Reprogramming Flashcards
What happens to axons when they reach intermediate targets?
They reprogramme
What happens to the neurons in the hindbrain when they cross the floor plate?
How is this different to in the spinal cord?
In the hindbrain: neurons continue straight on
In the spinal cord: neurons turn anteriorly
What happens to pre-crossing neurons if they are exposed to an ectopic floor plate?
They are attracted to the ectopic floor plate
What happens to post-crossing neurons if they are exposed to an ectopic floor plate?
They are not attracted to the ectopic floor plate
What happens to neuron sensitivity when they cross the floor plate?
Sensitivity changes:
- No longer attracted to netrins in the floor plate
- Now sensitive to repellants in the floor plate
How are semaphorins and slits expressed in the floor plate?
In 3 bands
Why do spinal cord axons turn after crossing the floor plate?
Repelled by slits and semaphorins in the floor plate
Why don’t the axons in the spinal cord go straight on once crossing the floor plate in the spinal cord? (like in the hindbrain)
Slits and semaphorins (repelllants in the floor plate) create a channel at which the axons grow
What is expressed in INSECT midline glial cells?
- Diffusible attractants (netrins)
- Cell surface repellants (slits)
What occurs in the roundabout mutant? (NO ROBO)
Neurons lose sensitivity to repellants in the midline:
- Axons cross the midline many times
What occurs in the commissurless mutant? (NO COMM)
Neurons are permanently sensitive to repellants in the midline:
- Axons don’t cross the midline
What happens when FORCE expression of comm?
What does this show?
Looks like robo mutant (no robo) - no longer sensitive to midline repellants
Shows that comm controls robo
Do all axons cross the midline?
NO some axons cross to form commissures that join with longitudinal pathways formed by axons that have not crossed the midline
What determines whether or not axons can cross the midline?
Levels of SLIT receptors at the cell surface
What type of molecule is comm?
Where is it found?
A membrane-associated protein
Found at the cell surface and on intracellular components
How does comm control robo?
Controls the intracellular trafficking of robo to the cell surface
What is Robo?
A receptor for the inhibitory protein SLIT
Where is robo expression high/low?
High on neurons that DON’T cross (repelled by the midline)
High on C neurons AFTER they have crossed (prevent crossing again)
Low on C neurons BEFORE crossing (so they can be attracted to the midline)
Why do the axons continuously cross the midline in robo mutants?
No robo, no slit detected, not repelled from the midline
Can constantly be attracted to the midline by netrins
Which 2 places is comm expressed?
1) In the MIDLINE
2) In neurons that normally cross the midline
Why do the axons not cross the midline in comm mutants?
Robo upregulated, higher sensitivity to slits, extend axons longitudinally
What are the 2 possible models of how Comm controls Robo?
1) Sorting model
2) Clearance model
Describe the sorting model of how comm controls robo
Presence of Comm forces the sorting of newly synthesised Robo into late endosomes:
Pre-crossing/during crossing - Robo recycled and degraded
Post-crossing - Robo upregulated
In neurons that DON’T cross - no Comm, no robo degredation
Describe the clearance model of how comm controls robo
- Robo goes down the axon with comm
- Robo gets to the surface but is CLEARED from the surface (during crossing)
- Following crossing, Robo is UPREGULATED
How is robo cleared from the cell surface during the crossing of a commisural axon?
Due to the HOMOPHILLIC interactions between comm in the axon and comm in the midline
In the clearance model, how is robo upregulated in post-crossing axons?
No longer homophillic interactions between comm in the axon and comm in the midline
What predictions distinguish between the clearance and the sorting model?
1) Only the clearance model requires comm expression in the midline
2) In the clearance model, robo is sent down the axon before the axon crosses, but in the sorting model, robo stays in the cell body before the axon crosses
Why is the clearance model an attractive model for how comm controls robo?
Suggests a mechanism for up regulation of robo in crossed axon segments
What 2 things are used to test if the clearance model or the sorting model is correct?
