spinal cord development

Post on 04-Dec-2014

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mid-term presentation given for capstone neuroscience class

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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