Molecular Biology of the Cell - Volume 3 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

From Cells to Multicellular Organisms
Cellular Mechanisms of Development
Morphogenetic Movements and the Formation of Overall Spatial Body Organization

In this and the following sections, we will discuss how the Spatial Organization of the early embryo arises and what physical forces are involved in its formation. As our primary example, we will look at the African clawed frog Xenopus laevis (Fig. 16-1), whose early development has been studied in exceptional detail. Like the embryos of other amphibians, Xenopus embryos are relatively resistant to external influences, making them a convenient model for experimental studies.

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Fig. 16-1. Selection/3.html">Stages of development of Xenopus laevis from a fertilized egg to a freely swimming, feeding tadpole. Top: mating of an adult male and female. Below: successive developmental stages (early stages shown in lateral view; 10-hour embryo in vegetal view; 19-hour embryo in animal view). All drawings (except the top one) are to the same scale. [P. D. Nieuwkoop, J. Faber, Normal Table of Xenopus laevis (Daudin), Amsterdam: North-Holland, 1956.]

To make the material easier to follow, vertebrate Embryogenesis (along with that of many other animal groups) is divided into three main periods. The first is Cleavage, during which the fertilized egg divides into numerous smaller Cells that form an epithelium-like layer; Gastrulation and neurulation then transform this layer into the archenteron (primitive gut) and the neural tube. This is followed by the period of Organogenesis, during which various Organs and body parts (limbs, eyes, Heart, etc.) emerge. The third developmental period is characterized by the growth of these newly formed rudimentary organs until they reach the proportions typical of the adult animal. These periods lack sharp boundaries and frequently overlap to a significant degree. We will trace the development from the fertilized egg to the onset of organogenesis using Xenopus embryogenesis as our primary example.

16.1.1. The polarity of the amphibian embryo is determined by egg polarity [2]

The amphibian egg is a relatively large Cell (about 1 mm in diameter) enclosed in a transparent, acellular capsule known as the jelly coat. Most of The Cell is packed with yolk platelets, which consist primarily of Proteins and Lipids. The yolk is concentrated in the lower half of the egg, termed the vegetal hemisphere, whereas the upper half is called the animal hemisphere. Fertilization initiates the transformation of this single cell into a multicellular larva (tadpole), in which the anterior-posterior axis, ventral-dorsal sides, and the median plane of Symmetry dividing the body into right and left halves must all be established. It is convenient to describe embryonic development using a system of three axes: anteroposterior (from HEAD to tail), dorsoventral (from back to belly), and mediolateral (from the midline to the left and right). This embryonic polarity is established at a very early stage of development in amphibians. Even before fertilization, the animal and vegetal poles of the egg contain distinct sets of mRNAs, as well as varying amounts of proteins and other cellular components, yet the egg remains symmetrical around the animal-vegetal axis. This initial animal-vegetal symmetry is sufficient to generate a cylindrical embryo possessing an anteroposterior axis.

Fertilization deepens the compositional differences within the amphibian egg, manifesting as the establishment of dorsoventral polarity. The outer, Actin-enriched layer of Cytoplasm, known as the cortical layer or cortex, suddenly rotates as a single unit relative to the internal cytoplasm, causing the animal pole of the cortex to shift relative to the animal pole of the inner contents toward the future ventral side (Fig. 16-2). The direction of this rotation is determined by the site of sperm entry; this is presumably mediated by the centrosome delivered into the egg by the sperm. Because this rotation displaces pigment granules within the egg, certain amphibians develop a faintly pigmented stripe known as the grey crescent. It forms on the side of the egg opposite the sperm entry point. The sperm entry point roughly corresponds to the ventral side, while dorsal structures, such as the Spinal Cord, develop on the opposite side (Fig. 16-3). Interventions that block cortical rotation result in so-called radialized animals with centrally located guts and a complete lack of dorsoventral Asymmetry.

Fig. 16-2. The first morphogenic movement following the fertilization of a frog egg. The egg cortex—a layer approximately a few micrometers thick—rotates by nearly 30° relative to the inner layers of the egg in a direction dictated by the site of sperm entry. In species with pigmented animal-pole cytoplasm, this rotation produces a clearly visible grey crescent located opposite the sperm entry point.

