After MT2 - Mesoderm Flashcards
1
Q
Previously we talked about morphogens involved in the differentiation of ectoderm (BMP and to an extent Wnt, when thinking of placodes). What morphogen across what axis is responsible for the different lineages of mesoderm?
A
BMP, differing across the D-V axis.
To some extend Wnt, when considering cardiogenic mesoderm.
2
Q
Name the major lineages of mesoderm, from structures found medially to structures found laterally.
A
The chordamesoderm
The paraxial mesoderm (somitic mesoderm)
The intermediate mesoderm
The lateral plate mesoderm
3
Q
What structure does the chordamesoderm form?
A
the notochord
4
Q
How can you stain for chordamesoderm?
A
Stain for chordin mRNA
5
Q
What structure does the paraxial mesoderm form?
A
The somites and the head
6
Q
How can you stain for paraxial/somitic mesoderm?
A
Stain for the gene Paraxis
7
Q
What structure does the intermediate mesoderm form?
A
The urogenital system
8
Q
What structure does the splanchnic mesoderm of the lateral plate mesoderm form?
A
The mesoderm of the circulatory system - th eheart, blood cells, vessels and linings of body cavities
9
Q
What structure does the somatic mesoderm of the lateral plate mesoderm form?
A
The skeleton of the pelvis and limbs.
10
Q
How can you stain for lateral plate mesoderm (splanchnic AND somatic)?
A
stain for the gene Pax2.
11
Q
The BMP gradient which we know increases medially to laterally also affects what mesoderm lineage you get.
High BMP → ______________.
Low BMP → ______________.
No BMP → _______________.
A
- Lateral plate mesoderm
- Paraxial mesoderm
- Chordamesoderm
12
Q
If you were to do an experiment and inject a BMP inhibitor (say, noggin) in the LPM (lateral plate mesoderm), what would happen?
A
The amount of BMP in that area where you injected noggin is no longer enough to specify lateral plate mesoderm. It will be at lower BMP levels, which may match what paraxial mesoderm is often exposed to, so you will get the induction of paraxial mesoderm/somites in that region where you’d usually find lateral plate mesoderm instead.
13
Q
Most of what we’ll discuss about mesoderm will be relevant to the _____ embryo.
A
chick
14
Q
Chordamesoderm forms the notochord; what is the function of the notochord?
A
To provide rigidity and structure for vertebrate embryos and to act as a messaging centre for tissue around it.
15
Q
The paraxial/somitic mesoderm at the anterior-most region of the embryo is not segmented but rather forms the _______mesoderm; which forms the skull, connective tissue and muscles of the face.
A
head mesoderm
16
Q
Does mesoderm differentiation into these different lineages happen after neurulation?
A
No, it can happen during neurulation.
17
Q
Intermediate mesoderm forms the ________ system - which accounts for the kidneys, gonads and associated ducts
A
urogenital system
18
Q
The dorsal end of the LPM (lateral plate mesoderm) is the ________ mesoderm, and the ventral end of the LPM is the ________mesoderm
A
Dorsal - somatic mesoderm
Ventral - splanchnic mesoderm
19
Q
What mesoderm lineage gives rise to the heart specifically?
A
The cardiogenic mesodermal cells that arise from splanchnic mesoderm of the lateral plate mesoderm
20
Q
The space between the somatic and splanchnic mesoderm form the body cavity and is called the ________.
A
Coelom
21
Q
Cardiogenic mesodermal cells are also called/can be interchangeably called as the heart ________ or heart ______
A
Cardiogenic mesodermal cells = heart anlagen = heart primordia.
22
Q
After cardiogenic mesodermal cells are induced, how does the heart form? (The answer shouldn’t involve any morphogens because I’m saying AFTER the cardiogenic mesoderm is induced…) in CHICK
A
After the cardiogenic mesodermal cells are induced, two heart primordia are formed on either side of the midline of the body.
Post gastrulation, the endoderm of the chick begins to pinch upward to form the gut, and this process fuses the two heart primordia together to form one primitive heart.
