Heredity and Development: Second Edition (1972)

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HEREDITY AND DEVELOPMENT: SECOND EDITION 242 11 Gastrulation and Organ Formation In this chapter we shall investigate the processes in Rana pipiens that convert a single-cell zygote into an embryo having the rudiments of the various organ systems. Structure of the Blastula. During the first day of development, the most obvious process that occurs is cell division. The mass of the embryo, which was originally in a single cell, is divided into many thousands of smaller cells. The result is a blastula, which is a spherical embryo with an internal cavity, the blastocoel (Fig. 11–1). The blastocoel is restricted to the animal hemisphere, and in the living embryo it contains a fluid. The cells near the animal pole are the smallest ones in the embryo and they become increas- ingly larger toward the vegetal pole. All cells of the blastula contain yolk granules, which are deposited in the ovum while it is being formed in the ovary. They serve as the food supply until the young tadpole is able to feed. The number and size of the yolk gran- ules per cell increase in relation to the nearness of the cell to the vegetal pole —the large cells of the vegetal hemisphere have their cytoplasm packed with yolk granules. Yolk granules are denser than ordinary cytoplasm and, as a result, the vegetal hemisphere, with its abundant supply of yolk granules, is heavier than the animal hemisphere. As a consequence of this density differ- ence the animal hemisphere is uppermost throughout the cleavage and blas- tula stages. The Three Embryonic Layers. We have learned previously that gastrula- 

HEREDITY AND DEVELOPMENT: SECOND EDITION 243 11–1 12-hour embryo. Early blastula. Cross section. tion involves a complete rearrangement of the parts of the embryo. The vege- tal hemisphere cells invaginate through the blastopore and the animal hemi- sphere cells spread over the entire embryo to form its outer covering. At the end of gastrulation, the cells frequently become arranged in three concentric layers, which from the outside to the center are the ectoderm (‘outer skin’), mesoderm (‘middle skin’), and endoderm (‘inner skin’). The ectoderm is the outer covering of the late gastrula in the frog. The epi- dermis and the nervous system develop from the ectoderm. The mesoderm is the second layer of the late gastrula in the frog, located under the ectoderm. It gives rise to muscles, the skeletal system, the dermis or inner layer of the skin, the circulatory, excretory, and reproductive systems. The endoderm is the inner layer of the late gastrula in the frog. It gives rise to the inner lining of the alimentary canal and the structures derived from it, such as lungs, the liver, pancreas, and the bladder. Fate Maps. The cells that form the ectoderm, mesoderm, and endoderm at the close of gastrulation can be located in the earlier stages. It is possible, for example, to map the early gastrula with reference to what its various parts will produce. We might call such a map a ‘fate map,’ since it will show the developmental fate of the parts of the gastrula. The technique for making a fate map of the amphibian gastrula was per- fected by the German embryologist W.Vogt. His method was as follows: Tiny pieces of agar are stained with vital dyes, which will not 

HEREDITY AND DEVELOPMENT: SECOND EDITION 244 harm living cells (Fig. 11–2). The stained agar pieces are then held against the embryo until some of the stain is absorbed by the surface cells. The result is a small colored spot on the embryo. The movement of this colored spot is then traced through development. If the spot is part of an area that invagi- nated, it is necessary to dissect the embryo to determine where it goes. Figure 11–3 is taken from Vogt’s work. In the first embryo there are eight zones indicated. These would have been colored in the living embryo, but are represented by dots of different sizes in the diagram. Embryo a is an early gastrula with the dorsal lip of the blastopore just formed; b is a middle gas- trula with a large yolk plug; c is a late gastrula with a small yolk plug. The eight colored areas undergo extensive movements during gastrulation. Regions 5, 6, and 8 are all invaginated to the interior of the embryo, and 1, 2, 3, and 4 are stretched to cover a large area of the surface. After performing numerous experiments of this sort, Vogt was able to pre- pare a fate map for the European toad, Bombinator (Fig. 11–4). The presump- tive ectoderm, that is, the area that will form the ectoderm later in develop- ment, occupies nearly half of the early gastrula. Two main subdivisions of the presumptive ectoderm are recognized: (1) the presumptive neural tube, which will form the brain, spinal cord, nerves, and some of the sense organs, and (2) the presumptive epidermis, which forms the outer layer of the skin. 11–2 Vogt’s diagram showing how embryos are stained (modified from Roux Arch. 106:565). 

