctrl). Ovary whole-mount staining with K8 and Laminin was performed (B–D) and the wound size in diameter was quantified (E). At 4.5 days (ovulation) Ctrl and Yap1-cKO ovaries had comparable wound size (* in B). At 6 days (OSE repair ongoing), the wounds in Ctrl ovary were significantly smaller than those in Yap1-cKO ovary ( * in C). At 7.5 days (repair completed), the wound was completely repaired in Ctrl, while the Yap1-cKO ovary still showed obvious wounds ( * in D). Scale bar, 100 μm. n = 3 pairs of mice. (F–H) Ctrl and Yap1-cKO mice were subjected to 12-hr 5-ethynyl-29-deoxyuridine (EdU) incorporation and were harvested at 4.5 days (ovulation stage). Representative images (F–G) and quantification (H) were showed. Out of 20 cells next to the rupture on one side, the numbers of EdU+ cells (arrowhead) in the OSE of rupture site decreased from 5.5 ± 0.3 cells in Ctrl to 1.4 ± 0.2 cells in Yap1-cKO. Scale bar, 100 μm for zoom out and 20 μm for zoom in. n = 3 pairs of mice. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. (I–K) Total OSE cells from Procr-CreER;Yap1fl/fl mice (Yap1-cKO), and Yapfl/fl mice (Ctrl) were isolated by FACS at 4.5 days (ovulation stage) (I), followed by culture in 3D Matrigel for 7 days. Representative brightfield and confocal images of K8 staining were shown (J). Colony sizes in diameter were measured (K). Scale bar, 20 μm. Data are pooled from three independent experiments and displayed as mean ± standard error of the mean (SEM). Unpaired two-tailed t-test is used for comparison. ***p < 0.001. (L) Illustration of lineage tracing, deletion of Yap1 and superovulation strategy. (M–O) Procr-CreER;Yap1fl/fl;R26-mTmG (Yap1-cKO) and Procr-CreER;R26-mTmG (Ctrl) mice were used. At 4.5 pi (ovulation), ovary whole-mount confocal imaging showed zones of concentrated GFP+ cells surrounding the rupture site in Ctrl, while fewer GFP+ cells were seen in Yap1-cKO ovary (M). At 7 pi (repair completed), ovary whole-mount confocal imaging showed large GFP+ patches located at corpus luteum (CL) in Ctrl, while rare GFP+ cells surrounding the unrepaired wound in Yap1-cKO ovary (N). Quantification showed significantly fewer GFP+ cells in Yap1-cKO compared with Ctrl in both ovulation stage and repair completed stage (O). Quantification showed an expansion of GFP+ cell numbers in Ctrl mice during the tracing and no expansion in Yap1-cKO (O). Scale bar, 100 μm. n = 3 pairs of mice. ***p < 0.001.

To analyze the proliferative capacity of Procr+ OSE cells, mice were subjected to 12 hr of 5-ethynyl-29-deoxyuridine (EdU) incorporation before harvesting the ovaries (Figure 2A). When analyzed at 4.5 dpi (ovulation), the number of proliferating OSE cells at rupture site (defined as 20 cells on one side from the opening) was significantly decreased from 5.5 ± 0.3 EdU+ in Ctrl to 1.4 ± 0.2 EdU+ in Yap1-cKO (Figure 2F–H). The impact to cell proliferation was further analyzed in vitro. Our previous study has established that Procr+, but not Procr−, OSE cells can form colonies in vitro (Wang et al., 2019). At 4.5 dpi, total OSE cells were isolated from both Ctrl and Yap1-cKO mice (Figure 2I), and placed in culture as previously described (Wang et al., 2019). Deletion of Yap1 in Procr+ cells drastically inhibited OSE colony formation (Figure 2J, K).

To visualize the contribution of Procr+ progenitors toward the repair in the presence or absence of Yap1, we performed in vivo lineage tracing. TAM was administered to 4-week-old mice to simultaneously delete Yap1 and initiate lineage tracing in Procr+ cells (Figure 2L). At 4.5 dpi, control (Procr-CreER;R26-mTmG) ovary displayed a zone of mGFP+ cells that are the progeny of Procr+ progenitors surrounding the rupture sites (Figure 2M). In contrast, Yap1-cKO (Procr-CreER;Yap1fl/fl;R26-mTmG) ovaries have markedly fewer mGFP+ cells around the wound (Figure 2M, O), supporting the notion that the activity of Procr+ progenitors was hampered at the beginning of the repairing process. At 7 dpi, control ovaries had generated patches of mGFP+ cells covering the newly formed corpus luteum (Figure 2N). However, Yap1-cKO ovaries still had obvious openings with few mGFP+ cells (Figure 2N, O). Together, these results suggest that YAP signaling activation is crucial for the proliferation of Procr+ progenitor cells and the timely repair of OSE after rupture.

An intrinsic lower level of Vgll4 in Procr+ cells is essential for their progenitor property and OSE rupture repair

Next, we investigated what could be the reason that YAP signaling is specifically activated in Procr+ cells. Vgll4 is a negative regulator of YAP by inhibiting the binding of YAP and TEAD4 (Feng et al., 2019). We FACS-isolated Procr+ cells and Procr− cells from the rupture sites (Figure 3A). qPCR analysis indicated that Procr+ cells have lower level of Vgll4 compared to Procr− cells (Figure 3B). This was further validated by Vgll4 immunostaining using Procr-rtTA;tetO-H2B-GFP reporter, in which H2B-GFP signals mark Procr-expressing cells. Consistent with the qPCR results, Procr+ cells also exhibited lower Vgll4 protein expression compared to Procr− cells (Figure 3C–E).

Figure 3 with 1 supplement see all
An intrinsic lower level of Vgll4 in Procr+ cells is essential for Procr+ cells’ stemness and ovarian surface epithelium (OSE) rupture repair.

At ovulation stage, Procr+ and Procr− OSE cells (Lin−, EpCAM+) were FACS isolated (A). qPCR analysis showed the lower Vgll4 level in Procr+ cells (B). Data are pooled from three independent experiments and presented as mean ± standard error of the mean (SEM). ***p < 0.001. (C–E) Procr-rtTA;TetO-H2B-GFP mice were administered with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) to induce superovulation, and fed with doxycycline for 3 days before harvest. Ovarian sections were stained with Vgll4 and K8. Representative images showed that at both rupture proximal region (C) and nonrupture region (D), H2B-GFP− (Procr−) OSE cells have high Vgll4 expression (arrows in C, D), while H2B-GFP+ (Procr+) OSE cells have no Vgll4 expression (arrowheads in C, D). Scale bar, 20 μm for zoom out and 5 μm for zoom in. Quantification of the staining was shown in (E). n = 3 mice and 15 images. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Targeting strategy and validation of TetO-Vgll4 knock-in mouse (F, G). A cassette of TetO-Vgll4-Flag-wpre-polyA was knocked in behind 3′UTR of Col1a1 gene (F). Immunohistochemistry staining of Vgll4 in the ovaries indicated more Vgll4+ OSE cells at the rupture sites (G). Scale bar, 10 μm. n = 3 pairs of mice. Illustration of superovulation and overexpression of Vgll4 in Procr+ cells (H). Ovary whole-mount confocal images of K8 and Laminin showed that at 4.5 days (ovulation), Ctrl (TetO-Vgll4) and Vgll4-OE (Procr-rtTA;TetO-Vgll4) ovaries have similar wound sizes (* in I). At 6 days (repair ongoing), the wound sizes in Ctrl mice were smaller than those in Vgll4-OE (* in J). At 7.5 days (repair completed), Ctrl ovary had completely repaired, while Vgll4-OE mice had obvious opening (* in K). Scale bar, 100 μm. The sizes of the wound in diameter were quantified (L). n = 3 pairs of mice. The mice were harvested at 4.5 days (ovulation) after 12-hr 5-ethynyl-29-deoxyuridine (EdU) incorporation. Representative images (M, N) and quantification (O) showed the number of EdU+ cells in the OSE surrounding the rupture site (arrowheads in M) decreased from 3.7 ± 0.3 in Ctrl to 1.0 ± 0.2 in Vgll4-OE (arrowheads in N). Scale bar, 100 μm for zoom out and 20 μm for zoom in. n = 3 pairs of mice. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Total OSE cells were isolated by FACS from Ctrl and Vgll4-OE at 4.5 days (ovulation) (P), and cultured in 3D Matrigel. At day 7 in culture, colony sizes were measured in diameter (Q), and representative images were shown (R) out of 15 images in each group. Scale bar, 20 μm. Data are pooled from three independent experiments and displayed as mean ± SEM. Unpaired two-tailed t-test is used for comparison. ***p < 0.001.

