Cell-specific metallothionein gene expression in mouse decidua and placentae

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.     

Zinc concentrations in mouse embryo and maternal plasma

The effect of a single teratogenic dose of the antiepileptic drug valproic acid and its nonteratogenic metabolite, 2-en-valproic acid, on zinc concentrations in mouse plasma, embryo, and decidua on d 9 of gestation was investigated. The substances were injected subcutaneously (sc) as their sodium salts. In this mouse model, valproic acid induced between 20% (400 mg/kg dose) and 60% (600 mg/kg dose) incidence of exencephaly in living fetuses; 2-en-valproic acid was not teratogenic at these dose levels. The zinc concentrations in plasma were significantly increased 1 and 2 h after administration of both substances. The embryonic zinc concentrations were increased 2 and 4 h after application of both substances. The concentrations of zinc in the decidua were not affected. The similarity of effects of valproic acid and its nonteratogenic analog on zinc concentrations in maternal plasma and embryo suggests that the teratogenicity of a single administration of valproic acid in the mouse is not owing to interference with the zinc metabolism in this species.     

Characterization of murine decidual natural killer (NK) cells and their relevance to the success of pregnancy

The identification of lytic cells in 6.5-day to 9.5-day murine decidua as NK cells has been extended. The cells with natural killer (NK) activity in early decidua were nonphagocytic and heterogeneous in size as assessed by velocity sedimentation at unit gravity. The numbers of lytic cells were reduced by treatment with anti-asialo GM1 in vivo and they were absent from the decidua of bg/bg mice. Thus, decidual NK cells were not distinct from NK cells in other tissues. The decline in the levels of decidual NK activity as pregnancy progressed was attributed to their regulation by other cells present in decidua by midgestation. The development of NK activity in decidua was dependent upon the presence of an embryo, however, decidual NK cells were not essential for successful pregnancy because viable offspring were obtained from mice lacking decidual NK activity. It was shown that NK cells from either spleen or decidua were unlikely to cause damage to embryos during the first half of pregnancy as freshly dissociated 9.5- and 11.5-day embryonic cells resisted NK lysis. Furthermore, blastocysts were not damaged by coincubation with splenic or decidual NK cells and were viable upon subsequent embryo transfer. These studies indicate that decidual NK cells are not essential for successful pregnancy and are not necessarily detrimental to early embryos. It is suggested that decidual NK cells may play other nonimmunological roles during embryonic development.     

A functional overlap of plasminogen and MMPs regulates vascularization during placental development

Both plasminogen activators and matrix metalloproteinases (MMPs) have been implicated in a variety of developmental processes in the mouse during embryo implantation and placentation. We show here that pharmacological treatment of plasminogen-deficient mice with the broad spectrum MMP inhibitor galardin leads to a high rate of embryonic lethality. Implantation sites from plasminogen-deficient galardin-treated mice at 7.5 days post coitus (dpc) showed delay in both decidualization and invasion of maternal vessels into the decidua. At 8.5 dpc, half of the embryos were runted and still at the developmental stage of a 7.5 dpc embryo. Most embryos that escaped these initial defects eventually died, probably from defective vascularization and development of the labyrinth layer of the placenta, although a direct role on embryo development cannot be ruled out. These results demonstrate that the combination of MMPs and plasminogen is essential for the proper development of the placenta. Plasminogen deficiency alone and galardin treatment alone had much less effect and there was a pronounced synergism on both placental vascularization and embryonic lethality, indicating a functional overlap between plasminogen and MMPs.     

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.     

Embryonic Expression of Vasoactive Intestinal Peptide (VIP) and VIP Receptor Genes

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a ³³P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.     

Distribution of hyaluronan in the mouse endometrium during the periimplantation period of pregnancy

The tissue distribution of stromal hyaluronan (HA) in the periimplantation mouse uterus was studied histochemically using a biotin-labelled HA-binding complex from cartilage proteoglycan. HA is present around proliferating stromal cells in both the pregnant and pseudopregnant mouse uterus prior to their differentiation into the decidualized phenotype. Decidualization is accompanied by clearance of HA from the extracellular matrix (ECM). This clearing is part of an intrinsic developmental program of the differentiating deciduum. A specific embryonic signal from the implanting conceptus is not required for this phenomenon to occur, since a similar response could be induced in deciduoma produced by artificial stimulation of a receptive uterus. Clearing of HA from the antimesometrial stroma is consistent with the hypothesis that the HA-negative decidual cell may be involved in restricting the invasion of trophoblast cells during embryo implantation. Retention of HA within angiogenic regions of the decidua basalis implies a functional role for this molecule in placental vascularization.     

Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development

The zebra finch (Taeniopygiaguttata) has become an increasingly important model organism in many areas of research including toxicology^1,2, behavior³, and memory and learning^4,5,6. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.     

