Date post: | 04-Dec-2014 |
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Education |
Upload: | marshall-benson |
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What happens to Rig-1 deficient mice?
What happens to Rig-1 deficient mice?
Predictions?
What happens to Rig-1 deficient mice?
Predictions?
•Homozygous deficient mice are born, but live only a few hours
•Suckling not observed
What’s going on here?
What’s going on here?
•Rig-1 deficient axons are unable to cross the ventral midline to form a commissure, in both the spinal cord and hindbrain
What’s going on here?
•Pre-crossing axons stall or turn away
•Rig-1 deficient axons are unable to cross the ventral midline to form a commissure, in both the spinal cord and hindbrain
OK, but how does it work?-- 2 models available:
A little simpler..
• Model 1: Rig-1 is an attractive receptor which binds a ligand (netrin?) necessary to enter the floor plate
A little simpler..
• Model 1: Rig-1 is an attractive receptor which binds a ligand (netrin?) necessary to enter the floor plate
• Model 2: Rig-1 inhibits the action of an axon growth cone repellant
How do we discern the mechanism?
• Rig-1 mutants (axons which lack Rig-1 receptors) grown in netrin-rich collagen showed an attractive response
How do we discern the mechanism?
• Rig-1 mutants (axons which lack Rig-1 receptors) grown in netrin-rich collagen showed an attractive response
--Therefore, Rig-1 does not specifically respond to netrins, an assumption of model 1
How do we discern the mechanism?
• Rig-1 mutants (axons which lack Rig-1 receptors) grown in netrin-rich collagen showed an attractive response
• However, Rig-1 mutants grown on the floorplate (netrin-rich) do not cross!
Something is antagonizing growth cone-netrin attractive response.
Slit is a candidate
• To show definitively that Slit-Rig-1 interaction is responsible for commissural outgrowth:
Slit is a candidate
• To show definitively that Slit-Rig-1 interaction is responsible for commissural outgrowth:
-- Knock out Rig-1
-- Provide antagonist to Slit
-- See if commissural outgrowth is rescued
Slit is a candidate
• To show definitively that Slit-Rig-1 interaction is responsible for commissural outgrowth:
-- Knock out Rig-1
-- Provide antagonist to Slit
-- See if commissural outgrowth is rescued
RESULT: Outgrowth is rescued, wildtype axons observed
More in vitro evidence
-- Wildtype dorsal spinal column axons cultured next to Slit2-expressing COS cells; no repelling action.
-- Rig-1 knockout DSC axons strongly inhibited by Slit2-expressing COS cells.
More in vitro evidence-- Wildtype dorsal spinal column axons cultured next to Slit2-expressing COS cells; no repelling action.
-- Rig-1 knockout DSC axons strongly inhibited by Slit2-expressing COS cells.
There is now enough evidence to accept Model 2: Rig-1 inhibits the action of Slit, an axon growth cone repellant found at the midline.
Loss of Slit function partially rescues crossing
Rig-1 = Robo3
• Robo1, Robo2, Robo3 are all in the same family of receptors with Slit ligand
• Robo1, Robo2 interact with Slit proteins and are repelled from the midline;
• Robo3/Rig-1 has opposite effect: interacts with Slit proteins to guide the axon across the midline.
Take-home message
• Rig-1 masks premature Slit responsiveness in commissural axons crossing the midline
• In vitro methodology is sufficient to show this