In Vivo Cell Fate Tracing Provides No Evidence for Mesenchymal to Epithelial Transition in Adult Fallopian Tube and Uterus

SUMMARY The mesenchymal to epithelial transition (MET) is thought to be involved in the maintenance, repair, and carcinogenesis of the fallopian tube (oviduct) and uterine epithelium. However, conclusive evidence for the conversion of mesenchymal cells to epithelial cells in these organs is lacking. Using embryonal cell lineage tracing with reporters driven by mesenchymal cell marker genes of the female reproductive tract (AMHR2, CSPG4, and PDGFRβ), we show that these reporters are also expressed by some oviductal and uterine epithelial cells at birth. These mesenchymal reporter-positive epithelial cells are maintained in adult mice across multiple pregnancies, respond to ovarian hormones, and form organoids. However, no labeled epithelial cells are present in any oviductal or uterine epithelia when mesenchymal cell labeling was induced in adult mice. Organoids developed from mice labeled in adulthood were also negative for mesenchymal reporters. Collectively, our work found no definitive evidence of MET in the adult fallopian tube and uterine epithelium.


INTRODUCTION
The mammalian female reproductive tract (FRT) is essential for fertility, and dysregulation in its functions contributes to many gynecological diseases, including ovarian cancer (Teixeira et al., 2001). The FRT develops from a pair of simple tubular structures known as müllerian ducts (MDs). The MDs are formed from the coelomic epithelium and, irrespective of genotypic sex, are present in both males and females (Mullen and Behringer, 2014;Teixeira et al., 2001). However, anti-Müllerian hormone (also known as Müllerian-inhibiting substance), secreted by the Sertoli cells of the fetal testes, causes regression of the MDs in males (Teixeira et al., 2001). In both humans and mice, the MD epithelium is mesoepithelial in nature because it expresses the mesenchymal markers, such as vimentin, in the epithelial cells. (Fritsch et al., 2013;Orvis and Behringer, 2007;Paranko and Virtanen, 1986). In females, the MDs undergo progressive differentiation with age in a caudal-to-cranial direction to acquire unique morphologic, organ-specific epithelial features and give rise to the fallopian tubes (FT) (oviducts in non-primates), uterus, cervix, and anterior portion of the vagina (Fritsch et al., 2013;Teixeira et al., 2001). Postnatally, the epithelium of the MDderived organs is more differentiated and expresses typical epithelial markers, such as cytokeratins (Fritsch et al., 2013;Peri et al., 2015;Song et al., 1998).
Mesenchymal to epithelial transition (MET) plays an essential role during embryonic development. Although there is limited evidence of MET in adult organs (Pei et al., 2019), there have been reports that suggest MET could be occurring in the FRT. For example, in the adult mouse uterus, stromal cells expressing the AMH type 2 receptor (AMHR2) or smooth muscle 22 α (SM22α, also called transgelin) have been shown to convert to epithelial cells during the repair and regeneration of the injured endometrium, and alterations in these cells contribute to endometrial hyperplasia (Huang et al., 2012;Owusu-Akyaw et al., 2019;Patterson et al., 2013;Yin et al., 2019). Interestingly, these AMHR2 + or SM22α + stromalcell-derived epithelial cells are generally not present in the normal endometrial epithelium and only appear after physical or postpartum injury (Huang et al., 2012;Patterson et al., 2013;Yin et al., 2019). Similarly, oviductal AMHR2 + stromal cells have been shown to undergo epithelialization and, upon oncogenic transformation, develop serous ovarian cancer (Hua et al., 2016;Kim et al., 2012). These studies appear to challenge the more traditional view of the adult epithelial regeneration and carcinogenesis in which epithelial cells are known to have a central role (Daikoku et al., 2008(Daikoku et al., , 2011Ghosh et al., 2017;Perets et al., 2013;Russo et al., 2018;Sherman-Baustet al., 2014;Wu et al., 2016). As many of these MET studies used mesenchymal-cell-specific Cre reporters that are activated in the MDs during early embryonic development when MD epithelial cells have mesoepithelial characteristics and express mesenchymal markers, it is unclear whether the subsequent appearance of the reporter-labeled epithelial cells is due to the embryonic expression of Cre in the MD epithelium or because of MET. Additionally, some of the Cre reporters are also expressed in the coelomic epithelium, the precursor of MDs (Guioli et al., 2007;Orvis and Behringer, 2007).
