Lecture 13 Axon guidance 3

Question 1

Which patterning factors are reused for axon guidance

Answer 1

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

Question 2

BMP7 and Netrin are examples of

Answer 2

Chemotropic factors

Question 3

What does BMP7 antagonise

Answer 3

Effects of Shh on DV axis

Question 4

What happens in a Netrin knockout?

Answer 4

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

Some still do due to BMP7

Question 5

What else can guide axons?

Answer 5

Shh

Question 6

Where is Shh expressed and effect on axons

Answer 6

In FP as C axons extending

COS cells expressing Shh attract C axons in explant assays

Question 7

What is SHh attraction blocked by?

Answer 7

Cyclopamine

Question 8

How does cyclopamine block SHH signalling?

Answer 8

binds to smo, from corn lillies

Question 9

What does cyclopamine not block and what does this show

Answer 9

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

Question 10

What is Cre and what is Lox P?

Answer 10

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

Question 11

What is the cre-lox system

Answer 11

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

Bacteriophage P1

Question 12

Length of LoxP

Answer 12

34 BP seq

Question 13

What can the cre-lox system be used for?

Answer 13

use this to specifically delete DNA lying between two loxP sites

Question 14

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

Answer 14

Wnt1

Question 15

A mouse WT mouse is crossed with what

Answer 15

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

Question 16

What is the cre-lox system used for

Answer 16

Tissue specific or inducible knock outs

Question 17

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

Answer 17

Intermediate targets = choice points

Question 18

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

Answer 18

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

Question 19

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

Answer 19

Netrin

Question 20

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

Answer 20

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

Question 21

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

Answer 21

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

Question 22

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

Answer 22

semaphorins and slits

Question 23

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

Answer 23

Repellants in FP = inhibitory molecules

Also expressed in Ventral spinal cord

Question 24

How doe repellants at FP affect axon guidance?

Answer 24

they create ‘channels’ that guide the axons

Question 25

Summary of sensitivity before/after midline

Answer 25

``` Before = sensitive to netrins After= sensitivity lost + become sensitive to semaphorins and slits ```

Question 26

How are drosophila similarly programmed to vertebrates FP

Answer 26

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

Question 27

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

Answer 27

``` attractant = netrins repellents = slits ```

Question 28

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

Answer 28

Roundabout (Robo) | commissureless (Comm)

Question 29

What does wildtype Robo encode?

Answer 29

Receptor slit

Question 30

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

Answer 30

axons that cross = LOW LEVELS OF ROBO | axons that don't cross = HIGH LEVELS OF ROBO

Question 31

Describe levels before and after Robo crosses midline

Answer 31

Low before, high after

Question 32

A mutant Robo would mean what?

Answer 32

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)

Question 33

What is expressed whilst axons cross the midline

Answer 33

Comm

Question 34

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

Answer 34

Comm is no longer expressed

Question 35

What does Comm control the levels of? how?

Answer 35

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

Question 36

High Comm = ? Robo

Answer 36

low Robo

Question 37

Vertebrate homolog of Robo

Answer 37

Robo1

Question 38

When is Robo1 expressed

Answer 38

Before and after crossing midline vs invert only high after

Question 39

Is there a homolog for Comm

Answer 39

No - ROBO like = Rig1

Question 40

When is Rig 1 expressed

Answer 40

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

Question 41

What happens in a KO Rig1?

Answer 41

axons don't cross the midline

Question 42

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

Answer 42

by CONTROLLING FASCICULATION | arrangement in bundles of axons

Question 43

What does controlling fasciculation involve

Answer 43

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

Question 44

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

Answer 44

Fasciclin II

Question 45

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

Answer 45

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

Question 46

Fas II also controls - overexpression means

Answer 46

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

Question 47

What are the two main types of target selection?

Answer 47

- discrete targets - cellular in DNS | - topographic map - multicellular

Question 48

What does the discrete targets suggest?

Answer 48

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

Question 49

What are address labels made of

Answer 49

Multiple cues combined

Question 50

Cues combined to make address label in insect muscles

Answer 50

Netrins = diffusible chemoattractant | Fas 3 = CAM

Question 51

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

Answer 51

Axons don't make synapses here

Question 52

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

Answer 52

Axons synapse to wrong muscles

Question 53

What is an example of a topographic map? Meaning

Answer 53

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

Question 54

Explain how topographic maps work proposed by Sperry

Answer 54

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

Question 55

Which hypothesis of topographic maps proposed by Sperry was wrong

Answer 55

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

Question 56

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

Answer 56

a non-permissive that repels temporal axons

Question 57

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

Answer 57

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

Question 58

Describe eph location in this system

Answer 58

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

Question 59

What do the ephrins do to temporal axons?

Answer 59

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

Question 60

Mutant experiments on ephrins A2 & A5

Answer 60

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

Question 61

What is also true about non-repellant factors

Answer 61

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