Growth and Guidance of Axons Flashcards
How many genes are there in the human genome?
Around 20,000
What are the names of the two people who made theories on how specific neuronal connectivity arises?
Wiess - Resonance Theory
Sperry - Chemoaffinity Hypothesis
Describe Weiss’s Resonance Theory
Stochastic/ random and diffuse neuronal outgrowth first occurs towards all targets, and then their is the elimination of non-functional connections
Describe Sperry’s Chemoaffinity Theory
Directed and specific outgrowth occurs through axons following ‘individual identification tags’ carried by cells and fibres of the embryo
There is no pruning back
Describe the basic anatomy of the newts visual system
They have a lens -> Retina -> Tectum
The nasal retina is more anterior on the eye but projects most posteriorly on the the tectum
The temporal retina of the eye is posterior on the eye but projects anteriorly onto the tectum
How did Sperry test which theory was right?
He cut the optic nerve and removed the temporal retina - this allowed only the nasal axons to grow back
If Sperry was right the nasal axons would go back and only go to the posterior end of the tectum
If Weiss was right there would be lots of growth first and the axons would be pruned back later
He found that the axons grew back directly to the right place
What did Sperry see in his newt experiment?
He saw that patterns of outgrowth were highly organised, sterotyped and reproducible
How do motor neurons develop in the chick embryo?
They are guided specifically to their targets
How can it be shown that motor neurons are guided in the chick embryo?
Normally the LS3 neuron innervates T7
If LS3 is removed and put in a different place in the embryo the neuron still finds its way to the target even though the motor neurons are in the wrong place
What are guidance cues?
Factors in the enviroment that axons can use to fins their correct target
What did Cajal propose?
That the growing tip of the axon - the growth cone - sensed cues in the enviroment
What could be the result of ablating cells?
Ablating cells can change the axon pathway
What is the labelled pathway hypothesis?
Axons can selectively fasiculate with other axons
Axon surfaces carry labels or cues
Different growth cones express different receptors for cues
What is the role of early axons
Pioneer axons form an axon scaffold which later neurons can follow and extend on
What is the role of subplate neurons in mammals?
They project from the cortex to the thalamus prior to the innervation of the cortex by the lateral geniculate nucleus neurons
What happens if you ablate the subplate neurons
The geniculate neurons fail to innervate the thalamus showing that the subplate neurons are required as a scaffold
What is the general pathway of the Til axon in the grasshopper ie where does it turn?
It turns at the the limb boundary (down) and then again as it approaches the specific cell Cx1
Ablation of Cx1 in the grasshopper has what effect on the Til axon?
It stalls at the other side of the limb boundary and does not cross it
What are the four ways in which guidance cues can act?
1) Contact attractant
2) Contact repulsion
3) Chemoattraction
4) Chemorepulsion
Why are aplysia growth cones ideal when studying growth cones?
They have flattened growth cones so it is easy to see their components
What are the components of the growth cone?
Central part - contains microtubule
Transitional part
Peripheral part - contains actin
Lamella and filopodia are made up of what?
F-actin
What is the structure of actin in lamella?
Actin bundles are cross-linked into a net
What is the structure of actin in filopodia?
The actin bundles are polarised to form larger bundles
Describe F actin treadmills in a resting growth cone
There is clear tracking of the actin from the periphery of the growth cone to the centre - the actin is recycled after being broken down into the actin subunits
What is the action of tubulin?
Tubulin is mainly centralised but will shoot up and down the back bone of the filopodia
What happens when a growth cone comes into contact with an attractive cue?
F-actin treadmilling slows and accumulates
This accumulation causes the filopodia to become stable and it drags microtubules to the back of the filopodia
What two key components lead to filopodial extension and reorganisation of microtubules?
The flow backwards is arrested by the engagement of a molecular clutch - receptor binds to cues which causes extension and results in the forward movement of the filopodia
Also an action-myosin-based actin-tubulin link pulls microtubules into the wake of the extending filopodia
What is growth cone collapse?
A growth cone comes into contact with an axon and collapses
A connection is maintained showing that a signal is being sent so the growth cones do not fall back due to the lack of adhesions
F-actin is destabilised
What are semaphorins?
A family of inhibitory guidance cues
They can be membrane bound or secreted
Secreted semaphorins can cause neurons to collapse effecting the f-actin
What are the two substrates that axons can put down and what are they like?
Poly-ornithine (porn, sticky)
Pallodium (non sticky)
Which substrate do growth cones not grow over?
Pallodium
Axon growth is better on what?
Laminin despite the adhesion to collagen is better
What does laminin do in relation to growth of axons and how can this be shown?