Here, we performed lineage tracing of both epithelial and mesenchymal cells by inducing permanent reporter expression in these cells during embryonic and adult stages. This technique is a gold standard approach to track the behavior of labeled cells and their subsequent progenies in their native environment in tissues and organs (Chari et al., 2017;Kretzschmar and Watt, 2012). To investigate comprehensively the extent of MET in the FRT organs, we analyzed 10 different mouse models. First, we analyzed Amhr2 Cre ;R26-lacZ fl/+ , Amhr2-lacZ, and Sm22 Cre ;R26-lacZ fl/+ mice to replicate the results of previous studies. We screened for the presence of any lacZ + cells in the oviduct and uterus after cutting the whole organs into 125-μm-thick, serial sections and also performed histopathological analysis using thin (5 μm) sections. Our analyses showed that lacZ + cells are indeed present in the oviductal and uterine epithelium of neonatal, prepubertal, and adult Amhr2 Cre ;R26-lacZ fl/+ and Sm22a Cre ;R26-lacZ fl/+ mice. To prove that these observations are not just limited to Amhr2 and Sm22a reporter mice, we analyzed three additional mouse models: Cspg4 Cre ;R26-lacZ fl/+ , Thy1 Cre ;lacZ fl/+ , and Acta1 Cre ;lacZ fl/+ . We chose these promoters because they are broadly expressed in mesenchymal cells during embryonic development, but their expression is limited to specific mesenchymal cells postnatally, such as, CSPG4 in pericytes and ACTA1 in heart and skeletal muscles (Smith et al., 2019). Because, reporter expression is not inducible in these models, the presence of reporter-labeled epithelial cells can be attributed to the mesoepithelial nature of the MDs rather than to MET. Therefore, we used Cspg4 CreER ;R26-lacZ fl/+. and Pdgfrβ rtTa ;tetO Cre ;R26-lacZ fl/+ mice to be able to induce reporter expression at different stages of development and, thereby, investigate the contributions of embryonic and adult mesenchymal cells separately. Furthermore, we used an alternative strategy of labeling epithelial cells in Pax8 rtTa ;tetO Cre ;R26-lacZ fl/+ mice, and investigated whether the labeled epithelial cells are replaced by unlabeled mesenchymal cells over time. We also developed organoids from these mouse models to test the contribution of any rare mesenchymal cells that might have been missed during our in vivo studies. Using these methodologies, we found no strong evidence supporting the role of MET in the adult oviduct and uterus during homeostasis and regeneration.

Amhr2Cre-Driven, LacZ-Labeled Cells Are Present in the Epithelium by Birth
During embryonic development, coelomic epithelium invaginates to form the Müllerian duct epithelium (MDE) (Guioli et al., 2007;Orvis and Behringer, 2007). Initially, MDE is mesoepithelial in nature. Eventually, it progressively acquires epithelial features and differentiates into the epithelium of MD-derived organs. The surrounding mesenchymal cells, Müllerian duct mesenchyme (MDM), also progressively differentiates to acquire FRT organ-specific features of stroma and muscularis layers. Immunostaining for different mesenchymal markers, such as vimentin, platelet-derived growth factor receptor beta (PDGFRβ), αSMA, and an epithelial marker cytokeratin 8 (CK8) on adult mouse oviduct and uterus showed that the expression of these mesenchymal markers is limited to stroma and muscularis layer, and CK8 is expressed only in the epithelium ( Figures 1A and 1B), suggesting that by adulthood, epithelial and mesenchymal cells are fully differentiated and express their respective markers. Amhr2 expression is present in the coelomic epithelium and MDM (Baarends et al., 1994;Teixeira et al., 1996). To determine the fate of AMHR2 + cells, we developed Amhr2 Cre ;R26-lacZ fl/+ mice in which lacZ-encoded β-galactosidase expression is specific to AMHR2 + cells and their progenies ( Figure 1C). Gross examination of reproductive tracts at postnatal day 1 (PND1) from these mice showed X-gal staining in FRT ( Figure 1C). Histological examination revealed the presence of X-gal-stained cells in both stroma and epithelium of the oviduct and uterus (Figures 1D and S1A-S1E). Quantification of X-gal staining in oviduct showed increased labeling with time, suggesting that the Amhr2-driven, lacZ-labeled epithelial cells actively contribute to oviduct development and are not vestigial cells ( Figure 1E). Using RNA in situ hybridization, we confirmed that the endogenous expression of Amhr2 is limited to coelomic epithelium, MDM, and oviduct stroma ( Figure 1F). We used adult mouse ovaries as a positive control in which Amhr2 expression was limited to stromal cells and ovarian surface epithelium ( Figure  1Fa). We also examined the endogenous Amhr2 expression using Amhr2-lacZ mice in which lacZ expression is knocked into the Amhr2 gene and is directly under the control of Amhr2 promoter (Arango et al., 2008). Grossly, X-gal staining was present in the ovary, oviduct, and uterus ( Figure 1Ga). Consistent with Amhr2 mRNA expression data, histological analyses confirmed that Amhr2-specific lacZ is expressed in adult FRT stroma but not in the epithelium (Figures 1Gb-d, S1F, and S1G). Collectively, these data showed the presence of Amhr2Cre-derived, lacZ-labeled cells in the epithelium of neonatal and adult FRT and the absence of Amhr2 mRNA and Amhr2-driven marker expression in adult FRT epithelium .

