Mouse Early Embryo Development
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Mouse Early Embryo Development
Embryonic development is the generation of a multicellular organism from a single cell. During this process, tissues and organs are differentiated and positioned at different parts of the organism. In animals, Embryonic development consists of 4 stages: Cleavage, Patterning, Differentiation and Growth. Mammalian Cleavage is strikingly different from most other patterns of Embryonic cell division. The mammalian oocyte is released from the ovary and swept by the fimbriae into the oviduct. The mature oocyte is surrounded by a protective coat of noncellular material (made of extracellular matrix and glycoproteins), called the Zona pellucida. Fertilization occurs in the ampulla of the oviduct, a region close to the ovary. 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 pro-nucleus with the oocyte pro-nucleus. After the sperm enters the ovum the nuclei of the sperm and nuclei of the ovum join to form the Zygote. Meiosis is completed at this time, and first Cleavage begins about a day later. Cleavages in mammalian eggs are among the slowest in the animal kingdom—about 12–24 hours apart. Meanwhile, the cilia in the oviduct push the embryo towards the uterus; the first cleavage occurs along this journey. The first cleavage is a normal meridional division; however, in the second cleavage, one of the two blastomeres divides meridionally and the other divides equatorially. This type of cleavage is called rotational cleavage. Most research on mammalian development has focused on the mouse embryo, since mice are relatively easy to breed throughout the year, have large litters, and can be housed easily (Ref. 1 & 2).

The first cleavage of mouse Zygote produces two identical cells, which then divides again to produce four cells. Usually the cells remain together, dividing asynchronously to produce 8 cells, 16 cells, and so on. In mice, at about the eight-cell stage, the embryo compacts. By the third day the embryo develops to a compact ball of 16-32 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 (Trophoectoderm) and the cells inside ICM (Inner Cell Mass). The Trophoblast cells secrete fluid into the Morula to create a Blastocoel. 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. At the Blastocyst stage the Embryo hatches from the surrounding Zona pellucida and subsequently implants in the Uterus. Mouse Embryos take about three and a half days to develop from the 1-cell stage to the Blastocyst stage containing 32 or more cells. The First (1 to 2) and Second (2 to 4) cell cycles of the Mouse Embryo take between 16-20 hrs and 18-22 hrs respectively, depending on the strain of Mice. The duration of certain phases of the cell cycle differ considerably between 1 and 2-cell Mouse Embryos (Ref. 3).

In Mouse, Blastocyst formation occurs at Day 4. At Day 4.5, the surface of ICM in the Blastocyst becomes the Primitive Endoderm while the remaining becomes Primitive Ectoderm or Epiblast. The implantation occurs and the Zona pellucida is discarded and Blastocyst attaches to Uterine wall. During the immediate Post-implantation period i.e at about Day 6, the Mouse Embryo changes dramatically in size and shape. The Embryonic tissue volume increases by about 40-fold. The Mural Trophectoderm gives rise to polyploid Trophoblast Giant cells. The rest of Trophectoderm becomes the Ectoplacental cone and the Extra-embryonic Ectoderm which give rise to the Placenta. Primitive Endoderm migrates to cover inner surface of Mural Trophectoderm to become the Parietal Endoderm and Epiblast to become the Visceral Endoderm. Epithelialization of the ICM into a layer of Epiblast cells occurs concomitantly with the formation of a central cavity called the Proamniotic cavity. The Embryo acquires the shape of a cup made up of two cell layers, the inner Epiblast and the outer Visceral Endoderm. Proamniotic cavity formation is believed to occur by cell death. This process appears to be regulated by the adjacent Visceral Endoderm. The Mouse Embryo at this stage, called the Egg Cylinder, has well delineated Extraembryonic and Embryonic regions that further define a polarized Proximal-Distal axis (Ref. 1 & 4).

The process of Gastrulation begins at about Day 6.5 in the Mouse. At that time, a Primitive Streak forms in a specific region of the Epiblast along the posterior axis of the Embryo. Little is known about the signals that regulate the generation of the Primitive Streak, although the genes Goosecoid, T (Brachyury) and Evx1 (Even skipped homeotic gene-1 homolog) are expressed. The forward migration of the Posterior Epiblast cells occurs as their cell-cell contacts break down, and they release enzymes that digest the basement membrane that lies underneath. Epiblast cells move through the Streak and spread forward and laterally between the Ectoderm and the Visceral Endoderm to form the Mesoderm. Later, the Definitive Endoderm (from Epiblast) replaces the Visceral Endoderm. The Primitive Streak first elongates, then at the anterior tip of the Primitive Streak, the Node forms. The Node, together with another important signaling center, the AVE (Anterior Visceral Endoderm), helps regulate the formation of the pattern of the Embryos body at this stage of development. Then Notochord and Somites form anterior to the Node. The Notochord is a temporary, rod-like structure that develops along the dorsal surface of the Embryo and ultimately connects the AVE and the Node. Cells at the anterior end of the Notochord eventually underlie the Forebrain. Endoderm, which gives 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 generates the Neural plate, which rounds up to form the Neural tube, the precursor to the CNS (Central Nervous System (Brain and Spinal cord)). Thus, by Day 8 in the Mouse Embryonic development, the Primitive Ectoderm of the Post-implantation Blastocyst generates the ectoderm, mesoderm, and endoderm of the Gastrula. These and other complex processes result in the formation of the tissues and organs that occur in an adult mammal. They require the activation and inactivation of specific genes at specific times, highly integrated cell-cell interactions, and interactions between cells and their noncellular environment, the extracellular matrix. In general, the Embryonic Ectoderm gives rise to the following tissues: CNS and outer surface or skin of the organism. The Embryonic Mesoderm gives rise to Skeletal muscles, Heart, Blood and other Connective tissues and the lining of the body cavity. The Endoderm, gives rise to the epithelium of the entire digestive tract, epithelium of the respiratory tract, structures associated with the digestive tract (Liver and Pancreas), epithelium of the reproductive ducts and glands. Besides somatic cells, Germ Cells are also formed during the development of embryo. Prior to Gastrulation, at about the time of Primitive Streak formation, Germ cell precursors 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 Primordial Germ Cells (Ref. 5, 6 & 7).