16.1.2. Cleavage converts a single cell into numerous cells [3]

Cortical rotation finishes about an hour after fertilization and sets the stage for cleavage, during which repeated mitoses convert the large zygote into a multitude of smaller cells called blastomeres, without changing the overall mass of the embryo. To survive, the embryo must rapidly reach a stage where it can feed, swim, and evade predators on its own; consequently, the initial cleavage divisions follow one another in rapid succession, with a Cell Cycle duration of only about 30 min (see Section 13.1.9). This high rate of METABOLISM/36.html">DNA Replication and mitotic cycling precludes Gene Transcription, making the cleaving embryo entirely dependent on the reserves of RNA, proteins, membranes, and other Materials accumulated in the egg during its maturation within the maternal Organism. Only DNA Biosynthesis remains critical at this stage, and its unusually rapid replication is made possible by a vast number of replication origins (see Section 9.3.9). The first cleavage furrow divides the egg vertically, that is, in a plane passing through the animal-vegetal axis, producing two symmetrical halves of the egg (Fig. 16-4). The next division is also vertical but occurs at a right angle to the first, resulting in four cells of equal size. The third cleavage furrow lies horizontally, slightly above the equatorial plane, so that the four upper cells formed are smaller than the lower ones; furthermore, the lower cells contain more yolk. After roughly 12 cell cycles, The rate of Cell Division drops sharply, division synchrony is lost, and Transcription of the embryonic genome begins. This transition, known as the mid-blastula transition (MBT), likely reflects the depletion of a specific maternal cellular component required for binding to newly synthesized DNA.

Fig. 16-3. The asymmetry of the Xenopus egg is shown in A. It determines the body axes of the future tadpole illustrated in B. Mapping the egg axes precisely onto the tadpole body is complex because gastrulation movements significantly alter the embryo's topology. As a first approximation, the animal pole corresponds to the anterior end (head) of the body, and the grey crescent corresponds to the dorsal side.

In other animals, the relative orientation of successive cleavage planes may differ. In eggs extremely rich in yolk, such as those of birds, cleavage furrows fail to divide the yolk entirely, leaving all nuclei concentrated at the animal pole; consequently, the embryo develops exclusively from a "cap" of cells sitting atop the yolk.

16.1.3. The blastula is a hollow sphere whose wall consists of a single cell layer [4]

From the very beginning of embryonic development, cells are interconnected not only mechanically but also via Gap Junctions, which allow ions and other low-molecular-weight substances to pass through, transmitting signals that (along with Other types of cues) play a vital role at later stages in coordinating cell behavior (see Section 14.1.7). At the periphery of the embryo, blastomeres are linked by tight junctions (see Section 14.1.1), which isolate the internal part of the embryo from the external environment. Around the 16-cell stage, the intercellular spaces in the central region of the embryo expand to form a single cavity, the blastocoel. This occurs because sodium ions are actively pumped into the internal intercellular spaces through cell membranes, raising the osmotic pressure inside the embryo and drawing Water in. The cells surrounding the blastocoel form an epithelium-like layer, and this developmental stage is now referred to as the blastula (Fig. 16-5). The organization of blastula cells into an epithelial-like layer is crucial for coordinating their subsequent behavior.

16.1.4. Following gastrulation, the hollow cellular sphere transforms into a three-layered Structure featuring a primitive gut [5, 6]

Once the blastula cells have formed an epithelial-like layer¹, the time arrives for coordinated movements that lead to The formation of the gastrula, a process known as gastrulation. This radical rearrangement converts the hollow spherical embryo into a multilayered, bilaterally symmetrical structure with a central gut tube. Through a complex process of invagination, a substantial patch of epithelium moves from the outer surface into the interior of the embryo. Subsequent development is driven by interactions among the internal, external, and middle layers generated during gastrulation. Gastrulation occurs in one form or another in all Multicellular animals. Because amphibian gastrulation is geometrically somewhat distorted, we will first examine this process in the sea urchin, a close relative of vertebrates (see Fig. 15-40, Section 15.4.1).