Cspg4Cre-Driven, LacZ-Labeled Cells Are Present in the FRT Epithelium
To confirm further our results in a different model system, we marked and traced the fate of chondroitin sulfate proteoglycan 4 (CSPG4)-expressing cells. During early embryonic development, CSPG4 is broadly expressed in mesenchymal cells, and later on, its expression is limited to a subset of mesenchymal cells known as pericytes (Smith et al., 2019). Therefore, we developed a mouse model in which lacZ expression is under the control of Cspg4 promoter-driven Cre. Grossly, X-gal staining was present in the MD (E14) and the FRT at PND1 and adult stages ( Figure 2A). Postnatal expression of CSPG4 is limited to perivascular cells ( Figure 2B), but histological analyses of Cspg4 Cre ;R26-lacZ fl/+ neonatal FTs revealed the presence of Cspg4Cre-driven, lacZ-labeled cells in both the epithelial and mesenchymal compartments (Figure 2Ca). Importantly, these labeled epithelial cells persisted in adulthood, and their number increased with age (Figures 2Cb and c and 2D). Moreover, immunostaining revealed that these lacZ-labeled epithelia consisted of both secretory (PAX8 + ) and ciliated (Ac-TUB + ) cells ( Figure 2E, arrowheads). To investigate whether these labeled cells survive pregnancy-induced remodeling, we collected Cspg4 Cre ;R26-lacZ fl/+ mice FRTs after three consecutive pregnancies and found lacZ + epithelial cells in the oviducts even after three cycles of pregnancy and involution ( Figures  2F-2H). Immunostaining confirmed that, similar to non-labeled cells, lacZ + cells also express estrogen receptor α (ERα) and respond to tamoxifen treatment, an estrogen agonist (Figures 2Ge and 2I-2K). Next, we isolated epithelium from Cspg4 Cre ;R26-lacZ fl/+ mice oviducts and demonstrated that both labeled and non-labeled epithelial cells can contribute to organoid development ( Figures 2L and 2M).
Similar to FTs, gross analysis of X-gal stained Cspg4 Cre ;R26-lacZ fl/+ uteri showed lacZ expression in uterine stroma and blood vessels, and the expression was absent in littermate controls ( Figure 3A). To screen extensively for any rare lacZ + cells, whole uterine arms of both control and mutant mice were cut into a series of 125-μm-thick sections and then analyzed ( Figure 3B). No staining was observed in any of the control mice ( Figure 3Ba). However, patches of lacZ + cells were present in the epithelium of some uterine sections at PND35 ( Figures 3B and 3C). These labeled cells were present in both uterine luminal and glandular epithelial cells and showed no regional bias toward any specific anatomical location ( Figure 3C). To ensure that the lacZ labeling is not because of CSPG4 expression in the uterine epithelium, we performed immunostaining for CSPG4 in X-gal stained neonatal (Figure 3Da-d) and adult nulliparous (Figure 3De and f) Cspg4 Cre ;R26-lacZ fl/+ uteri. At both these stages, CSPG4 expression was restricted to the perivascular cells ( Figure 3D). Contrary to our findings, previous reports have suggested that mesenchymal Cre-driven lacZ labeling appears in FRT epithelium only during post-injury and postpartum repair and regeneration (Huang et al., 2012;Patterson et al., 2013;Yin et al., 2019). This suggests that extensive remodeling and regeneration of epithelium during postpartum repair might allow for easier visualization of these labeled cells; otherwise, the mesenchymal Cre-derived lacZ + cells are also present in the FRTs of neonatal and nulliparous mice (Figures 1, 2, and 3). Tamoxifen treatment causes massive expansion of endometrial epithelium. Therefore, we injected adult Cspg4 Cre ;R26-lacZ fl/+ mice with this drug to cause expansion of rarely labeled epithelial cells for easy visualization ( Figure 3E). Both whole mount and histological sections revealed the presence of labeled cells in the uterine epithelium ( Figures 3F-3I). Overall, these data demonstrate the presence of embryonic mesenchymal-specific, Crederived lacZ + epithelium in postnatal oviduct and uterus. Consistent with the mesoepithelial nature of the MDE (Fritsch et al., 2013;Orvis and Behringer, 2007;Paranko and Virtanen, 1986), we observed the expression of vimentin but not CK8 in the undifferentiated MDE at the embryonic day 14.5 (E14.5; Figure S2A). Some MDE cells also expressed αSMA ( Figure S2A). These data suggest that embryonically active mesenchymal marker-specific Cre can cause recombination in the MDE cells resulting in the labeling of epithelial cells. Therefore, we sought to examine the pattern of mesenchyme-specific Cre-driven lacZ expression in the Mouse Genome Informatics (MGI) database (Smith et al., 2019). Data relating to the FRT organs from a total of three mouse models Sm22a Cre ; lacZ fl/+ (Boucher et al., 2003;Zhang et al., 2006), Thy1 Cre ;lacZ fl/+ (Dewachter et al., 2002), and Acta1 Cre ;lacZ fl/+ (Miniou et al., 1999) were available in this database, each reporting a distinct mesenchyme-specific marker-driven reporter expression profile in the FRT. SM22a, THY1, and ACTA1 are bona fide markers of mesenchymal cells and are not expected to be present in epithelial cells. A recent study has reported that Sm22α Cre -derived cells are only present in the stromal compartment of nulliparous uterus and that these cells transition to the epithelium for postpartum repair and regeneration (Yin et al., 2019), suggesting that a population of stromal stem cells contribute directly to the uterine epithelial regeneration. In contrast, our MGI database search revealed that Sm22α Cre -derived, lacZ-labeled cells are present in the FT and uterine epithelium of both pre-pubertal and adult Sm22a Cre ; lacZ fl/+ mice ( Figures S2B-S2I). A similar expression pattern was also observed in the two other mouse models: Thy1 Cre ;lacZ fl/+ and Acta1 Cre ;lacZ fl/+ ( Figures S3A and S3B). Collectively, these observations suggest that the presence of mesenchyme-specific, Cre-driven labeling in the FRT epithelial cells, as reported by earlier studies, is possibly a result of the recombination occurring in the MDE itself because of its mesoepithelial nature.

