The online version of this article (doi:10.1186/bcr2928) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
OW isolated RNA, performed in situ hybridisations, IHC, IF and carried out qRT-PCR analysis. AM carried out SAM analysis on the array data and processed the datasets. NK isolated, stained and analysed mammary primordial samples and performed gene set enrichment analyses. KD and CR contributed samples, antibody staining, and manuscript writing and review. HK provided samples and assisted with qRT-PCR analysis. JSR-F and MJS contributed to the design of the study, manuscript writing and review. CM and MZ carried out network analysis on array data. BAH conceived the study, collected primordial samples, analyzed the microarray data and wrote the manuscript. All authors have read and approved the manuscript for publication.
The mammary primordium forms during embryogenesis as a result of inductive interactions between its constitutive tissues, the mesenchyme and epithelium, and represents the earliest evidence of commitment to the mammary lineage. Previous studies of embryonic mouse mammary epithelium indicated that, by mid-gestation, these cells are determined to a mammary cell fate and that a stem cell population has been delimited. Mammary mesenchyme can induce mammary development from simple epithelium even across species and classes, and can partially restore features of differentiated tissue to mouse mammary tumours in co-culture experiments. Despite these exciting properties, the molecular identity of embryonic mammary cells remains to be fully characterised.
Here, we define the transcriptome of the mammary primordium and the two distinct cellular compartments that comprise it, the mammary primordial bud epithelium and mammary mesenchyme. Pathway and network analysis was performed and comparisons of embryonic mammary gene expression profiles to those of both postnatal mouse and human mammary epithelial cell sub-populations and stroma were made.
Several of the genes we have detected in our embryonic mammary cell signatures were previously shown to regulate mammary cell fate and development, but we also identified a large number of novel candidates. Additionally, we determined genes that were expressed by both embryonic and postnatal mammary cells, which represent candidate regulators of mammary cell fate, differentiation and progenitor cell function that could signal from mammary lineage inception during embryogenesis through postnatal development. Comparison of embryonic mammary cell signatures with those of human breast cells identified potential regulators of mammary progenitor cell functions conserved across species.
These results provide new insights into genetic regulatory mechanisms of mammary development, particularly identification of novel potential regulators of mammary fate and mesenchymal-epithelial cross-talk. Since cancers may represent diseases of mesenchymal-epithelial communications, we anticipate these results will provide foundations for further studies into the fundamental links between developmental, stem cell and breast cancer biology.
Additional file 1: Antibodies used in expression analysis experiments. Table gives details of antibodies used in this study. (XLS 26 KB)13058_2011_2728_MOESM1_ESM.XLS
Additional file 2: The mammary primordial transcriptome. Table A displays relative expression levels of 4,000 Affymetrix probes across three embryonic mouse mammary primordial populations. The spreadsheet gives the relative expression levels for all differentially expressed probes across all three populations. Expression levels are indicated by a relative abundance score for each population. A high positive value indicates expression at a high level, a low negative score indicates very low expression levels. The Affymetrix probe ID, Gene Symbol and q-value (indicating the % false discovery rate) are also indicated. Table B contains genes characteristic of mammary primordial epithelial cells. The table shows all 749 probes in the mammary primordial epithelial population with an abundance score of 1.5 or more when the differentially expressed gene set was sorted by descending abundance scores in the epithelial population. Such genes were considered population-enriched. The number of probes is indicated. Table C contains probes with known expression patterns that were detected as epithelial-specific or epithelial-enriched expression in the arrays. Table D contains probes induced or repressed by tissue separation. Table E contains genes characteristic of mammary primordial mesenchymal cells. The table shows all 642 probes in the mammary primordial mesenchymal population with an abundance score of 1.5 or more when the differentially expressed probe set was sorted by descending abundance scores in the primordial epithelial population. Such genes were considered population-enriched. (XLS 2 MB)13058_2011_2728_MOESM2_ESM.XLS
Additional file 3: Transcriptomic characteristics of the mammary primordial epithelium. (A) A figure summarising select genetic components of the mammary primordial epithelium detected by array analysis. (B) A figure depicting network analysis of the mammary primordial epithelium. (PDF 864 KB)13058_2011_2728_MOESM3_ESM.PDF
