Epiblast Of Embryo Research Articles

Revealing cell populations catching the early stages of human embryo development in naive pluripotent stem cell cultures.

Naive human pluripotent stem cells (hPSCs) are defined as the invitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period.

Asymmetry in the frequency and position of mitosis in the mouse embryo epiblast at gastrulation.

At gastrulation, a subpopulation of epiblast cells constitutes a transient posteriorly located structure called the primitive streak, where cells that undergo epithelial-mesenchymal transition make up the mesoderm and endoderm lineages. Mouse embryo epiblast cells were labelled ubiquitously or in a mosaic fashion. Cell shape, packing, organization and division were recorded through live imaging during primitive streak formation. Posterior epiblast displays a higher frequency of rosettes, some of which associate with a central cell undergoing mitosis. Cells at the primitive streak, in particular delaminating cells, undergo mitosis more frequently than other epiblast cells. In pseudostratified epithelia, mitosis takes place at the apical side of the epithelium. However, mitosis is not restricted to the apical side of the epiblast, particularly on its posterior side. Non-apical mitosis occurs specifically in the streak even when ectopically located. Posterior non-apical mitosis results in one or two daughter cells leaving the epiblast layer. Cell rearrangement associated with mitotic cell rounding in posterior epiblast, in particular when non-apical, might thus facilitate cell ingression and transition to a mesenchymal phenotype.

Myo/Nog cells are nonprofessional phagocytes.

Myo/Nog cells were discovered in the chick embryo epiblast. Their expression of MyoD reflects a commitment to the skeletal muscle lineage and capacity to differentiate into myofibroblasts. Release of Noggin by Myo/Nog cells is essential for normal morphogenesis. Myo/Nog cells rapidly respond to wounding in the skin and eyes. In this report, we present evidence suggesting that Myo/Nog cells phagocytose tattoo ink in tissue sections of human skin and engulf cell corpses in cultures of anterior human lens tissue and magnetic beads injected into the anterior chamber of mice in vivo. Myo/Nog cells are distinct from macrophages in the skin and eyes indicated by the absence of labeling with an antibody to ionized calcium binding adaptor molecule 1. In addition to their primary roles as regulators of BMP signaling and progenitors of myofibroblasts, Myo/Nog cells behave as nonprofessional phagocytes defined as cells whose primary functions are unrelated to phagocytosis but are capable of engulfment.

MLL1: the thin red line divides naïve and primed pluripotency.

From mouse pre-implantation and early post-implantation embryos, stem cells at different pluripotent states can be captured in in vitro culture. For example, naive embryonic stem cells (ESC) are derived from inner cell mass (ICM) of the blastocyst, while primed epiblast stem cells (EpiSC) are captured from the post-implantation embryo epiblast. Though both ESCs and EpiSCs are pluripotent stem cells, they have distinct characters in terms of pluripotent gene expression profile, epigenetic status, metabolic pathways, growth factor requirement, and in female, the X chromosome inactivation status (1). On the other hand, these two pluripotent states are interchangeable, once switching the culture condition of ESCs to that for EpiSCs, naive ESCs convert to EpiSCs. The efficiency of converting EpiSCs to ESCs, however, is much low, which usually requires over-expressing pluripotency-associated genes such as Nanog , Klf4 , Esrrb , Tfcp2l1 and Nr5a2 (2-6). Similar to somatic cell reprogramming to induced pluripotent stem cells, converting EpiSCs to ESCs is an epigenome-resetting process. Characterizing these epigenetic barriers represents an important approach to further improve reprogramming efficiency (7).

Genetic and Epigenetic landscape of Germline Stem Cells

Elucidating the critical epigenetics events involved in differentiation and reprogramming of cells to primordial germ cells (PGCs) is among the interesting issues in stem cell research. Here, I will talk about critical transcription factors and global hypomethylation in development of germ cells. Evidence strongly suggests that the earliest PGCs emerging in the E7.25 mouse embryo epiblast have a highly methylated genome, and high level of H3K9me2 in chromatin but during development, genome demethylated and patterns of histone codes changes dramatically. We designed a polycistroniclentiviral vector and overexpressed Stella, Oct4 and Nanos2 simultaneously in transduced cells; Increasing level of Prdm14, Nanog and decreasing of G9a expression is an interesting finding which might be considered as a primary step of reprogramming toward germline progenitor cells, here we propose decreasing H3K9me2 level as a consequence of G9a down regulation is a critical step which facilitated transition to different stemness state through creating a new epigenetic memory for the early germ cells. Keywords: Epigenetic, hypomethylation, Germ line Stem Cells, polycistroniclentiviral vector.

Surface markers in stem cells and cancer from the perspective of glycomic analysis

Most cancers are detected when patients present with symptoms, and at that point the disease is usually quite advanced and often not curable. Therefore, new biomarkers are needed for detection and therapy. The recent success of using monoclonal antibodies against nonprotein gangliosides for the treatment of high-risk neuroblastoma provides an incentive to search for new glycan-targeted immunotherapies for cancer using markers found through glycomic analysis as targets. Since more than 85% of cell surface components are glycosylated, glycomic analysis is useful to probe systematically the cancer cell surface, in search for novel glycoproteins and glycolipids. Furthermore, cancer cells tend to dedifferentiate and express many oncofetoproteins, since human embryonic stem cells (ESCs) are derived from epiblast of embryo, representing the early stage of normal embryonic development before gastrulation. Unique ESC surface markers are likely to be found in cancer cells, but not in normal mature tissues. Moreover, stem cells and cancer cells share several common features in related regulatory mechanisms and signaling pathways. Thus, identification of the cancer stem cells in cancer and definition of the glycoproteomic changes that accompany their transformation are important for the development of strategies for early detection and treatment of cancer.

