Lecture 17 - Drosophila appendage development Flashcards
(22 cards)
What are all limbs pattered by?
All limbs are complex 3D structures patterned by conserved signalling cascades
What can Polydactyl caused by?
due to Hedgehog signalling
Describe the life cycle of Drosophila
Fertilized egg –> (cleavage) syncytial blastoderm –> (gastrulation) embryo –> (hatching) 1st, 2nd, 3rd Instar Larva –> Pupa –> (metamorphosis) –> adult fly
Where do most adult cuticles come from?
from imaginal discs, which float around in body cavity of larva - epithelial structure - one per structure - e.g. 2 imaginal discs for 2 eyes.
How are imaginal discs are set aside during embryogenesis?
Imaginal discs come from embryo. Wing comes from the 2nd thoracic segment & is specified by UBX & ANTENOPEDIA activity. 2nd thoracic segment contains group of cells which form wing - invaginate & form wing imaginal disc.
Cells are set aside during embryogenesis.
describe features of wing imaginal disc?
30-50,000 in 4 days via proliferation using a patterning format.
Go from monolayer to a bilayer. The effect of this is that pouch cells get folded in half in middle & form wing proper.
Hinge cells form hinge which join wing blade to fly.
Notum is thorax of fly & is monolayer.
What is the role of morphogens?
- Morphogens allow the cells to know where they are
- Morphogens are expressed in patterns which are consistent with the axis of the tissue
- Wingless is expressed on the D/V boundary as well as A/P
- Patched is the Hh receptor & a target gene of Hh signalling.
- Wingless & Patched act as morphogens
How are X-Y axes established by morphogen signalling mechanisms?
Morphogen patterns sheets of cells & diffuses across tissue, creating concentration gradient. It must also define different cells’ fate with different concentration.
Bicoid is a morphogen
What is the role of the Engrailed transcription factor?
It defines the posterior compartment
engrailed (segment polarity gene) - presumptive imaginal discs ‘inherit’ their A/P identity from the segmentation machinery.
Engrailed is expressed in the posterior compartment (segment polarity gene).
How does the Engrailed transcription factor defines the posterior compartment?
- en-lacZ reporter gene, lacZ expressed in place of Engrailed (protein) but under the control of the en enhancers (these enhancers are themselves controlled by the segmentation system)
- lacZ (B-galactosidase) converts soluble colourless X-gal into insoluble blue precipitate.
- compartments in the developing fly are defined by differential cell affinities.
Posterior cells expressed engrailed - stay on until the time of adult tissue formation. It is a determinant for posterior fate.
What is the Hedgehog (Hh) signalling pathway?
Hh is a signalling molecule, a morphogen, it signals to the nucleus. If you signal to the nucleus, you require a TF - cubitus interruptus (ci) in flies, or Gli in vertebrates.
When pathway is off, Ci is degraded & forms a repressor. Active pathway = Ci in the nucleus & activates transcription. Ci is needed for Hh expression. Hh activates the pathway. Hh receptor Patched is usually repressing Smoothened. Smoothened causes Ci degradation.
Addition of Hh leads to Patched not repressing Smoothened, activating system. Removal Patched also leads to activation.
Ci activates signalling.
What are the key parts of the Hh signalling pathway?
- Engrailed suppresses expression of Ci
- Engrailed drives expression of Hh
- therefore Ci is only expressed in the absence of En
Some cells that lack Ci but have Hh.
Some cells that have Ci but have Hh
Hh can diffuse though as it is a morphogen.
Engrailed & Hh in posterior. Diffuses across boundary, meets cells that have Ci, turns on Hh & turns on Patched expression.
What is dpp?
Dpp is a transcriptional target of Hh signalling.
- Decapentaplegic (Dpp) is the fly TGF-B homologue
- Dpp moves to both the anterior & posterior forming a morphogen gradient
How is Dpp a secreted ligand of the TGF-beta superfamily?
Dpp (extracellular ligand) activates Tkv/Pnt (Type II & Type I receptor heterodimer). This activates a transcription factor - Mad. Mad activation occurs through phosphorylation.
Target genes:
- activates - omb, sal
- inhibits - brinker
Dpp acts as a morphogen.
Anterior compartment - high in Mad, gradient going out. Dip in centre, caused by high Hh signalling. High Mad in posterior.
What are the target genes that Dpp act on?
- high concentrations of Dpp induces Sal & Omb expression
- lower levels of Dpp induce only Omb expression
- wing structure is patterned & growth occurs
How an Dpp be detected at a distance from its expression?
Some people argue that morphogens cannot diffuse.
Morpho-trap has stopped Dpp diffusion. This means Mad is only activated in the centre of the disc. A lack of Dpp diffusion prevents Mad activation further away - altering the patterning of transcription factors downstream.
This shows we can alter downstream pattern by preventing the diffusion of Dpp, meaning Dpp is a morphogen in the wing disk.
Cells express Ci, as they lack engrailed. Cells express Cim so be able to respond to Hh. They should express Dpp.
What are the affects of Engrailed?
Engrailed –> Hedgehog –> Dpp –> Patterning
Presence of engrailed inhibits Ci expression, also increasing Hh. This leads to more Dpp & more patterning.
If Hh is active, turn off Dpp & therefore Dpp will diffuse out.
What axis are wingless on?
D/V axis
What is found in the dorsal part of the wing disc?
Expresses apterous
Wingless gets turned on, involved Notch signalling.
What is Wingless?
- Wg is a secreted signalling molecule
- Wingless drives expression of pathway target genes in a concentration dependent manner.
Describe the 2D co-ordinate system
Sensory organs not found in the 4 quadrants, so other information must be present, in addition to morphogens - this is selector genes - e.g. engrailed in posterior compartment, preventing sensory organs. Apterous also divides dorsal compartments.
What are the KEY POINTS of Drosophila appendage development?
Genetic control of pattern formation in Drosophila limb development:
- the segmentation system defined ‘patterning centres’
- Patterning by morphogen gradients
- A/P axis - Hh/Dpp; D/V axis - Wg/Wnt
Classic experimental techniques & evidence that demonstrates these concepts.
Segmentation system in the embryo, defining A/P compartments, makes Wingless active, is reused. Engrailed still used to define posterior & produce Hh, but instead of turning on Wingless, it turns on Dpp.
Wingless acts on different axis.
2 axis (A/P - Hh & Dpp, D/V - Wg/Wnt)
We can see Dpp needs to diffuse, through a morpho-trap
