Lecture 21- Axon guidance IV Flashcards
What is axon guidance important for?
-nervous system connectivity- precise wiring of the nervous system is important for neural function -axon’s finding their targets is incredibly important -the nerves have to find the target in a complex environment and very far e.g. the cyatic nerve -very important for Neuron migration – the cell will often follow the direction of the leading neurite to find the correct location (eg. Cortical neuron migration)
How are axons guided?
-guided to their target by environmental cues -Growing axons recognize various cues on the surface of other axons and cells -and respond to diffusible molecules such as Morphogens (these may be produced quite far away, also called ligands, diffusible molecules) - and use intermediate targets (cues on ther route -also cues on the target itself)
What are the environmental guidance cues sensed by on the axon?
-sensed by the growth cone -the growth cone is the one that senses most of the environmental guidance cues -they use receptors that are highly concentrated in the growth cone area, that activates signalling pathways resulting in cytoskeletal changes
What do the growth cones used to recognise guidance cues?
-environmental cues are detected by receptors on the growth cone -Ligand binding to receptors on the growth cone activates downstream signalling pathways which alter the actin cytoskeleton and change the direction of axon growth -cue= morphogens= ligand -generally affects Rho Rac pathway
What sort of cues can there be?
-Environmental cues may be attractive or repulsive -short range= usually contact dependent -long range= produced long way away, diffusible substances
How does the reaction to long range cues work?
-growth cones guided by cues released from a distance source (chemoattraction/ chemorepulsion) -Chemoattractants and repellents act in a gradient -the axon moves from low to high concentration (usually- if attractive) -growth cone can tell the difference in conc in 1 in a 1000 -attractive cue= there will be high conc close to the origin= -gradient of expression
What must happen for chemotaxis to occur?
- for chemotaxis to occur the growth cone must be positioned so that one side is exposed to a higher level of factor (to change direction) -e.g NGF (nerve growth factor) attracts axons, can see when in culture, always go to the source
What is the example of long range attractive cue in mice?
- Cues secreted from cells in the maxillary pad (whiskers)attract axons from the trigeminal ganglion
- tissue surrounding the maxillary pad has no affect on the axons
- the maxillary pad produces the neurotrophins, BDNF and NT-3 which attract the axons (two proteins that belong to the same family ad NGF)
- explant assay= take chunk of tissue from the origin and target
- take neighbouring tissue as well
Is repulsion as important as attraction in axon guidance?
-yes
What can contact with axons or cells result in in axons?
-contact with other axons or cells can repulse growth cones -contact dependent repulsion =causes the cytoskeleton to pull back quite quickly -e.g, sensory growth cone repelled by motor axon
Are axons growing from tissue explants repelled by the same tissues in vivo as in vitro?
-yes
What is the example of a repulsive cue?
- olfactory bulb neurons= grow along the septum, but do not grow into it
- something secerted by the septum to repulse them
- they head in the opposite direction to the septum
What is the example of a distant repellant cue?
-Growing axons may be repelled by distant factors (chemorepellents) -slit proteins are chemorepellents -act to repel axons
What are the main categories or guidance molecules?
- the main families
- ligands= environmental cues
- receptors that they will activate
- blue= the main function the pathway has -some are mainly involved in attraction and some in repulsion
- The Growth factors= acttraction
- Slit= repulsion -some can do both attraction and repulsion= netrin and ephrin
What is the example of an attractive guidance molecule?
- netrin signalling
- First identified in C.elegans (Unc6)
- in mammals, 3 secreted netrins (Netrin1,2,3) and 2 membrane bound netrins (Netrin G1 and G2)
- Receptors for secreted netrins: DCC, neogenin, Unc5A-5D and DSCAM (Unc40 in C. Elegans; Frazzled in Drosophila)
- Receptors for membrane bound netrins: NGL-1 and NGL-2(these are in the membrane)
- homologues in all species= in mammals multiple netrins secreted
- contact dependent molecules
- multiple receptors= initiate different UNC5 A- UNC5 B, UNC5 C , UNC5 D
- looking at the red ones
What is the downstream signalling pathway of netrin?
- the netrin:DCC signalling pathways
- netrin= binds to DCC -causes axon formation, cytoskeletal remodelling (axon guidance)
- transcription resulting in long term protein synthesis, microtubule dynamics
How did they discover the function of netrin?
