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Monitoring Stem Cell Research


Table of Contents

The President's Council on Bioethics
Washington, D.C.
January 2004
www.bioethics.gov


Appendix A:

Notes on Early Human Development

The term “embryo” refers to an organism in the early stages of its development. In humans, the term is traditionally reserved for the first two months of development. After that point, the term “embryo” is replaced by the term “fetus,” which then applies until birth. Some authors further reserve the term “embryo” for the organism only after it has implanted and established its placental connection to the pregnant woman. Similarly many also reserve the term “pregnancy” for the state of the woman only after implantation. At the beginning of the individual’s development, the entity is a single cell. After two months, it has limbs, distinct fingers and toes, internal development, and countless cells. So the term “embryo” applies to an individual throughout a vast range of developmental change. This document is a description of early human development, with emphasis on those events or structures that have figured most prominently in recent discussions of research using human embryos or their parts, especially for stem cell research.1

Development has fascinated centuries of observers, as they pursued deeper understanding of the stability of species characteristics at least from one generation to the next, as well as the uniqueness of each offspring. Uniqueness is especially marked in sexually reproducing organisms, that is, organisms where the genetic make-up of the offspring comes from a combination of maternal and paternal DNA, because a new genome is formed in each instance of conception. The stability reflects inheritance connecting one generation with the past and future members of its line.

Organisms and the processes of their development have evolved. As a result, the development of any organism has a species-specific pattern, but also shares many of the same developmental processes with other species related from its evolutionary origins. Many of the processes discussed here are common not just to all humans, or to all mammals, but to all vertebrates. In some cases they are shared even with invertebrates as well.

The process whereby a new individual of the species comes into being has been at the center of too many deep inquiries to list here, let alone discuss in the depth they deserve. But even in this short document it is important to note one question that is related to the connection of one generation to the next and previous generations. That is, how are we to understand the apparent directedness of development, following a complex network of pathways from a single cell to a multi-system, free-living, and even conscious being? This process occurs in a reliable pattern time after time, but also is sufficiently resilient to perturbations that developing entities can recover from significant disturbances. For example, at early stages of development an embryo may divide (or be cut) completely in half, and then each half recovers to form an entire offspring, resulting in identical twins.

Different notions of purposive directedness, functional explanation, and even vital forces have been invoked to explain development. One of the insights, from the relation of development to evolution, is that the development of an individual reflects the fact that it is descended from individuals that reproduced successfully and, like its forebears whose DNA it inherited, its development reflects their past survival with their particular characteristics. This legacy of ancestral success at survival is manifested in the new organism’s apparent directedness toward development along lines that enhance its own survival. Even very early embryos follow patterns of differentiation in the progeny of different cells. These patterns, in embryology, are called the fate of the progeny of a cell. The fate of the progeny of the newest single cell embryo is maximally broad—if it survives it will give rise to every type of cell of the species. But as the embryo becomes multicellular, its cells specialize and, in the absence of artificial perturbation, their progeny have increasingly specialized fates as well.

The evolved events and processes of development include some that reflect distant relations, such as the yolk sac that is conserved in placental mammals, including human beings. Other events or processes exhibit the evolution of more specific characteristics. In animals such as human beings, the specialized and complex membraneous structures that form the connection between the individual body of the pregnant woman and the developing individual body of the offspring begin to arise in the first week. Human embryos implant in the uterine wall starting at about the sixth day after conception, so of course they must arrive in the uterus with membranes capable of participating in that bond. They do not have a fully formed placenta at such an early stage, nor is the uterine wall unilaterally ready, but rather the contact of embryo and endometrium initiates complementary development finally resulting in the fully developed placenta. One way to look at it is that the early embryo’s very structure points to the future, showing its overall developmental fate to be connected to the maternal body. Another perspective is that this process reflects the past survival of many generations. In both senses, no moment of development can be understood in isolation from the context of the organism’s reflection of its predecessors in evolution, and its directed differentiation toward its future functioning.

I. GERM CELLS

For the beginning of an embryo, one can look both at the newly fertilized egg, and also further back, to embryos of the previous generation. The beginning of an individual is, of course, the union of egg and sperm, specifically the union of DNA in the nucleus of each, so as to form a new complete genome. But the egg and sperm in turn develop from primordial germ cells that were themselves developed when the parents of the new individual were embryos. This description starts at that point (Figure 1).