¹ The author uses a slightly imprecise term here by referring to the blastula wall as an epithelium or epithelial layer. Strictly speaking, an epithelium is a differentiated tissue that forms at later developmental stages from the ectoderm (the outer germ layer). The embryonic wall at the blastula stage is properly called the blastoderm, bearing only a superficial resemblance to true Epithelial Tissue. For purely practical reasons, we have not corrected this inaccuracy in every instance, and the reader should keep this in mind. — Note by the Editor.

Fig. 16-4. Cleavage stages in Xenopus (lateral view).

Fig. 16-5. The blastula. At this stage of development, cells form an epithelium-like layer enclosing a fluid-filled cavity, the blastocoel. Gap junctions provide electrical coupling between cells, while tight junctions on the outer surface isolate the embryo's internal space from the external environment. Note that in Xenopus, the blastula wall consists of multiple cell layers, with only the outermost cells tightly juxtaposed, as is typical of any epithelial type.

Fig. 16-6. Gastrulation in the sea urchin. Primary mesenchyme cells detach from the blastula wall at the vegetal pole (A). These cells crawl upward along the inner surface of the blastocoel wall (B). Meanwhile, the blastula wall in the region of the vegetal pole (the vegetal plate) begins to invaginate inward (C). Cells at the tip of the invaginating vegetal plate extend filopodia that attach to the inner wall of the blastocoel and pull it further inward, forming the archenteron or primitive gut (D, E). The tip of the gut tube makes contact with the blastula wall, and the Mouth ultimately forms at this site (F). (L. Wolpert, T. Gustafson, Endeavour, 26, 85-90, 1967.)

The sea urchin embryo is transparent, allowing both its external and internal development to be observed in living specimens; at the same time, this object is convenient for studying The activity of individual cells. The Starting Material for gastrulation is the blastula, which has a rather simple structure: it is a hollow sphere whose wall is formed by about 1,000 cells arranged in a single layer; the blastula cavity is called the blastocoel. This entire structure is covered by a thin layer of Extracellular matrix; one can also distinguish the vegetal and animal poles in the blastula. Gastrulation begins when several dozen so-called primary mesenchyme cells detach from the epithelium at the vegetal pole (Fig. 166, A). In all likelihood, these cells lose their ability to bind to other cells and to the extracellular matrix of the embryo's outer surface, and acquire an affinity for the Fibronectin-rich matrix (see Section 14.2.13) that lines the blastocoel. These cells migrate into the blastocoel and move along its wall, pulling themselves forward by means of long, thin extensions (filopodia) with "sticky" tips (Fig. 16-7). When the tip of a filopodium comes into contact with a surface to which it can firmly attach, the filopodium contracts and pulls the cell along. The formed filopodia are apparently retracted, and new ones emerge elsewhere, allowing the cell to move back and forth. Eventually, however, the cells occupy a well-defined position, which is presumably due to their specific affinity for certain Regions of the blastocoel surface. This has been demonstrated in experiments with Monoclonal Antibodies, which revealed highly specialized differences between cells in different regions of the embryo regarding their surface chemistry (we will return to this topic later—see Section 16.6.1). Once the primary mesenchyme cells have taken their place, they begin to form the Skeleton (16-6, E).

Fig. 16-7. Primary mesenchyme cells crawl along the inner surface of the blastula wall, extending contractile filopodia with "sticky" tips. (L. Wolpert, T. Gustafson, Endeavour, 26, 85-90, 1967.)

Fig. 16-8. This diagram illustrates a possible mechanism for the Initial Stages of cell invagination at the vegetal pole. The details of this process at THE MOLECULAR LEVEL are discussed in Sections 16.1.6 and 11.6.9.