23
Q
Cardiogenic mesodermal cells are only induced in splanchnic mesoderm above/dorsal to what structure?
A
Only in splanchnic mesoderm above pharyngeal endoderm
24
Q
Where is the pharyngeal endoderm located?
A
Beneath splanchnic mesoderm, found only in the ANTERIOR portion of the embryo.
25
The pharyngeal endoderm releases what two morphogens?
1. BMP
| 2. Crescent
26
Crescent is a ____ inhibitor
Wnt
27
True or false. All two morphogens released by the pharyngeal endoderm; BMP, crescent and the gene induced by BMP, FGF8, are essential for inducing cardiogenic mesoderm properly and getting the correct heart field
True
28
What are the two function Wnt has related to the induction of cardiogenic mesoderm?
1. Wnt promotes the formation of blood
| 2. Wnt INHIBITS the induction of cardiogenic mesoderm
29
Describe the induction of cardiogenic mesoderm.
In the anterior LPM...
- BMP is released by pharyngeal endoderm. Wnt is released by neural tube.
- Wnt activity is blocked because pharyngeal endoderm releases crescent, a Wnt inhibitor. This stops Wnt from inhibiting cardiogenic mesoderm induction.
- BMP induces FGF8 expression, which increases cardiac protein expression
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
At medial regions (i.e. near notochord)...
- notochord releases chordin, noggin to block BMP. This prevents FGF8 activation.
- Preventing activation of FGF8 in the medial region is why heart formation doesn't occur at the medial regions of the embryo.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
In the posterior LPM...
- No Wnt inhibitors (because no pharyngeal endoderm), therefore Wnt (a) inhibits cardiogenic mesoderm induction in the posterior LPM and (b) promotes blood formation.
30
What genes would you stain for to stain for cardiogenic mesodermal cells?
Mesp1 and Nkx2-5. When staining I recommend using Nkx2-5 since Mesp1 is produced by other tissues in the embryo as well.
31
If Drosophila have a mutation in Nkx2-5 (the homologue gene in Drosophila is called tinman), it results in what?
No heart formation
32
What is the anterior heart field?
The anterior heart field is the region of the embryo, often crescent-shaped, where cardiogenic mesoderm is induced.
33
What are two experiments that tested the importance of BMP in inducing cardiogenic mesoderm? these experiments did the same thing, just in different places in the embryo.
Experiment 1:
- Place a bead of BMP on one side of the embryo where the anterior heart field usually is.
- Stain for Nkx2-5, a gene produced by cardiogenic mesodermal cells.
- Notice that you'll get ectopic staining/Nkx2-5 expression around the bead, compared to the other side.
Experiment 2:
- Place a bead of BMP on one side of the embryo where the anterior heart field is usually not found (trunk region)
- Stain for Nkx2-5 to locate cardiogenic mesodermal cells
- Notice that you'll get no staining at all either on the side with the bead, or the side without the bead.
34
Why doesn't adding BMP induce cardiogenic mesoderm in the trunk region, where the heart field usually isn't?
Because the pharyngeal endoderm is not there, and this is what is releasing the crescent. In the trunk region (more posterior) you will have Wnt. Wnt is an inhibitor of cardiogenic mesoderm induction.
Similarly, this region has retinoic acid, which inhibits FGF8.
35
What determines how far back the anterior heart field extends?
Retinoic acid. Because it inhibits expression of FGF8, needed to induce cardiogenic mesodermal cells.
36
Retinoic acid is found in what region of the chick embryo? It is produced by what structures?
the trunk (not the caudal region!). Produced by the trunk somites!
37
What enzyme produces RA (retinoic acid)? What gene codes for this enzyme?
Retinaldehyde dehydrogenase-2 (RALDH2), which is an enzyme that is formed from the gene RADH2
38
What happens to the anterior heart field if you have a mutation in RADH2?
- RA levels drop in the trunk region
- Since RA can't inhibit FGF8, the anterior heart field will extend more posteriorly (towards the trunk) than normal.
- leads to an abnormally large heart
39
Does RA exist in the more caudal regions?
No.
40
The somites form in what direction?