Additional file 4: Mammary epithelial primordial core network members. Table contains mammary epithelial core network members. (XLS 46 KB)13058_2011_2728_MOESM4_ESM.XLS
Additional file 5: Transcriptomic characteristics of the mammary mesenchyme. (A) A figure summarising select genetic components of the mammary mesenchyme detected by array analysis. (B) A figure depicting network analysis of the mammary mesenchyme. (PDF 954 KB)13058_2011_2728_MOESM5_ESM.PDF
Additional file 6: Mammary mesenchymal primordial core network members. Table contains mammary mesenchymal core network members. (XLS 58 KB)13058_2011_2728_MOESM6_ESM.XLS
Additional file 7: List of candidate mammary inducing factors with Gene Ontology. This table contains expression patterns of genes enriched in primordial-associated mesenchyme when compared to postnatal mammary stroma in a previously published dataset [ 13]. Gene expression patterns were overlaid in order to discern enriched secreted factors present only in the mammary mesenchyme that could confer mammary cell phenotype to uninduced surface epithelium. (XLS 399 KB)
Additional file 8: Lists of candidate mammary lineage promoting/enforcing factors. Table A contains genes expressed by both the mammary mesenchyme and the mature mammary stroma in a previously published dataset [ 13]. These genes are candidates for promoting mammary differentiation or enforcing mammary lineage commitment. Table B contains genes expressed by human mammary stromal cells in a previously published dataset [ 55] which were also found expressed by the mammary mesenchymal cell populations in the current study. (XLS 266 KB)
Additional file 9: Prospective mediators of epithelial-mesenchymal interactions identified by network analysis. (A) A figure depicting the core interconnected network module. (B) A figure depicting how mesenchymal INHBB is poised to bind to epithelial ACVR1B and ACVR2B. (PDF 917 KB)13058_2011_2728_MOESM9_ESM.PDF
Additional file 10: Core interconnected network members with Gene Ontology. Table contains core interconnected network members with Gene Ontology. (XLS 440 KB)13058_2011_2728_MOESM10_ESM.XLS
Additional file 11: Multiclass SAM analysis of embryonic and postnatal mammary populations. This table displays relative expression levels of 16,382 Affymetrix probes across three embryonic mouse mammary primordial populations and three postnatal mammary epithelial populations. The spreadsheet gives the relative expression levels for all differentially expressed probes across all six populations. Expression levels are indicated by a relative abundance score for each population. A high positive value indicates expression at a high level, a low negative score indicates very low expression levels. The Affymetrix probe ID, Gene Symbol and q-value (indicating the % false discovery rate) are also indicated. (XLS 9 MB)13058_2011_2728_MOESM11_ESM.XLS
Additional file 12: Genes expressed by embryonic mammary cells and postnatal mammary cells. This table contains genes common to primordial cells and basal cell dataset and luminal cell datasets from [ 5]. The table lists those basal genes, LumER- genes, and LumER+ genes found in a previously published dataset [ 5] which were also found in the primordial epithelial or mesenchymal populations in the current study. A mouse stromal signature from [ 13] was used to determine which genes were also found in the primordial epithelial or mesenchymal populations. Genes expressed by embryonic mammary primordial cells and the cells contained within the terminal end bud and ductal microenvironments from [ 38] are also listed. (XLS 82 KB)
Additional file 13: Comparison of gene expression profiles of embryonic mammary cells with postnatal mammary cells. (A) Venn diagrams showing comparison of genes expressed by embryonic mammary primordial cells to those expressed by the cells contained within the terminal end bud and ductal microenvironments from [ 38]. (B) A heatmap showing the expression of a variety of key developmental, lineage, and progenitor markers in embryonic and postnatal mammary cell populations. (C) Venn diagram showing comparison of genes expressed by embryonic mammary primordial cells to those expressed by the conserved mouse/human MEC subpopulations and human fibroblasts from [ 13, 55]. (PDF 1 MB)
Additional file 14: Genes expressed by embryonic mammary cells and human breast cells. This table contains genes common to primordial cells and conserved mouse/human mammary stem/myoepithelial cells, luminal progenitors or mature luminal cell dataset from [ 13]. The table lists those conserved mouse/human genes expressed by mammary stem cell (MaSC) or myoepithelial cells, luminal progenitors (lum prog), and mature luminal (lum mature) cells found in a previously published dataset [ 13] which were also found expressed by the primordial epithelial (MBE) or mesenchymal (MM) populations in the current study. The human stromal signature defined in [ 55] was used to determine which genes were also found in the primordial epithelial or mesenchymal populations. (XLS 86 KB)