Induction of Cspg4Cre-Driven Recombination after MD Development and in Adult FRT Does Not Label Epithelial Cells
To label cells after MD development is completed at E14 (Orvis and Behringer, 2007), we developed a Cspg4 CreER ;R26-lacZ fl/+ mouse model in which lacZ expression is under the control of tamoxifen-inducible Cspg4 promoter-driven Cre. Time-mated, pregnant Cspg4 CreER ;R26-lacZ fl/+ mice were injected with a single dose of tamoxifen at E15.5 to induce Cspg4Cre-driven lacZ labeling in the fetuses ( Figure 4A). Subsequently, FRTs were collected postnatally at different chase points ( Figures 4B-4E). Gross analysis of both prepubertal and adult FRTs revealed lacZ labeling in the tamoxifen-treated, but not the vehicle-treated, mutants ( Figures 4B-4E). These lacZ-labeled cells were mainly present around blood vessels in both the oviduct and the uterus at different chase points ( Figures  4B-4E). Importantly, histologic analyses revealed the lack of any lacZ labeling in the epithelial cells of prepubertal or adult FRTs (Figures 4Cb and 4Eb). Instead, the expression was restricted to the stromal perivascular cells of these organs ( Figures 4Cb and 4Eb, arrowheads). Control oviducts and uteri did not show any X-gal staining (Figures 4Ba,4Ca,4Dc,and 4Ea). To investigate whether the progenies of Cspg4-expressing cells contribute to epithelium postnatally, we administered tamoxifen in adult Cspg4 CreER ;R26-lacZ fl/+ mice to induce labeling in CSPG4 + cells ( Figure 4F). Interestingly, after more than a year of chase period, both gross and histologic analyses of mutant FRTs showed the lack of any lacZ expression in the oviductal or the uterine epithelium ( Figures 4G and 4H). As expected, the lacZ expression was restricted to the stromal perivascular cells ( Figure 4H). Overall, these results suggest that after MD development, neither CSPG4-expressing cells nor their progenies contribute to the FRT epithelium.

Cell Fate Mapping in FRT Using Pdgfrβ Promoter-Driven Cre Reporter Mice
The main caveat of Cspg4 CreER ;R26-lacZ fl/+ , a tamoxifen-driven mouse model, is the inability to label most of the mesenchymal cells because tamoxifen treatment can have unwanted dose-dependent consequences on FRT organs. Therefore, one can argue that the absence of any lacZ-labeled epithelial cells in adult Cspg4 CreER ;R26-lacZ fl/+ mice (Figure 4) is because all CSPG4-expressing mesenchymal cells were not labeled during the tamoxifen pulse. To overcome that issue, we developed another mouse model in which lacZ expression is under the control of doxycycline-inducible Pdgfrβ-promoter-driven Cre ( Figure 5). We chose PDGFRβ because it faithfully marks the stromal cells of the oviduct and uterus (Figure 1). To label the maximum number of PDGFRβ + cells, doxycycline was continuously administered in drinking water to Pdgfrβ rtTa ;tetO Cre ;R26-lacZ fl/+ mice from E8.5 to PND1 to encompass the period of MD development and differentiation ( Figure 5A). Because PDGFRβ is also expressed in fat cells (Vishvanath et al., 2016), we used the ovarian fat pad as a positive control to check for the specificity of Pdgfrβ promoter-driven lacZ expression. Doxycycline administration resulted in the lacZ labeling of ovarian bursal fat pads in mutants but not in controls, thus confirming the specificity of this mouse model ( Figure 5B). Gross analysis of mutant FRTs revealed the presence of labeled cells in the oviduct as well as in the uterus ( Figure 5B, 5C, and 5Fa). Importantly, histologic analyses revealed the presence of lacZ + cells in the epithelia of these organs (Figure 5Db and 5Fb). Positive controls showed the presence of labeled cells in ovarian bursa and ovarian surface epithelium, which has a mesoepithelial character ( Figure 5Da). Next, we isolated whole FT and uterine epithelium from doxycycline-induced Pdgfrβ rtTa ;tetO Cre ;R26-lacZ fl/+ mice and developed organoids from these freshly isolated epithelial cells. X-gal staining of these organoids revealed the presence of both lacZ-labeled as well as unlabeled organoids that are morphologically similar ( Figures 5D, 5E, 5G, and 5H). Collectively, these results further confirm that mesenchyme-specific, Cre-driven, lacZ-labeled epithelial cells appear because of embryonic activity of the Cre in MDE.