It does not direct growth but it allows it
This can be seen because blocking the receptor for laminin does not change the direction of growth but does slow it
Gradients of laminin in vitro do not direct axon growth
Where is semaphorin 1 expressed?
Along the limb body boundary where Ti1 turns and avoids
What is the effect of using semaphorin 1 antibodies?
The Ti1 axon crosses the limb body boundary
What happens in mice lacking semaphorin 3A?
Axons will stray into the wrong territories of the limb
What are ephrins?
Non-permissive factors that cause contact repulsion
They are detected by cell surface receptors called ephs
They are tyrosine kinases
They cause repulsion
What does commisural mean?
It crosses the midline
What is the normal pathway of sensory relay neurons in the rodent spinal cord?
They are commisural, they grow from the roof plate towards the floor plate
What experiment did Placzek and Tessier Lavigne perform?
They took dorsal explants = normal growth in the culture initially
The axon will turn towards the floor plate as the floor plate secretes a chemoattractant
They purified the floor plate to find the chemoattractant netrin
What is netrin?
Netrin is secreted and is similar to laminin, which can associate within the extracellular matrix
Cells expressing netrin attract axons
The effect of netrin is only short
Cells expressing BMP repel or attract axons?
Repel
What does cyclpopamine block?
Shh signalling
What is cre recombinase?
It allows bacteriophage p1 to insert its DNA into the bacteria hosts genome
Its own DNA can then replicate
What does cre bind to?
A specific 34 base pair sequence called loxP which it can cut and rejoin to another loxP site
What is cre recombinase and loxP used for?
To delete DNA specifically between loxP sites - the removed gene is degraded
Gradients of morphogens are re used later for what?
To shape axons
Shh and BMPs specify neural plate
What happens if you knockout or lower the gradient of semaphorin 2?
Ti1 guidance is disrupted in a way that suggests that secreted sema2 directs Ti1 growth cones towards the body
When do growth cones re-programme?
When intermediate or choice points are encountered
When do commisural axons lose responsiveness to netrins?
After they cross the midline
This explains why in the hindbrain commisural axons are able to continue past the floor plate without turning
What are lipophilic dyes used for?
Image tracing, membrane tracking and axon tracing
When do commisural axons become sensitive to repellents?
After they cross the midline
What are the two key genes involved in preventing a an axon to recross the midline
Roundabout and commisureless
What are slit proteins?
Inhibitory proteins found in the floor plate
What does the roundabout gene code for?
A slit protein
Where is roundabout expressed?
It is expressed in high levels on axons that do not cross the midline
Commisural axons initially express low levels but they express high levels after they have crossed the midline
What is the result of a mutation in roundabout?
The axons cross the midline multiple times
Where is commisural expressed?
In axons that cross the midline - it is switched off after they have crossed
What happens if the commisural protein is missing?
Roundabout is expressed at much higher levels in the cells that would normally cross the midline and so the axons extend longitudinally so they do not cross the midline
How does commisural control roundabout?
At a post transcriptional level locally
Como encodes a trafficking protein which prevents robo from reaching the cell surface so that the growth cone can not receive slit inhibitory signals
What is the commisural homologue in vertebrates?
There is not one
However, there is another robo like gene called Rig1 which is expressed only in pre crossing fibres to block robo1
What is the roundabout homologue in vertebrates
Robo1
What is the role of fascicilin ii in insects?
It binds one cell surface to another cell surface
If expressed in cells that do not normally adhere, fascii can cause adhesion
It controls fasciculation in flies
It also controls defasciculation so that the right motor neuron can leave to innervate muscle
What is a discrete target?
A single cellular target
What does ablating specific muscle precursor cells show about the development of motor neurons?
The motor neurons fail to leave the main motor trunk at the appropriate branch points which suggests they are looking for specific labels
What occurs in topographic maps?
Neighbouring neurons send axons to neighbouring sites in their targets to maintain the topology in the target as in in the retinotectal system
What does the stripe assay show?
That cells from the posterior tectum make a non-permissive factor that repels temporal retinal axons
Nasal axons ignore whether they are in a posterior or anterior part but temporal axons avoid growing on posterior stripes so they must be avoiding a repellent factor
What happens if the posterior stripes are treated and what does this show?
The temporal axons will now grow on the posterior strips showing that a repellent protein must have been denatured
What are the inhibitory proteins in the posterior tectum?
Two ephrins - ephrin A2 and ephrin A5
What happens in mice if ephrin A2b and A5 is knocked out in mice?
The temporal axon grows to the posterior tectum so the topographic map is disordered
