Morpholinos are antisense molecules which are specific, stable, effective and nontoxic. They generally are comprised of about 25 nucleic acid bases linked by an uncharged synthetic backbone. They bind to complementary sequences of RNA by base pairing to prevent processes from happening at the bound sites. They are used in research to change the expression of proteins or to suppress the activity of noncoding RNA. Most commonly they are used in one of three ways:
* TRANSLATION BLOCKING: They can stop the progression of a ribosomal initiation complex toward the start codon of an mRNA, preventing translation of a protein.
* SPLICE MODIFICATION: They can interfere with splicing of pre-mRNA, changing the mRNA produced by the splicing process (usually by deletion of an exon).
* MICRO-RNA BLOCKING: They can suppress maturation of a primary miRNA by blocking the Drosha or Dicer cleavage sites, preventing the miRNA from reaching its mature and active form; alternatively, they can block the mRNA target of an miRNA.
Morpholinos are commonly used in zebrafish embryos, a system exquisitely sensitive to toxins and teratogens. They are also used in other embryonic systems, in cell cultures, in organ explants, and in intact adult organisms. Morpholinos have entered human clinical trials for a range of diseases. Morpholinos can block the replication of many kinds of viruses. Papers have reported successful knockdowns with Morpholinos in animals, protists, bacteria and plants.
Morpholinos are not recognized by cellular enzymes. The backbone of a Morpholino is sufficiently different from the backbone of a nucleic acid that the enzymes which recognize anionic nucleic acids will not interact with an uncharged Morpholino. Morpholinos are not degraded in cells or in organisms. Morpholinos do not bind to toll-like receptors or cause immune responses.
Generally one of several techniques must be used for delivering the Morpholinos to the cytosol of cells.
* For embryos Morpholinos are generally microinjected or electroporated.
* For cell cultures Morpholinos can be electroporated, scrape-loaded, or delivered with an endosomal escape reagent (e.g. Endo-Porter).
* For in vivo applications Morpholinos can be conjugated with arginine-rich cell-penetrating peptides or guanidinium dendrimers (e.g. Vivo-Morpholinos) for administration systemically by i.v. or i.p. or for localized administration by i.m., subcutaneous injection, intranasal administration or intracerebroventricular infusion.
As an experimental tool for manipulating cells or organisms at the RNA level, Morpholinos have been proven effective with many papers in the scientific literature describing their use (pubs.gene-tools.com). As a therapeutic, Morpholinos have shown great promise and established an excellent record of clinical safety (www.avibio.com). In my opinion, as existing techniques for cytosolic delivery of Morpholinos are improved and new techniques are developed, Morpholinos will be applied to new biological model systems in research and find their place as an accepted and powerful class of therapeutic compounds for humans.
| Marker Name | Mouse EC/ ES/EG cells |
Monkey ES cells |
Human ES cells |
Human EG cells |
Human EC cells |
|---|---|---|---|---|---|
| SSEA-1 | + | – | – | + | – |
| SSEA-3 | – | + | + | + | + |
| SEA-4 | – | + | + | + | + |
| TRA-1–60 | – | + | + | + | + |
| TRA-1–81 | – | + | + | + | + |
| Alkaline phosphatase | + | + | + | + | + |
| Oct-4 | + | + | + | Unknown | + |
| Telomerase activity | + ES, EC | Unknown | + | Unknown | + |
| Feeder-cell dependent | ES, EG, some EC | Yes | Yes | Yes | Some; relatively low clonal efficiency |
| Factors which aid in stem cell self-renewal | LIF and other factors that act through gp130 receptor and can substitute for feeder layer | Co-culture with feeder cells; other promoting factors have not been identified | Feeder cells + serum; feeder layer + serum-free medium + bFGF | LIF, bFGF, forskolin | Unknown; low proliferative capacity |
| Growth characteristics in vitro | Form tight, rounded, multi-layer clumps; can form EBs | Form flat, loose aggregates; can form EBs | Form flat, loose aggregates; can form EBs | Form rounded, multi-layer clumps; can form EBs | Form flat, loose aggregates; can form EBs |
| Teratoma formation in vivo | + | + | + | – | + |
| Chimera formation | + | Unknown | + | – | + |
KEYES cell = Embryonic stem cell |
TRA = Tumor rejection antigen-1 |
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Figure 21-83. The early stages of mouse development. The zona pellucida is a jelly capsule from which the embryo escapes after a few days, allowing it to implant in the wall of the uterus. (Photographs courtesy of Patricia Calarco.)