With the onset of primary mesenchyme cell migration, the epithelium in the region of the vegetal pole begins to invaginate, thereby forming the primitive gut (archenteron) (Fig. 16-6, B). Initially, the shape of the epithelial cells changes: the inner end of the cell facing the blastocoel becomes wider than the outer end, causing the cell layer to bend inward toward the blastocoel (Fig. 16-8). The next stage of invagination occurs through a different process—cell redistribution. The invaginating cells actively rearrange themselves, but their shape remains unchanged. As a result, the initially rather wide cavity of the gastrula transforms into a long, narrow intestinal tube. Simultaneously, certain cells at the tip of this intestinal tube extend long filopodia into the blastocoel, which contact the cavity walls, adhere to them, and contract, seemingly helping to guide the invagination process (Fig. 16-6, C, D). Movement ceases when the blind end of the intestinal tube comes into contact with the outer wall of the embryo at its opposite end (Fig. 16-6, E). Later, at the site of contact between the two touching layers, the embryo wall ruptures, and a secondary mouth is formed at the rupture site. Because the cells that guided invagination with their filopodia have completed their task, they detach from the epithelium, migrate into the space between the gut tube and the body wall, and transform into the so-called secondary mesenchyme, which eventually gives rise to the coelomic wall and musculature.

As a result of gastrulation, the hollow spherical blastula transforms into a three-layered structure: the inner layer, i.e., the wall of the primitive gut, is called the endoderm; the outer layer that remains on the outside is the ectoderm; and the intermediate loose tissue layer consisting of Primary and secondary mesenchyme is the mesoderm. These are the three primary germ layers characteristic of all higher animals. The ORGANIZATION OF THE triploblastic embryo roughly corresponds to the organization of the adult animal, featuring a digestive tube on the inside, an epidermis on the outside, and organs of Connective Tissue origin between them. As a first approximation, we can say that these three adult tissue types derive from the endoderm, ectoderm, and mesoderm, respectively, although exceptions do occur (see Sections 16.1.7, 16.1.8, and 16.1.9).

16.1.5. Gastrulation movements are based on clearly coordinated simple cell movements [1, 6, 7]

Gastrulation movements are based on a relatively simple set of fundamental cell movements. Cells can change shape through elongation or contraction; they adhere to or detach from other cells or the extracellular matrix; they can secrete extracellular matrix substances that restrict or direct their movements. Such types of cellular activity, along with Cell Growth and Division, underlie all morphogenetic movements of both individual cells and groups of cells, including, in particular, processes such as cell redistribution, which ensures invagination and the Formation of the primitive gut. Gastrulation, while being an exceptionally important event per se, also demonstrates diverse forms of cellular behavior.

Subsequent chapters will discuss the mechanisms used by cells to alter their shape or adhesive properties. Individual development presents us with a specific problem: we need to understand how elementary types of cellular activity are coordinated in time and space; it is this coordination that determines The behavior of each part of the embryo. At present, we do not yet fully understand these exquisitely orchestrated gastrulation movements. Nevertheless, experiments on amphibians help answer the question of which PARTS OF THE gastrulating embryo move under their own power and which parts are subject to external forces.

In amphibian embryos, the presence of a significant excess of yolk substantially slows down invagination, making the geometry of the process more complex than in the sea urchin. Invagination begins not at the vegetal pole, but slightly to the side, where a small depression called the blastopore first forms. The Cells of the prospective endoderm roll inward through this opening (Fig. 16-9). As the invagination deepens, the blastopore takes on a ring-like shape surrounding a portion of the endoderm visible on the outside. This part of the endoderm is called the yolk plug; it contains yolk-enriched cells (which will later enter the gut cavity and be digested). At the same time, the blastoderm at the animal pole actively expands and takes THE PLACE OF the cells rushing inward. Eventually, the blastoderm of the animal hemisphere covers the entire outer surface of the embryo, and the size of the blastopore decreases dramatically.

