Chapter 11 Organogenesis

Question 1

The limb bud arises from

Answer 1

The limb bud, which is rapidly dividing cells derived from the lateral plate mesoderm

Question 2

Do all cells within the limb bud give rise to nerve cells?

Answer 2

Skeletal and connective tissue is mesoderm from the limb bud, muscle comes from the somite via migration, and motor nerves will also appear via migration

Question 3

There are signaling centers essential for limb bud development, but how do these centers work? Science.

Answer 3

But more then just science. The initial induction of limb position is regulated by hox genes and is conserved. For example, we see that Hox c6 induces forelimb development in both the chick, frog and mouse.

Question 4

What proof is there that hox gene expression domains in any way effect the location of limb development?

Answer 4

Well… let’s say you treat a regenerating tadpole tail with retinoic acid. We will see… That’s right, limb formation. Retinoic acid is associated with the more anterior positions and so we will see anterior limbs develop, indeed we will see anterior limb development in the tail of a frog treated with retinoic acid. We actually see multiple ectopic leg buds form… (but we are also seeing hox gene induction)

Question 5

What specifically begins activation of limb buds?

Answer 5

activation of appropriate Hox genes by (anterior) retinoic acid FGF (posterior) gradient will lead to activation of the transcription factor Tbx4, 5. Because the world is an interesting place, Tbx4 is associated with development, and tbx5 is associated with forelimb development

Question 6

What is it the tbx4 and tbx5 do once these transcription factors are being produced?

Answer 6

They produce FGF ligand formation, and essentially the mesoderm starts talking to the ectoderm. The mesoderm makes FGF10 which activates FGF8 which activates FGF10. But the names could be anything. The important underlying principle, is that the activation of hox genes causes a production of tbx5 and tbx4 transcription factors, which in turn activate ligand signalling of FGF and Wnt. Do FGF and Wnt then proceed to mess around? No. No they do not. FGF and Wnt signaling causes activation of different genes in the ectoderm near this sight of activation, witch in turn communicates back with FGF and Wnt signalling, in a mutually activating system.

Question 7

How important is this production of FGF and its signalling?

Answer 7

SOOOO IMPORTANT!!! FGF signaling, implanted ectopically BY ITSELF is enough to induce limb formation. Whether this limb induced by FGF decides to be a forelimb, or hindlimb when it grows up will be based off its position on the A-P axis. This FGF (and also Wnt) reciprocating system between the ectoderm and mesoderm of the lateral plate is effectively forming a sight of cell proliferation, and maintain it.

Question 8

Apical Ectodermal Ridge (AER)

Answer 8

The AER is the tissue being induced in ectoderm. This ectodermal tissue in turn is essential for proximo-distal development. In other words, this apical ectodermal ridge (which signals using FGF) is in charge of telling the limb (“I’m distal! High guys, this way is distal. Hmmm… let’s grow distally, AER representing!”)

Question 9

AER is essential for proximal distal specification and uses predominately FGF as its signalling mechanism. We know this, because scientists told us so = false! We know this because a bead of FGF…

Answer 9

… let me explain. If you remove the apical ectodermal ridge, and then implant a beed soaked with FGF4 ( a specific FGF), then we will see development of the limb continue almost normally, forming humerus, radius, ulna and even a couple digits normally

Question 10

There are two models for specifying proximo-distal position, what are they?

Answer 10

Model one is that cells have a means of estimating the amount of time spent in the progress zone. This progress zone model states that cells have a molecular mechanism for telling how long they have stayed in the progress zone, if cells have stayed a short time in the progress zone, they will become proximal structures, if they stay for a long time they will become distal structures.

Question 11

What we do know regardless of the signalling system :

Answer 11

Cells as they leave the progress zone will drop their rate of division and begin differentiating.

Question 12

What is the second model for proximal distal specification?

Answer 12

This second model suggests that there is a proximal and distal signaling center (the distal center likely being AER), as these signals grow weak enough, due to seperation as they grow, they will allow the formation of intermediate structures between them. This model with two signals is known as the… two signal model…

Question 13

Name once again the two models for limb bud formation:

Answer 13

The two signal model.

The progress zone model.

Question 14

Name the three signaling centers of the fairly distal limb bud.

Answer 14

AER.
The progress zone.
The polarizing zone.

Question 15

The three zones of the limb bud are interdependent, what does this mean.

Answer 15

Interdependent: a mutual dependence, two or more things which require each other to work. If you remove one interdependent object, the rest of them fail.
If you remove the AER (Apical Ectodermal Ridge), you see a cessation of growth.
If you remove the non-limb mesenchyme, in other words, the progress zone, you see a cessation of growth

Question 16

Does the AER and the polarizing zone communicate.