Figure 21-84. Scanning electron micrographs of the early mouse embryo. The zona pellucida has been removed. (A) Two-cell stage. (B) Four-cell stage (a polar body is visible in addition to the four blastomeres—see Figure 20-22). (C) Eight-to-sixteen-cell morula—compaction occurring. (D) Blastocyst. (A-C, courtesy of Patricia Calarco; D, from P. Calarco and C.J. Epstein, Dev. Biol. 32:208–213, 1973. © Academic Press.)
Figure 21-85. A procedure for creating a chimeric mouse. Two morulae of different genotypes are combined.
Figure 21-86. Making a chimeric mouse with ES cells. The cultured ES cells can combine with the cells of a normal blastocyst to form a healthy chimeric mouse, and can contribute to any of its tissues, including the germ line. Thus the ES cells are totipotent.
Figure 8-70. Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured ES (embryonic stem) cells. Only a few rare ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells (indicated by orange) that carry the altered gene. Some of these mice will also contain germ-line cells that contain the altered gene. When bred with a normal mouse, some of the progeny of these mice will contain the altered gene in all of their cells. If two such mice are in turn bred (not shown), some of the progeny will contain two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such mice are missing genes that function during development, they often die with specific defects long before they reach adulthood. These defects are carefully analyzed to help decipher the normal function of the missing gene.
Figure 19-32. A summary of the junctional and nonjunctional adhesive mechanisms used by animal cells in binding to one another and to the extracellular matrix. The junctional mechanisms are shown in epithelial cells, while the nonjunctional mechanisms are shown in nonepithelial cells. A junctional adhesion is operationally defined as one that can be seen as a specialized region of contact by conventional or freeze-fracture electron microscopy. A nonjunctional adhesion shows no such obvious specialized structure. Note that the integrins and cadherins are involved in both nonjunctional and junctional cell-cell (cadherins) and cell-matrix (integrins) contacts. Cadherins generally mediate homophilic interactions, whereas integrins mediate heterophilic interactions (see Figure 19-26). The cadherins, integrins, and selectins act as transmembrane adhesion molecules and depend on extracellular divalent cations to function; for this reason, most cell-cell and cell-matrix contacts are divalent-cation-dependent. On blood cells, selectins and integrins can also act as heterophilic cell-cell adhesion molecules: the selectins bind to carbohydrate, while the cell-binding integrins bind to members of the Ig superfamily. The integrins and integral membrane proteoglycans that mediate nonjunctional adhesion to the extracellular matrix are discussed later. (Insert courtesy of Daniel S. Friend.)
| An Embryo is a multicellular diploid eukaryote in its earliest stage of development, from the time of first cell division until birth, hatching, or germination. In Humans, it is called an Embryo from the moment of Fertilization until the end of the 8th week of gestational age, whereafter it is instead called a Fetus. In organisms that reproduce sexually, once a Sperm fertilizes an Egg cell, the result is a cell called the Zygote. In animals, the development of the Zygote into an embryo proceeds through specific recognizable stages of Blastula, Gastrula, and Organogenesis. Little is known about the specific genes that regulate these early events or how interactions among cells or how cellular interactions with other factors in the three-dimensional environment of the early Embryo affect development. The processes, by which a fertilized Egg becomes an embryo, called embryogenesis, include coordinated cell division, cell specialization, cell migration, and genetically programmed cell death (Ref.1).
