Axon guidance Molecule - Netrins & Slits

Question 1

Name the secreted Netrins in the mammalian genome

Answer 1

• Netrin1
• Netrin2
• Netrin4

Question 2

Will neuritis grow in the absence of cues?

describe the study that tested this

Answer 2

In 2002, Goldberg et al:
1) Expressed the adenovirus coding for the anti-apoptotic protein ‘bcl-2’ in cultures RGCs (from rat embryos)

2) raised the cells in a serum free medium (containing no trophic factors)
3) They found that the cells survived but after 7 days no neurites had formed.
4) when the same experiment was done with trophic factors (BDNF & CDNF) neurites grew.

This shows that the extension of axons & dendrites is not intrinsically programmed into neurons. Thus neurites do require cues to initiate growth.

Question 3

Name the membrane bound netrins and their receptors:

Answer 3

ligand Receptor

Netrin G1 = NGL1
Netrin G2 = NGL2

Question 4

What are the Receptors for Netrin 1,2&4?

Answer 4

• DCC
• DSCAM
• UNC5

Question 5

What is the primary role of Netrins?

Answer 5

The Tessier-Lavigne group showed the primary role for Netrins is to guide commissural neurons centrally towards the midline

Question 6

Where is Netrin 1 & 2 expressed in a chick embryo?

Answer 6

Kennedy et al(1994) did insitu hybridisation for Netrin 1 & 2 mRNA.

They found that during periods of commissural axon outgrowth:
Netrin 1 was strongly expressed in the floor plate
Netrin 2 is diffusely expressed in the ventral spinal cord

Question 7

How was it discovered that Netrins control axon outgrowth?

Answer 7

(Kennedy et al, 1994)
1) They transfected COS cells with Netrin 1&2

2) They removed the dorsal spinal cord of the rat embryo and cultures these with the transfected COS cells
3) when they examined the axons growth patterns growth patterns they saw that axons grow towards COS cells expressing Netrin1 & 2 but not in control COS cells (that do not express Netrins)

This concluded that netrins are secreted diffusible molecules that influence axon outgrowth

Question 8

What happens to commissural axons in Netrin1 mutant mice?

Answer 8

Serafini et al (1996)
1)spinal cord sections of WT and mutant mice were labelled with TAG1 antibodies (a commissural axon marker)

2) in the WT; axons grow ventrally to reach the floorplate before crossing to the opposite side of the spinal cord
3) In the mutants; axons fail to reach the floorplate & often turn inward toward the ventricle or outward.

Question 9

Where are trochlear neurons locates?

Answer 9

At the midbrain-hindbrain boundary

Question 10

Describe the study that shows Netrin 1 to be a chemorepellant

Answer 10

Colamario & Tessier-Lavigne (1995)

1) transfected COS cells with Netrin 1
2) remove the ventral aspect of the hindbrain-midbrain boundary (which contains immature trochlear neurons)
3) culture the ventral hindbrain-midbrain boundary either with ‘control COS cells’ or ‘COS cells expressing Netrin1’. The COS cells are put below the ventral hindbrain-midbrain boundary, to mimic a dorsal region of Netrin1.
4) Stain the cultures with F84.1 to visualise the trochlear axons

Results:
Control - trochlear axons grew dorsally away from the floor plate
With Netrin1- axons did not grow

Conclusion:
Netrin1 repels trochlear axons

Question 11

What mediates whether Netrins are repulsive or attractive to an axon !

Answer 11

The type of Receptor on the growth cone

Question 12

Which receptor allows Netrin to act as a chemoattractive cue?

Answer 12

DCC Receptor

They activate signalling pathways that promote actin filament extension & stabilisation

Question 13

Which receptor allows Netrin to act as a chemorepellant cue?

Answer 13

UNC5 Receptor
The activate signalling pathways that results in actin depolymerisation & GC collapse. This, preventing the neurites extending in that region.

Question 14

What are the names of the secreted slits?

Answer 14

SLIT 1,2 &4

Question 15

What are the Receptors for slits?

Answer 15

The cell surface R ; Robo

Question 16

How many types of Robo R are there?

Answer 16

4

Robo 1,2,3&4

Question 17

Where are slit proteins expressed in Drosophila?

Answer 17

Along the ventral midline

Question 18

Where is Robo expressed in Drosophila?

Answer 18

In longitudinally projecting (ipsilateral) axons

Question 19

What occurs in Robo mutants ?

Answer 19

Neurons that normally project longitudinally now randomly cross the commissure

Question 20

Is Robo expressed in commissural axons?

Answer 20

Not before the commissural axons approach the midline

But as they cross the midline Robo is strongly expressed. This is because the axons wouldn’t cross if Robo was expressed prior to cross because Robo acts as a chemorepellant

Question 21

Why don’t ipsilateral axons cross the midline?

Answer 21

Ipsilateral axons always express Robo
Slit is always expressed at the ventral midline

It is likely slit is a chemorepellant for Robo so prevents the ipsilateral axons going near the midline

Question 22

What happens when slit is mutated

Answer 22

If an individual slit is mutated, very little occurs.

But Long et al, 2004 found that if all 3 slots are mutated the GC won’t go all the way across the midline or will recross the midline

Question 23

How do Robo levels change in the commissural axons during midline crossing?

Answer 23

The answer for this comes from Drosophila commissureless mutant studies.

Another protein ‘Comm’ is expressed and sorts Robo for degradation before and as commissural axons enter the midline so Robo & the slit present in the midline doesn’t repel