PDGFRβ + Cell-Derived Progenies in Adult FRT Are Restricted to the Mesenchymal Compartment and Do Not Contribute to Epithelial Cell Lineages
To complement the embryonic lineage tracing data from Pdgfrβ rtTa ;tetO Cre ;R26-lacZ fl/+ mice, we developed a Pdgfrβ rtTa ;tetO H2BJGFP mouse model in which doxycycline administration induces nuclear GFP expression exclusively in PDGFRβ + cells (Figures 5I and 5J). If PDGFRβ + cell-derived progeny contribute toward epithelial cell lineage maintenance, nuclear GFP expression will be present in both stromal and epithelial cells. Alternatively, nuclear GFP expression will be limited to stromal cells, suggesting the absence of any such contribution ( Figure 5I). Therefore, we treated adult Pdgfrβ rtTa ;tetO H2BJGFP mice with doxycycline for 3 days to induce nuclear GFP expression in PDGFRβ + cells and collected FRTs immediately for further analysis. Gross examination of FRT organs revealed the presence of GFP expression in doxycycline-treated mutants but not in controls, and the expression was consistent across the length of oviducts ( Figure 5J). Because we did not notice any differences in the labeling pattern between oviduct and uterus throughout this study using several different mouse models, here, we focused exclusively on oviducts. As expected, co-localization of GFP with CK8 (an epithelial marker), PAX8, and Ac-TUB revealed the expression of nuclear GFP specifically in stromal cells but not in the epithelial cells ( Figure 5K). A recent study has reported in vitro conversion of stromal cells into epithelial cells (Yin et al., 2019). Therefore, we co-cultured oviductal stromal and epithelial cells isolated from adult Pdgfrβ rtTa ;tetO H2BJGFP mice in the presence of doxycycline in the culture media ( Figure 5L). Live imaging of cell culture plates for 21 days did not show any GFP expression in oviduct epithelial organoids ( Figure 5L). However, intense nuclear GFP expression was present in the stromal cells ( Figure 5L). Furthermore, we isolated oviduct epithelial cells from adult Pdgfrβ rtTa ;tetO H2BJGFP mice and developed organoids in the absence of stromal cells ( Figures 5M and 5N). During the period of 21 days in culture and exposure to a low dose of doxycycline, we never observed GFP expression in oviduct organoids ( Figures 5M and 5N).
An alternative explanation to these results would be that the conversion of stromal cells to epithelial cells is a very slow process and occurs only in few cells. In such a model, stromal to epithelial conversion might escape detection in short-label-chase experiments as we did in Pdgfrβ rtTa ;tetO H2BJGFP mice. To rule out this possibility, we treated adult Pdgfrβ rtTa ;tetO Cre ;R26-lacZ fl/+ mice with doxycycline for 2 months to label the maximum number of PDGFRβ + cells and chased them for 1 year ( Figure 6A). Histological analysis of the oviducts and uteri collected from 1-year-old mutant mice showed that labeled cells were restricted to the stromal compartment and were completely absent in the epithelium ( Figures  6A-6C). Furthermore, we developed organoids from oviductal and uterine epithelial cells isolated from these mice and performed X-gal staining. No labeling was observed in either oviductal or uterine organoids ( Figures 6B and 6Cb). Collectively, these data suggested that mesenchymal lineages do not contribute to adult oviductal and uterine epithelial cells.