Developmental cycle of a frog

Figure 1: Developmental cycle, here of a frog. Note the continuity of germ plasm. [Figure 2.1, page 26, in Gilbert, S. Developmental Biology. 6th Edition. Sunderland, Mass.: Sinauer Associates Inc., 2000. Figure reproduced with permission of Sinauer Associates.]


The primordial germ cells are the cells that will give rise to either ova or sperm. They are large cells with some distinctive characteristics that make it possible to track them in development. Note that in Figure 1, they are highlighted throughout the life cycle of the animal. Primordial germ cells appear in embryonic development prior to the formation of the gonads (ovaries in female, or testes in a male). In humans and other mammals, the primordial germ cells actually develop first in the yolk sac. In either sex, the primordial germ cells migrate in through the developing gut of the embryo and then populate the new gonads of whichever type. In humans, the primordial germ cells first appear by the end of the fourth week of development, and begin their migration to the gonads. The primordial germ cells share certain characteristics with embryonic stem cells, including self-renewal and pluripotency. Primordial germ cells have been recovered from fetuses that were aborted (for reasons unrelated to research) and cell lines have been established from them, the progeny of which showed characteristics of multiple different types of cells.2

After the primordial germ cells populate the gonads, some continue to divide by mitosis, producing more like themselves. The primordial germ cells are diploid, meaning that they have all the normal chromosomes of the organism in pairs. In humans, this means that they have 22 pairs of autosomes, and one pair of sex chromosomes, or 46 total. Mitosis is the name of the process whereby the cell replicates its DNA and then divides equally to result in two cells, each cell including an entire complement of DNA just like the first cell before the division (in humans, that is the 46 total chromosomes mentioned) (Figure 2).

But if a cell is to become an ovum or sperm ready to combine with a gamete of the complementary type to produce a new organism (at first a zygote) containing the normal number of chromosomes, it must undergo a special type of cell division whereby each gamete acquires only half the diploid number. Each mature ovum or sperm must include only 23 single (not paired) chromosomes. Mature ova or sperm cells are haploid, indicating that their 23 chromosomes in their nuclei are unpaired (and after they combine, then the resulting single cell the zygote is again diploid). The process whereby the diploid primordial germ cells develop into haploid gametes is called meiosis (Figure 3). Mitosis is part of the life cycle of any cell, but meiosis or meiotic division occurs only in the development of haploid ova and sperm from diploid primordial germ cells. The process itself appears as though the cell nucleus is undergoing two rounds of mitosis, but omits the step of replicating DNA on the second cycle. In the “first round,” the differentiating primordial germ cell replicates its DNA, and then in the “second round” it divides again (without another replication). In the second division, the pairs of chromosomes separate, leaving each of the new cells with just one copy of each of the 22 (in humans) autosomes and just one sex chromosome.

Schematic summary of principal stages in mitotic division

Figure 2: Schematic summary of the principal stages in mitotic cell division, simplified to show the movement of just two pairs of chromosomes. [Figure 3-4, page 61, in Carlson, B.M. Patten’s Foundations of Embryology. 4th Edition. New York: McGraw-Hill, 1981. Figure(s) reproduced with permission of the McGraw-Hill Companies.]


This process does not always occur flawlessly. Errors, such as failure of the chromosomes to separate properly, sometimes produce new cells that have the wrong number of chromosomes, a condition called aneuploidy (that is, not the true number). One cell may have an extra copy of one of the chromosomes while the other cell is missing a copy. Such a condition can be detected in the lab by

Schematic summary of major stages of meiosis in germ cell

 


Figure 3: Schematic summary of the major stages of meiosis in a generalized germ cell, simplified to showing movement of two pairs of chromosomes at the start. [Carlson, 4th ed., Figure 3-6, p. 64.]

collecting some cells when they are about to go through mitosis so their chromosomes can be stained and be spread out so their number and appearance can be examined. A normal set of chromosomes produces a characteristic picture (22 recognizable pairs and a pair of sex chromosomes) called the normal karyotype. If a cell is aneuploid, it will produce an abnormal karyotype picture. If the aneuploid cell becomes an egg or sperm and is then involved in a conception, the embryo is also aneuploid. Aneuploidies are not uncommon events in germ cell development, but aneuploid survival is uncommon; nearly all aneuploidies are fatal very early in development.