Spatially and temporally controlled electroporation of early chick embryos

The introduction of in ovo electroporation a decade ago has helped the chick embryo to become a powerful system to study gene regulation and function during development. Although this is a simple procedure for embryos of 2-d incubation, earlier stages (from laying to early neurulation, 0-1 d) present special challenges. Here we describe a robust and reproducible protocol for electroporation of expression vectors and morpholino oligonucleotides into the epiblast of embryos from soon after laying (stage XI) to stages 6-7 (early neurulation), with precise spatial and temporal control. Within 3 h, about 12 embryos can be electroporated and set up for culture by the New technique; the effects of morpholinos can be assessed immediately after electroporation, and robust overexpression from plasmid DNA is seen 2-3 h after electroporation. These techniques can be used for time-lapse imaging, gain- and loss-of-function experiments and studying gene regulatory elements in living embryos.

Orphan Nuclear Receptor LRH-1 Is Required To Maintain Oct4 Expression at the Epiblast Stage of Embryonic Development

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.

Hepatocyte Growth Factor/Scatter Factor Promotes a Switch from E- to N-Cadherin in Chick Embryo Epiblast Cells

Epiblast cells downregulate E-cadherin and upregulate N-cadherin as they ingress through the primitive streak and when placed in culture. The factors that promote the alteration in cadherin expression during gastrulation are unknown. The effects of hepatocyte growth factor/scatter factor (HGF/SF) on cadherin expression were tested in cultures of prestreak epiblast cells. HGF/SF decreased the expression of E-cadherin and increased the percentage of cells with N-cadherin and sarcomeric myosin. Cells with N-cadherin but not E-cadherin differentiated into skeletal muscle. HGF/SF also stimulated proliferation and the formation of cellular aggregates. Sensitivity to HGF/SF in vitro depended on the original position of cells within the epiblast. More cells from the lateral epiblast switched cadherins and proliferated in response to HGF/SF than medial epiblast cells. HGF/SF may affect gastrulation by altering cadherin expression, modulating cell adhesion, and stimulating proliferation within the epiblast.

Labelling of basement membrane constituents in the living chick embryo during gastrulation

The basement membrane of the living chick embryo epiblast has been labelled with ultrastructural markers in order to study the movement and turnover of this structure during gastrulation. Two problems were addressed in these experiments. Firstly, to what extent does the basement membrane move medially with the epiblast during morphogenesis? Secondly, what is the relationship to the basement membrane of the so-called interstitial bodies? The ultrastructural markers used were concanavalin A conjugated to ferritin and fibronectin antibodies conjugated to peroxidase. Embryos were cultured using the technique of New, and the label was applied to the periphery of the basal surface of the epiblast through a hole in the endoblast at the early primitive streak stage of development. The embryos were then allowed to develop to the full primitive streak stage in the presence of the label. When the position of the label was determined after incubation, it was found to have accumulated in large amounts at the edge of the primitive streak at the point where the basement membrane is disrupted. This indicates that constituents of the basement membrane are transported medially with the epiblast cells and are sloughed off as the latter pass through the primitive streak. This movement of basement membrane constituents is counter to the direction of migration of the underlying mesoderm cells. When embryos are exposed to label for only 1 h, then washed and incubated for a further three hours, the marker was found in the interstitial bodies and not distributed throughout the basement membrane itself. This suggests that the interstitial bodies, which have been implicated in influencing the migration of the mesoderm cells, are turnover products of the basement membrane to which they are attached.

Studies on the Coleorhiza of Cereals. : III On coleorhiza in the germination of rice seeds.

Experiments were performed to investigate the nature of coleorhiza of rice seeds, and the following results were obtained : 1) The epiblast of rice embryo was very noticeable compared with those in the other cereals. It adhered closely to the coleorhiza. In the resting stage of the embryo the boundary between the coleorhiza and the epiblast were not clear, but became distinct at the germination, as the coleorhiza swelled and expanded toward the apex of the seminal root. Vertical sections of the embryos of 15 varieties of rice are shown in Fig 3. The epidermis of coleorhiza and epiblast consited of long-shaped cells and some of them grew into hairs. 2) The growth of the seminal root from which the coleorhiza and the epiblast were off was inferior to that of untreated ones, showing only 64% of growth rate of the control at the 24 th hour after the teatment. The coleorhiza and the epiblast in embryo seem to enact some roles in connection with the growth of the seminal root at the germination. 3) The depression of freezing point of coleorhiza tissue was altered by the concentration of artificial culture solution. 4) The absorption power of unhusked rice grain was equivalent to that of 0.5∼0.6 M sucrose solution, while that of the husked grain to that of 0.7∼0.8 M sucrose solution. In artificial culture solution of higher concentrations, the seminal roots stopped their growth, while the coleorhiza continued its elongation. The values of absorption power mentiond above are represented by SATO's 'Mittlere Saugkraft-maxima.'