Lecture 10 - Developmental Neurobiology - Growth cones

What are the growth cone domains?

• central - most inner

• transitional - middle

• peripheral - most outer

What is the anatomy of a growth cone?

• lamella and filopodium are made up of different kinds of f-actin

• they are both highly motile - move in environment

1. filopodium - finger like projections

   → the f-actin bundles are polarised to form larger bundles - actin added on at + end

2. lamella (podium) - webs between the fingers

   → the f-actin bundles are crosslinked into a net

How is f-actin in a resting growth cone?

• f-actin treadmills (not going anywhere, but instead flow is continuous)

• f-actin is chopped up as it moves in and is added to the +end

• tubulin is dragged sporadically into the filopodia

• this happens more dramatically when growth cone comes in contact with an attractive cue

what happens when a growth cone comes in contact with an attractive cue? (a bead that has been coated in an attractive molecule)

1. attractive cue binds to receptors on growth cone

2. f-actin treadmilling slows

3. this allows f-actin to accumulate in the filopodium touching the cue

4. f-actin builds up in that specific spot

   → f-actin treadmilling slows = f-actin accumulates

what does f-actin accumulation do?

1. stabilises the filopodia

2. drags microtubules into the back of the filopodia

3. causes extensions outwards

what would happen if the bead was immobile?

• the growth cone would reorganise its microtubules establishing a completely new growth direction

How does actin treadmilling control filopodia extension?

1. f-actin moves backwards during the resting state

2. receptors on tip of filopodia detect the signal/cue - they come in contact with it and a molecular clutch is engaged - which stops the backward flow of actin

3. this leads to the forward movement of filopodia

4. actomyosin-based actin-tubulin link pulls microtubules into the wake of extending filopodium

Can growth cones only be attracted to cues?

• they can also be repelled

• discovered when mixture of neurons - retinal ganglion and sensory - were found to fasciculate with only their own kind

• not due to attractive forces between the same neurons but instead due to repulsion of different types of neurons

What is a growth cone collapse?

• when a neuron’s growth cone suddenly stops growing and shrinks back in response to a repulsive signal

what is an example of a growth cone collapse?

• a sensory growth cone touches the retinal axon

• but then retreats and changes direction

• as it won’t want to be in contact (repels)

What happens to the actin during growth cone collapse?

• f-actin is destabilised, has the opposite effect to attractive cues

What does attraction involve?

• localised assembly of the actin cytoskeleton

• builds more actin filaments

What does repulsion involve?

• localised disassembly

• breaking down actin filaments

What are semaphorins?

• a family of inhibitory guidance cues

• they come in 2 different flavours:

  1. membrane-bound

  2. secreted (Sema3A)

• secreted/soluble semaphorins can cause growth cones to turn

• this has a collapsing effect primarily on f-actin

what are the 4 forces - the tactics of axon guidance?

1. contact attraction

2. contact repulsion

3. chemoattraction

4. chemorepulsion

What are permissive substrates?

• a substance that allows the growth cone to attach to it which enables the growth cone to grow along the substrate

what does a growth cone need?

• it needs substrates that are permissive for growth = substrates which allow growth

• growth is dependent on signals (attachment is not enough)

Why is adhesion/attachment not enough for axon growth?

• an axon can’t grow where it doesn’t attach, there’s no relationship between strength of adhesion and amount of axon growth

• eg. Laminin is less sticky than collagen but axons prefer to grow on laminin - growth is dependent on signals

Can permissive substrates dictate the direction of axon growth?

• no they cannot

• a permissive substrate (like laminin) does not dictate direction of axon growth - such substrates provide a surface that allows axons to grow

what is laminin?

• a growth promoting extracellular matrix protein localised in the optic nerve

• laminin is permissive for growth within a specific concentration gradient

• it does not dictate direction of axon growth - it is permissive not instructive

What is another name for permissive and non-permissive substrates?

