Heredity and Development: Second Edition (1972)

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HEREDITY AND DEVELOPMENT: SECOND EDITION 229 10 A Synopsis of Development of the Amphibian Embryo Frogs of one species or another are found on all of the major land masses. Their embryos are usually easy to collect and for more than a century they have been a favorite material for embryologists. In the cooler regions of the temperate zones there is usually one breeding season each year. Rana tempo- raria of Europe and Rana pipiens of North America lay their eggs in ponds during the spring. Those seeking to answer embryological questions by experimenting on living embryos previously could work only during the relatively short breed- ing season. Since the 1930s, however, it has been possible to obtain eggs from some species by injecting them with hormones. Thus eggs can be obtained from Rana pipiens females by injecting them with pituitary glands or with purified preparations of hormones. Most of the frogs used in this way, in the United States, are collected in the autumn, before they begin their hibernation, in northern Vermont or Wisconsin. Meiosis and Fertilization. When the ovum of the frog leaves the ovary, meiosis begins. The first meiotic division occurs and the first polar body is formed while the ovum is passing through the body cavity, or when it is in the upper portion of the oviduct. Metaphase of the second division is reached by the time the ovum enters the uterus. There are no further nuclear changes until fertilization. Fertilization occurs as the ova leave the body of the female. A single sperm enters each ovum. The head of the sperm contains the paternal nucleus with its haploid set of 13 chromosomes. Immediately behind the sperm head is a centriole. This will form an essential part of the mitotic apparatus of the embryo’s cells. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 230 Meiosis in the egg, which had stopped at metaphase of the second division, is resumed after fertilization. The second polar body is pinched off, leaving the maternal nucleus with the haploid set of 13 chromosomes. The maternal nucleus and the paternal nucleus then fuse to form the diploid zygote nucleus with 26 chromosomes. This nucleus divides by mitosis to form all the nuclei of the embryo and adult. Once fertilization has taken place, the development of the embryo begins. A sequence of definite stages is passed according to a timetable which is dependent on temperature. Our description will be based on Rana pipiens embryos developing at 20°C. At 25°, development would take approximately half as long and at 15° nearly twice as long. The Uncleaved Zygote. The just-fertilized ovum is a sphere approxi- mately 1.7 mm in diameter (Fig. 10–1, this figure and all of those in this chap- ter show the embryos magnified 25 times). Somewhat more than half of the embryo is a dark chocolate-brown and the remainder is almost white. The center of the dark area is the animal pole, the site where the polar bodies formed. The vegetal pole is 180° from the animal pole and in the center of the unpigmented area. The animal hemisphere, with the animal pole in its center, is the pigmented half of the embryo. The vegetal hemisphere is the unpig- mented half of the embryo that has the vegetal pole in its center. The entire embryo is surrounded by membranes of a jelly-like substance, which were secreted by the oviduct. (The jelly has been removed from the embryos used in the photographs.) Shortly after fertilization, the embryo rotates within its membranes so 10–1 0-hour embryo. 1-cell stage. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 231 that the animal hemisphere is uppermost. The orientation that one observes when examining an embryo under a microscope is shown in Figure 10–2. The animal pole is in the center. Since the pigmented area occupies slightly more than the animal hemisphere, a top view of the embryo shows only the heavily pigmented zone. The Early Cleavage Stages. Two and one-half hours after fertilization, the first spectacular event in development occurs (Fig. 10–3). A tiny groove appears in the animal hemisphere and this gradually enlarges to form the first cleavage furrow. This furrow slowly extends through the embryo until it is divided into two cells. Preceding this external indication of mitosis, the nucleus had gone through the usual prophase, metaphase, anaphase, and telophase stages, and each daughter cells receives a diploid set of chromosomes. The second cleavage occurs about 3 1/2 hours after fertilization (Fig. 10–4). The plane of this cleavage is vertical and at a right angle to the plane of first cleavage. It also begins at the animal pole and extends through the embryo to the vegetal pole. When this cleavage is complete, the embryo consists of four cells. The third cleavage occurs about 4 1/2 hours after fertilization (Fig. 10–5). The plane of this cleavage is perpendicular to the first two. Its position is somewhat above the equator of the embryo, with the result that there are pro- duced four smaller animal-hemisphere cells and four larger cells, which con- tain the lower part of the animal hemisphere and all of the vegetal hemisphere. The process of cleavage continues. The embryo becomes divided into smaller and smaller cells (Fig. 10–6). With each division of a cell there is a concomitant division of the nucleus, every daughter cell receiving the diploid set of 26 chromosomes. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 232 The Blastula Stages. The 9-hour embryo (Fig. 10–7) is an early blastula. The blastula stage is characterized by an internal cavity, the blastocoel, which cannot be seen from the exterior. More will be said about it later when we consider the internal events during early development. At 14 hours (Fig. 10–8), the embryo is a middle blastula. The rate of mito- sis of the cells of the animal hemisphere is more rapid than that of the cells of the vegetal hemisphere, so they are more numerous and smaller. If the embryo is turned upside down, the much larger vegetal hemisphere cells are visible (Fig. 10–9). During the next few hours, continuing cell division is the only visible event. The animal hemisphere cells become so small that they can be distin- guished only with difficulty (Fig. 10–10). In this 22-hour late blastula, the next major event of development is foreshadowed. If this embryo is rotated slightly and examined from the side, a special area of pigmentation will be observed (Fig. 10–11) slightly below the embryo’s equator. The blastopore will form at this point. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 233 Gastrulation. The special pigmented cells noticed at 22 hours gradually become a groove in the surface of the embryo (Fig. 10–12). This groove is the blastopore and its appearance marks the beginning of gastrulation. Gas- trulation is a process of development that leads to a complete reorganization of the embryo. All of the cells of the area corresponding roughly to the vege- tal hemisphere move to the interior of the embryo. The cells of the remainder of the embryo, corresponding roughly to the animal hemisphere, spread and cover the entire outer surface of the embryo. This process of cells turning into the interior is known as imagination. The cells are invaginated through the blastopore. The area immediately above the blastopore is known as the dor- sal lip of the blastopore. In a few hours the blastopore has changed from a small curved groove to a full semi-circle (Fig. 10–13). Cells are invaginating along the entire length of the blastopore. The overgrowth of the pigmented cells is re- 