1) Is comm required in the MIDLINE CELLS for correct crossing? (clearance models says should be)
2) Is robo PREVENTED from going to axons in the presence of comm? (sorting model says it should be)
How to test if comm required in the MIDLINE CELLS for correct crossing?
Use neuron-specific promoter to drive axon marker in a subset of cells:
- Use promoter to drive comm expression IN THE NEURONS in a comm mutant background
- Comm is in the neurons but NOT the midline
–> rescues the phenotype, in favour of the SORTING model
How to test if robo PREVENTED from going to axons in the presence of comm?
Use robo-GFP fusion protein in the fly:
Axon NOT expressing comm - robo moves down the axon
Axon expressing comm - robo remains in the CELL BODY
–> shows comm doesn’t ship robo down the axon, in favour of the SORTING model
What model explains how comm controls robo?
What are the problems with this model?
Sorting model
Problems, don’t understand why:
1) Comm is expressed in the midline
2) What controls upregulation of robo on the contalateral side (after crossing the midline)
What is the likely explanation of upregulation of robo after the midline?
Due to contact with the midline
When is robo made in the axon?
What does this allow?
BEFORE axons cross
Allows robo to rapidly appear on the cell surface once the midline is crossed
What type of molecules are TAG-1 and L1?
How are they expressed in commisural axons?
Cell adhesion molecules (on the cell surface) in MAMMALS
TAG-1:
High in axons pre-crossing, switched off post crossing
L1:
Up regulated in post-crossing axons
What is the comm homologue in vertebrates?
There isn’t one, instead:
- Another Robo-like receptor is expressed in pre-crossing axons
In vertebrates, what is the robo-like receptor expressed in pre-crossing axons?
Robo3/Rig1
- Also a receptor for slit
What happens in mice if KO robo3?
Why is this surprising?
PREVENTS floorplate crossing (axons aren’t attracted to the FP)
Surprising because robo is a receptor slit, if KO - assume will cross the floor plate (less sensitive to slits)
What are the 2 models of how Robo3 works?
1) Robo3 is a receptor for an ATTRACTANT not a repellant
2) Robo3 is an INHIBITOR of Robo
What does model 1 of how Robo3 works suggest when KO robo3?
Neurons are prematurely sensitive to slit and are not attracted to the floorplate
What happens in a mouse with Robo3KO AND blocked slit function?
What model is this consistent with?
Attraction to the floor plate is RESTORED
Consistent with model 2 (Robo3 is inhibitor of robo):
- NO inhibitor of Robo, robo can function and sense slits (doesn’t cross)
- BUT, no slits, robo cannot sense slits (axons cross)
How is slit function blocked?
Adding Robo1 ectodomain (part of receptor outside of the cell that usually binds to slit):
- Binds to slits and stops slits binding to Robo in the neurons
What experiments show that Robo3 function is to prevent premature slit sensitivity?
What does this show?
What is the control?
Take cells expressing slit in a bath with netrin:
- WT pre-crossing C axons IGNORE slits in presence of netrin (axons grow into netrin)
- Pre-crossing C axons LACKING Robo3 are REPELLED away from slit source (still grow into netrin, but away from slit)
Control: Not true for other inhibitory factors, only slit
When can netrin override effect of slit?
In WT neurons
But ONLY when ROBO3 is present
What prevents robo from functioning in the wrong part of the pathway?
Crosstalk between receptors
What is the role of Robo3 and Comm?
Robo3 (in vertebrates) is ANALOGOUS to comm (in flies) (similar function, different protein):
- Prevents Robo from signalling BEFORE the midline…
- Allowing netrin to signal through its receptor
What is the receptor of netrin?
Dcc
How does Robo3 and Comm prevent robo signalling?
Robo3:
Robo GETS TO the surface but Robo3 prevents its signalling
Comm:
PREVENTS robo from getting to the surface
How is the mechanism of how Robo3 works differnetly to comm?