Developmental expression of adenosine deaminase in placental tissues of the early postimplantation mouse embryo and uterine stroma

In this study, we have investigated the distribution of adenosine deaminase (ADA) in embryonic, extra-embryonic, and decidual tissues of the developing mouse embryo. ADA catalyzes a key step in purine metabolism converting adenosine to inosine. ADA specific activity (nmol/min/micrograms protein) was present at low levels in the embryo-decidual unit during the first 2 days of postimplantation development but then increased starting late on Day 6 of gestation (Day 0 plug). By Day 9, ADA specific activity was 80-fold higher than on Day 6. A histochemical staining method for ADA activity was applied to cryostat sections of the implantation site. The developmental increase localized primarily to the trophoblast/antimesometrial decidua interface between Days 7 and 9 of gestation, and decidua basalis and the metrial gland by Day 11. Immunofluorescent staining with sheep anti-mouse ADA antiserum confirmed the presence of ADA antigenicity in tissues forming the maternal/fetal interface. ADA specific activity was 19-fold higher in homogenates of the Day 11 decidua/parietal yolk sac than in the thymus, a tissue generally thought of as ADA-rich. High levels of ADA activity and immunoreactivity were also detected in the embryonal plasma during organogenesis, but the embryo proper showed only low levels. These results indicate that ADA is tightly regulated within tissues forming the maternal/fetal interface during early postimplantation stages of development.     

A uterine decidual cell cytokine ensures pregnancy-dependent adaptations to a physiological stressor

In the mouse, decidual cells differentiate from uterine stromal cells in response to steroid hormones and signals arising from the embryo. Decidual cells are crucially involved in creating the intrauterine environment conducive to embryonic development. Among their many functions is the production of cytokines related to prolactin (PRL), including decidual prolactin-related protein (DPRP). DPRP is a heparin-binding cytokine, which is abundantly expressed in uterine decidua. In this investigation, we have isolated the mouse Dprp gene, characterized its structure and evaluated its biological role. Dprp-null mice were made by replacing exons 2 to 6 of the Dprp gene with an in-frame enhanced green fluorescent protein (EGFP) gene and a neomycin (neo) resistance cassette. Heterozygous intercross breeding of the mutant mice yielded the expected mendelian ratio. Pregnant heterozygote females expressed EGFP within decidual tissue in locations identical to endogenous Dprp mRNA and protein expression. Homozygous Dprp-null mutant male and female mice were viable, exhibited normal postnatal growth rates, were fertile and produced normal litter sizes. A prominent phenotype was observed when pregnant Dprp-null mice were exposed to a physiological stressor. DPRP deficiency interfered with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure. Termination of pregnancy was associated with aberrations in mesometrial decidual cells, mesometrial vascular integrity, and disruptions in chorioallantoic placenta morphogenesis. The observations suggest that DPRP participates in pregnancy-dependent adaptations to a physiological stressor.     

Expression of a glucose-regulated cell surface protein in early mouse embryos

A 100,000-Da glucose-regulated surface protein (100K-GRP) has previously been isolated from the cell surface and culture medium of human fibroblasts. A rabbit antiserum directed against this protein reacts with the cell surface of both human and murine cultured cells and with a broad spectrum of mammalian tissues. It is shown, via indirect immunofluorescence, that this protein is also present on cells of the developing mouse embryo and can be detected as early as the 4-cell stage. The 8-cell embryo and morula show positive surface labeling; the inner cell masses of both the pre- and postimplantation blastocysts are also positive but the trophectoderm is not. At the 6-day egg cylinder stage, the embryonic and extra-embryonic ectoderm label intensely with the antiserum and visceral endoderm shows faint labeling. No labeling can be detected on parietal endoderm or on the trophoblastic giant cells invading the uterine decidua. However, the internal cells of the ectoplacental cone exhibit bright fluorescence. The same pattern is observed on 7- to 8.5-day embryos, except that at this stage no label is associated with the visceral endoderm. In addition, mesodermal cells emerging from the primitive streak are also labeled.     

Cell-type-specific expression of transforming growth factor-alpha in the mouse uterus during the peri-implantation period.

Immunohistochemistry as well as in situ and Northern blot hybridization were employed to determine temporal and cell-type-specific expression of transforming growth factor-alpha (TGF-alpha) in the mouse uterus during the peri-implantation period. The co-localization of TGF-alpha (by immunohistochemistry) with its mRNA (by in situ hybridization) in the luminal and glandular epithelia on Days 1-4 of pregnancy (Day 1 = vaginal plug) and also in many stromal cells on Days 3 and 4 indicates that these cells are the primary sites of TGF-alpha synthesis during the preimplantation period. The higher levels of TGF-alpha mRNA in total uterine RNA on Day 4, as shown by Northern blotting, is consistent with the recruitment of stromal cells expressing this gene. During the post-implantation period (Days 5-8), the co-localization of the mRNA and protein in the decidua at the implantation sites suggests that the decidualizing stromal cells synthesize TGF-alpha. Although in situ hybridization showed the presence of mRNA in embryos on Days 5-8, immunostaining was noted in the embryo only on Days 5 and 6. These results suggest that uterine and embryonic expression of TGF-alpha during the peri-implantation period could be involved in embryonic development, preparation of the uterus for implantation, and decidualization.     

Culture of mouse neural stem cell precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types. This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.