II. FERTILIZATION AND CLEAVAGE

Like the word “embryo,” the word “conception” refers to a series of events or processes, not an instantaneous occurrence. Human development begins after the union of egg and sperm cells during a process known as fertilization. Fertilization itself comprises a sequence of events that begins with the contact of a sperm cell with an egg cell and ends with the fusion of their two pronuclei (each containing 23 chromosomes) to form a new diploid cell, called a zygote. Fertilization normally occurs in the ampulla of the uterine tube 12-24 hours after ovulation (Figure 4).

Before that, however, sperm must travel through the vagina and the cervix, through the uterus, and then up the uterine tube. Smooth muscle contractions in the uterine tubes as well as ciliary activity (waving of hair-like structures) of the tube’s lining both are important in the transport of sperm up, and of the ovum into and then down, the uterine tube. Many more sperm, on the order of tens, or even hundreds, of millions, are ejaculated than reach the ovum. Those sperm that do come into the vicinity of the ovum must get through the material covering the ovum (the corona radiata and the zona pellucida) and finally contact and bind to the ovum’s membrane, by means of specialized structures in the head of the sperm cell. When a sperm does get into the ovum, then the ovum membrane changes so that other sperm cannot enter. Meanwhile, the sperm cell in the egg is also undergoing changes and its specialized structures fall away. The haploid nuclei of both the sperm and the egg are now called male and female pronuclei. Both swell, as their densely packed DNA loosens up prior to replication, and they also migrate toward the center of the ovum. Then their nuclear membranes disintegrate and the paternally and maternally contributed chromosomes pair up, an event called syngamy. In this integration, the diploid


Steps in process of fertilization

Figure 4A-E: Steps in the process of fertilization. The sequence of events begins with contact between a sperm and a secondary oocyte (a mature egg) in the ampulla of the uterine tube, and ends with formation of a zygote. [A-E: Fig. 1-1, page 3, in Moore, Essentials of Human Embryology, 1988, with permission from Elsevier.]

Figure 4F: (see following page) Shows fertilization, syngamy [from www.visembryo.com by Mouseworks, Inc.]

Fertilization, syngamyFigure 4F: Fertilization, Syngamy

syngamy. In this integration, the diploid chromosome number is restored, and a new complete genome comes into being.  The result of syngamy is an entity with an individual genome. Further, if all goes well, it is an entity that is capable of developing into a fully formed individual  of the species. The fertilized egg is now called a zygote. It is at this point already entering the first stage of its first mitotic division, and beginning cleavage (Figure 5).

Embryo after cleavage

Conception in the Lab

In Vitro Fertilization (IVF), literally “fertilization in glass” is the procedure of combining eggs and sperm outside the body in a dish. The zygotes that are the results of successful conception, if any, are grown in culture for a few days and then transferred to the uterus of the mother. It may be used when the prospective mother has damaged uterine tubes. Louise Brown, the first baby from IVF was born July 25, 1978, in the UK. IVF was put into practice in the U.S. starting in 1981 and there have since been over 114,000 U.S. IVF births.

Intracytoplasmic Sperm Injection (ICSI) is a variation on IVF. Instead of just allowing sperm and eggs to come into contact in a dish, a technician physically places a sperm cell inside the egg cell through the egg membrane. ICSI is used, among other reasons, when the prospective father has some condition affecting fertility, such as a low sperm count.

Usually more eggs are collected for fertilization than would be transferred at one time, both to increase likelihood of some successful conceptions, and because the process of collecting eggs involves hormonal treatments that can be uncomfortable and risky for the woman. Any early embryos that are not transferred right away are usually stored frozen for later transfer. But many of these are not transferred. In the U.S. as of June 2002 there were approximately 400,000 embryos in storage.

Figure 5: Embryo after cleavage.
[www.visembryo.com by Mouseworks, Inc.]


chromosome number is restored, and a new complete genome comes into being. The result of syngamy is an entity with an individual genome. Further, if all goes well, it is an entity that is capable of developing into a fully formed individual of the species. The fertilized egg is now called a zygote. It is at this point already entering the first stage of its first mitotic division, and beginning cleavage (Figure 5).

