Axon guidance Flashcards
what prevents growth cone collapse?
ligands for receptors on the regrowth cone
ere can axon guidance cues be found?
- on the ECM or diffuse from the target
what are the 4 families of axon cues?
- slits and their receptors
- ephrins and EPH receptors
- semaphorins (neuropilin and plexin)
- VEGF-A and its receptor
what do ROBO receptors bind to?
SLIT ligands
what so SLIT ligands bind to?
the robo receptors
what do EPH receptors bind to?
ephrins
what do ephrins bind to?
EPH receptors
what do semaphorins bind to?
neuropilin and an A-type plexi
what so neurpilin and A-type plexus bind to?
Semaphorins
what does the fact that both the EPH and the ephrins have intracellular domain mean?
there can be bidirectional signalling
what is so special about SEMA3E?
it can bind the A type plexin (plexinD1) without the neuropillin
what does the fact there are many combinations of the 3 semaphoring and 4 neuroplexins and 2 neuropillins mean?
many combinations can result for- when you have limited genes you can have lots of outcomes
what can neurpillin bind that isn’t semaphorin?
- VEGF164
when these bind what do they normally activate?
the PI3K pathways because this is involved in cytoskeletal
what is an in vivo approach to axon guidance studies?
- in situ hybridisation to monitor the expression of the candidate gene
- antibody labelling to visualise axons relative to their axon guidance cue
- axon tracing by lipophilic dye diffusion in axonal membranes
what is an ex vivo approach to looking at axon guidance cues?
- explant assays of neural tissues
- neurons differentiated from proliferative progenitor cell lines
what is an example of an ex vivo approach?
explant spinal cord tissue and explant a source of signal and see if the axons will grow towards it
what is a growth cone turning assay?
you can putt he axons in culture and then puff your guidance into the culture medium and then look at the turning towards the pipette and then can add an antibody and see if you get the same result or not
what is the dye used to retrogradely label the neurons?
DiI
what was an explant culture used to identify the correceptor of SEMA3A?
They placed sensory neurones from dorsal root ganglion and use COS cells that had been transfected with sema3a and you put them next to each other and if there nothing in the cells then there is no repulsion and the axons grow all the way around, but if you put SEMA3A in then the axons are repelled.
- then had a backer
- then they used DRGs from KO mice and they looked at different plexins KO
- they used a Plexina4 KO and there were partial repuslions
- then they sis plexina3 and there are some repulsion too but not completely
- but then they did a double KO and the axons were completley blind tot he SEMA3 so this showed that both of these are required int he cell for a repulsive affect
once they had done the explant culture and shown that plxn3A and plexin4A was needed in conjunction with neuropillin to bind semaphorin3A, how did they show that this was the case ex vivo?
they use embryonic KO mice and they strained for neurofilaments and they showed that normally the axons dont grow along the ridge of the arm but without plexin3A and the plexin4A there are axons growing on the ridge. They also looked at the neural tube and they found that normally there are no axons but there were in the double mutants
what are the two models that are used to look at the commissural axon guidance?
the drosophila and the mouse
how were the molecules involved in the formation of the commissural axons in the dorosphila nerve cord found, what were found and what were the phenotypes?
they did a screen and stained the commissary neurons and they found slit mutant which has no organisation of the axons and they were all bunched up like a slit (SLIT) and they also found the opposing phenotype where there were only commissural neurons going round and round in circles like a roundabout (ROBO)
what do the midline glial express at the commissures of the ventral nerve cord?
SLIT
what do the non crossing axons at the drosophila commissural nerve cord?
ROBO
explain how SLIT and ROBO regulate the crossing of axons at the nerve cord of the fly?
- in the wild type you have the midline expressing SLITS (glia)
- some axons have high levels of ROBO (non crossing) and others have low levels (crossing)
- the ones with low will not be repelled and will cross but to stop them crossing the axon upregulates ROBO once it has crossed
how can the ROBO mutants be explained?
- no ROBO means that axons aren’t stopped from crossing so they cross back
why is the role of SLIT and ROBO more ciliated in vertebrates than in flies?
there are 3 SLIT receptors and 3 ROBOs
how was the role of SLIT and ROBO in the commissure in the vertebrate looked at?
they did an in situ and look at where slits and robos were expressed.
- they found that ROBOs were expressed in the commissural neurons (overlap with TAG-1 staining)
- they found that the slits are all expressed at the midline in the same place and so they had to make tripled KOs and they found that too many axons were crossing and the commissure was very thick
- then they did an in vitro study and put COS cells transfected with SLIT2 and they used E11 spinal cord tissue and they showed that SLIT repels the axons
what makes the retinal chiasm so hard to look at the cross over of neurons?
- the cross over only occurs at a certain point along the AP axis
how did they find that SLITs positioned the optic chiasm?
- they did in situ for slit2 and found that slit2 is concentrated at a particular point and then when they do a stain for the axons you see that the slit is not where the axons are and just after there are axons and then there are slits again
- they form a corridor of crossing
how did they look at the result of mutating SLITs in the optic chiasm?
they used a DiI stain and inject into the eye and see the axons projecting. And when you look at the SLIT1/2 double mutant then there isn’t a specific place where axons are crossing so the commissure is not placed correctly
as well as SLIt and ROBO, what other guidance molecules in involved in the optic chiasm and why?
the eph/ephrinB1
what is the ventrotemporal crescent?
where all of the ipsilateral neurons that dont cross
what is expressed in the ventrotemporal crescent ?
ephb1
what ephb1 a receptor for?
ephrin B2
after finding that ephrin B1 was expressed in the ventrotemporal crescent, how did they go about investigating its role
they make a ephB1 KO and they used DiI and they found that the ipsilateral projection is completely gone - this shows that it is important to sort the ipsilateral tract
- then they did in situ for ephri B2 and they find that at E12.5-17.5 you see that most of the axons cross as this point and this is when ephrin B2 is at its highest
- then they made a ephB4 mRNA and swamped the system and this mopped up all of the ephrin B4 ligand and there was nothing for ephB1 endogenous to respond and this results in ipsilateral projection being missing
what is the general way that ephB1 and ephrin B2 interact at the optic chiasm?
- ephrin B2 is expressed at the chiasm and acts to repel the EPHB1 expressing neurons
what helps axons cross the optic chiasm?
VEGF- binds NRP1
what does in situ of VEGF show?
there is lots of VEGF around the optic chiasm
how did they study the role of VEGF?
- they deleted the neurpilin binding domain in the gene for VEGF
- then you look at the optic chiasm neurons and you see that the ipsilateral axons increase so too many axons go to the ipsilateral side
- do an expression analysis for nrp1 and for ipsilaterally expressing neurons in the RGC then you see there is no overlap
- they used the DiI labelling and looked where the neurons that express nrp1 originate from by putting it into the brain and you see that most goes contralaterally and a few ipsilaterally but then when you KO Nrp1 the axons dont know where to go so they dont cross and they stay ipsilaterllay
how did they use growth cone turning with VEGF?
they did a growth cone urning test with NRP1 expressing cells and you see that there is turning and when you add antibody you lose this
so what are the three things that happen int he optic chiasm?
- the SLITs and the robot position the chiasm
- te EPHB1 and EPRHINB2 are involved n ipsilateral repulsion
- VEGF and NRP1 for promoting crossing the midline
