Lecture 15- Vertebrate limb formation

Question 1

Define pattern formation

Answer 1

Coordinated differentiation and organisation of cells within the body in order to form structures that are specific size, shape and position within and organism

Question 2

Define positional information

Answer 2

Cells in a specific space within the developing embryo have intrinsic information that indicates that they must carry out a specific programme which is different to cells in different positions

Question 3

What are the 3 axes of the vertebrate limb?

Answer 3

• The P/D axis: the shoulder/humerus is proximal, the digits is distal
• The A/P axis: the thumb is anterior, the pinky digit is posterior
• The D/V axis: the palm is dorsal, the back of the hand is ventral

Question 4

What are the 3 skeletal elements of the vertebrate limb and what bones to they compose of?

Answer 4

1. Stylopod- humerus
2. Zeugopod- ulna and radius
3. Autopod- carpals and digits

Question 5

Which axis cannot be defined through the organisation of bones but instead defined through an alternative method?

Answer 5

The D/V axis cannot be defines through the organisation of the bones however the specific features of the epidermis vary on the dorsal and ventral sides

Question 6

Describe the first appearance of the limb bud

Answer 6

Protrusions from the flank at precise positions along the AP axis of the embryo

Question 7

What dictates the position of the limb fields and give an example?

Answer 7

Hox code

Anterior boundary of Hoxc6 specifies the forelimb field

Question 8

What 2 transcription factors specify limb identity?

Answer 8

Tbx4 and Tbx5

Question 9

Where is Tbx4 and Tbx5 expressed?

Answer 9

Tbx4 expressed in the hindlimb

Tbx5 expressed in the forelimb

Question 10

What drives the identity of Tbx4 and the hindlimb?

Answer 10

Pitx1

Question 11

What represses Tbx4?

Answer 11

Tbx5

Question 12

Describe 1 gain of function study for Tbx4/5

Answer 12

If you prepared a virus that will overexpress Tbx4 and inject the virus so that Tbx4 is expressed throughout the lateral mesoderm, the embryo will form a leg in the forelimb area. The opposite is observed when Tbx5 is overexpressed

Question 13

Describe 1 loss of function study for Tbx4/5

Answer 13

By blocking expression of Tbx5 (e.g through mouse KO), forelimb formation is lost and the hindlimb formation is not effected. Indicates that Tbx4 is sufficient to drive hindlimb formation and Tbx5 is required for forelimb bud formation

Question 14

What is observed the Pitx1 is KO or overexpressed?

Answer 14

KO: loose Tbx4 expression and hindlimb identifty

Overexpressed: Tbx4 expression is driven and hindlimb forms despite the region

Question 15

Describe the model for imitation of limb bud formation from Hox proteins to FGFs

Answer 15

Hox protein - RA - Tbx - FGF10 (mesoderm) - FGF8 (ectoderm)

Question 16

What regulates the expression of Tbx4/5?

Answer 16

RA

Question 17

Outline an experiment in which a limb is ectopically formed

Answer 17

FGF soaked bead loaded into the lateral mesoderm of the developing embryos leads to the ectopic formation of a limb

Question 18

What in the link between FGF and Tax gene expression in the developing limb?

Answer 18

Tbx4/5 drive the expression of FGF10 in the lateral mesoderm which drives the expression of FGF8 in the ectoderm

Question 19

Which 2 ways were embryos analysed after the ectopic expression of FGF10 in the limb?

Answer 19

1. insitu hybridisation

2. Visualise the morphology of the developing limb

Question 20

How does the type of limb formed change depending on the location of the ectopic FGF10 soaked bead>

Answer 20

• If the bead was implanted closer to the hindlimb, then hindlimb formed
• If the bead was implanted closer to the forelimb, then forelimb formed
• If the bead was implanted precisely between the two, then the developing limb partially expressed both Tbx4 (posteriorly) and Tbx5 (anteriorly). The ectopic limb developed morphologically features that were chimeric and resembled both a hindlimb and forelimb

Question 21

What conclusions were made from ectopic implantation of FGF10 soaked bead in the developing limb

Answer 21

Concluded that FGF10 is important for driving the expression of Tbx4/5 in the limb

Question 22

Outline the molecular expression that enabled forelimb formation initiation

Answer 22

1. RA and FGF10s important for expression of Tbx5
2. Wnt2b acts to restrict the expression pattern solely to the limb field
3. FGF10 in the lateral mesoderm establishes features within the limb field which are important for the later growth
4. FGF10 induces in the overlying ectoderm the expression of Wnt3a which in turn induces the expression of FGF8
5. Wnt3a and FGF8 together within the ectoderm define the AER/progress zone

Question 23

Outline the molecular expression that enabled hindlimb formation initiation

Answer 23

1. Pitx1 and Tbx4 together are important for the formation and identify of the hindlimb limb field
2. FGF10 and Tbx4 also lead to Wnt8c which acts to restrict the expression pattern solely to the limb field
3. FGF10 induces in the overlying ectoderm the expression of Wnt3a which induces the expression of FGF8 to define the AER/progress zone

Question 24

What are the 3 domains which interact with each other to drive the growth and patterning of the limb?

