Mouse Yolk Sac Research Articles

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells.

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using mouse yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

FOXO1 represses sprouty 2 and sprouty 4 expression to promote arterial specification and vascular remodeling in the mouse yolk sac.

Establishing a functional circulatory system is required for post-implantation development during murine embryogenesis. Previous studies in loss-of-function mouse models showed that FOXO1, a Forkhead family transcription factor, is required for yolk sac (YS) vascular remodeling and survival beyond embryonic day (E) 11. Here, we demonstrate that at E8.25, loss of Foxo1 in Tie2-cre expressing cells resulted in increased sprouty 2 (Spry2) and Spry4 expression, reduced arterial gene expression and reduced Kdr (also known as Vegfr2 and Flk1) transcripts without affecting overall endothelial cell identity, survival or proliferation. Using a Dll4-BAC-nlacZ reporter line, we found that one of the earliest expressed arterial genes, delta like 4, is significantly reduced in Foxo1 mutant YS without being substantially affected in the embryo proper. We show that FOXO1 binds directly to previously identified Spry2 gene regulatory elements (GREs) and newly identified, evolutionarily conserved Spry4 GREs to repress their expression. Furthermore, overexpression of Spry4 in transient transgenic embryos largely recapitulates the reduced expression of arterial genes seen in conditional Foxo1 mutants. Together, these data reveal a novel role for FOXO1 as a key transcriptional repressor regulating both pre-flow arterial specification and subsequent vessel remodeling within the murine YS.

Translational Comparison of the Human and Mouse Yolk Sac Development and Function.

The yolk sac (YS) is the oldest of the extraembryonic membranes in vertebrates. Considered a transitory structure in the human species, the importance of the YS for a successful pregnancy is often overlooked. Due to the general inaccessibility of healthy human YS tissue for research, the use of experimental animal models is of great value. In order to better understand whether the mouse could be used as a translational model for the study of the human YS under normal and pathological conditions, this review comprehensively describes key developmental aspects of the human and mouse YS, detailing their development and function. YS major similarities in both species comprise the following: (1) histological composition (both being composed of endoderm, mesoderm, and mesothelium layers); (2) endoderm endocytosis, synthesis, secretion, and transport capabilities; and (3) mesoderm onset of haematopoiesis and angiogenesis. Examples of main dissimilarities include (1) persistence across pregnancy (i.e. early pregnancy in humans vs term pregnancy in mice); (2) the existence of a secondary YS in humans; (3) the presence of proliferative primordial germ cells (PGCs) in the human versus their absence in mice; and (4) eversion of histological layers in the mouse. Although these differences should be considered when interpreting data from mouse-based studies, the overall morphofunctional similarities in the YS between these species indicate that the mouse can be potentially used as a translational model for the study of the human YS.

Modeling human yolk sac hematopoiesis with pluripotent stem cells.

In the mouse, the first hematopoietic cells are generated in the yolk sac from the primitive, erythro-myeloid progenitor (EMP) and lymphoid programs that are specified before the emergence of hematopoietic stem cells. While many of the yolk sac-derived populations are transient, specific immune cell progeny seed developing tissues, where they function into adult life. To access the human equivalent of these lineages, we modeled yolk sac hematopoietic development using pluripotent stem cell differentiation. Here, we show that the combination of Activin A, BMP4, and FGF2 induces a population of KDR+CD235a/b+ mesoderm that gives rise to the spectrum of erythroid, myeloid, and T lymphoid lineages characteristic of the mouse yolk sac hematopoietic programs, including the Vδ2+ subset of γ/δ T cells that develops early in the human embryo. Through clonal analyses, we identified a multipotent hematopoietic progenitor with erythroid, myeloid, and T lymphoid potential, suggesting that the yolk sac EMP and lymphoid lineages may develop from a common progenitor.