To examine whether the reduced level of Vgll4 is significant for the selective YAP signaling activation in Procr+ cells and rupture repair, we set to overexpress Vgll4 specifically in Procr+ cells. A new TetO-Vgll4 mouse line was generated, by inserting a tetO-Vgll4-Flag-wpre-polyA cassette behind the 3′UTR of the Col1a1 gene (Figure 3F and Figure 3—figure supplement 1A–C). Subsequently, Procr-rtTA;TetO-Vgll4 (Vgll4-OE) mice were generated by genetic crosses with TetO-Vgll4 as control (Ctrl). The efficacy of overexpression was validated by western blotting and qPCR, showing increased expression of Vgll4 and decreased expression of Ccn1 in Vgll4-OE cells (Figure 3—figure supplement 1D, E). Furthermore, immunostaining confirmed the increased number of Vgll4 high cells in the OSE layer of Vgll4-OE mice (Figure 3G). For this experiment, superovulation was performed to 4-week-old mice and doxycycline hyclate (DOX) was fed throughout the process (Figure 3H). The impact of Vgll4 overexpression was analyzed throughout the repairing process, at 4.5 dpi (ovulation), 6 dpi (OSE repair ongoing), and 7.5 dpi (OSE repair completed) by ovary whole-mount imaging. We found that the rupture in Ctrl and Vgll4-OE ovaries are comparable at 4.5 dpi (Figure 3I, L). At 6 dpi, while Ctrl ovaries had sights of wound closure, Vgll4-OE ovaries still showed larger areas of rupture (Figure 3J, L). At 7.5 dpi, Ctrl ovaries displayed complete OSE, whereas the repair in Vgll4-OE ovaries was obviously delayed (Figure 3K, L).

Next, we examined whether overexpression of Vgll4 affects progenitor proliferation. At 4.5 dpi (ovulation), the number of proliferating OSE cells at rupture site was significantly decreased from 3.7 ± 0.3 in Ctrl to 1.0 ± 0.2 in Vgll4-OE (Figure 3M–O). At 4.5 dpi, total OSE cells were isolated and cultured in vitro for 7 days (Figure 3P). Consistently, overexpression of Vgll4 inhibits cell proliferation and colony formation (Figure 3Q, R).Together, these results suggest that overexpression of Vgll4 in Procr+ cell impaired Procr+ cell proliferation and ovulatory rupture repair.

YAP signaling promotes Procr+ cells expansion at rupture site

We have previously found that Procr+ progenitor cells expand instantly at the periphery of the rupture site upon ovulation (Wang et al., 2019). To investigate whether YAP signaling activation is linked to the expansion of Procr+ progenitor cells, TAM was administered to 4-week-old Procr-CreER;Yap1fl/fl (Yap1-cKO) and Yap1fl/fl (Ctrl) mice for two times, followed by superovulation. At 4.5 dpi (ovulation), FACS analysis showed a dramatic decrease of Procr+ progenitor population when Yap1 was deleted (Figure 4A–C).

Figure 4 with 2 supplements see all
YAP signaling promotes Procr+ cells expansion at rupture sites through a combination of promoting cell division and enhancing Procr expression.

Illustration of superovulation and analysis strategy as indicated using Yap1fl/fl (Ctrl) and Procr-CreER;Yap1fl/fl (Yap1-cKO) mice (A). At ovulation stage, the percentage of Procr+ ovarian surface epithelium (OSE) cells in Ctrl and Yap1-cKO were FACS analyzed (B) and quantified (C). n = at least three mice in each group and displayed as mean ± standard error of the mean (SEM). Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Illustration of superovulation and analysis strategy as indicated using Procr-rtTA;TetO-H2B-GFP+/− (Ctrl) and Procr-rtTA;TetO-H2B-GFP+/−;TetO-Vgll4+/− (Vgll4-OE) mice (D). At ovulation stage, ovary section imaging showed that at the rupture sites, the number of H2B-GFP+ (Procr+) cells in Ctrl (arrowheads in E) is higher than those in Vgll4-OE (arrowheads in F). Scale bar, 100 μm. Quantification was shown in (G). n = 3 pairs of mice and 15 images in each group. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. The percentage of Procr+ OSE cells were analyzed by FACS at ovulation stage (H). The percentage of Procr+ cells in Ctrl are higher than that in Vgll4-OE (H, I). n = at least 3 mice and displayed as mean ± SEM. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Illustration of Tead4 motif in Procr promoter region (J). TEAD4 chromatin immunoprecipitation-qPCR (ChIP-qPCR) analysis using cultured primary OSE cells showed the enrichment of Procr promoter, and Ctgf promoter was used as positive control (K). n = 2 biological repeats. Unpaired two-tailed t-test is used for comparison. **p < 0.01, *p < 0.05, n.s., not significant. Analysis of luciferase reporter activity driven by WT (L) and Tead4 motif (−1486 to −1481 bp) deleted Procr promoter (M) in HEK293T cells transfected with increased amount of Yap1 overexpression plasmids. Data are pooled from three independent experiments and displayed as mean ± SEM. Unpaired two-tailed t-test is used for comparison. ***p < 0.001, **p < 0.01, n.s., not significant. (N) A proposed model of which YAP signaling promotes Procr+ cells expansion at rupture site through a combination of promoting cell division and enhancing Procr expression.

To better visualize the change ofProcr+ progenitor cells under the influence of YAP signaling, we generated Procr-rtTA;TetO-H2B-GFP+/−;TetO-Vgll4+/− mice (Vgll4-OE). Superovulation was performed to 4-week-old mice and DOX was fed throughout the experiments to maintain the expression of H2B-GFP in Procr+ cells (Figure 4D). When analyzed at 4.5 dpi (ovulation), at the wound edge (defined as 20 cells on one side from the opening) of control ovary (Procr-rtTA;TetO-H2B-GFP+/−), there were about 7.4 ± 0.3 H2B-GFP+ cells expressing the peak level of GFP (Figure 4E, G). In contrast, in Vgll4-OE ovary (Procr-rtTA;TetO-Vgll4;TetO-H2B-GFP+/−), only 2.4 ± 0.2 H2B-GFP+ cells were observed at the wound edge (Figure 4F, G). FACS analysis also showed that the percentage of Procr+ progenitor population decreased significantly from 18.5% ± 1.0% in Ctrl to 7.4% ± 1.2% in Vgll4-OE at ovulation stage (Figure 4H, I).

The proliferative activity of Procr+ cells was further evaluated in vitro. We isolated OSE cells from control (Procr-rtTA;TetO-H2B-GFP+/−) and Vgll4-OE (Procr-rtTA;TetO-Vgll4;TetO-H2B-GFP+/−) mice and placed in culture, followed by live imaging to document the division of H2B-GFP+ (Procr+) cells (Figure 4—figure supplement 1A, B). In control cells, we observed frequent division of Procr+ cells, and in most cases, it was one Procr+ cell dividing into two Procr+ cells (Figure 4—figure supplement 1A, C). But in Vgll4-OE, we could hardly observe cell division (Figure 4—figure supplement 1B, C). Together, these results suggest that inhibition of YAP signaling, by either Yap1-deletion or Vgll4-OE, impairs the expansion of Procr+ progenitors upon rupture.

YAP signaling enhances Procr expression

It is unclear how YAP maintains Procr expression during or after cell division. Thus, we investigated the association of YAP activation and Procr expression. OSE cells were isolated from Procr-rtTA;TetO-H2B-GFP+/− mice, and cultured on glass (YAP activation) or soft condition (0.48 kPa, YAP inactivation) (Figure 4—figure supplement 2A, B). DOX was added 2 days before harvest. Consistent with the notion, we found that, in soft condition, Yap1 was mostly cytoplasmic and most OSE cells are H2B-GFP− (Figure 4—figure supplement 2A). In contrast, most OSE cells are H2B-GFP+ in stiff condition and Yap1 was found in the nucleus (Figure 4—figure supplement 2B). These observations suggest that YAP activation might induce Procr expression. We verified by qPCR that Procr expression is upregulated in stiff conditions (Figure 4—figure supplement 2C). Our results support the notion that YAP activation induces Procr expression.

To further investigate whether Yap1 regulates Procr expression, we knocked down Yap1 by shRNA in OSE culture and found that this inhibits Procr expression (Figure 4—figure supplement 2D, E). Furthermore, blocking YAP activation by Verteporfin (VP) or Vgll4 overexpression also resulted in lower Procr expression (Figure 4—figure supplement 2F, G). These results suggest that inhibiting YAP signaling suppresses Procr expression.

