Lecture 22- Axon guidance V Flashcards
What are the main repulsive cues we discuss?
-Slit and Robo - slits are originally found in drosophila, secreted proteins, three versions Slit 1-3=4 receptors -Three vertebrate Slit ligands Slit1, Slit2 and Slit3 - Four Robo (roundabout) receptors Robo1, Robo2, Robo3 (Rig1) and Robo4 (magic roundabout)
What is the slit downstream signalling?
- Slit:Robo downstream signalling
- robo= activates kinases= all leads down to DCC

What do slit proteins do in vitro?
-Slit proteins repel axons in vitro, act as a chemorepellent -all slits: Slit 1, 2 and 3 act as a chemorepellent to the axons
In commisural axon guidance once the axons get to the flooorplate, the midline, why don’t they stay there?
-something must force them out! -they don’t stay in the floor plate as the Slit 1,2,3 are there and the Robo receptors are on the growth cone of the commissural axon -in drodophila= the ventral midline= floor plate -Slits are crucial in the axon guidance across the midline of the spinal cord
What happens in Slit mutants in Drosophila?
-the axons don’t exit the midline as there is nothing to push them out
What happens in Robo mutants?
-the axons enter the ventral midline region and exit but come back in again and then repeat= roundabout= hence the name

What is the normal movement of the commissural axons in Drosophila?
-

Which specific Robos control the axon repulsion at the midline (Drosophila)?
-Robo1 and Robo2 jointly control axon repulsion at the midline -when knockout both robo1 and robo2 more severe than just one of them, similar to slit
Is the repulsion of commisural axons at the midline the same in mice as in Drosophila?
-yes -Robo1 and Robo2 jointly control axon repulsion at the midline (mice)
What happens in robo mutant embryos in vertebrates?
-Axons aberrantly cross the midline in vertebrate robo mutant embryos -Slit expressed in the midline binds to Robo expressed on the growth cone and repels the axons from the midline

Why aren’t the axons stopped from entering the midline initially?
- 2 isoforms of Robo3 exist (Robo3A/3.1 and Robo3B/3.2)
- different functions
- Robo3A/3.1 is expressed before axons cross and desensitize axons to Slit allowing the axon to enter the midline
- Robo3B/3.2 is expressed as axons cross and allows Slit to activate Robo 1, which repels the axons from the midline

What controls the midline crossing?
-Robo3 isoforms -Overexpression of Robo3A/3.1 allows axons to cross (and recross) -Overexpression Robo3B/3.2 repels axons -expression depends on what the axons are doing
What are 3 more repulsive cues?
-Semaphorins (the ligand), -the receptors: Plexins and Neuropilin
How many semaphorin ligands are there?
- 21 Semaphorin ligands (Sema3A-3G, Sema4A-4G, Sema5A-5B, Seman 6A-6D and Sema7A)
Are semaphorins membrane bound or secreted?
-Semaphorins can be membrane bound or secreted
What do the Semaphorins bind to?
-Semaphorins bind to receptors: Plexins (Plxn) (Plexin A,B,C,D) and Neuropilins (Nrp1 and Nrp2)=co-receptor= must be bound to plexin= then can signal, only sema3 that bind to it)
What are the Neuropilin?
- Neuropilins act as co-receptors to plexin and only bind to class 3 Semaphorins (Sema3A-3G)
What happens when axons are exposed to Sema3A?
-Exposure to Sema 3A causes growth cone retraction
What are spinal cord axons repelled by?
-Spinal cord axons are repelled by Sema3B and Sema3F -Guidance of axons across the midline of the spinal cord – semaphorin is also involved
What happens to neuropilin 2 knockout (-/-) animals?
-Axon growth across the midline is disrupted in Neuropilin 2 knockout (-/-) animals -Axon growth in other areas is altered in neuropilin knockout mice: trigeminal projections, spinal nerve projections, limb projections
Are some molecules both chemoattractants and chemorepellents?
-yes -Netrin-1 can be attractive or repulsive depending on the type of neuron and the receptor expressed - DCC (Unc40) attractive signalling - Unc5 repulsive signalling
How can netrin be repulsive?
-Expression of Unc-5 protein converts netrin-1 attraction to repulsion
Where does netrin-1 act as a repulsive cue?
-Netrin-1 repels trochlear ventral hindbrain motor neurons from the indbrain -netrin as a repulsive cue in vivo -this is in the hindbrain, floor plate still there -trochlear motor neurons from the ventral part of the hindbrain section -axons move dorsally and cross over -the netrin in the floor plate is pushing them away from the floor plate
How did they check that it was Unc5 involved in the repelling behaviour in motor neurons?
-Motor neurons are repelled by Netrin-1 released from the vertebrate floor plate -to check that it is via Unc5 that netrin has the repulsive activity -figured it out via putting in an Unc5 antibody -Addition of Unc5 blocking antibody inhibits the ability of netrin-1 to activate Unc5 and therefore motor neuron axons grow towards the floorplate





