Estrogen receptor beta selectively restricts proliferation and favors surveillance in mammary epithelial cells

Estrogen (17β-estradiol) has paradoxical effects in both promoting and preventing breast cancer as estrogen activates proliferation, but also promotes p53-mediated surveillance pathways. Estrogen mediates its effects in target tissues through the activation of estrogen receptor subtypes: ERα and ERβ. To examine the capability of these receptors in mediating surveillance as opposed to proliferation, selective estrogen receptor agonists were compared with 17β-estradiol for induction of proliferation and radiation induced apoptosis in vivo. Transcriptional regulation of estrogen-responsive genes was also compared in mouse mammary epithelium in vivo and in the human mammary MCF7 cell line transduced with a repressible ERβ. Selective activation of ERβ with the agonist diarylpropionitrile (DPN) in vivo enhances p53-mediated apoptosis in the mouse mammary epithelium without stimulating proliferation. In addition, radiation-induced apoptosis is significantly reduced in mice lacking ERβ (βERKO). As expected, 17β-estradiol or selective activation of ERα with pyrazole triol (PPT) induced the expression of estrogen-response genes including progesterone receptor, amphiregulin and trefoil factor 1. DPN and ERβ failed to induce the expression of these genes. Interestingly, the ERβ agonist DPN selectively induced the expression of genes that repress proliferation including TGFβ2 while inhibiting proliferative canonical wnt signaling via beta-catenin by inducing WNT5a and AXIN2. DPN was also more potent in stimulating the expression of EGR1, a modulator of p53 activity. These results suggest that ERα and ERβ have distinct roles in gene regulation. In addition, the ability of DPN and ERβ to potentiate surveillance pathways while limiting proliferation suggests that ERβ agonists may have therapeutic and chemopreventive value in breast cancer

Designing New Approaches for the Study of Early Murine Endodermal Organogenesis using Whole Embryo Culture

This thesis investigates the applicability of novel approaches designed to study the molecular mechanisms required for the initiation of organogenesis within the early endoderm. The endoderm is the germ layer that gives rise to the gut-tube and associated organs including the thyroid, lung, liver and pancreas. Our laboratory focuses on understanding the molecular mechanisms governing the developmental transition from endoderm to liver and pancreas. Several signaling pathways including Wnt, Retinoic Acid (RA), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor-β (TGFβ) have been implicated in the emergence of the liver bud from the endoderm in the mouse or other vertebrate species. However, neither the exact signals nor the precise roles during budding process have been identified, due to the complexity of specifically altering these essential pathways using traditional genetic approaches during the earliest stages of endoderm organogenesis. These traditional techniques include transgenic, knockout or conditional knockouts strategies. To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture. The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis. The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos

A Fate Map of the Murine Pancreas Buds Reveals a Multipotent Ventral Foregut Organ Progenitor

<div><p>The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days <em>post coitum</em> (dpc) dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2–4 between the 2 and 11 somite stage (SS). The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL). Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification.</p> </div

Identification of ventral pancreas precursors.

<p><b>A–C.</b>A 6 SS embryo labeled in the lateral endoderm stretching from the right rostral edge of the AIP to the fourth somite, is depicted in the cartoon (A) and in a merged fluorescent/bright field view of the actual labeled embryo at the onset (red cells in B) and at the end of culture (C, 22 SS). <b>D–E.</b> DiI labeled cells (red, arrowheads) contribute to the liver bud (outlined by the dashed line) and adjacent gut tube (D) as well as in the lateral ventral pancreas (E, arrowheads) denoted by the prominent nuclear PDX1 immunofluorescence (green).</p

Summary of each VMEL labeled embryo’s contribution to the 9.5 dpc ventral foregut buds.

a<p>Somite Stage of embryo at onset of culture.</p>b<p>An “X” denotes the presence of DiI labeled cells in this structure.</p>c<p>A “–“ indicates that no DiI labeled cells were found in this structure.</p>d<p>ML = midline.</p

VMEL contribution to multiple foregut organ buds occurs in a temporally restricted manner.