In Mammals, including Human, Oocyte is released from the Ovary and swept by the fimbriae into the Oviduct. The mature Oocyte, a haploid cell that contains half the normal number of chromosomes, is surrounded by a protective coat of noncellular material called the Zona Pellucida. For Fertilization to occur, a haploid Sperm cell must bind to and penetrate the Zona pellucida, fuse with the cell membrane of the Oocyte, enter the Oocyte cytoplasm, and fuse its pronucleus with the Oocyte pronucleus. Fertilization occurs in the Ampulla of the Oviduct, a region close to the Ovary. In human, the Fertilization of an Egg by a Sperm generates a Zygote. After fertilization, the Zygote makes its way to the Uterus, a journey that takes five to seven days in humans. As it travels, the Zygote divides. The first cleavage produces two identical cells and then divides again to produce four cells. Usually the cells remain together, dividing asynchronously to produce 8 cells, 16 cells, and so on. In Humans and Mice, at about the eight-cell stage, the embryo compacts, meaning that the formerly loose ball of cells comes together in a tight array that is interconnected by gap junctions. These specialized membrane structures consist of an array of six protein molecules called Connexins, which form a pore that allows the exchange of ions and small molecules between cells. By the third to fourth day the embryo develops to a compact ball of twelve or more cells called a Morula. After several more divisions, the Morula cells begin to specialize and form a hollow sphere of cells called a Blastocyst or Blastula. The outer layer of the Blastocyst is named the TE (Trophectoderm) and the cells inside ICM (Inner Cell Mass). The cells of the ICM are Pluripotent stem cells that can give rise to all cell types of the three embryonic germ layers, i.e., Ectoderm, Mesoderm, and Endoderm, and the Germ Cell Lineage, as well as to the Nontrophoblast tissues that support the developing embryo. The latter are referred to as Extraembryonic tissues and include the Yolk sac, Allantois, and Amnion (Ref.2 & 3). Trophectoderm is the progenitor tissue of the entire outer epithelial component of the placenta, known as Trophoblast, and provides the functional bridge between the Fetus and the mother. Trophoblast, which ultimately consists of a range of terminally differentiated cell types, performs the majority of the absorptive, immunoprotective and endocrinological functions of the placenta. Although the initial Trophoblast cells of Humans divide like most other cells of the body, they give rise to a population of cells wherein nuclear division occurs in the absence of Cytokinesis. The original type of Trophoblast cells constitutes a layer called the Cytotrophoblast, whereas the multinucleated type of cell forms the Syncytiotrophoblast. By 5 to 7 days postfertilization in humans, the Blastocyst reaches the uterus. In the uterus, the Blastocyst hatches out of the Zona pellucida, the structure that originally surrounded the Oocyte and that also prevented the implantation of the Blastocyst into the wall of the oviduct. Many of the molecular and cellular events that occur during the second week of Human Embryonic development, help establish the Placenta. About post fertilization days 8 to 9 in humans, the ball-shaped embryo implants into the Uterine wall. The ICM of the human Embryo at this stage split into 2 layers. One is the Hypoblast, which lies next to the Blastocoel and gives rise to the Extraembryonic Endoderm, which forms the Yolk sac. The other cell layer that develops from the ICM is the Epiblast. The Epiblast cell layer is split by small clefts that eventually coalesce to separate the Embryonic Epiblast from the other Epiblast cells, which form the Amnionic cavity. Once the lining of the Amnion is completed, it fills with a secretion called Amnionic (Amniotic) fluid, which serves as a shock absorber for the developing Embryo while preventing its desiccation. The Embryonic Epiblast contains all the cells that will generate the actual embryo. At the start of the third week of Human development, the cells of the Embryonic Epiblast begin to differentiate. The process is known as Gastrulation and the Embryo at this stage is known as Gastrula. The process of Gastrulation begins between days 14 and 16 of Human development. At that time, a primitive streak forms in a specific region of the Epiblast along the posterior axis of the Embryo. By the end of the third week, they generate the three primary germ layers of the Embryo—Endoderm, Mesoderm, and Ectoderm. They require the activation and inactivation of specific genes at specific times, highly integrated cell-cell interactions, and interactions between cells and their non-cellular environment, the extracellular matrix. The forward-moving Epiblast cells spread laterally, a migration that induces the formation of the Mesoderm and the Notochord. The Notochord is a temporary, rod-like structure that develops along the dorsal surface of the embryo and will ultimately connect the AVE (Anterior Visceral Endoderm) and the node. Cells at the anterior end of the notochord eventually underlie the forebrain. At the anterior end of the primitive streak is the node, a two-layered structure and important signaling center in the embryo. The ventral layer of cells in the node comes from the Epiblast and generates the notochordal plate, which then forms the notochord. Endoderm, which will give rise to the Gut, also develops near the node, along the sides of the notochord. Meanwhile, the anterior region of the Mesoderm that develops from the primitive streak prepares to give rise to the heart. The Anterior Epiblast generates the Neuroectoderm and the Ectoderm that covers the surface of the Embryo. The Ectodermal tissue that lies dorsal to the Notochord will generate the Neural plate, which will round up to form the Neural tube, the precursor to the Central Nervous System (Brain and Spinal Cord) (Ref.4, 5 & 6). Thus, the Embryonic Ectoderm, gives rise to central nervous system (Brain and Spinal Cord) and Peripheral nervous system; outer surface or Skin of the organism; Cornea and lens of the eye; epithelium that lines the mouth and nasal cavities and the anal