BACKGROUND
the function of complex tissues, such as the mammary epithelium, is a product of the interactions between their constituent cell types.
in such tissues, disease states like cancer are essentially a failure of this cellular homeostasis and are characterised by insensitivity of cells to external regulatory factors and aberrant cell fate choices.
understanding the molecular regulation of the individual cell types in complex tissues is, therefore, a prerequisite for understanding disease states.
furthermore, advances in molecular pathology have demonstrated that different disease phenotypes correlate with different gene expression profiles  <cit> .
in a complex tissue, composed of different cell types with different molecular characteristics, the gene expression profiles of different diseases may reflect the contribution of different cell types to that disease.
therefore, a detailed molecular characterisation of the cell types in a complex tissue is essential for the interpretation of the molecular pathology of its diseases.
the resting adult mammary epithelium consists of two main structures, alveoli  and ducts   <cit> .
these two structures are themselves composed of two main epithelial cell layers, basal cells and luminal cells.
the basal cell layer mainly consists of myoepithelial cells which contract in response to oxytocin release during lactation to force milk down the ducts to the nipple.
recent work has demonstrated that the basal cell layer also contains the mammary epithelial stem cell compartment  <cit> .
the luminal cell layer has been shown to be composed of two functionally distinct lineages defined by expression of the cell surface proteins cd <dig> and sca- <dig>  cd24+/high sca-1+ luminal cells express estrogen receptor alpha , as well as receptors for prolactin and progesterone , while cd24+/high sca-1- luminal cells  express genes  for milk proteins even in the virgin and likely include alveolar progenitors  <cit> .
although it is known that the stem cells can generate all the myoepithelial, luminal er- and luminal er+ daughter cell types  <cit> , the mechanisms which control cellular homeostasis, fate determination and lineage commitment in the mammary epithelium are poorly understood.
they are likely to be a product of complex interactions between cell extrinsic paracrine influences, cell intrinsic transcriptional regulators and epigenomic modifications  <cit> .
some progress has been made towards understanding some of the cell intrinsic factors involved.
for instance, gata <dig> was recently identified as a transcription factor important in specifying commitment in the general luminal lineage  <cit>  and elf <dig> was shown to be a specifier of alveolar cell fate  <cit> .
a number of the cell extrinsic  factors operating within the mammary epithelium have also been characterised, such as wnt- <dig>  which acts downstream of progesterone signalling in ductal side-branching  <cit>  and the egf-family member amphiregulin  <cit> , which is produced by er+ cells in response to estrogen and stimulates mammary stem cell activity   <cit> .
the notch signalling pathway has also been shown to be an important determinant of luminal cell fate  <cit> .
however, the full extent and nature of paracrine interactions in the mammary gland, and the degree to which the different lineages contribute to them, and are defined by them, is still not fully understood.
gene expression patterns have been previously examined in the mouse mammary gland, either as changes in gene expression across the whole tissue in developmental timecourses  <cit>  or as comparisons between the total epithelium and the mammary stroma  <cit> .
in one report, gene expression patterns were examined in mouse luminal and myoepithelial cells purified by flow sorting as well as in stem cell enriched basal cell populations  <cit> .
however, these stem cell gene signatures were found to be not significantly different from the myoepithelial signatures, suggesting they were derived from cell populations dominated by myoepithelial cells.
the purity of the basal stem cell populations remains a persistent problem due the difficulty of isolating pure  stem cell fractions.
a number of gene expression studies have also been carried out on human breast cells.
the response of human breast epithelium to estrogen has been analysed at the gene expression level in breast cancer cell lines in vitro and as xenografts  <cit> , in normal breast tissue maintained as xenografts  <cit>  and in normal human er+ breast cells isolated by transduction of primary breast epithelial cells with a virus carrying an estrogen response element driving gfp expression  <cit> .
the comparative gene expression profiles of normal human myoepithelial  <cit> , basal non-myoepithelial   <cit>  and luminal epithelial cells  <cit>  have also been examined, as have the gene expression profiles of different in vitro progenitor  subpopulations of normal breast epithelial cells  <cit> .
however, to date no genome-wide transcriptome study has made a direct comparison between the two luminal epithelial populations  and the basal/myoepithelial cells, confounding the molecular characterisation of the luminal cells and preventing the analysis of the lineage commitment of, and interactions between, the two luminal cell types and the other cell types in the gland.
the aim of this study, therefore, was to carry out the first comprehensive gene expression study which examined gene expression patterns in the three distinct mouse mammary epithelial populations, basal/myoepithelial, luminal er- and luminal er+.
the analysis was to concentrate on three specific areas.
first, characterising cell-type specific patterns of gene expression which defined cell identity.
second, establishing a broad overview of the likely extent and nature of paracrine interactions amongst the populations.
last, defining cell intrinsic factors which may be important in determining lineage commitment and cell fate in the mammary epithelium.
the results of these analyses have, first, identified a novel potential function for a subpopulation of mammary luminal epithelial cells as non-professional immune cells; second, provided information on the large number of paracrine interactions yet to be fully characterised in the mammary gland and the likely complexity of their interactions; third, identified population-specific transcription factors which may have a role in lineage determination and fate specification in the mammary epithelium.
in particular, we have used in vitro and in vivo functional analyses to demonstrate that sox <dig> is a determinant of luminal cell fate.
RESULTS
identification of population-specific gene expression patterns in the virgin mouse mammary epithelium
to carry out a comprehensive, whole genome gene expression analysis of the epithelial cell populations in the virgin mammary gland , primary mouse mammary cells were isolated and stained with anti-cd <dig> and anti-sca- <dig> antibodies.
the cd24+/low sca-1-, cd24+/high sca-1- and cd24+/high sca-1+ cells were separated by flow cytometry  and used to prepare rna.
to demonstrate that these populations corresponded to basal/myoepithelial, luminal er- and luminal er+ cells respectively, as we have previously described  <cit> , relative gene expression levels of the basal cell marker keratin  <dig> , the luminal cell marker keratin  <dig>  and estrogen receptor α  were measured by quantitative real-time rtpcr .
the results showed that the cd24+/low sca-1- cells expressed krt <dig> but not krt <dig> and that the cd24+/high sca-1- and cd24+/high sca-1+ cells expressed krt <dig> but not krt <dig>  they also showed that the cd24+/high sca-1+ population was enriched for esr <dig> expression compared to the other two populations.
this agreed with previous data  <cit>  and with staining of sections through the mouse mammary gland  which showed that the basal cells were keratin 14+ , the luminal cells were k18+ and that er+ cells were found exclusively in the k18+ luminal cell layer.
therefore, these data confirmed the identity of the three populations   <cit> .
next, a whole transcriptome gene expression analysis of the three populations was carried out using the affymetrix platform .
to identify genes whose expression in one population was different from its expression in the other two, significance analysis of microarray  was applied  <cit> .
using this technique in a multiclass setting, the average gene expression within one group was compared with the average gene expression of all samples.
following repeated permutation of the data, the strength of the relationship between gene expression and its expressed group was established and a significance level determined.
when a fdr cutoff of  <dig> % was applied,  <dig> probe sets  were identified which showed significant differential expression across the three populations.
these  <dig> probes corresponded to  <dig> well known individual genes.
heat mapping and hierarchical cluster analysis of the genes identified  <dig> different gene clusters.
the cluster analysis showed that some gene sets were characteristic of only one population  whereas others were expressed in two of the three   .
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  <dig> 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  <dig>   <dig> and  <dig> genes as characteristic of basal/myoepithelial cells, luminal er- cells and luminal er+ cells, respectively .
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 krt <dig>  krt <dig> and esr <dig> expression in the populations , giving  <dig> genes in total .
the qpcr probes for  <dig> genes failed to amplify any product in any of the populations.
for a further  <dig> 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  <dig> genes which showed a differential expression pattern in both the array and qpcr analyses,  <dig> genes  showed identical expression patterns across the three populations in both sets of data.
with only  <dig> genes  was the pattern of differential expression suggested by the array data different to that suggested by the qpcr analysis .
