R-spondin1 is a novel hormone mediator for mammary stem cell self-renewal.

Signals from the niche play pivotal roles in regulating adult stem cell self-renewal. Previous studies indicated that the steroid hormones can expand mammary stem cells (MaSCs) in vivo. However, the facilitating local niche factors that directly contribute to the MaSC expansion remain unclear. Here we identify R-spondin1 (Rspo1) as a novel hormonal mediator in the mammary gland. Pregnancy and hormonal treatment up-regulate Rspo1 expression. Rspo1 cooperates with another hormonal mediator, Wnt4, to promote MaSC self-renewal through Wnt/ß-catenin signaling. Knockdown of Rspo1 and Wnt4 simultaneously abolishes the stem cell reconstitution ability. In culture, hormonal treatment that stimulates the expression of both Rspo1 and Wnt4 can completely substitute for exogenous Wnt proteins, potently expand MaSCs, and maintain their full development potential in transplantation. Our data unveil the intriguing concept that hormones induce a collaborative local niche environment for stem cells.

Amphiregulin mediates the hormonal regulation on Rspondin-1 expression in the mammary gland.

The steroid hormones are instrumental for the growth of mammary epithelial cells. Our previous study indicates that hormones regulate the expression of Rspondin-1 (Rspo1). Yet, the regulatory mechanism remains unknown. In the current study, we identify Amphiregulin (Areg) as a novel upstream regulator of Rspo1 expression mediating the hormonal influence. In response to hormonal signaling, Areg emanating from estrogen receptor (ER)-positive luminal cells, induce the expression of Rspo1 in ER-negative luminal cells. The paracrine action of Areg on Rspo1 expression is dependent on Egfr. Our data reveal a novel Estrogen-Areg-Rspo1 regulatory axis in the mammary gland, providing new evidence for the orchestrated action of systemic hormones and local growth factors.

Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium.

BACKGROUND: Protein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/ß-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins. RESULTS: We established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDß (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression. CONCLUSION: We established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.

Identification of multipotent mammary stem cells by protein C receptor expression.

The mammary gland is composed of multiple types of epithelial cells, which are generated by mammary stem cells (MaSCs) residing at the top of the hierarchy. However, the existence of these multipotent MaSCs remains controversial and the nature of such cells is unknown. Here we demonstrate that protein C receptor (Procr), a novel Wnt target in the mammary gland, marks a unique population of multipotent mouse MaSCs. Procr-positive cells localize to the basal layer, exhibit epithelial-to-mesenchymal transition characteristics, and express low levels of basal keratins. Procr-expressing cells have a high regenerative capacity in transplantation assays and differentiate into all lineages of the mammary epithelium by lineage tracing. These results define a novel multipotent mammary stem cell population that could be important in the initiation of breast cancer.

Activation of Wnt/ß-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/ß-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/ß-catenin signaling as a potential therapeutical approach.

Recent advances in tissue stem cells.

Stem cells are undifferentiated cells capable of self-renewal and differentiation, giving rise to specialized functional cells. Stem cells are of pivotal importance for organ and tissue development, homeostasis, and injury and disease repair. Tissue-specific stem cells are a rare population residing in specific tissues and present powerful potential for regeneration when required. They are usually named based on the resident tissue, such as hematopoietic stem cells and germline stem cells. This review discusses the recent advances in stem cells of various tissues, including neural stem cells, muscle stem cells, liver progenitors, pancreatic islet stem/progenitor cells, intestinal stem cells, and prostate stem cells, and the future perspectives for tissue stem cell research.

Endothelial Wnts control mammary epithelial patterning via fibroblast signaling.

Endothelial and fibroblast niches are crucial for epithelial organs. How these heterotypic cells interact is of great interest. In this study, we reveal an axis of signaling in which fibroblasts relay Wnt signals from the endothelial niche to organize epithelial patterning. We generate an Axin2-membrane GFP (mGFP) reporter mouse and observe robust Wnt/ß-catenin signaling activities in fibroblasts surrounding the mammary epithelium. To enable cell-type-specific gene manipulation in vitro, we establish an organoid system via coculture of endothelial cells (ECs), fibroblasts, and mammary epithelial cells. Deletion of ß-catenin in fibroblasts impedes epithelium branching, and ECs are responsible for the activation of Wnt/ß-catenin signaling in fibroblasts. In vivo, EC deletion of Wntless inhibits Wnt/ß-catenin signaling activity in fibroblasts, rendering a reduction in epithelial branches. These findings highlight the significance of the endothelial niche in tissue patterning, shedding light on the interactive mechanisms in which distinct niche components orchestrate epithelial organogenesis and tissue homeostasis.