<p><b>A–B, H–I, O–P.</b> Bright field/fluorescent merged views of embryos labeled with DiI (red) in the VMEL at the onset (A, H, O) and after ∼30 hours of culture (B, I, P). All embryos were sectioned as indicated (dashed lines in B, I and P) and immunofluorescence performed with PDX1 (green) to identify the ventral pancreas bud. Thyroid buds (C, J, Q) and different portions of the liver bud (rostral liver in D, K, R and liver bud in E, L, S) were identified based on morphology. All sections are presented at low magnification (C–F, J–M, Q–T) and a portion of this field (indicated by the boxed area) presented at high magnification (C’–F’, J’–M’, Q’–T’) where the indicated tissue bud has been outlined by a dotted line and arrowheads used to point to DiI labeled cells within the indicated tissue bud. <b>G, N, U.</b> Cartoons summarizing the VMEL contribution (red line) to the thyroid bud (thb), liver bud (lb) and ventral pancreas bud (vpb) for the embryo to the left of the cartoon. <b>A–G.</b> This 5 SS VMEL labeled embryo (red, A) was cultured through the 19 SS (B). Section analysis of this embryo revealed DiI contribution to the thyroid (C, C’), a swath of cells in the rostral liver (D, D’) and a limited number of cells in the midline of the liver (E, E’) and ventral pancreas buds (F, F’) as summarized in G. <b>H–N.</b> The VMEL labeled 5 SS embryo (H) was cultured through the 23 SS (I). Section analysis reveals no VMEL contribution to the thyroid bud (J, J’) but the presence of VMEL descendants in a swath of rostral liver bud (K, K’), in the midline of the liver bud (L, L’) and in the midline of the ventral pancreas bud (M, M’) as summarized (N). <b>O–U.</b> This 8 SS embryo (O) was DiI labeled in the VMEL and cultured through the 28 SS (P). Although labeled VMEL descendants were not found in the thyroid (Q, Q’) or rostral liver buds (R, R’) they were found in the midline of the liver (S, S’) and ventral pancreas buds (T, T’) as summarized (U). v = ventral and d = dorsal gut tube, h = heart.</p

Fate map of the 3–11 SS caudal foregut endoderm.

<p><b>A–C.</b> Fate maps summarizing the results obtained from DiI labeling the endoderm of individual 3–5 SS (A) 6–8 SS (B) and 9–11 SS (C) embryos and culturing through ∼9.5 dpc. Each shape represents the position and size of the DiI labeled endoderm from a single embryo at the stage indicated by each map. The color indicates which organ bud those labeled cells contributed to at the end of culture. DiI labeled cells that contributed to the dorsal pancreas bud are colored blue, those that contributed to the ventral pancreas bud are yellow, contributors to the liver bud are red while groups of cells which gave rise solely to gut tube are in white. Because multiple embryos were often labeled in similar domains, the color of each shape is transparent and thus darker shades of a color indicate overlapping labeled endoderm that gave rise to the same bud. Labeled groups of cells that gave rise to 2 organ buds are bi-colored and each color placed in the presumptive half that gave rise to the indicated organ. For ease of view, all the data points that gave rise to gut tube alone were placed on top of any colored shapes. A simple compass indicating the embryonic axis is provided. The orientation of the axis is preserved in all fate map cartoons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040707#pone-0040707-g002" target="_blank">Figures 2</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040707#pone-0040707-g003" target="_blank">3</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040707#pone-0040707-g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040707#pone.0040707.s001" target="_blank">Figure S1</a>).</p

Identification of the dorsal pancreas progenitors.