- Doesn’t affect the trafficking, it blocks the receptor at the CELL SURFACE
- Loss of Robo3 doesn’t lead to the upregulation of robo on pre-crossing axons, instead, allows robo to signal
What happens to the level of slits as the FP is encountered?
Levels of slit increase
How is robo stabilised in the membrane?
What does this cause?
When slit binds to robo as slit concentration increases
Causes:
- Amplification of the robo expression
- When above certain threshold this slit-induced robo signalling OVERWHELMS robo3 inhibition
- Through feedback, this TURNS OFF robo3 expression
- -> Fully stabilising Robo and slit repulsion
How do post-crossing axons respond to netrin in the presence of slit?
What does this show?
No longer respond to netrin (even though they do when slit is not present)
Shows that slits INHIBIT netrin
What are the 2 proposed models that suggest how slit inhibits netrin?
1) Model 1: Ligand-ligand interaction
(Slit and netrin physically interact)
2) Model 2: receptor-mediated silencing
(Ligands BIND to the receptor but the EFFECT of SLIT binding to ROBO silences signalling from the netrin receptor DCC
How can you test if it is ligand-ligand interaction or receptor-mediated silencing that blocks netrin signalling?
What were the results?
Create hybrid version of robo (intracellular domain - robo, extracellular domain Met) - test which part of the recpetor is important:
- If ligand-ligand interaction: extracellular should be important
- If receptor-mediated silencing: intracellular part should be important
Results:
- HGF ligand binds to HGF receptor extracellularly
- Turning response occurs
- Since HGF doesn’t do this normally, shows it is the intracellular domain of robo that is important (silencing is due to receptor interactions)
What causes the silencing of netrin once the floor plate is crossed?
Interactions between the cytoplasmic domains of DCC and Robo
As well as loss of sensitivity of the commisural axons for netrin when the floor plate is crossed, what else occurs?
Sensitivity to repellants (slits) increase
What are the 2 possibilites of the ‘switch’ in sensitivities as cross the floor plate?
Which model does the evidence suggest?
1) Contact with the floor plate as the trigger
2) Changes over TIME
Evidence for BOTH possibilities
What are the 2 contact/proximity with the floor plate triggering the switch idea?
1) As get CLOSER to the floor plate - increase in concentration of SLIT that binds to ROBO on the cell surface:
- Concentration too high for robo3 to inhibit
- Feedback (shutdown of robo3 when there is slit signalling through its receptor)
2) Something on the surface of the FP - when come into contact with it it flips the switch
- NrCAM
What is NrCAM and where is it present?
What does it interact with? What does this acheive?
Cell adhesion molecule ONLY expressed in the FP
Interacts with TAG1 on commisural axons to STABILISE robo on the commisural axons –> leads to increased sensitivity to sema –> repulsion
What does shh do in the floor plate?
Attracts neurons to the floor plate by binding to receptor and then SWITCHES to repulsion as C axons cross the floor plate, due to a cell INTRINSIC change in the C axons
Changes how axons respond to repellants, switches response to netrin
Repulsion of axons by shh makes the neurons turn LEFT to travel anteriorly
What happens if KO shh in the floor plate?
Confused neurons crossing of the floor plate and turning defects
What is 14-3-3?
What is it involved in?
Par-5 homolog which INHIBITS PKA and stops it from responding to [cAMP] which controls the polarity of response
Mediating the sensitivity switch
What revereses the response of C axons to shh?
Cell intrinsic levels of 14-3-3:
- Increases as C neurons get close to the floor plate
- Change in expression causes C axons to be repelled by the floor plate (initially attracted)
(PKA is normally activated by smo, which is activated when shh binds)
(Inhibit PKA - stop responding to cAMP, which controls the attractant response to a cue)
What makes post-crossing axons turn left and travel anteriorly
Repulsion by shh posteriorly
Attraction by wnt anteriorly (wnt4)