canal; epithelium of the pineal gland, pituitary gland, and adrenal medulla; and cells of the neural crest (which gives rise to various facial structures, pigmented skin cells called Melanocytes, and dorsal root ganglia, clusters of nerve cells along the spinal cord). The embryonic Mesoderm, gives rise to skeletal, smooth, and cardiac muscle; structures of the Urogenital system (kidneys, Ureters, gonads, and reproductive ducts); bone marrow and blood; fat; bone, and cartilage; other connective tissues; and the lining of the body cavity. The embryonic Endoderm, gives rise to the epithelium of the entire digestive tract (excluding the mouth and anal canal); epithelium of the respiratory tract; structures associated with the digestive tract (liver and pancreas); Thyroid, parathyroid, and thymus glands; epithelium of the reproductive ducts and glands; epithelium of the urethra and bladder. Another important type of cells in this developmental scheme is the PG (Primordial Germ) cells, which will give rise to Eggs and Sperm in the adult organism. Prior to gastrulation, at about the time of primitive streak formation, these precursor cells split off from the proximal region of the epiblast and migrate into the Extraembryonic Mesoderm. It is not until the proximal Epiblast cells reach the Extraembryonic Mesoderm that they are committed to becoming PG cells (Ref.7 & 8). The development of the mammalian embryo is controlled by regulatory genes, some of which regulate the transcription of other genes. These regulators activate or repress patterns of gene expression that mediate phenotypic changes during stem cell differentiation. Oct4 (Octamer Binding Transcription Factor-4) belongs to the POU (Pit-Oct-Unc) transcription factor family. The POU family of transcription factors can activate the expression of their target genes through binding an octameric sequence motif of an AGTCAAAT consensus sequence. Recent evidence indicates that Oct4 is almost exclusively expressed in ESCs (Embryonic stem Cells). During embryonic development, Oct4 is expressed initially in all Blastomeres. Subsequently, its expression becomes restricted to the ICM and down regulated in the Trophoectoderm. At maturity, Oct4 expression becomes confined exclusively to the developing Germ cells. Besides Oct4, other transcription factors like Nanog and SOX2 (SRY (Sex Determining Region-Y) Box-2) are also expressed in ICM and ESCs and are downregulated during differentiation. In Human, FGF2 (Fibroblast Growth Factor-2) maintains ESCs in the undifferentiated state. BMPs (Bone Morphogenic Proteins), on the other hand, induce differentiation into extraembryonic lineages, either in the presence or absence of serum. Several proteins take part in Embryonic differentiation. Much of the genetic and developmental information has been derived from the mouse where most information exists. In Human not much information is available. Some important proteins found in Trophoectoderm differentiation include GCM1 (Glial Cells Missing homolog-1), ID2 (Inhibitor of DNA binding-2, dominant negative helix-loop-helix protein) and Hash2. Hash2 gene is necessary for the specification of Spongiotrophoblast and Syncytiotrophoblast. Likewise, the gene, GCM1, must be active for Syncytiotrophoblast to develop from its precursors. The transcription factors believed to have a positive association with Trophectoderm specification have been inferred primarily in two ways: by their expression patterns in embryos, ES cells and TS cells and by the consequences of gene disruption on embryonic development. Many of these transcription factors also control the expression of genes characteristically expressed in trophoblast but not in the epiblast, primitive endoderm and their derivatives. ES and Trophoectoderm cells are beginning to provide insights into the changes in gene expression that accompany lineage specification and the subsequent post-specification events that lead to functional Trophoblast derivatives (Ref.9 &10). |
| References: |
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| Directional term | Defined Axis | Synonyms | Axis runs… |
|---|---|---|---|
| Anterior | Anteroposterior | Rostrocaudal1, Craniocaudal1, Cephalocaudal2 | …from head end to opposite end of body or tail. |
| Posterior | |||
| Dorsal | Dorsoventral | — | …from spinal column (back) to belly (front). |
| Ventral | |||
| Left (lateral) | Left-right | Dextro-sinister2, Sinistro-dexter2 | …from left to right sides of body. |
| Right (lateral) | |||
| Medial | Mediolateral3 | — | …from centre of organism to one or other side. |
| Left or right (lateral) | |||
| Proximal | Proximodistal | — | …from tip of an appendage (distal) to where it joins the body (proximal). |
| Distal | |||
| Notes: (1) Fairly common usage. (2) Uncommon usage. (3) Equivalent to one-half of the left-right axis. (The terms „intermediate“, „ipsilateral“, „contralateral“, „superficial“ and „deep“, while indicating directions, are relative terms and thus do not properly define fixed anatomical axes.) |
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Heparin
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Heparin, a highly-sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule.[1] It can also be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines. Pharmaceutical grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine (pig) intestine or bovine (cow) lung.[2]
Although used principally in medicine for anticoagulation, the true physiological role in the body remains unclear, because blood anti-coagulation is achieved mostly by endothelial cell-derived heparan sulfate proteoglycans (also expressed by the trophectoderm upon implantation (heparin receptors – on the endometrium)).[3] Heparin is usually stored within the secretory granules of mast cells and released only into the vasculature at sites of tissue injury. It has been proposed that, rather than anticoagulation, the main purpose of heparin is in a defensive mechanism at sites of tissue injury against invading bacteria and other foreign materials.[4] In addition, it is preserved across a number of widely different species, including some invertebrates that lack a similar blood coagulation system.
