Zebrafish Mutants and Embryogenesis Lab 2

Question 1

Why is the zebrafish a popular model organism?

Answer 1

Because of its transparent embryos and rapid development. Within just a few days, it develops a fully formed larva and its developmental stages are well-defined

Question 2

What are the 4 major periods of the lifecycle of the zebrafish?

Answer 2

Embryo, larvae, juvenile, adult.

Question 3

What are the 5 phases of the zebrafish in the first 24 hours?

Answer 3

zygote, cleavage, blastula, gastrula, segmentation

Question 4

What is the name of the membrane surrounding the embryo?

Answer 4

Chorion. Zebrafish usually keeps the chorion until 2-3 dpf.

Question 5

What happens in the zygote period?

Answer 5

Animal pole is formed. The animal pole is located at the top of the egg and contains a higher concentration of cytoplasm, with less yolk compared to the vegetal pole.
The animal pole is where most of the developmental activity will occur because it contains the egg’s nucleus and the cytoplasmic material that directs early development.
The animal pole is crucial for cellular processes such as division, protein synthesis, and early differentiation. The blastodisc is a small, disc-shaped region that forms at the animal pole of the fertilized egg. This is where the first developmental processes, such as cell division, will begin.
The blastodisc contains the first group of cells that are going to divide and form the early embryo. These cells will undergo mitotic divisions during the early stages of development.
In zebrafish, the blastodisc initially appears as a thin layer of cells on the surface of the yolk, and it is this region that will develop into the blastoderm, the outer layer of cells that forms the foundation of the embryo.

Question 6

What happens during the cleavage period?

Answer 6

During the cleavage period the blastodisc starts rapid and synchronous cell divisions and form blastomeres. These divisions occur without any growth, the overall size of the embryo doesn’t increase. The reason is the embryo in this stage relies on maternal RNA.

Question 7

What happens in the blastula period?

Answer 7

One of the things about this stage is zygotic transcription, meaning the embryo starts to express its own genes. The other one is epiboly begins. Epiboly is the movement.

Question 8

what happens in gastrula period?

Answer 8

When epiboly reaches %50, which means the cells cover about half of the yolk., gastrula period begins. In this period head to tail axis is established, and the cells begin to differentiate into future head and tail regions.
Gastrulation is the process where cells start moving inward.
You can think migration is from outer side to inner side. As a result of migration, 3 germ layers are formed. Ectoderm, mesoderm, endoderm.
Gastrulation is critical for setting embryos body plan and establishing the basic structure for future development.

Question 9

Explain the 3 germ layers role?

Answer 9

Ectoderm: skin, nervous system
Mesoderm: Muscle, bones, circulatory system
Endoderm: Digestive tract and internal organs

Question 10

What happens in segmentation?

Answer 10

In total it takes 10 hours. Mesoderm divides into somites. Somites are the segmented tissues. This somites start forming along the notochord and later on they will form vertebrae and muscles. In addition to that, the embryo elongates and neural tube is formed which becomes the central nervous system. Primary organs start differentiating. The head and tail are distinct. The heart initiates contractions toward the end of this stage.

Question 11

What happens in pharyngula period?

Answer 11

Embryos continue to grow, and brain, eyes, and muscles start to develop. The tail continues to grow, and body straightens. Heart beat becomes more regular, and blood vessels form. Melanocytes start to develop.

Question 12

What happens in the hatching period?

Answer 12

Pectoral fins become visible. They come out of their chorion.

Question 13

What happens in the larva period?

Answer 13

The larva swims freely, and the yolk sack is being depleted. The larva should be fed. Digestive track, liver and pancreas becomes functional. The larvae develops a swim bladder.

Question 14

What is the difference between a male and a female zebra fish?

Answer 14

Male have more pinkish color. Female have a yellow color. Female have a pale belly because it is filled with eggs. Female zebrafish have a larger and more rounded anal fin that is less pointed. Male zebrafish have a smaller, more pointed anal fin. In some cases, males may have a distinct white stripe on their anal fin that is more prominent during mating.

Question 15

What are some of the zebrafish mutants?

Answer 15

Wild type is AB.
Casper mutant is transparent because they lack pigment. They are actually generated from a combination of pigmentation mutants.
Nacre mutants lack stripes
Roy mutants lack iridophores

Question 16

What are the neural crest cells?

Answer 16

They are multi-potent cells which means they are a migratory cell population that contribute to the formation of various cell types including pigment cells.

Question 17

what are melanophpores?

Answer 17

Are pigment cells responsible for black and brown pigmentation.

Question 18

What are xanthophores?

Answer 18

They are yellow pigment cells.

Question 19

What are iridiphores?

Answer 19

They are the cells that reflect light, creating a metallic shiny appearance.

Question 20

Which gene is mutated in Nacre?

Answer 20

Mitfa Gene is critical for melanophore development. Which result in no stripes.

Question 21

Which gene is affected in Roy mutant.

Answer 21

İridophores

Question 22

What is defected in alb mutant?