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.
comparisons with previously published datasets
we compared our data with previous studies which have used a cell separation approach to isolate mouse  <cit>  or human  <cit>  mammary epithelial basal/myoepithelial and luminal subpopulations for gene expression profiling, to identify genes found in common between these datasets and our own data .
there was good concordance between the genes identified in our data and that of stingl and colleagues  <cit>  in their study of separated mouse epithelium.
they identified  <dig> probes, corresponding to  <dig> well-annotated genes, significantly upregulated in their myoepithelial/mammary stem cell enriched cell fraction compared to their mammary colony forming cells .
of these  <dig> genes,  <dig>  were significantly enriched in our basal/myoepithelial cell dataset, three were enriched in both the basal/myoepithelial cells and the luminal er+ cells and two were enriched in both the basal/myoepithelial cells and the luminal er- cells .
conversely, stingl and colleagues identified  <dig> probes, corresponding to  <dig> well-annotated genes, significantly downregulated in their myoepithelial/mammary stem cell enriched cell fraction compared to their mammary colony forming cells.
of these, which would correspond to genes enriched in the luminal epithelial fraction, none were enriched in our basal/myoepithelial population but  <dig>  were enriched in both our luminal populations, five were enriched only in the luminal er- cells and four only in the luminal er+ cells .
however, correspondence of our data with previously published datasets from separated human cells  <cit>  was lower, although it tended to be better between the mouse myoepithelial and human myoepithelial data sets than between the luminal data sets from the different species .
interaction mapping of genes differentially expressed in mammary epithelial subpopulations identifies key processes and novel functions
to get a better understanding of the key biological processes occurring in each of the three cell types, we generated network interaction maps for the differentially expressed genes .
interaction data derived from studies on human orthologues of the genes identified were used to create the network maps, as there is not enough data currently available purely from studies of mouse genes to make such an analysis meaningful.
when a basal/myoepithelial interaction map was constructed using both the differentially expressed genes and genes interpolated by the network mapping program , the resulting network was extraordinarily complex .
for ease of interpretation, therefore, a basal/myoepithelial network was constructed using only direct interactions between genes characteristic of this cell population, with no interpolations .
this network identified two major interaction 'modules' and three minor ones.
such interaction modules are indicative of important processes occurring within a cell, as they are composed of cell-type specific genes and defined by multiple interactions between those genes.
the two major interaction modules can be broadly characterised as an extracellular matrix module including multiple collagen genes and a cytoskeletal module including genes for the keratins, vimentin, and genes whose protein products are involved in regulation of cell shape, movement and contractility, such as the actin binding proteins myh <dig>  <cit>  and tpm <dig>  <cit>  and smooth muscle gamma actin actg <dig>  <cit> .
the minor modules indicated that the basal/myoepithelial cell population also has important processes based around platelet-derived growth factor , ephrin and insulin-like growth factor  signalling.
ephrins are mediators of contact-dependent communication between cells  <cit>  whereas both pdgf and igf <dig> signalling are involved in paracrine cell-cell communication  <cit> .
the luminal interaction maps were built using both cell-specific genes and interpolated genes .
as a result, it was less straightforward to define cell-specific interaction modules which would indicate the key cellular processes occurring in these cell types.
we therefore developed a mathematical approach to defining the modules which required the assignment of network hubs, node clusters and contiguous differentially expressed network paths.
to define network hubs , nodes were ranked by descending connectivity.
the minimum node connectivity in the top 10% of nodes was five for either network and this was therefore set as a threshold for identifying hubs.
differentially expressed hubs for the luminal er- and er+ networks are listed in tables  <dig> and  <dig> respectively.
there was limited overlap between the luminal er- and er+ network hubs with only three differentially expressed hubs being shared .
the majority of hubs in both networks had a high content of physical interactions with the exception of tnf, sp <dig>  fas, nfkb <dig> creb <dig>  egr <dig> and spi <dig>  which are almost exclusively transcriptional hubs.
in the luminal er- network tlr <dig>  ly <dig>  erbb <dig>  muc <dig> and cd <dig> were differentially expressed hubs with significant clustering character, although the strongest clustering was seen for the non-differentially expressed hubs egfr and erbb <dig>  clustering was less pronounced in the luminal er+ network with pgr, erbb <dig>  fgg, fgb, cd <dig> and col8a <dig> forming significantly clustered differentially expressed hubs.
with the exception of tlr <dig> and ly <dig> in the luminal er- network, clustering and connectivity were inversely correlated, with high connectivity nodes such as esr, ptn, ccl <dig>  tnf and bcl <dig>  exhibiting very little or no clustering.
*differentially-expressed genes.
knode, node connectivity, cnode, node clustering coefficient, calculated as cnode =  <dig> nnode/) where nnode is the number of interactions between the hub first neighbours.
hubs exhibiting clustering are shown in bold.
high content of physical or transcriptional interactions in the hub is shown in bold.
see legend to table  <dig> for details.
*differentially expressed genes.
to identify modules within the networks, a three-pass approach was adopted .
initially all differentially expressed hubs were identified and, where direct links existed between them in the network, these were used to provide the backbone for each module.
in the second pass, modules were allowed to expand by the addition of differentially expressed nodes with lower connectivity , if they were directly linked to differentially expressed hubs.
in the third pass, non-differentially expressed hubs were incorporated, if they were connecting at least two differentially expressed hubs.
this step allowed for bridging between modules, providing further information about their proximity and global topology within the luminal er- and luminal er+ networks .
following the initial two passes, four distinct modules were established for the luminal er- network : the tlr , kit , krt  and bcl <dig>  modules .
nodes tnf, fas, ccl <dig>  ccl <dig> and rps6ka <dig> were integrated in the bcl <dig> subnet only at the third pass through a network of transcriptional interactions, forming the tnf/fas module .
in addition, the krt module merged into the kit module at this stage.
the tlr and kit modules contained predominantly physical interactions whereas the tnf/fas module displayed a very strong transcriptional character .
the three modules exhibit topological proximity, defining a single subnetwork with  <dig> connections and  <dig> nodes .
compared to the luminal er- network overall, this subnetwork showed higher clustering and, whilst maintaining the average network connectivity, it exhibited a significantly shorter mean shortest path and a very low power exponent.
removing this subnetwork from the overall luminal er- network yielded a highly fragmented graph  with no clustering and very low connectivity .
for the luminal er+ network, six distinct modules were observed following the initial two passes : the esr , erbb <dig> , mllt <dig> , col8a <dig> , krt  and fgg  modules .
after the third pass, modules esr, erbb <dig>  krt, col8a <dig> and mllt <dig> were merged into a large subnetwork with differentially expressed hubs tgfb <dig>  areg, mbp, cav <dig>  eps <dig> and pcdha <dig> playing an interconnecting role .
unlike the luminal er- network, where all modules were proximal to each other, more fragmentation was observed for the luminal er+ network, with module fgg and differentially expressed hubs hist1h4h, hist1h4i and lnx <dig> not interconnected with the module subnetwork via any other hubs.
the luminal er+ subnetwork also had a much stronger physical interaction character than the luminal er- subnetwork.
compared to the overall luminal er+ network, its subnetwork showed high clustering and, whilst maintaining the average network connectivity, exhibited a significantly shorter mean shortest path.
as with the luminal er- subnetwork, removing the luminal er+ subnetwork from the overall luminal er+ network gave a highly fragmented graph with no clustering and very low connectivity .
the luminal er- and luminal er+ modules, their component nodes and potential functions are summarised in table  <dig>  they suggest that the major processes occurring in the luminal er- cells are tyrosine kinase cell signalling pathways in association with cellular cytoskeletal components  as well as passive immune signalling and inflammatory response processes.
the picture in the luminal er+ cells is less straightforward.
there is no obvious unifying theme to the major esr module, which leaves five minor modules involved in signal transduction, the cytoskeleton, cell adhesion and maintaining the extracellular matrix .
the tlr module within the luminal er- network was of particular interest   <cit> , as it indicated a novel role of these cells as non-professional immune cells which can respond to bacterial lipopolysaccharide.
to confirm that the luminal er- population was indeed enriched for cells expressing components of the cd14-tlr <dig> signalling complex, qpcr analysis of relative expression levels of the three components of the lps receptor, cd <dig>  ly <dig> and tlr <dig>  its downstream effecter irak <dig> and the lps receptor responsive gene tnf were carried out  <cit> .
the results  confirmed that all five genes were specifically enriched in the luminal er- cells compared to both the basal/myoepithelial and luminal er+ cells.
in particular, cd <dig>  tlr <dig> and tnf were expressed at 10-fold, 3-fold and 2-fold, respectively, higher levels in the luminal er- cells compared to the luminal er+ cells.
cd <dig> and tnf were expressed at 30-fold and 5-fold higher levels in the luminal er- cells compared to the basal/myoepithelial cells.
tlr <dig> expression was undetectable in the basal/myoepithelial cells.
to determine whether cd <dig> expression, and thus the potential to be able to respond to lps, was a general property of all luminal er- cells or only a subfraction, freshly isolated primary cells were stained with anti-cd <dig> and anti-sca- <dig> antibodies, to enable the three main cell compartments to be identified, as well as with anti-cd <dig> and anti-cd <dig>   <cit>  antibodies.
the results  showed that the cd24+/high sca-1- luminal er- population is itself composed of four different subpopulations, namely cd14- cd61- cells, cd14+ cells, cd61+ cells and cd14+ cd61+ cells.
interestingly, a small number of cd24+/low sca1- basal cells also showed elevated levels of cd <dig> expression.
the luminal er+ gene expression pattern is not an 'estrogen-responsive' gene expression signature
upon examination of the luminal er+ gene network, we noted that few of the genes enriched in the luminal er+ population were directly linked to esr <dig> by transcriptional interactions, with the exception of the progesterone receptor   <cit>  and the cytoskeletal protein keratin  <dig>   <cit> .
this suggested that the gene signature of the luminal er+ cells was not an 'estrogen responsive' gene signature.
rather, it was more likely to represent an underlying stable gene expression pattern characteristic of this differentiated cell population.
to investigate this further, we compared lists of estrogen responsive genes reported in studies of estrogen-stimulated normal human mammary epithelial cells  <cit>  and breast cancer cell lines with our lists of genes expressed in the epithelial subpopulations  <cit> .
the results  confirmed that there was little correlation between 'estrogen-responsive' signatures and the genes enriched in the luminal er+ population.
