Limits...
Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate.

Kendrick H, Regan JL, Magnay FA, Grigoriadis A, Mitsopoulos C, Zvelebil M, Smalley MJ - BMC Genomics (2008)

Bottom Line: One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns.These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK. howard.kendrick@icr.ac.uk

ABSTRACT

Background: Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out.

Results: A whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.

Conclusion: The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns. These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate. These data will form the basis for understanding not only cell fate determination and cellular homeostasis in the normal mammary epithelium but also the contribution of different mammary epithelial cell types to the etiology and molecular pathology of breast disease.

Show MeSH

Related in: MedlinePlus

Sox6 over-expression maintains luminal differentiation in mammary epithelial cells in vitro. A) qPCR analysis of Krt14, Krt18, Esr1 and Sox6 expression levels (mean levels from three independent experiments ± 95% confidence intervals) in cells transduced with virus expressing GFP only (light grey bars) or expressing both Sox6 and GFP (dark grey bars) compared to non-infected cells (open bars). B) Immunofluorescence staining for keratin 14 (upper two rows) and keratin 18 (lower two rows) expression in primary mouse mammary epithelial cells transduced with lentivirus expressing GFP only or Sox6 and GFP. Bars = 30 μm. Arrows indicate keratin positive cells in the Sox6 transduced cultures. These are common in the K18 stained cells but rare and only weakly stained in the K14 stained cells [see Additional file 20].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2629782&req=5

Figure 8: Sox6 over-expression maintains luminal differentiation in mammary epithelial cells in vitro. A) qPCR analysis of Krt14, Krt18, Esr1 and Sox6 expression levels (mean levels from three independent experiments ± 95% confidence intervals) in cells transduced with virus expressing GFP only (light grey bars) or expressing both Sox6 and GFP (dark grey bars) compared to non-infected cells (open bars). B) Immunofluorescence staining for keratin 14 (upper two rows) and keratin 18 (lower two rows) expression in primary mouse mammary epithelial cells transduced with lentivirus expressing GFP only or Sox6 and GFP. Bars = 30 μm. Arrows indicate keratin positive cells in the Sox6 transduced cultures. These are common in the K18 stained cells but rare and only weakly stained in the K14 stained cells [see Additional file 20].

Mentions: For in vitro analysis, cells were cultured for one week and then harvested, GFP+ cells were separated by flow cytometry and RNA isolated from them. The expression of Sox6, Krt14, Krt18 and Esr1 was examined by qPCR and compared to expression levels in cultured primary cells which had not been transduced with virus. The data (Figure 8A) demonstrated that Sox6 over-expression (approximately 800-fold over-expressed in Sox6 GFP virus-transduced cells compared to non-transduced cells) did not significantly alter Krt14 gene expression. However, Krt18 gene expression was significantly increased in Sox6 over-expressing cells compared to non-transduced cells or cells transduced with the control virus (an approximately 3-fold increase in expression levels). Esr1 expression levels were also increased in Sox6 over-expressing cells, although more modestly (1.7 fold; P < 0.05) [58]. Next, cultured virus-transduced primary cells were stained for keratin 14 (K14) and keratin 18 (K18) expression. When primary mouse mammary cells are isolated and grown in short-term culture, the majority of cells which proliferate are derived from the luminal epithelium. Within 48 hours in culture, these luminal origin cells (which are K14- K18+ in vivo) begin to promiscuously express K14 and acquire a K14+ K18+ phenotype [59-61]. It is thought that this represents a de-differentiation event resulting from the cells being removed from their normal environment [62]. Unsurprisingly, therefore, when non-transduced (GFP-) and control transduced primary cells were stained for K14 and K18 expression, they were found to be both K14+ and K18+. However, Sox6 transduced cells were K18+ but showed only weak K14 staining in occasional cells (Figure 8B) [see Additional file 20]. Therefore, Sox6 maintained mammary epithelial cells in the luminal epithelial lineage and blocked promiscuous K14 protein expression in vitro. However, as Sox6 over-expression did not block Krt14 gene expression, this cannot be a direct effect on Krt14 transcription.


Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate.

