Chapter 4 Early Embryogenesis PDF

2024 Chapter 4 Chapter 4: Early embryogenesis Key terms and concepts: Zygote Trophoblast (aka Trophectoderm) Histotroph Clone Blasto...

2024 Chapter 4 Chapter 4: Early embryogenesis Key terms and concepts: Zygote Trophoblast (aka Trophectoderm) Histotroph Clone Blastomere Chimera Cleavage division Monozygotic twin Morula Dizygotic twin Blastocyst, blastocoel Transgenic animal Compaction Knockout animal Inner cell mass (ICM) Embryonic stem cell Induced pluripotent stem (iPS) cell Learning objectives: By the end of this unit, you should be able to: 1. Define the key terms and concepts listed above and understand how each relates to developmental biology. 2. Describe the role of maternal messenger RNA from the egg cytoplasm in early zygotic development. 3. Describe the lineage potential of the blastomeres and the ICM and explain why this potency is important to cloning, twinning, and chimera formation. 4. Identify the first instance of lineage determination in the developing embryo. 5. Identify the cell types of the blastocyst and what they go on to form in later stage embryos. 6. Describe how transgenic and knockout animals are used to study the role of genes in development. 7. Understand the relationship between ES cells and the ICM of the blastocyst. I. Cleavage and Blastocyst Formation A. As the zygote (a single, very large cell with a diploid number of chromosomes), floats within the ampulla and begins to move down the uterine tube, it undergoes rapid mitotic divisions called cleavage divisions, producing cells called blastomeres. Unlike what we see in adult cells, little or no growth occurs between cleavage divisions, so although cell number increases, total cell mass does not. The zona pellucida continues to surround the dividing cell mass, limiting its enlargement, and preventing adhesion of the embryo to the walls of the uterine tube. 1. Because there is little growth between divisions, the original large size of the oocyte is reduced to that of typical cells of that organism. 2. In many non-mammalian species, specific cytoplasmic substances (such as yolk) are concentrated asymmetrically in the egg, and so are distributed to some cells but not others at this early stage. The future of the cells with yolk will be very different than that of cells without. In 1 2024 Chapter 4 mammals, all cells and their contents appear to be equivalent as far as we can tell at this time (regulative development). 3. The newly formed diploid nucleus of the zygote does not become active until several divisions have taken place in some species (in horses and sheep, major zygotic genome activation occurs at the 8- to 16-cell stages). In mice, the zygotic genome becomes active by the 2-cell stage. Until then, development is controlled by factors (RNA and protein) stored in the oocyte cytoplasm. The availability of the maternally derived precursors, enzymes and messenger RNA allows the zygote to synthesize DNA rapidly during cleavage with very little transcription of the embryonic genome. Once activation of the zygotic genome occurs, the genome of the zygote takes over the regulation of development. Activation of the zygotic genome in zebrafish and Xenopus (frog) occurs at more advanced stages of development. 4. Since most mammalian embryos contain little yolk, this floating ball of cells relies at first on uterine secretions (histotroph) for nutrition, and later on the placenta. 5. During the first few cleavage divisions, each blastomere has the potential to produce an entire organism and its associated membranes. Later, this totipotency appears to be lost under natural conditions. B. Once cleavage has produced a cluster of about 16 blastomeres, the embryo is called a morula. C. At about the 8-cell stage in mice, humans, and pigs; 16 cell stage in sheep; and 32 cell stage in cattle, the loosely arranged cluster of cells suddenly huddles together to form a compacted cluster. The cells now have maximum surface contact with each other. This process is called compaction. 1. Once the embryo is a compacted morula, it consists of two distinct cell populations; an inner core of cells surrounded by a superficial external layer that is flattened around the surface of the embryo. a. The superficial cells are flattened and are in contact with the external environment. They are linked by tight junctions and have epithelial characteristics. The superficial cells will give rise to the trophoblast (trophectoderm) cells. These outside future trophoblast (trophectoderm) cells will contribute to the future extraembryonic membranes, which will surround the embryo and help form the placenta. b. The cells that are in the interior of the ball are connected by gap junctions and seem to take a cue from their position and become "determined" as future embryonic cells. Soon, these cells look different and begin secreting different proteins than their neighbors. 