Answer 22

Melanin production also the retinal pigment epithelium

Question 23

What is Ache (Acetylcholinesterase)

Answer 23

Ache is an enzyme that is commonly found in neuromuscular junctions. Degrades a naturally occurring neurotransmitter called Acetylcholine (ACh). Acetylcholinesterase activity prevents accumulation of Acetylcholine in the post synaptic cleft.
Excessive accumulation of ACh results in over-stimulation of muscles, glands and central nervous system which can be fatal

Question 24

What is the Ache mutant zebrafish?

Answer 24

This mutant type carries a loss-of-function point mutation in the catalytic triad of AChE active site resulting in a null phenotype.
Mutant fish display dysfunctionalities in muscle fiber formation and their primary sensory neurons die prematurely.
Zebrafish ache homozygous mutants are initially motile but paralyzed at around 72 hours post-fertilization (hpf)

Question 25

what is a tail test?

Answer 25

Tail test is a method that is used to differentiate ache homozygous mutant larvae (ache -/-) from heterozygous and wt ones (ache +/- and ache +/+).
In this method, the tail of the fish is flicked to assess motility

Question 26

Why are we interested in DNA?

Answer 26

To identify the characteristics of genomic DNA.
To identify the gene mutations, sequence and functionality.
To study the genetic causes of disease and for the development of diagnostics and drugs.

Question 27

Explain genomic DNA extraction

Answer 27

1. Add 200 uL of DNA extraction buffer into every single-embryo-containing tube.
2. Add 2 uL of Proteinase K per tube.
3. Incubate o/n at 55°C for overnight.
4. Heat tubes in a heat block at 95°C for 20 minutes for inactivation of Proteinase K.
5. Centrifuge at 13000 rpm for 20 minutes at room temperature after samples cool down.
6. Transfer the supernatant into a new tube and add 175 uL of 100% isopropanol.
7. Mix and centrifuge at 13000 rpm for 20 minutes at +4°C.
8. Discard the supernatant and wash with 500 ul of 70% EtOH.
9. Centrifuge at 13000 rpm for 20 minutes at +4°C.
10. Discard the supernatant and leave it for air dry at RT.
11. Resuspend in 20 uL of ddH2O and store at +4°C for further experiments.

Question 28

Why do we use proteinase K?

Answer 28

Digest many contaminating proteins present. It also degrades nucleases that may be present in DNA sample and protects the nucleic acids from nuclease attack.

Question 29

Why do we use ısopropanol?

Answer 29

DNA is less soluble in solutions containing isopropanol than in solutions containing ethanol. In contrast to precipitation with ethanol, which requires 2-3 volumes of alcohol, precipitation with isopropanol is performed with 0.6-0.7 volume of alcohol.

Question 30

Why do we use ethanol?

Answer 30

Eliminates the solvation shell that surrounds the DNA, thus allowing the DNA to precipitate in pellet form. Additionally, ethanol helps to promote DNA aggregation.

Question 31

What is the Nanodrop Analysis?

Answer 31

The Nanodrop is a spectrophotometer commonly used in molecular biology and biochemistry labs to measure the concentration and purity of nucleic acids (DNA, RNA) and proteins in small sample volumes. The NanoDrop provides two different purity ratios A260/A280 and A260/A230.
An A260/A280 purity ratio of ~1.8 is generally accepted as “pure” for DNA and ~2.0 for RNA samples.
An A260/A230 purity ratio between 1.8 and 2.2 is generally accepted as “pure” for DNA and RNA. Higher A260/A280 might indicate protein contamination in the sample.

Question 32

How can PCR be used in the genotyping the Ache population?

Answer 32

PCR, using allele specific primers, allows for the identification ache (+/?) population as well.
The specificity of the primers ensures that amplification occurs only when there is a perfect match between the primer and the target DNA.

Question 33

Explain the steps of PCR

Answer 33

1. Initialization:
   Temperature: ~94–96°C for 1–5 minutes.
   Purpose: This optional step activates the DNA polymerase enzyme (if a hot-start polymerase is used) or ensures complete denaturation of the DNA.
2. Denaturation:
   Temperature: ~94–98°C for 20–30 seconds.
   Purpose: Heat separates the double-stranded DNA (template) into two single strands by breaking the hydrogen bonds between base pairs.
   Outcome: Single-stranded DNA serves as a template for primer annealing.
3. Annealing:
   Temperature: 50–65°C for 20–40 seconds.
   Purpose: Allows primers (short single-stranded DNA sequences) to bind (anneal) to their complementary sequences on the single-stranded DNA template.
   Key Point: The exact temperature depends on the primer’s melting temperature (Tm) to ensure specific binding.
4. Extension/Elongation:
   Temperature: 68–72°C (depends on the DNA polymerase used).
   Purpose: DNA polymerase synthesizes a new DNA strand complementary to the template strand by adding dNTPs (deoxynucleotide triphosphates).
   Key Points:
   Taq polymerase, commonly used in PCR, works optimally at ~72°C.
   The time depends on the length of the DNA sequence being amplified (~1 minute per 1,000 base pairs).
5. Final Elongation (Optional):
   Temperature: ~70–74°C for 5–10 minutes.
   Purpose: Ensures that any remaining single-stranded DNA is fully extended.

Question 34

What are needed for PCR?

Answer 34

Template DNA,
Primers (gene specific)
Buffer
DNA polymerase
dNTPs