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• W2021558950 abstract "•Aurora A kinase regulates mitotic spindle orientation in the mammary epithelium•Orientation perpendicular to the basement membrane is dependent on Notch activity•Blocking Aurora A-TPX2 interaction or Notch activity promotes parallel orientation•Parallel orientation promotes the basal, and perpendicular the luminal, cell lineages Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA) in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland. Cell fate determination in the progeny of mammary epithelial stem/progenitor cells remains poorly understood. Here, we have examined the role of the mitotic kinase Aurora A (AURKA) in regulating the balance between basal and luminal mammary lineages. We find that AURKA is highly expressed in basal stem cells and, to a lesser extent, in luminal progenitors. Wild-type AURKA expression promoted luminal cell fate, but expression of an S155R mutant reduced proliferation, promoted basal fate, and inhibited serial transplantation. The mechanism involved regulation of mitotic spindle orientation by AURKA and the positioning of daughter cells after division. Remarkably, this was NOTCH dependent, as NOTCH inhibitor blocked the effect of wild-type AURKA expression on spindle orientation and instead mimicked the effect of the S155R mutant. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between luminal and basal lineages in the mammary gland. The mammary epithelium consists of two main lineages: luminal epithelial cells, and basal myoepithelial cells. The former line the ducts and form the milk-secreting cells of the alveoli, whereas the latter are contractile and squeeze milk along the ducts during lactation. The luminal cells are themselves either estrogen receptor α positive (ER+) or negative (ER−). ER+ cells are hormone sensing and transduce systemic hormonal signals into localized control of epithelial function through paracrine interactions. The ER− cells include the milk secretory cells (Regan et al., 2012Regan J.L. Kendrick H. Magnay F.A. Vafaizadeh V. Groner B. Smalley M.J. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer.Oncogene. 2012; 31: 869-883Crossref PubMed Scopus (82) Google Scholar, Richert et al., 2000Richert M.M. Schwertfeger K.L. Ryder J.W. Anderson S.M. An atlas of mouse mammary gland development.J. Mammary Gland Biol. Neoplasia. 2000; 5: 227-241Crossref PubMed Scopus (309) Google Scholar). The myoepithelial, hormone-sensing ER+ and secretory ER− cells are considered to be the main differentiated populations in the mammary epithelium. Stem and progenitor cells have also been identified by functional assays and/or lineage tracing. Functional assays (the ability of stem and progenitor cells to transplant in vivo and proliferate in vitro, respectively) support a model in which stem cells are found in the basal layer, together with the myoepithelial cells, whereas cells with progenitor function are highly enriched in the luminal cell layer (Shackleton et al., 2006Shackleton M. Vaillant F. Simpson K.J. Stingl J. Smyth G.K. Asselin-Labat M.L. Wu L. Lindeman G.J. Visvader J.E. Generation of a functional mammary gland from a single stem cell.Nature. 2006; 439: 84-88Crossref PubMed Scopus (1624) Google Scholar, Sleeman et al., 2007Sleeman K.E. Kendrick H. Robertson D. Isacke C.M. Ashworth A. Smalley M.J. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland.J. Cell Biol. 2007; 176: 19-26Crossref PubMed Scopus (258) Google Scholar, Taddei et al., 2008Taddei I. Deugnier M.A. Faraldo M.M. Petit V. Bouvard D. Medina D. Fässler R. Thiery J.P. Glukhova M.A. Beta1 integrin deletion from the basal compartment of the mammary epithelium affects stem cells.Nat. Cell Biol. 2008; 10: 716-722Crossref PubMed Scopus (194) Google Scholar, Stingl et al., 2006Stingl J. Eirew P. Ricketson I. Shackleton M. Vaillant F. Choi D. Li H.I. Eaves C.J. Purification and unique properties of mammary epithelial stem cells.Nature. 