These models correspond to the protein data bank code 1HPN. 
From Wikipedia, the free encyclopedia
Organs derived from each germ layer. Image from NCBI.
A germ layer is a group of cells, formed during animal embryogenesis. Germ layers are particularly pronounced in the vertebrates; however, all animals more complex than sponges (eumetazoans and agnotozoans) produce two or three primary tissue layers (sometimes called primary germ layers). Animals with radial symmetry, like cnidarians, produce two germ layers (the ectoderm and endoderm) making them diploblastic. Animals with bilateral symmetry produce a third layer between these two layers (appropriately called the mesoderm) making them triploblastic. Germ layers eventually give rise to all of an animal’s tissues and organs through the process of organogenesis.
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[edit] Germ layers
Gastrulation of a diploblast: The formation of germ layers from a (1) blastula to a (2) gastrula. Some of the ectoderm cells (orange) move inward forming the endoderm (red).
Caspar Friedrich Wolff observed organization of the early embryo in leaf-like layers. Later, Heinz Christian Pander discovered germ layers while studying chick embryos.
Among animals, sponges show the simplest organization, having a single germ layer. Although they have differentiated cells (e.g. collar cells), they lack true tissue coordination. Diploblastic animals, Cnidaria and ctenophores, show an increase in complexity, having two germ layers, the endoderm and ectoderm. Diploblastic animals are organized into recognisable tissues. All higher animals (from flatworms to humans) are triploblastic, possessing a mesoderm in additition to the germ layers found in Diploblasts. Triploblastic animals develop recognisable organs.
[edit] Development
Fertilization leads to the formation of a zygote. During the next stage, cleavage, mitotic cell divisions transform the zygote into a tiny ball of cells, a blastula. This early embryonic form undergoes gastrulation, forming a gastrula with either two or three layers (the germ layers). In all vertebrates, these are the forerunners of all adult tissues and organs.
The appearance of the archenteron marks the onset of gastrulation.
In humans, after about three days, the zygote forms a solid mass of cells by mitotic division, called a morula. This then changes to a blastocyst, consisting of an outer layer called a trophoblast, and an inner cell mass called the embryoblast. Filled with uterine fluid, the blastocyst breaks out of the zona pellucida and undergoes implantation. The inner cell mass initially has two layers: the hypoblast and epiblast. At the end of the second week, a primitive streak appears. The epiblast in this region moves towards the primitive streak, dives down into it, and forms a new layer, called the endoderm, pushing the hypoblast out of the way (this goes on to form the amnion.) The epiblast keeps moving and forms a second layer, the mesoderm. The top layer is now called the ectoderm.
[edit] Endoderm
The endoderm is one of the germ layers formed during animal embryogenesis. Cells migrating inward along the archenteron form the inner layer of the gastrula, which develops into the endoderm.
The endoderm consists at first of flattened cells, which subsequently become columnar. It forms the epithelial lining of the whole of the digestive tube excepting part of the mouth and pharynx and the terminal part of the rectum (which are lined by involutions of the ectoderm). It also forms the lining cells of all the glands which open into the digestive tube, including those of the liver and pancreas; the epithelium of the auditory tube and tympanic cavity; the trachea, bronchi, and air cells of the lungs; the urinary bladder and part of the urethra; and the follicle lining of the thyroid gland and thymus.
The endoderm forms: the stomach, the colon, the liver, the pancreas, the urinary bladder, the lining of the urethra, the epithelial parts of trachea, the lungs, the pharynx, the thyroid, the parathyroid, and the intestines.
[edit] Mesoderm
The mesoderm aids in the production of cardiac muscle, skeletal muscle, smooth muscle, tissues within the kidneys, and red blood cells.
The mesoderm germ layer forms in the embryos of triploblastic animals. During gastrulation, some of the cells migrating inward contribute to the mesoderm, an additional layer between the endoderm and the ectoderm.
This key innovation evolved hundreds of millions of years ago and led to the evolution of nearly all large, complex animals. The formation of a mesoderm led to the development of a coelom. Organs formed inside a coelom can freely move, grow, and develop independently of the body wall while fluid cushions and protects them from shocks.
The mesoderm forms: skeletal muscle, the skeleton, the dermis of skin, connective tissue, the urogenital system, the heart, blood (lymph cells), and the spleen.
