Furley 3 - axon guidance

Question 1

What experiment was done to show there is a chemoattractive signal from the floor plate

Answer 1

In the dorsal spinal cord neurons were placed in culture near either control tissue or with the floor plate. Only in the floor plate was there movement of the neurons toward the tissue.

Question 2

What is the name of the chemoattractant found in the floor plate

Answer 2

Netrin

Question 3

How was netrin shown to be a secreted protein

Answer 3

Similar to laminin, which can associate with the ECM, cells expressing the netrin gene were placed away from the neurons but were still able to induce a chemoattractive response in the neurons.

Question 4

What was the evidence for a long range attractant in the floor plate

Answer 4

Used small vs large spinal cord explants in an open book confirmation. In small explants the diffusable gradient is lost and confusion in the neruons after crossing the FP is seen. In large explants, an anterior turn is seen. Wouldn’t be seen if the guidance factor was non diffusible.

Question 5

How was it determined that the long range diffusible cue was an attractant

Answer 5

In large explants: If the cue was attractive it would be anterior to the FP. But in the collagen matrix the signal diffuses faster than in the explant so a high gradient is seen in the middle. As a result a posterior turn would be seen in the anterior neurons compared to those that cross the FP more posteriorly. If it was a repellent signal it would be found highest posteriorly but again the diffusion of the signal would increase the concentration in the middle of the explant. As a result the most posterior neurons would turn posteriorly. The first prediction was seen

Question 6

How is Wnt4 expressed in the floor plate

Answer 6

Expressed in an anterior-posterior gradient and can turn commissural axons

Question 7

What is the result of Wnt4 transfected cos cells

Answer 7

Can turn post crossing axons in either direction

Question 8

What was the result of the Frizzled3 KO in axons after crossing the floor plate

Answer 8

Confusion of turns

Question 9

What length does the Wnt gradient act over

Answer 9

around 7mm (the length of the mouse spinal cord at day 11.5)

Question 10

How do gradients of morphogens that pattern the dorsal/ventral neural tube get reused in shaping axon paths

Answer 10

Shh and BMP specify a neural fate

Shh attracts commissural axons along with netrin whereas BMPs repel axons away from the roof plate.

Question 11

What are the two ways of measuring a gradient

Answer 11

Change of concentration over time (temporal)
OR
Change in concentration across the cell (spatial)

Question 12

Why is it likely that mechanisms are in place to enhance the range over which gradients are detected

Answer 12

Even relatively steep gradients require very small differences in receptor occupancy to be detected across the growth cone. In mid-gradients a growth cone this is even smaller.

Question 13

What is amplification

Answer 13

Requires local enhancement of signal together with inhibition of signal reception in other parts of the cell. One suggested mechanism involves clustering of receptors and/or signalling components in regions where receptors activated by transporting components from other parts of the cell.

Question 14

How were leukocytes and dictostelium used to investigate chemoattractant signal amplification

Answer 14

Chemoatteractant bound by G protein, this activates PI3K which phosphorylates PIP2 to PIP3. This provides a docking site for proteins containing pleckstrin homology domains (such as Akt) creating a localised signalling domain on the membrane

Question 15

How is PI3K activity regulated

Answer 15

PTEN phosphatase, ensures the signal is highly localised and labile, dependent on continued external activation

Question 16

What experiment was done to show the translocation of PH domain proteins

Answer 16

Dictyostelium cells carrying PH domain protein fused to GFP

A chemoattractant placed and concentration of GFP rose at the leading edge of the cell near to the source.

Question 17

What is the potential role of amplification in order for the machinery to test the attractive cue found

Answer 17

Cue is encountered by filopodium, this activates PI3K, cAMP, Calcium etc. MTs stabilise and are captured, filopodium recognised as good to go. MTs ship in specific Rho GEFs to test if the cue is right for the growth cone and amplify the signal. Depending on the cue and set of RhoGEFs activated; engorgement and extension may follow, or may not if the cue is repulsive

Question 18

What is the role of adaptation in gradient recognition

Answer 18

Mechanism to enhance the range over which gradients can be detected.

Question 19

How was adaptation shown in response to netrin

Answer 19

Netrin creates an exponential gradient when pulsed. Growth cones respond to this gradient by zigzagging. The alternating attractive and repulsive turning suggests cycles of desensitisation of the growth cone to netrin.

Question 20

What factor controls the distance a growth cone can be guided by a gradient

Answer 20

The shape of the gradient, which is dependent on the set up. i.e
A diffusible molecule like wnts (gradient along an axis) or Netrins (point source)
OR
Non diffusible molecules such as ephrins which are surface bound molecules expressed in gradients.

Question 21

Over what distance can a gradient from a point source act

Answer 21

1mm Typically an exponential gradient

Corresponds well to supposed point source ranges in vivo (roof plate to floor plate = around 1mm)

Question 22

What do point source gradients depend on

Answer 22

A limited diffusion rate
Some mechanism for removing the guiding molecule (a sink) otherwise gradient will flatten over time (depending on diffusion rates)

Question 23

What is an example of a substrate bound gradient and what are the benefits iof this

Answer 23

Ephrins.
Substrate bound gradients which are typically more linear in shape can act over a range of around 1cm, which again corresponds to in vivo observations