Introduction and Embryonic Development - Zebrafish (L3)

Question 1

Vertebrates

Answer 1

Phylum of organisms that possess a spinal chord supported by vertebrae. One example of a vertebrate is Danio rerio, the zebrafish, a teleost (bony fish).

Other examples are birds and 4-footed tetrapods (amphibians, reptiles, and mammals, including humans.)

Question 2

Dissecting microscopes

Answer 2

The Zebrafish embryo’s body and surrounding chorion membrane are almost transparent so that development is easy to observe using dissecting microscopes.

Question 3

Cartilage

Answer 3

To study development of the skeleton in more detail, we will stain the cartilage of fixed 7-day old embryos with alcian blue and examine the distribution of cartilage along the head and body.

Question 4

Alcian blue

Answer 4

Stain used to view cartilage of fixed 7-day old Zebrafish embryos.

Question 5

Fertilization

Answer 5

Occurs when a haploid sperm cell and a haploid egg cell fuse to become a zygote. After fertilization, the development of a zebrafish embryo is divided into 5 broad periods, each of which includes several visually distinct stages of development.

Question 6

Zygote

Answer 6

The first diploid cell of a new organism, results from fertilization of a haploid sperm and haploid egg.

Question 7

Chorion

Answer 7

Zebrafish zygotes are surrounded by a thin, transparent membrane called the chorion. The chorion lifts off the surface of a fertilized egg as a block to polyspermy.

Question 8

Animal pole

Answer 8

The dorsal surface of the zygote.

Question 9

Vegetal pole

Answer 9

The ventral surface of the zygote. Contains more yolk than the animal pole.

Question 10

Blastodisc

Answer 10

About 10 minutes after fertilization, the yolk-free cytoplasm of the single-cell zygote begins to move to the animal pole, where it is seen as a transparent bulge, the blastodisc. Subsequent development will occur in the blastodisc.

Question 11

Cleavage

Answer 11

Occurs about 45 minutes after fertilization. Cell division without an overall change in embryo size.

Question 12

Meroblastic vs. holoblastic cleavage

Answer 12

Cleavage is restricted to the blastodisc and does not penetrate the yolk (this is meroblastic cleavage).

If the cleavage plane cut through the entire cell including the yolk, it would be holoblastic cleavage.

Question 13

Blastomeres

Answer 13

The cells formed from cleavage.

Question 14

Cleavage period

Answer 14

Cleavage occurs synchronously during the cleavage period, with one cell division about every 15 minutes.

Question 15

Blastula period

Answer 15

From 128 cell stage until the onset of gastrulation, which occurs after 13 divisions.

Question 16

Blastulas

Answer 16

Ball of cells with irregular spaces in between them. The process of cell division becomes much less organized in blastulas. The cell cycle becomes longer, the organization of cleavage planes becomes indeterminate, and cell division begins at the animal pole and crosses the embryo in a wave.

Question 17

Yolk syncytial layer

Answer 17

AKA YSL. The blastomeres that sit on the surface of the yolk have remained connected to it throughout cleavage via the cytoplasm. These cells now fuse and release their cytoplasm and nuclei into the cytoplasm of the yolk cell to form the YSL.

The YSL nuclei continue to undergo mitotic divisions for ~3 cycles, but the cytoplasm of the YSL stays together and does not cleave.

YSL is a developmental structure characteristic of teleost fishes. At first, it forms a ring around the edge of the blastodisc, the ring then spreads inward until it forms a continuous border between the embryo and its yolk, eventually covering the entire surface of the yolk.

The YSL is a sort of membrane that separates the embryo proper from the yolk, which contains nutrients deposited by the embryo’s mother.

The correct expression of enzymes in the YSL cytoplasm and transporters at the YSL membrane is vital for the transport of minerals and food stores out of the yolk, until the embryo has developed mouth structures and swimming abilities that permit it to feed.

Question 18

“High stage”

Answer 18

c 3.3 hpf. The yolk is flattened and the blastodisc is a high mound of cells upon it. Subsequently, the animal-vegetal axis shortens, two stages pass, and the YSL surface begins to done towards the animal pole. This is the dome stage, an obvious sign that epiboly is beginning.

Question 19

Hours post fertilization

Answer 19

AKA hpf.

Zebrafish development is relatively rapid. At their preferred temperature of 28.5 degrees celsius, the first cell division occurs at 0.75 hours. Mid-blastula occurs at 3 hpf, gastrulation at 5 hpf, first body movements at 18 hpf. Most major organs are formed after 24 hpf and the fish begins to eat within 5 days.