indeed, many of the gene whose expression was stimulated by estrogen in the breast cancer cell lines were found to be enriched in our basal/myoepithelial population.
however, it should be noted that the well-known directly estrogen responsive genes krt <dig> and pgr, were not found to be upregulated in most of the published datasets.
identification of basal/myoepithelial cells as key mediators of paracrine signalling
having identified key processes likely to be occurring within each of the three populations, we next investigated how the populations might be interacting with each other.
mammary epithelial biology is characterised by the conversion of systemic hormone signals into local growth factor signals which stimulate stem cell proliferation and differentiation of daughter cell types.
whilst some of these paracrine interactions have been studied in depth  <cit> , the broad extent of paracrine networks within the mammary epithelium remains unknown.
we therefore queried the gene expression array data for genes potentially involved in paracrine signalling, either as receptors or ligands .
a number of genes were identified which fulfilled these criteria .
remarkably, they showed that the basal/myoepithelial cells have more than double the complement of cell-surface receptors and ligands than either of the two luminal populations.
of particular interest, the basal/myoepithelial cells expressed the genes for the notch family ligands, jag <dig>  jag <dig> and dll <dig>  <cit>  whilst the gene for the notch family receptor notch <dig>  <cit>  was expressed in both the luminal populations.
wnt family ligands were expressed by all three populations, although each cell type expressed a different complement of wnt genes and only the basal/myoepithelial cells expressed the genes for the frizzled receptors  <cit> .
it is well established that egf receptor family members and egf family ligands are important for mammary gland development and breast cancer  <cit> .
in particular, the paracrine role of amphiregulin  is well described  <cit> .
our analysis confirmed that luminal er+ mammary epithelial cells expressed the areg gene but also showed that the genes for two other family members, betacellulin   <cit>  and epigen   <cit> , were expressed in this cell type and btc was also expressed in the luminal er- cells.
interestingly, only one egf receptor family member, erbb <dig>  <cit> , was found by gene expression array and qpcr analysis to be differentially expressed in the normal mammary epithelium.
it was present in both the luminal epithelial populations but not in the basal/myoepithelial cells.
this analysis extends previous findings of paracrine signalling within the mammary epithelium and emphasises the complexity of the interacting signalling networks.
these include wnt and notch signalling, the egf family, fgf signalling, other receptor tyrosine kinases, g-protein coupled receptors, ligands for such receptors, integrins and ephrins/eph receptors all of which are differentially expressed between the cell populations.
in particular, the numbers of ligands and receptors expressed by the basal/myoepithelial cells indicates that this population is a key mediator of the paracrine signalling networks within the mammary epithelium.
identification of differentially regulated transcription factors within the mammary epithelium
transcriptional regulators are key cell-intrinsic factors in lineage selection and cell fate decisions in stem – differentiated cell hierarchies  <cit> .
therefore, the gene expression array data set was interrogated to identify differentially expressed factors which may regulate transcription .
the expression of a subset of these was analysed by qpcr .
as with the potential paracrine interactions, the basal/myoepithelial cells differentially expressed many more transcriptional regulators  than the other two populations.
the er- luminal cells differentially expressed twenty-one transcription factors but only seven transcription factors were differentially expressed in the luminal er+ cells.
three transcription factors were found to be expressed in both the basal/myoepithelial and luminal er- populations, one was common to both the basal/myoepithelial and luminal er+ cells and two were found in all three populations.
in common lineage progenitors, functional mutual repression and auto-stimulation by transcription factors can facilitate bilineage cell fate decisions  <cit> .
once the cell fate decisions have been executed, continued function of different subsets of the factors active in the progenitors are required to maintain the different differentiated cell lineages in stable fates  <cit> .
thus, modelling interactions between lineage-specific transcription factors can elucidate cell fate decision processes occurring in progenitor cells.
therefore, to begin to understand how interactions between lineage specific transcription factors may influence cell fate choices in the mammary epithelium, an interaction network was built.
transcription factors identified as lineage-specific but for which no interaction data exists were excluded.
the resulting network  was centred around two main hubs, namely myc and esr <dig>  suggesting that they are key regulators of mammary cell fate  <cit> .
clearly, a great deal of information exists on their interactions and this would bias any interaction network around these two key nodes.
nevertheless, the number of  interactions that myc makes with the basal-specific factors in particular suggests that it is a key factor in regulating mammary cell fate determination.
the small number of luminal er+ population specific factors and the fact that three of them, esr <dig>  pgr and foxa <dig>  <cit> , are closely connected also supports a key role for the estrogen receptor and its transcriptional network in regulating luminal er+ cell fate.
as described above, however, the luminal er+ transcriptional profile is not, in general, an estrogen responsive profile.
therefore, the activity of the luminal er+ specific transcription factors most likely leads to long term stable cell fate changes and not changes which are responsive to short-term fluctuations in estrogen signalling.
mutual repression and auto-stimulation loops may be one way to achieve this long term stability.
sox <dig> is a determinant of luminal cell fate
to demonstrate that the lineage specific transcription factors we identified can indeed influence cell fate and differentiation, we chose to investigate in more detail the function of sox <dig>  <cit> , the expression of which was only detectable in one population, the luminal er- cells .
primary mouse mammary epithelial cells were transduced in three independent experiments with either a control lentivirus carrying gfp only or a virus carrying the sox <dig> gene plus gfp.
the transduced cells were then split between in vitro and in vivo assays.
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 sox <dig>  krt <dig>  krt <dig> and esr <dig> was examined by qpcr and compared to expression levels in cultured primary cells which had not been transduced with virus.
the data  demonstrated that sox <dig> over-expression  did not significantly alter krt <dig> gene expression.
however, krt <dig> gene expression was significantly increased in sox <dig> over-expressing cells compared to non-transduced cells or cells transduced with the control virus .
esr <dig> expression levels were also increased in sox <dig> over-expressing cells, although more modestly   <cit> .
next, cultured virus-transduced primary cells were stained for keratin  <dig>  and keratin  <dig>  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  <dig> hours in culture, these luminal origin cells  begin to promiscuously express k <dig> and acquire a k14+ k18+ phenotype  <cit> .
it is thought that this represents a de-differentiation event resulting from the cells being removed from their normal environment  <cit> .
unsurprisingly, therefore, when non-transduced  and control transduced primary cells were stained for k <dig> and k <dig> expression, they were found to be both k14+ and k18+.
however, sox <dig> transduced cells were k18+ but showed only weak k <dig> staining in occasional cells  .
therefore, sox <dig> maintained mammary epithelial cells in the luminal epithelial lineage and blocked promiscuous k <dig> protein expression in vitro.
however, as sox <dig> over-expression did not block krt <dig> gene expression, this cannot be a direct effect on krt <dig> transcription.
for in vivo analysis, cells transduced with either gfp-only or sox6-gfp virus were transplanted into cleared mouse mammary fat pads.
after eight weeks, the transplanted fat pads were examined .
in nine out of eighteen fat pads transplanted in three independent experiments with cells transduced with the gfp-only virus, extensive mammary epithelial outgrowths were seen in which both ducts and alveolar buds were gfp labelled.
however, no outgrowths were observed in twenty fat pads transplanted in three independent experiments with cells carrying the sox6-gfp, although in two cases, cyst-like gfp-labelled structures were observed.
it was not possible to determine at the wholemount level whether fat pads which did not have extensive gfp+ outgrowths contained scattered gfp-labelled cells incorporated into non-gfp epithelium.
therefore, transplanted fat pads were processed to single cells, labelled with anti-sca- <dig> and anti-cd <dig> antibodies and analysed by flow cytometry to identify gfp+ cells and determine which of the mammary epithelial cell populations they segregated with .
transplanted fat pads from two experiments were pooled for this analysis whilst fat pads from the third transplant experiment were analysed independently.
as expected, gfp+ cells could be detected in the preparations derived from these control fat pads .
these control gfp+ cells mainly segregated with the cd24+/high sca-1- luminal er- population, although they could also be found in the cd24+/low sca-1- basal/myoepithelial and cd24+/high sca-1+ luminal er+ cells .
however, gfp+ cells could also be detected in preparations of cells from sox <dig> gfp virus transplants .
although only low numbers of gfp+ cells were present, their distribution was shifted towards the cd24+/high sca-1+ luminal er+ population .
discussion
we have described a comprehensive transcriptome analysis of three distinct mammary epithelial cell populations, basal/myoepithelial cells, estrogen receptor negative luminal epithelial cells and estrogen receptor positive luminal epithelial cells  <cit> .
these data provide new support for the distinct identities of these three populations and, in particular, justify distinguishing between two major subpopulations within the luminal epithelium.
we have termed these luminal er- and luminal er+ cells as it is expression of the estrogen receptor which makes them most easily distinguishable in tissue sections .
however, each of them expresses, in addition, a large number of unique genes which must be of relevance to their in vivo functions.
it is also clear that there are genes in common to the two luminal populations but which are not expressed by the myoepithelial cells.
expression of these genes, for cytoskeletal proteins  or tight junction components , for instance, must reflect common aspects of luminal epithelial cell function.
most obviously, they are for proteins important in maintaining the structure of the luminal cell layer and the integrity of the lumen itself.
