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Give an example of axon reprogramming

After transit of the midline commissural axons lose their responsiveness to netrins


What is the name of the point at which axons are reprogrammed

Choice points


Describe an experiment carried out to test that axons responses to attractants are reprogrammed

Rodent hindbrain neurons don’t turn immediately after reaching the floorplate. Red and green lipophilic dyes were injected either side of the midline at the roof plate of a spinal cord open book preparation. Next an ectopic floor plate was grafted onto one side of the preparation. It was expected and later confirmed that the neurons on the side of the midline where the ectopic floor plate was added would turn towards it. However it was observed that the axons from the opposite side of the preparation that have crossed the midline no longer responded to the ectopic floor plate. Hence they must have changed their behaviour to become no longer sensitive to chemoattractant netrins


In comparison to the mammal hindbrain spinal cord commissural axons turn after crossing the floor plate not because they are no longer attracted to it explain the significance of this

In fact the commissural axons have become sensitive to inhibitory molecules in the floor plate. These repellent molecules responsible for the change in direction are semaphorins and slits.


Describe the two mechanisms that act together to cause the change in direction/turning of the commissural axons after crossing the floor plate

Loss of attraction to chemoattractants and a gain of repulsion to chemorepellents


What are the two types of neurons seen in the ventral nerve cord of Drosophila

Commissural neurons and longitudinal neurons


In the developing Drosophila ventral nerve cord you can observe commissural and longitudinal neurons what factor is being secreted by the midline glia cells



What two unusual phenotypes are produced by genetic screens in Drosophila that exhibit problems with the neurons of the ventral nerve cord

Roundabout mutants – show no longitudinal neurons and are the result of robo mutation. Commissurless mutants – show no neurons crossing the midline and is due to comm mutation


What is the role of robo

Robo is a cell surface receptor for the inhibitory protein slit


Where is robo expressed

Expressed at high levels in the axons that don’t cross the midline


Robo is expressed at high levels at all times in commissural axons T or F

F – it is initially expressed at low levels in commissural neurons before crossing the midline. It is then upregulated and expressed at high levels after crossing the midline


What is seen in robo mutants

Insensitivity to slit so all the commissural neurons go back and forth across the midline forming roundabouts of neurons – they are constantly attracted to the Netrins produced by the midline glia cells and not repelled by the action of slit


Commissureless is only expressed in the commissural neurons T or F



Commissureless is expressed both before and after crossing the midline T or F

F – after crossing the midline comm is no longer expressed


What is seen in comm mutants

Robo protein is expressed at high levels in all cells that would normally cross the midline but which now project their axons longitudinally


What happens if comm expression is forced in all the neurons in the ventral nerve cord of Drosophila

Robo protein expression is lost everywhere resulting in a phenotype identical to that of the robo mutant


Explain how comm robo and slit interact in the invertebrate embryo

Slit binds to robo. Comm encodes a trafficking protein that prevents the vesicles containing robo from reaching the cell surface of the neurons. This in turn prevents the slit inhibitory signal from being received. After the axon crosses the midline comm expression is turned back off and robo-containing vesicles can reach the cell surface allowing the growth cone to respond to the inhibitory slit signal and thus change direction


Describe the two models for how slit and robo interaction

The sorting model suggests that in the presence of Comm newly synthesized Robo is trafficked into late endosomes instead of being shipped down the axon. In contrast the clearance model states that the homophilic binding of comm in midline glia with comm of the axon acts to cause removal of robo from the membrane


Which model of comm and robo interaction requires that commissureless is expressed in the midline glial cells

The clearance model


Which model of comm and robo interaction provides a mechanism by which robo can be upregulated after midline crossing

The clearance model


Describe the experiments carried out by Keleman et al. 2005 that investigated if comm was required in the midline cells for midline crossing

A neuron-specific promoter was used to drive an axonal marker in just a subset of crossing neurons. This same promoter was used to also drive Comm expression in comm loss of function mutants to try to rescue expression only in the neurons. This effects of this on rescue of axon guidance was tested and it was shown to not be a very good rescue of crossing. In addition this rescue was at least as good as when comm was turned on in midline cells only using the midline-specific promoter for Slit. This indicates that comm is not needed in the midline for correct crossing


What differences about the localisation and transport of robo are suggested by the two different models of robo/comm interactions

The sorting model suggests that robo should not be shipped down the axon in the presence of comm whereas the clearance model implies that it should be prior to its clearance from the membrane.


Describe the experiments by Keleman et al that tested whether robo was prevented from travelling down the axons in the presence of comm

Keleman created a Robo-GFP fluorescent fusion protein an expressed this protein in the neurons of the living embryo where it could be visualised by microscopy. In the absence of comm it was found that the robo-GFP fusion was transported along the axons and inserted into the membrane by visualising the packaged green fluorescence in vesicles moving towards the periphery indicative of kinesin activity. In the presence of comm the robo-GFP was detected in vesicles in the soma and there was no detection of its transport along the axons. This provides additional evidence for the sorting model


What are the two conclusions as a result of Keleman’s experiments and which model does this support

Midline crossing of commissural axons doesn’t require comm to be expressed in the midline cells and comm specifically affects the trafficking of robo. This validates the sorting model were in fact comm controls the sorting of Robo at the trans-Golgi network and targets it to the endosome and lysosome


What attributes of comm/robo activity are not explained by the sorting model

Why comm is on in the midline glial cells and what controls the upregulation of robo on the contralateral side


In vertebrates loss of response to netrins is thought to involve contact with the midline T or F



In vertebrates robo is expressed at high levels in the commissural axons before crossing the midlines and low levels after T or F

F – vice vera so as the same in flies low before crossing high after crossing


What is significantly different about robo/comm in vertebrates

Vertebrates don’t possess a comm homolog. Instead they express 3 different robos; robo1/2 are similar and are expressed on commissural axons but both before and after crossing the midline. Robo3/Rig1 then inhibits Robo1/2 from actin until the axons have crossed the midline


What is the phenotype of a robo1 knockout

Looks like a netrin knockout no axons cross the midline


What is surprising about a robo3/rig1 knockout

It prevents midline crossing of the floor plate. This is as if Rig1 is involved in stopping commissural axons from being sensitive to floor plate repellents