Lab
Chick Embryo Slides: The Early Embryology of the Chick
[Text adapted from "Structure and Development of the Vertebrates" by F. Moog and M. Krukowski. Figures from "Foundations of Embryology, 3rd Ed." by B.M. Patton and B.M. Carlson]
Introduction
The completely terrestrial vertebrates do not need to go to the water to reproduce, as amphibians do, but their embryos must develop in water nevertheless. The reptiles and birds meet this requirement by producing large eggs containing a considerable quantity of water. In the course of development the embryonic system produces a balloon-like membrane, the amnion, which fills with fluid so that the embryo itself floats in a private pool. An amnion is developed for the same purpose by the mammalian embryo. Because of the great significance of this structure in permitting their embryos to develop on land, the higher vertebrates are referred to collectively as "amniotes."
The egg of the chicken provides a convenient example of early amniote development. The embryo itself passes through the generalized chordate state, and we shall use it to see how the chordate form is arrived at, and how it is gradually elaborated as development proceeds. We shall also study the development of the special membranes that are produced: the amnion; the yolk sac, which digests the yolk; and the allantois, which is the respiratory organ of the embryo. These membranes, which are made up of living cells and grow like the embryo itself, are essential for maintaining life within the eggshell. In mammals, the same membranes are adapted to mediate between the embryo and the uterus.
In a bird's egg the "yolk" is a single cell consisting of a tremendous amount of food material and a tiny disc (called a "blastodisc" or "blastoderm") of living material that has the capacity to organize all the rest. The little blastodisc contains the nucleus, and a small amount of cytoplasm. This arrangement is different from that of amphibian eggs, in which the living material extends through the food mass, instead of being segregated in a local area. Because of its situation the blastodisc undergoes a highly modified pattern of cleavage and gastrulation, before the layers of tissue appear from which an embryonic body can be organized.
A slide showing an embryo at 18 hours of incubation will be on demonstration in the laboratory. Observe that slide sometime during today's laboratory and identify the following structures:
[Photos 3.71-3.74 in Schoenwolf Laboratory Studies of Vertebrate and Invertebrate Embryos, 8th ed are relevant to the following descriptions.]
Formation of the primitive streak and Hensen's node are critical for laying down the axes of the embryo. Important inductive interactions and cell movements are associated with these structures. The primitive streak first forms on the first day of incubation. Appearance of Hensen's node at the front of the streak heralds the onset of gastrulation. Up to 48 hours of incubation the primitive streak continues to retreat as the embryo progressively materializes in front of it. The principal outward sign of this development is the lengthening of the body axis as the notochord and neural plate grow longer, the neural folds close, and more somites are blocked out to each side of the neural tube. At some distance behind the advancing axis the ventral folds which complete the gut and body wall move gradually backward.
What happens between 18 and 48 hours of incubation is not solely enlargement of an embryo already laid down, but rather the actual laying-down process itself. It must be understood that the structures which are first seen at about 18 hours are not the primordia of the whole embryo but only of the front of the head. Similarly, the twelve somites which will be found in the 33-hour embryo are the somites which will appear in the neck of the future bird. Thus the 33-hour embryo is literally only a head and a neck, with the trunk just becoming manifest. A consequence of this mode of body formation is that for a considerable period the more anterior parts are much better differentiated than the more posterior parts. In the studies which follow, you will observe, for example, that the sense organs of the head are well developed before the tail has even appeared.
The closure of the gut allows the splanchnic (visceral) mesodermal layers of the two sides of the body to come together. This development enables the formation of a mesodermal tube, the heart, under the pharynx. The steps in heart formation are shown in Figures 9-6, 9-6 and 9-8 taken from Patton and Carlson (print out separately). As in the frog, the heart is two-layered at an early stage. The thick outer layer (myocardium, future cardiac muscle) is derived directly from mesoderm; the thin lining layer (endocardium) is composed of loose mesenchymal cells that bud off the myocardium. The heart forms, as the 33-hour preparation will show, under the hindbrain, and thus is a head structure at its first appearance. Much later it is shifted to its definitive position in the thorax.
A. Whole Mount of the 33-hour Chick Embryo
The specimens are whole embryos which have been cut off the surface of the yolk. The area pellucida and a part of the area opaca is included in each mount. The preparation is cleared, stained, and mounted entire either on a glass slide or in clear plastic. Examine the mount with the low-power objective only.
1. The 33-hour chick embryo. [Consult Schoenwolf photos 3.65-3.74.] Study first the neural tube. The forebrain is expanded laterally into the two optic vesicles. Immediately behind these is the midbrain, followed by series of subdivisions (neuromeres) which constitute the hindbrain. The future spinal cord begins at the level of the fifth somite. Observe that the tube is still unclosed at the posterior end; the primitive streak may be seen in this region. The notochord is under the neural tube; how far does is extend rostrally and caudally.
The outline of the broad, shallow pharynx may be faintly seen to each side of the midbrain and hindbrain. (To see the shape of the pharynx, consult the plastic model of the 33-hour chick on display in the laboratory.) Also ventral to the hindbrain at this early stage is the heart. Since the heart grows faster than the body itself, it has already curved somewhat. Posteriorly the heart has made connection with the two large omphalomesenteric veins which bring in blood from vitelline vessels extending over the yolk; anteriorly the heart continues into the short ventral aorta (hard to see).