2006; 439: 993-997Crossref PubMed Scopus (1258) Google Scholar). Luminal progenitors are mainly ER−, but a small subfraction of ER+ cells also have progenitor features (Regan et al., 2012Regan J.L. Kendrick H. Magnay F.A. Vafaizadeh V. Groner B. Smalley M.J. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer.Oncogene. 2012; 31: 869-883Crossref PubMed Scopus (82) Google Scholar, Beleut et al., 2010Beleut M. Rajaram R.D. Caikovski M. Ayyanan A. Germano D. Choi Y. Schneider P. Brisken C. Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland.Proc. Natl. Acad. Sci. USA. 2010; 107: 2989-2994Crossref PubMed Scopus (231) Google Scholar). During early mammary development and also when purified stem and progenitor subpopulations are transplanted, the basal stem cells have the potential to generate all the other cell types in the mammary epithelium with high efficiency (Shackleton et al., 2006Shackleton M. Vaillant F. Simpson K.J. Stingl J. Smyth G.K. Asselin-Labat M.L. Wu L. Lindeman G.J. Visvader J.E. Generation of a functional mammary gland from a single stem cell.Nature. 2006; 439: 84-88Crossref PubMed Scopus (1624) Google Scholar, Sleeman et al., 2007Sleeman K.E. Kendrick H. Robertson D. Isacke C.M. Ashworth A. Smalley M.J. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland.J. Cell Biol. 2007; 176: 19-26Crossref PubMed Scopus (258) Google Scholar, Stingl et al., 2006Stingl J. Eirew P. Ricketson I. Shackleton M. Vaillant F. Choi D. Li H.I. Eaves C.J. Purification and unique properties of mammary epithelial stem cells.Nature. 2006; 439: 993-997Crossref PubMed Scopus (1258) Google Scholar, Regan et al., 2012Regan J.L. Kendrick H. Magnay F.A. Vafaizadeh V. Groner B. Smalley M.J. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer.Oncogene. 2012; 31: 869-883Crossref PubMed Scopus (82) Google Scholar). In contrast, in situ lineage analysis of normal adult tissue suggests that in the resting postpubertal gland, the basal and luminal cell layers are maintained as separate lineages (Van Keymeulen et al., 2011Van Keymeulen A. Rocha A.S. Ousset M. Beck B. Bouvencourt G. Rock J. Sharma N. Dekoninck S. Blanpain C. Distinct stem cells contribute to mammary gland development and maintenance.Nature. 2011; 479: 189-193Crossref PubMed Scopus (622) Google Scholar, van Amerongen et al., 2012van Amerongen R. Bowman A.N. Nusse R. Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland.Cell Stem Cell. 2012; 11: 387-400Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). These findings argue that the myoepithelial and luminal lineages are maintained by separate stem/progenitor populations in the adult gland. However, at some adult stages, including alveolargenesis, basal stem cells may contribute to generating the luminal layer (van Amerongen et al., 2012van Amerongen R. Bowman A.N. Nusse R. Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland.Cell Stem Cell. 2012; 11: 387-400Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Furthermore, analysis of cap cells, the outermost cell layer of the specialized growth structure that drives ductal growth during puberty (the terminal end buds or TEBs), has suggested that they can contribute to both the myoepithelial and luminal lineages (Srinivasan et al., 2003Srinivasan K. Strickland P. Valdes A. Shin G.C. Hinck L. Netrin-1/neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis.Dev. Cell. 2003; 4: 371-382Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, Williams and Daniel, 1983Williams J.M. Daniel C.W. Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis.Dev. Biol. 1983; 97: 274-290Crossref PubMed Scopus (356) Google Scholar). Stem cells are defined by their potential to self-renew and to generate a defined set of differentiated progeny (Lechler and Fuchs, 2005Lechler T. Fuchs E. Asymmetric cell divisions promote stratification and differentiation of mammalian skin.Nature. 