To investigate whether YAP/TEAD4 directly regulate Procr expression, we analyzed the promoter of Procr. A Tead4-binding motif (5′-CATTCC-3′) was found at the proximal promoter of Procr (−1486 to −1481 bp) (Figure 4J). Chromatin immunoprecipitation-qPCR (ChIP-qPCR) showed that Tead4 could directly bind to the Procr promoter (Figure 4K). Therefore, we examined whether this Tead4-binding motif is responsible for induction of Procr expression by Yap1. While Yap1 induced the wild-type promoter luciferase in a dose-dependent manner (Figure 4L and Figure 4—figure supplement 2H, I), it could not activate the mutant reporter with the deletion of the Tead4-binding motif (Figure 4M and Figure 4—figure supplement 2H, I). These results suggest that Yap1 directly promotes Procr expression. Together, our data support a model that YAP signaling promotes expansion of Procr+ cells at rupture site through a combination of increased cell division and Procr expression (Figure 4N).

Procr is essential for the progenitor property

The upregulation of Procr expression coupled with YAP-induced cell division implies that the expression of Procr may be important for keeping the stem cell property in OSE. To assess the significance of Procr, we utilized a Procr-flox allele (Liu and Zeng, unpublished) and specifically deleted Procr in the progenitor using ProcrCreER/fl (Procr-cKO) mice. TAM was administered in 4-week-old mice for two times, followed by superovulation at 2 days after TAM injection (Figure 5A), and the phenotype was analyzed by ovary whole-mount imaging. Ctrl and Procr-cKO ovaries formed comparable ruptures at 4.5 dpi (ovulation) (Figure 5B, E). At 6 dpi (OSE repair ongoing), control ovaries showed smaller openings compared to Procr-cKO (Figure 5C, E). At 7.5 dpi (OSE repair completed), Ctrl ovaries were covered by complete OSE, whereas Procr-cKO ovaries still had regions with unrepaired OSE (Figure 5D, E). Furthermore, at 4.5 dpi (ovulation), the ovaries were harvested after 12 hr of EdU incorporation. The number of proliferated OSE cells at rupture site decreased from 4.7 ± 0.4 cells in control (Procrfl/+) to 1.6 ± 0.2 cells in Procr-cKO (Figure 5F–H). After deletion of Procr in vivo, total OSE cells were isolated and cultured in vitro for 7 days (Figure 5I). We found that deletion of Procr inhibits the proliferation of progenitor cells, resulting in reduced colony sizes (Figure 5J–L). Overall, these data suggest that Procr is essential for progenitor property upon rupture.

Procr is essential for the progenitor property.

(A–E) Illustration of superovulation and deletion of Procr in Procr+ cells using ProcrCreER/fl mice (Procr-cKO), and Procrfl/+ mice (Ctrl) (A). Ovary whole-mount confocal imaging of K8 and Laminin showed that at 4.5 days (ovulation), Ctrl and Procr-cKO have similar wound sizes (* in B). At 6 days (ovarian surface epithelium [OSE] repair ongoing), the wound sizes in Ctrl mice were smaller than those in Procr-cKO (* in C). At 7.5 days (repair completed), Ctrl ovary had completely repaired, while Procr-cKO remained obvious opening (* in D). Scale bar, 100 μm. Quantification of the wound size in diameter was shown in (E). n = 3 pairs of mice. Unpaired two-tailed t-test is used for comparison. ***p < 0.001. n.s., not significant. (F–H) Post 12-hr 5-ethynyl-29-deoxyuridine (EdU) incorporation, the mice were harvested at 4.5 days (ovulation) (A). Representative images showed the number of EdU+ cells (arrowhead) in the OSE surrounding the rupture site decreased from 4.7 ± 0.4 in Ctrl (arrowheads in F) to 1.6 ± 0.2 in Procr-cKO (arrowheads in G). Scale bar, 100 μm for zoom out and 20 μm for zoom in. Quantification of was shown in (H). n = 3 pairs of mice. Unpaired two-tailed t-test is used for comparison. ***p<0.001. Total OSE cells from Ctrl andProcr-cKO were isolated by FACS (I), followed by culture in 3D Matrigel. At culture day 7, representative brightfield and confocal images with K8 staining showed that OSE cells with Procr-cKO form markedly smaller colonies compared to Ctrl (J). Colony sizes were quantified in (K). qPCR analysis validated the deletion efficiency of Procr in OSE cells of Procr-cKO (L). Data are pooled from three independent experiments and displayed as mean ± standard error of the mean (SEM). Unpaired two-tailed t-test is used for comparison. ***p < 0.001. Scale bar, 20 μm. (M) A proposed model of YAP activation in Procr+ cells promoting OSE progenitor cell expansion. Procr+ OSE progenitors have intrinsically lower level of Vgll4 compared to Procr− OSE cells. At ovulation, cell–cell junctions at rupture site were disrupted, which induces the possibility of YAP activation in all OSE cells surrounding the rupture. However, the lower expression of Vgll4 in Procr+ cells allowed YAP activation in the progenitor cells at this area. YAP activation in Procr+ cells promoted cell division, and importantly, it directly upregulates Procr expression in the dividing cells, resulting in expansion of Procr+ progenitors around the wound.

Discussion

In this study, we addressed the molecular mechanism which links the ovulatory rupture to OSE stem/progenitor cells activation, thus promptly turning on proliferation and repairing the wound. Our findings support the following model. Procr+ OSE progenitors have intrinsically lower levels of Vgll4. Upon ovulatory rupture, the decreased adherent junction at the proximity of the rupture site promotes Yap1 nuclear localization. These intrinsic and extrinsic factors together lead to YAP signaling activation in Procr+ progenitors around the wound, which sequentially stimulates the proliferation of the progenitors. Importantly, YAP activation directly upregulates Procr expression in the dividing cells, resulting in the expansion of Procr+ progenitors around the wound (Figure 5M). Blocking YAP signaling in the progenitors by Yap1-cKO or Vgll4-OE impairs the progenitors’ activities and hinders OSE repair. Furthermore, Procr function is essential for these progenitors. When Procr was deleted, stem cell property was lost hindering OSE repair.

While we uncovered the significance of selective activation of YAP in OSE progenitors, it is still unclear how ovarian rupture is sensed and how YAP signaling is induced by injury. We observed reduced cell-adhesion protein E-cadherin is lowered at the site of the ovarian rupture, and it is previously known that E-cadherin signals through the Hippo pathway to block YAP (Yang et al., 2015). The current missing link is why and how E-cadherin would be reduced at the ovarian rupture site. We speculate that during the late stage of follicle development, the pre-ovulatory follicle forms a protrusion toward OSE. Subsequently, ovulation generates a rupture on OSE. These contiguous events likely induce the thinning of OSE surrounding the pre-ovulatory follicles and at the proximity of the rupture site, resulting in the reduced adherent junction proteins. Yet, there are other potentially more direct possibilities, i.e. mechanical stretching induces YAP (Halder et al., 2012). First, the pre-ovulatory follicle protrusion or the release of oocytes induces a mechanical force on the OSE surrounding the wound, activating YAP signaling. Second, at the rupture site, epithelial cells no longer become packed together because the epithelium has been denuded, therefore potentially cells flanking the rupture site would become ‘stretched’, consequently actives YAP.

YAP signaling promotes Procr+ cell expansion at the rupture site through a combination of increased cell division and Procr expression. In the current study, YAP is particularly activated in Procr+ progenitor cells at the rupture site. We observed that at the rupture sites, Vgll4 is highly expressed in Procr− cells, preventing YAP pathway activation in those cells around the rupture sites. Our findings demonstrate that the reduced levels of Vgll4 in Procr+ progenitors likely contribute to the selective activation of YAP signaling in these cells. Further study should investigate what mechanism determines the lower expression of Vgll4 in Procr+ progenitor cells.

In the current study, we generated a new TetO-Vgll4 mouse model that enables the overexpression of Vgll4 in a specific cell type. The overexpression of Vgll4 in the progenitor of OSE has been validated using Procr-rtTA. The advantages brought by our TetO-Vgll4 reporter will be of broad value in studies of Hippo-Yap signaling across all tissues.

Procr expression is initially found on the surface of vascular cells exerting an anticoagulation role, by binding and activating protein C (PC) in the extracellular compartment (Fukudome et al., 1998). More recently, studies from us and others have identified Procr as a stem cell surface marker in multiple tissues (Wang et al., 2015; Wang et al., 2020; Wang et al., 2019; Yu et al., 2016), but less is known regarding the function of Procr in stem/progenitor cells. In the current study, we demonstrate that, Procr is essential for the proliferation of Procr+ progenitor cells and OSE repair upon rupture. Our previous report indicated that PROCR concomitantly activates multiple pathways including ERK, PI3K–Akt–mTOR, and RhoA–Rock–P38 signaling in breast cancer cells (Wang et al., 2018). We speculate that similar intracellular pathways might be involved in the Procr+ OSE cells. Procr is regarded as a Wnt target gene from an in vitro screen in mammary stem cell culture (Wang et al., 2015). In this study, we identify YAP as a novel upstream regulator of Procr. ChIP-qPCR and promoter luciferase experiments demonstrate that Procr transcription can be directly upregulated by YAP activation.