PAX8 + Cells Self-Renew and Maintain Epithelium during Homeostasis and Regeneration
To investigate further the possibility of transdifferentiation of non-epithelial lineages to epithelial cells, we used an alternative approach of labeling most of the adult oviductal epithelial cells, instead of the stromal cells, and chased their fate. For that, we used Pax8 rtTa ;tetO Cre ;R26-lacZ fl/+ mice in which lacZ expression is under the control of the doxycycline-inducible Pax8 promoter ( Figure 6D). PAX8 is a specific marker of human and mouse oviductal epithelial progenitor cells (Ghosh et al., 2017;Perets et al., 2013;Yamamoto et al., 2016). PAX8 + cells self-renew and give rise to ciliated cells (Ghosh et al., 2017). If unlabeled stromal cells transdifferentiate into epithelial cells, one can expect a progressive decline in the labeling index of epithelial cells over time ( Figure 6D). Therefore, we induced epithelial cell labeling at saturation by treating Pax8 rtTa ;tetO Cre ;R26-lacZ fl/+ mice with doxycycline for 2 weeks ( Figure 6D). Histological examination of the oviducts collected at 210 days after label induction, revealed an increase in the number of lacZ + epithelial cells in distal oviducts in which Pax8 + -cell-derived progenies progressively give rise to ciliated cells. However, the number of lacZ + epithelial cells remains unchanged in the proximal oviduct, which mainly consists of Pax8 + secretory cells (Figures 6E and 6F). Furthermore, we chased the fate of these initially labeled epithelial cells across multiple pregnancies because pregnancy is associated with extensive degeneration and regeneration of cells in the FRT ( Figure 6G). Interestingly, even with this chase, the percentage of lacZlabeled oviductal epithelial cells either increased or remained stable over time ( Figures 6H  and 6I). We have also performed similar pulse-chase experiments in the uterine epithelium and found that the labeling index of uterine epithelial cells also remains stable over longer chases (Syed et al., 2020). These results further confirm that, in adult oviduct and uterus, stromal cells are unlikely to contribute to epithelial cell lineages during homeostasis or regeneration.

DISCUSSION
FRT consists of tubal organs, such as FTs and uterus, and provides a site for fertilization and subsequent embryonic and fetal development. Normal functioning of these organs is essential for female fertility, and defects in their development and/or functions are associated with many human diseases (Teixeira et al., 2001), including high-grade serous ovarian cancer, the deadliest gynecological cancer. Histopathological analysis of FTs collected from BRCA1/2 mutation-carrier patients undergoing risk-reducing salpingo-oophorectomy revealed the presence of ovarian cancer precursor lesions consisting of proliferating secretory cells in the tubes (Carcangiu et al., 2004;Medeiros et al., 2006;Piek et al., 2001) and suggesting that FT is a possible site of origin for serous ovarian cancer. Follow-up studies in mouse models targeting oncogenic mutations in the secretory cells of oviducts using Pax8 or Ovgp1 promoter provided experimental evidence that transformed secretory cells give rise to serous ovarian cancer (Perets et al., 2013;Sherman-Baust et al., 2014). Cell lineage tracing in mouse models and in in vitro analysis of human FT epithelial cells established that Pax8 + secretory cells act as stem/progenitor cells and give rise to ciliated cells through the modulation of the Wnt pathway (Ghosh et al., 2017;Yamamoto et al., 2016). Collectively, these studies firmly established that oncogenic transformation of PAX8 + epithelial cells lead to the development of serous ovarian cancer. In contrast, a study described that the deletion of Dicer and Pten in AMHR2 + oviductal stromal cells results in the development of serous ovarian cancer, suggesting the mesenchymal origin of these epithelial cancers (Hua et al., 2016;Kim et al., 2012). Here, we performed cell-lineagetracing analysis of oviductal stromal and epithelial cells and showed that mesenchymal cells do not transdifferentiate to epithelial cells and that the oviductal epithelium in adult mice is maintained by PAX8 + epithelial cells. During embryonic development, AMHR2 expression is first detected in coelomic epithelium, which is the precursor of the MDE (Zhan et al., 2006). Therefore, Amhr2Cre is expected to be expressed in the coelomic epithelium and, subsequently, in the MDE, resulting in the presence of Amhr2Cre-driven reporter-labeled cells in the FRT epithelium. These findings suggest that it is the endogenous expression of Amhr2 in the coelomic epithelium, rather than the process of MET, that is responsible for the presence of Amhr2Cre-derived, labeled cells in the oviductal epithelium.

STAR★METHODS LEAD CONTACT AND MATERIALS AVAILABILITY
Lead Contact-Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Assoc. Prof. Pradeep Tanwar (Pradeep.tanwar@newcastle.edu.au).
Materials Availability-All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.
Data and Code Availability-Quantitative data supporting the figures is described in the paper. Additional data files will be made available upon reasonable request from the corresponding author.
To determine embryonic age, the day of vaginal plug detection was considered as embryonic day 0.5 (E0.5) and the day of birth was considered as postnatal day 1 (PND1). For gross analyses, whole tissue images were taken by Nikon SMZ25 microscope with an attached Nikon DS-Fi2 camera.