Like other vertebrates, humans have polarity in three dimensions (head-tail, or back-front, and left-right). Establishing polarity is one
of the most basic manifestations of emerging specialization. But the egg is roughly spherical, and it is not readily apparent how polarity is established. Although it had been shown long ago that the point of sperm entry determines the plane of first cleavage (and thus subsequent ones) in amphibian eggs, mammals were believed until recently to remain spherically symmetrical until later in development. Recent data on mammalian zygotes, however, suggests that the point of sperm entry may similarly determine the cleavage plane.3

Even the first two cells resulting from the first cleavage may have different propensities, which persist through the next divisions as the progeny of one cell tend to become the body of the offspring and progeny of the other cell become the embryo’s contribution to the placenta and other supporting structures. The word “fate,” however, might be too strong, because the cells of such very early embryos are resilient to perturbations—if one cell is removed, the remaining ones can compensate.


III. Implantation

After fertilization, the zygote proceeds immediately to the first cleavage and subsequent cell divisions follow rapidly. The zygote is a very large cell, but the first waves of rapid cell division occur without increase in cell volume. The result is a closely bound mass of cells each of more typical cell size. At this stage the cells are called blastomeres, (“parts of the blast,” “blast” coming from the Greek for “bud” or “germ”) and the organism as a whole is called a morula (from the Latin for mulberry, descriptive of its appearance) from the time it has 16 blastomeres to the next stage. The morula is still encased in the zona pellucida. As it is undergoing this very rapid cell division, the organism is also migrating down the uterine tube toward the uterus. After it arrives in the uterus, at about day five after the initiation of fertilization, the zona pellucida breaks up; the process is called “hatching” and is a necessary prelude to implantation.

Many zygotes do not survive this long. Estimates vary widely of the rate of natural embryo loss prior to implantation or after implantation but still early in gestation. One study of healthy women trying to conceive found 22 percent of pregnancies (identified by sensitive hormone measures) were lost prior to becoming detectable clinically. Even after implantation, there is a substantial rate of loss, still not known precisely but estimated at 25 to 40 percent.4

When the morula enters the uterus, fluid starts to accumulate between its blastomeres. The fluid-filled spaces run together, forming a relatively large fluid-filled cavity. At the point when the cavity becomes recognizable, the organism is called a blastocyst (Figure 6). The outer cells of the blastocyst, especially those around the blastocyst cavity, assume a flattened shape. The flattened cells of the exterior blastocyst are the trophoblast. They become the embryo’s contribution to the placenta and other supporting structures. On one side of the blastocyst is a group of cells that project inside into the blastocyst cavity; this is the inner cell mass, or embryoblast, and its progeny form the body of the new offspring.


FORMATION OF THE BLASTOCYST

Three stages of mammalial blastocyst

Figure 6A-C: Three stages of the mammalial (blastocyst) of the pig, drawn from sections to show the formation of the inner cell mass.
[Carlson, 4th Ed., Fig. 4-10, page 124.]

Figure 6D: Early blastocyst (see following page).

Early blastocyst


The cells of the inner cell mass can give rise to progeny differentiating into all the types of cells in the adult body, so they are called pluripotent. They have not usually been described as totipotent because, the inner cell mass having already differentiated from trophoblast, the cells of the inner cell mass were believed to be no longer able to give rise to the cells of the trophoblast. Recent work, however, describes culture conditions under which human embryonic stem cells can differentiate to trophoblast cells.5


Although the new offspring itself develops only from the inner call mass, the trophoblast is not just passive padding. Its progeny are the essential and specialized connection between the embryonic and maternal systems. Embryonic stem cells can be isolated from the inner cell mass (see Chapter 4).