2005; 437: 275-280Crossref PubMed Scopus (772) Google Scholar). Stem cell homeostasis involves asymmetric divisions, with one daughter cell differentiating and one self-renewing. For stem cell expansion, cell divisions must be symmetric, with both daughters of the dividing cell assuming stem cell fate. The balance between asymmetric and symmetric division is regulated by both intrinsic and extrinsic mechanisms (Horvitz and Herskowitz, 1992Horvitz H.R. Herskowitz I. Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question.Cell. 1992; 68: 237-255Abstract Full Text PDF PubMed Scopus (438) Google Scholar). The former depends on the partitioning of determinants prior to mitosis that promote the stem cell fate, with equal distribution of the determinants between daughter cells leading to stem cell expansion, and unequal distribution to the daughters adopting different fates and stem cell homeostasis (Betschinger and Knoblich, 2004Betschinger J. Knoblich J.A. Dare to be different: asymmetric cell division in Drosophila, C. elegans and vertebrates.Curr. Biol. 2004; 14: R674-R685Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). In contrast, extrinsic mechanisms depend on interactions between stem cells and their daughters and the microenvironment or niche (Li and Xie, 2005Li L. Xie T. Stem cell niche: structure and function.Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (950) Google Scholar). In this case, the position of postdivision daughter cells, in or out of the niche, will determine whether they assume stem cell identity or an alternative fate. Aurora A (AURKA) is a centrosomal and mitotic spindle-associated kinase that coordinates mitotic events, regulates centrosome maturation and bipolar spindle formation, and may be involved in both stem cell fate control mechanisms. In Drosophila, AURKA can establish polarity during asymmetric cell division in a BORA-dependent manner (Hutterer et al., 2006Hutterer A. Berdnik D. Wirtz-Peitz F. Zigman M. Schleiffer A. Knoblich J.A. Mitotic activation of the kinase Aurora-A requires its binding partner Bora.Dev. Cell. 2006; 11: 147-157Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), thus determining the position of daughter cells with respect to the microenvironment. AURKA also regulates the partitioning of NUMB, an important cell fate determinant and negative regulator of NOTCH signaling, within the cytoplasm prior to division (Cayouette and Raff, 2002Cayouette M. Raff M. Asymmetric segregation of Numb: a mechanism for neural specification from Drosophila to mammals.Nat. Neurosci. 2002; 5: 1265-1269Crossref PubMed Scopus (158) Google Scholar). Here, we examine the role of AURKA as a regulator of mammary stem/progenitor cell behavior and cell fate determination. We show that AURKA can regulate the balance between the luminal and basal myoepithelial cell lineages by regulating the orientation of the mitotic spindle and thus the location of the postmitotic daughter cells. This mechanism is directly dependent on NOTCH signaling but is independent of NUMB localization. Rather, it requires activity of the NOTCH signaling pathway itself. These findings directly link AURKA, NOTCH signaling, and mitotic spindle orientation and suggest a mechanism for regulating the balance between mammary cell lineages. To determine the pattern of AurkA expression in stem, progenitor, and differentiated mammary epithelial cells, these subpopulations were freshly isolated by flow cytometry from 10-week-old virgin mice (Figures 1A, 1B, S1, and S2A ) according to previously defined markers (Regan et al., 2012Regan J.L. Kendrick H. Magnay F.A. Vafaizadeh V. Groner B. Smalley M.J. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer.Oncogene. 