Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland.

Recent lineage tracing studies have revealed that mammary gland homeostasis relies on unipotent stem cells. However, whether and when lineage restriction occurs during embryonic mammary development, and which signals orchestrate cell fate specification, remain unknown. Using a combination of in vivo clonal analysis with whole mount immunofluorescence and mathematical modelling of clonal dynamics, we found that embryonic multipotent mammary cells become lineage-restricted surprisingly early in development, with evidence for unipotency as early as E12.5 and no statistically discernable bipotency after E15.5. To gain insights into the mechanisms governing the switch from multipotency to unipotency, we used gain-of-function Notch1 mice and demonstrated that Notch activation cell autonomously dictates luminal cell fate specification to both embryonic and basally committed mammary cells. These functional studies have important implications for understanding the signals underlying cell plasticity and serve to clarify how reactivation of embryonic programs in adult cells can lead to cancer.Wellcome Trus

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2SAT transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells.</jats:p

Notch3 marks clonogenic mammary luminal progenitor cells in vivo

The identity of mammary stem and progenitor cells remains poorly understood, mainly as a result of the lack of robust markers. The Notch signaling pathway has been implicated in mammary gland development as well as in tumorigenesis in this tissue. Elevated expression of the Notch3 receptor has been correlated to the highly aggressive “triple negative” human breast cancer. However, the specific cells expressing this Notch paralogue in the mammary gland remain unknown. Using a conditionally inducible Notch3-CreERT2(SAT) transgenic mouse, we genetically marked Notch3-expressing cells throughout mammary gland development and followed their lineage in vivo. We demonstrate that Notch3 is expressed in a highly clonogenic and transiently quiescent luminal progenitor population that gives rise to a ductal lineage. These cells are capable of surviving multiple successive pregnancies, suggesting a capacity to self-renew. Our results also uncover a role for the Notch3 receptor in restricting the proliferation and consequent clonal expansion of these cells

Luminal progenitors restrict their lineage potential during mammary gland development.

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERαneg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes

Notch1 expression in the postnatal mammary gland is restricted to ERα^neg and PR^neg luminal cells.

<p><b>(A–B)</b> Representative sections of ducts from pubertal (6-wk-old) N1Cre^ERT2R26^mTmG females analyzed 24 h upon tamoxifen injection. Immunofluorescence was performed with anti-K5 antibodies (in red in A), anti-K8 (in red in B), anti-GFP (to reveal Notch1-marked cells in green) and DAPI stains DNA in blue; <i>n</i> = 3. <b>(C)</b> FACS plots of 6-wk-old N1Cre^ERT2R26^mTmG females analyzed 24 h upon tamoxifen injection. Dissociated mammary cells were gated as Lin^neg cells (CD45/CD31/Ter119)^neg, and then as mammary epithelial cells (MEC) using the CD24 and CD29 markers, allowing us to resolve luminal (CD24⁺CD29^low) and myoepithelial (CD24⁺CD29^high) populations. 98.24 ±0.4% of GFP^pos cells gated in MEC were found in the luminal subset by FACS analysis. Note that GFP^pos cells also display Tomato fluorescence 24 h after induction, as the Tomato protein is still present at this time point, even if recombination has occurred. Values are shown in average ± s.e.m, <i>n</i> = 6. (<b>D–E</b>) The expression of Notch1 in sorted luminal (CD24⁺CD29^low) (LUM) and myoepithelial cells (CD24⁺CD29^high) (MYO) mammary cells from 10-wk-old B6/N wild-type females was analyzed at the mRNA level by qRT-PCR in A and at the protein level by western blot in B. The relative mRNA expression was normalized to the housekeeping gene 18S in A, while lamin B1 was used as a loading control in E. For western blot analysis, we used anti-K8 and anti-K5 antibodies as controls for sorted luminal and myoepithelial cells, respectively. <i>n</i> = 2. <b>(F–G)</b> Representative sections of mammary ducts from 6-wk-old N1Cre^ERT2R26^mTmG females show that GFP-expressing cells (in green) are invariably negative for ERα (in red in F) and PR expression (in red in G). DAPI stains DNA in blue, <i>n</i> = 3. Scale bars correspond to 20 µm in C–D and F–G and 10 µm in the insets.</p

Dll1- and Dll4-mediated Notch signaling is required for homeostasis of intestinal stem cells

BACKGROUND & AIMS: Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors, due to their conversion into post-mitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SC), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiological ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4). METHODS: Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ERT2). RESULTS: Notch1 signaling was found to be activated in intestinal SC. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into post-mitotic goblet cells, concomitant with loss of SC (Olfm4+, Lgr5+ and Ascl2+). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut. CONCLUSIONS: Notch signaling in SC and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SC. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice

Micro-scale distribution of recently-detached Carybdea marsupialis box jellyfish along the coast of Denia (W. Mediterranean)