2 2024 Chapter 4 In mammalian development, this is the first example of determination and resulting differentiation. Organization of Cells: Within the embryo, cells organize themselves in two patterns: as epithelia and as mesenchyme. Epithelial cells are tightly connected to each other, sometimes tightly enough to preventing molecules from sneaking between them. This makes them excellent at separating one compartment (the outside environment) from another (the “inside” of the epithelium). Why might this be important? Mesenchymal cells are loosely arranged, separated from each other by some type of non-cellular substance (matrix). Most “filling-in” types of tissues in the embryo are mesenchymal in their arrangement until they differentiate into more organized tissues like fascia, cartilage or bone. D. Cleavage division begins while the embryo is in the oviduct. Divisions continue as the embryo moves through the oviduct to the uterus. The time it takes the embryo to reach the uterus varies among species; the embryo arrives in the uterus at a roughly characteristic stage of development in each species. Species Passage to the uterus (approximate) Pig 4-8 cell stage (day 2 post ovulation) Cattle 8-16 cell stage (day 3-3.5 post ovulation) Sheep 8-16 cell stage (day 3 post ovulation) Horse Morula (day 5-6 post ovulation) Dog Blastocyst (day 8 post ovulation) Table adapted from Hyttel et al (2010) “Domestic animal embryology” 1. While the embryo has been moving down the uterine tube, changes have occurred in the wall of the uterus in preparation for implantation. The time it takes for embryos to reach the uterus varies among species, from about 3-5 days in the cow and horse, to 7-8 days in the dog. By the time the embryo reaches the uterus, it is usually at the morula or blastocyst stage. E. During the next few days, division continues as this cluster of cells floats in the uterus. The outer (trophoblast or trophectoderm) cells are held together by tight junctions, which prevent ions and small molecules from leaking between cells. The trophoblast cells begin to pump Na+ ions and proteins into the interior of the compacted morula, causing water to be drawn in by osmosis. This creates an eccentric, fluid-filled space called a blastocoel. This now hollow cluster of cells is the blastocyst (blastula), formation (or blastulation), which is considered the end of cleavage. The zona pellucida, although not shown in the drawing, is still present at this stage. 1. The peripheral trophoblast (trophectoderm) cells will contribute to the formation of the extra-embryonic membranes and placenta. (The term "extra-embryonic" refers to tissues that develop from the blastula but are not part of the embryo itself. Instead they form supportive structures such as the amniotic sac, which will surround the embryo.) At one pole of the blastocyst is a cluster of cells just deep to the trophoblast, inner cell mass (ICM), from which the embryo will develop. These cells form from cells that were at the center of the morula. 3 2024 Chapter 4 2. Since inner cell mass cells can form all of the tissues of the body, but cannot form all of the extraembryonic membranes, they are considered pluripotent (less potential than totipotent cells, more than multipotent). Alone, they could not form a self-supporting embryo, since they have lost the potential to form the placental membranes. F. Following formation and swelling of the blastocyst, the zona pellucida ruptures (hatching). Actually, the most recent evidence is that the zona pellucida in vivo gradually dissolves, but everyone talks about hatching, and that is what is seen in vitro, so we will speak of it that way, too. The trophoblast cells that have covered the surface of the ICM degenerate and disappear in most domestic species, though not in mice, leaving the ICM directly exposed to the uterine environment. G. Following loss of the zona pellucida, the blastocyst is free to enlarge and change shape. It begins as a 2-3mm hollow sphere. In some species, such as the pig, opposite ends grow at an extremely rapid rate, forming a very long, closed tubular structure, with the embryonic disc located midway between the two ends. 1. Rupture of the zona pellucida also permits implantation of the embryo into the uterine lining. The embryos of some species implant very soon after hatching, while others, such as the horse, drift around the lumen of the uterus for weeks before implantation occurs. We will discuss implantation in the next chapter. II. Chimeras, Twins and Other Interesting Things! A. The cells of the early embryo are quite plastic and malleable in their ability to alter their relationship with each other. For example, if one cell is removed from a two-celled embryo, the remaining cell will form a whole, normal embryo. This is possible because each of these cells is totipotent. Even at the blastocyst stage, the cells of the inner cell mass will compensate for the removal of a cell or addition of several cells. This occurs because each of these cells is of equal potency and can produce any of the tissues of the embryo (only the trophoblast can’t be formed from these cells). 1. The ability to remove an ICM cell without harming the embryo has been utilized during in vitro fertilization to test for genetic diseases (or sex) before implanting the embryo into the mother. 2. If the cells of an early embryo (up until about the 8-cell stage) are experimentally separated, each is capable of forming a separate, genetically identical embryo since each is totipotent. This has been used experimentally to produce cloned animals, which is another way of saying they are identical twins (quadruplets, septuplets, whatever!). 