Stages Of Mammary Development Research Articles

Mutation of SOCS2 induces structural and functional changes in mammary development.

Lactation is an essential process for mammals. In sheep, the R96C mutation in suppressor of cytokine signaling 2 (SOCS2) protein is associated with greater milk production and increased mastitis sensitivity. To shed light on the involvement of R96C mutation in mammary gland development and lactation, we developed a mouse model carrying this mutation (SOCS2KI/KI). Mammary glands from virgin adult SOCS2KI/KI mice presented a branching defect and less epithelial tissue, which were not compensated for in later stages of mammary development. Mammary epithelial cell (MEC) subpopulations were modified, with mutated mice having three times as many basal cells, accompanied by a decrease in luminal cells. The SOCS2KI/KI mammary gland remained functional; however, MECs contained more lipid droplets versus fat globules, and milk lipid composition was modified. Moreover, the gene expression dynamic from virgin to pregnancy state resulted in the identification of about 3000 differentially expressed genes specific to SOCS2KI/KI or control mice. Our results show that SOCS2 is important for mammary gland development and milk production. In the long term, this finding raises the possibility of ensuring adequate milk production without compromising animal health and welfare.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.

The role of the IGF system in mammary physiology of ruminants

The IGF system plays a central role in all stages of mammary development, lactation and involution. IGFs exert their effects on the mammary gland through both endocrine and paracrine/autocrine mechanisms and the importance of circulating versus local IGF action remains an open question, especially in ruminants. At the whole organ level, a critical role for IGFs in ductal morphogenesis and lobuloalveolar development has been established, while at the cellular level the ability of IGFs to stimulate cell proliferation and control cell survival contributes to the number of milk-secreting cells in the gland. Much of this work has been conducted in rodents which provide an affordable research model and allow for genetic manipulation of specific components of the IGF system. Research into the role of the IGF system in dairy cows has generally supported information obtained with rodents though large gaps in our knowledge remain and species differences are not well defined. Examples include whether exogenous somatotropin exerts its effects on the mammary gland through local IGF-1 synthesis which is accepted dogma in rodents, what the role of IGF-1 versus IGF-2 is in the mammary gland, and how the IGFBPs regulate IGF bioactivity. This last area is particularly under-investigated in ruminants both at the whole animal and the cellular and molecular levels. Given that the IGF system may underlie many management practices that could contribute to enhancing productive efficiency of lactation, more research into the basic biology of this important system is warranted.

Transposable Element Dynamics and Regulation during Zygotic Genome Activation in Mammalian Embryos and Embryonic Stem Cell Model Systems.

Transposable elements (TEs) are mobile genetic sequences capable of duplicating and reintegrating at new regions within the genome. A growing body of evidence has demonstrated that these elements play important roles in host genome evolution, despite being traditionally viewed as parasitic elements. To prevent ectopic activation of TE transposition and transcription, they are epigenetically silenced in most somatic tissues. Intriguingly, a specific class of TEs—retrotransposons—is transiently expressed at discrete phases during mammalian development and has been linked to the establishment of totipotency during zygotic genome activation (ZGA). While mechanisms controlling TE regulation in somatic tissues have been extensively studied, the significance underlying the unique transcriptional reactivation of retrotransposons during ZGA is only beginning to be uncovered. In this review, we summarize the expression dynamics of key retrotransposons during ZGA, focusing on findings from in vivo totipotent embryos and in vitro totipotent-like embryonic stem cells (ESCs). We then dissect the functions of retrotransposons and discuss how their transcriptional activities are finetuned during early stages of mammalian development.

Role of Iron Metabolism-Related Genes in Prenatal Development: Insights from Mouse Transgenic Models.

Iron is an essential nutrient during all stages of mammalian development. Studies carried out over the last 20 years have provided important insights into cellular and systemic iron metabolism in adult organisms and led to the deciphering of many molecular details of its regulation. However, our knowledge of iron handling in prenatal development has remained remarkably under-appreciated, even though it is critical for the health of both the embryo/fetus and its mother, and has a far-reaching impact in postnatal life. Prenatal development requires a continuous, albeit quantitatively matched with the stage of development, supply of iron to support rapid cell division during embryogenesis in order to meet iron needs for erythropoiesis and to build up hepatic iron stores, (which are the major source of this microelement for the neonate). Here, we provide a concise overview of current knowledge of the role of iron metabolism-related genes in the maintenance of iron homeostasis in pre- and post-implantation development based on studies on transgenic (mainly knock-out) mouse models. Most studies on mice with globally deleted genes do not conclude whether underlying in utero iron disorders or lethality is due to defective placental iron transport or iron misregulation in the embryo/fetus proper (or due to both). Therefore, there is a need of animal models with tissue specific targeted deletion of genes to advance the understanding of prenatal iron metabolism.

