L4 - Reprogramming

Question 1

Axons reprogram when they reach

Answer 1

Choice points

Question 2

What happens to C-axons once they have crossed the floorplate

Answer 2

NO LONGER RESPOND to netrins

Question 3

Describe the experiment used to show that once the FP has been crossed C axons no longer respond to netrin

Answer 3

Ectopic grafting of the floorplate

Question 4

What also happens to c-axons once the floor plate has been crossed

Answer 4

Become sensitive to repellants as well

Question 5

What is the reason why c-axons become sensitive to repellants post crossing of the floor plate

Answer 5

Creates channels through which the c-axons can grow through

Question 6

Insect midline glial cells express ___________(e.g_________) and ___________(e.g_________)

Answer 6

diffusible attractants (e.g. netrins)
cell surface repellents (e.g. slits)

Question 7

What does robo encode

Answer 7

a receptor for the inhibitory slit protein

Question 8

Robo is expressed at high levels on _____

What is the logic behind this

Answer 8

Expressed at high levels on axons that DO NOT cross the midline

Robo is the receptor for slit, thus if Robo is high the cell is able to sense lots of the inhibitory slit molecule hence it is repelled from the midline and will not cross

Question 9

Describe how Robo expression on c-axons changes

Answer 9

Initially expressed at low levels - once the midline is cross the axons express Robo at high levels

Question 10

Describe the robo mutant

Answer 10

No robo = no receptor for slit = no slit detected

Axons go back and forth across the midline forms roundabouts

Question 11

Where is comm expressed (2)

Answer 11

Midline cells and axons that normally cross the midline

Question 12

What is the effect of lacking Comm

Answer 12

Robo protein expressed at high levels in those cells that would normally cross the midline and which extend longitudinally

Question 13

What happens when Comm expression is forced

Answer 13

Robo lost everywhere so no sensitivity to Slit

Roundabout (robo) phenotype

Question 14

What are the two models for how comm regulates Robo

Answer 14

Sorting

Clearance

Question 15

Describe the sorting model

Answer 15

Presence of comm forces sorting of newly synthesised Robo into late endosomes

Question 16

Describe the clearance model

Answer 16

Homophilic binding of Comm causes clearance of Robo from the cell surface

Question 17

What is an attraction to the clearance model

Answer 17

It suggests a mechanism for the upregulation of Robo in crossed axon segments

Question 18

Describe the results of Keleman 2005 expt to ask if Comm is required for midline cells for correct crossing

Answer 18

Neuron specific promoter used to drive an axonal marker
Same promoter was used to drive comm expression in comm mutants background

FOUND - rescue is also turned on in midline cells (using a midline specific promoter) - YOU DONT NEED COMM MIDLINE EXPRESSION TO RESCUE

Question 19

What is one of the differences between the sorting and clearance model

Answer 19

In SORTING Robo should not be shipped down the axon in the presence of Comm whereas it would be if it was cleared at the cell surface (CLEARANCE)

Question 20

How could you test to see if Robo protein is being transported down the axon

Answer 20

Robo-GFP fusion

Results showed Robo-GFP is found in an axon without comm - so robo transports along the axon

Question 21

What are the limitations of the sorting model even though it appears to be correct

Answer 21

Why comm is on the midline

What controls the upregulation of Robo on the contralateral side

Question 22

Describe how cell surface expression also changes in mammals

Answer 22

E.g. TAG-1 and L1

Question 23

Has a comm homolog been found in mammals

Answer 23

No

Question 24

What is found in mammals in place of comm

Answer 24

Another robo-like receptor (Robo3 AKA Rig-1)

Question 25

When is Robo3 expressed

Answer 25

Pre crossing axons

Question 26

What is the effect of knocking out in mice`

Answer 26

Prevents crossing of the floor plate

Question 27

Journal club paper for reprogramming lecture

Answer 27

Sabatier et al 2004

Question 28

What did Sabatier et al look to confirm in their paper

Answer 28

How Robo3 worked

Question 29

What were the two models from Sabatier et al for how Robo3 works

Answer 29

1) Rig-1 is an attractive receptor required for floor plate crossing 2) Rig-1 prevents sensitivity to a floor plate repellent

Question 30

Pre corssing C axons from ROBO3 knockouts are ...