The phenomena of YAP promoting stem/progenitor cell expansion have been reported in various tissues (Beverdam et al., 2013; Camargo et al., 2007; Cao et al., 2008; Ramos and Camargo, 2012; Schlegelmilch et al., 2011; Zhou et al., 2018). Yet, in this process, less is known about how YAP maintains stem cell properties. To the best of our knowledge, this is the first report illustrating a mechanism through which YAP promotes cell proliferation, and simultaneously upregulates the expression of an essential stemness gene to maintain cell fate, leading to a rapid expansion of stem cell numbers around the wound. In summary, our study provides new evidence and molecular insights into how ovulatory rupture triggers the activation of OSE stem cells, resulting inpromptly expanding their numbers for repair. This may have a broad implication to understand the action of tissue stem cells during would healing in other tissue.

Materials and methods

Lead contact and materials availability

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Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Yi Arial Zeng (yzeng@sibcb.ac.cn). All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Experiment animals

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TetO-H2B-GFP+/− (Stock: 005104), R26-mTmG+/− (Stock: 007576) from Jackson Laboratories, ProcrCreER (Wang et al., 2015), ProcrrtTA (Wang et al., 2019), YAPfl/+ (Feng et al., 2019), Procrfl/+(Liu and Zeng, unpublished), TetO-Vgll4 were used in this study. The TetO-Vgll4 mouse line was generated by knocking in a cassette of TetO-Vgll4-Flag-wpre-polyA behind 3′UTR of Col1a1 gene (Figure 3—figure supplement 1). All mice were housed in the SIBCB animal facility under IVC standard with a 12 hr light/dark cycle at room temperature. Both ovaries were used per mice and the number of mice per experiment was shown in figure legends. For targeted knockout in vivo, 4–5 weeks mice were administered with TAM diluted in sunflower oilby intraperitoneal (IP) injection at a concentration of 2 mg per 25 g body weight for two or three times (on every second day). For superovulation experiments, 4- to 5-week-old mice were injected with 10 IU of PMSG by IP, followed by IP injection of 10 IU of HCG about 48 hr later. For DOX feeding, DOX was dissolved in drink water at a concentration of 1 mg/ml. Experimental procedures were approved by the Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, with a project license number of IBCB0065.

OSE cells isolation and flow cytometry

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Ovaries from superovulated or 4- to 12-week-old female mice were isolated, and the oviduct and bursa were carefully cleared out under dissect microscope. The ovaries were minced into pieces as small as possible, and then placed in 10 ml digest buffer (RPMI 1640 with 5% fetal bovine serum (FBS), 1% penicillin–streptomycin, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 300 U/ml collagenase IV). After digestion at 37°C, 100 rpm for about 1 hr, ovarian cells were obtained after centrifugation at 1000 rpm for 5 min. The red blood cells were lysed with buffer at room temperature for 5 min, and then single cells were obtained with 0.25% trypsin treatment at 37°C for 5 min, followed by 0.1 mg/ml DNaseI incubation at 37°C for 5 min with gently pipetting before filtering through 70 μm cell strainers. The single cells were incubated on the ice and in dark with the following antibodies at a dilution of 1:200: FITC conjugated, PE conjugated, or biotinylated CD31, CD45, EpCAM-APC, Procr-PE, Procr-Biotin, Streptavidin-APC-Cy7, and Streptavidin-V450. All analysis and sorting were performed using a FACSJazz (Becton Dickinson). The purity of sorted population was routinely checked and ensured to be >95%.

OSE cells 3D culture assay

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FACS sorted OSE cells were resuspended with 60 μl 100% growth factor-reduced Matrigel and placed around the rim of a well of a 24-well plate, and allowed to solidify for at least 15 min at 37°C in a 5% CO2 incubator before adding 0.5–1 ml culture medium. Colonies were grown for 7–9 days and the medium was changed every other days. The culture medium was prepared by adding 5% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 10 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 5 mg/ml insulin, 5 mg/ml transferrin, 5 ng/ml sodium selenite, 0.1 mM Minimum Essential Medium (MEM) nonessential amino acids, 10−4 M 2-mercaptoethanol into Dulbecco’s modified essential medium (DMEM)/F12. The organoid images were captured by Zeiss inverted microscope at days 7–9.

Immunohistochemistry

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For section staining, ovarian tissues were fixed in 4% PFA at room temperature for 15 min, following by washed with phosphate-buffered saline (PBS) for three times, dehydrated in 30% sucrose at 4°C overnight and embedded with Optimum Cutting Temperature. 16–18 μm tissue sections were incubated in 0.1% or 0.5% Triton X-100 diluted with PBS (PBST) for 20 min and then 1 hr blocking using 10% FBS in PBST. Then sections were incubated with primary antibodies diluted in blocking buffer at 4°C overnight, followed by washes for three times (20 min per time). After wash, the sections were further incubated with secondary antibodies and 4′,6-diamidino-2-phenylindole (DAPI) diluted in blocking buffer for 2 hr at room temperaturein dark, followed by washes for three times (20 min per time) and mounted with mounting medium.

For staining of cultured colonies, colonies were released from Matrigel by incubating with dispase for 20–30 min. Then the colonies were fixed in 4% PFA on ice for 10 min, followed by cytospin (Thermo Fisher) into slides and staining protocol described above.

For whole mouse ovary immunohistochemistry, mouse ovaries that cleared without bursa and oviduct were fixed with fresh 4% PFA at room temperature for 15 min in 4 ml Eppendorf tubes, followed by washing with PBST for three times (20 min per time). The staining of whole ovaries was then transferred into the 2 ml Falcon tubes using a dropper carefully. Ovaries were blocked for 1 hr using 10% FBS in PBST. Then, the ovaries were incubated with primary antibodies diluted in blocking buffer at 4°C for 48 hr on a transference shaker with 10rpm, followed by washing for three times (20min per time) at room temperature. After washing, the ovaries were incubated with secondary antibodies diluted in blocking buffer for 24 hr at 4°C in dark, and counterstained with DAPI on a transference shaker with 10rpm, followed by washing for three times (20min per time) at room temperature. The ovaries could be stored in PBST at 4°C for at least 2 weeks.

For Yap1 staining in vivo, tyramide signal amplification assay (TSA staining) with Yap1 antibody from CST (Cat# 14074) was used. Briefly, paraffin sections were rehydrated in histoclear and gradual ethanol (100%, 100%, 95%, 85%, 75%, 50%, and 30%) and the TSA staining was performed using the Opal 4-Color Automation IHC Kit (PerkinElmer) following the manufacturer’s instructions. After TSA staining for Yap1, staining for GFP and Krt8 was performed following protocol described above.

Tissue sections and organoids fluorescent images were captured using Leica DM6000 TCS/SP8 laser confocal scanning microscope with a ×20/0.75 or ×40/0.75 or ×63/0.75 IMM objective with 1–3 μm z-step. Confocal images were processed with maximum intensity projections.

Whole mouse ovarian fluorescent images were captured with inverted Leica TCS SP8 WLL at a ×10/0.75 objective, z-stack was ~50–80 layers with 6–7 μm per layer, and the area was about 1.5 mm × 1.5 mm, which was about 1/6–1/4 of the ovary surface.

Western blotting

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Digested cells were lysed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer and boiled for 10 min. Proteins were separated by SDS–PAGE and transferred to nitrocellulose membrane (GE Company). Bolts were blocked with 3% Bovine Serum Albumin (BSA) in Tris-Buffered Saline with 0.5% Tween 20 (TBST) (50 mM Tris–HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 hr and incubated with primary antibodies at 4°C overnight, followed by incubated with secondary IgG-HRP antibodies for 2 hr at room temperature. Protein bands were visualized with chemiluminescent reagent and exposed to Mini Chemiluminescent Imager.

RNA in situ

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In situ hybridization was performed using the RNA scope kit (Advanced Cell Diagnostics) following the manufacturer’s instructions. Procr probes (REF#410321) and Ccn1 probes (REF#429001) were ordered from Advanced Cell Diagnostics. After in situ hybridization, TSA method was used for Krt8 staining following the manufacturer’s instructions using the Opal 4-Color Automation IHC Kit (PerkinElmer). The images were captured using Leica DM6000 TCS/SP8 laser confocal scanning microscope with a ×63/0.75 IMM objective.

EdU labeling assays

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The proliferation of OSE cells in vivo was measured by EdU uptake. Briefly, mice were injected with 100 μl EdU (2.5 mg/ml in dimethyl sulfoxide) for 12 hr. Then ovaries were harvested for section, following by EdU color staining using Click-iTEdU Alexa Fluor Imaging Kit (prepared according to the manufacturer’s instructions). After washed with PBS for three times (10 min per time), EdU color development was performed following the manufacturer’s protocol. After EdU signal developing, sections were blocked in blocking buffer for 1 hr at room temperature followed by antibody staining and mounted with mounting medium for imaging and quantification.

