Zebrafish Flashcards
Why is zebrafish a good model organism?
- Development is rapid
- It is transparent
- Good for genetic modification
- Easy to manipulate
- Many genes have conserved functions
What is the purpose of the yolk cell?
The larvae rely on it for nutrients until they are able to feed.
How many hours after fertilisation does the embryo begin to move?
16-18 hours.
What kind of cleavage does zebrafish undergo?
Discoidal meroblastic cleavage.
What is discoidal cleavage?
Only the cytoplasm of the blastodisc forms the embryo proper.
What does meroblastic mean?
The cleavage furrows do not completely divide the egg.
In what part of the egg does cleavage occur?
Only in the blastodisc.
Define the blastodisc.
A thin region of yolk-free cytoplasm at the animal pole.
What is the majority of the egg made from at the start of blastulation?
Yolk.
The egg becomes pear-shaped at the start of blastulation. Why is this? Give 3 steps.
- Calcium is released at fertilisation
- Calcium causes the actin cytoskeleton to contract
- This squeezes all non-yolk cytoplasm into the animal cap
Divisions are rapid. How long does each division take?
Approx. 15 mins.
How many divisions are synchronous?
The first 12.
What do the first 12 synchronous divisions form?
A mound of cells at the animal pole of the yolk cell - this will become the blastoderm.
What is the mid-blastula transition?
A switch from the transcription of maternal to zygotic genes.
When does the mid-blastula transition occur?
Approx. 10 divisions in.
There are 3 distinct cell populations at the mid-blastula transition. What are they?
- The YSL
- The EVL
- The deep cells
What is the YSL?
The yolk syncytial layer, formed from vegetal cells fused the to underlying yolk cell. This complex contains syncytial nuclei.
What is the EVL?
The enveloping layer, this becomes the periderm (protective layer that is shed).
What are the deep cells?
They give rise to the embryo proper.
Where are the deep cells found?
Between the YSL (inside) and EVL (outside).
Are the fates of the 3 distinct cell lineages determined at blastulation?
No.
When do cell fates become specified?
At the start of gastrulation.
What is epiboly?
Cellular movements that allow dramatic physical restructuring.
When does epiboly occur?
At the start of gastrulation.
What does epiboly occur?
The autonomous expansion of the YSL over the yolk cell. The YSL drags the deep cells and EVL with it.
What happens to the yolk at the end of gastrulation?
It becomes completely engulfed by the cells.
What happens to the blastoderm at the end of gastrulation?
It becomes completely internalised.
The YSL and EVL are tightly bound (with the deep cells in the middle). What happens if this connection is severed?
During epiboly the YSL expands on its own and the EVL and deep cells stay on top of the yolk.
How does the autonomous expansion of the YSL rely on?
A network of microtubules composed primarily of tubulin.
Involution occurs at 50% epiboly - what does this mean?
When 50% of the yolk is covered by the migrating YSL/EVL.
What is formed during involution?
The germ layers
What is the germ ring and what is it made of?
A thickening that occurs at the start of involution. It is made of 2 cell types; the epiblast and hypoblast.
Where does involution begin?
On the future dorsal side of the embryo.
What is the embryonic shield?
A thickening composed of intercalated epi and hypoblast cells.
The embryonic shield can organise a secondary axis in transplant experiments, meaning it is homologous to which structure in amphibians?
The dorsal blastopore lip.
What is the chordamesoderm and how is it formed?
A precursor to the notochord, formed from the convergent extension of the hypoblast.
In which direction does convergent extension of the hypoblast occur?
Anteriorward.
What is the paraxial mesoderm?
Cells next to the notochord that give rise to the somites.
What does the epiblast do?
It convergently extends to form the neural keel along the dorsal midline.
What is the neural keel?
A structure that eventually develops into the neural tube.
Where is the notochord?
Extends from the base of the head to the tail on the dorsal side.
What is the purpose of labelling cell nuclei?
To build fate-maps/trace cell lineages.
What is SPIM and what is it used for?
Selective Plane Illumination Microscopy, used to illuminate live specimens from the side.
Give 3 advantages of SPIM.
- High resolution
- High speed
- Non-invasive
Random mutagenesis is a forward genetic screen. What does this mean?
Random mutations are generated in individuals, these individuals then breed and mutations in the phenotype of the offspring are observed.
How are mutations generated in random mutagenesis? Give 3 methods.
With radiation, chemicals or by gene insertion.
Some phenotypic mutations are easily observed. Give an example.
Cyclopia.
How does random mutagenesis help us to understand zebrafish development?
When offspring are mutants can look at which genes have been disrupted and thus deduce function.
How many mutants has forward genetic screening by random mutagenesis found?
Approx. 10,000
What is the basic principle of genetic mapping?
Assesses how close 2 gene markers are by determining the recombination frequency at meiosis.
What are the 2 markers in genetic mapping?
A mutation and an identifiable genetic marker like an SSLP
What is an SSLP?
A simple sequence length polymorphism (also called microsatellites).
When can homozygosity mapping identify a mutation?
In a small, closed population.
What is the candidate gene approach?
Whereby a list of candidate genes is derived and tested to see whether they cause a mutation.
What 5 things must be considered in a candidate gene approach to mutation?
- Is the gene expressed in the mutant phenotype
- Is there in fact linkage to another mutated gene
- Is the gene mutated?
- Can wild-type RNA rescue the mutant phenotype?
