Axon outgrowth and Guidance

Question 1

What first left neuroscientists the impression that growth cones moved in an ordered and directed manner?

Answer 1

Golgi’s drawings.

Question 2

True or false: most retinal axons do not respond to more than one axon guidance cue to reach to optic tectum.

Answer 2

False, there are at least 10 steps in this process.

Question 3

Complete the information about the retinotectal pathway with the word bank.

dorsal
temporal
caudal
dorsal
rostral

Axons leaving the _____ part of the retina end in the ventral part of the optic tectum.

Axons leaving the ventral part of the retina end in the _____ part of the optic tectum.

Axons leaving the nasal of the retina end in the _____ part of the optic tectum.

Axons leaving the _____ part of the retina end in the _____ part of the optic tectum

Answer 3

Axons leaving the dorsal part of the retina end in the ventral part of the optic tectum.

Axons leaving the ventral part of the retina end in the dorsal part of the optic tectum.

Axons leaving the nasal part of the retina end in the caudal part of the optic tectum.

Axons leaving the temporal part of the retina end in the rostral part of the optic tectum.

Question 3

What does the random outgrowth theory has to say about axon pathfinding?

Answer 3

Axons would not follow cues to reach a specific target. Instead, there would be random axonal outgrowth in all directions, and only the relevant connections would be preserved by experience-dependent mechanisms.

Question 4

What is the chemoaffinity hypothesis postulating?

Answer 4

Neurons express different combinations of receptors and molecular markers, which allows them to follow specific cues and make specific connections with biochemically relevant targets.

Question 5

What famous experiment debunked the random outgrowth theory of neuronal development? Describe the experiment.

Answer 5

The Sperry experiment. The projections from the retina to the optic tectum were severed for one eye of a xenopus. The eye was then flipped 180 degrees. Projections from the flipped eye regrown to the same regions of the optic tectum, which supports the chemoaffinity hypothesis.

Question 6

Provide an example of transplant experiment in the visual system that proves the chemoaffinity theory.

Answer 6

When transplanting patches of neuroepithelium from a donor tadpole to the optic tract of a host, the axons will always grow toward the region of the patch where they bind under natural conditions.

Question 7

How can a growth cone be visualized?

Answer 7

By transfecting it with GFP-actin.

Question 8

True or false: in a typical growth cone, extensions are microtubule-rich while the core is actin-rich.

Answer 8

False: the opposite.

Question 9

What is driving the extension of the growth cone?

Answer 9

Actin polymerization.

Question 10

What makes growth cones sensible to their environment?

Answer 10

Presence of receptors.

Question 11

What are the two types of “extension” in a growth cone? What are they constituted from?

Answer 11

Lamellipodia (sheet-like) and filopodia (finger-like). Made from actin.

Question 12

What is controlling growth cone steering?

Answer 12

Microtubule depolymerization and polymerization.

Question 13

How is cytochalasin modulating actin polymerization?

Answer 13

Prevents actin polymerization by binding to + end of actin filament.

Question 14

Since actin is treadmilling in the extensions (filopodia and lamellipodia) of the growth cone, actin-poor spaces are created when actin polymerization stretches the plasma membrane forward. How are those spaces solidified?

Answer 14

The microtubules from the central core advance to occupy such spaces.

Question 15

What are the growth cone attractive and repulsive cues regulating specifically?

Answer 15

They are regulating the rates of actin polymerization and depolymerization.

Question 16

Which component of the extracellular matrix mainly promotes axon outgrowth ability?

Answer 16

Laminin.

Question 17

Propose a simple experiment to verify if a laboratory-made ECM promotes neurite outgrowth.

Answer 17

Culture neurons on a patterned substrate of the artificial ECM segmented in ECM-coated corridors and inert ECM-free patches. If the artificial ECM promotes neural outgrowth, growth cones should extend on the ECM-coated surface.

Question 18

True of false: integrins bind to laminins.

Answer 18

True.

Question 19

What is one characteristic that differs different types of integrin?

Answer 19

The laminin types to which they bind (determined by subunit composition).

Question 20

Give one documented example of laminin guiding axons in vivo.

