In vivo genetic cell lineage tracing reveals that oviductal secretory cells self-renew and give rise to ciliated cells.

The epithelial lining of the fallopian tube is vital for fertility, providing nutrition to gametes and facilitating their transport. It is composed of two major cell types: secretory cells and ciliated cells. Interestingly, human ovarian cancer precursor lesions primarily consist of secretory cells. It is unclear why secretory cells are the dominant cell type in these lesions. Additionally, the underlying mechanisms governing fallopian tube epithelial homoeostasis are unknown. In the present study, we showed that across the different developmental stages of mouse oviduct, secretory cells are the most frequently dividing cells of the oviductal epithelium. In vivo genetic cell lineage tracing showed that secretory cells not only self-renew, but also give rise to ciliated cells. Analysis of a Wnt reporter mouse model and various Wnt target genes showed that the Wnt signaling pathway is involved in oviductal epithelial homoeostasis. By developing two triple-transgenic mouse models, we showed that Wnt/ß-catenin signaling is essential for self-renewal as well as the differentiation of secretory cells. In summary, our results provide mechanistic insight into oviductal epithelial homoeostasis.

Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus.

In mice, implantation always occurs towards the antimesometrial side of the uterus, while the placenta develops at the mesometrial side. What determines this particular orientation of the implanting blastocyst remains unclear. Uterine glands are critical for implantation and pregnancy. In this study, we showed that uterine gland development and active Wnt signaling activity is limited to the antimesometrial side of the uterus. Dkk2, a known antagonist of Wnt signaling, is only present at the mesometrial side of the uterus. Imaging of whole uterus, thick uterine sections (100-1000µm), and individual glands revealed that uterine glands are simple tubes with branches that are directly connected to the luminal epithelium and are only present towards the antimesometrial side of the uterus. By developing a unique mouse model targeting the uterine epithelium, we demonstrated that Wnt/ß-catenin signaling is essential for prepubertal gland formation and normal implantation, but dispensable for postpartum gland development and regeneration. Our results for the first time have provided a probable explanation for the antimesometrial bias for implantation.

Epithelial Wnt/ßcatenin signalling is essential for epididymal coiling.

Organ shape and size are important determinants of their physiological functions. Epithelial tubes are anlagen of many complex organs. How these tubes acquire their complex shape and size is a fundamental question in biology. In male mice, the Wolffian duct (WD; postnatally known as epididymis) undergoes an astonishing transformation, where a straight tube only a few millimetres long elongates to over 1000 times its original length and fits into a very small space, due to extensive coiling of epithelium, to perform the highly specialized function of sperm maturation. Defective coiling disrupts sperm maturation and leads to male infertility. Recent work has shown that epithelial cell proliferation is a major driver of WD coiling. Still, very little is known about the molecular signals involved in this process. Testicular androgens are known regulators of WD development. However, epithelial androgen receptor signalling is dispensable for WD coiling. In this study, we have shown that Wnt signalling is highly active in the entire WD epithelium during its coiling, and is limited to only a few segments of the epididymis in later life. Pharmacological and genetic suppression of Wnt signalling inhibited WD coiling by decreasing cell proliferation and promoting apoptosis. Comparative gene expression analysis identified Fibroblast growth factor 7 (Fgf7) as a prime Wnt target gene involved in WD coiling and in vitro treatment with Fgf7 protein increased coiling of WDs. In summary, our work has established that epithelial canonical Wnt signalling is a critical regulator of WD coiling and its precise regulation is essential for WD/epididymal differentiation.

Proteomic and functional characterization of intra-tumor heterogeneity in human endometrial cancer.

Endometrial cancer is one of the most frequently diagnosed gynecological cancers worldwide, and its prevalence has increased by more than 50% over the last two decades. Despite the understanding of the major signaling pathways driving the growth and metastasis of endometrial cancer, clinical trials targeting these signals have reported poor outcomes. The heterogeneous nature of endometrial cancer is suspected to be one of the key reasons for the failure of targeted therapies. In this study, we perform a sequential window acquisition of all theoretical fragment ion spectra (SWATH)-based comparative proteomic analysis of 63 tumor biopsies collected from 20 patients and define differences in protein signature in multiple regions of the same tumor. We develop organoids from multiple biopsies collected from the same tumor and show that organoids capture heterogeneity in endometrial cancer growth. Overall, using quantitative proteomics and patient-derived organoids, we define the heterogeneous nature of endometrial cancer within a patient's tumor.