2012; 31: 869-883Crossref PubMed Scopus (82) Google Scholar) and AurkA gene expression levels determined by quantitative real-time reverse-transcription PCR. AurkA was expressed at significantly higher levels in the stem cells (CD45− CD24+/Low Sca-1− CD49fHigh c-Kit−) and luminal ER− progenitors (CD45− CD24+/High Sca-1− c-Kit+) compared to both the myoepithelial (CD45− CD24+/Low Sca-1− CD49fLow c-Kit−) and differentiated luminal ER+ (CD45− CD24+/High Sca-1+ c-Kit−) populations. Furthermore, expression of AurkA was significantly (p < 0.05) (Cumming et al., 2007Cumming G. Fidler F. Vaux D.L. Error bars in experimental biology.J. Cell Biol. 2007; 177: 7-11Crossref PubMed Scopus (608) Google Scholar) higher in the stem cells compared to the progenitors (Figure 1C). AurkA expression correlated with expression of Cyclin B (a G2/M cyclin) but not Cyclin D1 (a G1 cyclin), consistent with its role in mitosis (Figure 1C).Figure S1Full-Gating Cascade and Controls for Isolation of Mammary Epithelial Cell Subpopulations, Related to Figure 1Show full caption(A) Gating cascade. Samples were initially gated on forward and side scatter (FSC/SSC) to exclude debris, then single cells were isolated using time-of-flight analysis on both FSC and SSC (FSC-H versus FSC-W then SSC-H versus SSC-W). Dead cells were then excluded using DAPI staining. CD45+ leukocytes were next removed and finally the total epithelial cells were gated on a CD24 / Sca-1 plot as previously described (Britt et al., 2009Britt K.L. Kendrick H. Regan J.L. Molyneux G. Magnay F.A. Ashworth A. Smalley M.J. Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells.Breast Cancer Res. 2009; 11: R20Crossref PubMed Scopus (41) Google Scholar). Epithelial subpopulations were isolated from the total epithelial cells as shown in Figure 1.(B) Determination of background staining levels for CD24 / Sca-1, CD24 / CD49f and CD24 / c-Kit plots by comparison of fully stained samples (epithelial cells only) with control unstained samples.View Large Image Figure ViewerDownload (PPT)Figure S2AURKA Expression in Mammary Epithelial Cell Populations, Related to Figure 1Show full caption(A) Post-sort purity analysis of sorted cells from primary mouse mammary epithelial cell preparations in Figure 1. Note that the very small numbers of cells collected from the MaSC population meant that it was not possible to carry out post-sort analysis without losing those samples.(B and C) Representative histogram plots (B) and data from individual experiments (C) of analysis of levels of AURKA positive cells in mammary epithelial cell subpopulations determined by flow cytometry. (B) Gating to determine percentage of AURKA positive cells in MaSCs, myoepithelial cells, luminal ER negative cells and luminal ER positive cells separated using the strategy shown in Figure 1. The threshold for positivity was set independently for each experiment with reference to an unstained control as shown. x axis indicates AURKA staining (arbitrary units). y axis indicates cell counts. Two histograms are given for each population with the cell counts scaled to different levels appropriate to sizes of the least and most numerous populations. (C) Individual percentages of AURKA positivity in the four populations in five independent analyses. The histograms shown in (B) are from experiment 4.View Large Image Figure ViewerDownload (PPT) (A) Gating cascade. Samples were initially gated on forward and side scatter (FSC/SSC) to exclude debris, then single cells were isolated using time-of-flight analysis on both FSC and SSC (FSC-H versus FSC-W then SSC-H versus SSC-W). Dead cells were then excluded using DAPI staining. CD45+ leukocytes were next removed and finally the total epithelial cells were gated on a CD24 / Sca-1 plot as previously described (Britt et al., 2009Britt K.L. Kendrick H. Regan J.L. Molyneux G. Magnay F.A. Ashworth A. Smalley M.J. Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells.Breast Cancer Res. 2009; 11: R20Crossref PubMed Scopus (41) Google Scholar). Epithelial subpopulations were isolated from the total epithelial cells as shown in Figure 1. (B) Determination of background staining levels for CD24 / Sca-1, CD24 / CD49f and CD24 / c-Kit plots by comparison of fully stained samples (epithelial cells only) with control unstained samples. (A) Post-sort purity analysis of sorted cells from primary mouse mammary epithelial cell preparations in Figure 1. Note that the very small numbers of cells collected from the MaSC population