Answer 20

Laminin guiding retinal ganglion cells axons across the vitreal surface to the entrance of the optic nerve in the retina.

Question 21

How can antibodies be used to isolate cell adhesion molecules (CAMs)?

Answer 21

If antibodies are added to dispersed cells in suspension that supposedly express CAMs and those cells cannot cluster when brought in proximity, it is likely to imply that antibodies successfully bound CAMs and prevented cells to stick to each other.

Question 22

What type of calcium-dependent cell adhesion molecules is extensively found in the nervous system?

Answer 22

N-Cadherin.

Question 23

True or false: some CAMS that belong to the immunoglobulin family are calcium dependent.

Answer 23

False: all molecules are calcium-independent.

Question 24

Why do CAMs require intracellular domains?

Answer 24

To convey the extracellular signal intracellularly.

Question 25

True of false: since ephrins are ligands, they do not have intracellular domains.

Answer 25

False: some of them do, like ephrin B.

Question 26

In the case of ephrins, what is forward signaling? What is reverse signaling?

Answer 26

Forward signaling is the activation of signaling pathways downstream of Ephs (receptors) upon ligand binding.

Reverse signaling is the activation of signaling pathways downstream of Ephrins (ligand) upon binding to their receptors.

Question 27

What type of ephrin signaling prevents RGCs from entering the posterior region of the optic tectum? Is it repulsive or attractive?

Answer 27

Ephrin A - Eph A: repulsive.

Question 28

Complete the information about the axon guidance ecology of RGCs with the word bank.

ventral x 2
nasal
retina
posterior
anterior
EphrinB
dorsal
temporal
optic tectum

The _____ gradient in the retina goes ([high] to [low]) from _____ to dorsal.

The EphA gradient in the _____ goes ([high] to [low]) from _____ to _____.

The EphrinA gradient in the _____ goes ([high] to [low]) from _____ to _____.

The EphB gradient in the optic tectum goes ([high] to [low]) from _____ to _____.

Answer 28

The EphB gradient in the retina goes ([high] to [low]) from ventral to dorsal.

The EphA gradient in the retina goes ([high] to [low]) from temporal to nasal.

The EphrinA gradient in the optic tectum goes ([high] to [low]) from posterior to anterior.

The EphrinB gradient in the optic tectum goes ([high] to [low]) from dorsal to ventral.

Question 29

Are all ephrin signals repulsive?

Answer 29

No, EphrinBs are attractive for example.

Question 30

Give one documented example of ephrins guiding axons in vivo.

Answer 30

EphrinA-EphA (repulsive signaling) and EphrinB-EphB (attractive signaling) mediate the targetting of RGCs in the optic tectum.

Question 31

What are the two types of membrane attachment that we find in ephrins? Ephrins A and B rely on which types?

Answer 31

Ephrins are either GPI-anchored to the plasma membrane or have cytoplasmic domains. Ephrin As are GPI-achord while Ephrin Bs have cytoplasmic domains.

Question 32

Ephrin Bs or As are capable of reverse signaling?

Answer 32

Ephrin Bs.

Question 33

Why are some forms of semaphorin very efficient to establish a gradient?

Answer 33

Some forms (Sem3) are produced by the cells and can therefore easily diffuse.

Question 34

What is the consequence of eliminating the intracellular domain of neuropilins (Nrp)? What does that indicate?

Answer 34

No change. A coreceptor is needed (plexin).

Question 35

True of false: some semaphorins can signal directly through plexins without having to bind to Nrp.

Answer 35

True.

Question 36

What component of semamorphin systems mediates intracellular signaling?

Answer 36

Plexin coreceptors.

Question 37

What signal guides RGCs along the optic nerve? Is it attractive or repulsive?

Answer 37

Sema5A, repulsive.

Question 38

What signal is in charge of preventing axons from wandering in the optic chiasm?

Answer 38

Slit signaling.

Question 39

Describe the trajectory of commissural axon neurons in the neural tube of the mouse?

Answer 39

Roof plate of neural tube (caudal) -> floor plate of neural tube -> crossing midline -> projecting rostrally along the floor plate.

