furley 4 Flashcards
What do commissural axons lose their sensitivity to after crossing the midline - how was this shown
Netrins - ectopic floor plate placed beyond the endogenous floor plate. Axons exposed to ectopic floor plate before reaching the midline turn towards it. Axons exposed to ectopic floor plate only after crossing the midline no longer respond
Why dont commissural axons go straight on after crossing the floor plate like those in the hindbrain
The inhibitory molecules in the floor plate are semaphorins and slits. As well as being expressed in the floor plate these are also expressed in the ventral spinal cord thus creating a channel through which commissural axons can grow. Initially axons are sensitive to netrin and insensitive to sema/slits but after crossing the FP this sensitivity switches
What is the role of slit in flies
Slit encodes a secreted protein that is expressed by the midline glia and acts as a chemorepellent signal. It acts to repel commissural axons from the midline once they have crossed.
What is the role of robo in flies
Robo genes encode the receptor for slit. Robo protein is expressed at high levels on axons that don’t cross the midline. Commissural axons therefore initially express very little robo protein but following crossing of the ventral midline, they express high levels, become sensitive to slit and are therefore repulsed by the slit at the ventral midline.
What is the role of comm in flies
Comm is expressed in midline cells and in neurons that normally cross the midline. Found in comm neurons only before they have crossed the midline. Expression is down-regulated during crossing such that comm protein is absent from the post crossing segments of the commissural axons. Comm protein is thought to act to prevent robo from reaching the growth cones membrane, preventing sensitivity to slit and allowing for the crossing of the midline.
What is the result of a robo mutant
Slit is no longer detected, so all axons go back and forth across the midline forming a roundabout of axons
What is the result of comm mutant flies
Robo protein is expressed at high levels in those cells that would normally cross the midline, and which now extend their axons longitudinally.
What is the result of a slit mutant fly
Axons are attracted to the midline by netrin as normal but fail to move away from the midline on the other side. They appear to be stuck at the midline, collapsing into a single large bundle.
What is the sorting model
Comm prevents the trafficking of robo into the membrane, forcing newly synthesised robo into late endosomes
What is the clearance model
Homophilic binding of comm triggers clearance of robo from the cell surface.
What is the attraction of the clearance model over the sorting model
It suggests a mechanism for upregulation of robo in axons that have just crossed the midline
How did keleman test is comm is required in midline cells for correct crossing (clearance model suggests it should be)
Use neuron specific promoter to drive axon marker in just a subset of crossing neurons
Use the same promoter to also drive comm expression in comm mutants. Rescue us at least as good as when comm is also turned on in midline cells (using midline specific promoter of slit) i.e dont need comm midline expression to rescue.
Is robo prevented from going to axons in presence of comm
Visualising robo protein in axons in the living embryo using robo-GFP fusion. In the presence of comm robo persists in the cell body, whereas in an axon not expressing comm robo is transported along the axon.
What is the comm homologue seen in vertebrates
a 2nd robo-like receptor Rig-1 is expressed on pre crossing axons.
What is the result of a Rig-1 KO
It prevents FP crossing