The Mechanism of invagination in amphibians apparently relies on the same principles as in the sea urchin. It begins with A change in cell shape in the blastopore region. In amphibians, these are so-called bottle cells, characterized by a broad base and a long, narrow end that anchors the cells to the inner surface of the blastula wall (Fig. 16-10). These cells pull the epithelium inward, creating a longitudinal groove on the blastula surface. Following the formation of this groove, the cells continue their inward movement as a sheet that participates in gut formation. Just as in the sea urchin, this movement is driven by the active rearrangement (intercalation) of epithelial cells, especially in the marginal zone adjacent to the blastopore lip (Fig. 16-10, B). Small square pieces of isolated marginal zone tissue in culture can spontaneously elongate and widen due to cell rearrangement (Fig. 16-10, B), as if they were inside the embryo and rushing inward through the blastopore. The mechanism of cell redistribution underlying this convergent extension remains an enigma. A secondary guiding or driving force is provided by prospective mesodermal cells, which migrate along the inner surface of the blastocoel roof and pull the endoderm along with them—much like what happens in the sea urchin embryo, except that in the sea urchin, the driving force is provided by prospective secondary mesenchyme cells. The blastocoel roof is lined with a fibronectin-rich matrix, and when antibodies or Peptides that block fibronectin-cell surface receptor interactions are injected into the blastocoel (see Section 14.2.13), normal gastrulation is inhibited (particularly in newts and salamanders) (Fig. 16-11).

Fig. 16-9. A. Gastrulation in Xenopus. External view of the embryo (top) shown from the vegetal pole; transverse sections (bottom) taken in the plane indicated by the dashed line. The direction of cell movement is indicated by arrows. B. Fate map of an early Xenopus embryo (side view) at the onset of gastrulation, showing THE ORIGIN OF cells that will participate in the formation of the three germ layers through gastrulation movements. Various parts of the mesoderm (lateral plate, somites, notochord) originate from deeper-lying cells of the dotted region; other cells in this region, including the most superficial ones, will give rise to the ectoderm (grey, top) or endoderm (various shades of red, bottom). (R. E. Keller, J. Exp. Zool., 216, 81-101, 1981.)

In amphibians, as in sea urchins, gastrulation results in the formation of a three-layered embryo with an outer ectodermal layer, an inner endodermal tube representing the gut rudemic (primordium), and a mesoderm in between. The mouth opening forms at the anterior end of the embryo, where mesoderm is absent, allowing the ectoderm to come into direct contact with the endoderm.

Fig. 16-10. A. A section through a Xenopus embryo made during gastrulation in the same plane as in Fig. 16-9, showing the four MAIN TYPES OF movements underlying this process. B. A model of cell redistribution leading to convergent extension, which is likely the main driving force of gastrulation in Xenopus. (A — from R. E. Keller, J. Exp. Zool., 216, 81-101, 1981; B — from J. Gerhart, R. Keller, Annu. Rev. Cell Biol., 2, 201-229, 1986.)

16.1.6. Gastrulation movements are organized around the blastopore [2, 7, 8]

Cell movements during gastrulation are complex yet orderly, making it possible to construct a fate map On the surface of the embryo even before gastrulation begins (Fig. 16-9, B), which shows which cells will form the various parts of the adult organism. How is this entire complex array of gastrulation movements triggered and organized? In amphibians, an important preliminary step is the redistribution of egg cytoplasm immediately following fertilization. Invagination always begins at a Location corresponding to THE POSITION OF the grey crescent (see Section 16.1.1): here, in the region of the future dorsal blastopore lip, the fertilization-induced Rotation of the egg cortex relative to the internal cytoplasm (see Fig. 16-2) presumably creates a combination of cellular components with unique properties. If the dorsal blastopore Cytology/practical/96.html">Lip of a donor embryo is removed at THE START OF gastrulation and transplanted to another embryo, gastrulation in the recipient embryo will begin both in its own blastopore lip region and in the region where the donor lip was grafted (Fig. 16-12). As a result of this second gastrulation event, a second set of body structures forms, producing a double embryo (resembling Siamese twins). If cells with distinct pigmentation are used for the transplantation experiments, the recipient tissue can be easily distinguished from the implanted tissue. Using this method, it was shown that the implanted blastopore lip recruits the host epithelium into the invagination process it controls. We will show later that chemical and physical interactions between cells are exceptionally important for forming the three germ layers during gastrulation. But first, we will briefly review the subsequent Development of the endoderm, mesoderm, and ectoderm that make up the vertebrate embryo after gastrulation is complete.