comparison of our data set with other published data sets for separated myoepithelial and luminal cells  <cit>  showed good concordance between genes previously identified as enriched in mouse mammary myoepithelial and luminal cells  <cit> .
however, there was less agreement with genes previously identified as enriched in human myoepithelial and luminal cells.
a number of factors are likely to contribute to these differences.
clearly, species differences could be important.
for instance, it is known that while k <dig> is a basal/myoepithelial cell marker and k <dig> a luminal epithelial cell marker throughout the mouse mammary epithelium and in the ducts of the human breast, in the terminal ductal lobuloalveolar units of the human breast, k <dig> can be expressed by the luminal epithelial cells  <cit> .
furthermore, technical differences could influence the outcome of the analyses.
in particular, it should be noted that stingl and colleagues used the same affymetrix platform as ourselves for their mouse analysis  <cit> .
finally, comparing three distinct populations against each other, rather than just two, improves the contrasts between the populations and enables more population specific genes to be detected.
for example, a gene which is present in the basal/myoepithelial cells and one of the luminal populations, but not the other, may not be detected as being significantly differentially expressed when only myoepithelial and total luminal cells are compared.
however, when all three populations are compared against each other, the contrast with the luminal population from which the gene is absent enables the differential expression of the gene to be detected.
a novel functional cell type in the mammary epithelium
the nature of the luminal er- population as a discrete entity has been confirmed by its uniform staining profile with the 33a <dig> antibody  <cit> .
however, this population appeared to contain within it further distinct functional cell types.
use of network interaction mapping on the transcriptomic profiles of the luminal er- cells identified a toll-like receptor  signalling module including genes for the three components of the bacterial lipopolysaccharide  receptor, tlr <dig>  cd <dig> and ly <dig>  as well as downstream transducers of toll-like receptor signalling such as irak <dig> and the pro-inflammatory cytokine tnf, which is a tlr signalling target  <cit>  .
flow cytometric analysis of the mammary epithelium using anti-cd <dig> and anti-cd <dig>   <cit>  identified a small number of cd61+ cells in the cd24+/low sca-1- basal population and four distinct subpopulations within the luminal er- cells, namely cd61+, cd61+ cd14+, cd14+ and double negative cells.
this raises the possibility of a differentiation hierarchy in which basal stem cells generate basal cd61+ progenitors which then become cd61+ progenitors with luminal characteristics.
these then either lose cd61+ and become the double negative cells  or first acquire cd <dig> expression, becoming cd61+ cd14+ progenitors, and then lose cd <dig> expression to become the cd14+ population.
if this hypothesis is correct, it suggests that the luminal er- population contains two distinct functional cell types, the double negative cells and the cd14+ cells.
the function of the double negative cells remains unclear.
however, given that the expression of all components of the lps receptor can be found in the luminal er- population, it is likely that the cd14+ cells have a distinct function within the mammary epithelium as non-professional immune cells.
note that cd45+ cells were excluded from this analysis, so these are unlikely to be contaminating haematopoietic cells.
milk is an excellent growth medium for bacteria and it would be evolutionarily advantageous to have a cell type present in the breast capable of indicating the presence of bacterial contamination through toll-like receptor signalling pathways.
however, it is also likely that over-stimulation of this pathway in cd14+ mammary epithelial cells would lead to serious inflammation and would therefore be the cause of mastitis.
the presence of the distinct subpopulations within the luminal er- cells indicates that the gene expression profile of this population is derived from a mixture of different cell types.
however, as the luminal er- cells do all share at least one distinctive marker   <cit>  it is likely that the luminal er- profile includes genes whose expression is common to all the cells of this subpopulation, as well 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  <dig> and α-isoform smooth muscle actin and are thus differentiated myoepithelial cells  <cit> .
therefore, the gene expression profile of this population is largely a myoepithelial cell profile.
the luminal er+ population is also unlikely to represent a completely uniform cell population but as the majority of the cells are strongly keratin  <dig> positive and express the estrogen receptor  <cit> , the gene expression profile of this population will be dominated by this single cell type.
myoepithelial cells are key regulators of paracrine signalling
transcriptome and network interaction analysis of the basal/myoepithelial cells identified key processes of these cells as cytoskeletal function and extracellular matrix production and interactions.
given what is known about the contractile function of these cells in lactation and their position in the mammary gland between the luminal epithelium and the basement membrane around the ducts and lobulo-alveolar structures, these results were reassuring.
however, what was remarkable was that the genes characteristic of this population included more than double number of genes for proteins involved in cell – cell signalling than were characteristic of the two luminal populations combined.
this suggests a key role for myoepithelial cells in mediating paracrine and juxtacrine cell – cell interactions in the mammary epithelium.
of particular interest were the expression patterns of wnt and notch signalling pathway components, known to be important in mammary gland development  <cit> , which suggested a directionality of notch signalling from basal to luminal and a directionality of wnt signalling from luminal to basal.
notably, notch signalling was recently shown to be important for determining luminal cell fate  <cit> , possibly through regulation of luminal progenitors  <cit> .
activated wnt signalling has also been shown to increase the stem/progenitor fraction of basal mammary epithelial cells in mmtv-wnt <dig> transgenic mice  <cit> .
another gene from a family important for mammary development that, like notch <dig>  was expressed in both the luminal populations but not the basal/myoepithelial cells was erbb <dig>  a member of the epidermal growth factor receptor family, erbb <dig> most effectively binds the neuregulins nrg <dig> and nrg <dig>  but it has no intrinsic signalling activity of its own.
it must therefore operate as a heterodimer with other family members.
however, signalling complexes containing erbb <dig> have a strong propensity to activate the phosphoinositide-3-kinase  signalling pathway due to the presence of six binding sites for the p <dig> sh <dig> adaptor subunit of pi3k  <cit> .
erbb <dig> knockout animals were embryonic lethal but reduction of erbb <dig> expression in the mammary epithelium caused a reduction in terminal end bud numbers, branching and ductal density  <cit> .
previous reports of erbb <dig> expression have been inconsistent  <cit> , most likely due to variable antibody quality, however, the ductal outgrowth defects that occur when erbb <dig> expression is reduced, together with the observation that implantation of nrg1-soaked pellets induced ductal elongation at puberty  <cit> , support a role for erbb <dig> in pubertal mammary development.
whether or not erbb <dig> activation has the same consequences in both the luminal er+ and luminal er- cells remains to be determined and may depend on differential expression of dimerisation partners not detected by the microarray assays.
mammary epithelial cell subpopulations have distinct transcription factor profiles
mutual interactions between transcription factors associated with different cell lineages and involving positive and negative feedback loops have been demonstrated to be able to maintain haematopoietic cells in a small number of particular cell fates  when an apparently large number of potential intermediate fates are available  <cit> .
transcription factors in the mammary epithelium which have the potential to interact but which are apparently expressed in different populations are therefore of particular interest in the regulation of mammary epithelial cell fate.
we built a gene network to predict such interactions and identified a number of these which are potentially important.
the most obvious is between esr <dig>   <cit> , trp <dig>   <cit>  and myc   <cit>  and given the large number of transcription targets they share, it is likely that these three are key factors in determining cell fate in the mammary gland.
however, there are also other interacting factors of interest such as the runx <dig>   <cit>  – msx <dig>   <cit>  pair, the eya <dig>  – six <dig>  pair  <cit>  and the foxa <dig>   <cit>  – ar   <cit>  – etv <dig>   <cit>  triplet.
modelling these interactions in order to make predictions about how transcription factor behaviour determines mammary cell fate is an important challenge for the future.
sox <dig> is a determinant of luminal cell fate in the mammary gland
in order to model these interactions, functional data on each individual factor will be required.
given the large number of factors of interest, a relatively rapid throughput assay will be required, ruling out the use of transgenic or knockout mice.
therefore, to demonstrate that functional information on the role in determining mammary cell fate of the transcriptional regulators identified in this study can be relatively rapidly generated, and to provide at the same time the first data required for this modelling, we examined the function of sox <dig>  a member of the soxd group of the sry-related, high mobility group box transcription factor family, sox <dig> has two dimerisation domains and the hmg box domain, but no transactivation or transrepression domains  <cit> .
its action as an activator or repressor of transcription depends, therefore, on its binding partners  <cit>  and it has been shown both to repress specification and differentiation of oligodendrocytes during gliagenesis  <cit>  and promote differentiation and maturation of chondrocytes during skeletogenesis  <cit> .
sox <dig> has been shown to be upregulated in the mammary gland by 2-methoxyestradiol treatment  <cit>  but its role in mammary differentiation has not been previously investigated.
in this, study, sox <dig> was specifically expressed in the luminal er- cells and was undetectable in the basal and luminal er+ cells.
over-expression of sox <dig> in vitro caused an increase in krt <dig> luminal marker gene expression and a slight, but significant increase in esr <dig> expression.
it did not change krt <dig> gene expression.
however, staining of the cells with antibodies to either k <dig> or k <dig> showed that while control primary mammary cells in short-term culture promiscuously expressed both k <dig> and k <dig>   <cit> , sox <dig> over-expressing cells were k <dig> positive but k <dig> negative.
therefore, sox <dig> over-expression maintained the mammary epithelial cells in the luminal phenotype and prevented promiscuous k <dig> protein expression.
cleared fat pad transplant of sox <dig> over-expressing primary cells mixed with wild-type cells failed to generate extensive gfp+ outgrowths, suggesting that sox <dig> over-expression may block transplantation activity.
however, rare gfp+ cells could be detected in cells isolated from sox <dig> transplanted fat pads and analysed by flow cytometry.
the phenotype of these rare cells was biased toward the luminal er+ population.
it is unlikely that this was due to transduction of different cell populations by the control and sox <dig> viruses, as viral tropism is determined by envelope proteins and these are coded by the viral packaging plasmids, which were identical for the two viruses.
the small numbers of cells which could be detected and the caveats associated with over-expression studies mean that caution must be exercised in interpreting these data.
however, they are consistent with the in vitro data that sox <dig> is involved in promoting or maintaining a differentiated luminal phenotype, a corollary of which is that it blocks stem cell behaviour .
a more detailed understanding of the function and mechanism of sox <dig> action in the mammary epithelium must await knockdown and inducible over-expression studies.
nevertheless, our data are the first to suggest that sox <dig> has a role in cell fate determination in the mammary epithelium.