Anterior to posterior
41
True or false. In an embryo, the oldest/most mature somites are located at the more anterior end, and the newest ones are at the more posterior end.
True
42
True or false. New somites are not yet differentiated into their two different regions; the dermomyotome and the sclerotome.
true
43
Describe the nature of the cells that a new undifferentiated somite has.
New somites are undifferentiated (not yet differentiated into the dermomyotome and the sclerotome) and have an inner core of mesenchymal cells which are sourrounded by mesodermal cells that are epithelial in nature.
44
In a differentiated mature somite, the most dorsal end is called the ___________. The ventral end of the somite is called the __________.
dorsal region - dermomyotome
ventral region - sclerotome
45
The dermomyotome used to be called as two separate regions, with the ventral portion of the dermomytome being called the _______, and the dorsal-most surface being called the _________.
myotome
dermatome
46
Why do we just say dermomyotome now instead of dermatome and myotome separately?
Before it was believed that dermatome only gave rise to the dermis, and myotome only gave rise to muscles, but it turns out the dermatome can give rise to muscles and the myotome can give rise to dermis.
47
What does the sclerotome give rise to?
it gives rise to skeletal structures in the mid region - the the vertebrae, the tendons, and the rib cartilage.
48
We’ll use the ____ embryo to look at somite development/somitogenesis.
chick
49
True or false. Different groups of cells in the sclerotome of a somite give rise to different kinds of tissue, such as the spines of the vertebrae, the portions of the ribs, the intervertebral discs and the tendons
true
50
Somitogenesis in the chick happens during neurulation, which means somites are found ______(anterior/posterior) to the regressing Hensen's node.
Anterior to.
The Hensen's node should have already passed the region undergoing somitogenesis.
51
New somites pinch off off the PSM, which stands for the ___________-
pre-somitic mesoderm.
52
Unlike immature somites, which are mesenchymal in nature at the core and sourrounded by cells that are epithelial in nature around the outside, the cells of the pre-somitic mesoderm (PSM) are...
completely mesenchymal in nature.
53
Is the PSM also anterior to the regressing Hensen's node?
Yes
54
The first set of somites in any embryo form just posterior to the ______.
hindbrain
55
What are somitomeres?
Somite precursors, these are parts of the pre-somitic mesoderm (PSM) that have not yet separated into distinct blocks of tissue/somites
56
what is the anterior-most somitomere/anterior-most portion of the PSM called?
The S0 somitomere.
57
What is the newest somite called?
SI somite
58
What is the somitomere posterior to the S0 somitomere called?
The S-I somitomere
59
In order for the S0 somitomere to bud off the PSM, it must form an epithelial fissure between the somitomere and the PSM. What is an epithelial fissure?
It is a boundary that is formed as the outer cells of the S0 somitomere undergo MET (mesenchymal to epithelial transition) and separate from the completely mesenchymal PSM.
60
Describe how ephrin and eph receptors are responsible for triggering the MET that results in the separation of new somites from the PSM.
At the anterior half of the S-I somitomere:
- increased Mesp expression --> causes upregulation of eph receptors here
- At the same time, this is where pacemaker gene Hairy1 expression eventually ends up, overlapping with where we have Mesp expression.
At the posterior half of the S0 somitomere:
- the upregulation of the eph receptors in the anterior half of the S-I somitomere causes an upregulation of ephrin in the posterior half of the S0 somitomere.
- ephrin and eph interactions are repulsive, so the cells of the posterior half of the S0 somitomere will want to separate from the cells at the anterior half of the S-I somitomere.
- At the same time, an increase in ephrin results in the downregulation of Cdc42 in the cells of the S0 somitomere. This gene is involved in actin assembly, so downregulation of it will reduce motility of the cells of the S0 somitomere at the posterior end (MET).
After the new somite has formed:
- Once the S0 somitomere becomes the SI somite, the epithelial boundary is maintained by the expression of integrin on the cells of the outer layer of the SI somite, interacting with fibronectin in the sourrounding ECM.
61
True or false. The rate at which new somites bud off the PSM is species-specific.
