After Midterm 1 - Gastrulation and Body-axis Formation in the Xenopus

Question 1

While involuting, the xenopus embryo’s outer ectodermal tissue is going through epiboly. Compare and contrast epiboly in the xenopus and in the sea urchin.

Answer 1

• The ectodermal tissue in the sea urchin is only a single layer, so epiboly happens by cells flattening and expanding their shape
• The ectodermal tissue in the xenopus is multi-layered, so epiboly happens via radial intercalation.

Question 2

Cells of the organizer in the xenopus involute using an interaction between integrin and the extracellular matrix protein fibronectin. If integrin or fibronectin is blocked, so is involution.
The question here is, will this block epiboly of ectodermal tissue as well in the xenopus, if involution is blocked?

Answer 2

Cell movement is not independent. Yes, if involution is blocked, epiboly will be disrupted in some way, because cell movements are not independent of one another.

Question 3

Choose the correct response. During gastrulation, the blastopore lip forms…

a) In the marginal zone (zone between the vegetal and
animal half)

b) Just above the bottle cells

c) In the area of the blastula that will eventually form
the mesoderm

d) All of the above

Answer 3

answer is d) all of the above.

Question 4

If we divide a xenopus embryo in half, such that the two halves have a portion of the gray crescent, what happens to these two halves?

Answer 4

They both form into an intact embryo. This is because the location of the gray crescent roughly coincides with the organizer region. Organizer genes are expressed in both halves.

Question 5

If we divide a xenopus embryo in half, such that of the two halves only one of the halves contains the gray crescent (fully), what happens to these two halves?

Answer 5

The half with the gray crescent has the organizer region, organizer genes are expressed so it will form into an intact embryo.

The other half has no organizer region, therefore no organizer genes are expressed, and we won’t get the expression of dorsal structures. As a result, we get a belly piece for this half.

Question 6

What structure triggers gastrulation in the Xenopus and compare that to what triggers gastrulation in the sea urchin?

Answer 6

Xenopus - gastrulation is triggered by the SMO/organizer

Sea urchin - gastrulation is triggered by the micromeres

Question 7

What happens if you transplant cells of the organizer from one xenopus embryo to another embryo but on the side where the organizer usually does not form (the transient ventral side)?

Answer 7

The transient ventral side will begin to go through gastrulation, and form a second set of dorsal-related structures.

Question 8

What is the Nieuwkoop Centre?

Answer 8

A group of cells in the Xenopus that induce the region above to become the organizer region.

Question 9

The organizer triggers gastrulation in the Xenopus, and the Nieuwkoop centre triggers formation of the organizer.

Is transplanting just the Nieuwkoop centre enough to trigger gastrulation in the recipient embryo?

Answer 9

Yes. it will induce the formation of the organizer

Question 10

True or false. Vg1 and VegT are maternally-inherited factors in the xenopus egg.

Answer 10

true

Question 11

Where are Vg1 and VegT localized?

Answer 11

The vegetal half of the Xenopus embryo

Question 12

Describe, in detail, how the organizer is formed (the morphogens involved and everything)

Answer 12

There is a flowchart for this:

The area where VegT and veg1 overlap with B-catenin forms the Nieuwkoop centre

The Nieuwkoop centre forms an Xnr gradient. Xnr is highest at the transient dorsal side of the embryo and lowest at the transient ventral side.

Xnr creates a smad2 gradient (it activates smad2 with vg1)

Where Xnr is low - you either get low smad2, which induces lateral mesoderm, or no smad2, which induces ventral mesoderm.

Where Xnr is high - high smad2 - smad2 acts with twin and siamois (activated by B-catenin) to activate the organizer genes goosecoid, chordin and noggin. - the organizer region has now formed.

High smad2 also activates Hhex gene, which triggers formation of pharyngeal endoderm.

Question 13

What is the function of Hhex?

Answer 13

triggers formation of pharyngeal endoderm

Question 14

What does the pharyngeal endoderm do?

Answer 14

These are the cells that lead the involution in the Xenopus

Question 15

What kind of cells have the cells of the Nieuwkoop centre become by late gastrulation?

Answer 15

endoderm

Question 16

What process in the Xenopus does UV radiation stop?

Answer 16

UV radiation stops cortical rotation by blocking the formation of the MT tract that is used to rotate the cortex cytoplasm

Question 17

What happens to an embryo if it is UV irradiated?

Answer 17

The organizer fails to form, because the concentrations of the different factors have to be at specific concentrations at specific locations.
Because the organizer fails to form, we get a belly piece lacking the dorsal structures.

Question 18

As gastrulation continues, explain what happens to the location of the dorsal-ventral axis by the end of gastrulation?

Answer 18

By the end of gastrulation, the animal half of the embryo is now the dorsal end, and the vegetal half of the embryo is now the ventral end.

Question 19

Where is “anterior” and “posterior” by the end of Xenopus gastrulation?

Answer 19

Where the pharyngeal endoderm has settled after involuting - the anterior end

where the blastopore/anus is - the posterior end

Question 20

Body axes induction is, in general, driven by cell-to-cell __________.