<p><b>A–B.</b>A cartoon depicting a 9–11 SS embryo labeled over the third somite and slightly lateral on the right side (A) and a bright field/fluorescence merged image of the actual 9 SS embryo revealing the DiI (red) labeled cells (B). <b>C.</b> After culture, the merged image reveals that the resultant 25 SS embryo contained DiI labeled descendants in the dorsal gut (lower red) as well as a smaller patch of visible descendants in the ventral/lateral gut tube (upper red). <b>D–E.</b> Transverse sections as indicated by the dashed lines in (C) revealed the co-localization of DiI (red) in the PDX1-positive (green) dorsal pancreas bud (dp, E) but not in the ventral pancreas bud (vp, D). <b>F–H.</b> A cartoon representing a 5 SS embryo labeled over the third somite pair, as indicated in the cartoon (F) and shown in the actual labeled 5 SS embryo (G) that was cultured to the 20 SS (H). <b>I.</b> Section analysis of the embryo indicated in (H) with PDX1 (green) reveals DiI labeled descendants throughout the dorsal pancreas (arrowheads in I’, boxed region in I is magnified in I’). <b>J.</b> A cartoon of a 5 SS embryo labeled over the left first and second somite, cultured through the 20 SS and processed as above. <b>K–M.</b> Transverse sections through this embryo are aligned in a rostral to caudal manner as indicated by the arrow. DiI labeled descendants (red) are found in the dorsal endoderm rostral to the dorsal pancreas (K) and only slightly overlap with the rostral-most portion of the PDX1-positive dorsal pancreas bud (arrowhead in L). All of the more caudal sections of the dorsal pancreas (green) do not contain DiI labeled cells (M). <b>N.</b> A cartoon depicting a 5 SS embryo labeled in the endoderm over the caudal most fourth and much of the fifth somite. <b>O–Q.</b> Section analysis of the resultant 23 SS embryo, arranged in a rostral to caudal manner as indicated by the arrow, revealed that DiI labeled cells were absent from the rostral regions of the dorsal pancreas bud (green, O) but overlapped slightly with the caudal portion of the dorsal bud (arrowhead, P) and extend into gut tube caudal to the dorsal pancreas bud (red cells in Q). In all sections the arrowheads indicate region of DiI overlap with the dorsal pancreas bud.</p

Visceral endoderm expression of Yin-Yang1 (YY1) is required for VEGFA maintenance and yolk sac development.

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development

cKO embryos display a variety of defects in the yolk sac mesoderm.

<p>A–B, D–E) Whole mount immunofluorescence of 9.5 <i>dpc</i> WT (A–B) or mutant (D–E) yolk sacs (YS) using the endothelial marker PECAM (green) and the vascular smooth muscle marker (αSMA) demonstrates that the large disorganized vessels in the cKO (D) are not surrounded by αSMA (E). C, F) Section immunofluorescence of WT (C) and cKO (F) 9.5 <i>dpc</i> yolk sacs demonstrates loss of αSMA in the cKO. G–L) Section immunofluorescence of VEGFA (green) demonstrates relatively uniform VEGF levels in the 9.0, 9.25 and 9.5 <i>dpc</i> WT yolk sac (G–I) while VEGF distribution in the visceral endoderm of the mutant is progressively diminished at each stage (J–L). M) A Western blot of whole yolk sacs at the indicated stages. The ratio of VEGFA to GAPDH signal intensities for the cKO relative to each stage-matched WT control is displayed under each band. N) Cleaved Caspase-3 staining was used to assess the percentage of cell death in the yolk sacs layers of WT and cKO sections at 8.5 and 9.0 <i>dpc</i>. A significant increase in apoptosis was observed in the cKO mesoderm (ME) at 9.0 <i>dpc</i>. O) Phosphohistone-H3 (PH-3) staining was similarly used to assess proliferation and a significant decrease in proliferation was found in the cKO yolk sac mesoderm at 9.0 <i>dpc</i>. *** = p<0.001, ** = p<0.01; error bars = standard error; dotted line is drawn between the visceral endoderm (VE) and mesoderm derivatives (ME) on yolk sac sections.</p