Lecture 13 Axon guidance 3 Flashcards
Which patterning factors are reused for axon guidance
In spinal cord
BMP7 in RP = push
Netrin in FP = pull
BMP7 and Netrin are examples of
Chemotropic factors
What does BMP7 antagonise
Effects of Shh on DV axis
What happens in a Netrin knockout?
there is disruption at the floor plate where the C axons don’t cross the midline
Some still do due to BMP7
What else can guide axons?
Shh
Where is Shh expressed and effect on axons
In FP as C axons extending
COS cells expressing Shh attract C axons in explant assays
What is SHh attraction blocked by?
Cyclopamine
How does cyclopamine block SHH signalling?
binds to smo, from corn lillies
What does cyclopamine not block and what does this show
It doesn’t block netrin so 2 separate pathways guiding C axons
What is Cre and what is Lox P?
Cre - recombinase encoded by bacteriophage P1 (which inserts its DNA into host bacterial genome)
Lox P - the DNA sequence is can bind to - cut and rejoined to another Lox P site by Cre to create floxed gene
What is the cre-lox system
A type of recombination method in ES cells
You can manipulate genes at specific developmental points using an antibiotic controlled promoter
Bacteriophage P1
Length of LoxP
34 BP seq
What can the cre-lox system be used for?
use this to specifically delete DNA lying between two loxP sites
What promotor is used to drive expression in Cre of C axons derived from midline
Wnt1
A mouse WT mouse is crossed with what
Transgenic mouse expressing Cre under control of a specific promotor which creates a mouse with w/floxed gene in same cell. When specificall exp Cre turned on, deletes specific gene
What is the cre-lox system used for
Tissue specific or inducible knock outs
What are the different stages of growth cone navigation broken up by
Intermediate targets = choice points
What happens when axons reach intermediate targets (choice points)?
the axons reprogram depending on what information they receive here i.e sensitivity to specific guidance cues changes
What do axons lose sensitivity to when they cross the midline?
Netrin
What experiment shows this loss of sensitivity to netrin after crossing midline?
BY USING LIPOPHILIC DYES TO TRACE AXONS:
- if axons are exposed to floor plate cells before crossing then midline , they turn towards them
- if axons are exposed to floor plate cells after they cross the midline, they do not turn
What do experiment showing this loss of sensitivity to netrin after crossing midline not explain?
Explains in hindbrain axons C axons continue past FP without turning
Doesn’t explain why in spinal cord C axons turn sharply as pass FP
What inhibitory molecule repel axons after they have crossed the midline?
semaphorins and slits
When axons cross the midline, what do they become sensitive to?
Repellants in FP = inhibitory molecules
Also expressed in Ventral spinal cord
How doe repellants at FP affect axon guidance?
they create ‘channels’ that guide the axons
Summary of sensitivity before/after midline
Before = sensitive to netrins After= sensitivity lost + become sensitive to semaphorins and slits
How are drosophila similarly programmed to vertebrates FP
- Insect midline glial cells express diffusible attractants = netrins and cell surface repellants = slits
- some axons form commissures and then turn to form longitudinal pathways by axons that haven’t crossed
insect midline glial cells express diffusible attractants and cell surface repellants. what are they called?
attractant = netrins repellents = slits
Mutations that effected axons crossing the midline were found in the drosophila. Name the two most important ones that were found?
Roundabout (Robo)
commissureless (Comm)
What does wildtype Robo encode?
Receptor slit
How much Robo do axons crossing/not crossing the midline have?
axons that cross = LOW LEVELS OF ROBO
axons that don’t cross = HIGH LEVELS OF ROBO
Describe levels before and after Robo crosses midline
Low before, high after
A mutant Robo would mean what?
axons can keep crossing the midline as they have low levels
- this causes them to go in circles (explains why it is called roundabout mutant)
What is expressed whilst axons cross the midline
Comm
What happens when the axon crosses the midline in terms of Comm?
Comm is no longer expressed
What does Comm control the levels of? how?
Robo
- Comm controls trafficking of Robo to membrane (not TF as works in nucleus and would take too long)
Shown as forcing expression of COmm in all cells leads to Robo like mutant
High Comm = ? Robo
low Robo
Vertebrate homolog of Robo
Robo1
When is Robo1 expressed
Before and after crossing midline vs invert only high after
Is there a homolog for Comm
No - ROBO like = Rig1
When is Rig 1 expressed
Only in pre-crossing fibres. It blocks Robo1 until the midline is crossed
What happens in a KO Rig1?
axons don’t cross the midline
How to axons know whether to stay on scaffolds or get off?
by CONTROLLING FASCICULATION
arrangement in bundles of axons
What does controlling fasciculation involve
involves “homophilic” binding by cell adhesion molecules (CAMs)
What is an example of a CAM in insects that helps axon scaffolding?
Fasciclin II
Explain what happens in wildtype Fas II, overexpression of Fas II and loss of Fas II?
WT: controls fasiculation of ventral nerve cord longitudinal tracts
Overexpress = novel fasiculations
Loss = defasc
Fas II also controls - overexpression means
Defasiculation
overexpress = by pass mutant - motor axons fail to defasc at target
What are the two main types of target selection?
- discrete targets - cellular in DNS
- topographic map - multicellular
What does the discrete targets suggest?
that axons are “looking” for specific “labels” on their targets
e.g. ablation of target - fail to defasc
What are address labels made of
Multiple cues combined
Cues combined to make address label in insect muscles
Netrins = diffusible chemoattractant
Fas 3 = CAM
What does a loss of Netrin/Fas 3 at discrete targets cause?
Axons don’t make synapses here
What does ectopic Netrin/Fas 3 at discrete targets cause?
Axons synapse to wrong muscles
What is an example of a topographic map? Meaning
When neighbouring neurons send axons to neighbouring sites in their target to maintain the topology (order) in the target, e.g. retinotectal system
Explain how topographic maps work proposed by Sperry
a co-ordinate system, encoded by gradients of signalling molecules, stamps a “latitude and longitude” onto cells of the target
this tells axons where to jump off the main bundle
Which hypothesis of topographic maps proposed by Sperry was wrong
Each axon has a unique label complementary to a unique label in the target. (cf address labels in fly muscle) yet too many labels
What does a stripe assay on the retinotectal system shows the cells of posterior tectum make what?
a non-permissive that repels temporal axons
What non-permissive factor is released from posterior tectum cells?
Combo of 2 ephrins expressed high posteriorly –> low A in tectum
Describe eph location in this system
an Eph receptor for ephrins A2 & A5 is expressed in the retina in a counter gradient from temporal (hi) to nasal (lo)
What do the ephrins do to temporal axons?
cause them to collapse - repelling them from making synapses in posterior region
Mutant experiments on ephrins A2 & A5
In mice in which both Ephrin A2 and A5 are knocked out, temporal neurons project their axons into the posterior tectum and the topographic map is disordered
What is also true about non-repellant factors
non-permissive, repellant factors can be used instructively - ie they can direct growth cones to specific places - to form topographic maps
