L4 - Reprogramming Flashcards
Axons reprogram when they reach
Choice points
What happens to C-axons once they have crossed the floorplate
NO LONGER RESPOND to netrins
Describe the experiment used to show that once the FP has been crossed C axons no longer respond to netrin
Ectopic grafting of the floorplate
What also happens to c-axons once the floor plate has been crossed
Become sensitive to repellants as well
What is the reason why c-axons become sensitive to repellants post crossing of the floor plate
Creates channels through which the c-axons can grow through
Insect midline glial cells express ___________(e.g_________) and ___________(e.g_________)
diffusible attractants (e.g. netrins) cell surface repellents (e.g. slits)
What does robo encode
a receptor for the inhibitory slit protein
Robo is expressed at high levels on _____
What is the logic behind this
Expressed at high levels on axons that DO NOT cross the midline
Robo is the receptor for slit, thus if Robo is high the cell is able to sense lots of the inhibitory slit molecule hence it is repelled from the midline and will not cross
Describe how Robo expression on c-axons changes
Initially expressed at low levels - once the midline is cross the axons express Robo at high levels
Describe the robo mutant
No robo = no receptor for slit = no slit detected
Axons go back and forth across the midline forms roundabouts
Where is comm expressed (2)
Midline cells and axons that normally cross the midline
What is the effect of lacking Comm
Robo protein expressed at high levels in those cells that would normally cross the midline and which extend longitudinally
What happens when Comm expression is forced
Robo lost everywhere so no sensitivity to Slit
Roundabout (robo) phenotype
What are the two models for how comm regulates Robo
Sorting
Clearance
Describe the sorting model
Presence of comm forces sorting of newly synthesised Robo into late endosomes
Describe the clearance model
Homophilic binding of Comm causes clearance of Robo from the cell surface
What is an attraction to the clearance model
It suggests a mechanism for the upregulation of Robo in crossed axon segments
Describe the results of Keleman 2005 expt to ask if Comm is required for midline cells for correct crossing
Neuron specific promoter used to drive an axonal marker
Same promoter was used to drive comm expression in comm mutants background
FOUND - rescue is also turned on in midline cells (using a midline specific promoter) - YOU DONT NEED COMM MIDLINE EXPRESSION TO RESCUE
What is one of the differences between the sorting and clearance model
In SORTING Robo should not be shipped down the axon in the presence of Comm whereas it would be if it was cleared at the cell surface (CLEARANCE)
How could you test to see if Robo protein is being transported down the axon
Robo-GFP fusion
Results showed Robo-GFP is found in an axon without comm - so robo transports along the axon
What are the limitations of the sorting model even though it appears to be correct
Why comm is on the midline
What controls the upregulation of Robo on the contralateral side
Describe how cell surface expression also changes in mammals
E.g. TAG-1 and L1
Has a comm homolog been found in mammals
No
What is found in mammals in place of comm
Another robo-like receptor (Robo3 AKA Rig-1)
When is Robo3 expressed
Pre crossing axons
What is the effect of knocking out in mice`
Prevents crossing of the floor plate
Journal club paper for reprogramming lecture
Sabatier et al 2004
What did Sabatier et al look to confirm in their paper
How Robo3 worked
What were the two models from Sabatier et al for how Robo3 works
1) Rig-1 is an attractive receptor required for floor plate crossing
2) Rig-1 prevents sensitivity to a floor plate repellent
Pre corssing C axons from ROBO3 knockouts are …
Prematurely sensitive to slits
Attraction to the midline in ROBO3 KOs can be restored if
Slit function is blocked
What method would be used to restore slit function
Using soluble Robo
Soluble Robo binds to Slit in the culture prevetning it binding to Robo on the axons
Wild type pre-crossing axons _______ in the presence of netrin
Ignores slit
SO WHAT IS THE FUNCTION OF ROBO3
Prevent premature sensitivity to Slit
Role of Robo3 is ________ to the role of Comm in flies
This role is
Analogous
Prevents Robo from signalling before the midline which allows netrin to signal via its receptor
What is the receptor for netrin
DCC
What is different between Robo3 in mammals and comm in flies
The mechanism
Loss of Robo3 DOES NOT lead to an upregualtion of Robo on pre crossing axons
Losing comm would lead to the upregulation of robo - comm mutant - no axons cross the FP
What is thought to now be the effect of Slit binding Robo at the cell surface
Resulting signal amplifies Robo expression
Robo3 may be required to …
Prevent premature slit response as C axons approach the Fp
Evidence suggests that at ___________ slit-induced Robo signalling _______ Robo3 inhibition which then ________ robo3 expression __________ robo and _______ slit mediated repulsion
Certain threshold concentration Overwhelms Robo3 inhibition TUrns off Full stabilising Activating
The effect of turning to netrins is inhibited in what circumstances
When slit is present
What is the effect of slit on netrin
Slit inhibits netrin
What are the two models which could explain how Slit inhibits netrin
Model 1: Ligand-ligand interaction
Model 2: Receptor-mediated silencing
Describe model 1 - ligand ligand interaction
slit directly inhibits the netrin ligand
Describe model 2 - receptor mediated signalling
slit bidning to robo inhibits the compelx formed when netrin binds to DCC
What is the only thing that is required to block the response to netrin
Robo IC domain
How was it shown that it was the IC domain is required to block the response to netrin
Express hybrid version version of Robo
E.g. IC domain of Robo and the EC domain of Met (the HGF receptor)
When HGF applied it was seen that the turning response could be blocked
Since normally HGF doesnt could this response - isnt due to ligand but due to receptor interactions
So what is the mechanism for slit inhibitiing netrin
Silencing of the netrin response by Slit is due to an interaction between the cytoplasmic domains of DCC and Robo
BEFORE THE MIDLINE (ROBO3 AND ROBO)
Robo3 silences Robo
AFTER THE MIDLINE (ROBO AND SLIT)
Activation of Robo by slit silences DCC signalling
What throws the switch determine if Robo3 silences Robo or activation of Robo by slit silences DCC signalling
Contact with the floor plate triggers it
Changes over time in a cell intrinsic manner (essentially a clock)
What is the evidence that contact with the floorplate flips the switch
Robo story - suggests high conc slits will stabilise robo on cell surface TURNING ON slit senstivity as the FP is crossed
How does Sema sensitivity change as the floor plate is crossed
Contact mediated by Tag1 and NrCAM (on FP) to stabilise Sema receptors on C axons
What is the effect of treating c aoxns with Shh
Turns on Sema sensitivity
Shh initially also attracts
C axons
What changes with rgeards to Shh as C axons cross
Attraction of c axons to Shh changes to repulsion
what is the effect of a Smo KO
Confused crossing and turning defects
Response of C axons to Shh reversed by …
A cell intrinsic increase in 14-3-3 expression
How is Wnt4 an important factor for guidance
Wnt to Fzd to GSK3B and DVL - DVL affects the par 3 complex
14-3-3 is a ______ homolog
What is its function
Par 5
Inhibits protein kinase A
