Chapter 3 & 4 xenopus

Question 1

Vegetal - Endoderm -> mesoderm -> ectoderm - animal hemisphere`

Answer 1

yep

Question 2

Where can sperm enter the egg?

Answer 2

In the animal hemisphere.

Question 3

What does the entry point of the sperm dictate?

Answer 3

The first cleavage plane. The first cleavage plane will align with the line drawn from the top of animal hemisphere to the vegetal hemisphere, and the sperm entry point.

Question 4

Synchronous of the xenopus embryo is what?

Answer 4

Synchronous

Question 5

The entry point of the sperm sets what in the embryo?

Answer 5

It sets the first cleavage axis. It sets ventral portion of the egg. Ventral is the point of sperm entry.

Question 6

Gastrulation:
Blastocoel,
Blastula,
Archenteron,

Answer 6

Blastocoel: hollow made by pumping of ions
Blastula: the early embryo, has a hole in it.
Archenteron: early gastric tract.

Question 7

What is the blastopore?

Answer 7

The blastopore is the hole which will become the archenteron.

Question 8

What is the dorsal lip of the blastula?

Answer 8

The dorsal lip of the blastula is the cells on the blastopore just a bit closer to the animal pore then the vegital pore. These upper cells of the dorsal lip start to enter the blastopore, the pull in Endoderm and nearby mesoderm. This expands across the archenteron, until cells are entering from all directions. This pulls the ectoderm around the cell, coating the outside, and pulls the mesoderm within the cell. The endoderm becomes what is called the yolk plug, which blocks the whole left by blastopore becoming the archenteron. The cells of the dorsal lip are now ventral on the other side of the cell. The blastoceol still exists. but is now matched in size by the new, impressive archenteron.

Question 9

Notochord is what?

Answer 9

A cartilaginous skeletal rod. Not nervous tissue.

Question 10

Note: while the mesoderm has been concentrated on the dorsal side of the animal, it will do what?

Answer 10

It will settle down across the flanks of the animal, providing mesoderm between all the ectoderm and endoderm.

Question 11

What is VG1?

Answer 11

It is a TGF Beta, transforming growth factor, ligand. It is concentrated in the vegetal hemisphere

Question 12

cortical rotation

Answer 12

cortical rotation aligns the highest concentration of dorsal factors with the point directly opposite the point of sperm entry. This is achieved through cortical rotation, which is, the rotation of the outer membrane and cortex (cytoplasm immediately within the cell, next to the membrane), will rotate about thirty degrees.

Question 13

What does cortical rotation achieve?

Answer 13

It changes the location of and activates the dorsal factors of the early embryo. This signaling center will lead to the formation of a blastospore, and subsequent archenteron. It is aligned by cortical rotation exactly across from the point of sperm entry.

Question 14

Name two dorsalizing factor:

Answer 14

VG1, which is a ligand from the TGF beta (transforming growth factor) class

Wnt mRNAs. They will activate on the dorsal axis.

Question 15

Are all dorsalizing factors known?

Answer 15

No. They are not all known.

Question 16

Where does Wnt signalling activate?

Answer 16

On the future dorsal side of the cell.

Question 17

What is another name for the dorsal lip of the blastospore?

Answer 17

Spemann organizer.

Question 18

What causes cortical rotation?

Answer 18

Microtubule networks.

Question 19

How do we know that microtubules are necessary for the establishment of the dorsal axis/cortical rotation?

Answer 19

We know hey are involved in establishment of the dorsal axis because if we use UV radiation to destroy them, it ventralizes the embryo. We could probably see the lack of cortical rotation however if the vegetal hemisphere stay’s where it is and we can see dyed region move.

Question 20

What is the Wnt pathway, it has occurred twice now.

Answer 20

In the absence of the Wnt ligand, we see a protein complex destroying beta-catenin which is being constitutively produced. If Wnt is present it binds to frazzled which associates with disheveled, and another surface receptor to bind up the kinases which mark beta catenin for destruction. Therefore, since Wnt Mrna and wint in general is located in the dorsal region of the cell, beta catenin levels will be higher in dorsal region of the embryo namely the spemann organizer, the signaling center on the dorsal lip.

