Growth cones and Axonal guidance

Question 1

What do growth cones consist of to explore their environment?

Answer 1

‘Fingers’ (Filopodia/Filopodium) and lamella explore the area
- which are made up of different kinds of F-actin

Question 2

How are the actin bundles different in filolpodia and lamella?

Answer 2

In filopodia, the actin bundles are polarised but in lamella the actin bundles are cross linked to form a net

Question 3

How do growth cones move?

Answer 3

• F-actin ‘treadmills’ in constant polymerisation at the leading edge, adding actin on to the front and ‘chopping’ it off the back
• Microtubules extend through the whole growth cone for stability but also explore the back areas
• Retrograde flow (towards cell body) helps recycle actin and maintain dynamic stability

Question 4

How does the movement of growth cones change when it encounters an attractive cue?

Answer 4

1. Molecular cutch is engaged and rearward actin treadmilling slows
2. Results in forward movement of the filopodium
3. An actomyosin-based actin-tubulin link ‘pulls’ microtubules into extending filopodia

Question 5

What is needed to rearrange the cytoskeleton for forward movement?

Answer 5

The STIMULUS of the cue, attachment of the growth cone to a substrate is not enough

Question 6

How are growth cones repelled?

Answer 6

• Two types of neurons can be repulsed by each others axons, leading to a growth cone collapse
• They CAN attach but a signal is received that prevents the growth cone from travelling in that direction
• Growth cone collapse destabilises F-actin and the growth cone appears to retract

Question 7

What are semaphorins?

Answer 7

A family of inhibitory guidance cues
- Can be membrane-bound or secreted
- Secreted semaphorins can cause growth cones to have a collapsing effect on F-actin
- Can be used to channel axons and guide them where to go

Question 8

What do neurons prefer to grow on and why?

Answer 8

• Prefer to grow on laminin (a substrate) rather than collagen
• Laminin makes it easier for the substrates to bind to their growth cones which directs growth
• Laminin is a growth promoting ECM protein serving as a guidance cue, it also acts as a scaffold providing structural integrity for neuronal tissue
• Blocking receptors for laminin slows down the growth of retinal axons but does not change their direction
  (Laminin= permissive NOT guidance)

Question 9

What is axon growth a balance between?

Answer 9

permissive and non permissive factors

non-permissive factors can channel axons but axons still need permissive factors to grow

Question 10

What are Ephrins and Ephs and what do they do?

Answer 10

Ephrins = non-permissive contact repulsion factors
Ephs = molecules which detect ephrins (on axons)
GENERATES A REPULSIVE RESPONSE
- helps compartmentalise the embryo into discrete domains

Question 11

What are the four fources?

Answer 11

1. Contact attraction
2. Contact repulsion
3. Chemoattractant
4. Chemorepulsion

Contact attraction and repulsion tell them where they can and cannot go but chemoattractant and chemorepulsion tells them WHICH WAY they can and cannot grow

Question 12

What was Marysias work looking at chemoattractant and chemorepulsions?

Answer 12

• She looked at whether the organiser cells in the roof and floor plate influence axon growth
• Dorsal spinal chord was laid out and put in gel to observe axon growth
• Found that the floor plate attracts commissural axons towards it by secreting NETRIN (which is an ECM protein like laminin)
• Roof plate secretes BMPs which repel commissural axons (specifically BMP7)

Question 13

What is the role of Shh in guiding commissural axons?

Answer 13

• Same role as nectrin in that its secreted by the floor plate (as we know) and attracts commissural neurons
• This attraction can be blocked by CYCLOPAMINE which blocks Shh (doesn’t block nectrin)

Question 14

How was the role of Shh shown experimentally?

Answer 14

• Smoothened is a protein which acts as a receptor for the Shh ligands
• When Smoothened (Smo) is knocked out, Commissural axon guidance is disrupted

Question 15

What is cre recombinase and loxP

Answer 15

• Bacteriophage encodes a recombinase (cre) which enables it to insert its DNA into host bacteria’s genome
• Cre binds to loxP and can cut and rejoin to another loxP site
• This can be used to specifically delete DNA lying between two loxP sites e.g. in the mouse genome
• Known as a ‘floxed’ gene

Question 16

What is the benefits of cre recombinase and loxP in tissue specific knockouts and what are the stages?

Answer 16

• Conditional knockouts (gene deletion) at specific places and stages of development
  1. The target gene is modified by inducing LoxP sites
  2. Modified allele with LoxP is referred to as a floxed allele
  3. Mice expressing cre recombinase under the control of a tissue/promoter are then crossed with floxed LoxP mice
  4. Recombination occurs resulting in the deletion of the floxed region undergoing a knockout
  5. can knockout genes such as the smoothened gene

Question 17

What is an open book presentation?

Answer 17

• Cut open the hindbrain for example
• Lay it in a collagen gel
• After 18 hours or so add a lipophilic die
• Can be used to trace where axons have gone (anterogradely or retrogradely)

Question 18

How was an open book preparation used to look at the reprogramming of commissural axons?