True.
62
True or false. The PSM elongates at the same time that new somites bud off the PSM
True, these processes occur simultaneously
63
In the chick, the PSM continues to elongate because of a group of cells called _______, which are found in a region anterior to the finally fully regressed Hensen's node, and this region is called the ______________
The PSM elongates because of NMPs, and NMPs are found in the region of the embryo referred to as the Caudal Lateral Epiblast
64
NMPs, depending on what morphogens they are exposed to, can give rise to either ______ tissue or ______ tissue
Neural tissue or mesodermal tissue
65
Recall that we had mentioned that RA signalling is only found in the trunk region. In the anterior end of the embryo, RA is inhibited by FGF8. What is inhibiting RA signalling in the posterior end of the embryo?
FGF8 again!
66
Describe the FGF8 gradient as you go towards the posterior end of the embryo/ towards the caudal lateral epiblast.
The FGF8 concentration increases as you get closer to the posterior end of the embryo/closer to the caudal lateral epiblast.
67
In order for the PSM in the chick to elongate, NMPs in the caudal lateral epiblast must give rise to pre-somitic mesodermal cells rather than neural cells. Explain the process of how this happens.
The caudal lateral epiblast is in a region where FGF8 concentration is high.
FGF8 has many roles, we already discussed how it inhibits RA, but one role it has here is that it upregulates the gene Msgn1.
Msgn1 induces the expression of the gene Tbx6.
Tbx6 has two roles:
1. it induces NMPs to give rise to mesodermal PSM cells
2. it inhibits the expression of sox2, to prevent neural cell expression.
68
If you were to stain for Mesp, where would it be localized in the chick embryo?
Why is Mesp localized there?
What is this region called where Mesp is localized?
In the region where the anterior-most somitomere, S0, will bud off the PSM.
Mesp is localized there because this region is the transition point between where we have RA signalling to where we have FGF8 signalling.
As you move anteriorly from the region Mesp is localized, you get higher RA signalling. As you move posteriorly from the region Mesp is localized, you get higher FGF8 signalling.
The region where Mesp is localized, accounting for the region where new somites bud off the PSM, is called the "Determination Front."
69
True or false. The determination front is the transition point where we’re going from high FGF signalling to high RA signalling
True
70
True or false. In the chick embryo, the expression of the clockwork gene Hairy1 appears to move anteriorly in a wave-like fashion
True. Cells of the PSM are periodically turning on, and then off, the expression of Hairy1.
71
Why does Hairy1 expression turn on and then off?
This gene has a negative feedback loop that inhibits its regulator, whose gene is located upstream the DNA. The regulator turns on the gene, so inhibition causes Hairy1 expression to turn off.
72
True or false. The rate of somitogenesis, or number of somites, depends on the animal
True
73
What two factors dictate the rate of somitogenesis?
1. The rate of oscillation of pacemaker genes
| 2. The rate of elongation of the PSM.
74
Two embryos have the same rate of pacemaker gene oscillation, but embryo A has a greater rate of PSM elongation than embryo B. Which embryo will have a greater number of somites, that will also be smaller in size?
Embryo B which has a slower rate of PSM elongation will have more somites, and somites will also be smaller
75
Two embryos have the same rate of PSM elongation, but embryo A has a greater rate of pacemaker gene oscillation than embryo B. Which embryo will have a greater number of somites, that will also be smaller in size?
embryo A, which has a greater rate of pacemaker oscillation, will have a greater number of somites, and they will be smaller than the somites of embryo B.
76
True or false. Limb buds only form in the trunk region
True
77
Describe the axes involved when discussing a limb.
Anterior - where your thumb is, pointing to nose
Posterior - where your pinkie is, pointing to butt
Dorsal - back of hand
Ventral - palm of hand
Proximal - regions of the limb close to the site of attachment
Distal - regions of the limb far away from site of attachment
78
The components of a limb are broken down into three regions, name them from proximal to distal.
Stylopod
Zeugopod
Autopod
79
True or false. Though limbs differ in embryos, the three components - stylopod, zeugopod and autopod, occur across species.
