L14 - Drosophila Appendage Development Flashcards
What are limbs patterned by?
Conserved signalling cascades in imaginal discs
- 6 led discs
- 2 wing discs
What causes duplication of limbs?
All due to defects in the Hh signalling pathway
What are imaginal discs formed from?
Set aside during embryogenesis
What is found in thoracic segments 1-3 of the fly?
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
How do the imaginal discs develop?
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
How do the imaginal discs fold?
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
How is the wing disc divided?
These are patterned through morphogen gradients and signalling pathways
Link structures of 2D imaginal discs to structures they become in 3D
What does the central region of the wing disk become?
Wing blade
What does the surround of the wing disk become?
Hinge
What does the outer region of the wing disk become?
Noturn
Which molecules help define the four wing disc compartments?
Wingless and patched help define these compartments
Wingless – separates dorsal and ventral
Patched – separates posterior and anterior
Which cells in the wing disc help read out coordinates?
Sensory organ precursors
They are in a stereotypical position within the disc
What molecules help establish the X-Y axis?
Established by morphogen signalling mechanisms
- Engrailed, Hh, Dpp, Wg
How do imaginal discs get their A/P identity?
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
What is engrailed?
Defines the posterior compartment
- Segment polarity gene
- Transcription factor in the nucleus
- Made very early on development and is maintained throughout the flies life
How can the en-lacZ reporter gene be used to see where engrailed is expressed?
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
What does engrail tell cells about?
Engrail does not give cells any idea of their location within the cell – identity not position
What is the Hh signalling pathway controlled by?
Controlled by Ptc and Smo
- Ptc - receptor and target gene
- Ci – transcription factor activated
Engrailed suppresses expression of?
Ci
Engrailed - only in posterior end
Ci – only in anterior end
Engrailed drives expression of?
Hh
Where there is Ci there is no Hh
Hh - only in posterior end
Cells in anterior end can respond to Hh made?
In posterior end
Hh is secreted and diffusible
Concentration gradient of Hh anteriorly
In cells expressing Ci
What does engrail prevent posterior cells doing?
From responding to their own Hh
Hh drives expression of?
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
What is Dpp?
Hh target gene
Fly TGF-B homologue – another morphogen
Where is Dpp expressed?
Only expressed in strip up the midline
Then diffuses out
How is Dpp made?
If Hh signalling is active and Ci is present
Ptc and Dpp is made
How does Dpp create a morphogen gradient?
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
Dpp is a secreted ligand of the TGF-B superfamily. What is this pathway?
- Dpp – extracellular ligand
- Activates Tkv/Pnt – type II and type I receptor heterodimer
- Activates Mad – transcription factor
- Either
- Represses Brinker – target gene
- Activates Omb/Sal – target genes
High concentrations of Dpp induce?
Sal and Omb expression
Low concentrations of Dpp induce?
Only Omb expression
Overall, what does Dpp do to the wing?
Causes wing structure to be patterned and growth occurs
Dpp experimental evidence - disturbing normal development - membrane tethered anti-GFP – morpho-trap
Traps Dpp-GFP on the membrane disrupting gradient formation
It is trapped by the antibody
Dpp experimental evidence - disturbing normal development - anti-p-mad
A downstream transcription of Dpp
In experiment – gradient is lost as all Dpp is trapped in the middle
Dpp experimental evidence - disturbing normal development - anti-BRK
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
Summary of wing patterning
- Engrailed in posterior
- Activates Hh in posterior
- Hh diffuses to anterior where Ci expression allows Hh signalling
- Activates Dpp gradient
- Leads to patterning
What would happen if a clone of cells was added to the posterior end that lacked engrailed?
Hh repressed
Ci expressed
- Means they can respond to Hh diffusing in from surrounding area
Ptc and Dpp expressed
What would happen if a clone of cells was added to the anterior end that make engrailed?
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
Clones that change Ptc expression?
- Ptc binds Hh and is the pathway receptor
- If no Ptc you don’t have negative regulation of Smo
- Smo is activated
- Activation and expression of downstream Hh target genes
- Dpp expressed in cells
What would happen if a Ptc clone was induced in the anterior wing?
Production of dpp in clone has diffused out and patterned the surrounding area
Produces mirror image symmetry
- Due to induced morphogen gradients
What would a mutant region lacking the Ptc receptor cause?
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
