Lecture 11

Question 1

Contact of the growth cone with a cue is thought to first arrest actin before dragging the microtubules in T or F

Answer 1

F - Microtubules accumulate to a guidance cue before actin responds there is still retrograde actin transport at this point. Hence the idea that cue first arrests the actin before dragging microtubules is wrong and the microtubules in fact go in first. The first response of the growth cone is thus to uncouple the microtubules from F-actin. This is then followed by filopodial invasion by microtubules

Question 2

What is thought to be the purpose of initial microtubule invasion following cue contact

Answer 2

Invading microtubules may bring important signal transduction elements and/or factors required for membrane protrusion into area of contact with cue. This includes RhoGEFs which are known to associate with microtubule + end Tracking Proteins (+TIPs). Other componets such as monomeric actin and collapsin response mediator protein (CRMP) may also be recruited

Question 3

What is the name of the molecule that is secreted by the floor plate to direct axon growth towards it

Answer 3

Netrin

Question 4

What type of axon is guided by the floor plate

Answer 4

Commissural sensory relay neurons – interneurons that carry sensory information from the dorsal root ganglia up to targets in the thalamus

Question 5

What is the name of the chemorepellent secreted by the roof plate to direct axon growth away from it

Answer 5

BMP7

Question 6

Chemorepellents can induce growth cone collapse T or F

Answer 6

T

Question 7

Give examples of early patterning molecules that are later used to direct axon growth

Answer 7

BMP7 initially involved in dorsalisation of the neural tube later directs commissural sensory relay neurons away from the roof plate. Shh used to specify ventral neuronal fates in the embryo is also sued to guide neurons to the floor plate

Question 8

What are the major limitations that guidance signals need to overcome

Answer 8

Diffusion/transport barriers the extracellular matrix removal from the sink and saturation of the receptors

Question 9

What is behaviour is exhibited by the commissural axons of mammals once they cross the floor plate

Answer 9

They turn and project rostrally

Question 10

What two possibilities were proposed for how the commissural axons changed behaviour after crossing the midline

Answer 10

Possibility of an A-P gradient of either an attractant or a repellent acting as a long range diffusible cue. Or similarly the action of a short range non-diffusible cue

Question 11

Explain how the nature of the signals governing the directional change of commissural axons was determined by experimentation

Answer 11

Different size explants of spinal cord tissue were taken and compared by culturing in a collagen matrix. If you take a large spinal cord explant a long-range diffusible cue would take longer to diffuse out than it would if it was a smaller explant. By contrast a short range non-diffusible cue would remain stable in both large and small spinal cord explants. With this rationale the two spinal cord explants were taken and prepared in an open book configuration and injected with DiI. The lipophilic DiI was taken up into the cells and transported along the axon. In the larger explants the anterior/rostral turn made by the commissural axons as they crossed the floor plate could be seen however this is consistent with either a long or short-range cue. In contrast in the smaller explants it was found that the axons got knotted or stalled at the floor plate. This indicates that it’s a long-range diffusible cue and that the cue is diffusing out in the small explant and can’t cause commissural axons to change direction. This is because the diffusion of the molecule will be much faster in the collagen matrix than in the explant itself

Question 12

Describe how the nature of this long-range diffusible signal governing the change in direction of commissural axons was determined

Answer 12

DiI was injected into a spinal cord explant anteriorly centrally and posteriorly. Unusually this revealed that the posterior and middle axons turned anteriorly as would be expected for a chemoattractive signal however the anterior axons turned posteriorly. If the signal was a repellent secretion from a posterior source would lead to the diffusion of the molecule out of the explant near the edges hence shifting the highest point of the gradient towards the middle. If the signal was an attractant then the secretion from an anterior source would again lead to the diffusion of the molecule out of the explant near the edges. This would result in the highest point of the gradient shifting towards the middle too. Its this shift of the highest point of the gradient seen in vitro that accounts for the fact that the anteriormost axons turn caudally after crossing the floor plate and hence the signal is a chemoattractant