Protocol for In Vitro Establishment and Long-Term Culture of Mouse Vaginal Organoids.

Healthy vaginal epithelium is essential for normal reproductive functions and protects against infectious diseases. Here, we present a protocol for developing mouse vaginal organoids from single epithelial cells. These organoids recapitulate both functional and structural characteristics of vagina in situ. This model is a powerful tool for investigating how vaginal microbiome or chemicals in contraceptives and personal hygiene products interact with stem cells and alter the epithelial dynamics, which will lead to new insights into the pathogenesis of vaginal diseases. For complete details on the use and execution of this protocol, please refer to Ali et al. (2020).

Axin2+ endometrial stem cells: the source of endometrial regeneration and cancer.

Menstruation is one of the basic but poorly understood life processes in primates during which females shed inner uterine lining every month only to be completely regenerated back within a week. The definitive evidence for the existence and/or identity of stem cells responsible for this process has remained elusive for more than six decades now. Recently, we reported Axin2, a classical Wnt reporter gene, as a marker for endometrial stem cells that also serve as the cells of origin for endometrial cancer.

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

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 PDGFRß), 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.

Cell Lineage Tracing Identifies Hormone-Regulated and Wnt-Responsive Vaginal Epithelial Stem Cells.

The intact vaginal epithelium is essential for women's reproductive health and provides protection against HIV and sexually transmitted infections. How this epithelium maintains itself remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) to define the diverse cell populations in the vaginal epithelium. We show that vaginal epithelial cell proliferation is limited to the basal compartment without any obvious label-retaining cells. Furthermore, we developed vaginal organoids and show that the basal cells have increased organoid forming efficiency. Importantly, Axin2 marks a self-renewing subpopulation of basal cells that gives rise to differentiated cells over time. These cells are ovariectomy-resistant stem cells as they proliferate even in the absence of hormones. Upon hormone supplementation, these cells expand and reconstitute the entire vaginal epithelium. Wnt/ß-catenin is essential for the proliferation and differentiation of vaginal stem cells. Together, these data define heterogeneity in vaginal epithelium and identify vaginal epithelial stem cells.

Endometrial Axin2+ Cells Drive Epithelial Homeostasis, Regeneration, and Cancer following Oncogenic Transformation.

The remarkable regenerative capacity of the endometrium (the inner lining of the uterus) is essential for the sustenance of mammalian life. Over the years, the role of stem cells in endometrial functions and their pathologies has been suggested; however, the identity and location of such stem cells remain unclear. Here, we used in vivo lineage tracing to show that endometrial epithelium self-renews during development, growth, and regeneration and identified Axin2, a classical Wnt reporter gene, as a marker of long-lived bipotent epithelial progenitors that reside in endometrial glands. Axin2-expressing cells are responsible for epithelial regeneration in vivo and for endometrial organoid development in vitro. Ablation of Axin2+ cells severely impairs endometrial homeostasis and compromises its regeneration. More important, upon oncogenic transformation, these cells can lead to endometrial cancer. These findings provide valuable insights into the cellular basis of endometrial functions and diseases.

Data on the mRNA expression by in situ hybridization of Wnt signaling pathway members in the mouse uterus.

Wnt signaling plays an important role in uterine organogenesis and oncogenesis. Our mRNA expression data documents the expression of various Wnt pathway members during the key stages of uterine epithelial gland development. Our data illustrates the expression of Wnt signaling inhibitors (Axin2, Sfrp2, Sfrp4, Dkk1 and Dkk3) in mice uteri at postnatal day 6 (PND 6) and day 15 (PND 15). They also describe the expression pattern of the Wnt ligands (Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt5b, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a and Wnt10b) in mice uteri with or without progesterone treatment. Detailed interpretation and discussion of these data is presented in the research article entitled "Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus" [1].