Fig. 16-11. A. A normal newt embryo at the end of gastrulation; the endoderm and mesoderm have invaginated inward, and the blastopore has taken on a ring-like shape surrounding the large endodermal cells still remaining on the surface. This remaining patch of endoderm is called the yolk plug. B. A similar embryo in which gastrulation movements were blocked by injecting anti-fibronectin antibodies into the blastocoel cavity. The cells of the vegetal hemisphere are unable to move inward and remain on the outside (smooth rounded mass at the bottom of the figure); the cells of the animal hemisphere form a structure resembling an empty sack, which becomes folded due to the gastrulation movements that normally cause it to spread over the embryo's surface. (J. C. Boucaut et al., J. Embryol. Exp. Morphol., (Suppl.), 211-227, 1985.)

Fig. 16-12. A. Diagram of an experiment showing that the dorsal blastopore lip initiates and controls gastrulation movements and, when transplanted, organizes a second set of embryonic structures. B. Photograph of the resulting axolotl resulting from such an operation, featuring two heads and two tails. Similar results have been obtained in Xenopus, although the scale of duplication differs slightly. (B — courtesy of Jonathan Slack.)

16.1.7. The endoderm gives rise to the gut and its derivatives, such as the Lungs and Liver [9]

The endoderm forms the digestive tract tube rudiment, extending from the mouth to the anus. This tube gives rise not only to the Pharynx, Esophagus, Stomach, and intestines, but also to many glands: the Salivary Glands, liver, and Pancreas; the Trachea and lungs also form as outgrowths of the digestive tract wall, which is initially quite simple in structure. All these outgrowths enlarge and transform into a system of branching tubules emptying into the gut or Larynx. To be more precise, the endoderm forms only the internal epithelial components of these structures—the gut lining and secretory cells of the pancreas—while the supporting Muscle or connective tissue elements originate from the mesoderm.

16.1.8. The mesoderm gives rise to connective tissue and Muscles, as well as the cardiovascular and urogenital systems [9, 10]

Following gastrulation, the resulting middle germ layer, the mesoderm, divides into two parts for the right and left halves of the body. During this period, one region of the mesoderm specializes along the central body axis, determining the division of the body into right and left halves. This is the notochord, a thin cellular cord 80 µm in diameter; the ectoderm lies above it, the endoderm below it, and the mesoderm flanks it (see Fig. 16-15). Notochord cells swell and develop vacuoles, which causes the notochord to elongate and, consequently, the embryo to straighten. In the most primitive Chordates lacking a backbone, the notochord serves as a structural support. In vertebrates, the notochord acts as the axis around which mesodermal cells assemble to form the spinal Column. Thus, the notochord serves as a precursor to THE Vertebral Column both in evolution and during ontogeny.

The mesoderm primarily gives rise to Connective Tissues—initially the mesenchyme, whose cells form a loose network filling the spaces between other tissues (see Fig. 14-20), and subsequently bone, Cartilage, muscle, and fibrous tissues, including the inner layer of the Skin (the dermis). Muscle cells also originate from the mesoderm. It forms the bulk of the Urogenital System ducts, as well as The Cardiovascular system, including The Heart and Blood Cells.

16.1.9. The ectoderm gives rise to the epidermis and The Nervous system [9, 11]

After gastrulation, the outer wall of the embryo is represented by the ectodermal layer, which later forms the outer layer of the skin, the epidermis. However, the future of the ectoderm is not limited to this: the entire nervous system also forms from the ectoderm. The process of forming this system is called neurulation; it begins with the thickening of a broad dorsal region of the ectoderm, which then rolls up into a tube and separates from the rest of the cell layer. This transformation is induced by the notochord and mesoderm lying beneath this area of the ectoderm (see Section 16.6.3). The tube formed from the ectoderm is called the neural tube; during further development, it gives rise to the Brain AND SPINAL cord. Along the line where the neural tube separates from the future epidermis, a further number of ectodermal cells detach from it; these cells later migrate individually through the mesoderm. These are the neural crest cells, which give rise to virtually all Components of the Peripheral Nervous System (including sensory and sympathetic ganglia, and Schwann cells that form the myelin sheath of peripheral nerves) (see Section 19.1.1), as well as adrenaline-secreting cells of the Adrenal Glands and skin pigment cells. In the head region, many neural crest cells differentiate into cartilage, bone, and other types of connective tissue that form from the mesoderm in other parts of the body. This is one of the few exceptions to the general concept that the three germ layers correspond to three "concentric" layers of the adult organism. Sense Organs that transmit information about visual, auditory, olfactory, and other stimuli to the nervous system also develop from ectodermal primordia—some from the neural tube, others from the neural crest, and still others from the outer layer of the ectoderm (see Fig. 19-55, Section 19.7.1). For example, the retina develops as an outgrowth of the brain and is therefore a derivative of the neural tube, whereas olfactory cells differentiate directly from the ectodermal epithelium of the Nasal cavity.