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HEREDITY AND DEVELOPMENT: SECOND EDITION 235 stricting the area of light-colored cells to a zone of continually decreasing size. By 30 hours the blastopore is complete (Fig. 10–14). It is a 360° groove into which material is invaginating. The light-colored cells, which are now entirely surrounded by the blastopore, form the yolk plug. The blastopore constricts rapidly and the yolk plug becomes correspond- ingly smaller as gastrulation proceeds (Fig. 10–15). By 36 hours the yolk plug is very small and the embryo is nearly covered by the overgrowth of cells that were originally restricted to the animal hemisphere (Fig. 10–16). At the end of gastrulation, the yolk plug is drawn into the embryo and the blasto- pore remains as a tiny slit. At this time the entire outer surface of the embryo is covered by material that was part of the animal hemisphere at the begin- ning of gastrulation. The Neurula. The next prominent external change is the development of the nervous system. Approximately 40 hours after the beginning of develop- ment, the neural folds make their appearance on the top of the embryo (Fig. 10–17). These folds extend, as paired structures, from the blastopore region across the top of the embryo to a point where they join one another. These folds will eventually grow together, and in so doing they will form the neural tube. The neural tube will develop later into the brain and spinal cord. In the anterior region the neural folds are widely separated. This area will form the brain and the narrower posterior part will form the spinal cord. In Figure 10–17 the blastopore is not visible, being below the posterior edge of the embryo. The growth of the neural folds is a rapid process and by 47 hours the folds are much better developed (Fig. 10–18). The section that will form the brain is clearly separated from the section that will form the spinal cord. The area between the folds is the neural groove. The embryo has begun to elongate. At 50 hours, the two neural folds have come together and the neural groove closes off as an internal neural tube (Fig. 10–19). On the ventral side of this same embryo the area that will form the mucus glands has made its appearance (Fig. 10–20). The position of the blastopore is indicated by a pos- terior cleft. Later the cloacal opening will form where the blastopore closed. The Tailbud Stage. Figures 10–21 to 10–23 show three views of a 70- hour embryo of the tailbud stage. The dorsal view (Fig. 10–21) should be compared with the same view of the late neurula (Fig. 10–19). The neural folds have closed completely and a number of interesting looking bumps have made their appearance. Near the anterior end of the embryo there are prominent swellings in which the eyes are forming. 

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HEREDITY AND DEVELOPMENT: SECOND EDITION 237 10–17 42-hour embryo. Early neurula. Dorsal view. 10–18 47-hour embryo. Mid neurula. Dorsal view. Posterior to this is an area that will form the gills. Smaller bumps represent the beginnings of the pronephros, which is the embryonic kidney. A tiny tail is forming. In the lateral and ventral view, the mucus glands are seen as prominent structures. The cloacal opening (Fig. 10–23) is indicated by a mass of white material that is being extruded. The 100-hour Embryo. An embryo of 100 hours is the last stage we shall describe (Fig. 10–24) for by this time all of the main internal organ 

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HEREDITY AND DEVELOPMENT: SECOND EDITION 239 10–22 70-hour embryo. Tailbud. Lateral view. 10–23 70-hour embryo. Tailbud. Ventral view. 

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HEREDITY AND DEVELOPMENT: SECOND EDITION 241 systems have begun to form. Externally, the embryo has begun to resemble a tadpole. The eyes are present as bumps and the olfactory organs are pits on either side of the head. The gills have formed, and in a living embryo we would see blood corpuscles streaming through them. The embryo has a well- formed tail. It is at approximately this stage that the young tadpole hatches from the jelly membranes, in which it has been encased up to this time but not shown in the photographs. A ventral view of the head (Fig. 10–25) shows the paired olfactory organs, the mucus glands, the gills, and a median depression, the stomodaeum. Somewhat later the mouth forms at the inner end of the stomodaeum. In a period of four days the single-cell fertilized ovum has become a small tadpole with its organ systems functional. Many—in fact most—of the events that have occurred have been internal. Now that we have some knowl- edge of the external aspects of development, we can turn to the more com- plex internal events. Suggested Readings These books give more complete information on normal development of the amphibian embryo. These references apply to Chapter 11 as well. BALINSKY, B.I. 1970. An Introduction to Embryology. Third Edition. Philadelphia: W.B. Saunders. BODEMER, CHARLES W. 1968. Modern Embryology. New York: Holt, Rinehart and Winston. RUGH, ROBERTS. 1964. Vertebrate Embryology. The Dynamics of Development. New York: Harcourt, Brace & World.