IV. TROPHOBLAST TO PLACENTA

After the embyo covering degenerates, the blastocyst, now in the uterus, enlarges and its trophoblast attaches to the endometrium (the uterine lining) at about six days after fertilization. This begins the process of implantation, during which the blastocyst becomes integrated with the endometrium through specialized membranes. The embryo is now beginning its second week of development. The process of implantation takes three to four days, but is generally completed by day twelve. The trophoblast area that binds to the endometrium first differentiates into an inner layer of cells and an exterior layer in which the membranes dividing the cells degenerate and the cells fuse. As the blastocyst become more deeply embedded in the endometrium, the layered area expands until finally the whole trophoblast surface has divided into one layer or the other. Meanwhile, a sort of primitive circulation develops, supporting the embedded blastocyst while more complex structures continue to develop. The inner cell mass then separates itself from the overlying trophoblast. The resulting space is called the amniotic cavity and the layer of cells that forms its roof is called the amnion (Figure 7).

Another membrane called the chorionic sac develops from the trophoblast and nearby tissue. Finally, outgrowths of trophoblast from the chorion project into the endometrium and are called primary chorionic villi, later giving rise to the placenta. Although the blastocyst has become completely embedded in the endometrium and maternal blood bathes the chorionic villi, the maternal blood does not enter the blastocyst. Later, as the fetal circulation develops, the fetal and maternal blood systems still remain distinct and do not mingle. Nutrients, oxygen, and wastes diffuse in the appropriate direction across the placenta, but the two blood systems are individual and do not combine.

Completely implanted blastocysts


Figure 7A: Sections of completely implanted blastocysts at the end of the second week, illustrating how the secondary yolk sac forms. The presence of primary chorionic villi on the wall of the chorionic sac is characteristic of blastocysts at the end of the second week. A primitive uteroplacental circulation is now present. [Reprinted from Moore, Essentials of Human Embryology, 1988, Fig. 2-2, p. 13, with permission from Elsevier.]

micrographs of implantation

Figure 7B: Photo micrographs of implantation beginning and completed. [www.visembryo.com by Mouseworks, Inc.]

V. Twinning

The usual case for human beings is for one ovum to be released, and if all goes well, fertilized and developed to term. Less commonly, more than one ovum may be released and fertilized so that more than one embryo develops. These embryos would be genetically distinct, sharing the uterus during the same gestation period. They will have a family resemblance but no more genetic commonality than any other set of siblings, and they may be of the same or different sexes. These are called dizygotic twins (because they came from two zygotes). More rarely, a single zygote may, during its early cleavages, separate completely into two groups of cells. As discussed above, the two cells resulting from the first cleavage may already have different probable fates, the progeny of one contributing to the body and the other to the supporting structures. Both, however, at this stage are still totipotent and can, if disrupted, go on to generate a full individual organism. If this separation occurs, then monozygotic twins may be born (Figure 8). Monozygotic twins, two offspring coming from one zygote, have the same genome and are always of the same sex. When the twinning occurs in the first cleavages and there are not yet any extraembryonic membranes (Figure 8A), the two develop separately as do dizygotic twins, with separate amnions, chorions and eventually placentae. If an embryo should divide into two later in its development, between about days four and eight, the twins will share the same chorion and therefore eventually the same placenta, but a separate amnion will form around each (Figure 8B). Should an embryo divide later than this, between about the ninth and thirteenth days, the resulting twins will share the same amnion, chorion, and placenta. It is very rare for embryos to divide still later than this, but occasionally they do divide after the fourteenth day. These divisions may not be complete, and then the twins remain conjoined and can only be surgically separated after birth (Figure 8C). The twin birth rate in the U.S. has increased markedly in recent years, and was 30.1 per 1,000 live births in 2001.6 The rate of multiple births (most multiple births are twins; triplets and so on are more rare) is higher with assisted reproductive technologies and with higher maternal age. Dizygotic twins clearly can result in ART from transferring more than one embryo to the prospective mother. In addition, some assisted reproductive practices, like age of the embryo transferred, may be associated with more likelihood of monozygotic twinning,7 though in general the causes of monozygotic twinning are not known.

