Project description:Our study revealed that CUZD1 (CUB and zona pellucida-like domain containing protein-1) is a novel target of estrogen regulation in the mouse mammary epithelium. Mice lacking Cuzd1 exhibit delayed ductal outgrowth during puberty and a striking impairment in ductal branching and alveolar development during pregnancy. Ablation of Cuzd1 led to a marked reduction in steroid-induced proliferation of mammary ductal and alveolar epithelium. To identify the downstream targets of Cuzd1 in mammary gland development, we performed gene expression profling of mammary epithlial cells isolated from Cuzd1-null and its heterozygous litteremates on day 18 of pregnancy. The microarray results revealed downregulation of mRNAs corresponding to several members of the epidermal growth factor family in mammary epithelial cells of Cuzd1-null mice. Consequently, the activation of the ERBB receptors, ERBB1 and ERBB4, and their downstream target STAT5, was disrupted in Cuzd1-null mammary tissue. Collectively, these findings support a unique role for CUZD1 as a critical mediator of the steroid-induced proliferation and differentiation of ductal epithelium during pregnancy and lactation.
Project description:Our study revealed that CUZD1 (CUB and zona pellucida-like domain containing protein-1) is a novel target of estrogen regulation in the mouse mammary epithelium. Mice lacking Cuzd1 exhibit delayed ductal outgrowth during puberty and a striking impairment in ductal branching and alveolar development during pregnancy. Ablation of Cuzd1 led to a marked reduction in steroid-induced proliferation of mammary ductal and alveolar epithelium. To identify the downstream targets of Cuzd1 in mammary gland development, we performed gene expression profling of mammary epithlial cells isolated from Cuzd1-null and its heterozygous litteremates on day 18 of pregnancy. The microarray results revealed downregulation of mRNAs corresponding to several members of the epidermal growth factor family in mammary epithelial cells of Cuzd1-null mice. Consequently, the activation of the ERBB receptors, ERBB1 and ERBB4, and their downstream target STAT5, was disrupted in Cuzd1-null mammary tissue. Collectively, these findings support a unique role for CUZD1 as a critical mediator of the steroid-induced proliferation and differentiation of ductal epithelium during pregnancy and lactation. To investigate the functional role of Cuzd1, we created Cuzd1-null mice by homologous recombination using mouse embryonic stem cells. As severe defect was observed in alveolargenesis and lactation of Cuzd1 null mammary gland, we purified mammary epithlial cells from day18 pregnant mice (n=5 for each genotype), purified total RNA from these cells, pooled these samples and then hybridized to high density affymetrix microarrays.
Project description:Previoulsly expression profiling of the whole mammary gland across different stages of pregnancy and lactation has been performed on different strains of mice. Since mammary gland has both epithelial and stromal compartments, to specifically identify the genes involved in the transition from pregnancy to lactation a process termed as secretory activation, expression profiling of isolated mammary epithelial cells (MECs) from four CD1 mice each at Pregnancy day 14 (P14) and Lactation day 2 (L2) was performed in the current study. Statistical analysis of the mRNA changes between P14 and L2 identified 5,499 unique genes as being differentially expressed (5% FDR), of which, 2,902 genes and 2,604 genes were higher in P14 or L2 stages, respectively.
Project description:Previoulsly miRNA expression profiling of the whole mammary gland across different stages of pregnancy and lactation has been performed in mice. Since mammary gland has both epithelial and stromal compartments, to specifically identify the miRNAs involved in the transition from pregnancy to lactation a process termed as secretory activation, expression profiling of isolated mammary epithelial cells (MECs) from four CD1 mice each at Pregnancy day 14 (P14) and Lactation day 2 (L2) was performed in the current study. Statistical analysis of the miRNA changes between P14 and L2 identified 32 miRNAs to be differentially expressed with a fold change greater than or equal to 2, of which, the majority of them declinied at the onset of lactation.
Project description:To study the effect of pregnancy on mouse mammary epithelial subpopulations, epthelial cells derived from virgin or pregnant (12.5 day pregnant) mice were isolated using fluorescence-activated cell sorting. After elimination of haematopoietic and endothelial cells, two distinct epithelial subpopulations were sorted using antibodies against CD29 and CD24. Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as mammary stem cell enriched (CD29hiCD24+) and luminal (CD29loCD24+) respectively (ref: Shackleton et al, Nature 2006). Microarray profiling was used to compare gene expression profiles of the two subpopulations in 12.5 day pregnant and virgin mice.
Project description:This study examined the effect of early pregnancy on the gene expression profile of total isolated mammary epithelial cells in mice.
Project description:This study examined the effect of early pregnancy on the gene expression profile of total isolated mammary epithelial cells in mice. Total mammary epithelial cells were isolated from parous and age-matched virgin control mice. Four independent replicates were assessed per treatment group, resulting in a total of 8 samples.
Project description:RNA was isolated from mammary glands from 55 day old control mice, mice overexpressing the miR-200b/200a/429 cluster in mammary epithelial cells (MTB-200ba429) mice overexpressing the IGF-IR transgene in mammary epithelial cells (MTB-IGFIR), and mice overexpressing both the miR-200b/200a/429 cluster and the IGF-IR transgene in mammary epithelial cells (MTB-IGFIRba429)
Project description:To study the effect of pregnancy on mouse mammary epithelial subpopulations, epthelial cells derived from virgin or pregnant (12.5 day pregnant) mice were isolated using fluorescence-activated cell sorting. After elimination of haematopoietic and endothelial cells, two distinct epithelial subpopulations were sorted using antibodies against CD29 and CD24. Based on the immunohistochemical phenotype, and in vivo and in vitro functional assays, these subpopulations were identified as mammary stem cell enriched (CD29hiCD24+) and luminal (CD29loCD24+) respectively (ref: Shackleton et al, Nature 2006). Microarray profiling was used to compare gene expression profiles of the two subpopulations in 12.5 day pregnant and virgin mice. For each biological replicate, mammary gland from virgin or 12.5 day pregnant FVB/NJ mice were collected and digested to obtain a single cell suspension. CD45-CD31-TER119- cells were then sorted based on the expression of cell surface markers CD24 and CD29. There were 4 pools of pregant mice and 3 pools of virgin mice.
Project description:Stat1-null mice (129S6/SvEvTac-Stat1tm1Rds homozygous) uniquely develop estrogen-receptor-positive mammary tumors with incomplete penetrance and long latency. We studied the growth and development of the mammary glands in Stat1-null mice. Stat1-null MGs have faulty branching morphogenesis with abnormal terminal end buds. The Stat1-null MG also fails to sustain growth of 129S6/SvEv wild-type and null epithelium. These abnormalities are partially reversed by added progesterone and prolactin. Transplantation of wild-type bone-marrow into Stat1-null mice does not reverse the mammary gland developmental defects. Media conditioned by Stat1-null epithelium-cleared mammary fat pads does not stimulate epithelial proliferation whereas it is stimulated by conditioned media derived from either wild-type or progesterone and prolactin-treated Stat1-null epithelium-cleared mammary fat pads. Microarrays and multiplex cytokine protein assays showed that the mammary gland of Stat1-null mice had lower levels of growth factors that have been implicated in normal mammary gland growth and development. Transplanted Stat1-null tumors and their isolated cells also grow slower in Stat1-null mammary gland compared to wild-type recipient mammary gland. Stat1-null hosts responded to tumor transplants with granulocytic infiltrates while wild-type hosts show a mononuclear response. These studies demonstrate that growth of normal and neoplastic Stat1-null epithelium primarily depends on the hormonal milieu and factors, such as cytokines, from the mammary stroma.