CONCLUSIONS
this transcriptome analysis of mammary epithelial cell subpopulations has provided a framework for future studies of normal mammary epithelial cell homeostasis and the molecular pathology of breast disease.
first, it has confirmed the existence of distinct luminal epithelial cell lineages with distinct gene expression patterns.
second, it has identified a novel functional specialisation within the mammary epithelium, that of non-professional immune cell.
third, it has highlighted the complexity of the potential paracrine interactions occurring within the mammary gland.
fourth, it has identified cell-type specific transcriptional regulators with potential roles in mammary epithelial cell lineage specification and fate determination and has shown how these factors are likely to operate in a complex network.
last, it has shown that one of the factors identified, sox <dig>  may be a determinant of luminal cell fate in the mammary epithelium.
future studies will use these data to explore the contribution of the three epithelial cell types to different tumour phenotypes.
they will also focus on the role of the transcription factor network in cell fate choice and cellular homeostasis to model how perturbations in this network may lead to cancer.

 the function of complex tissues, such as the mammary epithelium, is a product of the interactions between their constituent cell types. in such tissues, disease states like cancer are essentially a failure of this cellular homeostasis and are characterised by insensitivity of cells to external regulatory factors and aberrant cell fate choices. understanding the molecular regulation of the individual cell types in complex tissues is, therefore, a prerequisite for understanding disease states. furthermore, advances in molecular pathology have demonstrated that different disease phenotypes correlate with different gene expression profiles. in a complex tissue, composed of different cell types with different molecular characteristics, the gene expression profiles of different diseases may reflect the contribution of different cell types to that disease. therefore, a detailed molecular characterisation of the cell types in a complex tissue is essential for the interpretation of the molecular pathology of its diseases. the resting adult mammary epithelium consists of two main structures, alveoli and ducts. these two structures are themselves composed of two main epithelial cell layers, basal cells and luminal cells. the basal cell layer mainly consists of myoepithelial cells which contract in response to oxytocin release during lactation to force milk down the ducts to the nipple. recent work has demonstrated that the basal cell layer also contains the mammary epithelial stem cell compartment. the luminal cell layer has been shown to be composed of two functionally distinct lineages defined by expression of the cell surface proteins cd and sca- cd24+/high sca-1+ luminal cells express estrogen receptor alpha, as well as receptors for prolactin and progesterone, while cd24+/high sca-1- luminal cells express genes for milk proteins even in the virgin and likely include alveolar progenitors. although it is known that the stem cells can generate all the myoepithelial, luminal er- and luminal er+ daughter cell types, the mechanisms which control cellular homeostasis, fate determination and lineage commitment in the mammary epithelium are poorly understood. they are likely to be a product of complex interactions between cell extrinsic paracrine influences, cell intrinsic transcriptional regulators and epigenomic modifications. some progress has been made towards understanding some of the cell intrinsic factors involved. for instance, gata was recently identified as a transcription factor important in specifying commitment in the general luminal lineage and elf was shown to be a specifier of alveolar cell fate. a number of the cell extrinsic factors operating within the mammary epithelium have also been characterised, such as wnt- which acts downstream of progesterone signalling in ductal side-branching and the egf-family member amphiregulin, which is produced by er+ cells in response to estrogen and stimulates mammary stem cell activity. the notch signalling pathway has also been shown to be an important determinant of luminal cell fate. however, the full extent and nature of paracrine interactions in the mammary gland, and the degree to which the different lineages contribute to them, and are defined by them, is still not fully understood. gene expression patterns have been previously examined in the mouse mammary gland, either as changes in gene expression across the whole tissue in developmental timecourses or as comparisons between the total epithelium and the mammary stroma. in one report, gene expression patterns were examined in mouse luminal and myoepithelial cells purified by flow sorting as well as in stem cell enriched basal cell populations. however, these stem cell gene signatures were found to be not significantly different from the myoepithelial signatures, suggesting they were derived from cell populations dominated by myoepithelial cells. the purity of the basal stem cell populations remains a persistent problem due the difficulty of isolating pure stem cell fractions. a number of gene expression studies have also been carried out on human breast cells. the response of human breast epithelium to estrogen has been analysed at the gene expression level in breast cancer cell lines in vitro and as xenografts, in normal breast tissue maintained as xenografts and in normal human er+ breast cells isolated by transduction of primary breast epithelial cells with a virus carrying an estrogen response element driving gfp expression. the comparative gene expression profiles of normal human myoepithelial, basal non-myoepithelial and luminal epithelial cells have also been examined, as have the gene expression profiles of different in vitro progenitor subpopulations of normal breast epithelial cells. however, to date no genome-wide transcriptome study has made a direct comparison between the two luminal epithelial populations and the basal/myoepithelial cells, confounding the molecular characterisation of the luminal cells and preventing the analysis of the lineage commitment of, and interactions between, the two luminal cell types and the other cell types in the gland. the aim of this study, therefore, was to carry out the first comprehensive gene expression study which examined gene expression patterns in the three distinct mouse mammary epithelial populations, basal/myoepithelial, luminal er- and luminal er+. the analysis was to concentrate on three specific areas. first, characterising cell-type specific patterns of gene expression which defined cell identity. second, establishing a broad overview of the likely extent and nature of paracrine interactions amongst the populations. last, defining cell intrinsic factors which may be important in determining lineage commitment and cell fate in the mammary epithelium. the results of these analyses have, first, identified a novel potential function for a subpopulation of mammary luminal epithelial cells as non-professional immune cells; second, provided information on the large number of paracrine interactions yet to be fully characterised in the mammary gland and the likely complexity of their interactions; third, identified population-specific transcription factors which may have a role in lineage determination and fate specification in the mammary epithelium. in particular, we have used in vitro and in vivo functional analyses to demonstrate that sox is a determinant of luminal cell fate.  identification of population-specific gene expression patterns in the virgin mouse mammary epithelium to carry out a comprehensive, whole genome gene expression analysis of the epithelial cell populations in the virgin mammary gland, primary mouse mammary cells were isolated and stained with anti-cd and anti-sca- antibodies. the cd24+/low sca-1-, cd24+/high sca-1- and cd24+/high sca-1+ cells were separated by flow cytometry and used to prepare rna. to demonstrate that these populations corresponded to basal/myoepithelial, luminal er- and luminal er+ cells respectively, as we have previously described, relative gene expression levels of the basal cell marker keratin, the luminal cell marker keratin and estrogen receptor α were measured by quantitative real-time rtpcr. the results showed that the cd24+/low sca-1- cells expressed krt but not krt and that the cd24+/high sca-1- and cd24+/high sca-1+ cells expressed krt but not krt they also showed that the cd24+/high sca-1+ population was enriched for esr expression compared to the other two populations. this agreed with previous data and with staining of sections through the mouse mammary gland which showed that the basal cells were keratin 14+, the luminal cells were k18+ and that er+ cells were found exclusively in the k18+ luminal cell layer. therefore, these data confirmed the identity of the three populations. next, a whole transcriptome gene expression analysis of the three populations was carried out using the affymetrix platform. to identify genes whose expression in one population was different from its expression in the other two, significance analysis of microarray was applied. using this technique in a multiclass setting, the average gene expression within one group was compared with the average gene expression of all samples. following repeated permutation of the data, the strength of the relationship between gene expression and its expressed group was established and a significance level determined. when a fdr cutoff of % was applied, probe sets were identified which showed significant differential expression across the three populations. these probes corresponded to well known individual genes. heat mapping and hierarchical cluster analysis of the genes identified different gene clusters. the cluster analysis showed that some gene sets were characteristic of only one population whereas others were expressed in two of the three. 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 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 and genes as characteristic of basal/myoepithelial cells, luminal er- cells and luminal er+ cells, respectively. 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 krt krt and esr expression in the populations, giving genes in total. the qpcr probes for genes failed to amplify any product in any of the populations. for a further 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 genes which showed a differential expression pattern in both the array and qpcr analyses, genes showed identical expression patterns across the three populations in both sets of data. with only genes was the pattern of differential expression suggested by the array data different to that suggested by the qpcr analysis. 