Axon Guidance and
Synaptogenesis
Module 404
Sean Sweeney
Aims and outcomes:
To understand how neurons develop from an
undifferentiated state to a complex morphology.
To understand the mechanisms that neurons use to
grow in appropriate directions to find the correct
partners and generate the ‘wiring diagram’ that
constitutes the functioning brain.
To be aware that different molecules expressed during
the process of neuronal differentiation generate
neuronal diversity AND molecular specificity to organise
the ‘wiring diagram’.
Undifferentiated neuronal
cells grow to become
morphologically distinct
and functioning nerves….
….making appropriate
connections with correct
synaptic partners in distinct
areas of the brain to form
circuits.
How do growing nerves generate the final wiring diagram?
Number of neurons in the human brain:
20,000,000,000 to 50,000,000,000
Number of synapses: 1014
Number of synapses per neuron: 2000 to 5000
How does a genetically programmed system organise this
complexity?
Neural induction, migration, determination and differentiation
(lectures in module 301)
Axon outgrowth (301)
Axon guidance (301)
Target selection
Synaptogenesis (formation and function)
Synapse refinement (addition and subtraction)
Behavioural development
Neuronal ‘stereotypy’ identified by Ramon y Cajal and
others (ca. 1890-1910)
Coghill and others (1929) ‘individuation vs integration in
the development of behaviour’ : Neurons, by their activity
and ‘learning’, select the correct connections during
development. ‘primitive thrashings of developing organisms’.
The Chemoaffinity Hypothesis:
Sperry, R.W. (1943) J. Expl. Zool. 92: 263-279 ‘Effect of 180
degree rotation of the retinal field on visuomotor coordination
The Chemoaffinity Hypothesis:
Severing the optic nerve, rotating the eye 180 degrees
and allowing the nerve to regenerate results in visuomotor
impairments in the frog (Sperry)
The Chemoaffinity Hypothesis of Sperry:
1. Axons have differential (biochemical) markers
2. Target cells have corresponding markers
3. Markers are the product of cellular differentiation
4. Axonal growth is actively directed by markers to
establish specific connections
It follows that: The code for axon guidance is ‘hard-wired’
(GENETIC!)
There is an order to the code
The Chemoaffinity Hypothesis Cont:
Uncrossing of optic
nerve fibres followed
by nerve regeneration
leads to
visuomotor defects
in frogs
(importance of a
‘midline’ choicepoint
the brain is bilaterally
symmetric)
Growth cones are active and dynamic projections rich in
microtubules and actin filaments
“The cone of growth is endowed with amoeboid movements. It could be
compared with a living battering ram, soft and flexible, which advances,
pushing aside mechanically the obstacles which it finds in its way,
until it reaches the area of its peripheral distribution.”
Santiago Ramon Y Cajal
Guidance Cues:
Target derived (positive and negative cues)
Local vs long range (diffusable vs cell attached in the
extra-cellular matrix)
Time dependent
Actin cytoskeleton
dynamics can be
regulated by
small monomeric
G-proteins
Rho - induces stress
fibres
Cdc42 - induces
filopodia
Rac - induces
lamellipodia
wild type
(untransfected)
Cdc42
Rac
Rho
Fibroblasts transfected
with a small G-protein
and stained for actin
(G-protein is
engineered so that it
cannot hydrolyse
GTP and is therefore
constitutively active)
Summary
Axons can use many
cues and combinations of
cues to guide them
to their correct location.
