Zebrafish and the Wnt-PCP Pathway Flashcards

1
Q

What makes zebrafish a useful model organism?

A

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.

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2
Q

What are forward and reverse genetic screens?

A

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.

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3
Q

What techniques are used to study zebrafish development?

A

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.

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4
Q

What are the disadvantages of zebrafish as a model organism?

A

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.

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5
Q

What is gastrulation?

A

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.

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6
Q

When does zebrafish gastrulation occur?

A

In zebrafish embryos kept at 28°C this occurs after 5½ hours after fertilisation.

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7
Q

What is the first stage of zebrafish embryo development?

A

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.

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8
Q

What happens to the zebrafish embryo after entering the sphere stage?

A

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).

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9
Q

How does gastrulation come to its point during epiboly?

A

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.

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10
Q

What wider form of cell movement is responsible for rostro-caudal axis formation?

A

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.

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11
Q

What cell migration types comprise convergent extension?

A

Collective migration and medio-lateral cell intercalation.

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12
Q

What is collective migration?

A

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.

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13
Q

In which parts of gastrulation is collective migration important?

A

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.

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14
Q

What is medio-lateral cell intercalation?

A

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.

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15
Q

In which parts of gastrulation is medio-lateral cell intercalation important?

A

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.

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16
Q

What is the Tubingen screen?

A

The first large scale forward genetic screen of zebrafish, which discovered several mutants that have a large effect on the convergent extension of cells in zebrafish embryos.

17
Q

What pathway was mutated in the mutant embryos with dysfunctional convergent extension?

A

The Wnt pathway.

18
Q

What Wnt pathway mutations did the Tubingen screen identify as having an effect on C-E?

A

The pipetail (ppt) is a knockout of the Wnt5 ligand

The knypek (kny) is a mutation of the Glypican cofactor

The trilobite (tri) is thought to be a mutation in either strabismus or Vangl2

The silberblick (slb) mutant is a mutation in Wnt 11

19
Q

Which was the first C-E mutant to be characterised, and what is its phenotype?

A

The first of these to be explained (in Heisenberg et al, 2000) was the silberblick (slb) mutant, so named as this is the German word for cross-eyed; later in development this mutation causes fusion of the eyes.

In situ hybridisation studies showed that this was caused by incomplete extension of the axial mesoderm, particularly the prechordal plate.

20
Q

How did Heisenberg et al, 2000 identify the mutated gene in the silberblick mutants?

A

The Wnt pathway was already suspected since it was thought to be involved in the movement of cells during gastrulation, injection of extra Wnt8 into Xenopus embryos causes them to develop two heads.

The was tracked to a chromosome, and then to a particular gene: Wnt11.

This was confirmed by injection of wildtype Wnt11 mRNA into the embryo, which rescued the phenotype.

21
Q

What effect on the gene does the silberblick mutation have?

A

The silberblick phenotype was shown to be caused by one of two single point mutations at 216 or 226 that both introduced an early stop codon in Wnt11, rendering the protein non-functional.

22
Q

What is the effect of the silberblick mutation on the expression of the gene?

A

In situ hybridisation studies showed that this also reduced expression of Wnt11 in the areas where it is normally expressed: the paraxial head mesoderm and the germ ring/bud.

23
Q

What was confusing about the silberblick mutation being found to be in Wnt 11?

A

The canonical Wnt pathway was not known to involve Wnt11.

24
Q

What does the Wnt pathway control?

A

The Wnt pathway controls Planar Cell Polarity (PCP), and is hence required to ensuring correct cell orientation and hence migration direction.

25
Q

How does signal transduction occur in the canonical Wnt pathway?

A

The canonical Wnt pathway begins with Wnt8 binding to Frizzled cell surface receptors.

This transmits the signal to a cytoplasmic signalling protein called Dishevelled.

26
Q

How does dishevelled relay the signal to the genome in the canonical Wnt pathway?

A

Dishevelled has three domains in the order DIX-PDZ-DEP.

The DIX-PDZ domains of this protein inhibit GSK3, relieving its nuclear inhibition of β-catenin which interacts with the transcription factor TCF3 to activate genes that lead to axis formation, such as sia.

27
Q

What is the sia gene responsible for?

A

Swinging from the chandelier.

28
Q

What role does Wnt 11 play in the canonical Wnt pathway, and how do we know this?

A

Wnt11 is not involved in the canonical Wnt pathway.

This was shown by injection of activated β-catenin into the cells failing to rescue the Slb mutants, hence lack of signalling in the canonical pathway could not have been responsible

29
Q

What similarities are there between the canonical and non-canonical Wnt pathways?

A

This is less well understood than the canonical pathway. Here Wnt11 is responsible for activating the Frizzled receptor which does again activate the dishevelled protein.

30
Q

How is the non-canonical Wnt pathway different from the canonical one?

A

The frizzled receptor is activated by Wnt11 rather than Wnt8.

In this instance it is not the DIX-PDZ Dishevelled domains acting upon GSK3, but the PDZ-DEP domains activating RhoA/Rac/Cdc42 that continues the pathway.

These proteins then activate the cell polarity genes through various pathways which are not yet elucidated, but are known to involve JNK signalling.

31
Q

What relation does the non-canonical Wnt pathway have with the slb mutants?

A

This is known to be the pathway that is inactivated by the slb mutation, as injection of a truncated version of dishevelled with only PDZ-DEP does rescue the slb phenotype.

Wildtype embryos injected with only the DEP domain (mimicking overexpression leading to decreased expression) produce a phenotype similar to the slb mutation.

32
Q

What is the Wnt/PCP pathway known to be involved in? How was its role demonstrated?

A

This flavour of the Wnt/PCP is known to be important in allowing cells to determine which way up they are, and so is vital for enabling proper migration by convergent extension.

The role of Wnt/PCP in cell orientation has been shown using knockouts in various species, which demonstrate a variety of cell polarity defects that are particularly easily visualised in the hair follicle orientations.

33
Q

How was the effect of the slb mutation on cell polarity investigated? What were the results of this?

A

This was tested in Heisenberg et al (2000) by cell transplantation.

They first showed that slb -/- cells do not undergo proper C-E migration, and then transplanted in cells from wildtype embryos into the slb hosts. These also failed to migrate properly due to the lack of Wnt signalling from the surrounding cells.

This was of particular issue for the prechordal plate migration, which was shown through time-lapse microscopy to be slower and more disordered in slb mutants.

34
Q

What are morphants?

A

Morphants are phenotypes caused by the introduction of antisense oligonucleotide ‘morpholinos’ that can target splice sites or transcription or translation start sites to reduce expression without totally knocking it out – hence this is often termed a ‘knockdown’.

35
Q

What disadvantages are there to morphant studies?

A

This method is quite controversial due to the off-target effects that the morpholinos can have. It is also now being superseded by genome editing techniques, i.e. CRISPR/Cas9.

36
Q

What did morphant screens elucidate concerning C-E mutations?

A

Knocking down Prickle with morpholinos also results in defective C-E migration, giving a similar phenotype to slb.

Knocking this down accentuates the phenotypes caused by Wnt mutants (slb, ppt, kny and tri) indicating that the prickle gene product interacts with the Wnt pathway.