Reconstructing Lineage Hierarchies of Mouse Uterus Epithelial Development Using Single-Cell Analysis

Abstract

The endometrial layer comprises luminal and glandular epithelia that both develop from the same simple layer of fetal uterine epithelium. Mechanisms of uterine epithelial progenitor self-renewal and differentiation are unclear. This study aims to systematically analyze the molecular and cellular mechanisms of uterine epithelial development by single-cell analysis. An integrated set of single-cell transcriptomic data of uterine epithelial progenitors and their differentiated progenies is provided. Additionally the unique molecular signatures of these cells, characterized by sequential upregulation of specific epigenetic and metabolic activities, and activation of unique signaling pathways and transcription factors, were also investigated. Finally a unique subpopulation of early progenitor, as well as differentiated luminal and glandular lineages, were identified. A complex cellular hierarchy of uterine epithelial development was thus delineated. Our study therefore systematically decoded molecular markers and a cellular program of uterine epithelial development that sheds light on uterine developmental biology.

Keywords: lineage hierarchy; luminal and glandular epithelia; single-cell RNA-seq; stem/progenitor cell; transcription factor; uterus.

Graphical abstract

Figure 1

Ultra-Shallow Single-Cell RNA-Seq Reveals Cell Heterogeneity during Uterine Epithelial Development (A) Schematic of the single-cell analysis of five developmental stages, Firstly, the tissues were digested into single-cell suspension; secondly, cells were labeled with CD326-APC antibody and sorted by FACS; thirdly, single-cell RNA-seq was conducted by CEL-Seq; Finally, the data were analyzed and validated. (B) Heatmap of 714 single cells from the five developmental stages by transcriptome analysis revealed four distinct populations. D, postnatal day. (C) t-SNE analysis identified cell heterogeneity during the development of uterine epithelia. PND, postnatal day. (D) Unique marker genes of the four main cell populations. D, postnatal day. (E) Representative markers for each cell population.

Figure 2

Cell Heterogeneity during Development Exhibits Characteristics of Progenitor and Mature States (A) Gene ontology (GO) analysis showed elevated cellular response to estrogen stimulus and estrogen receptor signaling during late development of uterine epithelia. (B) Expression of esr1 gene during development. (C) Gene ontology analysis showed that positive regulation of cyclin-dependent protein kinase activity was high during the early development of uterine epithelia. (D) Representative cyclin-dependent protein kinase gene expression during development. (E) Expression of Ki67 protein in the epithelia (CD326⁺) during development. Scale bar, 20 μm. (F) Gene ontology analysis showed that EMT was high during early development of uterine epithelia. (G) Expression of EMT-related genes during development. (H) Gene ontology analysis showed that telomerase activity was high during early development of uterine epithelia. (I) Expression of telomerase activity-related genes during development. (J) Specific telomerase inhibitors decreased cell proliferation of epithelial cell from early development. Data are presented as mean ± SEM, n = 6 independent experiments. ^∗∗p < 0.01, ^∗∗∗p < 0.001. PND, postnatal day.

Figure 3

Molecular Cascades Regulating the Development and Maturation of Uterine Epithelia (A) Differentially expressed transcription factors (TFs) of the respective cell cluster were selected and visualized using a heatmap. (B) Expression of GATA2 protein in uterine epithelia (CD326⁺) during development. Scale bar, 20 μm. (C–K) Sequential upregulation of signaling pathways (C), epigenetic activity (D), and metabolic activity (E) during development. Involvement of WNT (F) and RA metabolic processes (I) during early development of uterine epithelia. Expression of genes related to WNT (G) and RA metabolic processes (J) during development. Specific WNT (H) and ALDH1 (K) inhibitors decreased proliferation of epithelial cells from early development. (L) Expression of ALDH1A1 protein in the epithelia (CD326⁺) during development. Scale bars, 10 μm or 20 μm. PND, postnatal day. Data are presented as mean ± SEM, n = 6 independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S2.

Figure 4

Aldehyde Dehydrogenase 1A1 Enriches for an Epithelial Stem/Progenitor Subpopulation during Early Development of the Mouse Uterus (A) P7 endometrial epithelial cells were clustered into different subpopulations by hierarchical clustering. (B) Aldh1a1 is highly expressed in a unique cluster of cells. (C) Genes correlated with Aldh1a1 expression were ordered according to the respective correlation coefficients. (D) FACS isolation of CD326⁺ ALDH1A1-high cells from a single-cell suspension of P7 mouse uterus. (E) CD326⁺ ALDH1A1-high cells display high expression of aldh1a1-correlated genes. (F) CD326⁺ ALDH1A1-high cells possess high self-renewal capacity with more colonies formed. (G) Comparison of the colony formation efficiency of CD326⁺ ALDH1A1⁺ and CD326⁺ ALDH1A1⁻ cells. Data are presented as mean ± SEM, n = 3 independent experiments. ^∗∗p < 0.01. (H) ALDH1A1 is highly expressed by some epithelial cells within the colony in vitro. Scale bar, 100 μm. (I) ALDH1A1 is highly expressed in the glands as well as at the invagination sites responsible for gland formation, and is co-expressed together with high levels of Ki67, EZH2, PAX2, and VIMENTIN. Scale bar, 50 μm.

Figure 5

Differentiation of Luminal and Glandular Epithelia during the Maturation of Uterine Epithelia (A) Heatmap of single cells from the late developmental stages revealed six populations. (B) Unique marker genes of the six main cell populations, cluster 1 (C1) to cluster 6 (C6). (C) t-SNE analysis identified cell heterogeneity along the late developmental stages of uterine epithelia. PND, postnatal day. (D–I) Expression of representative markers selected for each cell population. (J) Differentiation of luminal (LE) and glandular (GE) epithelia occurs during the maturation of uterine epithelia.

Figure 6

Mapping Cellular Hierarchy of Uterine Epithelial Cells during Development and Maturation by Lineage Progression Analysis (A) Esr1⁺ lineage represents mature uterine epithelial lineage. (B and C) Hmga1 (B) and Tead2 (C) represent early progenitor lineage. (D) Hist1h2ao represents fast-proliferating lineage. (E) Aldh1a1 represents a subpopulation of stem/progenitor lineage of the early uterine epithelia. (F) Lpar3 represents differentiated luminal epithelial lineage during the maturation of uterine epithelia. (G) Gata2 is highly enriched in the mature Lpar3 lineage. (H) Prss29 represents differentiated glandular epithelial lineage during the maturation of uterine epithelia. (I) Reconstructed cellular lineage hierarchy from early stem/progenitor to late differentiated luminal and glandular epithelia. (J) A work model of the uterine epithelial lineage map.

Figure 7

Schematic Summary of Molecular Signatures Regulating Mouse Uterine Epithelial Development and Maturation The molecular cascades described here include developmental specific markers and TFs, stemness gene ontologies, signaling pathways, and epigenetic and metabolic regulation. PND, postnatal day; EMT, epithelial-to mesenchymal transition; GPCR, G-protein-coupled receptor; cGMP, cyclic guanosine monophosphate; BMP, bone morphogenetic protein; TLR, Toll-like receptor; NFkB, nuclear factor κB; TCA, tricarboxylic acid; RA, retinoic acid.