Lecture 12

Question 1

Give an example of axon reprogramming

Answer 1

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

Question 2

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

Answer 2

Choice points

Question 3

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

Answer 3

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

Question 4

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

Answer 4

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.

Question 5

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

Answer 5

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

Question 6

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

Answer 6

Commissural neurons and longitudinal neurons

Question 7

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

Answer 7

Netrins

Question 8

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

Answer 8

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

Question 9

What is the role of robo

Answer 9

Robo is a cell surface receptor for the inhibitory protein slit

Question 10

Where is robo expressed

Answer 10

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

Question 11

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

Answer 11

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

Question 12

What is seen in robo mutants

Answer 12

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

Question 13

Commissureless is only expressed in the commissural neurons T or F

Answer 13

T

Question 14

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

Answer 14

F – after crossing the midline comm is no longer expressed

Question 15

What is seen in comm mutants

Answer 15

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

Question 16

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

Answer 16

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

Question 17

Explain how comm robo and slit interact in the invertebrate embryo

Answer 17

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

Question 18

Describe the two models for how slit and robo interaction

Answer 18

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

Question 19

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

Answer 19

The clearance model

Question 20

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

Answer 20

The clearance model

Question 21

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

Answer 21

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

Question 22

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

Answer 22

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.

Question 23

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

Answer 23

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

Question 24

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

Answer 24

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

Question 25

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

Answer 25

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

Question 26

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

Answer 26

T

Question 27

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

Answer 27

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

Question 28

What is significantly different about robo/comm in vertebrates

Answer 28

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

Question 29

What is the phenotype of a robo1 knockout

Answer 29

Looks like a netrin knockout no axons cross the midline

Question 30

What is surprising about a robo3/rig1 knockout

Answer 30

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

Question 31

What were the two proposed models for the action of robo3/rig1 in vertebrates

Answer 31

Rig1 could be an attractive receptor required for floor plate crossing. Alternatively Rig1 may prevent premature sensitivity to a floor plate repellent

Question 32

What evidence confirms that Rig1 acts to prevent axons from being sensitive to slits

Answer 32

Pre-crossing commissural axons lacking Rig1 were not attracted to the floorplate. However surprisingly the commissural axons were attracted to the floor plate if slit function was block using a soluble robo receptor lacking its transmembrane domain. This indicates that normally Rig1 blocks robo function which in turn can be restored by blocking slit function. Secondly culturing commissural axons in a medium containing netrin resulted in axonal growth in all directions even if other cells in the culture are secreting slits. Hence in wild type axons if netrin is present the axons can ignore slit. In rig1 knockouts axons grow out but away from the slits. Hence Rig1 is prevent the axons from being sensitive to slits

Question 33

Netrin overrides the effects of slit but only if Rig1 is present T or F

Answer 33

T

Question 34

Robo3/Rig1 has an analogous role to commissureless in flies acting through the same mechanism T or F

Answer 34

F – although the role of Rig1 is analogous to comm it acts through a different mechanism

Question 35

What is significant about the response to netrin of post-crossing commissural axons to netrins and the influence of slits on this

Answer 35

Post-crossing commissural axons of Xenopus spinal cord neurons respond to netrin in axon turning assay. However this effect of netrin is negated when Slit is also present

Question 36

What were the two proposed models by which slits and netrins interact to negate the chemoattractive ability of netrins

Answer 36

Ligand-ligand interactions – slit and netrin interact and prevent netrin from binding to its receptor DCC. Receptor-mediated silencing – slit and netrin bind to their receptors and robo (slit receptor) silences the activity of DCC

Question 37

Describe the experiments used to determine which model of netrin silencing in the presence of slits was correct

Answer 37

The slit-binding extracellular domain of the robo receptor was removed and its intracellular domain was attached to the TrkA receptor. The generated a hybrid slit-NGF receptor where the activation of the robo intracellular domain is activated by NGF which importantly isn’t a chemorepellent. It was found that expression of this receptor and stimulation with NGF blocked the turning response. Since NGF does not do this normally this argues against the ligand-ligand interaction model. Hence it must be the receptor rather than the ligand blocking netrin which indicates that the silencing of the netrin response by slit is due to an interaction between the cytoplasmic domains of DCC and Robo

Question 38

Explain the current model of how netrin/DCC slit/robo and Rig1 interact to direct commissural axons

