laboratory

Development to larval stage in sea urchins

In all animal embryos, the initial divisions of the zygote are called cleavages. As cleavage proceeds, the mitotic divisions are rapid with no time for cell growth between divisions. Thus the cells become smaller after each cleavage. As the blastula begins to form, the cleavage rate slows down. Future cell divisions are slower, allowing the cells to grow. The formation of a mature blastula marks the end of the cleavage period. Observe cleavage and blastula formation in the video below.

video - cleavage and blastula formation of a sea urchin embryo

When the blastula is fully formed, gastrulation begins. In almost all animal embryos, gastrulation forms a new internal cavity that becomes the digestive tract, and additional cells move inside the embryo to form mesoderm. The mesoderm eventually gives rise to internal organs such as the heart, kidneys, and reproductive tract. Gastrulation is accomplished in various ways by different animal groups. In echinoderms (such as the sea urchin), it is a 2-step process. Study the following video and micrographs to learn how mesoderm and the digestive tract are formed in the sea urchin.

video - gastrulation of a sea urchin embryo

When gastrulation is complete, a mouth forms at the end of the digestive tract opposite to the anus, and spicules (the larval skeleton) are secreted by mesoderm cells. The embryo then changes into the larval body form which is known as a pluteus larva. The larva can swim and feed. After a few weeks of growth and further morphological changes, it undergoes metamorphosis to the adult sea urchin body form. Observe transformation of the gastrula into a pluteus in the animation and further growth of the larva in the micrographs below.

animation - development from gastrula to a pluteus larva

young pluteus older pluteus
Young pluteus larva
2-Week old pluteus larva (1 mm in length)

Note the differences in the pluteus vs. trochophore larval forms. Then answer questions 7 and 8.

PART 2. DEVELOPMENT OF AQUATIC VERTEBRATE EMBRYOS

Amphibian embryos

All animal embryos go through cleavage in which the cells are reduced in size. In eggs that have a large amount of unequally distributed yolk, the cleavage pattern is asymmetrical and not all cells are the same size. In amphibian embryos, such as the frog, those cells containing mainly yolk divide more slowly and thus are larger than cells contain mainly cytoplasm during most of the cleavage period.

cleavage
8-Cell frog embryo: " a" is viewed from the top and "b" from the side. Cells containing yolk are lighter in color.

Study the following video of frog development from the first cleavage division to the gastrula stage. Note the similarities and differences between frog and sea urchin gastrulation. Examine the micrographs of the blastula and gastrula stages to compare an external vs. internal view of the embryo. The blastula stage is difficult to detect unless the blastocoel cavity can be seen within the embryo.

video - development of the frog embryo to the gastrula stage

blastula
Blastula (external view)
Cut blastula showing blastocoel cavity
gastrula gastrula cut
Gastrula (external view)
Cut gastrula showing new internal cavity

Now we will examine further development of the frog embryo to the neurula stage and then to a hatched tadpole larva. First view the short black-and-white video that shows gastrulation with the blastopore facing forward, followed by formation of the neural tube. Remember that the neural tube is extremely important in vertebrate animals because it forms the brain and spinal cord. Then study the longer color video that begins with neurulation and ends with a tadpole larva. Note that development of the embryo occurs within the tough vitelline membrane which can be clearly seen at later developmental stages. The emergence of the larva from the vitelline sac constitutes "hatching".

video - gastrulation and neurulation in a frog embryo

video - development of a frog embryo from neurulation to a hatched tadpole larva.

Now view this image and answer question 9. You may use this chart of frog development to review the embryonic stages.

Fish embryos

Zebrafish
The embryos of fish develop from eggs with so much yolk that cytoplasm is segregated into a small patch at the egg periphery. This is the only part of the egg that divides during the cleavage period. As a result, the developing embryo lies on top of the yolk mass throughout development.

fish 2-cell stage
Adult zebrafish in an aquarium
2-Cell stage of zebrafish embryo

Now view this time lapse video of zebrafish development. The entire embryonic period from the 2-cell stage to larva takes only 48 hours in this fast-developing species.

video - development of zebrafish embryo from 2-cell stage to a few hours before hatching

When you understand the development of Zebrafish embryos, view this image and answer question 10.

Medaka

The living embryos of a small fish (the Japanese Medaka) will be used to visualize development from neural tube to organ rudiment stage in an egg containing a large amount of yolk. Examine the living fish embryos at your table using your microscopes with the 4X objective only. Remove the lid from a dish and place the dish on the stage (move the slide holder out of the way and position the dish in the center of the stage). You can move the embryos within the dish with a clean toothpick.

The blastula stage is represented by a flat cap of cells on top of the large yolk. By the neural tube stage, the embryonic axis (neural tube and associated structures) may be seen as a narrow streak on top of the yolk. The rest of the egg consists of a mass of yolk surrounded by a the vitelline membrane (called a chorion in fish) to which chorionic filaments are attached. These filaments serve to attach the eggs to the ventral body surface of the female fish. The yolk contains one or more large lipid droplets which serve as an additional energy source for the developing embryo. A thin layer of cells has grown from the embryo to surround most of the yolk at this stage, but is transparent and very difficult to see. It will become the yolk sac and functions to absorb and transport yolk into the embryonic body. The embryonic body develops on top of the yolk.

Carefully observe the following stages of Medaka development:

1. Neural tube stage
Neurulation is complete and the anterior end of the embryo; you should see a thin, tube-like line on one side of the yolk. The anterior end of the neural tube is beginning to expand to form the brain. Small blocks of tissue, the somites, may be forming on either side of the neural tube.
Capture an image: Label neural tube, brain region, somites (if visible), and yolk.
Submit to WebAssign for question 11.

3. Optic cup stage
The embryonic body is clearly visible on the surface of the yolk. The neural tube is well defined and may now be called the spinal cord in the middle and posterior regions of the embryo. Somites are visible along the sides of the spinal cord. Note the appearance of the 5-part brain. The lateral outgrowths from the forebrain have invaginated to form optic cups. The optic cups will develop into the retina of the eyes, as in all vertebrate embryos. Note also the presence of a lens within the optic cup.
Capture an image: Label spinal cord, somites, brain, optic cups and lens.
Submit to WebAssign for question 12.

4. Beginning of circulation
The embryo is now much larger; body length wraps around the yolk. Note the heart (located beneath the head) which is strongly beating. Observe the blood vessels lying both within the embryo and on the yolk surface (within the yolk sac). Nutrients from the yolk are being absorbed and transported to the embryonic body by these vessels. Observe blood moving through the blood vessels. The somites and brain are more fully developed than in the previous embryonic stage, and the optic cups are larger. The tail of the embryo is conspicuous.
Capture an image: Label optic cup, lens, heart, and blood vessels (on the yolk). Submit to WebAssign for question 13.

5. Beginning of organ rudiment stage (90-100 hours at 25°C)

The eyes are now well formed; the optic cups contain black pigment. The brain is larger with well defined regions. Note the heart, which is beating more rapidly than in the previous stage, and the more complex pattern of blood vessels surrounding the yolk. Limb buds which will form the pectoral fins may be visible.
Capture an image of the head from the dorsal side: label optic cup, lens, and brain. Submit to WebAssign for question 14.