[edit] Ectoderm
The ectoderm produces tissues within the epidermis, aids in the formation of neurons within the brain, and constructs melanocytes.
The ectoderm is the start of a tissue that covers the body surfaces. It emerges first and forms from the outermost of the germ layers.
The ectoderm forms: the central nervous system, the lens of the eye, cranial and sensory, the ganglia and nerves, pigment cells, head connective tissues, the epidermis, hair, and mammary glands.
[edit] Neural crest
Because of its great importance, the neural crest is sometimes considered a fourth germ layer. It is, however, derived from the ectoderm.
[edit] References
- Evers, Christine A., Lisa Starr. Biology:Concepts and Applications. 6th ed. United States:Thomson, 2006. ISBN 0-534-46224-3.
[edit] See also
The Cell Surface and the Mechanism of Compaction
Compaction creates the circumstances that bring about the first differentiation in mammalian development: the separation of trophoblast from inner cell mass. How is this done? There is growing evidence that compaction is mediated by events occurring at the cell surfaces of adjacent blastomeres. In the first stage of compaction, each of the eight blastomeres interacts with its neighbors to undergo membrane polarization. Different components of the cell surface migrate to different regions of the cell (see Figure 1; Ziomek and Johnson, 1980).
 |
| Figure 1 Compaction and the formation of the mouse blastocyst. (A,B) 8-cell embryo. (C) 16-cell morula. (D) 32-cell blastocyst. The left side represents the entire organism or its cross section. The right side details the changes associated with the maturation of the trophoblast. (Right-hand figures after Fleming, 1992.) |
This polarity can be seen by tagging certain cell-surface molecules with fluorescent dyes. One such tag, which recognizes a class of glycoproteins, shows that at the 4-cell stage these glycoproteins are randomly distributed throughout the membrane (Figure 2A). However, at the mid-8-cell stage, these molecules are found predominantly at the poles farthest away from the center of the aggregate (Figure 2B). Membrane polarization is influenced by cell-cell interactions, because it takes place only when the cells are in contact with at least one other blastomere. If a blastomere is separated from the rest of the embryo, it loses its polarization.
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| Figure 2 Polarization of membrane components in 8-cell mouse blastomeres. (A) Homogeneous, nonpolar distribution of membrane components labeled with fluorescent concanavalin A at the 4-cell stage. (B) Heterogeneous, polar distribution of these components at the 8-cell stage. (A from Fleming et al., 1986; B from Levy et al., 1986. Photographs courtesy of the authors.) |
Specific cell surface proteins play a role in compaction. One such molecule, E-cadherin (also known as uvomorulin), a 120-kDa adhesive glycoprotein, is synthesized at the 2-cell stage and is uniformly spread throughout the cell membrane. However, as compaction occurs, E-cadherin becomes restricted to those sites on cell membranes that are in contact with adjacent blastomeres. Antibodies to this molecule cause the decompaction of the morula (Figure 5.25; Peyrieras et al., 1983; Johnson et al., 1986). The carbohydrate portion of this glycoprotein may be essential to its function, as tunicamycin (a drug that inhibits the glycosylation of proteins) also prevents compaction.
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| Figure 3 Prevention of compaction by antiserum directed against the cell-surface adhesion glycoprotein E-cadherin. (A) Normal compaction occurring in the absence of antiserum. (B) Proliferation without compaction occurring in the presence of antibodies to E-cadherin. (Photographs courtesy of C. Ziomek.) |
The phosphatidylinositol pathway may also be important for initiating compaction. If 4-cell mouse embryos are placed into media containing drugs that activate protein kinase C, premature compaction occurs. Similarly, diacylglycerides can transiently cause these 4-cell embryos to undergo compaction. When this occurs, the E-cadherin accumulates specifically at the junctions between the blastomeres (Winkel et al., 1990). These results suggest that the activation of protein kinase C may initiate compaction by shifting the localization of E-cadherin.
Finally, the cell membrane may also be modified during compaction by cytoskeletal reorganization. Microvilli, extended by actin microfilaments, appear on adjacent cell surfaces and attach one cell to the other. These microvilli may be the sites where E-cadherin is functioning to mediate intercellular adhesion. The flattening of the blastomeres against one another may therefore be brought about by the shortening of the microvilli through actin depolymerization (Pratt et al., 1982; Sutherland and Calarco-Gillam, 1983).
Thus, there is growing evidence that compaction is caused by changes in the architecture of the blastomere cell surface. It is not certain, though, how these events relate to one another or how they are coordinated into the integrated network of events that causes compaction.