Answer 38

Before getting to midline Rig1 is expressed at the same time as Robo and prevents robo from signalling in response to slits. This allows netrin to attract the axons to the midline. After crossing the floor plate Rig1 expression has disappeared due to an unknown mechanism. Now slit can bind to robo and it can signal. Robo then interacts with DCC to silence its activity rendering the axon unable to respond to the netrin attraction. Thus changes in Rig-1 expression underlie the changes in sensitivity to both Netrin and DCC as the midline is crossed

Question 39

Describe the CAMs that are thought to play a role in switching the sensitivity of commissural axons and the significance of their interactions

Answer 39

The floor plate expresses NrCAM and the commissural axons express TAG-1. Interfering with the interaction between NrCAM and TAG-1 disrupts turning at the floor plate as antibodies against these molecules caused aberrant turning of commissural axons

Question 40

What changes are seen in terms of the semaphorin receptor expressed by commissural axons as they come into contact with the floor plate

Answer 40

Prior to arrival at floor plate the semaphorin receptors neuropilin-1 neuropilin-2 and plexins are degraded by calpain. However when the neuron comes into contact with the floor plate the action of calpain is inhibited and the cells are now responsive to semaphorins

Question 41

Describe the role of sonic hedgehog in the response to semaphorins at the floor plate

Answer 41

Once the commissural axons reach the floor plate they will also be exposed to high levels of sonic hedgehog. In the presence of sonic hedgehog and semaphorins there is a repulsion of the neurons. The presence of sonic hedgehog actually switches on the sensitivity to semaphorins

Question 42

What is seen in commissural axons if they reach the floor plate with no sonic hedgehog

Answer 42

With sonic hedgehog absent there is in fact an attraction because the axons aren’t sensitive to the chemorepellent semaphorins

Question 43

What is seen in a netrin knockout with regards to axonal crossing of the floor plate

Answer 43

Axons project towards the floor plate and some still cross it

Question 44

What is seen in a conditional smo knockout in the commissural neurons

Answer 44

A large number of axons navigate normally most likely due to the chemoattraction of netrins. However a small number of axons stray away and don’t reach the floor plate

Question 45

What evidence is there to suggest that there is a cell intrinsic/temporal component to the changes in sensitivity of the commissural axons

Answer 45

Firstly commissural axons also change their character with time and the sensitivity of these commissural neurons changes at a particular age. In addition cells change their expression of CAMs overtime in culture in the absence of the floor plate. For example over time TAG-1 expression drops and L1 expression rises. This indicates a change that this is intrinsic to these cells. Finally the initial response of commissural axons to shh is attraction but then turns to repulsion.

Question 46

What mediates the change in response to sonic hedgehog and what evidence showed this

Answer 46

The change in response to sonic hedgehog is mediated by the Par-5-like protein 14-3-3. Knockdown of 14-3-3 stops reversal and prevents repulsion by sonic hedgehog and in fact leads to enhanced attraction. Prior to crossing very little 14-3-3 is expressed but it then gets upregulated once the axons cross the floor plate. In addition 14-3-3 overexpression leads to premature turning of commissural axons

Question 47

What are the three roles of sonic hedgehog in guidance of the commissural axons

Answer 47

Firstly sonic hedgehog is involved in attracting commissural axons to the floor plate. Secondly it switches on sensitivity of commissural axons to semaphorins hence becoming repelled. Then finally sonic hedgehog expression in the midline is also graded and plays a role in the anterior turning of the post-crossing axons accounting for some anterior-posterior patterning.

Question 48

Thus what are the three effects of a loss of sonic hedgehog in the axons of the spinal cord

Answer 48

A failure of commissural axons to be attracted to the FP a failure to become repelled by the semaphorins and the FP and finally a failure to become repelled and turn anteriorly

Question 49

Outline the multiple mechanisms acting to direct the commissural axons in the spinal cord

Answer 49

BMPs repel the dorsal spinal cord axons ventrally. Netrin and sonic hedgehog attract the axons towards the floorplate. Subsequent to crossing there is a change in 14-3-3 expression from low to high a change in PKA activity from high to low a switch from attraction to repulsion and the combined effects of wnts and sonic hedgehog which cause post-crossing axons anteriorly

Question 50

Describe how wnts and sonic hedgehog act to direct post-crossing commissural axons

Answer 50

Wnt is expressed in a gradient from high to low as you move posteriorly and hence attracts axons anteriorly. Sonic is expressed high posteriorly and low anteriorly and acts as a repellent to also direct the axons anteriorly