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Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium.

ABSTRACT: Fibroblasts have been shown to be one of the essential players for mammary gland organization. Here, we identify two major types of mouse mammary gland fibroblasts through single-cell RNA sequencing analysis: Dpp4 ⁺ fibroblasts and Dpp4 ^- fibroblasts. Each population exhibits unique functional characteristics as well as discrete localization in normal mouse mammary glands. Remarkably, estrogen, a crucial mediator of mammary gland organization, alters the gene expression profiles of fibroblasts in a population-specific manner, without distinct activation of estrogen receptor signaling. Further integrative analysis with the inclusion of five other publicly available datasets reveals a directional differentiation among the mammary gland fibroblast populations. Moreover, the combination with the mouse mammary epithelium atlas allows us to infer multiple potential interactions between epithelial cells and fibroblasts in mammary glands. This study provides a comprehensive view of mouse mammary gland fibroblasts at the single-cell level.

SUBMITTER: Yoshitake R

PROVIDER: S-EPMC8923650 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium.

Yoshitake Ryohei R Chang Gregory G Saeki Kohei K Ha Desiree D Wu Xiwei X Wang Jinhui J Chen Shiuan S

Frontiers in cell and developmental biology 20220301

Fibroblasts have been shown to be one of the essential players for mammary gland organization. Here, we identify two major types of mouse mammary gland fibroblasts through single-cell RNA sequencing analysis: Dpp4 + fibroblasts and Dpp4 - fibroblasts. Each population exhibits unique functional characteristics as well as discrete localization in normal mouse mammary glands. Remarkably, estrogen, a crucial mediator of mammary gland organization, al ...[more]

PMID: 35300413

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Project description:INTRODUCTION: Parity-identified mammary epithelial cells (PI-MECs) are an interesting cellular subset because they survive involution and are a presumptive target for transformation by human epidermal growth factor receptor 2 (HER2)/neu in mammary tumors. Depending on the type of assay, PI-MECs have been designated lobule-restricted progenitors or multipotent stem/progenitor cells. PI-MECs were reported to be part of the basal population of mammary epithelium based on flow cytometry. We investigated the cellular identity and lineage potential of PI-MECs in intact mammary glands. METHODS: We performed a quantitative and qualitative analysis of the contribution of PI-MECs to mammary epithelial cell lineages in pregnant and involuted mammary glands by immunohistochemistry, fluorescence-activated cells sorting (FACS), and quantitative polymerase chain reaction. PI-MECs were labeled by the activation of Whey Acidic Protein (WAP)-Cre during pregnancy that results in permanent expression of yellow fluorescent protein. RESULTS: After involution, PI-MECs are present exclusively in the luminal layer of mammary ducts. During pregnancy, PI-MECs contribute to the luminal layer but not the basal layer of alveolar lobules. Strikingly, whereas all luminal estrogen receptor (ER)-negative cells in an alveolus can be derived from PI-MECs, the alveolar ER-positive cells are unlabeled and reminiscent of Notch2-traced L cells. Notably, we observed a significant population of unlabeled alveolar progenitors that resemble PI-MECs based on transcriptional and histological analysis. CONCLUSIONS: Our demonstration that PI-MECs are luminal cells underscores that not only basal cells display multi-lineage potential in transplantation assays. However, the lineage potential of PI-MECs in unperturbed mammary glands is remarkably restricted to luminal ER-negative cells of the secretory alveolar lineage. The identification of an unlabeled but functionally similar population of luminal alveolar progenitor cells raises the question of whether PI-MECs are a unique population or the result of stochastic labeling. Interestingly, even when all luminal ER-negative cells of an alveolus are PI-MEC-derived, the basal cells and hormone-sensing cells are derived from a different source, indicating that cooperative outgrowth of cells from different lineages is common in alveologenesis.

Project description:The CCND1 gene, which is frequently overexpressed in cancers, encodes the regulatory subunit of a holoenzyme that phosphorylates the retinoblastoma protein. Although it is known that cyclin D1 regulates estrogen receptor (ER)α transactivation using heterologous reporter systems, the in vivo biological significance of cyclin D1 to estrogen-dependent signaling, and the molecular mechanisms by which cyclin D1 is involved, are yet to be elucidated. Herein, genome-wide expression profiling conducted of 17β-estradiol-treated castrated virgin mice deleted of the Ccnd1 gene demonstrated that cyclin D1 determines estrogen-dependent gene expression for 88% of estrogen-responsive genes in vivo. In addition, expression profiling of 17β-estradiol-stimulated cyclin D1 small interfering RNA treated MCF7 cells shows cyclin D1 is required for estrogen-mediated gene expression in vitro. Genome-wide chromatin immunoprecipitation-Seq analysis revealed a cyclin D1-DNA bound form associated with genes that were regulated by estrogen in a cyclin D1-dependent manner. The cyclin D1-dependent estrogen signaling pathways identified in vivo were highly enriched for extracellular membrane-associated growth factor receptors (epidermal growth factor receptor, ErbB3, and EphB3) and their ligands (amphiregulin, encoded by AREG gene), and matrix metalloproteinase. The AREG protein, a pivotal ligand for epidermal growth factor receptors to promote cellular proliferation, was induced by cyclin D1 via the AREG promoter. Chromatin immunoprecipitation analysis demonstrated the recruitment of cyclin D1 to the breast cancer 1 (Brca1)/ERα binding site of the Areg gene. Cyclin D1 genetic deletion demonstrated the in vivo requirement for cyclin D1 in assembling the estrogen-dependent amplified in breast cancer 1-associated multiprotein complex. The current studies define a requirement for cyclin D1 in estrogen-dependent signaling modules governing growth factor receptor and ligand expression in vivo and reveal a noncanonical function of cyclin D1 at ERα target gene promoters. Cyclin D1 mediates the convergence of ERα and growth factor signaling at a common cis-element of growth factor genes.

Dataset Information

Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium.

Publications

Single-Cell Transcriptomics Identifies Heterogeneity of Mouse Mammary Gland Fibroblasts With Distinct Functions, Estrogen Responses, Differentiation Processes, and Crosstalks With Epithelium.

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