16.1.10. The neural tube is formed as a result of coordinated cell shape changes [6, 12]

The formation of the neural tube is a remarkably noteworthy event (Fig. 16-13). Initially, The surface of the gastrula appears quite uniform, but some changes are already taking place: the ectoderm near the midline begins to rise, forming the neural plate. The lateral edges of the neural plate thicken; these thickenings, called neural folds, gradually approach each other, and a groove forms along the midline of the plate itself. Eventually, the folds converge above it and fuse, resulting in a hollow neural tube covered on top by a continuous layer of ectoderm. As with gastrulation, all these processes are driven by the elongation, adhesion, and contraction of individual cells within the epithelial layer. The cells of the neural plate are interconnected by strong lateral junctions. Initially, the cells elongate in a direction perpendicular to the cell layer. This cell elongation is associated with the lengthening of microtubules and is necessary for neurulation to proceed: Treatment with colchicine, a drug that disrupts microtubules, prevents the neural folds from forming altogether or causes them to disappear if they have already formed. The elongated cells then acquire a wedge shape with narrow ends directed toward the upper (apical) surface of the cell layer. Because the cells are tightly connected along their lateral surfaces and their width at the base remains unchanged, the cell sheet as a whole bends (Fig. 16-14). Apical constriction of the cells occurs due to the contraction of bundles of actin filaments running beneath the apical cell surface, where the cells are joined by adhesion belts (see Section 14.1.3).

Fig. 16-13. Formation of the neural tube in Xenopus. External dorsal view. Sections are taken in the plane indicated by the dashed line. (T. E. Schroeder, J. Embryol. Exp. Morphol., 23, 427-462, 1970.)

16.1.11. Mesodermal cell clusters segment to form somites on either side of the body's longitudinal axis [13]

Flanking the newly formed neural tube are extensive regions of mesoderm (Fig. 16-15). The thickened medial region of the mesoderm gives rise to the vertebrae, Ribs, skeletal muscles, and the connective tissue layer of the skin. Initially, the mesoderm on each side is a single mass of tissue, but it soon divides into "blocks" called somites (Fig. 16-16). Each somite corresponds to a single skeletal element resulting from differentiation and will subsequently divide into three parts. The part of the somite facing the notochord is called the sclerotome and is the source of cells that form the ribs and vertebrae; the outward-facing part adjacent to the dorsal surface of the embryo is called the dermatome and is the source of cells forming the connective tissue layer of the skin, or dermis; and the remaining part of the somite is called the myotome (located between the sclerotome and dermatome) and is the source of Skeletal Muscle cells.

Fig. 16-14. Bending of a cell sheet driven by cell shape changes mediated by microtubules and actin filaments. As the apical ends of the cells constrict, the membranes of their outer surfaces become concave. This may be a symptom of rapid shape change driven by contractions occurring under conditions of tight cell-cell contact along lateral surfaces, where the flow of excess membrane along intercellular junctional complexes is restricted.

Fig. 16-15. Schematic cross-section through the trunk region of a frog embryo after closure of the neural tube. (T. Mohun, R. Tilly, R. Mohun, and J. M. W. Slack, Cell, 22, 9-15, 1980.)

Fig. 16-16. Somite formation in Xenopus. Top: external LATERAL VIEW OF the embryo. The dashed line indicates the plane of the horizontal section shown in the middle panel. Bottom: diagram of mesodermal cell rearrangement during somite formation (high magnification). In Xenopus, all cells that will subsequently form somites are initially oriented at right angles to the body axis. During somite formation, all cells in each group rotate simultaneously.