meant that it was not possible to carry out post-sort analysis without losing those samples. (B and C) Representative histogram plots (B) and data from individual experiments (C) of analysis of levels of AURKA positive cells in mammary epithelial cell subpopulations determined by flow cytometry. (B) Gating to determine percentage of AURKA positive cells in MaSCs, myoepithelial cells, luminal ER negative cells and luminal ER positive cells separated using the strategy shown in Figure 1. The threshold for positivity was set independently for each experiment with reference to an unstained control as shown. x axis indicates AURKA staining (arbitrary units). y axis indicates cell counts. Two histograms are given for each population with the cell counts scaled to different levels appropriate to sizes of the least and most numerous populations. (C) Individual percentages of AURKA positivity in the four populations in five independent analyses. The histograms shown in (B) are from experiment 4. To test comparative AURKA protein expression in the four populations, fresh samples were isolated by flow cytometry, fixed, stained for AURKA expression, and then analyzed again by flow cytometry in five independent experiments. Percentages of AURKA-positive cells across the experiments varied quite widely; however, in all experiments, the stem cell population contained the highest percentage of AURKA positive cells (Figure S2). To investigate the function of AURKA in mammary epithelial cells in vitro, freshly harvested primary mammary epithelial cells were transduced with lentiviruses driving expression of GFP only (empty vector control; EV), wild-type AURKA plus GFP (WT), or a mutant form of AURKA plus GFP (S155R) and placed in monolayer culture at identical plating densities. S155R cannot bind to its regulator TPX2 nor can it be targeted by PP1 phosphatase. As a result, it forms a constitutively, but weakly, active protein that associates only with the centrosomes of mitotic cells and not the mitotic spindles (Bibby et al., 2009Bibby R.A. Tang C. Faisal A. Drosopoulos K. Lubbe S. Houlston R. Bayliss R. Linardopoulos S. A cancer-associated aurora A mutant is mislocalized and misregulated due to loss of interaction with TPX2.J. Biol. Chem. 2009; 284: 33177-33184Crossref PubMed Scopus (38) Google Scholar). The TPX2-AURKA interaction is required to form mitotic spindles of the correct length (Bird and Hyman, 2008Bird A.W. Hyman A.A. Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A.J. Cell Biol. 2008; 182: 289-300Crossref PubMed Scopus (151) Google Scholar). To confirm that the expressed proteins localized as expected, epithelial cell colonies were fixed after 10 days and stained for AURKA, TPX2, and EG5, which associates with the AURKA-TPX2 complex on mitotic spindles (Ma et al., 2011Ma N. Titus J. Gable A. Ross J.L. Wadsworth P. TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle.J. Cell Biol. 2011; 195: 87-98Crossref PubMed Scopus (69) Google Scholar) (Figure S3). As expected, TPX2 could be seen decorating the mitotic spindles of dividing cells in all cultures. AURKA and EG5 also decorated the mitotic spindles in EV- and WT-transduced cultures. However, in S155R-transduced cells, both AURKA and EG5 were restricted to the centrosomal region, demonstrating that their localization to the mitotic spindle was dependent on the TPX2-AURKA interaction. Next, transduced colonies were fixed and stained for the basal lineage marker keratin 14 (K14) and the luminal lineage marker keratin 18 (K18) (Figure 2A). For each sample, the mean number of GFP+-transduced cells per field of view was determined (Figure 2B). There was no significant difference in the number of GFP+ cells in WT cultures compared to EV-transduced cells (mean ± SD, 83.22 ± 2.75 versus 76.4 ± 8.5 GFP+ cells per field, respectively). However, the mean number of GFP+ S155R cells was significantly lower (46.42 ± 11.83 GFP+ cells per field; p < 0.05 t test versus EV and WT-transduced cells). When primary mouse mammary cells are isolated and grown in short-term culture under standard conditions, the majority of cells that proliferate are derived from the luminal ER− progenitor population (Sleeman et al., 2007Sleeman K.E. Kendrick H. Robertson D. Isacke C.M. Ashworth A. Smalley M.J. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland.J. Cell Biol. 2007; 176: 19-26Crossref PubMed Scopus (258) Google Scholar, Regan et al., 2012Regan J.L. Kendrick H. Magnay F.A. Vafaizadeh V. Groner B. Smalley M.J. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer.Oncogene. 2012; 31: 869-883Crossref PubMed Scopus (82) Google Scholar). In monolayer culture, these luminal cells, which express low levels of K14 in vivo (Regan et al., 2012Regan J.L. Kendrick H. Magnay F.A. Vafaizadeh V. Groner B. Smalley M.J. c-Kit is required for growth and survival of the cells of origin of Brca1-mutation-associated breast cancer.Oncogene. 2012; 31: 869-883Crossref PubMed Scopus (82) Google Scholar), upregulate K14 expression and acquire a K14+ K18+ phenotype (Sleeman et al., 2007Sleeman K.E. Kendrick H. Robertson D. Isacke C.M. Ashworth A. Smalley M.J. Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland.J. Cell Biol. 2007; 176: 19-26Crossref PubMed Scopus (258) Google Scholar). To examine whether AURKA expression affected this promiscuous expression of lineage markers, the number of cells expressing K14 only, K18 only, or coexpressing both K14 and K18 in the lentivirus-transduced cultures was determined (Figure 2C). A total of 95.32% ± 3.79% (mean ± SD) of EV- and 99.15% ± 1.47% of WT-transduced cells stained for both K14 and K18. However, only 68.18% ± 8.23% of S155R-transduced cells were double positive for K14 and K18 (p < 0.05 t test on Log10-transformed data versus EV and WT-transduced cells). The remaining S155R-transduced cells were K14+ only (32.9% ± 10.07%) and had a more flattened, spread appearance than K14+/K18+ cells (Figure 2A, arrowheads). Furthermore, staining of the cultures with two markers of mammary myoepithelial cells, keratin 5 (K5) and α-isoform smooth muscle actin (SMA), demonstrated that whereas S155R-transduced cells could express both K5 and SMA, EV and WT-expressing cells were K5 and SMA negative (Figure S4). Therefore, these data not only demonstrated that S155R AURKA reduced proliferation of mammary epithelial progenitors in vitro but also suggested that it promoted differentiation along the basal myoepithelial lineage. To examine the role of AURKA in mammary epithelial cell fate determination in vivo, freshly harvested primary mouse mammary epithelial cells were transduced with the EV, S155R, or WT lentiviruses and transplanted at equal numbers into cleared mammary fat pads of syngeneic mice. Eight weeks after transplantation, the fat pads were removed and examined (Figures 3A and 3B). GFP-labeled mammary epithelial outgrowths were observed in 10 out of 30 fat pads transplanted with EV or WT-expressing cells and in 9 out of 30 fat pads transplanted with S155R-expressing cells. However, whereas EV and WT outgrowths were extensive and filled ≥50% of the fat pad in six and seven outgrowths, respectively, S155R outgrowths were rudimentary (Figure 3A). Analysis of sections of EV, WT, and S155R outgrowths stained for myoepithelial (K5 and SMA) and luminal (K18 and K19) markers also revealed morphological differences (Figures 3C–3E, S5A, and S5B). EV and WT outgrowths displayed a typical normal mammary epithelial morphology with distinct myoepithelial (K5 and SMA positive) and luminal (K18 and K19 positive) layers. In contrast, the S155R outgrowths typically consisted of only a single layer of myoepithelial cells that expressed little or no luminal keratin.Figure S5Transplantation of AURKA-Transduced Mammary Epithelial Cells, Related to Figure 3Show full caption(A and B) Sections through cleared fat pad outgrowths transduced with Empty Vector (EV), S155R or WT viruses. Sections were stained for K18 (A) or SMA (B) expression and counterstained with DAPI. Note the predominantly single layer of epithelium in the S155R outgrowths. Bar = 40 μm.