Lecture 14- Drosophila appendage development

Question 1

What can be observed and concluded when a region of posterior limb bud expressing Shh is transplanted to the anterior?

Answer 1

Mirror image duplicated lib is produced indicating Shh acts as a morphogen in patterning structures

Question 2

What can cause defects and limbs duplications?

Answer 2

Defect in Hh signalling

Question 3

What is the name of the tissues in which many adult drosophila structures derive from?

Answer 3

Imaginal discs

Question 4

Which imaginal disc gives rise to the wings?

Answer 4

Haltere disc

Question 5

At what stage of Drosophila development do imaginal discs become patterned?

Answer 5

During metamorphosis

Question 6

Give 3 example of how gene expression can be visualised through staining in the dissected imaginal disc

Answer 6

1. Antibody staining
2. Reporter lines (lacz)
3. Molecular markers

Question 7

What imaginal discs are observed in T1/2/3 segments?

Answer 7

T1 = leg disc
T2 = leg and wing disc
T3 = leg and haltere disc

Question 8

What are the 3 fates for wing imaginal discs?

Answer 8

1. Wing blade
2. Hinge
3. Notum

Question 9

How many cells does a wing disc contain and what is the thickness by the end of the third larval instar and

Answer 9

50,000 columnar epithelial cells

2 cell thick

Question 10

What is the process called in which imaginal discs unfold into their adult shape?

Answer 10

Eversion

Question 11

What 4 compartments does the wing imaginal disc divide into?

Answer 11

Dorsal, ventral, anterior and posterior

Question 12

What cellular properties help maintain compartment boundaries?

Answer 12

Different cell-cell affinities promoting local cell-cell interactions and inhibiting cell movement and intermixing

Question 13

What type of gene is engrailed and where is it expressed?

Answer 13

Engrailed is a segment polarity gene expressed exclusively in the posterior compartment

Question 14

Outline how engrailed expression in the posterior compartments

Answer 14

1. Use Engrailed-lacz reporter gene
2. lacZ is expressed in place of engrailed but under the control of engrailed enhancers
3. lacZ converts soluble colourless X-gal into insoluble blue precipitate allowing expression to be visualised

Question 15

How does Hh expression compare to Engrailed in the wing imaginal disc and why is this?

Answer 15

Both expressed at high levels posteriorly but not anteriorly as engrailed drives Hh expression

Question 16

Why is Ci expressed anteriorly in the wing imaginal disc?

Answer 16

Because engrailed is expressed posteriorly and surpresses Ci expression

Question 17

What is the movement of Hh in the wing imaginal disc?

Answer 17

Diffuses away from the posterior compartment and slightly into the anterior

Question 18

Describe the expression of Ptc in the ing imaginal disc

Answer 18

Low levels anteriorly and posteriorly but high levels along the Hh pathway boundary

Question 19

Why is there low levels of Ptc posteriorly despite high levels of Hh?

Answer 19

Because there are low levels of Ci (due to engrailed suppression) and Hh pathway requires Ci

Question 20

Describe the Hh pathway when no Hh is present

Answer 20

When there is no Hh present, Ptc suppresses Smo which causes Ci to become phosphorylated and cleaved

Ci acts as a repressor of gene expression

Question 21

Describe the Hh pathway when Hh is present

Answer 21

When Hh is present, Hh binds to Ptc so no longer supresses Smo which eventually leads to the switching on of target genes

Question 22

Given 3 examples of target genes of Hh signalling involved in the wing imaginal disc

Answer 22

Dpp, Wg and En

Question 23

Where is Dpp expressed in the wing imaginal disc?

Answer 23

Along the A/P border

Question 24

What causes Dpp expression to be high in the wing imaginal disc?

Answer 24

High levels of Hh lead to the Hh pathway stimulation and leads to high levels of Dpp expression as Dpp is a Hh pathway target gene

Question 25

Describe the pattern of diffusion for Dpp in the wing imaginal disc

Answer 25

Expressed along the A/P border so diffuses in both directions (anterior and poster) down a concentration gradient

Question 26

What is expressed downstream of Dpp?

Answer 26

Dpp - Tkv/Pnt - Mad - Brinker or Omb and Sal

Question 27

What do high concentrations of Dpp induce the expression of?

Answer 27

Induce Sal and Omb expression

Question 28

What do low concentrations of Dpp induce the expression of?

Answer 28

Induce Omb expression

Question 29

Which target gene does expression of Dpp turn off?

Answer 29

Brinker

Question 30

How can Dpp expression be detected?

Answer 30

Using GFP

Question 31

What is a morphotrap?

Answer 31

Expresses nanobodies which bind to morphogens and prevent its diffusion

Question 32

Explain the morphotrap experiment which proves Dpp is a morphogen and diffuses

Answer 32

1. Take a Dpp mutant but knock-in a eGFP:Dpp fusion protein to function like ‘normal’ Dpp
2. Dpp, anti-phos Mad and anti-brinker are all visualised where you would expect
3. Now take eGFP:Dpp fusion protein but also express a morphotrap
4. This traps Dpp-GFP on the membrane so disrupts gradient formation
5. Therefore the expression domain of Dpp is much tighter and gradient of Dpp, anti-mad and anti-brinker is not observed

Question 33

Outline the pathway of expression in the wing imaginal disc

Answer 33

En - Hh - Dpp - A/P patterning

Question 34

Name the method used to induce clones of mutant cells?

Answer 34

FLP/FRT

Question 35

What happens if we made a clone of cells in the posterior compartment that were engrailed mutants?

Answer 35

1. If there is no engrailed, Ci would be expressed and Hh around the clone would be able to activate Hh signalling
2. This would lead to expression of Dpp which would diffuse into the clone and into the surroundings

Question 36

What happens if we make a clone of cells that express engrailed in the anterior compartment?

Answer 36

1. If you have engrailed you make Hh
2. Hh means you don’t make Ci but Hh would diffuse out of the clone and get target gene expression around the outside of the clone

Question 37

What happens if we make patched mutant clones?

Answer 37

1. If you don’t have Ptc, you don’t negatively regulated Smo
2. This means Hh signalling is switched on within the patch of clones
3. Ultimately switches on Dpp expression within the close and Dpp protein diffuses out into the surroundings

Question 38

What happens if we make a clone of cells that are mutants of Ptc and Dpp?

Answer 38

1. Ptc mutant means there is no receptor for Hh so Smo isn’t repressed so Hh pathway is active
2. But there is also no Dpp so no gene to express Dpp
3. Despite the Hh pathway being active, no Hh is being made
4. There is no receptor to bind the Hh produced
5. Therefore Dpp is made on the anterior side of the clone as Hh can diffuse past the cloned cells and act at a longer distance when its Ptc receptor is no longer present

Question 39

What signaling molecules pattern the D/V axis of the wing imaginal disc?

Answer 39

Wingless and Apterous

Question 40

Give 3 examples of Wg target genes

Answer 40

Ac, DII and Vg

Question 41

Where is apterous expressed in the wing imaginal disc?

Answer 41

Exclusively in the dorsal comparment

Question 42

Where is Wg expressed in the wing imaginal disc?

Answer 42

At the D/V boundary

Question 43

What signalling molecules establish the A/P and D/V axis?

Answer 43

• A/P axis is set up by Hh/Dpp gradients

* D/V axis established by gradients of Wg/Wnt