Answer 20

communication

Question 21

What is juxtacrine signalling?

Answer 21

Cell to cell communication where communication is shorter range and contact-mediated, such that the communicating cells are either contacting each other via contact, or through contact with the surrounding ECM.

Question 22

What is paracrine signalling?

Answer 22

Cell to cell communication over a longer range, that involves one cell releases a signal, which diffuses a distance away and is picked up by receptors of another cell.

Question 23

What is a morphogen?

Answer 23

Any signalling factor that induces a change in the morphology of an organism.

Question 24

True or false. Morphogens usually set up a concentration gradient that establishes differential gene expression in different cells, leading to the formation of a diverse array of structures.

Answer 24

true

Question 25

Morphogens are involved in the formation of the organizer. Name the two?

Answer 25

Xnr and Smad2

Question 26

Remember that we said that the cells of the organizer have high levels of Smad2. But even in these organizer cells, smad2 occurs in different concentrations. Is this true or false?

Answer 26

True. Even though what causes organizer cells to be organizer cells is high smad2 expression, smad2 levels still differ in certain subset of organizer cells, which causes them to have differential gene expression and therefore induce the formation of different tissues.

Question 27

What are the different tissue types that differential high concentrations of smad2 give rise to?

Answer 27

• Pharyngeal endoderm of the organizer induces the formation of the forebrain
• The prechordal plate induces the formation of the midbrain
• The notochord induces the formation of the hindbrain and trunk
• The cells of the dorsal blastopore lip induce the formation of the tip of the tail

Question 28

UV radiation in the Xenopus blocks cortical radiation. How does this affect wnt signalling and beta-catenin localization

Answer 28

Wnt-signalling is supposed to be localized at the transient dorsal side of the embryo, at the marginal zone, but is now localized at the vegetal half of the embryo, and that is also where beta-catenin is localized, because wnt signalling is what stops beta-catenin degradation.

Question 29

What happens to the Xenopus embryo as you increase the amount of UV radiation it is exposed to?
Make sure to include what morphogen’s levels are disrupted

Answer 29

The embryo becomes increasingly ventralized (lacks a nervous nervous system and associated dorsal structures).

Since UV blocks cortical rotation, we don’t get the formation of the organizer.

No organizer = no organizer genes = no goosecoid, chordin and noggin

Organizer genes inhibit the morphogen BMP.

BMP determines the identity of ectodermal tissue. When BMP inhibition is low (i.e. BMP levels are high), the ectoderm forms epidermis.

BMP occurs throughout the embryo pre-gastrulation.

Because we aren’t inhibiting BMP, the entire embryo’s ectoderm is signalled to become epidermis. There is no formation of the Central Nervous system, making the embryo be ventralized (lack dorsal structures). Similarly, high levels of BMP leads to the expression of ventral mesoderm, and no expression of dorsal mesoderm.

Question 30

What function does LiCl have on the Xenopus embryo?

Answer 30

Just like with UV radiation, LiCl disrupts the proper formation of the organizer region. Except here we have beta-catenin localized everywhere, even in places that should not have beta-catenin (LiCl is a GSK3 blocker, preventing the formation of the degradation complex which is responsible for removing B-catenin in certain cells).

Question 31

What happens to the Xenopus embryo as you increase the amount of LiCl it is exposed to?

Make sure to include what morphogen’s levels are disrupted

Answer 31

With increased levels of LiCl you get lots of B-catenin activity, since the degradation complex cannot form.

An overabundance of B-catenin means an overabundance of the twin and siamois

An overabundance of twin and siamois works with smad2 to create an overabundance of organizer genes/BMP inhibitors

Excess BMP inhibition means more and more of the ectoderm is becoming neural ectoderm, this means the embryo is getting exceedingly dorsalized (dorsal features are becoming dominant) and less ectoderm is made into epidermis (ventral features are getting minimized). Low BMP also means more of the mesoderm is becoming dorsal mesoderm.

You get embryos with the heads and dorsal regions getting more and more prominent.

Question 32

Which organizer gene is NOT a BMP inhibitor?

Answer 32

Goosecoid.

Question 33

What are our three BMP inhibitors?

Answer 33

Chordin, noggin, follistatin.

Question 34

Is BMP a maternally-deposited factor?

Answer 34

No, it’s just a protein secreted early on in development

Question 35

True or false. BMP signalling happens throughout the Xenopus blastula (i.e. everywhere, it’s not localized) and only the organizer genes can block BMP signalling at the organizer.

Answer 35

True.

Question 36

How would you rescue a belly piece that has been formed by UV radiation?

Answer 36

By adding (the correct concentration) of a BMP inhibitor.

Question 37

BMP determines the identity of ectodermal tissue. When BMP inhibition is low (i.e. BMP levels are high), the ectoderm forms epidermis. Explain the biochemical pathway that causes this.

Answer 37

BMP is a ligand that, after binding to its receptor, results in the expression of smad1 and smad5.

smad 1 and smad5 work with smad4 as transcription factors to activate epidermal genes.

