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Dev Neurobiology extra > Lecture 13 Axon guidance 3 > Flashcards

Lecture 13 Axon guidance 3 Flashcards

1
Q

Which patterning factors are reused for axon guidance

A

In spinal cord
BMP7 in RP = push
Netrin in FP = pull

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2
Q

BMP7 and Netrin are examples of

A

Chemotropic factors

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3
Q

What does BMP7 antagonise

A

Effects of Shh on DV axis

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4
Q

What happens in a Netrin knockout?

A

there is disruption at the floor plate where the C axons don’t cross the midline

Some still do due to BMP7

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5
Q

What else can guide axons?

A

Shh

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6
Q

Where is Shh expressed and effect on axons

A

In FP as C axons extending

COS cells expressing Shh attract C axons in explant assays

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7
Q

What is SHh attraction blocked by?

A

Cyclopamine

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8
Q

How does cyclopamine block SHH signalling?

A

binds to smo, from corn lillies

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9
Q

What does cyclopamine not block and what does this show

A

It doesn’t block netrin so 2 separate pathways guiding C axons

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10
Q

What is Cre and what is Lox P?

A

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

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11
Q

What is the cre-lox system

A

A type of recombination method in ES cells
You can manipulate genes at specific developmental points using an antibiotic controlled promoter

Bacteriophage P1

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12
Q

Length of LoxP

A

34 BP seq

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13
Q

What can the cre-lox system be used for?

A

use this to specifically delete DNA lying between two loxP sites

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14
Q

What promotor is used to drive expression in Cre of C axons derived from midline

A

Wnt1

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15
Q

A mouse WT mouse is crossed with what

A

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

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16
Q

What is the cre-lox system used for

A

Tissue specific or inducible knock outs

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17
Q

What are the different stages of growth cone navigation broken up by

A

Intermediate targets = choice points

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18
Q

What happens when axons reach intermediate targets (choice points)?

A

the axons reprogram depending on what information they receive here i.e sensitivity to specific guidance cues changes

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19
Q

What do axons lose sensitivity to when they cross the midline?

A

Netrin

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20
Q

What experiment shows this loss of sensitivity to netrin after crossing midline?

A

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
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21
Q

What do experiment showing this loss of sensitivity to netrin after crossing midline not explain?

A

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

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22
Q

What inhibitory molecule repel axons after they have crossed the midline?

A

semaphorins and slits

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23
Q

When axons cross the midline, what do they become sensitive to?

A

Repellants in FP = inhibitory molecules

Also expressed in Ventral spinal cord

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24
Q

How doe repellants at FP affect axon guidance?

A

they create ‘channels’ that guide the axons

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25
Q

Summary of sensitivity before/after midline

A 
Before = sensitive to netrins
After= sensitivity lost + become sensitive to semaphorins and slits
26
Q

How are drosophila similarly programmed to vertebrates FP

A
  1. Insect midline glial cells express diffusible attractants = netrins and cell surface repellants = slits
  2. some axons form commissures and then turn to form longitudinal pathways by axons that haven’t crossed
27
Q

insect midline glial cells express diffusible attractants and cell surface repellants. what are they called?

A 
attractant = netrins
repellents = slits
28
Q

Mutations that effected axons crossing the midline were found in the drosophila. Name the two most important ones that were found?

A

Roundabout (Robo)

commissureless (Comm)

29
Q

What does wildtype Robo encode?

A

Receptor slit

30
Q

How much Robo do axons crossing/not crossing the midline have?

A

axons that cross = LOW LEVELS OF ROBO

axons that don’t cross = HIGH LEVELS OF ROBO

31
Q

Describe levels before and after Robo crosses midline

A

Low before, high after

32
Q

A mutant Robo would mean what?

A

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)

33
Q

What is expressed whilst axons cross the midline

A

Comm

34
Q

What happens when the axon crosses the midline in terms of Comm?

A

Comm is no longer expressed

35
Q

What does Comm control the levels of? how?

A

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

36
Q

High Comm = ? Robo

A

low Robo

37
Q

Vertebrate homolog of Robo

A

Robo1

38
Q

When is Robo1 expressed

A

Before and after crossing midline vs invert only high after

39
Q

Is there a homolog for Comm

A

No - ROBO like = Rig1

40
Q

When is Rig 1 expressed

A

Only in pre-crossing fibres. It blocks Robo1 until the midline is crossed

41
Q

What happens in a KO Rig1?

A

axons don’t cross the midline

42
Q

How to axons know whether to stay on scaffolds or get off?

A

by CONTROLLING FASCICULATION

arrangement in bundles of axons

43
Q

What does controlling fasciculation involve

A

involves “homophilic” binding by cell adhesion molecules (CAMs)

44
Q

What is an example of a CAM in insects that helps axon scaffolding?

A

Fasciclin II

45
Q

Explain what happens in wildtype Fas II, overexpression of Fas II and loss of Fas II?

A

WT: controls fasiculation of ventral nerve cord longitudinal tracts
Overexpress = novel fasiculations
Loss = defasc

46
Q

Fas II also controls - overexpression means

A

Defasiculation

overexpress = by pass mutant - motor axons fail to defasc at target

47
Q

What are the two main types of target selection?

A
  • discrete targets - cellular in DNS

- topographic map - multicellular

48
Q

What does the discrete targets suggest?

A

that axons are “looking” for specific “labels” on their targets
e.g. ablation of target - fail to defasc

49
Q

What are address labels made of

A

Multiple cues combined

50
Q

Cues combined to make address label in insect muscles

A

Netrins = diffusible chemoattractant

Fas 3 = CAM

51
Q

What does a loss of Netrin/Fas 3 at discrete targets cause?

A

Axons don’t make synapses here

52
Q

What does ectopic Netrin/Fas 3 at discrete targets cause?

A

Axons synapse to wrong muscles

53
Q

What is an example of a topographic map? Meaning

A

When neighbouring neurons send axons to neighbouring sites in their target to maintain the topology (order) in the target, e.g. retinotectal system

54
Q

Explain how topographic maps work proposed by Sperry

A

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

55
Q

Which hypothesis of topographic maps proposed by Sperry was wrong

A

Each axon has a unique label complementary to a unique label in the target. (cf address labels in fly muscle) yet too many labels

56
Q

What does a stripe assay on the retinotectal system shows the cells of posterior tectum make what?

A

a non-permissive that repels temporal axons

57
Q

What non-permissive factor is released from posterior tectum cells?

A

Combo of 2 ephrins expressed high posteriorly –> low A in tectum

58
Q

Describe eph location in this system

A

an Eph receptor for ephrins A2 & A5 is expressed in the retina in a counter gradient from temporal (hi) to nasal (lo)

59
Q

What do the ephrins do to temporal axons?

A

cause them to collapse - repelling them from making synapses in posterior region

60
Q

Mutant experiments on ephrins A2 & A5

A

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

61
Q

What is also true about non-repellant factors

A

non-permissive, repellant factors can be used instructively - ie they can direct growth cones to specific places - to form topographic maps

Dev Neurobiology extra (6 decks)

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