Answer 18

• Commissural axons cross the midline
• Experiment wanted to look at whether reprogramming changes once midline is crossed
• This makes sense because floor plate midline secretes chemoattractants (nectrin and Shh) and if it crossed the midline and was still responsive, it would just turn back
• They added an ectopic floor plate before the midline and found through the OBP that the axons are no longer responsive to the original and they just continue past the original floor plate without turning

THIS IS IN A RAT HINDBRAIN

Question 19

How do commissural axons act differently in the same scenario (open book reprogramming) in the spinal chord?

Answer 19

• So in the hindbrain the axons carry through the original floor plate without turning (already spoken ab)
• But in the spinal chord with the same situation, the commissural axons become sensitive to something INHIBITORY in the floor plate
• The inhibitors are semaphorins and slits

Question 20

What is the underlying message from the open book presentation experiments for commissural axons in the spinal chord?

Answer 20

Initially, the axons must be sensitive to nectrin but not to semaphorin or slits, once the floor plate has been crossed the sensitivities switch

Question 21

Explain the robo mutant found in drosophila that effect the commissural pathway?

Answer 21

• Dorsophila mechanisms is highly conserved to the other species
• ‘Roundabout/Robo’ gene encodes a cell surface receptoron axons for slit
• The axons that do not cross the FP have high expression of Slit (obvs to stop them from crossing).
• Commissural axons that do cross the midline low levels before they cross (so they arent repelled from crossing) but high levels after they cross (so they cant recross)
• In Robo mutants, slit is no longer detected by the receptors so all axons go back and forth across the midline hence the name roundabout

Question 22

Explain the effects of Comm in drosophila

Answer 22

• Comm usually expressed in those neurons that normally cross the midline and is switched off after they cross
• Downregulated once the neuron has crossed
• When Comm is missing, Robo protein is at high levels everywhere and everything goes longitudinally
• Comm encodes a trafficking protein that prevents Robo from reaching the cell surface so the growth cone cannot receive slit inhibitory signals before it leaves

Question 23

What is axon fasciculation?

Answer 23

Nerves ‘bundled’ together to form a fascicle

Question 24

How does fasciculation occur?

Answer 24

‘Homophillic’ binding by cell adhesion molecules e.g. faciclin II
Cells expressing faciclin II two bind as faciclin II sticks to itself

Question 25

Where is Faciclin II expressed in drosophila?

Answer 25

In the longitudinal midline tracks, can observe bundles
KO shows axons all falling apart and not stuck to each other
Overexpression leads to inducing of fasciculations that aren’t meant to happen xx

Question 26

What happens when axons want to innervate e.g. a muscle

Answer 26

The axons defasciculate (unbundle) to go and innovate different muscle blocks

Question 27

What is a ‘By pass’ phenotype

Answer 27

Overexpression of Fasciculin II where the axons fail to defasciculate and miss their targets

Question 28

What does BEAT do?

Answer 28

Interferes with the cell adhesion molecules that usually keep fasciculation intact
Therefore, expression of BEAT can cause defasciculation

Question 29

What are the two targets of axons?

Answer 29

1. Discrete targets (cellular)
2. Topographic maps (multicellular)

Question 30

What happens if you ablate a cell that was a discrete target for an axon?

Answer 30

Failure of relevant motor axons to leave the main motor trunk at appropriate branch points
This implies there’s labels in the target cell for guidance

Question 31

What are the ‘address’ labels that discrete targets carry to direct the axon to them?

Answer 31

-** Netrins** (expressed in specific muscles) , loss of netrin is similar to ablating the whole cell showing its importance
- Netrin importance also shown by GOF experiments where the wrong cells have nectrin and are therefore innovated by the axon

• Faciclin 3 is another address label that is expressed in specific muscles and the axons that normally innervate them

Question 32

What were the two possibilities that Sperry proposed for topographic maps?

Answer 32

1. Each axon has a unique label complementary to a unique label in the target. (cf address labels in fly muscle)
2. That a co-ordinate system, encoded by gradients of signalling molecules, stamps a “latitude and longitude” onto cells of the target. This would be read by complementary gradients of receptors expressed in the retinal ganglion cells.

Question 33

What is a stripe assay?

Answer 33

lab technique that involves creating a pattern or stripe of a particular substance and observing the response of axons or cells to this pattern

Question 34

Which of Sperrys theories did he think was the most probable for topographic mappingand how did he test it?

Answer 34

The gradient one
- Took stripes of anterior and posterior membranes and laid them out to see different reactions in the retinal ganglion cells
- Found nasal neurons didn’t care whether they grew anterior or posterior but the temporal axons did
- This is due to the temporal axons avoiding a repellent factor in the posterior stripes

SHOWS THERE ARE GRADIENTS OF EPHRINS ON MULTICELLULAR TARGETS WHERE TOPOGRAPHIC MAPS ARE TO BE FORMED

Question 35

How was it found that cells from the posterior tectum makes a non-permissive factor that repels temporal axons?

Answer 35

• When heat abolishes posterior there is no T axon activity, when heat abolishes the anterior there is still activity.
• Posterior membranes cause temporal growth cones to collapse in vitro
• The ‘repellent’ is two inhibitory factors which are two ephrins expressed in a gradient from posterior to anterior
• In KO experiments with the two ephrins (A2 and A5) the temporal neurons project axons into the posterior tectum