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. comparisons with previously published datasets we compared our data with previous studies which have used a cell separation approach to isolate mouse or human mammary epithelial basal/myoepithelial and luminal subpopulations for gene expression profiling, to identify genes found in common between these datasets and our own data. there was good concordance between the genes identified in our data and that of stingl and colleagues in their study of separated mouse epithelium. they identified probes, corresponding to well-annotated genes, significantly upregulated in their myoepithelial/mammary stem cell enriched cell fraction compared to their mammary colony forming cells. of these genes, were significantly enriched in our basal/myoepithelial cell dataset, three were enriched in both the basal/myoepithelial cells and the luminal er+ cells and two were enriched in both the basal/myoepithelial cells and the luminal er- cells. conversely, stingl and colleagues identified probes, corresponding to well-annotated genes, significantly downregulated in their myoepithelial/mammary stem cell enriched cell fraction compared to their mammary colony forming cells. of these, which would correspond to genes enriched in the luminal epithelial fraction, none were enriched in our basal/myoepithelial population but were enriched in both our luminal populations, five were enriched only in the luminal er- cells and four only in the luminal er+ cells. however, correspondence of our data with previously published datasets from separated human cells was lower, although it tended to be better between the mouse myoepithelial and human myoepithelial data sets than between the luminal data sets from the different species. interaction mapping of genes differentially expressed in mammary epithelial subpopulations identifies key processes and novel functions to get a better understanding of the key biological processes occurring in each of the three cell types, we generated network interaction maps for the differentially expressed genes. interaction data derived from studies on human orthologues of the genes identified were used to create the network maps, as there is not enough data currently available purely from studies of mouse genes to make such an analysis meaningful. when a basal/myoepithelial interaction map was constructed using both the differentially expressed genes and genes interpolated by the network mapping program, the resulting network was extraordinarily complex. for ease of interpretation, therefore, a basal/myoepithelial network was constructed using only direct interactions between genes characteristic of this cell population, with no interpolations. this network identified two major interaction'modules' and three minor ones. such interaction modules are indicative of important processes occurring within a cell, as they are composed of cell-type specific genes and defined by multiple interactions between those genes. the two major interaction modules can be broadly characterised as an extracellular matrix module including multiple collagen genes and a cytoskeletal module including genes for the keratins, vimentin, and genes whose protein products are involved in regulation of cell shape, movement and contractility, such as the actin binding proteins myh and tpm and smooth muscle gamma actin actg. the minor modules indicated that the basal/myoepithelial cell population also has important processes based around platelet-derived growth factor, ephrin and insulin-like growth factor signalling. ephrins are mediators of contact-dependent communication between cells whereas both pdgf and igf signalling are involved in paracrine cell-cell communication. the luminal interaction maps were built using both cell-specific genes and interpolated genes. as a result, it was less straightforward to define cell-specific interaction modules which would indicate the key cellular processes occurring in these cell types. we therefore developed a mathematical approach to defining the modules which required the assignment of network hubs, node clusters and contiguous differentially expressed network paths. to define network hubs, nodes were ranked by descending connectivity. the minimum node connectivity in the top 10% of nodes was five for either network and this was therefore set as a threshold for identifying hubs. differentially expressed hubs for the luminal er- and er+ networks are listed in tables and respectively. there was limited overlap between the luminal er- and er+ network hubs with only three differentially expressed hubs being shared. the majority of hubs in both networks had a high content of physical interactions with the exception of tnf, sp fas, nfkb creb egr and spi which are almost exclusively transcriptional hubs. in the luminal er- network tlr ly erbb muc and cd were differentially expressed hubs with significant clustering character, although the strongest clustering was seen for the non-differentially expressed hubs egfr and erbb clustering was less pronounced in the luminal er+ network with pgr, erbb fgg, fgb, cd and col8a forming significantly clustered differentially expressed hubs. with the exception of tlr and ly in the luminal er- network, clustering and connectivity were inversely correlated, with high connectivity nodes such as esr, ptn, ccl tnf and bcl exhibiting very little or no clustering. *differentially-expressed genes. knode, node connectivity, cnode, node clustering coefficient, calculated as cnode = nnode/) where nnode is the number of interactions between the hub first neighbours. hubs exhibiting clustering are shown in bold. high content of physical or transcriptional interactions in the hub is shown in bold. see legend to table for details. *differentially expressed genes. to identify modules within the networks, a three-pass approach was adopted. initially all differentially expressed hubs were identified and, where direct links existed between them in the network, these were used to provide the backbone for each module. in the second pass, modules were allowed to expand by the addition of differentially expressed nodes with lower connectivity, if they were directly linked to differentially expressed hubs. in the third pass, non-differentially expressed hubs were incorporated, if they were connecting at least two differentially expressed hubs. this step allowed for bridging between modules, providing further information about their proximity and global topology within the luminal er- and luminal er+ networks. following the initial two passes, four distinct modules were established for the luminal er- network: the tlr, kit, krt and bcl modules. nodes tnf, fas, ccl ccl and rps6ka were integrated in the bcl subnet only at the third pass through a network of transcriptional interactions, forming the tnf/fas module. in addition, the krt module merged into the kit module at this stage. the tlr and kit modules contained predominantly physical interactions whereas the tnf/fas module displayed a very strong transcriptional character. the three modules exhibit topological proximity, defining a single subnetwork with connections and nodes. compared to the luminal er- network overall, this subnetwork showed higher clustering and, whilst maintaining the average network connectivity, it exhibited a significantly shorter mean shortest path and a very low power exponent. removing this subnetwork from the overall luminal er- network yielded a highly fragmented graph with no clustering and very low connectivity. for the luminal er+ network, six distinct modules were observed following the initial two passes: the esr, erbb, mllt, col8a, krt and fgg modules. after the third pass, modules esr, erbb krt, col8a and mllt were merged into a large subnetwork with differentially expressed hubs tgfb areg, mbp, cav eps and pcdha playing an interconnecting role. unlike the luminal er- network, where all modules were proximal to each other, more fragmentation was observed for the luminal er+ network, with module fgg and differentially expressed hubs hist1h4h, hist1h4i and lnx not interconnected with the module subnetwork via any other hubs. the luminal er+ subnetwork also had a much stronger physical interaction character than the luminal er- subnetwork. compared to the overall luminal er+ network, its subnetwork showed high clustering and, whilst maintaining the average network connectivity, exhibited a significantly shorter mean shortest path. as with the luminal er- subnetwork, removing the luminal er+ subnetwork from the overall luminal er+ network gave a highly fragmented graph with no clustering and very low connectivity. the luminal er- and luminal er+ modules, their component nodes and potential functions are summarised in table they suggest that the major processes occurring in the luminal er- cells are tyrosine kinase cell signalling pathways in association with cellular cytoskeletal components as well as passive immune signalling and inflammatory response processes. the picture in the luminal er+ cells is less straightforward. there is no obvious unifying theme to the major esr module, which leaves five minor modules involved in signal transduction, the cytoskeleton, cell adhesion and maintaining the extracellular matrix. the tlr module within the luminal er- network was of particular interest, as it indicated a novel role of these cells as non-professional immune cells which can respond to bacterial lipopolysaccharide. to confirm that the luminal er- population was indeed enriched for cells expressing components of the cd14-tlr signalling complex, qpcr analysis of relative expression levels of the three components of the lps receptor, cd ly and tlr its downstream effecter irak and the lps receptor responsive gene tnf were carried out. the results confirmed that all five genes were specifically enriched in the luminal er- cells compared to both the basal/myoepithelial and luminal er+ cells. in particular, cd tlr and tnf were expressed at 10-fold, 3-fold and 2-fold, respectively, higher levels in the luminal er- cells compared to the luminal er+ cells. cd and tnf were expressed at 30-fold and 5-fold higher levels in the luminal er- cells compared to the basal/myoepithelial cells. tlr expression was undetectable in the basal/myoepithelial cells. to determine whether cd expression, and thus the potential to be able to respond to lps, was a general property of all luminal er- cells or only a subfraction, freshly isolated primary cells were stained with anti-cd and anti-sca- antibodies, to enable the three main cell compartments to be identified, as well as with anti-cd and anti-cd antibodies. the results showed that the cd24+/high sca-1- luminal er- population is itself composed of four different subpopulations, namely cd14- cd61- cells, cd14+ cells, cd61+ cells and cd14+ cd61+ cells. interestingly, a small number of cd24+/low sca1- basal cells also showed elevated levels of cd expression. the luminal er+ gene expression pattern is not an'estrogen-responsive' gene expression signature upon examination of the luminal er+ gene network, we noted that few of the genes enriched in the luminal er+ population were directly linked to esr by transcriptional interactions, with the exception of the progesterone receptor and the cytoskeletal protein keratin. this suggested that the gene signature of the luminal er+ cells was not an'estrogen responsive' gene signature. rather, it was more likely to represent an underlying stable gene expression pattern characteristic of this differentiated cell population. to investigate this further, we compared lists of estrogen responsive genes reported in studies of estrogen-stimulated normal human mammary epithelial cells and breast cancer cell lines with our lists of genes expressed in the epithelial subpopulations. the results confirmed that there was little correlation between'estrogen-responsive' signatures and the genes enriched in the luminal er+ population. indeed, many of the gene whose expression was stimulated by estrogen in the breast cancer cell lines were found to be enriched in our basal/myoepithelial population. however, it should be noted that the well-known directly estrogen responsive genes krt and pgr, were not found to be upregulated in most of the published datasets. identification of basal/myoepithelial cells as key mediators of paracrine signalling having identified key processes likely to be occurring within each of the three populations, we next investigated how the populations might be interacting with each other. mammary epithelial biology is characterised by the conversion of systemic hormone signals into local growth factor signals which stimulate stem cell proliferation and differentiation of daughter cell types. whilst some of these paracrine interactions have been studied in depth, the broad extent of paracrine networks within the mammary epithelium remains unknown. we therefore queried the gene expression array data for genes potentially involved in paracrine signalling, either as receptors or ligands. a number of genes were identified which fulfilled these criteria. remarkably, they showed that the basal/myoepithelial cells have more than double the complement of cell-surface receptors and ligands than either of the two luminal populations. of