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Background: Cow’s milk is a highly-nutritious dairy product that is widely consumed worldwide. It is secreted by the developed mammary gland (MG) of dairy cattle. However, a comprehensive understanding of cell-type diversity and cell function within bovine MG is lacking. In the current study, we used single-cell RNA sequencing to investigate the transcriptome of 24,472 high-quality MG cells isolated from newborn and adult cows.
Results: Unbiased clustering analysis revealed the existence of 24 cell types, which could be divided into four categories: 9 immune, 3 epithelial, 9 fibroblast, and 3 endothelial cell types. Other cell subtypes were further identified based on re-clustering and pseudotemporal reconstruction of epithelial cells that included 3 mature luminal epithelial, 1 intermediate, and 2 progenitor cell subtypes. The individual top marker genes of these 3 mature luminal epithelial cell subtypes (L0, L1, and L5) were APOA1, STC2, and PTX3, which were further validated using immunofluorescence. Based on functional analysis, the L0, L1, and L5 cell subtypes were all involved in the upregulation of lipid metabolism, protein and hormone metabolism, and the immune response, respectively. Furthermore, we discovered a novel myofibroblast that expresses COL1A1 and CSN3, has visible epithelial-like characteristics, and shows the potential to differentiate into luminal epithelial cells, especially immune-sensing luminal cells (L5).
Conclusions: We constructed the first single-cell atlas of the dairy cow MG, and our new findings of epithelial-like myofibroblast cells and their differentiation trajectories into luminal cells may provide novel insights into the development and lactogenesis in dairy cattle MGs.
Keywords
single-cell RNA-sequencing, mammary gland, dairy cows, epithelial cells, myofibroblast
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTables.xlsx
Additional file 1: Table S1. Marker gene list of 24 clusters of mammary gland of dairy cows identified by single-cell RNA sequencing. Table S2. Immune cell types and key genes in the mammary gland of dairy cows. Table S3. The marker gene list of 6 epithelial sub-clusters identified in the mammary gland of dairy cows. Table S4. The branch-specific expression genes and clusters of epithelial sub-cell types changing over pseudotime. Table S5. The co-expressed genes in different modules that generated by WGCNA. Table S6. The marker gene list of sub-fibroblast cells identified in the mammary gland of dairy cows using single-cell RNA sequencing.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
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This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
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History
CURRENT STATUS: Posted
Version 1
Posted 13 Nov, 2020
No community comments so far
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HTML Views: ...
Subject Areas
Animal Science
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Cow’s milk is a highly-nutritious dairy product that is widely consumed worldwide. It is secreted by the developed mammary gland (MG) of dairy cattle. However, a comprehensive understanding of cell-type diversity and cell function within bovine MG is lacking. In the current study, we used single-cell RNA sequencing to investigate the transcriptome of 24,472 high-quality MG cells isolated from newborn and adult cows.
Results: Unbiased clustering analysis revealed the existence of 24 cell types, which could be divided into four categories: 9 immune, 3 epithelial, 9 fibroblast, and 3 endothelial cell types. Other cell subtypes were further identified based on re-clustering and pseudotemporal reconstruction of epithelial cells that included 3 mature luminal epithelial, 1 intermediate, and 2 progenitor cell subtypes. The individual top marker genes of these 3 mature luminal epithelial cell subtypes (L0, L1, and L5) were APOA1, STC2, and PTX3, which were further validated using immunofluorescence. Based on functional analysis, the L0, L1, and L5 cell subtypes were all involved in the upregulation of lipid metabolism, protein and hormone metabolism, and the immune response, respectively. Furthermore, we discovered a novel myofibroblast that expresses COL1A1 and CSN3, has visible epithelial-like characteristics, and shows the potential to differentiate into luminal epithelial cells, especially immune-sensing luminal cells (L5).
Conclusions: We constructed the first single-cell atlas of the dairy cow MG, and our new findings of epithelial-like myofibroblast cells and their differentiation trajectories into luminal cells may provide novel insights into the development and lactogenesis in dairy cattle MGs.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTables.xlsx
Additional file 1: Table S1. Marker gene list of 24 clusters of mammary gland of dairy cows identified by single-cell RNA sequencing. Table S2. Immune cell types and key genes in the mammary gland of dairy cows. Table S3. The marker gene list of 6 epithelial sub-clusters identified in the mammary gland of dairy cows. Table S4. The branch-specific expression genes and clusters of epithelial sub-cell types changing over pseudotime. Table S5. The co-expressed genes in different modules that generated by WGCNA. Table S6. The marker gene list of sub-fibroblast cells identified in the mammary gland of dairy cows using single-cell RNA sequencing.
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