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spinal cord development

Date post: 04-Dec-2014
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mid-term presentation given for capstone neuroscience class
20
What happens to Rig-1 deficient mice?
Transcript
Page 1: spinal cord development

What happens to Rig-1 deficient mice?

Page 2: spinal cord development

What happens to Rig-1 deficient mice?

Predictions?

Page 3: spinal cord development

What happens to Rig-1 deficient mice?

Predictions?

•Homozygous deficient mice are born, but live only a few hours

•Suckling not observed

Page 4: spinal cord development

What’s going on here?

Page 5: spinal cord development

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

Page 6: spinal cord development

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

Page 7: spinal cord development

OK, but how does it work?-- 2 models available:

Page 8: spinal cord development

A little simpler..

• Model 1: Rig-1 is an attractive receptor which binds a ligand (netrin?) necessary to enter the floor plate

Page 9: spinal cord development

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

Page 10: spinal cord development

How do we discern the mechanism?

• Rig-1 mutants (axons which lack Rig-1 receptors) grown in netrin-rich collagen showed an attractive response

Page 11: spinal cord development

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

Page 12: spinal cord development

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.

Page 13: spinal cord development

Slit is a candidate

• To show definitively that Slit-Rig-1 interaction is responsible for commissural outgrowth:

Page 14: spinal cord development

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

Page 15: spinal cord development

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

Page 16: spinal cord development

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.

Page 17: spinal cord development

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.

Page 18: spinal cord development

Loss of Slit function partially rescues crossing

Page 19: spinal cord development

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.

Page 20: spinal cord development

Take-home message

• Rig-1 masks premature Slit responsiveness in commissural axons crossing the midline

• In vitro methodology is sufficient to show this


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