Formation of the Inner Cell Mass
The creation of an inner cell mass distinct from the trophoblast is the crucial process of early mammalian development. How is a cell directed into one or the other of these paths? How is a cell informed that it is either to give rise to a portion of the adult mammal or to give rise to a rather remarkable supporting tissue that will be discarded at birth? Observations of living embryos suggest that this momentous decision is merely a matter of a cell’s being in the right place at the right time. Up through the 8-cell stage, there are no obvious differences in the biochemistry, morphology, or potency of any of the blastomeres. However, compaction forms inner and outer cells with vastly different properties. By labeling the various blastomeres, numerous investigators have found that the cells that happen to be on the outside will form the trophoblast, whereas the cells that happen to be inside will generate the embryo (Tarkowski and Wróblewska, 1967; Sutherland et al., 1990). The inner cells have been found to come most frequently from the first cell to divide at the 2-cell stage. This cell usually produces the first pair of blastomeres to reach the 8-cell stage, and these cells usually divide in such a way that they are inside the loosely aggregated cluster of blastomeres (Graham and Kelly, 1977).
Hillman and co-workers (1972) have shown that when each blastomere of a 4-cell mouse embryo is placed on the outside surface of a mass of aggregated blastomeres, the external, transplanted cells only give rise to trophoblast tissue. Therefore, it seems that whether a cell becomes trophoblast or embryo depends on whether it was an external or an internal cell after compaction.
How many cells form the inner cell mass?
If most of the cells of the blastocyst form the trophoblast, then how many cells of the compacted embryo actually form the inner cell mass? Beatrice Mintz (1970) solved this problem by making chimeric mice, wherein two mouse embryos are fused together during early stages of development. She took two 4-celled embryos, one from homozygous black-furred parents and one from homozygous white-furred parents, and she placed them together so that the cells integrated to form one eight-cell embryo. This embryo underwent compaction and was implanted into the uterus a foster mother mouse (see p. 364). Each cell can become a part of the trophoblast or a part of the inner cell mass with equal frequency. Mintz performed this experiment hundreds of times. If only one cell formed the inner cell mass, then each mouse pup should be either all white or all black. 0% should be mixed. If the inner cell mass was composed of two cells, one-quarter of them should be white, one-quarter black and 50% should be mixed (1WW: 2WB : 1BB). If there were three cells in the ICM, the percentage of mixed colored mice should be 75% (1WWW; 3WWB; 3WBB: 1BBB); and if there were four cells, the frequency of chimeric coat patterns should be 87.5%. Her figures showed that her allophenic mice had mixed pigmentation 73% of the time. Therefore, it appears that three cells make the inner cell mass of the compacted embryo.
Literature Cited
Fleming, T. P. 1992. Trophectoderm biogenesis in the preimplantation mouse embryo. In T. P. Fleming, (ed.) Epithelial Organization and Development. Chapman and Hall, London, pp. 111–134.
Fleming, T. P., Pickering, S. J., Qasim, F. and Maro, B. 1986. The generation of cell surface polarity in mouse 8-cell blastomeres: The role of cortical microfilaments analyzed using cytochalasin D. J. Embryol. Exp. Morphol. 95: 169–191.
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http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.2615
Figure 11.22. The cleavage of a single mouse embryo in vitro. (A) 2-cell stage. (B) 4-cell stage. (C) Early 8-cell stage. (D) Compacted 8-cell stage. (E) Morula. (F) Blastocyst. (From Mulnard 1967; photographs courtesy of J. G. Mulnard.)
Figure 11.23. Scanning electron micrographs of (A) uncompacted and (B) compacted 8-cell mouse embryos. (Photographs courtesy of C. Ziomek.)
Figure 11.24. Implantation of the mammalian blastocyst into the uterus. (A) Mouse blastocysts entering the uterus. (B) Initial implantation of the blastocyst in a rhesus monkey. (A from Rugh 1967; B courtesy of the Carnegie Institution of Washington, Chester Reather, photographer.)
Figure 11.25. Mouse blastocyst hatching from the zona pellucida. (Photograph from Mark et al. 1985, courtesy of E. Lacy.)
Figure 11.26. Schematic diagram showing the derivation of tissues in human and rhesus monkey embryos. (After Luckett 1978; Bianchi et al. 1993.)