Ppp2r2a Knockout Mice Reveal That Protein Phosphatase 2A Regulatory Subunit, PP2A-B55α, Is an Essential Regulator of Neuronal and Epidermal Embryonic Development.

The serine/threonine protein phosphatase 2A (PP2A) is a master regulator of the complex cellular signaling that occurs during all stages of mammalian development. PP2A is composed of a catalytic, a structural, and regulatory subunit, for which there are multiple isoforms. The association of specific regulatory subunits determines substrate specificity and localization of phosphatase activity, however, the precise role of each regulatory subunit in development is not known. Here we report the generation of the first knockout mouse for the Ppp2r2a gene, encoding the PP2A-B55α regulatory subunit, using CRISPR/Cas9. Heterozygous animals developed and grew as normal, however, homozygous knockout mice were not viable. Analysis of embryos at different developmental stages found a normal Mendelian ratio of Ppp2r2a–/– embryos at embryonic day (E) 10.5 (25%), but reduced Ppp2r2a–/– embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live Ppp2r2a–/– pups were observed at birth. Ppp2r2a–/– embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. Ppp2r2a–/– embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in Ppp2r2a–/– embryos were highly disorganized, with reduced immunolabeling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the Ppp2r2a–/– embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Thus PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis.

Signaling pathways in mammalian preimplantation development: Linking cellular phenotypes to lineage decisions.

The first stages of mammalian development, before implantation of the embryo in the maternal uterus, result in the establishment of three cell populations in the blastocyst: trophectoderm, epiblast, and primitive endoderm. These events involve only a small number of cells, and are initiated by morphological differences among them related to cell adhesion and polarity. Much attention has been paid to the master transcription factors that are critical for establishing and maintaining early lineage choices. Nevertheless, a large body of work also reveals that additional molecular mechanisms are involved. Here, we provide an updated view of the role of different signaling pathways in the first stages of mouse development, and how their cross-talk and interplay determine the initial lineage decisions occurring in the blastocyst. We will also discuss how these pathways are critical for translating cellular phenotypes, the product of the morphogenetic events occurring at these stages, into transcriptional responses and expression of lineage-specifying transcription factors. Developmental Dynamics 246:245-261, 2017. © 2016 Wiley Periodicals, Inc.

Abstract 657: N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation

Abstract N-Myc and STAT Interactor (NMI) is an evolutionarily conserved protein that is widely expressed in fetal and adult tissues. Early studies implicated its role in regulating the activities of transcription factors (such as MYC, STATs, BRCA1, TIP60 etc.) critical to tumor progression and metastasis. However, the functional relevance of these regulatory activities of NMI remains unknown. Recent findings from our lab have revealed that NMI protein expression is decreased by 70% in primary tumor specimens from patients with metastatic breast cancer. Most recently, we have found that lack of NMI expression in breast cancer cells confers resistance to chemotherapy by blocking autophagy-induced cell death. Thus the status of NMI expression in breast cancer patients may be an important clinical consideration. Additionally, our functional studies have demonstrated that loss of NMI expression allows manifestation of TGFβ and Wnt driven EMT that results in increased invasion and metastatic dissemination. Overall, we have noticed a profound impact of NMI on multiple developmental signaling pathways that are essential for mammary development as well as tumor progression. To further elucidate the biological role of NMI, we have created a genetically engineered mammary specific Nmi knock out model. We observed that in normal murine mammary tissue Nmi is expressed in the mammary epithelium during all stages of mammary development. However, it's expression is strikingly induced at the onset of pregnancy, implicating an important role of NMI in mammary ductal development and/or lactation. Remarkably, the Nmi knock out mice exhibit distinctly increased number of alveolar structures (30% more than in control mice) during lactation. Moreover, these Nmi-/- mammary glands also show 20% more proliferating mammary epithelial cells (MECs) compared to respective littermate controls. In addition, 3D-alveologenesis of Nmi knock out MECs is highly responsive to induction by growth factors such as TGFα and FGFs when compared to MECs from littermate controls. Hyper-proliferative phenotype has been implicated as one of the key factors for breast cancer initiation as well as progression. Overall, our work describes a direct evidence for the role of NMI as a key regulator of mammary epithelial cell proliferation. Citation Format: Hawley C. Pruitt, Brandon J. Metge, Sarah K. Bailey, Lalita A. Shevde, Rajeev S. Samant. N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 657.

Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

BackgroundWnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data.ResultsA single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport.ConclusionsThis integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling.

IN VITRO PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM A BULGE REGION OF WHISKER FOLLICLE

71 The neural crest (NC), a transient structure in embryo development, is unique to vertebrates. NC is derived from the ectoderm and formed at the border of neuroectoderm and somatic ectoderm. Originally NC is represented by a cell population lying in the form of epithelial cords emerging from both sides of the neural tube. At the neurulation process end the NC cells undergo an epithelialmesenchymal transition (EMT) and migrate distantly in various tissues and organs of the developing embryo, where they give rise to the multiple differentiated cell types [1, 2]. In adult vertebrate organism the NC derivatives are represented by the following cell types: neurons and glial cells of the peripheral nervous system, the majority of sensory neurons, melanocytes, Merkel cells, odontoblasts, stromal and endothelial cells of cornea, C cells of thyroid gland, the adrenal medulla, a part of thymic stroma [1, 2]. The most plastic are the cells of cranial NC. In addition to the above mentioned differentiated cell types, they form a part of the bone, cartilage and connective tissues of the face, forehead and ventral neck [3, 4]. Unusually wide differentiation potential of the NC cells suggests an existence of multipotent stem cells. Stemple and Anderson firstly revealed the existence of multipotent stem cells in the NC in early embryonic stage of mammalian development [5]. A long time it was thought that, like NC per se, neural crestderived multipotent stem cells (NC-MSCs) have a transient nature. Consequently, the NCMSCs exist only at the premigratory (from the NC specification moment until the beginning of its cell migration) and migratory stages (from the NC cell delamination until their incoming into the sites of final destination), and further, within migration to sites of its final localization, undergo a process of commitment, turning into the progenitor cells with limited proliferative and/or differentiation potential [6, 7]. An example is the transition of NC-MSCs into the melanocyte precursors — melanoblasts. In the last decades, NC-derived cells have been isolated from a number of tissues and organs of the adult mammalians. These cells demonstrate the ability to self-renewal and multilineage differentiation, at least in vitro, i.e., they are multipotent stem cells [8–11]. The origin of these cells from NC was proved by creating special lines of transgenic mice [12, 13], as well as by means of their transplantation in the developing chick embryo, where the NC-MSCs migrated via the UDC 576.3+612.014.2/3

The genome-defence gene Tex19.1 suppresses LINE-1 retrotransposons in the placenta and prevents intra-uterine growth retardation in mice.

DNA methylation plays an important role in suppressing retrotransposon activity in mammalian genomes, yet there are stages of mammalian development where global hypomethylation puts the genome at risk of retrotransposition-mediated genetic instability. Hypomethylated primordial germ cells appear to limit this risk by expressing a cohort of retrotransposon-suppressing genome-defence genes whose silencing depends on promoter DNA methylation. Here, we investigate whether similar mechanisms operate in hypomethylated trophectoderm-derived components of the mammalian placenta to couple expression of genome-defence genes to the potential for retrotransposon activity. We show that the hypomethylated state of the mouse placenta results in activation of only one of the hypomethylation-sensitive germline genome-defence genes: Tex19.1. Tex19.1 appears to play an important role in placenta function as Tex19.1−/− mouse embryos exhibit intra-uterine growth retardation and have small placentas due to a reduction in the number of spongiotrophoblast, glycogen trophoblast and sinusoidal trophoblast giant cells. Furthermore, we show that retrotransposon mRNAs are derepressed in Tex19.1−/− placentas and that protein encoded by the LINE-1 retrotransposon is upregulated in hypomethylated trophectoderm-derived cells that normally express Tex19.1. This study suggests that post-transcriptional genome-defence mechanisms are operating in the placenta to protect the hypomethylated cells in this tissue from retrotransposons and suggests that imbalances between retrotransposon activity and genome-defence mechanisms could contribute to placenta dysfunction and disease.