Kendrick H, Regan JL, Magnay FA, Grigoriadis A, Mitsopoulos C, Zvelebil M, Smalley MJ - BMC Genomics (2008)

Sox6 over-expression maintains luminal differentiation in mammary epithelial cells in vitro. A) qPCR analysis of Krt14, Krt18, Esr1 and Sox6 expression levels (mean levels from three independent experiments ± 95% confidence intervals) in cells transduced with virus expressing GFP only (light grey bars) or expressing both Sox6 and GFP (dark grey bars) compared to non-infected cells (open bars). B) Immunofluorescence staining for keratin 14 (upper two rows) and keratin 18 (lower two rows) expression in primary mouse mammary epithelial cells transduced with lentivirus expressing GFP only or Sox6 and GFP. Bars = 30 μm. Arrows indicate keratin positive cells in the Sox6 transduced cultures. These are common in the K18 stained cells but rare and only weakly stained in the K14 stained cells [see Additional file 20].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629782&req=5

Figure 8: Sox6 over-expression maintains luminal differentiation in mammary epithelial cells in vitro. A) qPCR analysis of Krt14, Krt18, Esr1 and Sox6 expression levels (mean levels from three independent experiments ± 95% confidence intervals) in cells transduced with virus expressing GFP only (light grey bars) or expressing both Sox6 and GFP (dark grey bars) compared to non-infected cells (open bars). B) Immunofluorescence staining for keratin 14 (upper two rows) and keratin 18 (lower two rows) expression in primary mouse mammary epithelial cells transduced with lentivirus expressing GFP only or Sox6 and GFP. Bars = 30 μm. Arrows indicate keratin positive cells in the Sox6 transduced cultures. These are common in the K18 stained cells but rare and only weakly stained in the K14 stained cells [see Additional file 20].
Mentions: For in vitro analysis, cells were cultured for one week and then harvested, GFP+ cells were separated by flow cytometry and RNA isolated from them. The expression of Sox6, Krt14, Krt18 and Esr1 was examined by qPCR and compared to expression levels in cultured primary cells which had not been transduced with virus. The data (Figure 8A) demonstrated that Sox6 over-expression (approximately 800-fold over-expressed in Sox6 GFP virus-transduced cells compared to non-transduced cells) did not significantly alter Krt14 gene expression. However, Krt18 gene expression was significantly increased in Sox6 over-expressing cells compared to non-transduced cells or cells transduced with the control virus (an approximately 3-fold increase in expression levels). Esr1 expression levels were also increased in Sox6 over-expressing cells, although more modestly (1.7 fold; P < 0.05) [58]. Next, cultured virus-transduced primary cells were stained for keratin 14 (K14) and keratin 18 (K18) expression. When primary mouse mammary cells are isolated and grown in short-term culture, the majority of cells which proliferate are derived from the luminal epithelium. Within 48 hours in culture, these luminal origin cells (which are K14- K18+ in vivo) begin to promiscuously express K14 and acquire a K14+ K18+ phenotype [59-61]. It is thought that this represents a de-differentiation event resulting from the cells being removed from their normal environment [62]. Unsurprisingly, therefore, when non-transduced (GFP-) and control transduced primary cells were stained for K14 and K18 expression, they were found to be both K14+ and K18+. However, Sox6 transduced cells were K18+ but showed only weak K14 staining in occasional cells (Figure 8B) [see Additional file 20]. Therefore, Sox6 maintained mammary epithelial cells in the luminal epithelial lineage and blocked promiscuous K14 protein expression in vitro. However, as Sox6 over-expression did not block Krt14 gene expression, this cannot be a direct effect on Krt14 transcription.

Bottom Line: One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns.These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate.

View Article: PubMed Central - HTML - PubMed

Affiliation: Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London, UK. howard.kendrick@icr.ac.uk

ABSTRACT

Background: Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out.

Results: A whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.

Conclusion: The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns. These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate. These data will form the basis for understanding not only cell fate determination and cellular homeostasis in the normal mammary epithelium but also the contribution of different mammary epithelial cell types to the etiology and molecular pathology of breast disease.

Show MeSH
Related in: MedlinePlus