Drosophila appendage development Flashcards
How are limbs patterned?
By signalling cascades
What do disruptions in the hh signalling pathway do in limbs?
Duplications of digits and wings
Where do the adult tissues of the fly develop from?
Imaginal discs
What are imaginal discs?
Small structures of epithelial cells that are set aside during embryogenesis (originate in the embryo)
When can the imaginal discs be seen?
At the segmentation stage of development
Where are the leg imaginal discs?
T1-T3
Where are the wing imaginal discs?
T2
Where are the haltare imaginal discs?
T3
What happens to the wing imaginal disc during larval development?
Increases in size from 30 cells to about 50,000 columnar cells by the end of the 3rd instar
What patterns the wing?
Signalling transduction pathways and morphogens (not pre detemined)
What is the wing split into?
4 quadrants:
- Anterior
- Posterior
- Dorsal
- Ventral
What does fate mapping of the 2D wing imaginal disc show?
- Centre of the 2D structure becomes the wing blade
- Surrounding part of the 2D that becomes the wing blade - becomes the HINGE
- Surrounding the part that becomes the hinge - becomes the NOTUM
What does the hinge in the wing do?
Translates the muscular movement into the movement of the wing
What is the unfolding of the 2D imaginal disc to the 3D structure called?
What does the middle of the disc become?
Eversion
Middle of disc become edge of the wing
What does the patterning of the wing disc start with?
Axis formation
How do the cells in the wing disc know where they are?
Wing disc is divided into 4 compartments - producing ‘coordinates’ in the disc (map)
What are the 2 main molecules that define the quadrants in the disc and give the cells positional info?
Wingless and patched
Where is wingless expressed in the wing disc?
What does this become?
On the BOUNDARY between the dorsal and ventral disc
Becomes the EDGE of the wing
Where is patched expressed in the wing disc?
On the boundary between the anterior and posterior disc
What is patched?
A receptor for the hh signalling pathway
Where are sensory organ precursors present?
What are they?
Have stereotypical positions within the wing disc
Precursors for the sensory bristles that the fly uses on its wing
What are the 4 stages of establishing the XY axis of the wing disc?
1) Engrailed
2) Hedgehog
3) Decapentaplegic
4) Wingless
What does engrailed define?
The posterior compartment of the wing disc
What type of molecules is engralied?
Segment polarity gene
Transcription factor - sits inside the nucleus
Why is engrailed expressed in the posterior half of the wing disc?
It is inherited from the segmentation machinery
Disc is made of a group of cells following segmentation - some of these cells already express engrailed
How can engrailed be visualised using reporter genes?
Where does this show engrailed is expressed?
- LacZ expressed in the place of the Engrailed DNA sequence
- BUT under the control of the enhancers that control engrailed
- When add colourless X-gal gas - lacZ converts the gas into blue precipitate
Shows engrailed to be expressed in the posterior of:
- Segments
- Wings
- Legs
How does engrailed affect hh signalling?
What does this cause?
Represses Ci (In Hh signalling, Ci is changed from a repressor to an activator)
Causes:
- Ci to be active in the anterior of the disc
- Ci to be repressed in the posterior of the disc
What drives the expression of Hh?
What does this mean?
Engrailed
The cells where hh is expressed cannot respond to the hh as engrailed is expressed and represses ci
Where is Hh high/low in the wing disc?
Why?
High in the posterior (made in the cells that express engrailed)
Low in the anterior (concentration gradient - hh is a morphogen, diffuses away from source)
What is patched?
Where is it expressed?
Hh receptor
AND
Hh TARGET gene
Patched receptor expressed on the boundary between anterior and posterior cells
Where can cells respond to hh signalling?
Only on the boundary between anterior and posterior and onwards (anteriorly)
What is dpp a transcriptional target of?
hh
Where is dpp expressed?
Why?
In a STRIPE along the anterior posterior boundary of the wing disc
These cells can respond to hh signalling as there is no engrailed expressed that represses ci - target genes can be expressed
Why is dpp only expressed in a small stripe at the anterior posterior boundary?
Hh morphogen doesn’t travel that far as the hh protein is is heavily modified by lipids
What is the homologue of dpp in drosophila?
TGF-beta
What type of molecule dpp?
Morphogen
What happens after dpp is made at the AP boundary of the wing disc?
What does this form?
Dpp diffuses from the boundary into both the anterior and posterior
Forms a gradient in both directions
What does the gradient of dpp tell the cells?
Telling the cells how far they are away from the midline (fixed reference point)
What signal transduction cascade is dpp the extracellular ligand of?
The TGF-beta signalling cascade
What 2 receptors does dpp interact with?
Type I and II receptors (Tkv an Pnt)
What happens when dpp binds to type II receptor?
- Forms a heterodimer with type I receptor
- Phosphorylates and activates Mad (transcription factor)
What does Mad do when it is activated?
It switched genes on/off:
- Omb, sal = on
- Brinker = off
When is sal induced?
What is the boundary of sal?
What does this show about the threshold of sal?
At very high levels of p-Mad (at AP the boundary)
Very sharp boundary
High threshold
What is the threshold for omb?
How is this seen?
Broad threshold
Broad pattern of expression
What do the cells in the middle of disc (in the AP direction) express?
Why is this unusual?
BOTH sal and omb
Normally, target genes of morphogens switch off other genes to ensure that only ONE target gene is on
What do LOW levels of dpp induce?
What do HIGH levels of dpp induce?
How does this give cells positional information?
Omb expression only
Sal AND OMB
Different types of cells define different areas of the map
What is a morpho trap?
Membrane tethered anti-GFP
What happens when express a morphotrap in the middle of the wing disc after adding dpp-GFP?
- Traps dpp-GFP
- Preventing it from diffusing
- Lack of dpp gradient
- p-Mad is trapped in the same cells
- Brinker is active in more cells and is closer to the midline (no dpp signalling)
What happens if make a clone of cells in the posterior compartment that are LACKING in ENGRAILED?
- No hh made
- But can make Ci (not blocked by engrailed)
- SO, can respond to hh made by surrounding cells that diffuse in)
- These cells then express patched and dpp
- Forms an ectopic midline
- Another reference point in the map, defining the middle of the wing
What is the midline of the wing disc defined as?
The boundary between dpp-expressing and dpp-non-expressing cells
What happens if make a clone of engrailed expressing cells into the anterior?
- Express hh
- Induces dpp in the SURROUNDING CELLS (can respond to hh as Ci is not repressed)
What happens is make a clone of patch LACKING cells in the ANTERIOR (away from the midline)?
- No inhibition of smoothened
- Downstream target genes are activated
- Dpp activated inside of the clone
- Artificial midline
- Production of dpp in the clone
- Diffuses out and patterns the cells around the clone
- Mirror image of symmetry of ectopic wing
- Formation of triple anterior wing
What happens is prevent patched receptor expression IN the midline?
Hh moves PAST this region (not bound by the receptor) until find a receptor past the patched mutant