Abstract PD09-10: DNA Damage and Repair in Mammary Gland Development

Abstract Numerous factors are linked to breast cancer risk including reproductive history, early exposure to radiation, age and genetic background. Events that occur during specific stages of mammary development are believed to alter the lifetime risk of breast cancer. Epidemiological studies over the past 3 decades have suggested an association between parity and decreased lifetime breast cancer risk. Studies have also shown that breast cancer risk is significantly higher in women who received radiotherapy to the chest as adolescents compared to those who received chest radiation as adults. These associations implicate development specific changes in mammary epithelium that affect the molecular mechanisms whereby cells respond to DNA damage and, importantly, are at subsequent cancer risk. Because many of the genes linked to hereditary breast cancer are associated with DNA double-strand break (DSB) repair, we hypothesize that there are differences in overall DSB repair, homologous recombination (HR), and cell fate following damage in mammary epithelium at different stages of mammary gland development—puberty, pregnancy, lactation and post-involution. To assess these differences we are using mice containing the DR-GFP reporter targeted to chromosome 17 to study HR at chromosome breaks induced by the transient expression of I-SceI in mammary epithelial cells. DR-GFP is a widely used repair reporter in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats. We have observed that the efficiency of HR in primary mammary epithelial cells derived from 8-week old virgin mice is similar to that observed in other primary cells of somatic origin (approximately 0.65% of cells heterozygous for the DR-GFP reporter). Interestingly, the amount of HR is increased in mammary epithelium derived from pregnant mice compared to age-matched virgin controls (0.98 versus 0.65%). This 1.5 fold increase in HR suggests that in pregnancy, mammary epithelium is more proficient in the accurate repair of DNA DSBs. We are also assessing DNA damage signaling in different periods of mammary gland development. Preliminary analysis of histone H2AX phosphorylation following whole-body irradiation suggests more rapid kinetics of DNA damage signaling response in mammary tissue derived from pregnant as compared to age-matched virgin mice. We have recently generated DR-GFP containing mice that express I-SceI from a randomly integrated I-SceI transgene under the control of a tetracycline-inducible promoter. We are currently using these mice to assess HR in an entirely in vivo system and are in the process of breeding this reporter line with mice carrying mutations in Brca1 or Brca2. This mouse model will be used to assess HR in vivo in the mammary tissue of these mice during different stages of mammary gland development. This project will provide valuable insight into how developmental changes unique to mammary gland epithelium affect cellular responses to DNA damage. We hope to consider therapeutic manipulation of identified stage-specific differences to decrease breast cancer susceptibility. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD09-10.

Intracellular and Extracellular miRNAs in Regulation of Angiogenesis Signaling.

Angiogenesis, the process by which new blood vessels are formed, is a critical phenomenon that is activated during various stages of mammalian development. MicroRNAs (miRNAs), a class of short, single stranded, non-coding RNAs, are recognized as important regulators of angiogenesis, and the role of intracellular miRNAs in modulating angiogenesis signaling has been identified. The recent discovery of extracellular and circulating miRNAs has sparked new questions regarding their potential in modulating angiogenesis signaling not only within cells but also between cells. In this review, we discuss the characteristics of intracellular and extracellular miRNAs and decipher the potential functional roles for these molecules in regard to the angiogenic process. We summarize what is currently known about circulating miRNAs in distinct clinical populations and discuss evidence that implicates extracellular miRNAs as novel mediators of angiogenesis-associated intercellular signaling. Lastly, we offer a new perspective on the functional role of vesicle-encapsulated circulating miRNA in modulating angiogenesis signaling pathways.

Wnt Signalling Pathway Parameters for Mammalian Cells

Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report.