Modes of monozygotic twinning

Figure 8A-C: Modes of monozygotic twinning. [Carlson, 4th Ed., Fig. 1-12, page 23]

VI. The primitive streak and Gastrulation

While implantation is occurring, the inner cell mass is also undergoing changes. First, the inner cell mass separates into two layers, the epiblast, which is next to the amniotic cavity, and the hypoblast, which is next to what was the blastocyst cavity but is by this stage called the primary yolk sac. The epiblast thus forms the floor of the amniotic cavity (as the amnion forms the roof) and is connected with the amnion around the edges. The hypoblast is connected around its edges with the exocoelomic membrane or primary yolk sac. Thus, the supporting structures, collectively called the extraembryonic membranes, are outside of the body that is starting to develop and that will eventually be born, but during embryonic development the membranes are also continuous with that body. By the end of the second week, the hypoblast has developed a thickened area, called the prochordal plate, that is located at what will be the cranial (head) end of the individual. In fact, the prochordal plate shows where the mouth will develop.

As the third week of development begins, dividing cells pile up in a line to form a thicker band in the epiblast. The line or band starts nearly directly across from the prochordal plate, and extends from the edge toward the center of the embryonic disc. The band is called the primitive streak. In many policy discussions, the appearance of the primitive streak is an important boundary. This summary will continue just a little longer, in order to discuss briefly the nature of the primitive streak.

The end of the primitive streak that is toward the middle of the disc (nearer the prochordal plate marking the mouth) is the cranial end, and this end thickens more as more cells divide. This especially thick end is called the primitive knot (formerly called Henson’s node). The end of the primitive streak near the edge is the caudal (or tail-ward) end. As a model, think of the primitive streak as a zipper: the epiblast cells that made the thickness now start to migrate across the surface and into the zipper of the primitive streak. As the cells enter the primitive streak, they do a U-turn around the edge and continue to migrate back the way they came but underneath the surface, displacing the hypoblast cells. This movement results in three layers, all of epiblast origin: what was the epiblast on top, the cells that used to be part of the epiblast but are now underneath it, and the cells that remain in between (Figure 9).

These three layers get new names, and they also get newly specified fates for their progeny. In the same order as above, they are the ectoderm, mesoderm, and endoderm. The completion (during the third week after fertilization) of forming these three layers is called gastrulation. The ectodermal layer gives rise to progeny fated to become the skin, the nervous system, and sensory structures of the eye, ear, and nose; mesoderm gives rise to the skeletal and muscular systems, connective tissue and blood vessels, and endoderm gives rise to epithelial parts (e.g., the linings) of the digestive and respiratory systems.

Gastrulation is a crucial event in the development of the body plan of the individual, and it is a stage of development common to all vertebrates. Our understanding of the significance of establishing the three germ layers has grown more complex and subtle over the years. Once interpreted as three completely separate paths or compartments of development, we now know that the progeny of the three layers are not totally isolated in their fates. Cartilage, for example, was once thought to be entirely of mesodermal origin, but now we know that some cartilaginous structures of the head and neck come from ectoderm. Even more recently, work with certain adult stem cell populations in culture and under special conditions has suggested plasticity of cell progeny from one germ layer to develop characteristics of cells typically from another germ layer, long after gastrulation has assigned the cells of different germ layers their different fates. Gastrulation is not the first differentiating event: cells begin to acquire fates for different parts of the developing embryo before the inner cell mass separates into epiblast and hypoblast, indeed some results suggest even before the blastocyst develops an inner cell mass and trophoblast. Yet these findings in no way detract from the significance of gastrulation. They rather facilitate our understanding of gastrulation by placing it in the context of the entire process of differentiation, beginning from the very earliest stages.



Scematic Drawings of Embryonic Disc

Figure 9A-C: Schematic drawings of the embryonic disc and its associated extraembryonic membranes during the third week. A: the amniotic cavity has been opened to show the primitive streak, a midline thickening of the epiblast. Part of the yolk sac has been cut away to show the bilaminar embryonic disc (epiblast and hypoblast). The transverse section (lower right of A) illustrates the proliferation and migration of cells from the primitive streak to form embryonic mesoderm. B and C: drawings illustrating early formation of the notochordal process from the primitive knot of the primitive streak. In the longitudinal sections on the right side, note that the notochordal process grows cranially in the median plane between the embryonic ectoderm and endoderm. [Reprinted from Moore, Essentials of Human Embryology, 1988, Fig. 3-1, page 17, with permission from Elsevier.]


Figure 9D: Photo micrograph of Primitive Streak
[www.visembryo.com by Mouseworks, Inc.]