Question 40

In commissural guidance, how is netrin involved?

Answer 40

Attractive signal of the floor plate.

Question 41

Is netrin GPI-anchored or does it have a cytoplasmic domain?

Answer 41

Neither, not bound to plasma membrane.

Question 42

What kind of response does netrin binding to DCC receptors provoke? Under which conditions is that reversed?

Answer 42

Attractive. Reversed when Unc5 is coexpressed.

Question 43

What is the receptor of slit?

Answer 43

Robo.

Question 44

Name two morphogens involved in the patterning of the nervous system.

Answer 44

Shh and Wnt.

Question 45

In what other process are involved morphogens?

Answer 45

Cellular migration.

Question 46

What intracellular family of proteins is of particular importance for controlling growth cone morphology by translating extracellular signals into intracellular events?

Answer 46

Rho GTPases.

Question 47

Rac, Cdc-42 and Rho all regulate different aspects of the growth cone morphology. For which aspect is each Rho protein GTPase responsible for?

Answer 47

Activation of Rac: promotes expension of lamellipodia.
Activation of Cdc-42: promotes expension of filopodia.
Activation of Rho: promotes actin depolymerization.

Question 48

What are doing GDP dissociation inhibitors?

Answer 48

Bind to the Rho GTPase protein in its GDP form and lock it into that state.

Question 49

What is ephexin?

Answer 49

A GEF for multiple Rho GTPases (promotes GTP form of the latter).

Question 50

What Rho GTPases is ephexin activating?

Answer 50

Mainly Rho and Cdc-42 (= collapse).

Question 51

The Slit-Robo pathway has which impact on the Rho GTPase system?

Answer 51

Promotion of GAP activity in the Cdc-42 GTPase cycle (= deactivation).

Question 52

List three broad factors that determine the response of neurons to axon guidance cues.

Answer 52

1. Neuronal type.
2. Environment.
3. Developmental stage.

Question 53

Are membrane receptors associated with axon guidance cues usually directly interacting with Rho GTPase proteins? If not, what is the intermediate step?

Answer 53

No, they usually modulate the activity of GEFs, GAPs and GDIs, and those directly interact with Rho GTPases.

Question 54

Explain the paradigm-shifting experiment that was interested in understanding how neurons adapt their responses to axon guidance cues in an appropriate spatio-temporal manner during development.

Answer 54

Addition of a PKA inhibitor (major downstream effector of cAMP) to a petri dish containing Xenopus spinal neurons and netrin reversed the neurons’ valence to netrin.

Question 55

If you wanted to change the valence (not eliminate) of a neuron to a guidance cue, what would you target intracellularly?

Answer 55

The ratio of intracellular cAMP to cGMP.

Question 56

Netrin binding to a dimer of DCC and Unc-5 receptors leads to what kind of response for the growth cone?

Answer 56

Repulsion.

Question 57

What simple experiment could be done to show that protein synthesis is necessary for RGC responsiveness to chemotropic cues?

Answer 57

Add reagents impairing the protein synthesis machinery.

Question 58

True or false: components of translational machinery are only found in the nucleus of a neuron.

Answer 58

False: they are at least also found in the growth cone during development.

Question 59

What is one signal (guidance cue) that prevents commissural neurons from re-entering the floor plate once they’ve crossed it?

Answer 59

EphrinA-EphA.

Question 60

What could be a mechanism to regulate receptor expression in a spatio-temporal manner for neurons in outgrowth?

Answer 60

Environmentally-induced transcription of dormant mRNA in the growth cone.

Question 61

What extracellular post translational mechanism can modify the responsiveness of neurons to guidance cues?

Answer 61

Metalloproteases by clipping off receptors from the neuron’s surface.

Question 62

What is preventing commissural neurons from retrogradely crossing the midline once they’ve crossed it? Isn’t netrin (floor plate signaling) still attracting them towards the midline?

Answer 62

Once commissural neurons cross the midline, Robo receptors are upregulated and their intracellular domains interact with DCC’s domains to silence them. Therefore, there is no more competing cuing activity and neurons respond almost exclusively to Slit signaling.