Living image of cultured OSE cells

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OSE cells were isolated from the mice and cultured on glass for 3–4 days. DOX was added into the medium 1 day and Hoechst 33342 was added 30 min before image. Live-cell imaging was performed at 37°C on a Zeiss Cell discoverer seven with perfect focus system. Cells were imaged at 1 time per 5 min for 24 hr with 70% laser power.

Chromatin immunoprecipitation-qPCR

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Cultured primary OSE cells were crosslinked in a final concentration of 1% formaldehyde (Sigma) PBS buffer for 15 min at 37°C, then added glycine to stop crosslinking. Chromatin from nuclei was sheared to 200–600 bp fragments using ultrasonic apparatus, then immunoprecipitated with antibody of TEAD4 (ab58310, Abcam) or normal mouse IgG (sc-2025, Santa Cruz) overnight. Antibody/antigen complexes were recovered with Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology) for 2 hr at 4°C. After washing, the chromatin was eluted, decrosslinked and digested. The immunoprecipitated DNA was collected with QIAQIUCK PCR Purification Kit (QIAGEN). Purified DNA was performed with ChIP-qPCR. Assessing the enrichment of the proteins of interest on the targeting region by calculating the value of ‘fold over IgG’.ChIP-qPCR primers used were as follows.

  • Negative Ctrl CHIP-R, TATCCCCACTGCCCAGAAGA.

  • Negative Ctrl CHIP-F, GATCAACGCAGGGGAGAGAG.

  • Procr CHIP-R, GTGAATGCACACACACACCC.

  • Procr CHIP-F, ATATCCGAGCTACACACGGC.

  • Ctfg CHIP-R, GAACTGAATGGAGTCCTACACA.

  • Ctfg CHIP-F, TGTGCCAGCTTTTTCAGACG.

Preparation of Procr promoter luciferase reporter and luciferase assay

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The DNA sequence of Procr promoter containing TEAD4-binding sites (about 2 kb before the initiation codon) were amplified by PCR, separated by agarose gel, purified by Gel Extraction Kit, and then cloned into pGL3-promoter vector. Luciferase assays were performed in 293T cells with the pGL3-Procr promoter luciferase reporter described above 0.2 mg reporter plasmid were transfected together with CMV-Renilla (0.005 mg) to normalize for transfection efficiency. For luciferase assays in overexpression plasmid-transfected cells, cells were transfected with the indicated plasmids and reporter plasmid together, and then the luciferase activity was measured 36 hr later using Dual-Luciferase Reporter Assay System Technical Manual kit following the manufacturer’s protocol.

Cell culture, viral production, and infection

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HEK 293T was obtained from American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 10% FBS plus 1% penicillin and streptomycin antibiotics at 37°C in 5% CO2 (vol/vol). For cells cultured on different modulus of elasticity, hydrogel substrates with tunable mechanical properties were prepared following the previous protocol (Tse and Engler, 2010), and the glass was as solid control. HEK 293T cells were used to produce lentivirus. When cells were up to 80%–90%, indicated constructs and packaging plasmids transfection was performed in Opti-MEM, and the media were replaced 12 hr later. Viral supernatants were collected 48–72 hr after medium change and filtered through a 0.45-μm filter, followed by concentration. For primary OSE cells infection, concentrated virus was diluted in the culture medium along with 1:100 polybrene.

RNA isolation and quantitative real-time PCR

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Total RNA was isolated from fresh OSE cells or cultured cells lysed with Trizol according to the manufacturer’s instructions. The cDNA was generated from equal amounts of RNA using the SuperScriptIII kit. qPCR was performed on a StepOne Plus (Applied Biosystems) with Power SYBR Green PCR Master Mix. RNA level was normalized to Gapdh. The cycling condition was as 10 min at 95°C for initial denaturing, 40 cycles of 15 s at 95°C for denaturing, 1 min at 60°C for annealing and extension, following by melt curve test.

Quantification and statistical analysis

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For quantification of nuclear Yap1+, Vgll4+, and EdU+ cells, 40 OSE cells at the both edges of ruptured sites (20 OSE cells at one side of rupture site) was identified as rupture regions, while other regions as nonrupture regions. At least 30 rupture regions and 30 nonrupture regions were counted. For quantification of the diameter of rupture, the longest diameter was counted, and at least 20 rupture sites were counted. For quantification of mG+ clone sizes, about 0.3 mm2 circle centered on ruptured sites was identified as rupture regions. At least 30 rupture regions were counted. For quantification of colonies size, diameters of the colonies were measured using Zeiss software.

Statistical analyses were calculated in GraphPad Prism (Student’s t-test or one-way analysis of variance). For all experiments with error bars, the standard error of measurement was calculated to indicate the variation within each experiment. All the p values were calculated using GraphPad PRISM six with the following significance: n.s. p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Statistical details for each experiment can be found in the figures and the legends.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent (M. musculus)Mouse: Procr-CreERProcrCreERN/AWang et al., 2015
Genetic reagent (M. musculus)Mouse: Procr-rtTAProcrrtTAN/AWang et al., 2019
Genetic reagent (M. musculus)Mouse: R26-mTmGThe Jackson LaboratoryJax: 007576
Genetic reagent (M. musculus)Mouse: TetO-H2B-GFPThe Jackson LaboratoryJax: 005104
Genetic reagent (M. musculus)Mouse: Yap1fl/+Yap1fl/+N/AFeng et al., 2019
Genetic reagent (M. musculus)Mouse: Procrfl/+Liu and Zeng, unpublishedN/ALiu and Zeng, unpublished
Genetic reagent (M. musculus)Mouse: ICRSLACN/AFemale
Genetic reagent (M. musculus)Mouse: C57BL/6SLACN/AFemale
Genetic reagent (M. musculus)Mouse: TetO-Vgll4-FlagThis paperN/ASee Materials and methods
Cell line (Homo-sapiens)Human: HEK 293T cellsATCCCat# CRL-3126
Antibodyanti-CD31, Biotin (Rat monoclonal)BDCat# 553371; RRID:AB_394817FACS (1:200)
Antibodyanti-CD45, Biotin (Rat monoclonal)BDCat# 553080; RRID:AB_394610FACS (1:200)
Antibodyanti-Ter119, Biotin (Rat monoclonal)BDCat# 553672; RRID:AB_394985FACS (1:200)
Antibodyanti-CD31, FITC (Rat monoclonal)BDCat# 553372; RRID:AB_394818FACS (1:200)
Antibodyanti-CD45, FITC (Rat monoclonal)BDCat# 553079; RRID:AB_394609FACS (1:200)
Antibodyanti-Ter119, FITC (Rat monoclonal)BDCat# 557915; RRID:AB_396936FACS (1:200)
Antibodyanti-EpCAM, APC (Rat monoclonal)Thermo FisherCat# 17-5791-82; RRID:AB_2716944FACS (1:200)
AntibodyStreptavidin-apc-Cy7BioLegendCat# 405208; RRID:N/AFACS (1:500)
AntibodyStreptavidin-V450BDCat# 560797; RRID:AB_2033992FACS (1:500)
Antibodyanti Procr, Biotin (Rat monoclonal)Thermo FisherCat# 13-2012-82; RRID:AB_657694FACS (1:200)
Antibodyanti Procr, PE (Rat monoclonal)Thermo FisherCat# 12-2012-82; RRID:AB_914317FACS (1:200)
Antibodyanti-Krt8 (Rat monoclonal)DSHBCat# TROMA-I; RRID:AB_531826IHC (1:250)
Antibodyanti-E-Cadherin (Mouse monoclonal)BDCat# 610181; RRID:AB_397581IHC (1:100)
Antibodyanti-Yap1 (Rabbit monoclonal)CSTCat# 14074; RRID:AB_2650491IHC (1:100)
Antibodyanti-Yap1 (Rabbitpolyclonal)ABclonalCat# A1002; RRID:AB_2757539IHC (1:200)WB (1:200)
Antibodyanti-GFP (Chicken polyclonal)Thermo fisherCat# A10262; RRID:AB_2534023IHC (1:500)
Antibodyanti-Vgll4 (Rabbit polyclonal)Self-madeCat# N/A; RRID:N/AIHC (1:100)
Antibodyanti-Flag (Mouse monoclonal)SigmaCat# F1804; RRID:AB_262044WB (1:100)
Antibodyanti-Vgll4 (Rabbitpolyclonal)ABclonalCat# A18248; RRID:AB_2862024IHC (1:100)WB (1:100)
Antibodyanti-Laminin (Rabbit polyclonal)SigmaCat# L9393; RRID:AB_477163IHC (1:500)
Antibodyanti-a-E-catenin (Rabbit polyclonal)ProteintechCat# 12831-1-AP; RRID:AB_2087822IHC (1:200)
Antibodyanti-ZO-1 (Mouse monoclonal)Thermo FisherCat# 33-9100; RRID:AB_2533147IHC (1:100)
AntibodyNormal mouse IgGSanta cruzCat# sc-2025; RRID:AB_737182ChIP (1:100)
Antibodyanti-TEAD4 (Mouse monoclonal)AbcamCat# ab58310; RRID:AB_945789ChIP (1:100)
Recombinant DNA reagentpLKO.1-EGFP-shYap1This paperPlasmidPol III-based shRNA backbone
Recombinant DNA reagentpcDNA3.1-Yap1This paperPlasmidpcDNA3.1 backbone
Recombinant DNA reagentPGL3.1 Procr promoterThis paperPlasmidPGL3.1 Promoter backbone
Recombinant DNA reagentPGL3.1 Procr promoter TEAD4-binding motif mutationThis paperPlasmidDeletion of 5′CATTCC3′ at the −1486 to −1481 bp in Procr Promoter region
Sequence-based reagentProcr-FThis paperqPCR primers5′CTCTCTGGGA AAACTCCTGACA3′
Sequence-based reagentProcr-RThis paperqPCR primers5′CAGGGAGCAGCT AACAGTGA3′
Sequence-based reagentVgll4-FThis paperqPCR primers5′ATGAACAACAATA TCGGCGTTCT3′
Sequence-based reagentVgll4-RThis paperqPCR primers5′GGGCTCCATGCT GAATTTCC3′
Sequence-based reagentYap1-FThis paperqPCR primers5′GCCATGCTTTCG CAACTGAA3′
Sequence-based reagentYap1-RThis paperqPCR primers5′CAAAACGAGGGT CCAGCCTT3′
Sequence-based reagentCcn1-FThis paperqPCR primers5′TCGCAATTGGAA AAGGCAGC3′
Sequence-based reagentCcn1-RThis paperqPCR primers5’CCAAGACGTGG TCTGAACGA3’
Sequence-based reagentBirc5-FThis paperqPCR primers5′AGAACAAAATTG CAAAGGAGACCA3′
Sequence-based reagentBirc5-RThis paperqPCR primers5′GGCATGTCAC TCAGGTCCAA3′
Commercial assay or kitClick-iTTMEdU Cell Proliferation Kit for ImagingThermo FisherCat# C10337
Commercial assay or kitDual-Luciferase Reporter Assay System Technical ManualPromegaCat# E1910
Commercial assay or kitOpal 4-Color Automation IHC KitPerkinElmerCat# NEL8200001KT
Commercial assay or kitRNAscope Multiplex Fluorescent Detection Kit v2ACDCat# 323,110
Commercial assay or kitSYBR green MixRocheCat# 04913914001
Commercial assay or kitPrimeScript RT master MixTakaraCat# RR036A
Chemical compound, drugHoechst33342Thermo FisherCat# H21492
Chemical compound, drugTamoxifenSigma-AldrichCat# T5648
Chemical compound, drugDoxcyclinehyclateSigma-AldrichCat# D9891
Chemical compound, drugVerteporfinMCECat# HY-B0146
Software, algorithmFlow Jo vXFlow Johttps://www.flowjo.com
Software, algorithmGraphPad Prism 6GraphPad softwarehttps://www.graphpad.com
OtherDNase ISigma-AldrichCat# D4263
OtherType IV collagenaseWorthingtonCat# LS004189
OtherMatrigelBDCat# 354,230
OtherDispaseBDCat# 354,235
OtherDAPIThermo FisherCat# D1306
OtherMEM Non-Essential Amino Acids SolutionThermo FisherCat# 11140050
OtherL-GlutamineThermo FisherCat# 25030081
OtherITSThermo FisherCat# 41,440
Other2-MercaptoethanolMilliporeCat# ES-007-E
OtherHydrocortisoneSigma-AldrichCat# 614,517
OtherSodium pyruvateThermo FisherCat# 11360070

