Modeling mammalian gastrulation with embryonic stem cells

Understanding cell fate patterning and morphogenesis in the mammalian embryo remains a formidable challenge. Recently, in vivo models based on embryonic stem cells (ESCs) have emerged as complementary methods to quantitatively dissect the physical and molecular processes that shape the embryo. Here we review recent developments in using embryonic stem cells to create both two and three-dimensional culture models that shed light on mammalian gastrulation.Comment: 18 pages, 1 figur

SL-07 Competency of Diploid Parthenogenesis Embryonic Stem Cell (pESC) for Cell Therapy

Naturally, mammalian embryo resulting from the fertilized oocytes by sperm and develops to the zygote with diploid chromosome. Through the embryo engineering, mammalian embryo also could be produce by: nuclear transfer and parthenogenesis. Embryo produce by nuclear transfer method could develop to term with very limited results. However, parthenogenetic embryos could not develop to term because of the incorrect expression of imprinted genes (genomic imprinting)........

Biosynthesis and expression of zona pellucida glycoproteins in mammals

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three glycoproteins (ZPA, ZPB, ZPC), products of the gene families ZPA, ZPB and ZPC that have been found to be highly homologous within mammalian species. Most data on the structure and function of the ZP are obtained from studies in mouse. New data from pig and other domestic animals, however, indicate that the mouse model does not hold for all other species. Whereas in the mouse ZPB is the primary sperm receptor, in the pig ZPA has been shown to possess receptor activity. Contrary to the mouse, where the growing oocyte is the only source of zona glycoproteins, in domestic animals these proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play also a role in granulosa cell differentiation. In several mammalian species, the epithelial secretory cells of the oviduct synthesize and secrete specific glycoproteins (oviductins) that become closely associated with the ZP of the ovulated oocyte. Once bound to the ZP, oviductin molecules could act as a protective layer around the oocyte and early embryo by virtue of their densely glycosylated mucin-type domains. Copyright (C) 2001 S. Karger AG, Basel

Simulating the Mammalian Blastocyst - Molecular and Mechanical Interactions Pattern the Embryo

Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v) and embryonic-abembryonic (eb-ab) axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1) The position determines gene expression, and (2) the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1) A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM). In this case the blastocoel simply acts as a static boundary. (2) The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial mechanical simulations with genetic networks to explain mammalian embryogenesis. Such a framework provides the means to test hypotheses in a controlled in silico environment

A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

A 3D mechanical model of the early mammalian embryo

The early development of the mammalian embryo leads to the formation of a structure composed by an outer layer of polarized cells surrounding an inner mass of nonpolarized cells. Experimental biology has shown that this organization results from changes in cell polarity, cell shape and intercellular contacts at the 8 and 16-cell stages. In order to examine how the physical properties of embryo cells (adhesion, cortical tension) influence the organization of the cells within the embryo, our team has developed a 3D mechanical model of the dividing early embryo, based on cellular Potts models. In this paper we will present the principles of our simulations, the methodology used and we will show that a very simple mechanical model can reproduce the main structural features (geometry, cell arrangement) of the mammalian embryo during its early developmental stages, up to the 16-cell stage

Building an apical domain in the early mouse embryo: lessons, challenges and perspectives

Cell polarization is critical for lineage segregation and morphogenesis during mammalian embryogenesis. However, the processes and mechanisms that establish cell polarity in the mammalian embryo are not well-understood. Recent studies suggest that unique regulatory mechanisms are deployed by the mouse embryo to establish cell polarization. In this review, we discuss current understanding of cell polarity establishment, focusing on the formation of the apical domain in the mouse embryo. We will also discuss outstanding questions and possible directions for future study.Wellcome Trust: 207415/Z/17/Z Leverhulme Trust: RPG-2018-085 (RG92356

Embryo metabolism : what does it really mean?

The study of early embryo metabolism has fascinated researchers in the field for nearly a century. Herein, we give a brief account of the general features of embryo metabolism and some consideration of the research performed to reach such conclusions. It is becoming increasingly obvious that metabolism informs many fate decisions and outcomes beyond ATP generation, such as DNA methylation, Reactive Oxygen Species generation and cell signaling. We discuss the reasons for studying metabolism in the face of our current knowledge of the effect that the culture environment on the developing embryo and the downstream effects that can cause. The study of in vitro embryo metabolism can also give us insight into developmental perturbations in vivo. The strengths and limitations of the methods we use to study metabolism are reviewed with reference to species-specific fundamental biology and plasticity and we discuss what the future holds for metabolic studies and the unanswered questions that remain