(C) Whole mounts of fat pads from the first round of transplantation with RFP+ mammary epithelial cells transduced with GFP+ EV (Bar = 4 mm), S155R (Bar = 2 mm) and WT virus (Bar = 8 mm) showing GFP signal, RFP and overlay. Note colocalization of the signals demonstrates that the transplanted outgrowths are derived from the originally transplanted GFP+/RFP+ cells.(D and E) Sections through outgrowths from first round transplantation of mRFP+ mammary epithelial cells transduced with EV (D) or WT (E) virus. Note that the RFP was driven from a Krt14 promoter, hence the mRFP expression is limited to the basal cell layer. Bar = 15 μm.View Large Image Figure ViewerDownload (PPT) (A and B) Sections through cleared fat pad outgrowths transduced with Empty Vector (EV), S155R or WT viruses. Sections were stained for K18 (A) or SMA (B) expression and counterstained with DAPI. Note the predominantly single layer of epithelium in the S155R outgrowths. Bar = 40 μm. (C) Whole mounts of fat pads from the first round of transplantation with RFP+ mammary epithelial cells transduced with GFP+ EV (Bar = 4 mm), S155R (Bar = 2 mm) and WT virus (Bar = 8 mm) showing GFP signal, RFP and overlay. Note colocalization of the signals demonstrates that the transplanted outgrowths are derived from the originally transplanted GFP+/RFP+ cells. (D and E) Sections through outgrowths from first round transplantation of mRFP+ mammary epithelial cells transduced with EV (D) or WT (E) virus. Note that the RFP was driven from a Krt14 promoter, hence the mRFP expression is limited to the basal cell layer. Bar = 15 μm. The limited extent of the outgrowths produced by the S155R-transduced cells suggested that whereas these cells were as transplantable as the control cells, their ability to proliferate and/or properly differentiate was impaired. To further characterize differentiation defects in the outgrowths, cells isolated from transplanted fat pads were analyzed by flow cytometry to determine the proportions of the different mammary epithelial cell types (Figures 3F–3I). GFP+ cells isolated from control fat pads (Figure 3G) had a flow cytometric profile similar to GFP− cells from the same preparations or to transplanted nontransduced epithelial cells (Figure 3F). However, GFP+ cells isolated from S155R fat pads (Figure 3H) were significantly shifted into the CD24+/Low Sca-1− basal myoepithelial population (which formed 9.3% ± 1% of the GFP+ control cells but 40.4% ± 5.4% of the GFP+ S155R cells; mean ± SD, n = three independent transplant experiments; p < 0.0001 t test on Log10-transformed data) (Figure 3J). There was no significant difference between the size of the basal myoepithelial GFP+ populations isolated from WT (Figure 3I) and control fat pads. However, the size of the differentiated luminal CD24+/High Sca-1+ population was significantly decreased in the WT outgrowths compared to EV controls (42.13% ± 1.35% of GFP+ EV cells and 20.33% ± 0.58% of GFP+ WT cells; mean ± SD, n = three independent transplant experiments; p < 0.0001 t test on Log10-transformed data), and the size of the luminal CD24+/High Sca-1− progenitor-enriched population was significantly increased (32.36% ± 4.07% of GFP+ EV cells and 43% ± 4.8% of GFP+ WT cells; mean ± SD, n = three independent transplant experiments; p < 0.05 t test on Log10-transformed data) (Figure 3J). Next, to determine whether AURKA not only regulated the fate of differentiating mammary cells but also self-renewal of mammary stem cells, serial transplants were carried out. Primary mammary epithe" @default.
• W2021558950 created "2016-06-24" @default.
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• W2021558950 date "2013-07-01" @default.
• W2021558950 modified "2023-10-17" @default.
• W2021558950 title "Aurora A Kinase Regulates Mammary Epithelial Cell Fate by Determining Mitotic Spindle Orientation in a Notch-Dependent Manner" @default.
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