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Development > Xenopus development > Flashcards

Xenopus development Flashcards

1
Q

List 4 qualities that make xenopus a model organism.

A
  1. We can induce egg laying by injecting HCG, not restricted to breeding seasons
  2. Large embryos, esp. with X. laevis, so easy to work with
  3. Can culture fragments independently
  4. Can use different species for different things, e.g. X. tropicalis for genetics, X. laevis for manipulations etc.
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2
Q

The amphibian egg is composed of 2 hemispheres. What are they?

A
  1. Animal

2. Vegetal

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3
Q

When is egg polarity established?

A

In oogenesis.

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4
Q

What kind of symmetry is there around the animal-vegetal axis?

A

Radial.

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5
Q

What 4 things does the animal hemisphere contain?

A
  1. Pigment
  2. The germinal vesicle (nucleus)
  3. Small yolk platlets
  4. Lots of cytoplasm
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6
Q

What 3 things does the vegetal hemisphere contain?

A
  1. Large yolk platlets
  2. Less cytoplasm
  3. Vegetal specific mRNAs
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7
Q

a) Which vegetal specific mRNAs are found in the vegetal hemisphere?
b) Are they present in all amphibians?
c) Are they zygotic or maternal?

A

a) Vg1, vegt and wnt11.
b) These are specific to anurans only.
c) Maternal

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8
Q

How long after fertilisation does cleavage begin?

A

90 minutes.

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9
Q

There are 2 distinct phases of cleavage. In the fertilisation-mid blastula stage…

a) What are divisions like and why?
b) What drives development?
c) What kind of divisions occur?
d) What is the point of this phase?

A

a) Divisions are extremely rapid as G1 and G2 have been removed and S-phase is shortened.
b) The proteins and mRNAs stockpiled in the egg cell are used for development.
c) Synchronous
d) To generate large volumes of cells quickly

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10
Q

There are 2 distinct phases of cleavage. In the mid blastula-early gastrula phase…

a) What are divisions like and why?
b) What drives development?
c) What kind of divisions occur?

A

a) Divisions slow down as G1 and G2 are reintroduced to the cell cycle.
b) Transcription of the zygotic genome begins and drives development
c) Divisions become asynchronous

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11
Q

During gastrulation, where does the dorsal blastopore appear?

A

In the vegetal hemisphere.

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12
Q

How can the dorsal blastopore be identified?

A

By darkly pigmented bottle cells.

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13
Q

Define epiboly.

A

A process whereby the blastopore extends around the circumference of the embryo and closes over the vegetal hemisphere.

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14
Q

Which germ layers involute in gastrulation?

A

Mesoderm and endoderm involute and migrate below the dorsal ectoderm.

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15
Q

What does the blastopore form in xenopus?

A

The anus: xenopus is a deuterostome

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16
Q

During gastrulation the blastocoel is destroyed and replaced by…?

A

The archenteron.

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17
Q

How are fate maps created?

A

Dyes are injected into cells to track their migration during development.

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18
Q

What do animal cells become?

A

Ectoderm

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19
Q

What do vegetal cells become?

A

Endoderm

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20
Q

What do equatorial cells become?

A

Mesoderm

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21
Q

What is the neural plate?

A

A flat, epithelial sheet of cells on the dorsal side of the embryo.

22
Q

What happens to the neural plate after gastrulation?

A

The lateral edges curl up to meet each other and fuse at the dorsal midline to form the neural tube.

23
Q

Does the neural tube sit above or beneath the dorsal epidermis?

A

Beneath

24
Q

What will the lateral plate form?

A

The heart, connective tissue and smooth gut muscle.

25
Q

There are 3 mechanisms of fate determination. What are they?

A
  1. Localised determinants in the cytoplasm (autonomous specification)
  2. Embryonic induction (conditional specification via cell-cell signalling)
  3. Morphogenic gradients
26
Q

Xenopus is difficult to genetically manipulate. How is embryonic manipulation achieved?

A

By depleting mRNA and thus reducing transcription of desired genes.

27
Q

Describe the process of mRNA depletion.