Additional file 15: Gene set enrichment analysis of mammary primordial gene expression data. This file contains the gene sets (with a nominal P-value of < 0.05 in order of significance) enriched in primordial cells and postnatal cells when the two data sets were compared. Gene sets with similarity to progenitor populations are in italics and underlined. Gene sets with a FDR q value of < 0.50 are in bold. (XLS 64 KB)13058_2011_2728_MOESM15_ESM.XLS
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Klinowska TC, Streuli CH: Analyzing cell-ECM interactions in adult mammary gland by transplantation of embryonic mammary tissue from knockout mice. Methods Mol Biol. 2000, 139: 345-358. PubMed
Sakakura T, Nishizuka Y, Dawe CJ: Capacity of mammary fat pads of adult C3H/HeMs mice to interact morphogenetically with fetal mammary epithelium. J Natl Cancer Inst. 1979, 63: 733-736. PubMed
Sahlberg C, Mustonen T, Thesleff I: Explant cultures of embryonic epithelium. Analysis of mesenchymal signals. Methods Mol Biol. 2002, 188: 373-382. PubMed
Mouse Genome Informatics website. [ http://www.informatics.jax.org]
Mailleux AA, Spencer-Dene B, Dillon C, Ndiaye D, Savona-Baron C, Itoh N, Kato S, Dickson C, Thiery JP, Bellusci S: Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo. Development. 2002, 129: 53-60. PubMed
Foley J, Dann P, Hong J, Cosgrove J, Dreyer B, Rimm D, Dunbar M, Philbrick W, Wysolmerski J: Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development. Development. 2001, 128: 513-525. PubMed
Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M, Forrest NC, Hartley L, Robb L, Grosveld FG, van der Wees J, Lindeman GJ, Visvader JE: Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol. 2007, 9: 201-209. 10.1038/ncb1530. CrossRefPubMed
Pispa J, Mustonen T, Mikkola ML, Kangas AT, Koppinen P, Lukinmaa PL, Jernvall J, Thesleff I: Tooth patterning and enamel formation can be manipulated by misexpression of TNF receptor Edar. Dev Dyn. 2004, 231: 432-440. 10.1002/dvdy.20138. CrossRef
Cytoscape 2.8.0: An Open Source Platform for Complex Network Analysis and Visualization. [ http://www.cytoscape.org]
Robinson GW, Hennighausen L: Inhibins and activins regulate mammary epithelial cell differentiation through mesenchymal-epithelial interactions. Development. 1997, 124: 2701-2708. PubMed
Dunbar ME, Dann PR, Robinson GW, Hennighausen L, Zhang JP, Wysolmerski JJ: Parathyroid hormone-related protein signaling is necessary for sexual dimorphism during embryonic mammary development. Development. 1999, 126: 3485-3493. PubMed
Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA: Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005, 121: 335-348. 10.1016/j.cell.2005.02.034. CrossRefPubMed
Cunha GR, Young P, Christov K, Guzman R, Nandi S, Talamantes F, Thordarson G: Mammary phenotypic expression induced in epidermal cells by embryonic mammary mesenchyme. Acta Anat (Basel). 1995, 152: 195-204. 10.1159/000147698. CrossRef
Oakes SR, Naylor MJ, Asselin-Labat ML, Blazek KD, Gardiner-Garden M, Hilton HN, Kazlauskas M, Pritchard MA, Chodosh LA, Pfeffer PL, Lindeman GJ, Visvader JE, Ormandy CJ: The Ets transcription factor Elf5 specifies mammary alveolar cell fate. Genes Dev. 2008, 22: 581-586. 10.1101/gad.1614608. CrossRefPubMedPubMedCentral
Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008, 133: 704-715. 10.1016/j.cell.2008.03.027. CrossRefPubMedPubMedCentral
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005, 102: 15545-15550. 10.1073/pnas.0506580102. CrossRefPubMedPubMedCentral
Gritli-Linde A, Hallberg K, Harfe BD, Reyahi A, Kannius-Janson M, Nilsson J, Cobourne MT, Sharpe PT, McMahon AP, Linde A: Abnormal hair development and apparent follicular transformation to mammary gland in the absence of hedgehog signaling. Dev Cell. 2007, 12: 99-112. 10.1016/j.devcel.2006.12.006. CrossRefPubMedPubMedCentral
Heckman BM, Chakravarty G, Vargo-Gogola T, Gonzales-Rimbau M, Hadsell DL, Lee AV, Settleman J, Rosen JM: Crosstalk between the p190-B RhoGAP and IGF signaling pathways is required for embryonic mammary bud development. Dev Biol. 2007, 309: 137-149. 10.1016/j.ydbio.2007.07.002. CrossRefPubMedPubMedCentral
Taylor RA, Wang H, Wilkinson SE, Richards MG, Britt KL, Vaillant F, Lindeman GJ, Visvader JE, Cunha GR, St John J, Risbridger GP: Lineage enforcement by inductive mesenchyme on adult epithelial stem cells across developmental germ layers. Stem Cells. 2009, 27: 3032-3042. PubMed
Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, Asselin-Labat ML, Gyorki DE, Ward T, Partanen A, Feleppa F, Huschtscha LI, Thorne HJ, Fox SB, Yan M, French JD, Brown MA, Smyth GK, Visvader JE, Lindeman GJ: Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med. 2009, 15: 907-913. 10.1038/nm.2000. CrossRefPubMed
- Transcriptome analysis of embryonic mammary cells reveals insights into mammary lineage establishment
Jorge S Reis-Filho
Matthew J Smalley
Beatrice A Howard
- BioMed Central
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