Dietary regulation of developmental programming in ruminants: epigenetic modifications in the germline

Ruminants have been utilised extensively to investigate the developmental origins of health and disease, with the sheep serving as the model species of choice to complement dietary studies in the rat and mouse. Surprisingly few studies, however, have investigated delayed effects of maternal undernutrition during pregnancy on adult offspring health and a consistent phenotype, together with underlying mechanistic pathways, has not emerged. Nevertheless, when broad consideration is given to all studies with ruminants it is apparent that interventions that are initiated very early in gestation, and/or prior to conception, lead to greater effects on adult physiology than those that are specifically targeted to late gestation. Effects induced following dietary interventions at the earliest stages of mammalian development have been shown to arise as a consequence of alterations to key epigenetic processes that occur in germ cells and pluripotent embryonic cells. Currently, our understanding of epigenetic programming in the germline is greatest for the mouse, and is considered in detail in this article together with what is known in ruminants. This species imbalance, however, looks set to change as fully annotated genomic maps are developed for domesticated large animal species, and with the advent of 'next-generation' DNA sequencing technologies that have the power to globally map the epigenome at single-base-pair resolution. These developments would help to address such issues as sexually dimorphic epigenetic alterations to DNA methylation that have been found to arise following dietary restrictions during the peri-conceptional period, the effects of paternal nutritional status on epigenetic programming through the germline, and transgenerational studies where, in future, greater emphasis in domesticated ruminants should be placed on traits of agricultural importance.

Wnt1 Overexpression Leads to Enforced Cardiomyogenesis and Inhibition of Hematopoiesis in Murine Embryonic Stem Cells

Recent findings emphasized a critical role for the Wnt signaling pathway during the early steps of embryogenesis, including the development of the hematopoietic system and cardiac development. To date, the role of Wnt in promoting or inhibiting development of both tissues was discussed controversially, dependent on species and time point of expression. Differentiation of embryonic stem cells (ESC) recapitulates early stages of mammalian development. In the present study, we generated murine ESC lines overexpressing Wnt1 (Wnt1 ES). When induced to differentiate toward the cardiomyocytic lineage, Wnt1 ES showed a significant increased ability to generate cardiomyocytes when compared with a control ESC (control ES) line. In addition, Wnt1 ES cells were unable to form hematopoietic cells, whereas development of endothelial cells, a cell type closely associated with blood during embryogenesis, was comparable to control ES. Finally, cardiac differentiation was markedly decreased by the addition of the Wnt antagonist Dkk-1 to the culture medium. These findings suggest that Wnt1 may regulate differentiation of immature mesodermal cells in a tissue-specific manner.

Abstract 3397: The role of Pdef in mammary development, tumorigenesis and metastasis

Abstract ETS proteins represent one of the largest families of transcription factors with diverse functions that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. The ETS family gene, PDEF (prostate derived ets factor), is expressed in normal epithelial tissues including prostate, breast, colon and bladder. Significantly, PDEF protein is present in non-invasive cancers but reduced or absent in most invasive cancers. By re-expression and knock-down of PDEF in cancer cell lines, we and others have shown that PDEF target genes control aspects of the metastatic process, including cell growth, migration and invasion. We hypothesize that PDEF is important for normal developmental processes and that loss of PDEF regulatory networks is a key event in the development of invasive cancer. To study the function of Pdef in vivo, we generated mice with constitutive Pdef knockout (Pdef −/−). Specific stages of mammary development were examined, including early-pubescent (5 week), virgin adult (8 week), pregnancy (days 7.5 and 13.5), lactation (days 1 and 7) and involution (day 7). Inhibition of the development of the ductal tree and an increase in ductal side branching and terminal end bud proliferation was observed in 5-8 week old virgin Pdef −/− mice. TGFβ1 and ERα are required for development of the ductal tree during puberty and mRNA levels for both are reduced in the Pdef −/− mice at 5 and 8 weeks of age. Incomplete lobuloalveoli development was observed in Pdef −/− pregnant and lactating mice, although no phenotypic effects were observed in the resultant pups. To explore the role of Pdef in mammary tumor progression and metastasis, we generated mice expressing the MMTV-PyMT or MMTV-Neu oncogene in wild type and Pdef −/− backgrounds. No impact of Pdef dosage upon tumor incidence or growth was observed in the PyMT model; however, initial tumor formation was delayed by 4 weeks (8.9 vs. 4.8) and time from tumor initiation to sacrifice was 3 weeks longer (29.4 vs 26.4) in the Pdef −/− Neu model. Significantly, an increase in lung metastasis was observed in the Pdef −/− mice in both the PyMT and Neu models. Thus, loss of Pdef in vivo is associated with defects in mammary development and increased metastasis in mouse models of breast cancer, suggesting Pdef is a metastasis suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3397.

Abstract A130: Effect of high-dose estrogen exposure in adolescence on mammographic density in adulthood.