5th International Jellyfish Bloom Symposium, 30 May to 3 June 2016, Barcelona.-- 1 pageCarybdea marsupialis have been found in high densities (~90 ind m3 juveniles, ~51 ind m3 adults) in shallow waters along the coast of Denia (W. Mediterranean) since summer 2008, varying significantly in abundance from year to year. In order study the role of juvenile stages in population dynamics, we analyzed the seasonal and micro-scale spatial distribution of recently-detached cubomedusae (~0.5 mm DBW). The main objective was to determine whether their distribution was focused or dispersed, as a means of revealing the location of the polyps. We also tested the correlation between environmental variables such as nutrient (P, N, Si), Chl-a and zooplankton abundance. During 2015 we collected samples from 33 points: 11 points along 15km of coastline at 3 distances from the coast (0m, 250m and 500m). Recently-detached C. marsupialis were almost only present at a depth of 0m in the samples collected from May to July. Maximum densities (~5 ind m3) were recorded in June in the centre of the study area. We hypothesized that the polyps would be located where the adults had mated the previous year. However, the distribution of recently-detached individuals did not coincide with the higher 2014 adult densities. This might be ascribed to the advection caused by the currents, which alternate along the coast. Abundance was not directly correlated with either zooplankton, nutrients or Chl-a. The next step would be to model the currents pattern and its effect in the dispersion and survival of juvenilesPeer Reviewe

Notch1-expressing cells contribute to alveologenesis at pregnancy and expand in response to hormonal stimulation.

<p><b>(A)</b> N1Cre^ERT2R26^mTmG females were induced with a low dose of tamoxifen (1µg/g of mouse body weight) at 4 wk of age and analyzed as virgins (left) or at mid-pregnancy (14.5 d post coitum, dpc) (right panel). Representative pictures of whole mount mammary gland ducts are shown, where red denotes tomato fluorescence, while green indicates Notch1-derived GFP^pos cells. <i>n</i> = 3. <b>(B)</b> Quantification by flow cytometry of the percentage of GFP^pos luminal cells (Lin^-CD24⁺CD29^low) from N1Cre^ERT2R26^mTmG females induced at 4 wk of age (0.1mg/g of mouse body weight) and analyzed at virgin or pregnant state (14.5 dpc). An extensive increase in the amount of GFP^pos cells is evident at pregnancy in A and B. <i>n</i> = 7. (**) <i>p</i> = 0.001 with <i>t</i> test. <b>(C)</b> Left, representative mammary section of N1Cre^ERT2R26^mTmG females injected with tamoxifen at mid-pregnancy (14.5 dpc) and analyzed 24 h later (pulse). Right, mammary section of N1Cre^ERT2R26^mTmG female injected at 4 wk of age and analyzed at mid-pregnancy (chase). <b>(D)</b> Quantification by flow cytometry of the percentage of GFP^pos cells luminal cells (Lin^-CD24⁺CD29^low) from N1Cre^ERT2R26^mTmG females injected at mid-pregnancy (pulse: 69.75 ±3.55%) compared to females injected at 4 wk of age and analyzed at mid-pregnancy (chase: 86.1 ±2.01%). Values are shown as percentages ±s.e.m. of <i>n</i> = 5. <b>(E)</b> 3-D organotypic cultures from adult N1Cre^ERT2R26^mTmG females were induced with 4-hydroxytamoxifen (4-OHT) in vitro and treated for 10 d with different hormones, as indicated. Red denotes tomato fluorescence and green indicates Notch1-marked cells at Day 0 and progeny at Day 10; <i>n</i> = 4. <b>(F)</b> Quantification of the clonal expansion of GFP^pos cells in 3-D organoids, classified as single cells (one cell, in light green) or clones (two or more cells, in dark green) indicates a significant expansion only in response to estradiol (β-2E bars). (***) <i>p</i> < 0.001 with <i>t</i> test. Scale bars correspond to 40 µm in A and E and 20 µm in C.</p

Proposed model for luminal cell hierarchy during mammary gland development.

<p><b>(A)</b> Lineage tracing from embryos reveals that Notch1-expressing (N1^pos) multipotent stem cells exist only during embryonic mammary development, when they co-express myoepithelial cytokeratin (K5) and luminal cytokeratin (K8). These multipotent stem cells can generate all mammary lineages (labeled in green postnatally). <b>(B)</b> After birth, luminal stem cells resolve in two distinct luminal progenitors: ERα^pos and ERα^neg cells, which maintain exclusively their own lineage throughout adulthood. Unipotent progenitors lacking ERα expression (ERα^neg) marked by Notch1 are responsible for generating milk-producing alveoli at pregnancy and define self-renewing luminal cells able to survive mammary gland involution.</p

Notch1 is expressed in multipotent embryonic stem cells.

<p><b>(A)</b> Schematic representation of the Notch1-CreERT2^SAT knock-in mice (referred to as N1Cre^ERT2) and Rosa26^mTomato/mGFP reporter mice (called R26^mTmG) used in this study. Pregnant females were induced with tamoxifen to label their embryos at embryonic day E15.5 and double transgenic N1Cre^ERT2R26^mTmG littermates were analyzed 24 h later <b>(B–D)</b> or 5 wk after birth (<b>E–G</b>). <b>(B–D)</b> Representative embryonic mammary bud sections show that Notch1^pos cells (marked by GFP in green) express both myoepithelial (K5, in red in B) and luminal markers (K8, in red in C) and they are negative for ERα (in red in D), <i>n</i> = 2. (<b>E–G</b>) Representative pubertal mammary gland sections show that Notch1-derived clones (in green) contain myoepithelial (K5^pos in red in E) and luminal cells (K8^pos in red in F) as well as ERα^pos and ERα^neg cells (ERα in red in G), <i>n</i> = 3. 4',6-diamidino-2-phenylindole (DAPI) stains DNA in blue. Scale bars correspond to 20 µm in B–G, and 10 µm in the magnifications of the insets.</p