Watching zebrafish embryos; lifting and counting cells

Question 1

Which part of the fertilised zebrafish embryo contains genetic material?

Answer 1

The single cell. During fertilization, the DNA from the sperm and egg combine to form a single cell with a complete set of chromosomes, which will then divide and differentiate into all the specialized cells that make up the developing zebrafish embryo.

Question 2

What are the advantages of using Zebrasfish as an animal model?

Answer 2

1. Transparency: Zebrafish embryos are transparent, which allows researchers to observe and track the development of organs and tissues in real-time, including the formation of the heart, brain, and other organs.
2. Reproduction: Zebrafish reproduce quickly and in large numbers, which makes them easy to maintain in the laboratory and ideal for genetic studies and drug screenings.
3. Genetic similarity: Zebrafish have a high degree of genetic similarity to humans, with about 70% of their genes being similar to human genes. This means that research on zebrafish can provide insights into human disease and biology.
4. Regenerative abilities: Zebrafish have a remarkable ability to regenerate damaged tissues and organs, making them an excellent model for studying tissue regeneration.
5. Small size: Zebrafish are small and easy to handle, which makes them suitable for high-throughput studies and drug screenings.
6. Cost-effective: Zebrafish are relatively inexpensive to maintain in the laboratory, which makes them a cost-effective animal model for research.

Question 3

What is the structure of a fertilised zebrafish egg?

Answer 3

single cell, chorion and yolk

Question 4

What are the steps of Zebrafish embryo development?

Answer 4

1.Fertilization: Sperm and egg unite to form a single cell called a zygote.
Cleavage: The zygote undergoes multiple rounds of rapid cell division to form a ball of cells called a blastula.
2. Blastula: The blastula has two types of cells: the outer layer of cells, called the blastoderm, and the yolk cell, which is located in the center of the embryo.
3. Gastrulation: The blastoderm cells migrate to the bottom of the embryo, forming a structure called the embryonic shield. The cells then rearrange themselves to form three distinct cell layers: the endoderm, mesoderm, and ectoderm. Establishment of the anterior-posterior, dorsal-ventral and medio-lateral axes
4. Segmentation: The mesoderm cells are organized into segments, or somites, which will eventually give rise to the muscles and bones of the embryo.
5. Organogenesis: The major organs and tissues begin to form, including the heart, brain, eyes, and gut.
6. Hatching: The embryo breaks out of its protective membrane and hatches from its egg.
7. Larval stage: The embryo transforms into a larva and begins to feed and swim independently.
8. Juvenile stage: The larva grows and matures into a juvenile fish.

Question 5

What is the first organ that develops in zebrafish?

Answer 5

The first organ to develop in a zebrafish embryo is the heart. The heart starts to form during the gastrulation stage of embryonic development, which occurs around 10 hours post-fertilization in zebrafish. During this process, a group of mesodermal cells migrate to the midline of the embryo and form a linear heart tube. The heart tube then undergoes looping, which is a process of twisting and folding, to form a four-chambered heart with distinct chambers and valves.

Question 6

What is the structure that the Zebrafish breaks out from?

Answer 6

Zebrafish embryos develop inside a protective membrane called the chorion, which is a transparent, proteinaceous layer that surrounds the embryo and the yolk. After the embryonic development is complete, the zebrafish embryo breaks out of the chorion to hatch from its egg.

Question 7

What is mid blastula transition?

Answer 7

This is the stage at which the embryo starts transcribing its own genome. Up to then, the embryo was only using the mRNAs deposited in the egg by the mother prior to fertilization.

Question 8

What is the name of the three layers formed during gastrulation?

Answer 8

Ectoderm, mesoderm, endoderm.

Question 9

What are the names of the developmental stages and its correct order?

Answer 9

1. Segmentation
2. Blastula
3. Cleavage
4. Gastrula
5. Organogenesis

Question 10

When does the heart start beating in a zebrafish embryo?

Answer 10

The heart starts beating at around 36 to 48 hours post-fertilisation (hpf). Sometimes you can already see it beating as early as 28 hpf!

Question 11

When does the embryo start moving?

Answer 11

Before coming out of the chorion

Question 12

Name 4 other animal models used in research.

Answer 12

mouse, chick, fruit fly (Drosophila), nemoatode (C. elegans), frog (Xenopus).

Question 13

Name the components of a fully labelled 24hpf zebrafish embryo.

Answer 13

Question 14

What are the cell movements during gastrulation?

Answer 14

Invagination: A group of cells at the dorsal side of the embryo start to buckle inward, forming a depression called the blastopore. This is the site where the endoderm and mesoderm will later form.
Epiboly: The cells on the surface of the embryo start to spread and thin out, like a sheet of cells, to cover the entire surface of the embryo.
Convergent extension: The cells in the mesoderm and endoderm start to intercalate, which means they elongate and narrow in a direction perpendicular to their original position. This causes the cells to move towards the blastopore and converge at the midline, while also extending the length of the embryo.
Delamination: A group of cells in the ectoderm separates from the overlying epidermis and migrates inward to form the neural crest cells.

Question 15

What is the general protocol for lifting and counting cells?

Answer 15

1. Prepare a trypsin-EDTA solution: Mix 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline (PBS) to make a working solution. Adjust the concentration of trypsin and EDTA as needed for your cells.
2. Add trypsin-EDTA to the cells: Remove the culture medium from the cells and rinse them with PBS. Add enough trypsin-EDTA solution to cover the cells and incubate them at 37°C for a few minutes, depending on the cell type and the density of the cells. Monitor the cells under a microscope to check for detachment from the culture surface.
3. Neutralize the trypsin-EDTA: Once the cells have detached from the culture surface, add an equal volume of culture medium to the trypsin-EDTA solution to neutralize the trypsin activity.
4. Collect the cells: Transfer the cell suspension to a centrifuge tube and centrifuge the cells at 300-500 g for a few minutes to pellet the cells. Discard the supernatant and resuspend the cells in a suitable buffer or medium.
5. Count the cells: Dilute the cell suspension to an appropriate concentration, depending on the counting method and the experimental conditions. Use a hemocytometer or an automated cell counter to count the cells and calculate the cell density.

Question 16

What is a hemocytometer?

Answer 16

A hemocytometer, also known as a counting chamber, is a specialized glass slide that is used for manual cell counting under a microscope. The grid pattern on the hemocytometer slide creates a known volume and area for counting cells, and the total number of cells in the sample can be calculated based on the number of cells counted and the dilution factor of the sample.

Question 17

Why do some researchers add trypan blue to the sample before counting?

Answer 17

A dye that can selectively stain dead or damaged cells in a sample, while leaving viable cells unstained.When added to a cell suspension, trypan blue can help distinguish between live and dead cells, allowing researchers to accurately determine the viability and concentration of viable cells in the sample.

Therefore, some researchers add trypan blue to the sample before counting to selectively stain and identify dead cells in the cell suspension. The dead cells will appear blue under the microscope, while the viable cells will remain unstained.