Abstract A130 Background Since adolescence is an important stage of mammary development, exposure to high-dose estrogens at this time may have long-term effects on breast tissue and therefore mammographic density. It is well-established that women with greater mammographic density for age and BMI are at an increased risk of breast cancer. Methods We conducted a retrospective cohort study of women from the Australian Tall Girls Study who were 40 years or older and had been assessed for tall stature during adolescence between 1959 and 1993. Eligible women included 263 who had been treated during adolescence with high-dose estrogens and 254 who had not been treated. Treated women had received one of two types of estrogen: 3mg diethylstilbestrol (DES) daily, or 150 μg ethinyl estradiol (EE). A mammogram within the previous two years was obtained from 167 treated women (mean age 48.4 yrs) and 142 untreated women (mean age 46.2 yrs). The total area of the breast image and the area of mammographically dense tissue (dense area), and hence non-dense area and percent mammographic density (PMD), were calculated from digitally scanned cranio-caudal mammographic films using a computer thresholding technique. Reproductive history, lifestyle factors, hormone exposure, treatment and anthropometric data were collected from medical records and by telephone interview using a structured questionnaire. Results After adjusting for age and BMI, treated women had, on average, 17% lower dense area (p=0.03); adjusted means for treated and untreated women were 24.5 cm2 (95% CI: 21.8, 27.2) and 29.1 cm2 (95% CI: 26.0, 32.4), respectively. There was no difference in adjusted means between treated and untreated women for non-dense area [71.7 cm2 (95% CI: 66.2, 77.7) versus 70.5 cm2 (64.7, 76.9); p=0.78], PMD [ 24.8 % (95% CI: 22.4, 27.4) versus 27.7% (95% CI: 24.8, 30.7); p=0.16], or total area [105.6 cm2 (95%CI: 100.1,111.4) vs 109.3 cm2 (95%CI: 103.1,115.8); p=0.41]. Conclusion High-dose estrogen treatment for tall stature in adolescence is unlikely to increase risk of breast cancer through mechanisms related to mammographic density. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A130.

Differential regulation of the Oct‐3/4 gene in cell culture model systems that parallel different stages of mammalian development

Oct-3/4 is an essential transcription factor that regulates stem cell fate during embryogenesis. Previous reports have shown that the Oct-3/4 gene utilizes different enhancers to regulate its expression as development proceeds. However, the cis-elements contributing to the differential activity of these enhancers require further study. Here, we investigated the function of the HMG/POU cassette and LRH-1 site present in the distal enhancer (DE) and the proximal enhancer, respectively. F9 and P19 EC cells were the focus of this study because their differential utilization of Oct-3/4 enhancers parallels the use of these enhancers during different stages of development. We determined that the LRH-1 site functions as a positive and a negative cis-regulatory element in P19 and F9 EC cells, respectively. Furthermore, we determined that the HMG/POU cassette in the DE strongly activates the Oct-3/4 promoter in F9 cells, but is a much weaker positive regulatory element in P19 cells. Given that HMG/POU cassettes play key roles in the regulation of at least seven essential genes, the Oct-3/4 HMG/POU cassette was examined more closely by focusing on Sox2, which can bind to HMG/POU cassettes. Although chromatin immunoprecipitation demonstrated that Sox2 binds to the Oct-3/4 gene equally well in both EC cell lines, tethering Sox2 to the region of the HMG/POU cassette only activated the Oct-3/4 promoter in F9 EC cells. These and other findings suggest that the differential activity of the HMG/POU cassette of the Oct-3/4 gene in EC cells is due to differential action of Sox2 and its associated co-factors.

Myb proteins regulate expression of histone variant H2A.Z during thymocyte development

The c-myb gene encodes a transcription factor required for the normal development of T cells in the thymus, and for subsequent peripheral T-cell activation and survival. However, the profile of genes known to be transcriptionally regulated by c-Myb in T cells does not adequately explain the pleiotrophic nature of the effects of c-Myb. We present here a detailed molecular characterization of the regulation of a novel target gene, the histone variant H2A.Z. We show that c-Myb is able to bind to and activate the H2A.Z promoter in T cells both in vitro and in vivo, and present evidence that perturbation of Myb activity during T-cell development results in reduced H2A.Z expression. As H2A.Z is absolutely required for the early stages of mammalian development, and plays essential roles in the regulation of chromatin structure in gene promoters in yeast, its regulation by c-Myb is likely to be of some importance during T-cell development.