A
  1. Embryo is injected with antisense oligonucleotides.
  2. Antisense trands bind to mRNA
  3. The RNA/DNA hybrid is detected and degraded by RNAse H
  4. The oocyte is then transferred to a surrogate female who lays them with a jellycoat for fertilisation
28
Q

Describe the process of mRNA depletion.

A
  1. Embryo is injected with antisense oligonucleotides.
  2. Antisense strands bind to mRNA
  3. The RNA/DNA hybrid is detected and degraded by RNAse H
  4. The oocyte is then transferred to a surrogate female who lays them with a jellycoat for fertilisation
29
Q

Describe an experiment with vegT whereby it is injected into the animal cap. What happens?

A

Vegt is localised to the vegetal cap. A normal animal cap produces ectoderm only.
When injected into the animal cap, vegt causes the formation of meso and endoderm.

30
Q

Describe an experiment with vegT whereby it is injected into the animal cap. What happens?

A

Vegt is localised to the vegetal cap. A normal animal cap produces ectoderm only. A normal vegetal cap produces endoderm only.
When injected into the animal cap, vegt causes the formation of meso and endoderm.

31
Q

In experiments with antisense oligonucleotides for vegt, what happens? What does this suggest?

A

Embryos given low levels of AOs display normal mesoderm but defective endoderm.
Embryos given high levels of AOs lack both normal meso and endoderm.
This suggests vegt is important for both meso and endoderm specification. Specification is non-autonomous.

32
Q

Vegt is required for endo and mesoderm specification in all deuterostomes. True or false?

A

False: vegt is specific to xenopus.

33
Q

What is vegt?

A

A t-box TSF

34
Q

What does vegt do?

A

Activates zygotic genes in the vegetal hemisphere

35
Q

Vegt’s targets are all other TSFs. True or false?

A

False: they also include signalling molecules.

36
Q

Bix1/2/3/4, mix1/2 and sox17a are all examples of what?

A

TSFs required for endoderm formation.

37
Q

Nodal1/2/4/5/6 and gdf3 are all examples of what?

A

Signalling molecules required for mesoderm formation.

38
Q

What is cortical rotation?

A

The process that forms the DV axis in xenopus.

39
Q

When does cortical rotation occur?

A

In the first stage of cleavage.

40
Q

Describe the process of cortical rotation.

A
  1. Outer cortex of egg rotates 30 degrees
  2. Vegetal pole migrates to future dorsal surface
  3. Animal pole migrates to future ventral surface
41
Q

During cortical rotation, microtubules form in parallel arrays. Where is the a) minus and b) plus end of each MT and in which direction is polymerisation?

A

a) At the centriole. Polymerisation is towards the animal pole.
b) Away from the centriole. Polymerisation is towards the vegetal pole.

42
Q

Kinesin is a motor protein that migrates from the minus to plus ends of the MTs. What does it bind to?

A

Dsh and GSK3β

43
Q

The MTs depolymerise at the end of the first cell cycle, causing Dsh and GSK3β to be released on the dorsal side of the embryo. What are they involved in?

A

Wnt signalling.

44
Q

What does wnt signalling inhibit?

A

GSK3β

45
Q

What does GSK3β do?

A

Phosphorylates β-catenin, leading to its degradation.

46
Q

Thus when wnt signalling is active what is β-catenin able to do?

A

It enters the nucleus and binds to TSFs.

47
Q

What does the β-catenin-TSF complexes do?

A

Activate dorsal specific genes.

48
Q

What happens when AOs for β-catenin are injected into an embryo?

A

No dorsal-specific genes are activated and the embryo becomes ventralised.

49
Q

So basically what is the purpose of wnt signalling?

A

To activate transcription of dorsal-specific genes via β-catenin.

50
Q

Why is cortical rotation essential for wnt signalling?

A

It displaces Dsh and GSK3β to the dorsal side of the embryo ready for wnt signalling.

51
Q

What happens if you inject the ventral side of an embryo with β-catenin?

A

It forms a secondary dorsal axis.

Development (18 decks)

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