Transcriptome analysis of mammary epithelial subpopulations ...

BMC Genomics 2008, 9:591http://www.biomedcentral.com/1471-2164/9/591well as genes expressed in subsets of its cells. The basal/myoepithelial cell population is also a mixed population.However, > 90% of these cells express both keratin 14 andα-isoform smooth muscle actin and are thus differentiatedmyoepithelial cells [5]. Therefore, the gene expressionprofile of this population is largely a myoepithelialcell profile. The luminal ER + population is also unlikely torepresent a completely uniform cell population but as themajority of the cells are strongly keratin 18 positive andexpress the estrogen receptor [5], the gene expression profileof this population will be dominated by this single celltype.Myoepithelial cells are key regulators of paracrinesignallingTranscriptome and network interaction analysis of thebasal/myoepithelial cells identified key processes of thesecells as cytoskeletal function and extracellular matrix productionand interactions. Given what is known about thecontractile function of these cells in lactation and theirposition in the mammary gland between the luminal epitheliumand the basement membrane around the ductsand lobulo-alveolar structures, these results were reassuring.However, what was remarkable was that the genescharacteristic of this population included more than doublenumber of genes for proteins involved in cell – cell signallingthan were characteristic of the two luminalpopulations combined. This suggests a key role formyoepithelial cells in mediating paracrine and juxtacrinecell – cell interactions in the mammary epithelium. Ofparticular interest were the expression patterns of Wnt andNotch signalling pathway components, known to beimportant in mammary gland development [64,65],which suggested a directionality of Notch signalling frombasal to luminal and a directionality of Wnt signallingfrom luminal to basal. Notably, Notch signalling wasrecently shown to be important for determining luminalcell fate [19], possibly through regulation of luminal progenitors[18]. Activated Wnt signalling has also beenshown to increase the stem/progenitor fraction of basalmammary epithelial cells in MMTV-Wnt1 transgenic mice[3].Another gene from a family important for mammarydevelopment that, like Notch3, was expressed in both theluminal populations but not the basal/myoepithelial cellswas Erbb3. A member of the epidermal growth factorreceptor family, Erbb3 most effectively binds the neuregulinsNrg1 and Nrg2, but it has no intrinsic signallingactivity of its own. It must therefore operate as a heterodimerwith other family members. However, signallingcomplexes containing Erbb3 have a strong propensity toactivate the phosphoinositide-3-kinase (PI3K) signallingpathway due to the presence of six binding sites for thep85 SH3 adaptor subunit of PI3K [51]. Erbb3 knockoutanimals were embryonic lethal but reduction of Erbb3expression in the mammary epithelium caused a reductionin terminal end bud numbers, branching and ductaldensity [51,66]. Previous reports of Erbb3 expression havebeen inconsistent [67,68], most likely due to variableantibody quality, however, the ductal outgrowth defectsthat occur when Erbb3 expression is reduced, togetherwith the observation that implantation of Nrg1-soakedpellets induced ductal elongation at puberty [69], supporta role for Erbb3 in pubertal mammary development.Whether or not Erbb3 activation has the same consequencesin both the luminal ER + and luminal ER - cellsremains to be determined and may depend on differentialexpression of dimerisation partners not detected by themicroarray assays.Mammary epithelial cell subpopulations have distincttranscription factor profilesMutual interactions between transcription factors associatedwith different cell lineages and involving positive andnegative feedback loops have been demonstrated to beable to maintain haematopoietic cells in a small numberof particular cell fates ('stable states') when an apparentlylarge number of potential intermediate fates are available[54]. Transcription factors in the mammary epitheliumwhich have the potential to interact but which are apparentlyexpressed in different populations are therefore ofparticular interest in the regulation of mammary epithelialcell fate. We built a gene network to predict such interactionsand identified a number of these which arepotentially important. The most obvious is between Esr1(luminal ER + ) [56], Trp63 (basal) [70] and Myc (basal andluminal ER - ) [55] and given the large number of transcriptiontargets they share, it is likely that these three are keyfactors in determining cell fate in the mammary gland.However, there are also other interacting factors of interestsuch as the Runx2 (basal) [71] – Msx2 (luminal ER + ) [72]pair, the Eya2 (basal) – Six4 (luminal ER - ) pair [73] andthe Foxa1 (luminal ER + ) [57] – AR (basal and luminalER + ) [74] – Etv5 (basal) [75] triplet. Modelling these interactionsin order to make predictions about how transcriptionfactor behaviour determines mammary cell fate is animportant challenge for the future.Sox6 is a determinant of luminal cell fate in the mammaryglandIn order to model these interactions, functional data oneach individual factor will be required. Given the largenumber of factors of interest, a relatively rapid throughputassay will be required, ruling out the use of transgenic orknockout mice. Therefore, to demonstrate that functionalinformation on the role in determining mammary cellfate of the transcriptional regulators identified in thisstudy can be relatively rapidly generated, and to provide atthe same time the first data required for this modelling,Page 20 of 28(page number not for citation purposes)