Oviductal organoids-Oviducts from adult female mice were collected for epithelial isolation. Briefly, both oviducts were placed on a clean Petri dish containing Dulbecco phosphate buffer saline (DPBS) to remove any excess vascular and connective tissues. The oviducts were then uncoiled under stereo microscope and microdissecetd into 1-2 mm small pieces and digested in enzyme solution (10 mg/ml Pronase from Streptomyces griseus (Sigma-Aldrich) and 0.5 mg/ml DNase I (Sigma-Aldrich) at 4°C for 13-14 hours. Enzymatic digestion was stopped by adding 10% v/v fetal bovine serum (FBS) (Bovogen) in the enzyme solution and the epithelium was then squeezed out from the oviductal pieces by using fine needles under stereo microscope. Isolated epithelial tubes were collected in the collection tube and a single cell suspension was prepared by repeated pipetting. These epithelial cells were then washed twice with DMEM-F12 media containing 5% FBS, 1% Lglutamine (HyClone) and 1% penicillin-streptomycin (Thermo Fisher Scientific). After washing, differential attachment was performed by incubating cells in a 10 cm cell culture plate containing the above mentioned DMEM-F12 media in an incubator at 37°C with 5% CO2. After 2 hours of incubation, media was collected in a 15 mL tube and centrifuged at 1500 rpm for 5 minutes to pellet the epithelial cells. The epithelial cell pellet was resuspended in 30% media and 70% Matrigel and was placed as 50 μl droplets in each well of a 24-well cell culture plate. The Matrigel with cells was allowed to solidify by at 37°C for 20 minutes. Each well of the culture plate was overlaid with 75% oviductal epithelial (ode) culture media (Dulbecco's modification of Eagle's medium/Ham's F-12 50/50 mix media (DMEM-Ham's F12) without , supplemented with 2% Ultroser G serum substitute (USG; Pall Corporation), 1% penicillin-streptomycin (Thermo Fisher Scientific) and 25% WNT3A-RSPO3-NOGGIN conditioned media (WRN-CM). WRN-CM was prepared from L-WRN cells (ATCC® CRL-3276) by following previously described protocol (Miyoshi and Stappenbeck, 2013). This media was further supplemented with several signaling modulators and growth factors, such as mouse epidermal growth factor (EGF; 100 ng/ml; Sigma-Aldrich), fibroblast growth factor 10 (FGF 10, 100ng/ml; Peprotech), nicotinamide (1mM; Sigma) and TGFBR1 kinase inhibitor IV (SB-431542; 0.5 μM; Selleckchem). ROCK inhibitor (Y-27632; 10 μM; TOCRIS) was added to the media only during the first 3 days of culture and passaging. Media was changed every 3 days. After 21 days in culture, organoids were harvested for further processing. During organoid development the images were captured by JuLi™ Stage Real-Time Cell History Recorder (NanoEnTek).
Uterine epithelial organoids-The uteri collected from adult female mice were washed with Mg 2+ and Ca 2+ free Hank's Balanced Salt Solution (HBSS). Uterine epithelium was isolated as described previously (Syed et al., 2020). Briefly, uterine arm was slit open and cut longitudinally into 3-4 mm pieces. The tissue fragments were then incubated in Pronase/ DNase enzyme solution [10mg/ml Pronase (Sigma-Aldrich) from Streptomyces griseus and 0.5 mg/ml DNase I (Sigma-Aldrich)] at 4°C on a shaker for 13-14 hours. Enzymatic digestion was stopped by adding 10% v/v fetal bovine serum (FBS) (Bovogen), and uterine epithelium was collected after passing through 70 μm cell strainer. Cells were then washed with DMEM-F12 media containing 5% FBS, 1% L-glutamine (Sigma-Aldrich) and 1% penicillin-streptomycin (Thermo Fisher Scientific) by centrifugation at 1500 rpm for 5 minutes. After washing cells were incubated for 2 hours in a cell culture plate with abovementioned FBS containing DMEM-F12 media for differential attachment.

METHOD DETAILS
Embryonic and adult cell lineage tracing experiments-For embryonic labeling of the Müllerian duct mesenchyme with lacZ in Cspg4 CreER ;R26-lacZ fl/+ mice, pregnant R26-lacZ fl/fl females mated with Cspg4 CreER males were administered with a single intraperitoneal tamoxifen injection (40 mg/kg B.W. in sesame oil at a concentration of 20 mg/ml) at gestation day 15.5 (equivalent to E15.5). Female reproductive tracts from these tamoxifen exposed pups were collected at postnatal day 14 (PND14) and 56 (PND56). For embryonic induction of lacZ-encoded β-galactosidase in Pdgfrb rtTA ;tetO Cre ;R26-lacZ fl/+ mice, pregnant females were administered with 5 mg/ml doxycycline (Sigma) in drinking water (supplemented with 1% sucrose) from embryonic day 8.5 (E8.5) to postnatal day 1 (PND1). Female reproductive tracts were dissected out and tissues collected at 12 months age.