Answer 16

Ummm… since this is biology… Yes. They do. The AER produces FGF predominately. The polarizing zone produces SHH (Sonic Hedge Hog), and there is molecular interaction between theses zones.

Question 17

SHH production from the polarizing zone sets the A-P axis

Answer 17

We know this because addition of an agarose bead filled with SHH placed on the other side caused formation of the same structure as is normally seen in the posterior

Question 18

SHH causes activation of hox genes.

Answer 18

We know this because if you place a source of SHH, and measure the concentrations of SHH you will see the hox gene expressed which would normally be expressed in that region given normal expession. Loss of SHH likely results in loss of Hox activation.

Question 19

Let’s talk about ZRS, ZRS is the

Answer 19

SHH distal control region, there ZRS controls expression levels of SHH.

Question 20

How far away is ZRS from the SHH coding region?

Answer 20

1 Mb away. Which could mean it produces a diffusible protein or that there is a very particular pattern of folding.

Question 21

ZRS is the distal regulatory domain which regulates the expression of sonic hedgehog in distal tissues

Answer 21

But we can have a mutation of the ZRS regulator, this mutation causes an overexpression of SHH and the formation of 6th digit. This same locus and mutation also exists in cats and mice.

Question 22

What do we see when we see a loss of function of the ZRS?

Answer 22

If the ZRS is lost, we see a failure of limb formation. This shows that the three regulatory regions
progress zone
Apical Ectodermal Ridge
and the polarizing zone
are all indeed interdependent, and that the signalling between SHH and the AER is essential for growth to be maintained.

Question 23

Wnt-7a and engrailed specify the dorsal to ventral axis. Dorsal expression is dorsally located. This makes sense. Tell me why.

Answer 23

This makes sense as the dorsal expression of Wnt signals is also seen with the formation of the spinal cord. We see engrailed expression dictate the ventral portion of the limb. If we see loss of engrailed, dorsalization will occur, and Wnt signalling will spread into the region normally occupied by engrailed. If we loose Wnt, we will see a spread of engrailed and ventralization of the embryo.

Question 24

Dorsal and ventral regions are developing as separate compartments.

Answer 24

Due to engrailed and Wnt signalling.

Question 25

What evidence is there for the conservation of developmental mechanisms in terms of AER and Polarizing zone/limb development?

Answer 25

If we perform an ectopic transfer of the polarizing zone or the AER into a chick embryo wing bud from a mouse we will see… Formation of a chick digits in the chicken, which means the cells perform the same mechanism needed here.

Question 26

If you transfer presumptive thigh tissue into the wingbud distal region of a chick leg bud, what will you see?

Answer 26

You will see wings with claws, in other words, the tissue knew that it was now distal tissue, but it did not know that it was no longer leg tissue, it had already been specified as leg not wing, it had not yet been specified whether it was distal or proximal, which kind of pokes a hole in the proximal model and provides descent evidence for the two signals model.

Question 27

When he says that hox gene expression unfolds in the P-D axis and in the A-P what does he mean?

Answer 27

He points out that while hoxa genes specify the proximal to distal axis, hoxd genes specify the AP axis.

Question 28

Hox gene expression unfolds over time… meaning?

Answer 28

It means that we see progressive expression, we see hox-9, and d-10 for the humerus, and we see differential expression latter for the radius and ulna, and once again a divergence of expression for the hand.

Question 29

More then this change of hox expression as we move from proximal to distal, we also see that different hox genes are associated with different morphological regions of the arm.

Answer 29

Different hox genes are active in the humerus, then those which are active in the radius and ulna, or in the carpals, or the phalanges.

Question 30

So given that we see a specific hox gene expressed in a specific morphological region, what would happen if we knocked out the homologs of the hox gene being produced in that region?

Answer 30

His example is the knockout of hox11 and we see the complete loss of the radius and ulna, with the humerus attaching directly to the hand. (done in a mouse)

Question 31

Three fingers are fused together if there is a homozygous knockout of the hoxd-13 allele

Answer 31

this shows that hox genes are also specifying AP patterning of the digits.

Question 32

Cell death is as important as cell growth, and it is controlled by the mesoderm.

Answer 32

Specification is due to BMP signaling by the mesoderm, this makes sense as this is what we see in mesoderm induction by the lateral mesoderm. We see that placement of BMP antagonist causes the retention of webbing between digits in chickens.

Question 33

MC, ED = non webbed

MC, EC = non webbed

Answer 33

EC, MD = webbed

EC, MC = webbed