Single-cell RNA-seq of de-differentiated mammary adipocytes during lactation
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PROVIDER: PRJEB27175 | ENA |
REPOSITORIES: ENA
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Project description:Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα+ preadipocyte- and fibroblast-like cells during pregnancy, and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. In order to determine the molecular signature of these de-differentiated adipocytes in the mammary gland, we compared these cells with classical adipocytes. Using the AdipoChaser-mT/mG system, we pre-labeled mature adipocytes with GFP expression to characterize the features of these de-differentiated adipocytes (Figure 4A), and then purified CD31-/CD45-/PDGFRα+/Tomato+ and CD31-/CD45-/PDGFRα+/GFP+ cells from the stromal vascular fraction (SVF) of lactating mammary gland at the peak of lactation through FACS. Gene expression analyses showed that the CD31-/CD45-/PDGFRα+/Tomato+ cells were indeed enriched with Tomato expression, while the CD31-/CD45-/PDGFRα+/GFP+ cells were enriched with GFP expression (Figure 4C). We then collected CD31-/CD45-/PDGFRα+/GFP+ cells as single cells for subsequent single cell RNA-sequencing analysis (Figure 4D-G, Supplemental. Figure S1A-G). After the flow sorting and single cell RNA amplification, 26 CD31-/CD45-/PDGFRα+/GFP+ cells passed the quality control, and these cells were used for single-cell RNA-sequencing analysis. Due to technical difficulties in sorting single mature white adipocyte through flow cytometry, adipocytes differentiated from the immortalized murine-derived brown pre-adipocyte cell line were used as mature adipocyte control (Pradhan et al., 2017). Additionally, we also included population RNA-seq experiments, i.e. three mature white adipocyte samples, two GFP+, and six GFP- ones.
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Project description:Studying composition and developmental mechanisms in mammary gland is crucial for healthy growth of the newborn. Here, we constructed the largest transcriptomic dataset of mammary gland cells thus far. The dataset captured 126,829 high-quality nuclei from adipocytes, endothelial cells, epithelial cells, fibroblasts cells, immune cells, myoepithelial cells and precursor cells at five timepoint of mammary development. Remarkably, adipocytes were annotated in the mammary gland for the first time at both snRNA-seq and spatial transcriptome levels. Histological observation and cell type annotations indicated that the living space of adipocytes was compressed to a great extent during lactation. The cell-cell interaction pathway indicated that to maintain their own survival, adipocytes in lactation eliminated chemokines and avoided the phagocytosis of immune cells through the chemokine receptor-ligand pairs, such as CCL21-ACKR4. In the period of natural involution, we speculated that adipocytes inhibited apoptosis based on the activation of ADI-POQ-ADIPOR2 pairs, which was further confirmed in the spatial transcriptome level. Meanwhile, we found that the dedifferentiation of epithelial cells was initiation, that is, they were converted into mesenchymal stem cells. Another vital feature of remodeling mammary gland was that other cells seem to be actively cleared by immune cells via the IL34-CSF1R pathway. Our cell transcriptomic profile constitutes an essential reference for future studies in the development and remodeling of the mammary gland.
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