Several Signaling molecules and transcription factors act within each cell type to give them their characteristic properties like maintenance or protection of the Pluripotent state of the ICM and to provide the Paracrine signals to adjacent cell lineages for their survival. Two ideal candidates for this are the transcription factors Oct4 (Octamer Binding Transcription Factor-4) and SOX2 (SRY (Sex Determining Region-Y) Box-2), which cooperates in the transcriptional activation of FGF4  (Fibroblast Growth Factor-4) in Embryonal cells. Oct4, a POU domain protein, is expressed in the Zygote, Morula, ICM, Epiblast, and later in Germ cells. It is essential for establishing and maintaining Pluripotency of the ICM, which in its absence differentiates into Trophectoderm. Besides Oct4, other transcription factors like Nanog and SOX2 are also expressed in ICM and ESCs (Embryonic Stem Cells) and are down regulated during differentiation. The transcription factor CDX2 (Caudal type Homeobox-2) is required for the maintenance of Trophectoderm fate, making CDX2 the earliest known determinant of this lineage. Formation of the Trophectoderm does not require CDX2, suggesting that factors upstream of CDX2 regulate Trophectoderm differentiation prior to Blastocyst formation. Other 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 Mash2 (Mammalian Achaete Scute Homolog-2). CDX2, Eomes (Eomesodermin homolog (Xenopus laevis)) and ESRRB (Estrogen Related Receptor-Beta) are required early in the Mouse Extraembryonic Ectoderm development from Trophoectoderm. Mouse TSCs (Trophoblast Stem Cells) are generally derived from Blastocysts and early post implantation Trophoblasts and grow in the presence of FGF4. Removal of FGF4 from the TSCs causes them to differentiate into Trophoblast Giant cells and other Trophoblast subtypes. ID2 is expressed in Mouse Chorionic Trophoblast, while Mash2 is expressed in Murine Spongiotrophoblast. Hand1 (Heart and Neural crest Derivatives expressed transcript-1) is necessary for the formation of Mouse Giant cells but not for the specification of Spongiotrophoblast and Syncytiotropphoblast. By contrast, Mash2 has the opposite effect to Hand1. The gene mSNA, which represses the transcription of genes that promote the transition from mitotic to endoreplicative cell cycles in Mouse Trophoblast, becomes down regulated during Giant cell differentiation. Expression of GCM1 in Mouse labyrinth is consistent with structural homology of the tissues. Several proteins are also involved in the formation of Epiblast or Primitive Endoderm from ICM. Recent evidence suggests that the precursors of Primitive Endoderm in Mouse can be identified on Blastocysts between day 3.5 and 4.5 embryos as a group of cells segregating from the rest of the ICM, that express the transcription factor GATA6. Transcription factors like LRH-1 (Liver Receptor Homolog-1), Oct4  and SOX2, on the other hand are involved in Epiblast differentiation. LRH-1 is required for maintenance of Oct4 expression in the Epiblast. Similarly, the differentiation of Embryonic ectoderm, mesoderm and endoderm requires another transcription factor GCNF (Germ Cell Nuclear Factor) (Ref. 8, 9 & 10).

Besides transcription factors, other genes, notably BMP4 (Bone Morphogenic Protein-4), also help shape the Mouse Embryo prior to Gastrulation. BMP help regulate the differentiation of Mesenchymal cells, which are derived from mesoderm, including bone-forming osteoblasts, and adipocytes, which are fat cells. They also play a role in CNS development. BMP4, which is expressed in the Extraembryonic Ectoderm next to the Epiblast and also in the ICM of the E3.5 and E4.5 Mouse Blastocyst, may activate genes in Epiblast cells that then migrate to form the Primitive streak. WNT3 (Wingless-Type MMTV Integration Site Family Member-3) apparently helps induce the formation of both the Primitive Streak and the Node in mammals, although there is no evidence indicating that WNT3 expression is required for Mesoderm induction. Thus, discovery of several genes and proteins involved in Mouse Embryonic development has made Mouse a powerful model to study Mammalian Embryo development, specially to investigate implantation owing to the ability to control Uterine physiology through exogenous stimuli, and more recently, the ability to manipulate gene expression (Ref. 1 & 11).