HEREDITY AND DEVELOPMENT: SECOND EDITION 245 11–3 One of Vogt’s experiments with vital stains. a is an early gastrula with the dorsal lip below 6. b is a mid gastrula. Spots 5, 6, 7, and 8 have been invaginated. The yolk plug is on the ventral side. c is a late gastrula. Spots 1, 2, 3, and 4 have spread to cover more of the surface than they occupied at the beginning of gastrulation (modified from Roux Arch. 120:568). The presumptive mesoderm forms a band surrounding the embryo near the equator. It also is divided into two main areas. The presumptive notochord is composed of cells that will form the notochord, a rod of tissue that extends along the dorsal side of the embryo beneath the neural tube. The great impor- tance of the notochord for development will be explained at a later time. The remainder of the presumptive mesoderm will form the other structures derived from this layer, such as the muscular, skeletal, circulatory, reproduc- tive, and excretory systems. The presumptive endoderm is restricted to the ventral portion of the vege- tal hemisphere. This area will form the lining of the alimentary canal and structures derived from it, such as the liver, pancreas, and bladder. Gastrulation Movements. During gastrulation all of the material below the line separating the ectoderm and mesoderm is invaginated. The cell movements involved can be understood by referring to the diagrams of sec- tions of progressively older embryos (Figs. 11–5 to 11–10). A convenient stage with which to begin is a very late blastula that has the faintest indication of the place where the dorsal lip will appear. An embryo of this stage is shown in Figure 10–11. A median longitudinal section of such an embryo is shown in Figure 11–5a, and an interpretative diagram is given in Figure 11–5b. The roof of the blastocoel consists of two regions of ectoderm: the presumptive epidermis and the presumptive neural tube. The portion of the blastocoel roof above the point where the dorsal lip of the blastopore will form is the 

HEREDITY AND DEVELOPMENT: SECOND EDITION 246 11–4 Vogt’s fate map for Bombinator. a is a lateral view. b is a view toward the dorsal lip (modi- fied from Roux Arch. 120:638). 

HEREDITY AND DEVELOPMENT: SECOND EDITION 247 11–5 22-hour embryo. Late blastula. Cross section (a) and diagram showing the presumptive regions (b). presumptive notochord. During gastrulation one should pay special attention to the movements of the presumptive notochord and the presumptive neural tube areas. Gastrulation begins with the formation of the dorsal lip of the blastopore at about 22 hours after fertilization. Figure 11–6 shows a section of a somewhat older 30-hour gastrula. This should be compared to Figure 10–14 of the whole embryo. Invagination has produced a tiny cavity, the archenteron (‘primitive gut’). The opening of the archenteron to the outside is the blasto- pore. The invaginating cells are beginning to obliterate the blastocoel. In this embryo the blastopore is complete, so the ventral lip is seen as a slight imagi- nation. Part of the presumptive notochord cells have turned in to form the roof of the archenteron. These are rolled in over the dorsal lip in a manner analogous to a rope moving 

HEREDITY AND DEVELOPMENT: SECOND EDITION 248 11–6 30-hour embryo. Mid gastrula. Longitudinal section and diagram of the presumptive regions. over a pulley. The presumptive neural tube and presumptive notochord areas are expanding to cover larger portions of the surface. Four hours later, the gastrula shows important changes (Fig. 11–7, which should be compared with the whole embryo in Fig. 10–15). The archenteron has become larger and there is a corresponding reduction in the blastocoel. (Note that in this figure, as in many others, there are spaces such as the one in the endoderm of the yolk plug. This was not present in the living embryo, but is an artifact resulting from the technique employed in making the preparation.) In the 36-hour embryo the archenteron has nearly reached its full size (Fig. 11–8, which should be compared with the whole embryo in Fig. 10–16). The embryo is now covered entirely by ectoderm, except for a small amount of endoderm protruding as the yolk plug. All of 

HEREDITY AND DEVELOPMENT: SECOND EDITION 249 11–7 34-hour embryo. Late gastrula. Longitudinal section and diagram of the presumptive regions. the presumptive notochord cells have invaginated. They form the roof of the archenteron and are situated beneath the portion of the ectoderm that later will form the neural tube. The section shown is not exactly along the mid-line of the embryo, which is why the blastocoel does not show: its position is indi- cated by dashed lines. The Neurula. The further history of the presumptive regions will be shown in two older embryos. Figure 11–9 shows a neurula of the same stage as the whole embryo of Figure 10–18. This is a section along the mid-line, so the neural folds are seen only in the anterior portion of the embryo. The archenteron occupies nearly half of the embryo. It has a ventral outgrowth, the liver diverticulum, which will form the liver. The blastopore does not appear in the section, but its position is indicated in 