• permissive substrates = contact attractants

• non-permissive substrates = contact repellants

• there is no simple relationship between adhesiveness and permissiveness

Why do growth cones need permissive substrates?

They need permissive substrates so that they can grow

Why do growth cones need non-permissive factors?

so that they can be kept out of regions of the embryo

Why is a combination of permissive and non permissive factors used?

To channel axons into particular areas

How can non-permissive factors channel axon growth?

• semaphorin I is expressed in discrete bands in the grasshopper limb

• blocking semaphorin I with antibodies removes its non-permissive/repulsive function

• without this function the axons cross boundaries they usually avoid and enter main body of embryo in the wrong place

• shows that semaphorin I is critical for axon guidance and path restrictions

• non-permissive factors restrict axon growth

• axons still rely on permissive factors to actually grow

Axon growth is a balance between what?

Permissive and non permissive

What are ephs and ephrins?

• major family of non-permissive factors

• ephrins - membrane bound ligands that act as contact repulsion factors

• ephs - cell surface receptors that detect ephrins

  → ephs have tyrosine kinase domains on the inside of the cells

• both ephs and ephrins are membrane bound

• they both cause repulsion between cells which helps:

  → to compartmentalise the embryo into discrete domains

  → keeps axons out of specific areas

How do ephs and ephrins have a reciprocal pattern of expression?

• they are part of the compartmentalisation of the embryo - you can see spikes of them in the hindbrain

• important for axon guidance as we can use this to guide axons into the embryo

overall explain the main points of growth cones

1. they need permissive substrates to grow

2. they can be kept out of regions of the embryos by non-permissive factors

3. can be channeled by a combination of permissive and non-permissive factors

how do growth cones know where they can and cannot grow?

• largely by contact with short range cues

• short range cues like: contact attraction and contact repulsion forces

• they know which way to go by long range cues like: chemoattractants and chemorepellents

what are the 2 guidance factors?

1. chemoattractants

2. chemorepellants

What are commissural neurons?

• commissural sensory relay neurons = second order neurons that synapse information up to the thalamus

• they cross the midline contralaterally - onto the opposite side

• they extend from dorsal to ventral and cross the floor plate to the contralateral side (opposite)

How was it discovered that the floor plate influences direction of axon growth?

1. control: dorsal spinal cord alone

   • explants of dorsal spinal cord placed on collagen gel to observe axon growth in vitro

   • axons extended straight out from the dorsal side to the ventral

2. floor plate placed nearby:

   • when floor plate explant was placed adjacently to the dorsal explant

   • axons within a certain distance would turn and grow towards the floor plate

   • suggesting a chemoattractant is being secreted in floor plate

What did this experiment suggest about the chemoattractant responsible?

• it must be a diffusing molecule because as the floor plate was moved away from the explant the axons kept growing towards it

How was the chemoattractant responsible found?

• biochemical purification led to cloning of the gene encoding the floor plate chemoattractant protein - expressed along midline of vertebrate NS

• chemoattractant = netrin - a secreted protein that can associate with the extracellular matrix

• this gene was transferred into cells that don’t normally have a chemoattractant effect but they began to as they began to secrete netrin

What effect do roof plate BMPs have on commissural axons?

• the commissural axons are repelled by BMPs

• dorsal spinal cord axons placed adjacent to the roof plate (expressing BMPs) = axons move away from the roof plate

What does the effect of BMPs on commissural axons tell us about axons pathway patterning?

• cells expressing BMP7 mimic repulsion of roof plate

• C axon guidance to FP is due to combination of push and pull from chemotropic factors

• early patterning factors can be used to layer guide axons

How do long and short range guidance cues work together to guide axons to targets?

• Sema1 acts as a short range cue

• Sema2 acts as a long range cue

  → this creates a gradient

• if Sema1 blocked = axons in wrong area

• Sema2 blocked = axons stray before they get to the boundary

• so combination of the four forces are used to guide axons at different stances in their pathway

What is netrin?

A secreted protein which can associate with the extracellular matrix