Breast cancer resistance protein (Bcrp/Abcg2) is selectively modulated by lipopolysaccharide (LPS) in the mouse yolk sac

Bacterial infection alters placental ABC transporters expression. These transporters provide fetal protection against circulating xenobiotics and environmental toxins present in maternal blood. We hypothesized that lipopolysaccharide (LPS-bacterial mimic) alters the yolk sac morphology and expression of key ABC transporters in a gestational-age dependent manner. Yolk sac samples from C57BL/6 mice were obtained at gestational ages (GD) 15.5 and GD18.5, 4 or 24 h after LPS exposure (150ug/kg; n = 8/group). Samples underwent morphometrical, qPCR and immunohistochemistry analysis. The volumetric proportions of the histological components of the yolk sac did not change in response to LPS. LPS increased Abcg2 expression at GD15.5, after 4 h of treatment (p < 0.05). No changes in Abca1, Abcb1a/b, Abcg1, Glut1, Snat1, Il-1β, Ccl2 and Mif were observed. Il-6 and Cxcl1 were undetectable in the yolk sac throughout pregnancy. Abca1, breast cancer resistance protein (Bcrp, encoded by Abcg2) and P-glycoprotein (P-gp/ Abcb1a/b) were localized in the endodermal (uterine-facing) epithelium and to a lesser extent in the mesothelium (amnion-facing), whereas Abca1 was also localized to the endothelium of the yolk sac blood vessels. LPS increased the labeling area and intensity of Bcrp in the yolk sac’s mesothelial cells at GD15.5 (4 h), whereas at GD18.5, the area of Bcrp labeling in the mesothelium (4 and 24 h) was decreased (p < 0.05). Bacterial infection has the potential to change yolk sac barrier function by affecting Bcrp and Abcg2 expression in a gestational-age dependent-manner. These changes may alter fetal exposure to xenobiotics and toxic substances present in the maternal circulation and in the uterine cavity.

Isolation of High-Molecular-Weight DNA from Mouse Yolk Sacs and the Like.

Often, genotyping of mouse embryos is required, and a small part, such as the yolk sac, can be used for this purpose. Here, DNA samples are prepared from extra-embryonic tissues by digestion with Proteinase K and subsequent extraction. The yolk sac of mid-gestation or later-stage embryos provides a sufficient amount of DNA for Southern analysis. Small tissues of a few hundred cells are used for genotyping early postimplantation- and preimplantation-stage embryos by polymerase chain reaction (PCR).

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.

Mouse Yolk Sac Hematopoiesis.

The yolk sac is the first observed site of hematopoiesis during mouse ontogeny. Primitive erythroid cells are the most well-recognized cell lineages produced from this tissue. In addition to primitive erythroid cells, several types of hematopoietic cells are present, including multipotent hematopoietic progenitors. Yolk sac-derived blood cells constitute a transient wave of embryonic and fetal hematopoiesis. However, recent studies have demonstrated that some macrophage and B cell lineages derived from the early yolk sac may persist to adulthood. This review discusses the cellular basis of mouse yolk sac hematopoiesis and its contributions to embryonic and adult hematopoietic systems.

Yolk sac macrophage progenitors traffic to the embryo during defined stages of development

Tissue macrophages in many adult organs originate from yolk sac (YS) progenitors, which invade the developing embryo and persist by means of local self-renewal. However, the route and characteristics of YS macrophage trafficking during embryogenesis are incompletely understood. Here we show the early migration dynamics of YS-derived macrophage progenitors in vivo using fate mapping and intravital microscopy. From embryonic day 8.5 (E8.5) CX3CR1+ pre-macrophages are present in the mouse YS where they rapidly proliferate and gain access to the bloodstream to migrate towards the embryo. Trafficking of pre-macrophages and their progenitors from the YS to tissues peaks around E10.5, dramatically decreases towards E12.5 and is no longer evident from E14.5 onwards. Thus, YS progenitors use the vascular system during a restricted time window of embryogenesis to invade the growing fetus. These findings close an important gap in our understanding of the development of the innate immune system.

Visualizing the Vascular Network in the Mouse Embryo and Yolk Sac.

Whole mount immunofluorescence is a valuable technique that can be used to visualize vascular networks in early developing embryonic tissues. This technique involves the permeabilization of fixed mouse embryos and yolk sacs, and primary antibody tagging of the endothelial cell marker platelet endothelial cell adhesion molecule 1 (Pecam-1). A secondary antibody tagged with a fluorophore targets the primary antibody, fluorescently labeling endothelial cells and revealing vascular networks.

Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Vitamin B12 deficiency causes megaloblastic anemia and neurologic disorder in humans. Gene defects of transcobalamin (TC) and the transcobalamin receptor (TCblR), needed for cellular uptake of the TC-bound B12, do not confer embryonic lethality. TC deficiency can produce the hematologic and neurologic complications after birth, whereas TCblR/CD320 gene defects appear to produce mild metabolic changes. Alternate maternofetal transport mechanisms appear to provide adequate B12 to the fetus. To understand this mechanism, we evaluated the role of TC, TCblR/CD320, and megalin in maternofetal transport of B12 in a TCblR/CD320-knockout (KO) mouse. Our results showed high expression of TCblR/CD320 in the labyrinth of the placenta, embryonic brain, and spinal column in wild-type (WT) mice. Megalin expression was about the same in both WT and KO mouse visceral yolk sac, brain, and spinal column. Megalin mRNA was down-regulated in the KO embryonic spinal cord (SC) and kidneys. Megalin expression remained unaltered in adult WT and KO mouse brain, SC, and kidneys. Injected dsRed-TC-B12 and TC-57CoB12 accumulated in the visceral yolk sac of KO mice where megalin is expressed and provides an alternate mechanism for the maternofetal transport of Cbl during fetal development.-Arora, K., Sequeira, J. M., Quadros, E. V. Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Distinct regulatory networks control the development of macrophages of different origins in zebrafish

AbstractMacrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors.

Identification of novel genetic markers for mouse yolk sac cells by using microarray analyses

The mouse embryonic yolk sac consists of a visceral yolk sac (VYS) and parietal yolk sac (PYS), and may function as a materno-fetal exchange system for nutrients and wastes, and physical protector for the embryo/fetus. The present study was undertaken to characterize gene expression of the VYS and PYS endodermal cells, and to identify their novel genetic markers from microarray data. Apoa4, Lrp2, Fxyd2, Slc34a3 and Entpd2 were predominantly expressed in VYS epithelial cells. Gkn2 and Pga5 were selected as markers for PYS cells. These genetic markers may be useful for characterization of murine yolk sacs during development.

Distribution and accumulation of 10 nm silver nanoparticles in maternal tissues and visceral yolk sac of pregnant mice, and a potential effect on embryo growth

We examined the distribution of silver in pregnant mice and embryos/fetuses following intravenous injections of 10 nm silver nanoparticles (AgNPs) or soluble silver nitrate (AgNO3) at dose levels of 0 (citrate buffer control) or 66 µg Ag/mouse to pregnant mice on gestation days (GDs) 7, 8 and 9. Selected maternal tissues and all embryos/fetuses from control, AgNP- and AgNO3-treated groups on GD10 and control and AgNP-treated groups on GD16 were processed for the measurement of silver concentrations, intracellular AgNP localization, histopathology and gross examination of tissue morphology. Inductively-coupled plasma mass spectrometry revealed silver in all examined tissues following either AgNP or AgNO3 treatment, with highest concentrations of silver in maternal liver, spleen and visceral yolk sac (VYS), and lowest concentrations in embryos/fetuses. For VYS, mean silver concentration following AgNO3 treatment (4.87 ng Ag/mg tissue) was approximately two-fold that following AgNP treatment (2.31 ng Ag/mg tissue); for all other tissues examined, mean silver concentrations following either AgNP or AgNO3 treatment were not significantly different from each other (e.g. 2.57 or 2.84 ng Ag/mg tissue in maternal liver and 1.61 or 2.50 ng Ag/mg tissue in maternal spleen following AgNP or AgNO3 treatment, respectively). Hyperspectral imaging revealed AgNP aggregates in maternal liver, kidney, spleen and VYS from AgNP-treated mice, but not AgNO3-treated mice. Additionally, one or more embryos collected on GD10 from eight of ten AgNP-treated mice appeared small for their age (i.e. Theiler stage 13 [GD8.5] or younger). In the control group (N = 11), this effect was seen in embryos from only one mouse. In conclusion, intravenous injection of 10 nm AgNPs to pregnant mice resulted in notable silver accumulation in maternal liver, spleen and VYS, and may have affected embryonic growth. Silver accumulation in embryos/fetuses was negligible.

