The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.

Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental -what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells? -to the detailed -what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdUimmunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25-500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 minutes, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the G 2 /M phase protein pHisH3 maximally colocalized 8 hr after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 hr. In addition, triple labeling with BrdU and PCNA and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.

SUMMARYFOXA1, estrogen receptor a (ERa) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERa and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERa expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERa and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERa expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERa-positive breast cancers, at least in part, through its control of ERa expression.

Summary In the field of breast biology, there is a growing appreciation for the “gatekeeping function” of basal cells during development and disease processes; yet, mechanisms regulating the generation of these cells are poorly understood. Here, we report that the proliferation of basal cells is controlled by SLIT/ROBO1 signaling and that production of these cells regulates outgrowth of mammary branches. We identify the negative regulator TGF-β1 upstream of ROBO1 and show that it induces Robo1 expression specifically in the basal layer, functioning together with SLIT2 to restrict branch formation. Loss of SLIT/ROBO1 signaling in this layer, alone, results in precocious branching due to a surplus of basal cells. SLIT2 limits basal cell proliferation by inhibiting canonical WNT signaling, increasing the cytoplasmic and membrane pools of β-catenin at the expense of its nuclear pool. Together, our studies provide mechanistic insight into how specification of basal cell number influences branching morphogenesis.

Once thought to produce global, nonspecific brain injury, drugs of abuse are now known to produce selective neuro-adaptations in particular brain regions. These neuro-adaptations are being closely examined for clues to the development, maintenance, and treatment of addiction. The hippocampus is an area of particular interest, as it is central to many aspects of the addictive process, including relapse to drug taking. A recently appreciated hippocampal neuro-adaptation produced by drugs as diverse as opiates and psychostimulants is decreased neurogenesis in the sub-granular zone (SGZ). While the role of adult-generated neurons is not clear, their functional integration into hippocampal circuitry raises the possibility that decreased adult SGZ neurogenesis may alter hippocampal function in such a way as to maintain addictive behavior or contribute to relapse. Here, we review the impact of opiates and psychostimulants on the different stages of cell development in the adult brain, as well as the different stages of the addictive process. We discuss how examination of drug-induced alterations of adult neurogenesis advances our understanding of the complex mechanisms by which opiates and psychostimulants affect brain function while also opening avenues for novel ways of assessing the functional role of adult-generated neurons. In addition, we highlight key discrepancies in the field and underscore the necessity to move "beyond BrdU"--beyond merely counting new hippocampal cells labeled with the S phase marker bromodeoxyuridine--so as to probe mechanistic questions about how drug-induced alterations in adult hippocampal neurogenesis occur and what the functional ramifications of alterations in neurogenesis are for addiction.

Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (Experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (Experiment 2). In Experiment 1, mice received sham or morphine pellets (subcutaneous, 0 and 48 hrs) and bromodeoxyuridine (BrdU) 2 hrs prior to sacrifice (24, 72 or 96 hrs). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 hrs of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 hrs. In Experiment 2, nestin-GFP mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 hrs, sacrifice 96 hrs) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells were less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were Type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

Recent evidence suggests that mu opioid receptors (MOR) are key regulators of hippocampal structure and function. For example, exogenous MOR agonists morphine and heroin negatively impact hippocampal function and decrease adult hippocampal neurogenesis. Here we explored the role of MOR in the birth and survival of hippocampal progenitor cells by examining adult neurogenesis in mice that lack MOR. Adult male mice lacking exon 1 of MOR were injected with the S phase marker bromodeoxyuridine (BrdU) and sacrificed either two hours or four weeks later to evaluate proliferating and surviving BrdU-immunoreactive (IR) cells, respectively, in the adult hippocampal granule cell layer. WT, heterozygote, and homozygote mice did not differ in the number of BrdU-IR cells at a proliferation time point. However, four weeks after BrdU injection, heterozygote and homozygote mice had 57% and 54% more surviving BrdU-IR cells in the hippocampal granule cell layer as compared to WT mice. A decrease in apoptosis in the heterozygote and homozygote mice did not account for the difference in number of surviving BrdU-IR cells since there were no alterations in number of pyknotic, TUNEL-positive, or activated caspase 3-IR cells compared to WT. In concordance with the increased numbers of granule cells maturing into neurons, heterozygote and homozygote mice had larger hippocampal granule cell layers and increased numbers of granule cells. These findings indicate that MOR may play a role in regulating progenitor cell survival and more generally encourage further exploration of how MOR activation can influence hippocampal structure and function.

Slit, Netrin, Ephrin, and Semaphorin’s roles in development have expanded greatly in the past decade from their original characterization as axon guidance molecules (AGMs) to include roles as regulators of tissue morphogenesis and development in diverse organs. In the mammary gland, AGMs are important for maintaining normal cell proliferation and adhesion during development. The frequent dysregulation of AGM expression during tumorigenesis and tumor progression suggests that AGMs also play a crucial role as tumor suppressors and oncogenes in breast cancer. Moreover, these findings suggest that AGMs may be excellent targets for new breast cancer prognostic tests and more effective therapeutic strategies.