Question 38

Does BMP also affect mesoderm?

Answer 38

Yes!

High levels of BMP in mesodermal cells = that mesoderm becomes ventral mesoderm

Low levels of BMP in mesodermal cells = that mesoderm becomes dorsal mesoderm

Question 39

From all this, we conclude that the morphogen _______ is responsible for establishing the true dorsal-ventral axis of the Xenopus

Answer 39

BMP

Question 40

Imagine you’ve got a Xenopus blastula, and you remove the animal cap. The cells of the animal cap are soon-to-be ectodermal cells.

1) what is the fate of this animal cap if all cells of the animal cap are kept together in the explant?
2) what is the fate of animal cap cells if each individual cell is isolated from one another?

Answer 40

1) The whole animal cap isolated will give rise to epidermal cells. This is because of the presence of BMP everywhere in the Xenopus blastula, pre-organizer formation. BMP signalling is high everywhere, so this induces epidermal genes and therefore induces formation of epidermal cells.
2) Each individual cell becomes a neural cell. This is because isolating each individual cell blocks BMP cell-to-cell communication. The BMP produced by each cell is too low, and diffuses away from the cell towards the outer edges of the petri dish. So you have an ectodermal cell that is low in BMP, this allows for neural genes to be expressed.

Question 41

True or false. As organizer mesodermal precursor cells involute and migrate along the animal pole, they are releasing the organizer genes/BMP inhibitors, causing local changes in the ectoderm lying above, making the ectoderm of the now dorsal half neural ectoderm.

Answer 41

True.

Question 42

True or false. By the end of gastrulation, we have the formation of the true dorso-ventral axis in the Xenopus, but also the anterior-posterior axis (posterior is where the blastopore formed, anterior is where the migrating pharyngeal endoderm eventually ended up.

Answer 42

True.

Question 43

What is one lab technique you could use to block a certain gene, say for example, you want to block the expression of chordin?

Answer 43

A morpholino. A morpholino is something that binds to mRNA of target genes, and prevents translation, in order to do Loss-of-Function experiments

Question 44

How can you stain for neural cells/neural tissue specifically?

Answer 44

Stain for Sox2. Sox2 is a gene only expressed by neural cells.

Question 45

The anterior-posterior axis is dependent not on the BMP gradient, but on the _____ gradient

Answer 45

wnt gradient

Question 46

Which two tissues, arising from cells of the organizer, express wnt inhibitors?

Answer 46

1. The prechordal plate or “head mesoderm” as it is sometimes called
2. The pharyngeal endoderm

Question 47

How is Smad2 related to wnt inhibition?

Answer 47

High Smad2 = high wnt inhibitor expression

Question 48

What are the three wnt inhibitors?

Answer 48

1. Cerberus
2. Dickkopf
3. Frzb

Question 49

True or false. Wnt inhibitors are transcribed AFTER the organizer is formed, otherwise it would’ve prevented the expression of twin and siamois by B-catenin

Answer 49

True.

Question 50

True or false. The role of the wnt inhibitors become important after the pharyngeal endoderm and prechordal plate cells have migrated to the anterior portion of the embryo

Answer 50

True.

Question 51

Why is staining for B-catenin a way to visualize wnt-inhibition?

If you were to do this in a Xenopus embryo at late gastrulation, where would you find low stainage of B-catenin?

Answer 51

If wnt is inhibited, that means the degradation complex is formed successfully. B-catenin levels drop drastically. If you stain for B-catenin, areas with less pigment are areas where wnt is inhibited.

Because wnt inhibitors are produced at the anterior end, where the pharyngeal endoderm and prechordal plate have finished migrating, this is where you’ll see the lowest stainage for B-catenin.

Question 52

The formation of anterior structures is induced where wnt inhibition is ________(low/high)

Answer 52

wnt inhibition must be high (i.e. wnt levels are low)

Question 53

What happens if you inject wnt inhibitors into a 32-cell embryo?

Answer 53

The embryo will form a second set of anterior structures

Question 54

What happens if you block wnt inhibitors and therefore block wnt inhibition?

Answer 54

The embryo will not form any anterior structures, no head.

Question 55

a) The area where wnt inhibition (anterior) overlaps with BMP inhibition (dorsal) coincides with the formation of the ________.
b) Where we have BMP inhibition (dorsal) but no wnt inhibition (posterior), this coincides with the formation of the _________.
c) Where we have BMP (ventral) and where we have wnt signalling (posterior), this coincides with the _______.

Answer 55

a) the brain
b) spinal cord
c) epidermis

Question 56

Choose the correct response.

Cerberus, Frzb and Dickkopf are:

a) Wnt antagonists blocking all beta-catenin signalling,
including the activation of twin and siamois

b) Capable of restorying dorsal ectoderm in a UV
irradiated embryo

c) Induced by high Smad2 expression at the organizer
cells

d) Expressed in the dorsal blastopore lip in the late
gastrula

e) Wnt antagonists that induce neural tissue via graded
nodal expression

Answer 56

answer is c.