Zebrafish and the Wnt-PCP Pathway Flashcards
What makes zebrafish a useful model organism?
Zebrafish are ideal for study as they produce huge clutches of eggs – up to 1500 but usually 200 – in a non-seasonal manner.
The embryos are both external (no opaque womb in the way or complex culture condition requirements) and transparent, allowing them to be easily visualised and the cells within them to be tracked.
The embryos also develop very quickly, turning from a single cell to a free swimming fish within a week and reaching sexual maturity in 2-3 months.
Genetic modification of these cells is also easy. Transgenes can be easily introduced through microinjection, and the cells can be used in CRISPR/Cas9 editing and reverse genetics techniques.
Convenient for both forward and reverse genetic screening, which is more difficult in other genetic mammals.
What are forward and reverse genetic screens?
Forward genetic screening is when the genes responsible for a phenotype are identified.
In a reverse genetic screen a gene is mutated and the phenotype is observed.
What techniques are used to study zebrafish development?
Fluorescence microscopy can be used for a variety of techniques, including In situ hybridisation to track expression of particular genes or wholemount immunohistochemistry to track proteins/cells.
Light-sheet microscopy also allows for individual cell tracking. Time-lapse microscopy can similarly be used to produce video that tracks development.
Cells can also be transplanted from one area to another to study migration and development.
What are the disadvantages of zebrafish as a model organism?
The embryos of zebrafish can be tricky to manipulate due to their small size. They are also slow to reach sexual maturity compared to worms, flies and yeast.
Their sex determination mechanism is not X/Y which can lead to problematic sex ratios, and due to a genome duplication there can be redundancies between the ohnologous pairs of genes that comprise the genome.
Their genome is also highly polymorphic, and no inbred lines exist yet. The cryopreservation of genetic lines is difficult for the fish, large aquaria are necessary.
Not being amniotes/mammals, their findings often require further validation to show their relevance to higher organisms. Their behavioural repertoire is similarly limited, at least compared to rodents; it is far richer than worms, flies and yeast.
What is gastrulation?
Gastrulation is the developmental stage in which the three germ layers form; ectoderm, mesoderm and endoderm.
This is a process that varies hugely in topology between different species, but is very well characterised in zebrafish.
When does zebrafish gastrulation occur?
In zebrafish embryos kept at 28°C this occurs after 5½ hours after fertilisation.
What is the first stage of zebrafish embryo development?
The zebrafish embryo is initially just a single blastomere situated above a large yolk cell.
This divides rapidly to form a blastoderm that remains at the animal pole of the yolk.
Once this blastoderm becomes large enough (approximately 100 cells), it is said to have entered the sphere stage. The blastoderm is coated in a monolayer of flattened cells known as the outer enveloping layer.
What happens to the zebrafish embryo after entering the sphere stage?
The embryo undergoes epiboly, in which the blastoderm spreads out very thinly until it has engulfed the entire yolk cell.
This is led by a ring of thicker cells that spread out to form an equator. It is this line – the germ ring – that forms the length of the fish (the rostro-caudal axis).
How does gastrulation come to its point during epiboly?
When the epiboly has spread over half the yolk cell, this is known as the shield stage.
The cells in the bulging ends of the line (embryonic shields) begin to involute and travel backwards along the line, forming separate layers.
The cells that are ingressing here (yellow) will form the mesendoderm which will later split into the mesoderm and endoderm, while the remaining outer epiblast cells (pink) and the enveloping layer cells (blue) will form the ectoderm.
What wider form of cell movement is responsible for rostro-caudal axis formation?
Many different mechanisms of cell movement occur during zebrafish gastrulation, but one the most important is convergent extension (AKA convergence and extension, C-E).
This is what is mainly responsible for the rostro-caudal axis formation.
What cell migration types comprise convergent extension?
Collective migration and medio-lateral cell intercalation.
What is collective migration?
Collective migration is characterised by a large number of cells moving together in concert, largely maintaining each of their cell-cell contacts and so overall shape.
In which parts of gastrulation is collective migration important?
This drives C-E along the anterior-posterior axis via migration of the prechordal plate, a small grouping of cells that migrates in front of the embryonic shield.
What is medio-lateral cell intercalation?
Several cell layers merge to form fewer layers in a longer axis. This therefore affects the cell group’s dimensions in both the A-P axis and the M-L axis.
In which parts of gastrulation is medio-lateral cell intercalation important?
These cell movements are important in the formation of key signalling centres produced by the axial mesoderm. .
Medio-lateral intercalation is responsible for reorganising the mesenchymal mesoderma (somite) cells towards the midline, producing the notochord.