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figure 1-source data 1, Figure 2-source data 1, Figure 3-source data 1, Figure 4-source data 1, Figure 5-source data 1, Figure 1-figure supplement 2-source data 1, Figure 2-figure supplement 1-source data 1,Figure 2-figure supplement 1-source data 2,Figure 4-figure supplement 1-source data 1;Figure 4-figure supplement 2-source data 1;Figure 4-figure supplement 2-source data 2.

References

Decision letter

  1. Lynne-Marie Postovit
    Reviewing Editor; University of Alberta, Canada
  2. Ricardo Azziz
    Senior Editor; University at Albany, SUNY, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Ricardo Azziz as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

The reviewers were generally positive about this manuscript. The requested revisions are mostly editorial and involve tempering claims and/or editing the text or figures. Simple experiments, designed to strengthen conclusions are also requested.

1) The authors failed to identify how stem/progenitor cells sense ovarian rupture to then trigger rupture repair through inducing pathways such as Yap. It is not necessary that the authors identify what senses the rupture for publication, as identifying the sensors that detect tissue wounding is a major challenge in regenerative biology. However, the authors claimed to have resolved this point in the abstract, introduction and discussion, and therefore should be more precise in describing their main findings.

2) If the ovulation hormones used to induce ovarian tissue rupture can potentially induce Yap signaling, the authors should explicitly discuss this potential experimental limitation in their manuscript.

3) The authors conclude that YAP signalling mediates its effects on healing at least in part by enhancing the expression of Procr in OSE. However, as it stands the results remain correlative. The authors should thus determine whether Procr overexpression can overcome YAP KO or Vgll4 overexpression, at least using in vitro models.

4) YAP pathway activation was extrapolated using in situ hybridization for one target (Cyr61). If feasible, the authors are encouraged to examine additional YAP signalling target genes by the same or another independent method (such as qPCR), to strengthen their main conclusion.

5) In Line 115, the authors state "various adherent or tight junction components" were examined. However, the results showed the effect on E-cadherin only. Did the authors examine multiple components or just one? Additional adherens junction proteins besides E-cadherin (such as a-catenin) would be helpful to reinforce their conclusion.

6) Please further characterise or describe the sorted OSE cells (perhaps with additional markers) to ensure that the Procr+ and Procr- cells are of the same lineage.

7) For figure 4 l and m, please show IF and/or Westerns to validated increasing YAP expression.

8) Resolution of the images on YAP immunostaining (Figure 1d, 1e) was too poor to discriminate between nuclear or cytoplasmic staining patterns. Please improve these images.

9) Please quantify proliferation shown in Figure S3b.

10) Line 303, "bigger openings compared to control" the subject was reversed in Figure legends "would size in Ctrl were smaller". Please correct

11) Line 303, 7d pi did not match Figure 5e, which was labeled as 7.5d. Please correct.

12) Results in lines 307 and 308 (5.5, 1,4) did not match legends of Figure 5h (4.73, 1.62. Please correct.

Reviewer #1:

This is a well conducted and interesting study that increases our knowledge of mechanisms governing healing after ovarian rupture. This study specifically demonstrates the importance of Procr+ progenitors that are positively regulated by YAP signaling. The study uses elegant mouse models and for the results are convincing. A few revisions would enhance the study and enable the authors to more strongly conclude that YAP promotes healing at least in part by increasing the expression of Procr.

Col1a1 is not specific for Procr+ OSE cells. I do not think this will affect the results terribly, but should be acknowledged.

The authors conclude that YAP signalling mediates its effects on healing at least in part by enhancing the expression of Procr in OSE. However, as it stands the results remain correlative. The authors should thus determine whether Procr overexpression can overcome YAP KO or Vgll4 overexpression, at least using in vitro models.

The authors should attempt to determine whether there are human correlates to the phenomena observed in the mouse models.

Please further characterise the sorted OSE cells (perhaps with additional markers) to ensure that the Procr+ and Procr- cells are pf the same lineage.

For figure 4 l and m, please show IF and/or Westerns to validated increasing YAP expression.

Please quantify proliferation shown in Figure S3b.

Reviewer #2:

The ovary is a fascinating yet understudied organ in the field of stem cell biology: every reproductive cycle, it is ruptured as eggs are released. The ovary thus undergoes successive waves of injury. The mechanisms through which the ovary's surface epithelium is repaired remain incompletely understood. Multiple candidate stem and progenitor cells have been described for the ovarian surface epithelium (Ng and Barker, 2015; Nat Rev Mol Cell Biol), and the authors of the present report have previously demonstrated that Procr+ progenitors contribute to ovarian rupture repair (Wang et al., 2019; Nature Communications).