particular interest, the basal/myoepithelial cells expressed the genes for the notch family ligands, jag jag and dll whilst the gene for the notch family receptor notch was expressed in both the luminal populations. wnt family ligands were expressed by all three populations, although each cell type expressed a different complement of wnt genes and only the basal/myoepithelial cells expressed the genes for the frizzled receptors. it is well established that egf receptor family members and egf family ligands are important for mammary gland development and breast cancer. in particular, the paracrine role of amphiregulin is well described. our analysis confirmed that luminal er+ mammary epithelial cells expressed the areg gene but also showed that the genes for two other family members, betacellulin and epigen, were expressed in this cell type and btc was also expressed in the luminal er- cells. interestingly, only one egf receptor family member, erbb, was found by gene expression array and qpcr analysis to be differentially expressed in the normal mammary epithelium. it was present in both the luminal epithelial populations but not in the basal/myoepithelial cells. this analysis extends previous findings of paracrine signalling within the mammary epithelium and emphasises the complexity of the interacting signalling networks. these include wnt and notch signalling, the egf family, fgf signalling, other receptor tyrosine kinases, g-protein coupled receptors, ligands for such receptors, integrins and ephrins/eph receptors all of which are differentially expressed between the cell populations. in particular, the numbers of ligands and receptors expressed by the basal/myoepithelial cells indicates that this population is a key mediator of the paracrine signalling networks within the mammary epithelium. identification of differentially regulated transcription factors within the mammary epithelium transcriptional regulators are key cell-intrinsic factors in lineage selection and cell fate decisions in stem – differentiated cell hierarchies. therefore, the gene expression array data set was interrogated to identify differentially expressed factors which may regulate transcription. the expression of a subset of these was analysed by qpcr. as with the potential paracrine interactions, the basal/myoepithelial cells differentially expressed many more transcriptional regulators than the other two populations. the er- luminal cells differentially expressed twenty-one transcription factors but only seven transcription factors were differentially expressed in the luminal er+ cells. three transcription factors were found to be expressed in both the basal/myoepithelial and luminal er- populations, one was common to both the basal/myoepithelial and luminal er+ cells and two were found in all three populations. in common lineage progenitors, functional mutual repression and auto-stimulation by transcription factors can facilitate bilineage cell fate decisions. once the cell fate decisions have been executed, continued function of different subsets of the factors active in the progenitors are required to maintain the different differentiated cell lineages in stable fates. thus, modelling interactions between lineage-specific transcription factors can elucidate cell fate decision processes occurring in progenitor cells. therefore, to begin to understand how interactions between lineage specific transcription factors may influence cell fate choices in the mammary epithelium, an interaction network was built. transcription factors identified as lineage-specific but for which no interaction data exists were excluded. the resulting network was centred around two main hubs, namely myc and esr suggesting that they are key regulators of mammary cell fate. clearly, a great deal of information exists on their interactions and this would bias any interaction network around these two key nodes. nevertheless, the number of interactions that myc makes with the basal-specific factors in particular suggests that it is a key factor in regulating mammary cell fate determination. the small number of luminal er+ population specific factors and the fact that three of them, esr pgr and foxa, are closely connected also supports a key role for the estrogen receptor and its transcriptional network in regulating luminal er+ cell fate. as described above, however, the luminal er+ transcriptional profile is not, in general, an estrogen responsive profile. therefore, the activity of the luminal er+ specific transcription factors most likely leads to long term stable cell fate changes and not changes which are responsive to short-term fluctuations in estrogen signalling. mutual repression and auto-stimulation loops may be one way to achieve this long term stability. sox is a determinant of luminal cell fate to demonstrate that the lineage specific transcription factors we identified can indeed influence cell fate and differentiation, we chose to investigate in more detail the function of sox, the expression of which was only detectable in one population, the luminal er- cells. primary mouse mammary epithelial cells were transduced in three independent experiments with either a control lentivirus carrying gfp only or a virus carrying the sox gene plus gfp. the transduced cells were then split between in vitro and in vivo assays. 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 sox krt krt and esr was examined by qpcr and compared to expression levels in cultured primary cells which had not been transduced with virus. the data demonstrated that sox over-expression did not significantly alter krt gene expression. however, krt gene expression was significantly increased in sox over-expressing cells compared to non-transduced cells or cells transduced with the control virus. esr expression levels were also increased in sox over-expressing cells, although more modestly. next, cultured virus-transduced primary cells were stained for keratin and keratin 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 hours in culture, these luminal origin cells begin to promiscuously express k and acquire a k14+ k18+ phenotype. it is thought that this represents a de-differentiation event resulting from the cells being removed from their normal environment. unsurprisingly, therefore, when non-transduced and control transduced primary cells were stained for k and k expression, they were found to be both k14+ and k18+. however, sox transduced cells were k18+ but showed only weak k staining in occasional cells. therefore, sox maintained mammary epithelial cells in the luminal epithelial lineage and blocked promiscuous k protein expression in vitro. however, as sox over-expression did not block krt gene expression, this cannot be a direct effect on krt transcription. for in vivo analysis, cells transduced with either gfp-only or sox6-gfp virus were transplanted into cleared mouse mammary fat pads. after eight weeks, the transplanted fat pads were examined. in nine out of eighteen fat pads transplanted in three independent experiments with cells transduced with the gfp-only virus, extensive mammary epithelial outgrowths were seen in which both ducts and alveolar buds were gfp labelled. however, no outgrowths were observed in twenty fat pads transplanted in three independent experiments with cells carrying the sox6-gfp, although in two cases, cyst-like gfp-labelled structures were observed. it was not possible to determine at the wholemount level whether fat pads which did not have extensive gfp+ outgrowths contained scattered gfp-labelled cells incorporated into non-gfp epithelium. therefore, transplanted fat pads were processed to single cells, labelled with anti-sca- and anti-cd antibodies and analysed by flow cytometry to identify gfp+ cells and determine which of the mammary epithelial cell populations they segregated with. transplanted fat pads from two experiments were pooled for this analysis whilst fat pads from the third transplant experiment were analysed independently. as expected, gfp+ cells could be detected in the preparations derived from these control fat pads. these control gfp+ cells mainly segregated with the cd24+/high sca-1- luminal er- population, although they could also be found in the cd24+/low sca-1- basal/myoepithelial and cd24+/high sca-1+ luminal er+ cells. however, gfp+ cells could also be detected in preparations of cells from sox gfp virus transplants. although only low numbers of gfp+ cells were present, their distribution was shifted towards the cd24+/high sca-1+ luminal er+ population. discussion we have described a comprehensive transcriptome analysis of three distinct mammary epithelial cell populations, basal/myoepithelial cells, estrogen receptor negative luminal epithelial cells and estrogen receptor positive luminal epithelial cells. these data provide new support for the distinct identities of these three populations and, in particular, justify distinguishing between two major subpopulations within the luminal epithelium. we have termed these luminal er- and luminal er+ cells as it is expression of the estrogen receptor which makes them most easily distinguishable in tissue sections. however, each of them expresses, in addition, a large number of unique genes which must be of relevance to their in vivo functions. it is also clear that there are genes in common to the two luminal populations but which are not expressed by the myoepithelial cells. expression of these genes, for cytoskeletal proteins or tight junction components, for instance, must reflect common aspects of luminal epithelial cell function. most obviously, they are for proteins important in maintaining the structure of the luminal cell layer and the integrity of the lumen itself. comparison of our data set with other published data sets for separated myoepithelial and luminal cells showed good concordance between genes previously identified as enriched in mouse mammary myoepithelial and luminal cells. however, there was less agreement with genes previously identified as enriched in human myoepithelial and luminal cells. a number of factors are likely to contribute to these differences. clearly, species differences could be important. for instance, it is known that while k is a basal/myoepithelial cell marker and k a luminal epithelial cell marker throughout the mouse mammary epithelium and in the ducts of the human breast, in the terminal ductal lobuloalveolar units of the human breast, k can be expressed by the luminal epithelial cells. furthermore, technical differences could influence the outcome of the analyses. in particular, it should be noted that stingl and colleagues used the same affymetrix platform as ourselves for their mouse analysis. finally, comparing three distinct populations against each other, rather than just two, improves the contrasts between the populations and enables more population specific genes to be detected. for example, a gene which is present in the basal/myoepithelial cells and one of the luminal populations, but not the other, may not be detected as being significantly differentially expressed when only myoepithelial and total luminal cells are compared. however, when all three populations are compared against each other, the contrast with the luminal population from which the gene is absent enables the differential expression of the gene to be detected. a novel functional cell type in the mammary epithelium the nature of the luminal er- population as a discrete entity has been confirmed by its uniform staining profile with the 33a antibody. however, this population appeared to contain within it further distinct functional cell types. use of network interaction mapping on the transcriptomic profiles of the luminal er- cells identified a toll-like receptor signalling module including genes for the three components of the bacterial lipopolysaccharide receptor, tlr cd and ly as well as downstream transducers of toll-like receptor signalling such as irak and the pro-inflammatory cytokine tnf, which is a tlr signalling target. flow cytometric analysis of the mammary epithelium using