The placenta is the culprit in programming chronic disease

s / Placenta 36 (2015) A1eA60 A2 IFPASA. FETAL MEMBRANES e A FOCUS FOR THE FUTURE OR A LEGACY FROM THE PAST? A.M. Carter. University of Southern Denmark, Denmark The fetal membranes of amniotes are allantois, amnion, chorion and yolk sac. The manner of their development and contribution to placentation varies greatly acrossmammals. This is true also of the cavities they enclose, which are the allantoic sac, amniotic sac and exocoelom. In human development, there is an allantoic stalk, but no allantoic sac; the yolk sac is a short-lived structure and the exocoelom is obliterated in the second trimester by expansion of the amnion. The mouse is commonly used as a model of human placentation. Here, too, there is no allantoic sac and the exocoelom does not persist to term. In contrast, the inverted vascular yolk sac supports the embryo until the chorioallantoic placenta is formed and it acts as an accessory placenta through term. It has a string of important functions yet frequently is discarded or ignored either through ignorance or because it has no obvious equivalent in human pregnancy. In ruminants, which are important to veterinary research and as models in fetal physiology, there is yet another pattern that includes transient yolk sac placentation and a persistent allantoic sac. Embryologists of the late 19C, like Hubrecht and Selenka, uncovered the role of fetal membranes in early development and placentation. They feature prominently in the mid-20C accounts of Amoroso and Mossman. This legacy seems largely to have slipped from the collective consciousness of 21C placentologists. Yet the mouse yolk sac may harbour clues to overall placental function and human yolk sac play a critical role in embryonic development. Medical as well as veterinary research might be well served by a closer focus on fetal membranes. KEY1. THE PLACENTA IS THE CULPRIT IN PROGRAMMING CHRONIC DISEASE K. Thornburg, S. Louey. Melinda Pierce and Kevin Kolahi Center for Developmental Health, Knight Cardiovascular Institute, School of Medicine, Oregon Health and Science University, Portland, USA For the first time in US history, a sustained decrease in life expectancy is predicted. The abrupt change in the upward trajectory in life expectancy is related to increases in the prevalence of obesity, diabetes and uncontrolled hypertension, all of which have been increasing since the ‘90s. Children in the US are now the 3rd generation of people who have consumed mostly processed foods to satisfy their energy requirements. Processed foods are generally high in calories and low in nutrients. It is now well known that babies born at the extremes of birthweights are especially prone to developing chronic disease later in life. Because the placenta is the gatekeeper for nutrients entering the fetus and because the vascular bed of the placenta sets the loading conditions of the embryo/fetal heart, the placenta plays a central role in determining risk for later disease. The size and shape of the placenta interact with maternal phenotypic features to determine risk of coronary heart disease, heart failure and sudden death. Lung and colorectal cancers are predicted by placental size and shape. The number of placental cotyledons predicts blood pressure in children and umbilical cord length is associated with an elevated risk for rheumatic heart disease. Placental size, shape and efficiency in a population vary over time for unknown reasons. One can fairly speculate that population level dietary changes over time are the drivers of placentation; animal data are sparse but support the idea. Sex-specific features of placental function have been shown in humans and animals but the role of diet in altering functional changes is unknown. Understanding the interactions between maternal diet, stress, phenotype and sex-specific placental form and function should be a high priority for scientists and funding agencies. Healthy placentas make healthy babies. Healthy babies are the foundation of a healthy population KEY3. AGING AND THE PLACENTA Roger Smith. Mothers and Babies Research Centre, HMRI, Newcastle, NSW,

Trophoblast expression dynamics of the tumor suppressor gene gastrokine 2.

