Lecture 14- Drosophila appendage development Flashcards
What can be observed and concluded when a region of posterior limb bud expressing Shh is transplanted to the anterior?
Mirror image duplicated lib is produced indicating Shh acts as a morphogen in patterning structures
What can cause defects and limbs duplications?
Defect in Hh signalling
What is the name of the tissues in which many adult drosophila structures derive from?
Imaginal discs
Which imaginal disc gives rise to the wings?
Haltere disc
At what stage of Drosophila development do imaginal discs become patterned?
During metamorphosis
Give 3 example of how gene expression can be visualised through staining in the dissected imaginal disc
- Antibody staining
- Reporter lines (lacz)
- Molecular markers
What imaginal discs are observed in T1/2/3 segments?
T1 = leg disc T2 = leg and wing disc T3 = leg and haltere disc
What are the 3 fates for wing imaginal discs?
- Wing blade
- Hinge
- Notum
How many cells does a wing disc contain and what is the thickness by the end of the third larval instar and
50,000 columnar epithelial cells
2 cell thick
What is the process called in which imaginal discs unfold into their adult shape?
Eversion
What 4 compartments does the wing imaginal disc divide into?
Dorsal, ventral, anterior and posterior
What cellular properties help maintain compartment boundaries?
Different cell-cell affinities promoting local cell-cell interactions and inhibiting cell movement and intermixing
What type of gene is engrailed and where is it expressed?
Engrailed is a segment polarity gene expressed exclusively in the posterior compartment
Outline how engrailed expression in the posterior compartments
- Use Engrailed-lacz reporter gene
- lacZ is expressed in place of engrailed but under the control of engrailed enhancers
- lacZ converts soluble colourless X-gal into insoluble blue precipitate allowing expression to be visualised
How does Hh expression compare to Engrailed in the wing imaginal disc and why is this?
Both expressed at high levels posteriorly but not anteriorly as engrailed drives Hh expression
Why is Ci expressed anteriorly in the wing imaginal disc?
Because engrailed is expressed posteriorly and surpresses Ci expression
What is the movement of Hh in the wing imaginal disc?
Diffuses away from the posterior compartment and slightly into the anterior
Describe the expression of Ptc in the ing imaginal disc
Low levels anteriorly and posteriorly but high levels along the Hh pathway boundary
Why is there low levels of Ptc posteriorly despite high levels of Hh?
Because there are low levels of Ci (due to engrailed suppression) and Hh pathway requires Ci
Describe the Hh pathway when no Hh is present
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
Describe the Hh pathway when Hh is present
When Hh is present, Hh binds to Ptc so no longer supresses Smo which eventually leads to the switching on of target genes
Given 3 examples of target genes of Hh signalling involved in the wing imaginal disc
Dpp, Wg and En
Where is Dpp expressed in the wing imaginal disc?
Along the A/P border
What causes Dpp expression to be high in the wing imaginal disc?
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
Describe the pattern of diffusion for Dpp in the wing imaginal disc
Expressed along the A/P border so diffuses in both directions (anterior and poster) down a concentration gradient
What is expressed downstream of Dpp?
Dpp - Tkv/Pnt - Mad - Brinker or Omb and Sal
What do high concentrations of Dpp induce the expression of?
Induce Sal and Omb expression
What do low concentrations of Dpp induce the expression of?
Induce Omb expression
Which target gene does expression of Dpp turn off?
Brinker
How can Dpp expression be detected?
Using GFP
What is a morphotrap?
Expresses nanobodies which bind to morphogens and prevent its diffusion
Explain the morphotrap experiment which proves Dpp is a morphogen and diffuses
- Take a Dpp mutant but knock-in a eGFP:Dpp fusion protein to function like ‘normal’ Dpp
- Dpp, anti-phos Mad and anti-brinker are all visualised where you would expect
- Now take eGFP:Dpp fusion protein but also express a morphotrap
- This traps Dpp-GFP on the membrane so disrupts gradient formation
- Therefore the expression domain of Dpp is much tighter and gradient of Dpp, anti-mad and anti-brinker is not observed
Outline the pathway of expression in the wing imaginal disc
En - Hh - Dpp - A/P patterning
Name the method used to induce clones of mutant cells?
FLP/FRT
What happens if we made a clone of cells in the posterior compartment that were engrailed mutants?
- If there is no engrailed, Ci would be expressed and Hh around the clone would be able to activate Hh signalling
- This would lead to expression of Dpp which would diffuse into the clone and into the surroundings
What happens if we make a clone of cells that express engrailed in the anterior compartment?
- If you have engrailed you make Hh
- 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
What happens if we make patched mutant clones?
- If you don’t have Ptc, you don’t negatively regulated Smo
- This means Hh signalling is switched on within the patch of clones
- Ultimately switches on Dpp expression within the close and Dpp protein diffuses out into the surroundings
What happens if we make a clone of cells that are mutants of Ptc and Dpp?
- Ptc mutant means there is no receptor for Hh so Smo isn’t repressed so Hh pathway is active
- But there is also no Dpp so no gene to express Dpp
- Despite the Hh pathway being active, no Hh is being made
- There is no receptor to bind the Hh produced
- 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
What signaling molecules pattern the D/V axis of the wing imaginal disc?
Wingless and Apterous
Give 3 examples of Wg target genes
Ac, DII and Vg
Where is apterous expressed in the wing imaginal disc?
Exclusively in the dorsal comparment
Where is Wg expressed in the wing imaginal disc?
At the D/V boundary
What signalling molecules establish the A/P and D/V axis?
- A/P axis is set up by Hh/Dpp gradients
* D/V axis established by gradients of Wg/Wnt