Primitive Streak


VII. Neurulation

Neurulation is the series of developmental events that result in the beginnings of the central nervous system (Figure 10). From the cranial end of the primitive streak, a long stiff structure develops in the mesoderm, elongating still further in the cranial direction. This becomes the notochord, which marks the head/tail axis of the embryo. Later, the vertebral column develops around it. But at this time, the notochord and its adjacent tissue exert influence called primary induction on the ectoderm lying over them, such that the ectoderm thickens and becomes the neural plate.

The neural plate then actually pushes up to form folds (called the neural folds) along each side of the tissue over the notochord. The neural folds then meet and fuse to enclose the neural tube, beginning at the middle of the (future) tube, like a zipper closing from the middle toward each end. This process is completed by the end of the third week. Some cells along the crests of the folds migrate through the embryo. They are called neural crest cells, and they give rise to a variety of nerve cells including dorsal root (spinal) and autonomic nervous system ganglia, and some other nervous system and endocrine structures.

Schematic drawings of human embryo during third and fourth weeks


Figure 10A-H: Schematic drawings of the human embryo during the third and fourth weeks. Left side: Dorsal views of the developing embryo illustrating early formation of the brain, intraembryonic coelom, and somites. Right side: Schematic transverse sections illustrating formation of the neural crest, neural tube, intraembryonic coelom, and somites. [Reprinted from Moore, Essentials of Human Embryology, 1988, Fig. 3-3, page 20, with permission from Elsevier.]

Neurulation and Notochordal Process

Figure 10I: Neurulation and Notochordal Process
[www.visembryo.com by Mouseworks, Inc.]


The mesoderm still adjacent to the neural tube resolves into the form of paired blocks on either side of the tube, which are called somites. The first pair of somites appears at about the twentieth day after fertilization, at the cranial end of the neural tube. More pairs appear in the caudal direction, up until about the thirtieth day. Mesodermal cells from the somites give rise to most of the skeleton and skeletal muscle.

Blood cell and blood vessel formation actually start at the beginning of the third week after fertilization, first in the supportive structures of the yolk sac and chorion. Blood vessel formation begins in the embryo body about two days later, although blood is not formed in the embryo itself until the fifth week. The heart begins as a wide blood vessel, which later folds up to develop the chambers of the fully formed heart. But even as a tube, the membranes of its cells have the electrical and contractile capacity to begin beating in the third week, and thus to begin primitive circulatory function with blood. During this time the primary chorionic villi elaborate branches and form capillary networks and vessels connected with the embryonic heart. Oxygen and nutrients diffuse from the maternal blood to the embryonic blood through these capillaries, while carbon dioxide, urea, and other metabolic wastes diffuse from the embryonic blood into the maternal blood. Meanwhile, even firmer connections form between embryonic supporting membranes and the endometrium, finally completing the development of the placenta.

VIII. Organogenesis

The basic structures and relations of all the major organ systems of the body emerge during the fourth through the eighth weeks of embryonic development. First, the embryo folds in several ways so that the flat linear structure distinguished by neural tube flanked by somites become roughly C-shaped. The effect of this is to bring the regions of the brain, gut, and other internal organs into their familiar anatomical relations. During the fourth week the neural pores, the ends of the neural tube “zipper,” close. First the one at the cranial or head end, which is called the anterior or rostral pore, closes, and later the caudal or tail-ward pore closes. Closure of the neural pores completes the closure of what will become the central nervous system. Also during the fourth week, limb buds become visible, first buds for arms and later for legs. Further, two accumulations of cells along the neural tube become distinguishable: the alar plate and the basal plate. Cells of the alar plate go on to become mostly sensory neurons, while basal plate cells give rise mostly to motor neurons. Already while the neural tube is closing, its walls along the cranial area are thickening to form early brain structure. Cranial nerves, for example the nerves for the eye and for the muscles of the face and jaw, also are beginning to develop at this time. The embryonic brain develops rapidly in both size and structure especially during the fifth week, and the optic cup that will form the retina of the eye becomes visible as well.

IX. Conclusion and Continuation

Embryonic development continues with the emergence and differentiation of organs, the skeleton, limbs, and digits, and with the development of the face and further differentiation and integration throughout the body. The development discussed above is summarized briefly in Table 1.8 But development continues, and is a continuous process, past the eight-week mark, when the organism is no longer called an embryo and instead is called a fetus. Although the basic elements of the body plan have been established during embryogenesis, a great deal of development of that body plan, refinement and integration, continues in the fetal stage, also called phenogenesis (emergence of the normal appearance of the body). Development continues after birth as well.