Question 21

Evidence for maternal determinants, how do we know.

Answer 21

If you cut the embryo in half on its second cleavage plane, we see a complete that the ventral half forms a ventralized embryo and the dorsal half forms a dorsazlied embryo.

Question 22

What would happen if you cut on the first cleavage plane instead of the second?

Answer 22

The first cleavage plane would lead to normal embryos, albiet smaller. This is because the necessary determinants are still in each half the embryo to develop ventral and dorsal axis.

Question 23

What is the nieuwkoop center (key initial signalling center)? what does it do? How do you know?

Answer 23

It induces the formation of Spemann organizer in the region above it. If you transfer a nieuwkoop center from one embryo to another which already has one, it will form too spemann centers and establish two dorsal axis with no ventral one. You see formation of a siamese twin with only dorsal tissue

Question 24

How do we know that the early embryo already has differentiated nieuwkoop center?

Answer 24

Because ectopic transfer of the nieuwkoop center will induce another dorsal axis

Question 25

in VG1 what does it seem like vg stands for? implications?

Answer 25

vg seems to stand for vegetal, like vegetal hemisphere. So vg’s in the early embryo will be dorsalizing factors.

Question 26

What causes formation of the nieuwkoop center?

Answer 26

All factors are likely not known. Beta catenin is necessary but not suffiscient for formation, VGt and likely VG 1 are also necessary. I would guess beta catenin gardient is being caused by Wnt signalling but I don’t know for certain

Question 27

Dorsal terminants must be complexed with the microtubule network. Why do you think this?

Answer 27

Because these factors shift with cortical rotation.

Question 28

What does direct injection of beta-catenin into the vental portion of the vegetal hemisphere result in?

Answer 28

We see formation of the nieuwkoop center on the vegetal side as well as the dorsal side (dorsal side is the natural formation), the fact this only works in the ventral hemisphere is evidence that dorsal determinants of the vegetal hemisphere are also important for the formation of the nieuwkoop center

Question 29

What is an additional way to determine importance of beta catenin without utilizing exogenous addition of beta-catenin

Answer 29

perform a knock out of GSK (glycogen synthase kinase) 3, GSK3 is phosphorylates beta-catenin marking it for degredation, which is necessary in order to degrade beta-catenin. This causes dorsalization of the embryo. This combined with the effect exogenous beta catenin implies it is key to dorsalization.

Question 30

Spemann organizer is induced by the nieuwkoop center.

Answer 30

True. so true. If you perform an ectopic transfer of the spemann organizer from one embryo to another it will induce the native tissue to form a dorsal side.

Question 31

Which of the primary layers are maternally determined?

Answer 31

Ectoderm and endoderm

Mesoderm will be induced.

Question 32

Where is mesoderm induced from? by what interactions?

Answer 32

Ectoderm is induced to become mesoderm via signals from the vegetal cells, also known as endoderm.

Question 33

If I cut up an late blastula, where would I see formation of mesoderm?

Answer 33

I would see it only in tissue which included endoderm and ectoderm.

Question 34

Animal caps on top of the vegetal cells will lead to:

Answer 34

Induction of mesoderm by the vegetal cells of the ectoderm.

Question 35

How long can induction occur from?

Answer 35

Hours 4 - 11 after fertilzation ectoderm cells are competent to be induced into mesoderm. This requires a 2 hour period of contact. Outside of this window and conditions then cells will default to ectoderm.

Question 36

When does control transition from maternal to zygotic control in the developing embryo?

Answer 36

After the 12th division, mid blastula. It is theorized that there is an inhibitor to zygotic gene transcription, and at the point 12th division the inhibitor is now diluted enough to allow transcription of the zygotic genes. Threshold DNA/cell. Ok. so we have a certain amount of initial DNA, and a set amount of inhibitor. As we increase DNA concentration, due to mitotic divisions, we see more and more DNA, and the same amount of inhibitor, eventually inhibitor per cell is present at a much lower concentration compared to the amount of DNA to be inhibited and inhibition of DNA fails. Zygotic control is now in place.