In the present report, the authors attempt to answer the fascinating question of how progenitors sense tissue injury in order to effect regeneration; broadly speaking, how tissues sense injury is one of the biggest questions in the field of stem cell biology. The authors show that the transcriptional coactivator Yap is expressed by Procr+ ovarian epithelial progenitors, and is necessary for rupture repair. This is demonstrated through two parallel methods: directly deleting Yap (Figure 2) or overexpressing Vgll4, which encodes a Yap antagonist (Figure 3). Both of these genetic manipulations are specifically conducted in Procr+ ovarian epithelial progenitors in vivo.

While the authors convincingly show that Yap is necessary for ovarian rupture repair, the biggest unanswered question in the paper is, mechanistically, how the rupture is actually sensed (i.e., what turns on Yap?) Yap is a transcriptional coactivator that is repressed by the Hippo mechanotransduction pathway, and there are multiple mechanical inputs (e.g., cell adhesion, cell stretching, extracellular matrix stiffness, and cell density) that can all regulate the Hippo pathway (Halder et al., 2012; Nat Rev Mol Cell Biol). The authors claim that the E-cadherin cell-adhesion protein is lowered at the site of the ovarian rupture (Figure 1), and it was previously known that E-cadherin signals through the Hippo pathway to block Yap (Yang et al., 2015; PNAS).

However the missing link in the paper is why E-cadherin would be reduced at the ovarian rupture site. There are other potentially more direct possibilities. For instance, it is known that mechanical stretching induces Yap (Halder et al., 2012; Nat Rev Mol Cell Biol). First, during the process of egg release, overlying epithelial cells will possibly become "stretched", which would induce Yap? Second, at the rupture site, epithelial cells will no longer become packed together because the epithelium has been denuded: so potentially cells flanking the rupture site would become "stretched", which would also activate Yap? These ideas are speculative, but are intended to try to assist the authors in formulating a mechanistic link between ovarian ruptures and Yap activation. For instance, the authors could consider looking at cell stretching/roundness/morphology by imaging.

In summary, the authors show that Yap is required for ovarian regeneration, consistent with the well-known roles of Yap in cell proliferation and tissue repair in other contexts (Ramos and Camargo, 2012; Trends in Cell Biology). However the authors may not have fully addressed the question they posed in the Introduction, namely how is ovarian rupture sensed and how is Yap itself induced by injury.

Comments for the authors:

There are multiple merits to this paper, including the demonstration that Yap is required in Procr+ ovarian progenitors to drive rupture repair. However the missing link in the manuscript is how tissue rupture is sensed and how Yap is turned on: something that the authors raised in the Introduction of their paper. The authors show E-cadherin is decreased at the rupture (Figure 1), but it is unclear why E-cadherin should be decreased.

An open question remains whether the authors need to find the "rupture sensing mechanism" (i.e., what is upstream of Yap) to warrant publication in eLife. It is also possible that without finding this mechanism that the manuscript could be published. But in that case, the authors may want to tone down their claims that they discovered how "stem cell senses the rupture and promptly turns on proliferation".

Strengths:

1. The ovary is an understudied organ, and this study sheds light onto its regenerative powers.

2. The authors specifically examine the role of Yap in a defined cell population (Procr+ progenitors), instead of perturbing Yap in the whole organ.

3. Two parallel approaches are used to block Yap: directly deleting Yap (Figure 2) or overexpressing Vgll4, which encodes a Yap antagonist (Figure 3). Both of these perturbations compromise effective rupture repair. Yap deletion also reduces the frequency of Procr+ progenitors (Figure 4).

Weaknesses:

1. Why is E-cadherin decreased at rupture sites? (Figure 1a)

2. The authors suggest that nuclear Yap is induced in superficial epithelial cells nearby the rupture (Figure 1b). While the higher-magnification images are a bit easier to interpret, the zoomed-out image (leftmost image in Figure 1b) is not that clear. Yap seems to be expressed in many cells, including deeper cells (i.e., not superficial epithelial cells) and even some cells quite far away from the rupture.

3. The authors claim that reduced Vgll4 levels in Procr+ progenitors (as opposed to Procr- non-progenitors) renders Procr+ progenitors hyper-sensitive to Yap activation. However there is a fairly modest ~2-fold difference in Vgll4 mRNA between Procr+ vs. Procr- cells (Figure 3b) – is this enough to substantially change a cell's receptiveness to Yap signaling?

4. The notion that Procr itself is required for ovarian regeneration is potentially interesting (Figure 5). However, Procr is expressed in multiple cell-types within the ovary; the authors actually showed that vascular cells are a major population of Procr+ cells (Wang et al., 2019; Nature Communications). Is it possible that when Procr is deleted that the epithelial progenitors themselves are not significantly impacted, but rather the impaired regeneration can be ascribed to vascular (or other) defects? Procr-/- mice die of vascular defects (Gu et e al., 2002; J Biol Chem).

Reviewer #3:

The wound healing process during the normal ovulation process is a very interesting but poorly understood process. Wang et al. intend to identify molecular mechanisms by which tissue-resident stem/progenitor cells engage in response to epithelium rupture. Wang et al. made use of a marker gene, Procr, to investigate stem/progenitor population in the ovarian epithelium that was described in their earlier publication. With the advantage of being able to isolate and characterize this population, strengths of the methods employed in the manuscript involves the usage of several novel mouse strains to perform lineage tracing or genetic ablation specifically in the Procr+ cells versus other epithelial cells that lack Procr expression. The strengths of the results in this study revealed compelling functional phenotypes in respect to loss of function (YAP, Procr) and gain of function (Vgll4) in the context of ovarian rupture repair process.

The weakness of the methods is the means of inducing ovarian epithelium rupture requires the usage of two hormones, which can also activate YAP pathway independently. Although this does not have a large impact on the conclusions, it is important to recognize the observation on YAP pathway is associated with this specific experimental method. However, multiple methods were used to identify functional consequences of ovarian rupture repair; the conclusions are well supported by the results. In addition, the authors generated several new transgenic mouse strains (Procr-rtTA, TetO-Vgll4, Procrflox) to address the functional relevance of cell type of origin as well as the roles of YAP signaling pathway. These strains will be useful resources for the community.

1. The title and main conclusion of the study on selective activation of the YAP pathway within Procr+ progenitors were supported by the detection of a single target Cyr61 examined by RNA in situ hybridization. If feasible, the authors are encouraged to examine additional target genes by the same or another independent method (such as qPCR), to strengthen their main conclusion.

2. In Line 115, the authors state "various adherent or tight junction components" were examined. However, the results showed the effect on E-cadherin only. Did the authors examine multiple components or just one? Additional adherens junction proteins besides E-cadherin (such as a-catenin) would be helpful to reinforce their conclusion.

3. Resolution of the images on YAP immunostaining (Figure 1d, 1e) was too poor to discriminate between nuclear or cytoplasmic staining patterns.

https://doi.org/10.7554/eLife.75449.sa1

Author response

Essential revisions:

The reviewers were generally positive about this manuscript. The requested revisions are mostly editorial and involve tempering claims and/or editing the text or figures. Simple experiments, designed to strengthen conclusions are also requested.

1) The authors failed to identify how stem/progenitor cells sense ovarian rupture to then trigger rupture repair through inducing pathways such as Yap. It is not necessary that the authors identify what senses the rupture for publication, as identifying the sensors that detect tissue wounding is a major challenge in regenerative biology. However, the authors claimed to have resolved this point in the abstract, introduction and discussion, and therefore should be more precise in describing their main findings.

We apologize for the confusion caused. Following the suggestion, we have rephrased “senses” into “is triggered by” in the abstract, into “the molecular mechanism in which linking the ovulatory rupture to OSE stem/progenitor cells activation” in the introduction, and discussion. We especially appreciate the reviewers’ insights on the possible reasons for turning on Yap, and we have included them in the discussion (p.13-14).

“While we uncovered the significance of selective activation of Yap in OSE progenitors, it is still unclear how ovarian rupture is sensed and how Yap signaling is induced by injury. […] Second, at the rupture site, epithelial cells no longer become packed together because the epithelium has been denuded, therefore potentially cells flanking the rupture site would become "stretched", consequently actives YAP.”

2) If the ovulation hormones used to induce ovarian tissue rupture can potentially induce Yap signaling, the authors should explicitly discuss this potential experimental limitation in their manuscript.

Following the advice, we found two pieces of literature. One reports that HCG down-regulates YAP1 protein level and target gene Ctgf expression in granulosa cells 1; another one shows that HCG downregulates YAP1 expression and upregulates p-YAP in cumulus-oocyte complexes 2. Thus, both papers suggest ovulation hormones suppress Yap signaling in granulosa cells. We have not come across previous reports about Yap and OSE.

3) The authors conclude that YAP signalling mediates its effects on healing at least in part by enhancing the expression of Procr in OSE. However, as it stands the results remain correlative. The authors should thus determine whether Procr overexpression can overcome YAP KO or Vgll4 overexpression, at least using in vitro models.

