Single-cell transcriptomic profiling of the neonatal oviduct and uterus reveals new insights into upper Müllerian duct regionalization

Abstract

The upper Müllerian duct (MD) is patterned and specified into two morphologically and functionally distinct organs, the oviduct and uterus. It is known that this regionalization process is instructed by inductive signals from the adjacent mesenchyme. However, the interaction landscape between epithelium and mesenchyme during upper MD development remains largely unknown. Here, we performed single-cell transcriptomic profiling of mouse neonatal oviducts and uteri at the initiation of MD epithelial differentiation (postnatal day 3). We identified major cell types including epithelium, mesenchyme, pericytes, mesothelium, endothelium, and immune cells in both organs with established markers. Moreover, we uncovered region-specific epithelial and mesenchymal subpopulations and then deduced region-specific ligand-receptor pairs mediating mesenchymal-epithelial interactions along the craniocaudal axis. Unexpectedly, we discovered a mesenchymal subpopulation marked by neurofilaments with specific localizations at the mesometrial pole of both the neonatal oviduct and uterus. Lastly, we analyzed and revealed organ-specific signature genes of pericytes and mesothelial cells. Taken together, our study enriches our knowledge of upper MD development, and provides a manageable list of potential genes, pathways, and region-specific cell subtypes for future functional studies.

Keywords: Müllerian duct; craniocaudal patterning; oviduct; regionalization; uterus.

Figure 1.. Single-cell transcriptional atlas of PND3 oviduct and uterus.

(A) Tissue sample surveyed in this study. The dotted line indicated where the oviduct and the uterus were separated. Scale bar=2.5 mm. (B) Uniform manifold approximation and projection (UMAP) plot featuring general cell clusters from both the oviduct and uterus. In the lower right, the same UMAP was annotated according to cell’s organ origin. (C) UMAPs featuring cell type markers, Hoxc8 for oviductal mesenchyme, Hoxa10 for uterine mesenchyme, Top2a for proliferating cells, Epcam for epithelium, Msln for mesothelium, Pecam1 for endothelium, Rgs5 for pericytes, and Lyz2 for myeloid cells. (C) Heatmap of top five marker genes for each cluster.

Figure 2.. Characterization of mesenchymal subpopulations in PND3 oviduct and uterus.

(A) UMAP featuring the mesenchymal subpopulation after re-clustering with higher resolution. In the lower right, the same UMAP was annotated according to cell’s organ origin. (B) UMAPs feature marker genes for cell subtypes: Cnn1 for smooth muscle cells in both the oviduct and uterus, Tcf21 for uterine smooth muscle, Nrp2 for oviductal smooth muscle, Pkib & Pcdh10 for two uterine mesenchymal subtypes, Wfdc1 & Angptl7 for two oviductal mesenchymal subtypes, and Nefm for a mesenchymal subtype found in both the oviduct and uterus. (C) mRNA expression of four markers for mesenchymal subpopulations, Wfdc1, Angptl7, Nefm and Tcf21, by RNAscope. Ms (mesometrial side), Anti-Ms (antimesometrial side). N=3. Scale bar=50 μm.

Figure 3.. Region-specific epithelial subtypes in PND3 oviduct and uterus.

(A) UMAP featuring oviductal and uterine epithelial cells extracted from the overall dataset in Fig 1B. (B) UMAPs featuring marker genes Msx1 and Lrpap1 for uterine and oviductal epithelium, respectively. (C) UMAP featuring oviductal epithelial cells extracted from Fig 3A and then re-clustered. (D) Violin plot showing Gsap expression in two subclusters in Fig 3C. (E) Immunofluorescent staining of MSX1 and RNAscope staining of Gsap in the cranial oviduct, caudal oviduct and uterus at PND3. Ms (mesometrial side), Anti-Ms (antimesometrial side). N=3. Scale bar: 25 and 50 μm for immunofluorescence and RNAscope, respectively.

Figure 4.. Mesenchymal-epithelial crosstalk represented by region-specific ligands that are potentially functional.

Only region-specific ligands whose knockout mice display embryonic lethality and/or reproductive phenotype were included. Please see all ligand-pair pairs in Fig S4 and supplementary files 7-12.

Figure 5.. Gene expression comparison between oviductal and uterine pericytes.

(A) Violin plots and UMAPs of higher expressed genes (Cebpb, Rgs16, Nr2f1, Hoxc8 and Cryab) in oviductal pericytes. (B) Violin plots and UMAPs of higher expressed genes (Amer1, Plac8, Hoxd11, Lsp1, and Hoxa10) in uterine pericytes

Figure 6.. Gene expression comparison between oviductal and uterine mesothelium.

(A) UMAPs featuring expressions of epithelium-specific (Epcam, Mecom), mesothelium-specific (Upk3b), and commonly shared (Krt8, Krt18 and Krt19) marker genes. (B) Violin plots of higher expressed genes in the oviductal mesothelium (up row: Lgals7, Igf1, Mt1, Ccl2, and Nrgn) and uterine mesothelium (bottom row: Alcam, Rspo3, Fxyd3, Tcf21, and Hoxd11).