Question 37

If I take dorsal vegetal cells, and dorsal ectoderm and put them together, animal cap cells are induced to become muscle tissue.
If I perform the same procedure ventrally, I get blood cells induced. Implications go. What does that mean. Think son.

Answer 37

This means that even at the stage of the blastula, there is a distinct difference between dorsal and ventral axis, which we knew from our nieuwkoop centers experiments. This shows mesoderm induction is specified, at least early on, by the dorsal ventral axis, prior to migration of mesoderm cells around the embryo.

Question 38

Does the specifier for dorsal induce dorsal tissue or inhibit formation of ventral tissue?

Answer 38

It inhibits formation of ventral tissue, which implies that dorsal is default, because an inhibitor is necessary to make default occur…

Question 39

Why do we see organizer formation on the dorsal side of the embryo.

Answer 39

It is theorized that the formation of this tissue on the dorsal side results from a diffusible ligand signal from the vegetal region of the embryo (future endoderm). It is theorized that whatever is diffusing is diffusing in greater concentration from dorsal region. Or.. and this seems less idiotic and more achievable, dorsal factors work synergistically with whatever is diffusing from the vegetal hemisphere, so it only matters if both these factors overlap in sufficient concentrations.

Question 40

Where is the nieuwkoop center?

Answer 40

It is located in the future endoderm, and it is inducing cells to form an organizer from the future mesoderm.

Question 41

Nodal related proteins are what?

Answer 41

The are TGF-beta.

Question 42

Where is nodal expression concentrated?

Answer 42

Nodal expression is concentrated in the neiuwkoop center, but is found throughout all future endoderm at some low level.

Question 43

So if we have VegT and Vg1 alone, what do we see?

Answer 43

We see low level production of nodal, this is what we would see in ventral vegetal cells. (ventral mesoderm)

Question 44

What if we have high VegT and Vg1 (vegetal TGF-beta) and we have a high level of beta catenin, focused on the dorsal side of the embryo?

Answer 44

We have a high level of nodal related protein produced. This region, where beta-catenin and VegT and Vg1 overlap is the nieuwkoop center, this will induce the spemann organizer by high production of nodal (a TGF-beta). The spemann organizer will now, in theory, signal not to become ventral to the mesoderm near it, causing harmonic convergence.

Question 45

How does TGF beta signaling work?

Answer 45

Essentially TGF beta ligand binds. It will lead to a phosphorylation of some form of smad. Smad’s will work together and complex in a phosphorylation cascade which leads to activation of a target gene. Controlling smad’s is therefor an excellent means of regulating TGF - beta activity. I like smad 4 since he seems to be last and actually get transcription done.

Question 46

What is a BMP?

Answer 46

Bone morphogenic protein, it is a type of TGF-beta, and therefor uses smad’s and therefore smad - 4 is still my favorite out of a sample size of two.

Question 47

What is activin, answer honestly:

Answer 47

Activin is a TGF-b (long live smad’s) which is mesoderm inducing.

Question 48

A bead of activin where placed in side of animal cap cells. A low concentration yields expression of some, high affinity binding site genes, near the beads. A high concentration of activin however, yields activity of a high expression low affinity binding site genes, in a cluster around the beads, and the low concentration high affinity expression farther out. What does this imply?

Answer 48

We see that a single TGF-b like Activin is capable of causing gradient based signalling and could therefore cause multiple levels of expression in mesoderm cells. 3 specifically:
cells not touched by the signalling of activin
cells with only low-response genes active
and cells with high response genes active.

Question 49

What is a dominant negative receptor, how are they used?