We appreciate the reviewer’s suggestion. We would like to clarify that in the proposed model, YAP signaling activation at the rupture site drives cell division and simultaneously enhances Procr expression (Figure 5m). Therefore, we reasoned that overexpression of Procr alone would not be sufficient to rescue YAP-cKO or Vgll4-OE.

Nevertheless, we understand the reviewers’ concern and performed relevant experiments, in which we overexpress YAP and knockdown Procr in a human OSE cell line, OVCAR3 cells. Overexpression of YAP promoted the in vitro sphere formation and in vivo xenografts growth. While knockdown of PROCR blocked the sphere formation and xenografts growth induced by YAP-OE. Those data suggest YAP signaling mediates its effects in part by PROCR (see Author response image 1).

Author response image 1

4) YAP pathway activation was extrapolated using in situ hybridization for one target (Cyr61). If feasible, the authors are encouraged to examine additional YAP signalling target genes by the same or another independent method (such as qPCR), to strengthen their main conclusion.

We appreciate the suggestion. We included qPCR analyses in the revised Figure S2c, showing that Procr+ OSE cells have a higher level of Birc5 expression, another YAP target gene.

5) In Line 115, the authors state "various adherent or tight junction components" were examined. However, the results showed the effect on E-cadherin only. Did the authors examine multiple components or just one? Additional adherens junction proteins besides E-cadherin (such as a-catenin) would be helpful to reinforce their conclusion.

Following the suggestion, we included the immunostaining of adherents and tight junction proteins in the revised Figure S1c-d, showing a decreased α-catenin and ZO-1 in the OSE of rupture proximal regions compared to rupture distal and non-rupture regions.

6) Please further characterise or describe the sorted OSE cells (perhaps with additional markers) to ensure that the Procr+ and Procr- cells are of the same lineage.

Following the suggestion, we performed Krt8 (K8) staining with the sorted Procr+ and Procr- OSE cells, and the results indicated that both Procr+ and Procr- OSE are K8+ epithelial cells. These results have been included in revised Figure S2a-b.

7) For figure 4 l and m, please show IF and/or Westerns to validated increasing YAP expression.

Following the suggestion, we performed immunostaining and Western blotting, and the results verified increasing YAP expression. These data have been included in the revised Figure S5h-i.

8) Resolution of the images on YAP immunostaining (Figure 1d, 1e) was too poor to discriminate between nuclear or cytoplasmic staining patterns. Please improve these images.

Following the suggestion, we have replaced them with images with higher magnification in revised Figure1d-e.

9) Please quantify proliferation shown in Figure S3b.

Following the suggestion, we have quantified the proliferation of Figure S4a-b (previous S3a-b) and shown in the revised Figure S4c.

10) Line 303, "bigger openings compared to control" the subject was reversed in Figure legends "would size in Ctrl were smaller". Please correct

We apologize for the confusion caused. It has been revised to “control ovaries showed smaller openings compared to Procr-cKO (Figure 5c, 5e)” in Line 341 (previous Line 303).

11) Line 303, 7d pi did not match Figure 5e, which was labeled as 7.5d. Please correct.

We apologize for the mistake. It has been revised to 7.5d.

12) Results in lines 307 and 308 (5.5, 1,4) did not match legends of Figure 5h (4.73, 1.62. Please correct.

We apologize for the mistake. It has been revised to (4.7±0.4, 1.6±0.2).

Reference:

1. Ji, S.-Y. et al. The polycystic ovary syndrome-associated gene Yap1 is regulated by gonadotropins and sex steroid hormones in hyperandrogenism-induced oligo-ovulation in mouse. MHR: Basic science of reproductive medicine 23, 698-707, doi:10.1093/molehr/gax046 (2017).

2. Sun, T. and Diaz, F. J. Ovulatory signals alter granulosa cell behavior through YAP1 signaling. Reproductive Biology and Endocrinology 17, doi:10.1186/s12958-019-0552-1 (2019).

https://doi.org/10.7554/eLife.75449.sa2

Article and author information

Author details

  1. Jingqiang Wang

    1. Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, National Children’s Regional Medical Center, Hangzhou, China
    2. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Validation, Writing - original draft, Writing – review and editing
    Contributed equally with
    Chunye Liu and Lingli He
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1670-7759
  2. Chunye Liu

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Data curation, Project administration, Resources, Supervision, Validation
    Contributed equally with
    Jingqiang Wang and Lingli He
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7167-6735
  3. Lingli He

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Data curation, Investigation
    Contributed equally with
    Jingqiang Wang and Chunye Liu
    Competing interests
    No competing interests declared
  4. Zhiyao Xie

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Data curation, Investigation
    Competing interests
    No competing interests declared
  5. Lanyue Bai

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Data curation, Resources
    Competing interests
    No competing interests declared
  6. Wentao Yu

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Data curation, Investigation
    Competing interests
    No competing interests declared
  7. Zuoyun Wang

    Human Anatomy & Histoembryology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  8. Yi Lu

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  9. Dong Gao

    State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    Contribution
    Writing – review and editing
    Competing interests
    No competing interests declared
  10. Junfen Fu

    Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, National Children’s Regional Medical Center, Hangzhou, China
    Contribution
    Funding acquisition, Supervision
    For correspondence
    fjf68@zju.edu.cn
    Competing interests
    No competing interests declared
  11. Lei Zhang

    1. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    2. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, China
    3. School of Life Science and Technology, ShanghaiTech University, Shanghai, China
    Contribution
    Funding acquisition, Project administration, Resources, Supervision, Writing – review and editing
    For correspondence
    rayzhang@sibcb.ac.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2566-6493
  12. Yi Arial Zeng

    1. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
    2. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, China
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Writing - original draft, Writing – review and editing
    For correspondence
    yzeng@sibcb.ac.cn
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1898-8099

Funding

National Key Research and Development Program of China (2019YFA0802000)

  • Yi Arial Zeng

National Key Research and Development Program of China (2020YFA0509002)

  • Yi Arial Zeng

National Natural Science Foundation of China (31625020)

  • Yi Arial Zeng

Chinese Academy of Sciences (XDA16020200)

  • Yi Arial Zeng

National Natural Science Foundation of China (32030025)

  • Lei Zhang

National Natural Science Foundation of China (31625017)

  • Lei Zhang

Fundamental Research Funds for the Central Universities (2020XZZX002-22)

  • Junfen Fu

National Key Research and Development Program of China (2021YFC2701901)

  • Junfen Fu

China Postdoctoral Science Foundation (2020TQ0260)

  • Jingqiang Wang

Zhejiang Provincial Preferential Postdoctoral Foundation (ZJ2020150)

  • Jingqiang Wang

Postdoctoral Exchange Fellowship Program 2021 (No. 160 Document of OCPC 2021)

  • Jingqiang Wang

The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

Acknowledgements

Thank to Dr. Chi Chung Hui of University of Toronto for helpful comments on the manuscript. This research was supported by grants from National Key Research and Development Program of China (2019YFA0802000 and 2020YFA0509002 to YAZ); National Natural Science Foundation of China (31625020 to YAZ; 32030025 and 31625017 to LZ); the Chinese Academy of Sciences (XDA16020200 to YAZ); Fundamental Research Funds for the Central Universities (2020XZZX002-22 to JF); the National Key Research and Development Programme of China (2021YFC2701901 to JF); China Postdoctoral Science Foundation (2020TQ0260 to JW); Zhejiang Provincial Preferential Postdoctoral Foundation (ZJ2020150 to JW); Postdoctoral Exchange Fellowship Program 2021 (No.160 Document of OCPC, 2021) to JW; Shanghai Leading Talents Program to LZ.

Ethics

All mice were housed in the SIBCB animal facility under IVC standard with a 12-hr light/dark cycle at room temperature. Experimental procedures were approved by the Animal Care and Use Committee of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, with a project license number of IBCB0065.

Senior Editor

  1. Ricardo Azziz, University at Albany, SUNY, United States

Reviewing Editor

  1. Lynne-Marie Postovit, University of Alberta, Canada

Publication history

  1. Received: November 10, 2021
  2. Preprint posted: November 18, 2021 (view preprint)
  3. Accepted: February 24, 2022
  4. Version of Record published: March 14, 2022 (version 1)

Copyright

© 2022, Wang et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jingqiang Wang
  2. Chunye Liu
  3. Lingli He
  4. Zhiyao Xie
  5. Lanyue Bai
  6. Wentao Yu
  7. Zuoyun Wang
  8. Yi Lu
  9. Dong Gao
  10. Junfen Fu
  11. Lei Zhang
  12. Yi Arial Zeng
(2022)
Selective YAP activation in Procr cells is essential for ovarian stem/progenitor expansion and epithelium repair
eLife 11:e75449.
https://doi.org/10.7554/eLife.75449

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