Table1: Summary of Developmental Timecourse
Stage
Week after fertilization
Days after fertilization
Event
Pregenesis:  develop-ment of parents 4th week develop-ment of parents 24 Parents' primordial germ cells (PGCs) begin their migration to parents' gonads

Blastogenesis

1st week, embryo is unilaminar 1 Fertilization
1.5-3 1st cleavages, move to uterus
5 Free blastocyst in uterus
5-6 Hatching, start implantation
2nd week, embryo is bilaminar 7-12 Fully implanted
13 Primary stem villi and primitive streak appear
3rd week, embryo is trilaminar 16 Gastrulation begins, notochord forms
18 Primitive pit, neural plate, neural groove
20 First somites, primitive heart tube
4th week 22 Neural folds fuse, pulmonary primordium,
24 PGCs begin migration, Cranial neuropore closes, optic vesicles and pit form
26 Caudal neuropore closes, arm limb buds
28 Leg limb buds, more brain, eye/ear devel.
Organogenesis 5th - 8th weeks 29-56  
Phenogenesis 9th - 38th weeks    

_________________

ENDNOTES

1. There are many fine embryology texts, and the reader is urged to consult one or more for deeper, broader and more extended treatment of embryology. The following references are samples only, not a comprehensive bibliography, selected in part for accessibility to the general though committed reader, and in part for recent publication. A few examples concentrating on human embryology would include Larsen, W. J., Essentials of Human Embryology. New York: Churchill Livingstone, 1988; Sadler, T. W., Langman’s Medical Embryology 8th Edition, Philadelphia: Lippincott Williams and Wilkens, 2002; or Sweeney, L. J., Basic Concepts in Embryology: A Student’s Survival Guide, New York: McGraw-Hill, 1988. For a more comparative approach consider Carlson, B. M., Patten’s Foundations of Embryology. 6th Edition, New York: McGraw-Hill, 1996; and for more comparison and inclusion of related topics, see Gilbert, S. J., Developmental Biology, 6th Edition, Sunderland, MA: Sinauer Associates Inc., 2000. In addition, there are many fine web-based resources, which the reader is encouraged to visit, for example http://anatomy.med.
unsw.edu.au/cbl/embryo/Embryo.htm
and http://www.visembryo.com/ and to accompany Gilbert’s text, http://www.devbio.com/. These sites provide links to further resources as well.

2. Shamblott M.J., et al., “Derivation of pluripotent stem cells from cultured human primordial germ cells” Proceedings of the National Academy of Sciences. 95(23): 13726-13731 (1998). [Erratum in: Proc Natl Acad Sci USA 96(3): 1162 (1999).]

3. Pearson, H., “Your destiny, from day one.” Nature 418(6893): 14-15 (2002).

4. Wilcox, A. J., et al., “Incidence of early loss of pregnancy,” New England Journal of Medicine 319(4): 189-194 (1988). See also this review article: Norwitz, E. R., et al., “Implantation and the survival of early pregnancy,” New England Journal of Medicine 345(19): 1400-1408 (2001). Some estimates are indeed much higher (as high as 80 percent for embryo loss before and after implantation).

5. Xu, R.H., et al., “BMP4 initiates human embryonic stem cell differentiation to trophoblast” Nature Biotechnology 20(12): 1261-1264 (2002).

6. National Center for Health Statistics, “Births: Final Data for 2001.” National Vital Statistics Reports 51(2) (2002), available at http://www.cdc.gov/nchs/data/nvsr/ nvsr51/nvsr51_02.pdf.

7. Milki, A.A. et al., “Incidence of monozygotic twinning with blastocyst transfer compared to cleavage-stage transfer” Fertility and Sterility 79(3): 503-506 (2003).

8. Table 1 follows closely the table of events shown in Larsen (1998), p. xi, and also the table presented by John M. Opitz, MD, at the January 16, 2003, meeting of the Council. Not all the events listed in Larsen’s table were included in the Table 1 above, however.

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