True
80
What type of germ tissue gives rise to the limb?
In the region where limbs develop, the somatic mesoderm (the dorsal part of the lateral plate mesoderm) will have cells that undergo EMT.
These mesenchymal somatic mesodermal cells will cluster and proliferate with the ectoderm overlying it.
81
How does the embryo know where limb formation will occur and whether the limbs will be forelimbs or hindlimbs?
This information comes from Hox genes expressed by somites. The somites near the site where limbs should form express Hox genes that contain that information
82
True or false. Hox genes are expressed along the entire length of the embryo, but different groups of hox genes specify specific areas of the embryo’s body plan, like “this is the head” and “this is the forelimb.”
True
83
What is an experiment that proved that hox genes were responsible for planning out where limb formation would occur?
In one experiment, they took somites occurring at the limb region from one side of an embryo and transplanted it to the other side, but in a way that replaced some of the non-limb somites beneath that area where limb formation usually grows. What we saw was a wider limb, a larger limb bud than normal.
In that same experiment, they took somites occurring at non-limb regions of the trunk, and transplanted that to replace some somites near where limb formation usually occur (on the other side of the embryo). The result was a much narrower and smaller limb bud than normal. This is because the information specifying limb bud formation has shortened.
84
Describe the morphogenetic cues that specify FORElimb formation.
RA from the trunk somites work together with Hox genes to upregulate Tbx5 in the mesenchymal lateral plate mesodermal cells.
Tbx5 induces the expression of FGF10.
FGF10 further maintains the expression of Tbx5, which maintains expression of FGF10, through a positive feedback loop.
FGF10 upregulates FGF8 at the most distal region of the ectoderm of the limb, and FGF8 induces the formation of the AER.
FGF8 also maintains FGF10 levels, via Wnt3, which in turn maintain FGF8 levels, through a positive feedback loop.
85
How does the morphogenetic cues that specify HIND limb formation differ from that specifying FORElimb formation? (two differences)
- RA has no involvement in hindlimb formation. Whereas for forelimbs, RA works with hox genes to activate Tbx5
- Instead of Tbx5, what gets activated is actually Tbx4.
86
True or false. The signal that triggers hindlimb formation with the Hox gene is not known
True
87
If we put a bead, or a cluster of cells, secreting FGF10 and place that in a region where you usually do not see the formation of limb bud, like for example in the trunk region between the forelimb and hindlimb, what kind of limb do we see?
This will result in a limb that has both forelimb and hindlimb features.
On the anterior side, because it is close to Hox genes specifying forelimb development, we will get a forelimb anterior side because this side expresses Tbx5.
On the posterior side, because it is close to Hox genes specifying hindlimb development, we will get a hindlimb part on the posterior side because this side expresses Tbx4.
88
The expression of FGF8 is expressed at the ectoderm at the most distal end of the limb, forming the ______________________ (AER)
apical ectodermal ridge (AER)
89
What process maintains the expression of FGF8 at the AER
The positive feedback loop of FGF8 with FGF10 and Wnt3
90
Why is FGF8 important to be maintained?
This gene drives mitosis and therefore the outgrowth of the limb. If we stop FGF8, we will not have the AER anymore, and limb growth/development will stop.
91
You can stain for the AER by staining for____.
FGF8
92
What are the four functions of the AER?
1. Helps to identify the anterior-posterior, and dorsal-ventral, axes of the limb
2. Helps maintain the mesenchymal, plastic, nature of the cells of the progress zone, and makes them continue to proliferate
3. Controls the expression of the genes that set the proximal-distal axis of the limb, generating the stylopod, zeugopod and autopod.
4. FGF8 triggers mitosis for limb growth
93
What would happen to cells of the progress zone if the AER were to be removed or stopped in some way?
They would lose their mesenchymal nature and begin to differentiate.
94
What is the "progress zone"?
The UNDIFFERENTIATED mesenchymal cells of the LPM that are beneath/behind the AER.
