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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.

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qPCR analysis validates gene expression array analysis of virgin mammary epithelial cell gene expression. Data from qPCR analysis of expression of 55 genes in triplicate independent samples of basal/myoepithelial cells, ER- luminal cells and ER+ luminal cells. Each data point is the mean level of expression, ± 95% confidence intervals, across the three samples of that population relative to the comparator sample. A 'round robin' comparison analysis was used as described in the Methods. Genes determined by this method to be characteristic of the comparator population are indicated below each pair of graphs.
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Figure 3: qPCR analysis validates gene expression array analysis of virgin mammary epithelial cell gene expression. Data from qPCR analysis of expression of 55 genes in triplicate independent samples of basal/myoepithelial cells, ER- luminal cells and ER+ luminal cells. Each data point is the mean level of expression, ± 95% confidence intervals, across the three samples of that population relative to the comparator sample. A 'round robin' comparison analysis was used as described in the Methods. Genes determined by this method to be characteristic of the comparator population are indicated below each pair of graphs.

Mentions: To identify genes whose expression characterised the three subpopulations, the list of genes was split into three sets on the basis of relative abundance of expression. Any gene with a relative abundance of 2 or higher in a population was considered as population-specific. If a gene was represented by more than one probe set, the average of all probe sets was used for further analysis. This analysis identified 861, 326 and 488 genes as characteristic of basal/myoepithelial cells, luminal ER- cells and luminal ER+ cells, respectively [see Additional files 4, 5, 6]. To confirm our approach, a subset of genes specific for each of the three populations, as well as some which were common to two of the populations, were selected for qPCR validation. Furthermore, a number of genes previously shown to be relevant in mammary biology were included in this analysis, as was the data collected on Krt14, Krt18 and Esr1 expression in the populations [see Additional file 1], giving 58 genes in total (Figure 3). The qPCR probes for 3 genes failed to amplify any product in any of the populations. For a further 10 genes, there was no differential expression pattern in the array analysis. Technical problems, such as poor signal strength from the Affymetrix probe, cannot be ruled out in these cases. Of the remaining 45 genes which showed a differential expression pattern in both the array and qPCR analyses, 40 genes (88.9%) showed identical expression patterns across the three populations in both sets of data. With only 5 genes (11.1%) was the pattern of differential expression suggested by the array data different to that suggested by the qPCR analysis [see Additional file 7]. Overall, therefore, the qPCR data showed that the gene expression array dataset could be relied upon for a global picture of the biology of the three populations.


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)

qPCR analysis validates gene expression array analysis of virgin mammary epithelial cell gene expression. Data from qPCR analysis of expression of 55 genes in triplicate independent samples of basal/myoepithelial cells, ER- luminal cells and ER+ luminal cells. Each data point is the mean level of expression, ± 95% confidence intervals, across the three samples of that population relative to the comparator sample. A 'round robin' comparison analysis was used as described in the Methods. Genes determined by this method to be characteristic of the comparator population are indicated below each pair of graphs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: qPCR analysis validates gene expression array analysis of virgin mammary epithelial cell gene expression. Data from qPCR analysis of expression of 55 genes in triplicate independent samples of basal/myoepithelial cells, ER- luminal cells and ER+ luminal cells. Each data point is the mean level of expression, ± 95% confidence intervals, across the three samples of that population relative to the comparator sample. A 'round robin' comparison analysis was used as described in the Methods. Genes determined by this method to be characteristic of the comparator population are indicated below each pair of graphs.
Mentions: To identify genes whose expression characterised the three subpopulations, the list of genes was split into three sets on the basis of relative abundance of expression. Any gene with a relative abundance of 2 or higher in a population was considered as population-specific. If a gene was represented by more than one probe set, the average of all probe sets was used for further analysis. This analysis identified 861, 326 and 488 genes as characteristic of basal/myoepithelial cells, luminal ER- cells and luminal ER+ cells, respectively [see Additional files 4, 5, 6]. To confirm our approach, a subset of genes specific for each of the three populations, as well as some which were common to two of the populations, were selected for qPCR validation. Furthermore, a number of genes previously shown to be relevant in mammary biology were included in this analysis, as was the data collected on Krt14, Krt18 and Esr1 expression in the populations [see Additional file 1], giving 58 genes in total (Figure 3). The qPCR probes for 3 genes failed to amplify any product in any of the populations. For a further 10 genes, there was no differential expression pattern in the array analysis. Technical problems, such as poor signal strength from the Affymetrix probe, cannot be ruled out in these cases. Of the remaining 45 genes which showed a differential expression pattern in both the array and qPCR analyses, 40 genes (88.9%) showed identical expression patterns across the three populations in both sets of data. With only 5 genes (11.1%) was the pattern of differential expression suggested by the array data different to that suggested by the qPCR analysis [see Additional file 7]. Overall, therefore, the qPCR data showed that the gene expression array dataset could be relied upon for a global picture of the biology of the three populations.

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