L14 - Drosophila Appendage Development Flashcards
Limbs are __________ patterned by ____________
Complex 3D structures patterend by conserved signalling cascades
Duplications of limbs is result of mutations due to a …
Defect in Hh signalling
What can be said about Hh signalling
Highlty conserved signalling pathway
Used in early patterning events and then again in limb development
How many instar stagges
3
Most of the adult tissue in drosophila is derived from
Imaginal discs
What are the imaginal discs made from
Diploid epithelial cells
What are the different immaginal discs
Mouthparts Antenna Eye Leg Haltare Wing Genitalia
What does the number of discs depend on
The number of strcutures they go on to form
How many legs in drosophila
How many immaginal discs for the legs
6
6
When are the immaginal discs set aside
During embryogenesis
How can you see what the immaginal discs are
Using stains for specific marker genes
Describe what immaginal discs are seen in segments t1 - T3`
Leg - in all
Win - in T2
Haltare - in T3
Wing immaginal discs begins as a ____________
How many cells are they formed from?
2D epithelium
50000 cells
When do cells of the imaginal disc stop proliferating
At the third instar stage
What are the four segments of the wing disc
A P D V
The central region of the wing disc goes on to form the
Wing blade
The surrounding region of the wing blade goes on to form the
Hinge
What is the function of the wing hinge
Translate the force of the muscles which change the shape of the thorax into movements of the wing
What is responsible for forming the boundary between anteiror and posteiror
Patched gene expression (Hh)
What is reponsible for the formation of the boundary between dorsal and ventral
Wg gene expression
Nubbin gene expression is localised to the
Wing pouch
What type of gene is engraield
Segement polarity gene
Where is engrailed expressed
In the posterior of each segement
Describe the process used to visualse engrailed expression
LacZ expressed under the engrailed pormotoer
Use X gal
If there is LacZ present (forming B-galactosidease) Xgal gel goes from colourless to blue (insoluble)
Results of the En-LacZ show that
En expressed in the posterior of all sgements
Very sharp boundary
Describe Hh signalling when there is no Hh ligand
Ptc represses Smo Smo unable to signal Ci to SuFu Ci to Cos 2 and Fu - interacts with 3 kinases - cleaved to a shorter repressive form Enters nucelus - represses target genes
Describe the Hh signalling pathway when Hh is bound
No repression of Smo Smo is phosphriylated Interaction with Ci and SuFU blocked Interaction with Ci and 3 kinases blocked Full length Ci enters the nucelus Acts as a transcritpional activator Causes transcription of target genes
What are some examples of Hh target genes
DPp
Wg
Engrialed
What is the function of en
On ci
On Hh
Causes promotion of Hh expresision
Blocks expression of Ci
When is Ci expressed
In the absence of engrailed
Pathched is a …. and a ….
Receptor for Hh
Also a TARGET gene for Hh
What is the effect of Hh on Ptc expresison
What is this known as
Increases expression of Ptc
Positive feedback
Where is engrailed expression hi
P
Where is Ci expression hi
A
Where there is no En
Descirbe the expression of Hh about the AP axis
High posterior
Diffusible molecule so diffuses into the anterior and a concnetration gradient is fomred
Where is engrailed not expressed
Anteriorly
To be competent to respond to hedgehog what must cells also be expressing
Ci
What is the effect of Hh on Ptc
Upregualtion
Dpp is a target …
Of Hh signalling
What is the effect of Hh signalling of Dpp
Increased expression of dpp
What is the homologue of Dpp
TGF-B
Where is response to Hh seen
anterior compartment as these cells express Ci - only the ones that are in reach of Hh diffusion from the posterior
When Dpp is transcribed what is the effect of this
Dpp is also diffusible so another concentration gradient is establsihed going both anterior and posterior
Desribe the pathway of Dpp
Dpp –> Tkv/pnt –> Mad
Mad –/ brn
Mad –> Omb,sal
What is the active version of mad known as
p-mad
What genes do mad activate
Omb, Sal
What genes does mad repress
Brinked
What is Tkv/Pnt
Type1/type 2 receptor heterodimer
Describe the distribution of hh mRNA
Hh is localised to cells in the posterior that are seeing engrailed
What is the expression of Dpp mRNA
Expressed in a gradient based on Hh concentration gradient eastblished
What is the expression of p-MAd
Expressed in a concentration gradient since Dpp diffuses both anteriorly and posteriorly
How can you detect Dpp expression
Using a GFP fusion protein
Dpp acts as a ___________
Morphogen
What is the effect of high levels of Dpp
Sal and Omb expression
What genes are expressed by low Dpp levels
Only induce expression of Omb expression
What is a morphotrap
Expressing nanobodies which bind to a part of the morphogen preventing diffusion of the morphogen
Describe what morphotrap is used in the investigation of Dpp
Express GFP-Dpp
Anti-GFP nanobodies (single chain AB) which are tethered to the membrane
This traps dpp at the membrane where it is produced preventing the formation of a gradient
Describe the effects of GFP-dpp and anti-GFP which is membrane bound
Shows dpp expressed in the centre of the disc
No gradient formed
What was seen on the morphotrap when probing for p-mad
Dpp is held in the centre - cant diffuse away
Lack of a gradient of p-mad trapped in cells
What is the effect of p-mad on brinker
Repression of brinker
What is seen from the morphotrap when probing for anti-brk
Dpp expression represses the expression of Brk
Brk expression domains is expanded horizontally
Pathway from En to patterning
En –> Hh –> Dpp –> Patterning events
Describe what happens when a group of cells are transplanted into the anterior of the wing which are engrail (-/-)
Ci produced in group of cells (due to effect on engrailed from nearby cells) Hh from surrounding cells diffuses over Expression of Ptc and Dpp Dpp defines the midline Formation of an ectopic midline
What is the effect of transplanting a group of cells that express engrailed into the anterior
Engrailed - makes Hh
No Ci
Cells transplanted dont respond to Hh
Hh diffuses to neighbouring cells (which express Ci and no Engrailed) dpp expresion and ectopic midline
What is the effect of putting a group of Ptc (-/-) into the anteiror
No supresison of smo by Ptc
Hh signalling consitiutively active
Even without the ligand still active signalling
Act Ci enters nucleus in this group of cells - dpp induced - ectopic axis
Where there is an actopic activation and formation of a midline we see
Duplication of sturcutures
Describe the effect of putting Ptc KO cells at the boundary where Dpp is usuaully expressed
Ptc normally binds Hh
Without ptc Hh can diffuse further
Dpp expression shifted anteriorly