Fig. 16-17. Comparison of embryogenesis in fish, amphibians, birds, and mammals. Early stages of development (top) are very similar; later stages (bottom) differ significantly. The earliest stages are shown at approximately the same scale, whereas later stages are shown at different scales. (E. Haeckel, Anthropogenie oder Entwickelungsgeschichte des Menschen. Leipzig, Engelmann, 1874.)

Somites do not all differentiate at once, but rather sequentially, one after another from head to tail. Segmentation is accompanied by changes in mutual cell-cell interactions within the mesoderm. In the early, unsegmented mesoderm, electrical coupling between cells is maintained via gap junctions, which disappear just before or during somite formation. Evidently, The Nature of cell communication changes first, after which cells aggregate into compact groups to form somites (see Fig. 16-16). As we will see later, physical segmentation correlates with the appearance of chemical differences between adjacent groups of somite cells (see Section 16.5.20), a segmentation pattern reminiscent of zebra stripes. Most likely, selective adhesion based on differences in cell-surface chemistry drives the division of the cell mass into physically distinct somites.

16.1.12. The vertebrate body plan is established in miniature at an early stage and maintained as the embryo grows [9]

During the stage of somite formation, the embryo is only a few millimeters long and typically consists of 10⁵ cells. Thus far, we have focused on Xenopus, but the shape and size of the embryo are remarkably similar in the salamander, fish, chicken, and human (Fig. 16-17). At later stages, embryos differ significantly in size and shape, but at this stage it is clearly visible that their body plan is identical. Details are added later as the embryo grows. Meanwhile, the Central Nervous System is represented by a neural tube with a thickened anterior end that will subsequently develop into the brain; the gut and its derivatives are laid down as an endodermal tube, and the trunk segments correspond to the somites. Certain connective tissue types of the cardiovascular system are represented by the more peripheral unsegmented mesoderm, and the epidermal skin layer by the ectoderm. During subsequent development, the linear dimensions of all these components may increase by a factor of 10, 100, or more, and their total volume and cell number by millions, yet the overall body plan remains unchanged.

Conclusions

The eggs of most animal species are relatively large cells containing stores of nutrients and other cellular components whose synthesis is regulated by the maternal genome. In amphibians, the first and most critical movement following fertilization is the rotation of the egg cortex relative to its internal cytoplasm. The asymmetry generated by this rotation, as well as the pre-existing asymmetry in cytoplasmic distribution within the egg prior to fertilization, establishes the future anteroposterior and dorsoventral body axes. During subsequent cleavage divisions, the egg divides into numerous smaller cells, yet the overall volume of the embryo does not increase. Cells in the outer layers of the embryo form tight junctions with one another, isolating the internal Contents of the embryo from the external environment. Fluid penetrates into the embryo, forming a cavity (the blastocoel) within it. The wall of such an embryo is called the blastoderm, and the embryo itself is referred to as a blastula.

During gastrulation, the blastoderm at one end of the embryo begins to invaginate as a result of cell shape changes. The driving force behind the later stages of gastrulation is thought to be the convergent extension of the invaginating epithelium driven by changes in cell packing, which transforms the embryo into a three-layered structure comprising an internal tube (endoderm), an outer covering (ectoderm), and an intermediate layer of cells that has detached from this ectoderm (mesoderm). The endoderm will subsequently form the lining of the gut and its derivatives, the ectoderm will form mainly the epidermis and nervous system, and the mesoderm will form the bulk of the muscles and connective tissue, the cardiovascular system, and the urogenital tract. As a result of gastrulation, groups of cells that were previously distant from one another are brought into close proximity and interact. For instance, the dorsal mesoderm induces the thickening of the overlying ectoderm; this region then rolls up and detaches, forming the neural tube and neural crest. This process, termed neurulation, also depends on changes in ectodermal cell shape. Located in the middle of the dorsal mesoderm is a cord of specialized cells called the notochord, which forms the central axis of the embryo. The elongated masses of mesoderm flanking the notochord fragment to give rise to somites, from which the vertebrae and skeletal musculature develop.