Answer 24

1. Apical Ectodermal Ridge (AER): will become specified under the action of FGF10. Area forms through the thickening of ectoderm and expresses a high number of FGFs
2. Progress zone: made of mesenchymal cells that are actively dividing and promoting the growth of the limb and located behind the AER
3. Zone of Polarising Activity (ZPA): region expresses high levels of Shh

Question 25

Experimentally removing the AER confirms the AER is necessary for what?

Answer 25

The progress zone and limb growth

Question 26

What is the resulting phenotype when the AER is removed at 3, 3.5 and 4 days?

Answer 26

* If the AER was removed at 3 days, the resulting embryo developed stylopod, some zeugopod but no autopod * If the AER was removed at 3.5 days, the resulting embryo limb developed stylopod, zeugopod but again, no autopod * If the AER was removed at 4 days, the resulting limb developed stylopod, zeugopod and some autopod

Question 27

What are the 2 models for P/D patterning of the vertebrate limb?

Answer 27

Model 1: progress zone model of proximal-distal patterning Model 2: two-signal model of proximal-distal patterning

Question 28

Describe the progress zone model of P/D patterning

Answer 28

1. The AER was driving the outgrowth of the limb by acting on the progress zone by promoting the proliferation of the cell 2. Cells were adopting a specific cell fate according to the time spent dividing in the progress zone 3. The time spent in the progress zone directly correlates with how distal the elements forming were 4. The more proximal elements were specified early, and the cells that spent longer in the progress zone would develop into more distal elements

Question 29

Describe the two signal model of P/D patterning

Answer 29

1. Two antagonising gradients that are established early which act together to specify different regions within the developing limb and ultimately results in the formation of different regions that are patterned 2. The cells position along the opposing signals gradient will determine there proximal or distal fate 3. RA and FGF were the two signals antagonising each other

Question 30

Describe the RA and FGF gradients in the limb bud

Answer 30

* High levels of RA in the proximal part of the limb and decreases distally * High levels of FGF in the distal part of the limb and decreases proximally

Question 31

What genes are expressed where due to FGF/RA in the limb bud?

Answer 31

* Proximally Meis genes are activated by high RA and are required to form the stylopod * Distally, Hox13 genes are activated by high FGFs and required to form the autopod * In the middle (neither proximal or distal with mixed levels of RA and FGFs), Hox11 is activated and required to form the zeugopod

Question 32

What can be observed and concluded from implanting Shh beads/Shh expressing cells/ZPA grafts?

Answer 32

Mirror image duplication of the limb Concludes that ZPA which drives A/P patterning canoe entirely due to Shh activity

Question 33

What zones pattern the A/P and P/D axis of the limb?

Answer 33

``` A/P = ZPA D/P = AER and progress zone ```

Question 34

How does Shh concentrations control the cell types specified?

Answer 34

1. In the ZPA, there are high levels of Shh which decrease as you move further away 2. Therefore the most posterior digits are specified close to the ZPA where Shh is at high concentrations 3. Anterior digits are specified further away from the ZPA where Shh is at lower concentrations

Question 35

What is observed in a Shh mutant limb?

Answer 35

A complete loss of the distal most skeletal elements but the most proximal elements develop as can develop without Shh present Also, loss of identity of the zeugopods

Question 36

Describe the feedback loops established between the AER and ZPA that allows the limb to develop along both the A/P and D/P axis

Answer 36

ZPA is important for maintaining FGF4/8 expression in the AER FGF8 in the AER is important for maintain Shh signalling in the ZPA

Question 37

What 3 genes are essential for D/V patterning of the limb and where are they expressed?

Answer 37

* Wnt7a is specifically expressed in the dorsal ectoderm * En1 is specifically expressed in the ventral ectoderm * Lmx1b is specifically expressed in the mesenchyme of the dorsal limb

Question 38

What is observed and concluded when Lmx1b is knocked out in the limb mesenchyme?

Answer 38

* Causes the ventilation of the limb and the loss of dorsal features * Confirming that Lmx1b is required for specifying the dorsal pattern of the limb and in the absence the limb mesenchyme adopts a alternative ventral fate * The ventral identify is therefore the default pathway

Question 39

What is the proposed model for controlling the D/V patterning of the limb?

Answer 39

* BMP signalling occurs in the ventral part of the limb which drives the ventral expression of En1 * En1 expression inhibits and restricts the expression of Wnt7a to the limbs dorsal ectoderm * Wnt7a drives the expression of Lmx1b which is only expressed in the dorsal half of the limb

Question 40

What is observed if the AER is removed at an early stage of limb development?

Answer 40

A limb without a humerus/radius/ulna etc Limb development ceases

Question 41

What is observed if an additional AER is grafted anterior to the existing one at an early stage of limb development?

Answer 41

A limb with mirror image duplication of digits as the extra AER FGFs induce the formation of another ZPA

Question 42

What is observed if a leg mesenchyme is grafted in place of the limb mesenchyme at an early stage of limb development?

Answer 42

A limb with characteristics of a leg

Question 43

What is observed if a non-limb mesenchyme is grafted in place of the wing mesenchyme at an early stage of limb development?

Answer 43

A truncated limb

Question 44

What is observed if the AER was replaced by FGF bead at an early stage of development?

Answer 44

A normal limb develops