Axon guidance Flashcards
Evidecne that neurite relies on extracellular signals?
In absence of trophic factors, no neurites formed after 7 days in culture
- Neurites formed in presence of trophic factors (brain derived and ciliary derived neurotrophic factors).
- Therefore, immature neurons are not intrinsically programmed to extend neurites.
- Rather external signals are necessary to induce neurite growth.
Most neurons grow best in the presence of a combination of different growth factors.
Role of Netrins in axon guidance with reference to relevant expeirment
Tessier-Lavigne group showed that a primary role for netrins is to guide commissural neurons ventrally towards the midline.
During periods of commissural axon outgrowth, netrin-1 is strongly expressed in the floor plate and netrin-2 is diffusely expressed above the floor plate in the ventral spinal cord.
What are slits?
- Slits are secreted axon guidance molecules.
- The mammalian genome codes for 3 secreted slits (slit 1,2 & 4).
- Slits bind to the Robo cell surface receptors expressed by neurites.
- Four Robo receptors in the vertebrate genome:
- Robo1
- Robo2
- Robo3
Robo4
describe roel of slit-robo signalling
- In developing Drosophila, Slit proteins are expressed along the ventral midline.
- Robo is expressed in longitudinally projecting (ipsilateral) axons.
- Longitudinal axons aberrantly cross the midline in Robo mutants
In Robo mutants, neurons that normally project longitudinally now randomly cross commissure.
Describe slit-robo signalling in vertebrates
- Slit-Robo interactions prevent neurons from aberrantly crossing midline.
- Longitudinal axons express high levels of Robo and so steer away from Slit-rich midline.
- Commissural axons initially don’t express Robo, so can freely cross midline.
However, once they have passed through the midline, Robo is upregulated in commissural axons. This prevents re-crossing and may also provide the initial repulsive force to drive newly crossed axons away from midline, ensuring they join the contralateral fascicle.
Evidence for slit-robo signalling in vertebrates
- Labelling commissural neurons by implanting lipophilic fluorescent dye DiI into dorsal spinal cord.
- In control, commissural axons grow ventrally, cross the midline and then run longitudinally in a fascicle.
- In Slit triple mutants, growth cones often stall at midline or cross and then re-cross midline.
Evidence showing how Robo levels change in commissural axons during midline crossing?
- Immunohistochemical evidence shows that prior to and at onset of commissure crossing, Robo is downregulated in commissural axons.
However, neurites progress through the commissure, Robo becomes upregulated.
Detailed analysis of Comm expression reveals that it is upregulated in commissural axons during and after midline crossing
Evidence that netrins can act as chemeorepellant
- COS cells transfected with netrin-1.
- Result: in control, trochlear axons grow dorsally away from floor plate. However, in the presence of a dorsal source of netrin-1, axons do not grow.
Conclusion, netrin-1 repels trochlear axons.
Evidence that effects of Netrins-1 is selective for commisural axons
- Neurofilament antibody staining of motor neuron (mn) axons (that innervate the muscle) and dorsal root ganglia (d) sensory neurons (that project into the dorsal spinal cord).
- Axon trajectories of these neurons are unaffected by netrin-1 mutation.
Netrin functions are evolutionarily conserved
Explanation for the difference in effect of netrins-1 on commisural and trochlear neurons
- Commissural neurons are attracted to ventral aspect of cord by netrin.
- Trochlear neurons are repelled from midline by netrin.
Activation of diff receptor subtypes.
Attraction- DCC receptors, actin filament extension and stabilisation pathways activated
Repulsion-UNC-5 receptors; signalling pathways of actin depolymerisation and growth cone collapse
How do Robo levels change in commissural axons during midline crossing?
Via Comm- TM protein expressed in commissural axons; not expressed in longitudinal axons.
Comm expression is upregulated in commissural axons during midline crossing.
When Comm is expressed, it associates with Robo and recruits it to endosomes
Comm expression is extinguished after commissural axons cross midline
Comm homolog in mammals- USP33
USP33 co-immunoprecipitates with Robo.
Therefore, when USP33 is present, it prevents Robo from being targeted for degradation
Evidence of importance of USP33 in comisural axons and crossing the midline.
Knockdown of USP33: commissural axons fail to cross midline
Loss of USP33= degradation of ROBO.
Growth cones will become insensitive to Slit.
Stall at midline.
Knockdown of USP33 prevents Slit-mediated growth cone collapse