HEREDITY AND DEVELOPMENT: SECOND EDITION 250 11–8 36-hour embryo. Late gastrula. Longitudinal section and diagram of the presumptive regions. the diagram of the presumptive regions. The yolk plug has been incorporated in the main mass of endoderm. The blastocoel is but a shadow of its former self. In the late neurula (Fig. 11–10) the neural tube has formed and the anterior portion is enlarged and bends ventrally. This is the brain. The neural tube is hollow throughout its length, as shown in the diagram. Only a portion of the cavity of the neural tube is visible, because the section is not exactly longitu- dinal and median. The epidermis covers the entire embryo. The mesoderm is represented in this section by the notochord and the ventral mesoderm. The archenteron is now bounded by endoderm on all sides. Earlier, the presump- tive notochord formed the archenteron roof, but by the late neurula stage the endoderm has moved up from the sides and formed a layer beneath the notochord. Some of the details of neural tube formation can be illustrated better 

HEREDITY AND DEVELOPMENT: SECOND EDITION 251 with cross sections of the embryos. Figure 11–11 shows an embryo shortly before the closure of the neural folds. The ectoderm on the dorsal side has formed two ridges, the neural folds, with the neural groove between them. The remaining surface of the embryo is covered with epidermis, which is also ectoderm. Beneath the ectoderm there is a continuous layer of mesoderm, which forms the notochord on the dorsal mid-line. The remaining portion of the mesoderm is a thin layer, which is difficult to see in the photograph, but is recognizable by the numerous darkly stained nuclei. Most of the embryonic mass is endoderm. This innermost layer forms the thin sides and roof of the archenteron and the thick ventral portion. The embryo just described shows the characteristic distribution of 11–9 47-hour embryo. Mid neurula. Longitudinal section and diagram of the presumptive regions. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 252 11–10 55-hour embryo. Late neurula. Median longitudinal section and diagram of the parts. the three embryonic layers. The outer layer is the ectoderm, beneath this is the mesoderm, and the innermost one is the endoderm. We might visualize them as three tubes of different sizes fitting into one another. A few hours later the two neural folds meet and fuse, forming the neural tube with its central cavity (Fig. 11–12). This cavity extends throughout the neural tube, being especially wide in the brain region. Apart from the closure of the neural folds, this stage is not much different from the preceding one. The embryo consists of the three embryonic layers with little cell differentiation. The 80-hour Embryo. For our purposes it will be necessary to consider one older stage, an embryo of 80 hours. A section in the anterior end 

HEREDITY AND DEVELOPMENT: SECOND EDITION 253 11–11 47-hour embryo. Late neurula. Cross section. 11–12 50-hour embryo. Late neurula. Cross section. 11–13 80-hour embryo. Cross section in eye region. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 254 of an embryo of this age shows interesting changes in the neural tube (Fig. 11–13). This structure has enlarged to form the brain, and from its ven- tro-lateral portion the optic cups have grown out. The optic cups will form the retina, which is the portion of the eye that is sensitive to light. The epi- dermis adjacent to the optic cup forms the lens. Both the optic cup and lens are derived from ectoderm. Note that this section is anterior to both the archenteron and notochord. A study of Figure 11–10 will show why this is so. Figure 11–14 is a more posterior section of the same embryo showing other structures. The neural tube at this level is the hindbrain, which is the portion that forms the medulla later in development. The otic vesicles, which were pinched off from the layer of ectoderm covering the embryo, are lateral to the hindbrain. They will be the ears. The archenteron is present in this sec- tion, surrounded by its layer of endodermal cells. The heart is forming from mesodermal cells below the archenteron. The epidermis on the ventral side has formed the mucus glands. A section in the mid-region of the body shows the beginnings of the excre- tory system (Fig. 11–15). The mesoderm at the sides is forming the pronephros. The mesoderm above the pronephros, which is known as the myotome, will form most of the voluntary muscles of the body. Later the mesoderm ventral to the pronephric region will split to form a double layer and the space between will be the coelom. 11–14 80-hour embryo. Cross section of heart region. 

HEREDITY AND DEVELOPMENT: SECOND EDITION 255 11–15 80-hour embryo. Cross section of mid body region. The coelom was mentioned earlier, but not defined. Now we have the background for an adequate definition. A coelom is a body cavity lined by thin membranes (epithelia) derived from mesoderm. This brief survey of early development in the amphibian embryo was designed to provide a background for the consideration of the problems of embryology. Now that we have learned something of how an embryo devel- ops we can consider some of the controlling processes that are responsible for embryonic differentiation. Suggested Readings The readings suggested for Chapter 10 will serve for this chapter as well.