Question 13

What is the nature of the chemoattractant molecule responsible for the anterior turning of the commissural axons after cross the floor plate

Answer 13

Wnt4 is expressed in an anterior-posterior gradient in the floor plate and acts as the chemoattractant signal responsible for the commissural axons turning anteriorly

Question 14

How was the nature of the chemoattractant signal responsible for the change in commissural axon direction determined

Answer 14

Wnt4 was found to attract axons in vitro. In addition frizzled 3 knockout results in confusion when the commissural axons reach the floor plate

Question 15

Over what sort of range were the chemoattractant signals directing commissural axon direction found to be acting

Answer 15

Distances of at least several millimetres

Question 16

What two methods are there for detecting a gradient

Answer 16

Temporal detection - change in concentration over time where cells compare the amount of ligand at two different time points. Spatial detection – where cells compare the amount of ligand at different points on its surface

Question 17

Which type of gradient interpretation mechanism is used by neurons

Answer 17

Spatial detection

Question 18

At what point is the accuracy of orientation of a growth cone towards its source most accurate

Answer 18

The absolute concentration of the chemoattractant is most accurate closest to the KD of the receptor

Question 19

What level of gradient is most suitable for growth cones to respond accurately to

Answer 19

2% change in ligand concentration over 10µm (0.2% per micron)

Question 20

Larger diameter cells orient more accurately that small cells in shallow gradients T or F

Answer 20

T

Question 21

Amplification mechanisms are required for a growth cone to respond accurately to small differences in receptor occupancies. What is required of these amplification mechanisms

Answer 21

Amplification requires a local enhancement of the signal together with inhibition of signal reception in other parts of the cell

Question 22

How is it suggested that amplification of guidance cues is achieved

Answer 22

Clustering of receptors and/or signalling components in regions where receptors activated by transporting components from other parts of the cell

Question 23

Describe how signal amplification occurs in Dictyostelium

Answer 23

Chemoattractants in leukocytes and Dictyostelium activate PI3-K via a GPCR. This chemoattractant is detected by a GPCR which activates PI3-K which in-turn phosphorylates PIP2 to PIP3 . This provides a docking site for proteins such as Akt which contains PH domains. This creates a localised signalling domain on the membrane. PI3-K activity is antagonised by PTEN phosphatase which acts to ensure signals are highly localised and labile dependent on continued external activation

Question 24

Amplification mechanisms involve receptor clustering and sequestration of components T or F

Answer 24

T

Question 25

As well as amplification of guidance cues describe the need for adaptation by the growth cone

Answer 25

If growth cones operate most discriminatively when the chemoattractant concentration is closest to the receptor KD then the receptors will saturate as concentration increases. Even the shallowest gradients required to turn cells (0.2% per µm) will result in 90% receptor occupancy just 250µm from the optimal ligand concentration. Hence a way to modulate the response of the growth cone to different concentrations of the cue is essential

Question 26

Outline the experiments of Μu-Ming Poo in implying the need of axons to adapt to guidance cue concentration

Answer 26

Axons of neurons isolated from early embryonic Xenopus spinal cord were found to respond to netrin pulsed into a dish from a pipette. This pulsed netrin forms a (exponential) gradient which is revealed by including a fluorescent marker in the pipette. Growth cones responding to such a gradient initially exhibit a zig-zagging trajectory which implies axons are sensitising and de-sensitising as they go along

Question 27

How was the de-sensitisation and re-sensitisation of axons later proven

Answer 27

To determine whether growth cones adapt to increasing attractant concentrations axons were pre-treated with differing concentrations of netrin. Immediately (30min) after treatment growth cones no longer responded to the guidance cue. However after a longer period (90mins) growth cones became resensitized. This resensitization was dependent upon MAPK activation and local protein synthesis in the growth cone