Gastrokines (GKNs) were originally described as stomach-specific tumor suppressor genes. Recently, we identified GKN1 in extravillous trophoblasts (EVT) of human placenta. GKN1 treatment reduced the migration of the trophoblast cell line JEG-3. GKN2 is known to inhibit the proliferation, migration and invasion of gastric cancer cells and may interact with GKN1. Recently, GKN2 was detected in the placental yolk sac of mice. We therefore aimed to further characterize placental GKN2 expression. By immunohistochemistry, healthy first-trimester placenta showed ubiquitous staining for GKN2 at its early gestational stage. At later gestational stages, a more differentiated expression pattern in EVT and villous cytotrophoblasts became evident. In healthy third-trimester placenta, only EVT retained strong GKN2 immunoreactivity. In contrast, HELLP placentas showed a tendency of increased levels of GKN2 expression with a more prominent GKN2 staining in their syncytiotrophoblast. Choriocarcinoma cell lines did not express GKN2. Besides its trophoblastic expression, we found human GKN2 in fibrotic villi, in amniotic membrane and umbilical cord. GKN2 co-localized with smooth muscle actin in villous myofibroblasts and with HLA-G and GKN1 in EVT. In the rodent placenta, GKN2 was specifically located in the spongiotrophoblast layer. Thus, the gestational age-dependent and compartment-specific expression pattern of GKN2 points to a role for placental development. The syncytial expression of GKN2 in HELLP placentas might represent a reduced state of functional differentiation of the syncytiotrophoblast. Moreover, the specific GKN2 expression in the rodent spongiotrophoblast layer (equivalent to human EVT) might suggest an important role in EVT physiology.

Corrigendum: Vascular development and hemodynamic force in the mouse yolk sac

The article “Vascular development and hemodynamic force in the mouse yolk sac” that is part of the research topic “Mechanotransduction and Development of Cardiovascular Form and Function” published 20 August 2014, is missing an Acknowledgment section.Acknowledgment This work is supported by the National Institutes of Health (R01HL120140 and U54HG006348) as well as by the Optical Imaging and Vital Microscopy core at Baylor College of Medicine.

Gastrokine-2 is transiently expressed in the endodermal and endothelial cells of the maturing mouse yolk sac

The yolk sac (YS) is a thin, bi-layered membrane encompassing the developing mammalian embryo in utero. The outer layer of the YS is composed of visceral endodermal cells derived from the primitive endoderm. The inner mesenchymal layer is highly vascularised and the first source of haematopoietic cells. YS haematopoiesis takes place from shortly after gastrulation (E7.5) until after midgestation (~E12.5) when the placenta and sites within the embryo proper predominate as sites of blood cell production. Here, we have assessed gene expression in the developing YS to determine what factors are associated with the end of haematopoietic cell production in this extra-embryonic tissue. We have observed that transcripts encoding gastrokine-2 (Gkn2) are up-regulated in the YS at E11.5, peak at E12.5 and are then reduced. Low levels of Gkn2 transcript were detected in purified YS endothelial cells. Gastrokine-2 protein was detected in the EpCAM-expressing visceral endodermal layer. Gastrokine-2 protein predominantly clustered at the basal side of the visceral endodermal cells facing the endothelial cell layer though did not appear to be strongly associated with secretory lysosomes. Gastrokine-1 protein binds F-actin. In contrast, gastrokine-2 in the YS is predominantly expressed at the basal side of the visceral endodermal cells, whereas F-actin in the YS is mostly found in the inner leaf of the luminal side of visceral endoderm. We have therefore identified expression of Gkn2 transcript and protein in the visceral endodermal layer and at lower levels in YS endothelial cells. This previously unreported expression of a “stomach-specific” secreted protein may be linked to the changes in the YS niche coinciding with the shift of haematopoiesis away from the YS into the embryo.

Vascular development and hemodynamic force in the mouse yolk sac.

Vascular remodeling of the mouse embryonic yolk sac is a highly dynamic process dependent on multiple genetic signaling pathways as well as biomechanical factors regulating proliferation, differentiation, migration, cell-cell, and cell-matrix interactions. During this early developmental window, the initial primitive vascular network of the yolk sac undergoes a dynamic remodeling process concurrent with the onset of blood flow, in which endothelial cells establish a branched, hierarchical structure of large vessels and smaller capillary beds. In this review, we will describe the molecular and biomechanical regulators which guide vascular remodeling in the mouse embryonic yolk sac, as well as live imaging methods for characterizing endothelial cell and hemodynamic function in cultured embryos.