A cell transcriptomic profile reveals the molecular mechanisms of adipocytes in mammary gland across development

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. we used ten swine mammary glands from five developmental stage to generate the largest swine cell transcriptomic dataset thus far, in which adipocytes in mammary gland were identified for the first time.

研究乳腺的细胞组成与发育机制，对于新生儿的健康成长至关重要。本研究构建了迄今为止规模最大的乳腺细胞转录组数据集。该数据集包含了乳腺发育五个时间点下，来源于脂肪细胞、内皮细胞、上皮细胞、成纤维细胞、免疫细胞、肌上皮细胞及前体细胞的126829个高质量细胞核。值得注意的是，本研究首次在单细胞核RNA测序（snRNA-seq）与空间转录组（spatial transcriptome）层面完成了乳腺脂肪细胞的注释。组织学观察与细胞类型注释结果显示，泌乳期脂肪细胞的生存空间被大幅压缩。细胞间互作通路分析显示，泌乳期脂肪细胞可通过CCL21-ACKR4等趋化因子受体-配体对清除趋化因子，从而规避免疫细胞的吞噬作用以维持自身存活。在乳腺自然复旧阶段，我们基于ADI-POQ-ADIPOR2通路的激活情况推测，脂肪细胞可抑制细胞凋亡，该结论在空间转录组层面得到了进一步验证。与此同时，本研究发现上皮细胞的去分化是乳腺重塑的起始事件，即上皮细胞可转化为间充质干细胞。乳腺重塑的另一重要特征是，其余细胞可通过IL34-CSF1R通路被免疫细胞主动清除。本研究的细胞转录组图谱可为未来乳腺发育与重塑相关研究提供关键参考依据。此外，本研究采集了5个发育阶段共10个猪乳腺样本，构建了迄今为止规模最大的猪乳腺细胞转录组数据集，其中首次完成了猪乳腺脂肪细胞的鉴定。