Answer 30

Prematurely sensitive to slits

Question 31

Attraction to the midline in ROBO3 KOs can be restored if

Answer 31

Slit function is blocked

Question 32

What method would be used to restore slit function

Answer 32

Using soluble Robo Soluble Robo binds to Slit in the culture prevetning it binding to Robo on the axons

Question 33

Wild type pre-crossing axons _______ in the presence of netrin

Answer 33

Ignores slit

Question 34

SO WHAT IS THE FUNCTION OF ROBO3

Answer 34

Prevent premature sensitivity to Slit

Question 35

Role of Robo3 is ________ to the role of Comm in flies This role is

Answer 35

Analogous Prevents Robo from signalling before the midline which allows netrin to signal via its receptor

Question 36

What is the receptor for netrin

Answer 36

DCC

Question 37

What is different between Robo3 in mammals and comm in flies

Answer 37

The mechanism Loss of Robo3 DOES NOT lead to an upregualtion of Robo on pre crossing axons Losing comm would lead to the upregulation of robo - comm mutant - no axons cross the FP

Question 38

What is thought to now be the effect of Slit binding Robo at the cell surface

Answer 38

Resulting signal amplifies Robo expression

Question 39

Robo3 may be required to ...

Answer 39

Prevent premature slit response as C axons approach the Fp

Question 40

Evidence suggests that at ___________ slit-induced Robo signalling _______ Robo3 inhibition which then ________ robo3 expression __________ robo and _______ slit mediated repulsion

Answer 40

``` Certain threshold concentration Overwhelms Robo3 inhibition TUrns off Full stabilising Activating ```

Question 41

The effect of turning to netrins is inhibited in what circumstances

Answer 41

When slit is present

Question 42

What is the effect of slit on netrin

Answer 42

Slit inhibits netrin

Question 43

What are the two models which could explain how Slit inhibits netrin

Answer 43

Model 1: Ligand-ligand interaction | Model 2: Receptor-mediated silencing

Question 44

Describe model 1 - ligand ligand interaction

Answer 44

slit directly inhibits the netrin ligand

Question 45

Describe model 2 - receptor mediated signalling

Answer 45

slit bidning to robo inhibits the compelx formed when netrin binds to DCC

Question 46

What is the only thing that is required to block the response to netrin

Answer 46

Robo IC domain

Question 47

How was it shown that it was the IC domain is required to block the response to netrin

Answer 47

Express hybrid version version of Robo E.g. IC domain of Robo and the EC domain of Met (the HGF receptor) When HGF applied it was seen that the turning response could be blocked Since normally HGF doesnt could this response - isnt due to ligand but due to receptor interactions

Question 48

So what is the mechanism for slit inhibitiing netrin

Answer 48

Silencing of the netrin response by Slit is due to an interaction between the cytoplasmic domains of DCC and Robo

Question 49

BEFORE THE MIDLINE (ROBO3 AND ROBO)

Answer 49

Robo3 silences Robo

Question 50

AFTER THE MIDLINE (ROBO AND SLIT)

Answer 50

Activation of Robo by slit silences DCC signalling

Question 51

What throws the switch determine if Robo3 silences Robo or activation of Robo by slit silences DCC signalling

Answer 51

Contact with the floor plate triggers it Changes over time in a cell intrinsic manner (essentially a clock)

Question 52

What is the evidence that contact with the floorplate flips the switch

Answer 52

Robo story - suggests high conc slits will stabilise robo on cell surface TURNING ON slit senstivity as the FP is crossed

Question 53

How does Sema sensitivity change as the floor plate is crossed

Answer 53

Contact mediated by Tag1 and NrCAM (on FP) to stabilise Sema receptors on C axons

Question 54

What is the effect of treating c aoxns with Shh

Answer 54

Turns on Sema sensitivity

Question 55

Shh initially also attracts

Answer 55

C axons

Question 56

What changes with rgeards to Shh as C axons cross

Answer 56

Attraction of c axons to Shh changes to repulsion

Question 57

what is the effect of a Smo KO

Answer 57

Confused crossing and turning defects

Question 58

Response of C axons to Shh reversed by ...

Answer 58

A cell intrinsic increase in 14-3-3 expression

Question 59

How is Wnt4 an important factor for guidance

Answer 59

Wnt to Fzd to GSK3B and DVL - DVL affects the par 3 complex

Question 60

14-3-3 is a ______ homolog What is its function

Answer 60

Par 5 | Inhibits protein kinase A