Figure 11.27. Tissue formation in the human embryo between days 7 and 11. (A, B) Human blastocyst immediately prior to gastrulation. The inner cell mass delaminates hypoblast cells that line the blastocoel, forming the extraembryonic endoderm of the primitive yolk sac and a two-layered (epiblast and hypoblast) blastodisc similar to that seen in avian embryos. The trophoblast in some mammals can be divided into the polar trophoblast, which covers the inner cell mass, and the mural trophoblast, which does not. The trophoblast divides into the cytotrophoblast, which will form the villi, and the syncytiotrophoblast, which will ingress into the uterine tissue. (C) Meanwhile, the epiblast splits into the amnionic ectoderm (which encircles the amnionic cavity) and the embryonic epiblast. The adult mammal forms from the cells of the embryonic epiblast. (D) The extraembryonic endoderm forms the yolk sac. (After Gilbert 1989; Larsen 1993.)
Figure 11.28. Amnion structure and cell movements during human gastrulation. (A) Human embryo and uterine connections at day 15 of gestation. In the upper view, the embryo is cut sagittally through the midline; the lower view looks down upon the dorsal surface of the embryo. (B) The movements of the epiblast cells through the primitive streak and Hensen’s node and underneath the epiblast are superimposed on the dorsal surface view. At days 14 and 15, the ingressing epiblast cells are thought to replace the hypoblast cells (which contribute to the yolk sac lining), while at day 16, the ingressing cells fan out to form the mesodermal layer. (After Larsen 1993.)
Figure 11.29. Formation of the notochord in the mouse. (A) The ventral surface a the 7.5-day mouse embryo, seen by scanning electron microscopy. The presumptive notochord cells are the small, ciliated cells in the midline that are flanked by the larger endodermal cells of the primitive gut. The node (with its ciliated cells) is seen at the bottom. (B) The formation of the notochord by the dorsal infolding of the small, ciliated cells. (From Sulik et al. 1994; photograph courtesy of K. Sulik and G. C. Schoenwolf.)
Figure 11.30. Human embryo and placenta after 40 days of gestation. The embryo lies within the amnion, and its blood vessels can be seen extending into the chorionic villi. The small sphere to the right of the embryo is the yolk sac. (The Carnegie Institution of Washington, courtesy of C. F. Reather.)
Figure 11.31. Relationship of the chorionic villi to the maternal blood in the uterus.
Figure 11.34. The two signaling centers of the mammalian embryo. (A) In the day 7 mouse embryo, the dorsal surface of the epiblast (embryonic ectoderm) is in contact with the amnionic cavity. The ventral surface of the epiblast contacts the newly formed mesoderm. In this cuplike arrangement, the endoderm covers the surface of the embryo. The node is at the bottom of the cup, and it has generated chordamesoderm. The two signaling centers, the node and the anterior visceral endoderm, are located on opposite sides of the cup. Eventually, the notochord will link them. The caudal side of the embryo is marked by the presence of the allantois. (B) By embryonic day 8, the anterior visceral endoderm lines the foregut, and the prechordal mesoderm is now in contact with the forebrain ectoderm. The node is now farther caudal, due largely to the rapid growth of the anterior portion of the embryo. The cells in the midline of the epiblast migrate through the primitive streak (white arrows). (Photographs courtesy of K. Sulik.)
Figure 11.41. Relationship between the animal-vegetal axis of the egg and the embryonic-abembryonic axis of the blastocyst. The polar body marks the animal pole of the embryo. The dorsal-ventral axis of the embryo appears to form at right angles to the animal-vegetal axis of the egg.
Figure 11.42. Left-right asymmetry in the developing human. (A) Abdominal cross sections show that the originally symmetrical organ rudiments acquire asymmetric positions by week 11. The liver moves to the right and the spleen moves to the left. (B) Not only does the heart move to the left side of the body, but the originally symmetrical veins of the heart regress differentially to form the superior and inferior venae cavae, which connect only to the right side of the heart. (C) The right lung branches into three lobes, while the left lung (near the heart) forms only two lobes. In human males, the scrotum also forms asymmetrically. (After Kosaki and Casey 1998.)
Figure 11.44. Situs formation in mammals. (A) Proposed pathway for left-right axis formation in the mouse. The leftward movement of cilia in the node activates some as yet unidentified factor (possibly the product of the inv gene). This product activates the nodal and lefty2 genes. The diffusion of Nodal and Lefty2 proteins to the right-hand side is restricted by the product of the Lefty1 gene which coats the bottom of the neural tube on the left side. Nodal activates Pitx2, the gene whose product activates left-sided properties in the various organs containing it. Either Nodal or Lefty2 (perhaps both) repress the Snail gene whose product is needed to instruct right-sidedness. (B) Ciliated cells of the mammalian node. This photograph is a close-up of the node seen in Figure 11.29A. (Photograph courtesy of K. Sulik and G. C. Schoenwolf.)
