L3 - Gradients in Axon Guidance Flashcards
What is the gradient guiding c-axons from the roofplate to floorplate
ChemoREPELLENT BMPs from the roofplate
ChemoATTRACTANT Netrins from the floorplate
What is dorsal
Roof plate
What is ventral
Floor plate
Cells that express the NETRIN gene are able to
Turn c-axons
Most of the c-axons in mammals turn …
Rostrally
A rostal turn is from _____ to _______
Posterior to anterior
What are the two possibilities for WHY the c-axons make the rostral turn
AP gradient of chemoattractant vs repellent
Short range, non diffusible cue
What would be the prediction that could be made if the cue was diffusible vs non diffusible
A small explant will lose its gradient if the chemotrophic agent is diffusible (large explant won’t)
If the guiding factor was non diffusible then this would not be the case
Evidence from Journal club paper
What are the questions to identifying the candidate molecule
Right place right time
Suff
Nesc
How did the journal club paper identify Wnts as the candidate molecule
Only Wnts had any effect
Wnt4 expressed in a AP gradient in the FP
Wnt4 transfected cos cells can turn post crossing axons in either directions
Fz3 KO mouse has confused turns post floor plate
Wnt4 binds to Fzd3
Wnt gradients act over distances of …
At least few mm
What are the two ways a cell can detect gradients
Temporal detection
Spatial detection
What is temporal detection
Change in concentration over time
Cell must compare ammount of ligand at two different time points
What is spatial detections
Change in concentration across the cell (or growth cone)
Cell must compare the ammount of ligand at two different points on its surface
What mech of detection is likely to be most useful for SLOW MOVING EUKARYOTES
Spatial detection
What is the accuracy of orientation towards a source dependent on
The absolute concentration
Steepness of the concnetration gradient
When is the most accourate response seen. Why is this?
When absolute conc closest to Kd
Since will have half of the receptors occupied and will be easiest to detect change
What is the gradient expressed as
% change per um
A gradient of 0.2% is equivilant to a ____ change per 10um
2%
Cells with a ______ diameter oritent better than _______ cells in shallower gradeints
LARGER
SMALLER
What is amplification used for
To enhance sensitivity to gradients
What does amplificiation require
Local enhancement of signal together wirh inhibition of signal reception in other parts of the cell
Describe one suggest mechanism of receptor enhancement
Clustering of receptors/signalling components in region where receptors are activated by transporting components from another part of the cell
Chemoattractants is leukocytes and dictostellium activate ____
PI3K via a GPCR
What are the keypoints from the chemoattractant induced signalling pathway in leukocytes and dictostellium
Chemoattractant detected by a GPCR (7TMD protein)
Activation of PI3K causes phos of PIP2 –> PIP3
Provides a docking site for proteins with a PH domains such as Akt - localised signalling domain on the membrane
What is the action of PTEN
WHy is this important
Antagonises action of PI3K
Phosphatase that acts on PIP3 –> PIP2
Ensures signals highly localised and dependent on constant external activation
PHs domains are recruited to which part of a chemotaxing cell
LEading edge
What is the effect of a PI3 K inhibitor in neurones
Disruption of growth cone turning induced by netrin
Recent studies have shown PH domain proteins also localise in
Growth conesq
Describe the results of the experiment that showed PH domains proteins during growth cone stimulation
xenopus SC neurone expressing Akt-PH-GFP
Turns in response to BDNF
Can see fluctuations which shows the PIP3 labile nature to maintain its responsiveness
What type of vesicular transport occurs in growth cones in response to cues
Asymmetric
Describe the experiments that showed asymmetric vesicle transport was involved
Vesicles tagged with FM1 membrane dye
Growth cone loaded with caged Ca
UV flash causes release of Ca
Vesicular transport to the source of Ca
Vesicles are seen associated with _____- suggesting …
Associated with MT
Suggeests they are activeyl transported along MT
What is the second mechanism to enhance the range over which cues can be detected
Adaptation
Why is adaptation needed
Receptors saturate as concentration rises
Even the shallowed gradients required to turn a cell will result in 90% receptor occupancy
What is an example of adaptation as seen in normal physiology
Synaptic transmission signal saturation leads to sensitisation and the signal falls
Describe the frog axon turning assay
Using a Xenopus spinal cord neurone turning assay
Axons of isolated neurone from early emrbyonic Xenopus spinal cord respond to netrin pulsed into a dish from a pipette
Which lab performed the frog axon turning assay
Mu ming poo
Evidence for adaptation in the response to netrin
Pulsed netrin creates an exponential gradient revealed by including a fluro marker in pipette
Growth cones zig zag
Suggests an alternative pattern of attraction and repulsion = cycles of desenstisation and sensitisation of the grwoth cone to netrin
How was the pulsing netrin expeiment taken forward
What (first) was the aim
To determine if GCs adapt to increasing attractant concentrations Ming et al.
Pretreated axons with different concentrations of netrin
What were the results of Ming et al study pre treating the axons with netrin
Imm after treatment growth cones fail to respond
After a longer period GCs resensitised
Resensitisation dep on MAPK activation and local protein synthesis in the growth cone
what other molecule elicits a similar response of adaptation ….
Sema3A
what determines the distance that growth cones could be guided by gradients
Type of molecule and how the gradient is set up
Examples of diffusible molecules forming gdts
Wnt - expression gdt along an axis
Netrins - point source from the FP
Examples of non diffusible molecules forming gdts
Ephrins - surface bound molecules expressed in gradients
Poiint source (diffusible) gdts are typically __________ and have a theoretical max range of ________
Expo
1mm
How does the theoretical max range of a point source grf correspond to what is seen in vivo
Roof plate (BMPs) and floor plate (Netrins) act over this distance
What do point sources depend on
Limited diffusion rate
Some mechanism for removing the guiding molecule
What happens if there isnt a mechanism for removing the molecule
Gdt will flatten over time
What is different in the linear substrate bound gradient
Can be much longer and has a theoretical max range of 1cm
How does the theoretical max range of a linear substrate bound gradient correspond to what is seen in vivo
Ephrins in the tectum
Describe the distribution of Netrin mRNA in the chick
Netrin1 localised to the floorplate
BUT netrin2 expressed throughout
How many netrin genes in mouse
How does its expression pattern compare to that of the chick
Only single netrin gene
mRNA pattern is a combination of the chick genes with notable expression in the ventricular zone
What is the structure of the netrin protein, what does this suggest about it … how is this confirmed by what is seen in vivo
lamin like structure might be bound in the ECM
Found associated with basement membrane of the spinal cord
What has recent evidence shown about the nessecity of Netrin
May not be required in the FP for C axon guidance
Is Wnt4 a point source
No likely to be graded expression - expression likely to vary temporally
Expression likely to change from rostra to caudal (A-P) as dev proceeds