How many somites does your mount have? Although all embryos in this series were incubated for 33 hours, there may be more or less than twelve somites present in different individuals. As pointed out before, the somites present at 33 hours represent only the neck region of the future bird. The trunk somites will come from the still-unsegmented posterior mesoderm known as the segmental plate.
The embryonic body proper is surrounded by a clear area pellucida outside of which is the area opaca; the difference in appearance is caused by the fact that yolk adheres to the latter. Is there anything comparable with these areas in the frog? The dark blotches on the opaca are blood islands. These produce blood, which begins to circulate at about 40 hours in vessels that also form in this region. Thus, this part of the area opaca constitutes the area vasculosa; the unvascularized peripheral part of the opaca is called the area vitellina. (The vitelline area is not included in all mounts.) Around the head is an area which is clearer than the rest of the pellucida. The greater transparency of this region is due to the fact that it consists of only two layers, not yet having been invaded by mesoderm.
Draw this mount in pencil. Make the drawing of the body about 6 inches long and of commensurate width. Be careful to show accurately the relative proportions of all named structures.
B. Serial cross sections of the 33-hour chick embryo
To study the internal anatomy of the chick embryo we will use serial cross sections of entire embryos. In preparing these sets the embryos are cut into a series of thin sections, and all the sections are mounted in consecutive order on glass slides. The sections are laid in horizontal rows, from left to right, so that they may be "read" like lines of print. Thus if an embryo were cut into 45 sections, it might appear like this one on the slides:
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | Chick | |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 33hr. | |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | #1 |
| 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | Chick | |
| 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 | 33hr. | |
| 41 | 42 | 43 | 44 | 45 | #2X |
In working with your serial sections, make a survey of all the sections and try to build up a three-dimensional picture of the embryo as you do so. Keep your whole mount drawing in front of you to use as a guide in interpreting the cross sections. You will be asked to find and draw certain representative sections of each embryo, but do not focus your attention on these sections exclusively. The practical exam will aim to test your understanding of the entire embryo.
The 33-hour chick embryo.
Start at the beginning of the first row and run along until you find a solid mass of tissue. This mass is the tip of the head. It is separate from the extraembryonic membranes below because the head is already lifting itself off the flat blastoderm by means of the backwardly directed head fold (examine this on the model in the lab). Proceeding farther back you will find that the head consists of an outer ectodermal layer (future epidermis) and an inner brain, with loose mesenchyme between. The forebrain is expanded laterally into the two optic vesicles. Observe that the membranous tissue under the head consists only of ectoderm and endoderm; is the clear area noticed around the head on the whole mount. To each side of this region, two layers of mesoderm are present.
Passing the optic vesicles you will come to the narrower midbrain. The crescent-shaped opening ventral to the brain is the pharynx. At one point the pharyngeal endoderm comes close to the outer ectoderm on the ventral side; this is the oral plate, where the gut will later open into the mouth. The blood vessels under the pharynx are the ventral aortae, coming forward from the heart; those above the pharynx are the dorsal aortae. Find the first aortic arch running around the edge of the pharynx to connect the ventral and dorsal aortae. (Blood vessels have extremely thin walls at this stage and consequently appear simply as spaces in the mesenchyme.) In this region you will probably find the point where the head is still connected to the extraembryonic membranes. Is the notochord present?
Following the ventral aortae back you will observe that they merge into a single vessel which leads into the heart. Identify the myocardium (muscular outer wall), endocardium (thin lining membrane), and the dorsal mesocardium (the suspensory lining from the coelom). Observe that the pericardial cavity is open and continuous with the extraembryonic coelom. How will the pericardial cavity become closed? Note the appearance of the hindbrain in these sections.
Shifting your attention to the extraembryonic area, identify the area pellucida and the area opaca (yolk clings to the inner surface of the latter). The upper layer of the extraembryonic membrane is composed of ectoderm continuous with the future epidermis of the body and of somatic mesoderm; these two layers constitute the somatopleure. Below is the splanchnic mesoderm and the endoderm constituting the splanchnopleure; the extraembryonic part of this membrane is of course the yolk sac. Observe that there are blood vessels (vitelline vessels) in the yolk sac. What will the function of these vessels be for?
Continue back to the end of the heart, where the omphalomesenteric veins enter. Find the point closest to the head at which the gut is closed on the ventral side; this is the anterior intestinal portal. Around this region you will find the first somite. Count to the sixth somite. Here identify the three regions of the mesoderm (see Fig. 12-9 taken from Patten and Carlson). You may be able to see a small, thin-walled pronephric tubule or duct in the nephrotome. The duct grow back beyond the pronephric region and eventually opens into the hindgut (at about 60 hours of incubation). The pronephros is much better developed in the frog embryo, in which it functions for a time, than in the chick, in which it never functions.
Find the region where the neural folds are still open. Is the mesoderm segregated into parts in this region? (Recall the whole mount.) Still farther back find the place where the ectoderm and mesoderm merge into a common mass. This is the primitive streak.
Draw sections through:
Under the title for each drawing note the number of the slide, the number of the line, and the number of the section. Thus the eighth section in the first row in the second slide of a set would be labeled "2(1,8)." This identification of the sections will help you relocate structures when you are reviewing and studying at later times.
This page was posted 3 March 2003