L14 - Drosophila Appendage Development

Question 1

What are limbs patterned by?

Answer 1

Conserved signalling cascades in imaginal discs

• 6 led discs
• 2 wing discs

Question 2

What causes duplication of limbs?

Answer 2

All due to defects in the Hh signalling pathway

Question 3

What are imaginal discs formed from?

Answer 3

Set aside during embryogenesis

Question 4

What is found in thoracic segments 1-3 of the fly?

Answer 4

Regions of epithelial cells expressing markers that shows they will become limbs
Just T3 – cells that will form haltare
Just T2 – cells that will form wings

Question 5

How do the imaginal discs develop?

Answer 5

Begin as just 30 cells
As larvae develops they undergo replication
Wing discs contains 50,000 columnar epithelial cells by third larval instar
- This is normally when proliferation stops

Question 6

How do the imaginal discs fold?

Answer 6

What was the middle of the disk becomes the edge of the wing
Transforms it from 2D to 3D
Discs fold into their adult shape via eversion

Question 7

How is the wing disc divided?

Answer 7

These are patterned through morphogen gradients and signalling pathways
Link structures of 2D imaginal discs to structures they become in 3D

Question 8

What does the central region of the wing disk become?

Answer 8

Wing blade

Question 9

What does the surround of the wing disk become?

Answer 9

Hinge

Question 10

What does the outer region of the wing disk become?

Answer 10

Noturn

Question 11

Which molecules help define the four wing disc compartments?

Answer 11

Wingless and patched help define these compartments
Wingless – separates dorsal and ventral
Patched – separates posterior and anterior

Question 12

Which cells in the wing disc help read out coordinates?

Answer 12

Sensory organ precursors

They are in a stereotypical position within the disc

Question 13

What molecules help establish the X-Y axis?

Answer 13

Established by morphogen signalling mechanisms

- Engrailed, Hh, Dpp, Wg

Question 14

How do imaginal discs get their A/P identity?

Answer 14

Segmentation pattern at the beginning of imaginal discs occurs when there is already clear segments
Presumptive imaginal discs inherit their A/P identity from segmentation machinery
- Half express engrailed, half do not

Question 15

What is engrailed?

Answer 15

Defines the posterior compartment

• Segment polarity gene
• Transcription factor in the nucleus
• Made very early on development and is maintained throughout the flies life

Question 16

How can the en-lacZ reporter gene be used to see where engrailed is expressed?

Answer 16

lacZ expressed in place of Engrailed protein but under the control of the en enhancers
lacZ converts soluble colourless X-gal into insoluble blue precipitate
- Posterior wing expresses engrail
- Posterior parts of abdomen and thorax segments express engrail
- Compartments in the developing fly are defined by differential cell affinities

Question 17

What does engrail tell cells about?

Answer 17

Engrail does not give cells any idea of their location within the cell – identity not position

Question 18

What is the Hh signalling pathway controlled by?

Answer 18

Controlled by Ptc and Smo

• Ptc - receptor and target gene
• Ci – transcription factor activated

Question 19

Engrailed suppresses expression of?

Answer 19

Ci
Engrailed - only in posterior end
Ci – only in anterior end

Question 20

Engrailed drives expression of?

Answer 20

Hh
Where there is Ci there is no Hh
Hh - only in posterior end

Question 21

Cells in anterior end can respond to Hh made?

Answer 21

In posterior end
Hh is secreted and diffusible
Concentration gradient of Hh anteriorly
In cells expressing Ci

Question 22

What does engrail prevent posterior cells doing?

Answer 22

From responding to their own Hh

Question 23

Hh drives expression of?

Answer 23

Ptc
Pathway activated
Ci is present so downstream targets switched on
- One of these is Ptc itself
- Due to feedback loop
Ptc - only expressed in stripe up the middle

Question 24

What is Dpp?

Answer 24

Hh target gene

Fly TGF-B homologue – another morphogen

Question 25

Where is Dpp expressed?

Answer 25

Only expressed in strip up the midline

Then diffuses out

Question 26

How is Dpp made?

Answer 26

If Hh signalling is active and Ci is present

Ptc and Dpp is made

Question 27

How does Dpp create a morphogen gradient?

Answer 27

Dpp moves to both the anterior and posterior ends
Transcribed at midline but protein diffuses
Messages from midline sent in both directions
Tells cells their location relative to a fixed reference point

Question 28

Dpp is a secreted ligand of the TGF-B superfamily. What is this pathway?

Answer 28

1. Dpp – extracellular ligand
2. Activates Tkv/Pnt – type II and type I receptor heterodimer
3. Activates Mad – transcription factor
4. Either
   - Represses Brinker – target gene
   - Activates Omb/Sal – target genes

Question 29

High concentrations of Dpp induce?

Answer 29

Sal and Omb expression

Question 30

Low concentrations of Dpp induce?

Answer 30

Only Omb expression

Question 31

Overall, what does Dpp do to the wing?

Answer 31

Causes wing structure to be patterned and growth occurs

Question 32

Dpp experimental evidence - disturbing normal development - membrane tethered anti-GFP – morpho-trap

Answer 32

Traps Dpp-GFP on the membrane disrupting gradient formation

It is trapped by the antibody

Question 33

Dpp experimental evidence - disturbing normal development - anti-p-mad

Answer 33

A downstream transcription of Dpp

In experiment – gradient is lost as all Dpp is trapped in the middle

Question 34

Dpp experimental evidence - disturbing normal development - anti-BRK

Answer 34

In experiment - Brk domain expands sideways
Thinks the cells aren’t seeing any Dpp
Brk found in highest concentrations at edges where p-mad isn’t

Question 35

Summary of wing patterning

Answer 35

1. Engrailed in posterior
2. Activates Hh in posterior
3. Hh diffuses to anterior where Ci expression allows Hh signalling
4. Activates Dpp gradient
5. Leads to patterning

Question 36

What would happen if a clone of cells was added to the posterior end that lacked engrailed?

Answer 36

Hh repressed
Ci expressed
- Means they can respond to Hh diffusing in from surrounding area
Ptc and Dpp expressed

Question 37

What would happen if a clone of cells was added to the anterior end that make engrailed?

Answer 37

Hh expressed
Ci repressed
- Cant respond to their own Hh
- Respond to Hh diffusing in from surrounding area
Dpp expressed around cells where there is Ci
Artificial midline made in anterior compartment

Question 38

Clones that change Ptc expression?

Answer 38

1. Ptc binds Hh and is the pathway receptor
2. If no Ptc you don’t have negative regulation of Smo
3. Smo is activated
4. Activation and expression of downstream Hh target genes
5. Dpp expressed in cells

Question 39

What would happen if a Ptc clone was induced in the anterior wing?

Answer 39

Production of dpp in clone has diffused out and patterned the surrounding area
Produces mirror image symmetry
- Due to induced morphogen gradients

Question 40

What would a mutant region lacking the Ptc receptor cause?

Answer 40

No inhibition of Smo so Hh downstream targets activated
Hh can move region to an area with a Ptc receptor
Dpp expression extends around region