95
True or false. As the limb bud grows, the AER drags the progress zone with it, such that the progress zone is always at the more distal end
True
96
True or false. The cells at the most proximal regions of the progress zone leave first, and the ones at the more distal regions of the progress zone leave last.
True.
97
Cells that leave the progress zone FIRST will differentiate to form the ____________,
Stylopod
98
Cells that leave the progress zone FIRST will differentiate to form the ____________, this is because of the expression of the ______ gene, which happens where RA levels are _____ and FGF8/Wnts levels are _____
Stylopod.
Meis gene.
RA levels are high, FGF8/Wnts levels are low.
99
Cells that leave the progress zone SECOND will differentiate to form the ____________, this is because of the expression of the ______ gene, which happens where RA levels are _____ and FGF8/Wnts levels are _____
Zeugopod.
Hox11 gene.
RA levels are intermediate, FGF8/Wnts levels are intermediate.
100
Cells that leave the progress zone LAST will differentiate to form the ____________, this is because of the expression of the ______ gene,which happens where RA levels are _____ and FGF8/Wnts levels are _____
Autopod.
Hox13 gene.
RA levels are low, FGF8/Wnts levels are high.
101
If an extra AER is grafted on a developing limb that already has an AER, what do we expect to see?
We will see a duplication of the limb, mainly the distal features of the limb because the progress zone likely is carrying cells that will eventually differentiate into the distal features after leaving.
102
If we take mesenchyme from the hindlimb progress zone and transplant that under the forelimb AER, what do we expect to see?
Progress zone cells of the hindlimb express Tbx4, so when transplanted to the forelimb, they will continue to express Tbx4 (which signifies hindlimb features) because of the positive feedback loop between FGF10 and Tbx4. So the forelimb will be expressing hindlimb features, and these will be distal hindlimb features because the cells remaining in the progress zone will be the ones that will later differentiate into distal features after leaving, the other proximal cells have already left.
103
If we replace progress zone mesenchyme with nonlimb mesenchyme, and transplant that nonlimb mesenchyme beneath the AER of a forelimb or hindlimb, what do we expect?
In order for the AER to be maintained, the positive feedback loop between the FGF8, expressed at the AER, and the FGF10, expressed by progress zone mesenchymal cells, needs to be maintained. Nonlimb mesenchyme is not expressing FGF10, so the positive feedback loop doesn't exist to maintain FGF8 in the AER. FGF8 is what drives mitosis of the limb, so in this case, limb growth will stop.
104
If we take an FGF8 secreting bead and replace the forelimb AER with this bead, will this prevent limb growth?
No, because the progress zone is still intact and can maintain that FGF8 level through the positive feedback loop with FGF10. The bead has assumed the role of the AER, because all the AER needs to do is secrete FGF8
105
If we were to take mesenchyme from a younger embryo’s progress zone, then transplant that onto an older embryo’s limb bud, beneath it's AER, what would we expect?
The limb will start to grow proximal features again! This is because cells that differentiate to later give rise to proximal features, like the stylopod and zeugopod, had not yet migrated out of the younger embryo's progress zone. They have ended up migrating after transplantation
106
If you were to take an older embryo's progress zone and transplant onto young limb bud, beneath the AER, what would you expect?
The limb will start to grow mainly distal features such as the autopod. This is because an older embryo's progress zone only contains the cells that will eventually leave and differentiate to make the distal portions of the limbs. The group of cells that, after leaving, differentiate into the more proximal regions have already left the progress zone of the older embryo before transplantation.
107
How is the AER formed?
The AER is formed after mesenchymal cells of the LPM express FGF10, which induces the expression of FGF8 in the OVERLYING ECTODERM, and this overlying ectoderm becomes the AER.
I added this because it is important in an exam to mention the OVERLYING ectoderm part.
108
Why, in a limb, is RA restricted to the more proximal regions, and FGF8 and Wnts are restricted to the more distal regions? In other words, how does RA inhibit FGF8, and how does FGF8 inhibit RA, creating these gradients?
1. RA inhibits FGF8 by directly inhibiting it's transcription
2. FGF8 inhibits RA via a CYP26-mediated RA degradation