Answer 49

A TGF-b receptor is made from a complex of type 1 and type two receptors. Let us say however that I take type one receptors and make them lack a cytoplasmic domain. This means that even though type two receptors are working normally, we will see no signalling activity. This is why this is dominant. Only one receptor type (1 or 2) must lack a cytoplasmic domain to stop a sweet, sweet, smad phosphorylation cascade. Dominant negatice receptors therefor allow for exploration of what would happen without a specific signal type. Say, what would happen without activin for example. Oh… and BMP (bone morphogenic proteins) are involved in ventral mesoderm… Activin and nodal, are of course dorsal mesoderm. Nodal being the TGF-b that first establishes the Spemann center. Nodal is produced by the nieuwkoop center which in term is established by beta catenin and Vg1 and VegT

Question 50

How does the organizer result in dorsalization of regions?

Answer 50

It antagonizes/inhibits BMP-4 and wnt signaling, which is causing ventralization of the embryo. If you knock out either BMP-4 or the appropriate wnt, what do you see? well you will see dorsalization, which is the default state of the embryo.

Question 51

How is inhibition of Wnts and BMPs achieved?

Answer 51

B a large variety of signalling molecules, sometimes multiple inhibit a single pathway., other times, cerberus for example inhibits three ventralizing factors, BMP, Wnt, and the other one…

Question 52

Where are all ventralizing factors antagonists produced?

Answer 52

They are all produced within the Spemann organizer. Because it does not mess around. Cerberus is cool.

Question 53

Sog works by binding to the ligand DPP and stopping it from binding. Then a protease comes along and cleaves Sog bound to dpp. How does this relate to the activity of chordin and BMP-4?

Answer 53

Chordin is an ortholog to sog. It is produced in the spemann organizer, it binds to BMP-4, which is a ortholog to DPP, and inhibits DMP. Another analog to tolloid is involved in cutting Chordin. Chordin is an inhibitor of BMP-4, and does so by one to one stoichiometric binding of BMP-4. Go Chordin!

Question 54

When does the AP axis arise? when does the orthogonal

Answer 54

AP axis arises during gastrulation, this is also the moment where dorsal and ventral become orthogonal to the AP axis.

Question 55

What occurs first Dorsal ventral patterning or AP patterning?

Answer 55

You even asking this shiz? Nuclear b-catenin levels are dictating the Dorsal ventral axis, and that happens in the freakin blastula! You’ve studied it. AP is only appearing during gastrulation, and it is set by Wnt signalling gradients, with Wnt being highest in the posterior.

Question 56

Wnt signalling is highest in the posterior, why does this make sense?

Answer 56

Because Wnt was located on the posterior region to begin with… The archenteron started at the but and went to find the mouth. So Wnt must necessarily be located at the point of the blastopore.

Question 57

Wnt’s and BMP help make dorsal ventral patterns. Wnt alone settles the AP axis (this second part may not be true), alright, so how is Wnt setting the Dorsal ventral axis?

Answer 57

Well Beta catenin specifies the axis? but we know beta catenins is a result of the cesation of the proteolysis of beta cetanin caused by the canonical Wnt signal pathway’s activation of its Ligand. So… boom roasted! Beta-catenin is set up by Wnt. Begins signalling, set DV axis. Then Wnt is free to set the AP axis. BMP’s are also involved in setting Dorsal ventral axis.

Question 58

Neural ectoderm appears after gastrulation above what tissue?

Answer 58

Above the outer segment of the spemann organizer.

Question 59

What is inducing the ectoderm to become neural?

Answer 59

It is using the outer region of the Spemann organizer. This organizer is causing the ectoderm above it to a different quantity of determing signals then the ectoderm beneath it, and to have different signals to some degree.

Question 60

In the absence of the organizer BMP is expressed how?

Answer 60

BMP is expressed throughout the entire embryo without an organizer.

Question 61

The organizer does what for the embryo in terms of formation of neural tissue?

Answer 61

It acts as an inhibitor of BMP signalling, which we knew. (Chordin for example is a antagonist of BMP-4 produced by the organizer) repeat, chordin is an antagonist of BMP-4 in the organizer)

Question 62

What is necessary for the creation of the neural tissue?