anti-cd and anti-cd identified a small number of cd61+ cells in the cd24+/low sca-1- basal population and four distinct subpopulations within the luminal er- cells, namely cd61+, cd61+ cd14+, cd14+ and double negative cells. this raises the possibility of a differentiation hierarchy in which basal stem cells generate basal cd61+ progenitors which then become cd61+ progenitors with luminal characteristics. these then either lose cd61+ and become the double negative cells or first acquire cd expression, becoming cd61+ cd14+ progenitors, and then lose cd expression to become the cd14+ population. if this hypothesis is correct, it suggests that the luminal er- population contains two distinct functional cell types, the double negative cells and the cd14+ cells. the function of the double negative cells remains unclear. however, given that the expression of all components of the lps receptor can be found in the luminal er- population, it is likely that the cd14+ cells have a distinct function within the mammary epithelium as non-professional immune cells. note that cd45+ cells were excluded from this analysis, so these are unlikely to be contaminating haematopoietic cells. milk is an excellent growth medium for bacteria and it would be evolutionarily advantageous to have a cell type present in the breast capable of indicating the presence of bacterial contamination through toll-like receptor signalling pathways. however, it is also likely that over-stimulation of this pathway in cd14+ mammary epithelial cells would lead to serious inflammation and would therefore be the cause of mastitis. the presence of the distinct subpopulations within the luminal er- cells indicates that the gene expression profile of this population is derived from a mixture of different cell types. however, as the luminal er- cells do all share at least one distinctive marker it is likely that the luminal er- profile includes genes whose expression is common to all the cells of this subpopulation, as well 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 and α-isoform smooth muscle actin and are thus differentiated myoepithelial cells. therefore, the gene expression profile of this population is largely a myoepithelial cell profile. the luminal er+ population is also unlikely to represent a completely uniform cell population but as the majority of the cells are strongly keratin positive and express the estrogen receptor, the gene expression profile of this population will be dominated by this single cell type. myoepithelial cells are key regulators of paracrine signalling transcriptome and network interaction analysis of the basal/myoepithelial cells identified key processes of these cells as cytoskeletal function and extracellular matrix production and interactions. given what is known about the contractile function of these cells in lactation and their position in the mammary gland between the luminal epithelium and the basement membrane around the ducts and lobulo-alveolar structures, these results were reassuring. however, what was remarkable was that the genes characteristic of this population included more than double number of genes for proteins involved in cell – cell signalling than were characteristic of the two luminal populations combined. this suggests a key role for myoepithelial cells in mediating paracrine and juxtacrine cell – cell interactions in the mammary epithelium. of particular interest were the expression patterns of wnt and notch signalling pathway components, known to be important in mammary gland development, which suggested a directionality of notch signalling from basal to luminal and a directionality of wnt signalling from luminal to basal. notably, notch signalling was recently shown to be important for determining luminal cell fate, possibly through regulation of luminal progenitors. activated wnt signalling has also been shown to increase the stem/progenitor fraction of basal mammary epithelial cells in mmtv-wnt transgenic mice. another gene from a family important for mammary development that, like notch was expressed in both the luminal populations but not the basal/myoepithelial cells was erbb a member of the epidermal growth factor receptor family, erbb most effectively binds the neuregulins nrg and nrg but it has no intrinsic signalling activity of its own. it must therefore operate as a heterodimer with other family members. however, signalling complexes containing erbb have a strong propensity to activate the phosphoinositide-3-kinase signalling pathway due to the presence of six binding sites for the p sh adaptor subunit of pi3k. erbb knockout animals were embryonic lethal but reduction of erbb expression in the mammary epithelium caused a reduction in terminal end bud numbers, branching and ductal density. previous reports of erbb expression have been inconsistent, most likely due to variable antibody quality, however, the ductal outgrowth defects that occur when erbb expression is reduced, together with the observation that implantation of nrg1-soaked pellets induced ductal elongation at puberty, support a role for erbb in pubertal mammary development. whether or not erbb activation has the same consequences in both the luminal er+ and luminal er- cells remains to be determined and may depend on differential expression of dimerisation partners not detected by the microarray assays. mammary epithelial cell subpopulations have distinct transcription factor profiles mutual interactions between transcription factors associated with different cell lineages and involving positive and negative feedback loops have been demonstrated to be able to maintain haematopoietic cells in a small number of particular cell fates when an apparently large number of potential intermediate fates are available. transcription factors in the mammary epithelium which have the potential to interact but which are apparently expressed in different populations are therefore of particular interest in the regulation of mammary epithelial cell fate. we built a gene network to predict such interactions and identified a number of these which are potentially important. the most obvious is between esr, trp and myc and given the large number of transcription targets they share, it is likely that these three are key factors in determining cell fate in the mammary gland. however, there are also other interacting factors of interest such as the runx – msx pair, the eya – six pair and the foxa – ar – etv triplet. modelling these interactions in order to make predictions about how transcription factor behaviour determines mammary cell fate is an important challenge for the future. sox is a determinant of luminal cell fate in the mammary gland in order to model these interactions, functional data on each individual factor will be required. given the large number of factors of interest, a relatively rapid throughput assay will be required, ruling out the use of transgenic or knockout mice. therefore, to demonstrate that functional information on the role in determining mammary cell fate of the transcriptional regulators identified in this study can be relatively rapidly generated, and to provide at the same time the first data required for this modelling, we examined the function of sox a member of the soxd group of the sry-related, high mobility group box transcription factor family, sox has two dimerisation domains and the hmg box domain, but no transactivation or transrepression domains. its action as an activator or repressor of transcription depends, therefore, on its binding partners and it has been shown both to repress specification and differentiation of oligodendrocytes during gliagenesis and promote differentiation and maturation of chondrocytes during skeletogenesis. sox has been shown to be upregulated in the mammary gland by 2-methoxyestradiol treatment but its role in mammary differentiation has not been previously investigated. in this, study, sox was specifically expressed in the luminal er- cells and was undetectable in the basal and luminal er+ cells. over-expression of sox in vitro caused an increase in krt luminal marker gene expression and a slight, but significant increase in esr expression. it did not change krt gene expression. however, staining of the cells with antibodies to either k or k showed that while control primary mammary cells in short-term culture promiscuously expressed both k and k, sox over-expressing cells were k positive but k negative. therefore, sox over-expression maintained the mammary epithelial cells in the luminal phenotype and prevented promiscuous k protein expression. cleared fat pad transplant of sox over-expressing primary cells mixed with wild-type cells failed to generate extensive gfp+ outgrowths, suggesting that sox over-expression may block transplantation activity. however, rare gfp+ cells could be detected in cells isolated from sox transplanted fat pads and analysed by flow cytometry. the phenotype of these rare cells was biased toward the luminal er+ population. it is unlikely that this was due to transduction of different cell populations by the control and sox viruses, as viral tropism is determined by envelope proteins and these are coded by the viral packaging plasmids, which were identical for the two viruses. the small numbers of cells which could be detected and the caveats associated with over-expression studies mean that caution must be exercised in interpreting these data. however, they are consistent with the in vitro data that sox is involved in promoting or maintaining a differentiated luminal phenotype, a corollary of which is that it blocks stem cell behaviour. a more detailed understanding of the function and mechanism of sox action in the mammary epithelium must await knockdown and inducible over-expression studies. nevertheless, our data are the first to suggest that sox has a role in cell fate determination in the mammary epithelium.  this transcriptome analysis of mammary epithelial cell subpopulations has provided a framework for future studies of normal mammary epithelial cell homeostasis and the molecular pathology of breast disease. first, it has confirmed the existence of distinct luminal epithelial cell lineages with distinct gene expression patterns. second, it has identified a novel functional specialisation within the mammary epithelium, that of non-professional immune cell. third, it has highlighted the complexity of the potential paracrine interactions occurring within the mammary gland. fourth, it has identified cell-type specific transcriptional regulators with potential roles in mammary epithelial cell lineage specification and fate determination and has shown how these factors are likely to operate in a complex network. last, it has shown that one of the factors identified, sox may be a determinant of luminal cell fate in the mammary epithelium. future studies will use these data to explore the contribution of the three epithelial cell types to different tumour phenotypes. they will also focus on the role of the transcription factor network in cell fate choice and cellular homeostasis to model how perturbations in this network may lead to cancer. 
the mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns. 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 and genes as highly differentially expressed in the three cell types, respectively. 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. 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. 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. 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. 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. one of these, sox 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. a number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. however, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out.
 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.  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 and 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, sox 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. 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. 