Author Manuscript
Author Manuscript Author Manuscript Author Manuscript 6-8 weeks of age for two months or two weeks duration, respectively. Female reproductive tracts from Pdgfrb rtTA ;tetO Cre ;R26-lacZ fl/+ mice were collected 12 months post-induction and from Pax8 rtTA ;tetO Cre ;R26-lacZ fl/+ mice at 7 months post-induction. To induce GFP expression in Pdgfrb rtTA ;tetO H2BJGFP female mice, 5 mg/ml doxycycline was given in drinking water (supplemented with 5% sucrose) for two days in 6-8 weeks old mice. Female reproductive tracts were collected 24 hours later to visualize GFP expression.
Doxycycline treatment to induce GFP expression in oviductal organoids-Pdgfrb rtTA ;tetO H2BJGFP female mice express H2BJGFP in PDGFRβ-expressing cells specifically. However, tetO H2BJGFP expression is terminated upon withdrawal of doxycycline, such that with each cell division the label intensity is diluted to half. Thus, for continuous GFP expression in PDGFRβ-expressing cells, we added and maintained 1 mg/ml doxycycline in oviductal organoid culture medium during the whole 21-day culture period. For the co-culture of oviductal organoids with PDGFRβ-expressing stromal fibroblasts, native GFP fluorescence images were taken using JuLi™ Stage Real-Time Cell History Recorder (NanoEnTek) every 3 days.
Tamoxifen treatment-To investigate the effect of tamoxifen administration on lineage labeled cells, 10mg of tamoxifen (Sigma, diluted in sesame oil) was administered by intraperitoneal injection (five injections of 2mg each; every 24 hours) to 5-6 weeks old Cspg4 CreER ;R26-lacZ fl/fl female mice, and tissues were collected one week after the last tamoxifen injection.
RNA in situ hybridization-Müllerian ducts and adult mouse oviducts were collected at E15.5 and PND28, respectively, for RNA in situ hybridization. Briefly, the tissues were fixed in 10% buffered formalin for 24 hours at room temperature. Tissues were then embedded in paraffin and sectioned at 4μm thickness for further processing. RNA in situ hybridization was performed using RNAscope 2.5 High Definition Red Kit (Advanced Cell Diagnostics, Hayward, CA) as described (Goad et al., 2017).
X-gal/β-galactosidase staining-X-gal/β-galactosidase staining was performed by following previously described protocol (Tanwar et al., 2012). Briefly, intact MD and oviducts were pre-fixed in 4% paraformaldehyde at 4°C for 1 hour before washing with rinse buffer (0.1% sodium deoxycholate, 0.2% NP40, 2mM magnesium chloride in 0.1M phosphate) 3 times for 30 minutes each at room temperature. Tissues were incubated in Xgal (Bioline) solution post-washing for 8-10 hours at room temperature. X-gal/βgalactosidase stained, or unstained tissues were then fixed with 4% paraformaldehyde at 4°C overnight. For, whole mount imaging, uterine arms were sliced at 125 μm transverse sections using Mcllwain Tissue Chopper. Paraffin embedded tissue sections were cut at 5-8 μm thickness and processed for further immunostaining or nuclear fast red (Sigma) staining.
X-gal/β-galactosidase staining of organoids was also performed as described above, except for pre-fixation, which was done for 15 minutes at room temperature, and washing, which was done in rinse buffer without NP40. X-gal staining of Matrigel-embedded organoids was performed in 24-well cell culture plates to minimize the loss of organoids during the staining process.
Immunofluorescence (IF), immunohistochemistry (IHC) and nuclear fast (NF) staining-Tissues and organoids were fixed in 4% (w/v) paraformaldehyde at 4°C overnight, before processing for paraffin embedding and sectioning. For IF, IHC and nuclear fast staining, 6 μm thick tissue sections were deparaffinized followed by rehydration and processed separately according to respective protocols. For nuclear fast staining, rehydrated sections were incubated in nuclear fast red solution (Sigma) at room temperature for 4 minutes. Subsequently, the tissue sections were washed and dehydration for long term storage. For IF and IHC staining, antigen retrieval was done on rehydrated tissue sections by heating them at 98°C for 30 minutes in EDTA buffer (1mM; pH 8.0). After blocking, sections were incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit anti-PAX8 (1:1000; catalog number 10336-1-AP; Proteintech), mouse anti-Acetylated-Tubulin (1:1000; catalog number T7451, clone 6-11B-1, Sigma-Aldrich), rabbit anti-β-galactosidase (

QUANTIFICATION AND STATISTICAL ANALYSIS
Image analysis and quantification was done using ImageJ software (NIH) and statistical analyses were performed on GraphPad Prism (v.7.04). Statistical analyses were done using two-way ANOVA and unpaired t test. Data are presented as mean ± SEM. P values less than 0.05 were considered as statistically significant.   Data represent means ± SEM; p values are indicated; two-way ANOVA with Sidak's test. White arrowheads point to Cspg4 + pericytes in (B). In (E), PAX8 + , βgal + , βgal + AcTub + , and βgal + AcTub + cells are marked with white, blue, yellow, and pink arrowheads, respectively.