A novel function of R-spondin1 in regulating estrogen receptor expression independent of Wnt/ß-catenin signaling.

R-spondin1 (Rspo1) has been featured as a Wnt agonist, serving as a potent niche factor for stem cells in many tissues. Here we unveil a novel role of Rspo1 in promoting estrogen receptor alpha (Esr1) expression, hence regulating the output of steroid hormone signaling in the mouse mammary gland. This action of Rspo1 relies on the receptor Lgr4 and intracellular cAMP-PKA signaling, yet is independent of Wnt/ß-catenin signaling. These mechanisms were reinforced by genetic evidence. Luminal cells-specific knockout of Rspo1 results in decreased Esr1 expression and reduced mammary side branches. In contrast, luminal cells-specific knockout of Wnt4, while attenuating basal cell Wnt/ß-catenin signaling activities, enhances Esr1 expression. Our data reveal a novel Wnt-independent role of Rspo1, in which Rspo1 acts as a bona fide GPCR activator eliciting intracellular cAMP signaling. The identification of Rspo1-ERα signaling axis may have a broad implication in estrogen-associated diseases.

Author Correction: Hormones induce the formation of luminal-derived basal cells in the mammary gland.

In the initial published version of this article, there was a mistake in one author name (Jingsong Li). The correct name should be "Jinsong Li". This correction does not affect the description of the results or the conclusions of this work.

Hormones induce the formation of luminal-derived basal cells in the mammary gland.

In the mammary gland, it is widely believed that the luminal cells are unipotent after birth, contributing only to the luminal compartment in normal development. Here, by lineage tracing, we uncovered an unexpected potential of luminal cells that can give rise to basal cells during pregnancy. These luminal-derived basal cells (LdBCs) persisted through mammary regression and generated more progeny in successive rounds of pregnancies. LdBCs express basal markers as well as estrogen receptor α (ERα). In ovariectomized (OVX) mice, stimulation with estrogen and progesterone promoted the formation of LdBCs. In serial transplantation assays, LdBCs were able to reconstitute new mammary glands in a hormone-dependent manner. Transcriptome analysis and genetic experiments suggest that Wnt/ß-catenin signaling is essential for the formation and maintenance of LdBCs. Our data uncover an unexpected bi-potency of luminal cells in a physiological context. The discovery of ERα+ basal cells, which can respond to hormones and are endowed with stem cell-like regenerative capacity in parous mammary gland, provides new insights into the association of hormones and breast cancer.

Endothelial cell clonal expansion in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics.

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells.

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

Mammary Development and Breast Cancer: A Wnt Perspective.

The Wnt pathway has emerged as a key signaling cascade participating in mammary organogenesis and breast oncogenesis. In this review, we will summarize the current knowledge of how the pathway regulates stem cells and normal development of the mammary gland, and discuss how its various components contribute to breast carcinoma pathology.

Identification of blood vascular endothelial stem cells by the expression of protein C receptor.

Vascular growth and remodeling are dependent on the generation of new endothelial cells from stem cells and the involvement of perivascular cells to maintain vessel integrity and function. The existence and cellular identity of vascular endothelial stem cells (VESCs) remain unclear. The perivascular pericytes in adult tissues are thought to arise from the recruitment and differentiation of mesenchymal progenitors during early development. In this study, we identified Protein C receptor-expressing (Procr+) endothelial cells as VESCs in multiple tissues. Procr+ VESCs exhibit robust clonogenicity in culture, high vessel reconstitution efficiency in transplantation, long-term clonal expansion in lineage tracing, and EndMT characteristics. Moreover, Procr+ VESCs are bipotent, giving rise to de novo formation of endothelial cells and pericytes. This represents a novel origin of pericytes in adult angiogenesis, reshaping our understanding of blood vessel development and homeostatic process. Our study may also provide a more precise therapeutic target to inhibit pathological angiogenesis and tumor growth.