3. “Twins” are classified as either monozygotic (identical) from one fertilization event producing a single embryo which later splits or 4 2024 Chapter 4 dizygotic (fraternal) from separate egg fertilizations producing separate embryos carried together in the uterus. Monozygotic twins are not very common among the domestic animals we study. It is estimated that about 8-10% of twins born to cows are monozygotic (it’s about 30% in humans), and it’s even more uncommon in the other domestic animals. Consider a litter of seven kittens. Do they all have the same genome? Are they "septuplets?" Could any of them be twins? Monozygotic or dizygotic? Must they all have been sired by the same male? 4. Monozygotic twins can be formed by the splitting of the early cleavage- stage embryo, but occurs even more often at the blastocyst stage. In this case, the embryonic disc splits, but the trophoblast does not, resulting in shared extraembryonic membranes. The nine-banded armadillo embryo typically undergoes splitting at this stage, resulting in four identical embryos (of course, all of the same sex!). 5. Conjoined twins are the result of incomplete splitting of an early embryo, generally at early gastrulation (the next stage we will discuss- coming up in chapter 4). There are many possible types of conjoined twins, depending on the degree to which the ICM has split and where the split occurs. B. Not only can the embryo reprogram itself if it loses cells, it also can reprogram if it gains cells. Two early embryos, each of about 8-cells and each with its own genotype, can fuse at this early stage, and form one organism with a mixture of cells of each genotype (each individual cell is normal, but there are two different populations of cells in the same animal). This animal is then called a chimera. The inner cell mass of a blastocyst can also incorporate additional cells to generate a chimera. 1. Experimentally, Embryonic Stem (ES) cells, which are pluripotent, can be added to the inner cell mass of a blastocyst to create a chimera. This technique is used frequently in mice for research purposes. Mouse ES cells can be genetically modified in culture and then used to create chimeric mice that are at least partially composed of these manipulated cells. If the manipulated cells contribute to the germ cells of the chimera, the chimera can pass the mutation on to progeny. This technique is generally used to generate animals with specific (targeted) mutations because it is possible to specifically target desired areas of DNA modification using the ES cell technique. The technique is sometimes referred to as “gene knock-out or “knock-in” because specific genes are targeted and alternative DNA is “knocked in” or “knocked out” in ES cells to create specific desired mutations. C. Transgenic animals: A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. One method by which this is done (routinely in research mice) is by injecting DNA into the male pronucleus of fertilized eggs. The male pronucleus is larger in mice and easier to inject. The 5 2024 Chapter 4 injected DNA can then incorporate into the genome, generally randomly, and be part of the genetic material of the resulting adult animals. Genetically engineered mice (knock-out mice, knock-in mice, gene targeted mice) Transgenic mice and other genetically engineered mice are routinely used these days to study how genes are regulated during development. There are also lots of possible commercial applications for transgenesis. For example, manufactured DNA could be added to a cow so it was capable of producing high levels of human therapeutic proteins in its milk. But with the longer gestation and other costs, it’s no wonder that companies that are exploring these techniques would then like to be able to clone the resulting animals! Embryonic Stem Cells: Embryonic stem (ES) cells were first derived from mice in 1981 separately by Evans and Kaufman, and Gail Martin. The ES cells were derived from the inner cell mass of the mouse blastocyst and were able to give rise to all the cell lineages of the embryo proper. When these cells were transplanted to blastocysts, they were able to aggregate with the cells of the inner cell mass and generate chimeric animals composed of both inner-cell mass and ES cell derived cells. Once techniques to manipulate the genome of the ES cells were developed, these cells were used extensively in to generate genetically engineered mice for research purposes. In 1998, Dr. James Thomson, at the time an associate research veterinarian at the UW Primate Research Center, made history by isolating pluripotent stem cells from the inner cell mass (ICM) of human embryos at the blastocyst stage. He was able to grow these ICM cells indefinitely in culture without having them differentiate, and was able to prove that they were capable of differentiating into any cell type in the body. One way he did this was by producing teratomas (tumors composed of cells derived from all three of the primitive cell types of the embryo) from them! Other laboratories have produced multipotent (more restricted) stem cells from germ cells in fetal gonads. Specific types of multipotent adult stem cells (i.e. bone marrow) have also been isolated and cultured, but appear to be limited in the types of tissues they can produce. Since the isolation of human ES cells, Dr. Thomson’s lab and others have been able to generate induced Pluripotent Stem (iPS) cells. These are pluripotent cells derived from differentiated cell types (generally fibroblasts); they are rendered pluripotent by expression of specific combinations of genes. Eventually iPS cells may be useful in therapeutic applications, but several obstacles must be overcome before they can be employed. Clearly, there are many ethical questions regarding these techniques, in addition to the scientific ones. 6