Question 20

Epiboly

Answer 20

4.33-9 hpf. “Around the ball.” A process of cell migration which begins as the blastodisc things and moves with the YSL to spread completely over the surface of the yolk cell. The yolk cell itself bulges toward the animal pole.

Question 21

Blastoderm

Answer 21

As epiboly begins, the blastodisc becomes called blastoderm. During the process, embryo stages are often described in terms of blastoderm coverage. If 50% of the distance between the poles along the animal-vegetal axis is covered with blastoderm, then the embryo’s stage is 50% epiboly.

Question 22

50% epiboly stage

Answer 22

At 50% epiboly stage, epiboly pauses for about 90 minutes as gastrula begins.

Question 23

Gastrula period

Answer 23

Populations of cells move to the interior of the embryo and three tissue layers and the embryonic axis (anterior-posterior) form.

Gastrulation ends when epiboly is complete at 10 hpf.

Question 24

Three tissue layers formed during gastrulation

Answer 24

Endoderm, mesoderm, and ectoderm

Question 25

Germ ring

Answer 25

Cells begin to migrate under the leading edge of the blastoderm during germ ring stage

Question 26

Embryonic shield

Answer 26

As gastrulation proceeds, the organization of the body plan of the zebrafish begins with a bulge of cells called the embryonic shield that forms at one side of the blastoderm.

Once the embryonic shield forms, epiboly continues at a rate of ~15% p/h while gastrulation continues.

Question 27

Shield stage

Answer 27

The embryonic shield is visible on one side of the blastoderm.

Question 28

Epiblast

Answer 28

The upper layer, which sends cells into the hypoblast. Becomes the ectoderm layer and its derived structures.

Question 29

Hypoblast

Answer 29

The lower layer. Becomes mesoderm and endoderm.

Question 30

Neural plate

Answer 30

Formed at 90% epiboly, when a part of the ectoderm glattens in the head region near the animal pole of the embryo forming the neural plate. This will fold into a tube becoming the first part of the nervous system.

Question 31

Segmentation period

Answer 31

10.3-24 hpf. As segmentation progresses, the embryo lengthens, then the tail begins to form. An obvious feature is the development of somites. The embryo begins to move during this period. Organs including the brain, blood, blood vessels, and kidney also form during this period.

Question 32

Somites

Answer 32

Body segments which form during segmentation. Compartments of mesoderm that will give rise to the muscles and vertebrae.

Question 33

Notochord

Answer 33

Somites form around the notochord, a long, rod-shaped bundle of mesodermal cells that form the primitive head to tail axis in the embryo.

Question 34

Brain

Answer 34

In segmentation, becomes clearly visible as a series of 5 lobes in the head.

Question 35

Spinal chord

Answer 35

Nerves extend posterior from the brain. Dorsal to the notochord, which supports it from underneath, connected by the floor plate.

Question 36

Heart

Answer 36

At approximately 24 hpf, the fish’s heart begins to beat and to circulate erythroblast.

Question 37

Erythroblast

Answer 37

Embryonic blood cells which appear clear when the heart begins to beat.

Question 38

Pharyngula period

Answer 38

24-48 hpf. Pharyngeal arches formed. Embryo continues to lengthen and straighten out, the pectoral fins begin to form, and pigment cells become visible in retina and in patchy stripes along the body.

Will also see color from erythroblasts, which now have a bit of hemoglobin protein for gas transport and exchange.

Question 39

Pharyngeal arches

Answer 39

Made of cartilage structures in the head, later form the jaw and gills. Form during the pharyngula period.

Question 40

Melopores

Answer 40

AKA pigment cells. Congregate to form dark brown patches on the skin and embryo’s eyes begin to darken. This happens from around 28 hpf.

Question 41

Hatching glands

Answer 41

Around the same time as pigment cells congregate, hatching glands anterior to the embryo’s heart secrete a proteolytic enzyme that begins degrading the chorion membrane, and as the embryo’s body movements become increasingly coordinated and strong, it will hatch out of the chorion, approximately from 48-72 hpf.

Question 42

Gut and liver

Answer 42

During the 3rd day of development, 48-72 hpf, the embryos continue to grow, and complete organ development of endodermal structures like the gut and liver form.

Question 43

Pectoral fins

Answer 43

Begin to extend in length around 60 hpf.

Question 44

Swim bladder

Answer 44

Develops on the 4th day of development. Resembles an air bubble located between the embryo trunk and the remaining yolk ball.

The inflation of a swim bladder allows the embryos to rise or fall vertically while swimming. This is necessary for the embryo to hunt for external food sources (as its yolk supplies are rapidly dwindling by day 4, and they begin to eat on day 5).