-Growth cone turning assay –the growth cone goes toward sthe netrin origin= towards high concentration -showed in a time lapse series of a growth cone exposed to a gradient of Netrin-1 created by injection of concentrated solution from a micropipette
What are the dorsal commissural neurons?
- dorsal commissural neurons= dorsal column of the spinal cord where the cell bodies are, need to send the axons ventrally and to cross over the floor plate
- then head down anteriorly top form a white matter tract that runs longitudinally along the spinal cord
What was the experiment using dorsal commissural neurons?
- attraction of dorsal commissural axon guidance in the spinal cord, attracted by netrin-1 to the floorplate
- netrin is expresed by the floor plate region -growth cone expresses DCC (receptor for Netrin1)
- makes the axons grow towards the floor plate
- they also performed an explant assay= to make sure that it was netrin that was involved
- this confirmed that it is specifically netrin acting on the axons
What happens when you remove netrin 1 in axon guidance of the spine?
-Removal of Netrin-1 disrupts axonal outgrowth from the dorsal spinal cord (if you remove netrin= the dorsal spinal cord neurons are then not doing anything= disruption!) -Commissural axon guidance is disrupted in Netrin-1 knockout mice (they form a commisure at the floor plate region in normal mice -in knockouts= try to cross too early, disrupted growth)
What happens when you disrupt DCC?
-Disruption of DCC interactions blocks Netrin-1 dependent axon outgrowth -Addition of DCC blocking antibody inhibits the ability of netrin-1 to activate DCC and therefore axon growth is prevented -this is how they figured out that the DCC recptor was the responsible receptor -blocking the DCC receptor results in dirsuption in the dorsal spinal cord formation -get neurite still but don’t know where to go -Commissural axon guidance is disrupted in DCC knockout mice (they do not cross over )
Are other axonal pathways disrupted in netrin knockout mice?
-Other axonal pathways are disrupted in Netrin-1 knockout mice -also disrupted in corpus collosum -it doesn’t form! -they do not cross over and the communication between two hemispheres does not occur
What is the second example of attractive molecule signalling we talk about?
-Ephrin signalling -this is short range, contact dependent cue (like netrin was)
Are Ephrin ligands membrane bound?
-yes, thus the signalling is contact dependant
What are the two types of ephrins?
Class A and Class B
What are the Class A ephrins like?
- Class A ephrins (A1-A5) are tethered to the membrane by a GPI-link -usually bind to EphA… –class A do not have a transmmembrane link
What are class B ephrins like?
– Class B ephrins (B1-B3) have a transmembrane and cytoplasmic domain -usually will bind to V
What are the receptors that Ephrins act on?
- 14 Eph receptors, EphA1-A8, EphA10 and EphB1-B4, EphB6 – general rule ephrinA’s bind EphA and ephrinB’s bind EphB
What is the Eph-Ephrin signalling like?
- activation of Eph receptor results in receptor phosphorylation, Rho GTPase activation and remodelling of the actin cytoskeleton (forward signalling)
- ephrins are also able to signal (reverse signalling) (kinase activation and other signalling pathways)
- both signalling events can happen simultaneously (can result in a reaction on both cells)
What was an experiment showing the ephrin signalling as an attractive cue?
-Activation of ephrin by EphA7-Fc induces spreading of the growth cone -growth cone will grow towards the cell with EphA7¨-EphA7-FC = a fusion protein containing the extracellular domain of EphA7 attached to the Fc domain of an antibody -Addition of EphA7-Fc to neurons in culture activates ephrin on the growth cone -to make them soluble= take the extracellular domain= and bind to antibody= so can isolate it -attractive type of resposne of the growth cone
Another experiment with ephrins?
-Activation of ephrin by EphA7-Fc induces motor neuron axon outgrowth -ephA7 makes the axons grow
What does the activation of ephrin signalling cause?
-Activation of ephrin signalling causes growth cone attraction -when have an EphA receptor= binds to ephrin A= will make the axon grow = attractive cue
Summary?
- Axons are guided to their targets by environmental cues - Environmental cues bind to receptors on the growth cone which activates downstream signalling events leading to changes in the actin cytoskeleton - Environmental cues may be short range or long range (for example Eph/ephrin or netrin) - Environmental cues may be attractive or repulsive - Netrin/DCC signalling and Eph/ephrin signalling act as chemoattractants