Answer 62

Inhibition of Wnt and BMP in the prospective ectoderm. So let’s take a look at this. Inhibition of Wnt and BMP in the mesoderm stops Wnt and BMP from specification of ventral and causes formation of dorsal tissues accordingly via antagonists which participate in one on one binding with Wnt and BMP-4 (this includes chordin, an antagonist of BMP-4). Now we look higher, the ectoderm and the formation of the brain. This we see utilizes the same mechanism. Inhibition of Wnt and BMP-4, which now causes specification of neural tissue. Therefore the organizer, followed by gastrulation allows us to specify subtypes of all three primary cell types in the embryo, gastrulation then aligns it. One organizer from one center. Damn.

Question 63

Inhibition of Wnt and BMP is necessary to make neural ectoderm. How would you test this?

Answer 63

Well if I blocked BMP everywhere I would expect neural tissue to overproduced, which is seen via beta cetanin staining. I would expect over expression of BMP in the cell to overwhelm inhibition of BMP expression, and cause a decrease in the amount of neural tissue. Loss of the Spemann organizer should result in loss of neural tissue. Addition of spemann organizer should result in addition of neural tissue. Misplacement of spemann organizer inside ectodermal tissue may induce formation of neural tissue, if and only if sustained nodal/ other TGF-b signaling from the nieuwkoop center is not required to maintain the Spemann center. I do not know if that is necessary. Use of beta galactosidase staining linked to genes known to be expressed in neural tissue would of course be a means of testing this.

Question 64

The open window, and the 2 hours of exposure (7 hour window) is necessary for the induction of ectoderm to mesoderm. I do not know if this independent of the formation of the spemann center. I believe however that we see formation of the nieuwkoop center prior to formation of the blastula. Indeed we seem to

Answer 64

Mesoderm seems to be specified before formation of blastula/blastocyst. Therefore it is likely that the specification of the mesoderm via induction is a necessary requisiute to the production of the spemann center. I don’t know though.

Question 65

What does FGF do? Be honest. We don’t have time for messing.

Answer 65

FGF is… A promoter of the formation of neural tissue. Likely induces neural tissue formation unless blocked by the BMP pathway. We therefor see neural tissue where
- BMP concentration are low due to inhibition by the spemann center
- Where FGF levels are high.
It follows that FGF levels must be higher in the vegetal hemisphere or in the dorsal region.

Question 66

What does FGF stand for?

Answer 66

Fibroblast growth factor. This makes sense because how would something other than fibroblast growth factor promote the induction of neural ectoderm? This is especially obvious when we consider the Bone morphogen proteins are inhibiting formation of neural tissue. That is why bone heads are considered stupid. BMPs are winning. Note: none of this is scientifically proven, while fibroblast growth factor is necessary for neural induction of ectoderm. BMP does inhibit.

Question 67

How could we test that FGF is activating BMP.

Answer 67

One: we could look at a quantification of their protein gradients through some form of antibody tagging flourescence. We would expect to see High levels of FGF where there is neural tissue. We wouold expect that given the same level of FGF, but increasing levels of BMP, we would eventually see the loss of neural tissue induction. Furthermore we would expect that given the same level of BMP, and increasing levels of FGF, we would eventually see neural induction of ectoderm as FGF concentrations approach infinity.

Question 68

Graph comparison.

Answer 68

I see FGF concentrated in the vegetal hemisphere. I see BMP concentrated in the ventral hemisphere. So. I expect the ventral hemisphere to have a slanted line, where neural tissue is induced more in the lower regions because of higher and higher FGF concentrations. But is far harder to induce the further we go towards the ventral hemisphere, where BMP is less inhibited. That is indeed what we see.

Question 69

How does a FGF signal work in three words?

Answer 69

Receptor tyrosine kinase.
FGF binds to RTK. Afterwards we see a phosphorylation cascade, ending with phosphorylation of a transcription factor and activation of a gene. In our case. Involves Ras. Sweet neural inducing genes.

Question 70

What is FGF?

Answer 70

FGF is a ligand, furthermore it is a ligand in the RTK family.