These cues are interpreted
by the growth cone as the
perceived cues act to
regulate the actin
cytoskeleton and
determine the direction
of the growing axon
Neural induction, migration, determination and differentiation
(lectures in module 301)
Axon outgrowth (301)
Axon guidance (301)
Target selection
Synaptogenesis (formation and function)
Synapse refinement (addition and subtraction)
Behavioural development
Guideposts/choicepoints
Dictinct identifiable cells
act as local routemarkers
to give direction to
growing pioneer axons
Ti1 pioneer axons in
grasshopper embryo
(Bentley and Caudy 1983)
Contact mediated attraction
Growth cones adhere to substrate
cell upon detection of a positive
cue (a cell surface molecule)
Mediated by:
CAMs (IgG superfamily proteins)
cadherins
ephrins/Eph receptors
integrins
Contact mediated
repulsion
growth
Growth cones retreat from
a cell upon detection of a
negative cue (a cell
surface molecule)
Mediated by:
collapsins/semaphorins
growth
The collapsins/semaphorins
Chemoattraction
Long distance cue
Secreted
Mediated by:
Nerve Growth Factor
Netrin/DCC/unc5 interaction
Gradient of secreted cue
The Netrins/DCC/unc5
Chemorepulsion
Long distance cue
Secreted
Mediated by:
slit/roundabout interaction
semaphorins/collapsins
Gradient of secreted cue
Slit/roundabouts
The Drosophila embryonic
ventral nerve cord
anterior
posterior
ventral view
dorsal
ventral
Drosophila embryo
side view
Fasciculation:
pioneers vs followers
Followers can fasciculate
and de-fasciculate and use
complex combinations of cues
to do so
Trophic support, a mechanism for regulating
numbers and direction of growth cones
growth
Target cell
Secreting NGF
Competing growth cones
Gradient of Nerve Growth Factor
Trophic support, a mechanism for regulating
numbers and direction of growth cones
growth
Target cell
Secreting NGF
Competing growth cones
Gradient of Nerve Growth Factor
Growing nerves that receive insufficient NGF die by a process
of programmed cell death (aka apoptosis)
The Nerve Growth Factors/Trk receptors
Neural induction, migration, determination and differentiation
(lectures in module 301)
Axon outgrowth (301)
Axon guidance (301)
Target selection
Synaptogenesis (formation and function)
Synapse refinement (addition and subtraction)
Behavioural development
Dendritogenesis: 1st step, determine polarity:
One neurite predominates
and becomes the axon,
others become the dendrites.
Thereafter, guidance cues
may be similar to those
guiding axons, growth occurs
in similar timewindow
Dendrites may also utilise
‘tiling’.
The Drosophila
larval body wall
is innervated
by sensory dendrites
of many different
classes
(Grueber et al., 2002
Development, 129;
2867-78)
Sensory dendrites
occupy territories that
Exclude dendrites of the
same sensory
class. Ablation identifies a
mutual inhibition
that ensures efficient ‘tiling’
of the body wall surface.
Also occurs in zebrafish
‘Heteroneural Tiling’
Target selection and synaptogenesis.
Dscam: determining adhesivity and diversity
In Dscam nulls, all terminal arbours fail to
develop. In mutants lacking various splice
forms, many terminal arbours are lacking.
Dscam generates diversity and specificity of
connections (Bharadwaj and Kolodkin (2006)
Cell 125, 421-424)
Each neuron expresses a small and distinct
subset of alternatively spliced DSCAM
isoforms required for the recognition of ‘like’
targets.
Grueber paper DSCAM
Dendrite ‘self’-avoidance contributes to
efficient tiling: isoneuronal recognition
DSCAM mediates isoneuronal
recognition by an inhibitory mechanism
regulated by the C-terminal of the
protein (see Zinn, K. (2007) Cell 129,
455-456
Neural induction, migration, determination and differentiation
(lectures in module 301)
Axon outgrowth (301)
Axon guidance (301)
Target selection
Synaptogenesis (formation and function)
Synapse refinement (addition and subtraction)
Behavioural development
Synaptogenesis: what are the cues that induce a synapse
to form from a growth cone?
Many of the molecules regulating guidance are also
involved in synaptogenesis: are these cues inductive?
Partner recognition (cessation in growth)?: adhesion molecules
sidekicks, flamingo, DSCAM, SYG1, SYG2
Shen (2004) Molecular mechanisms of target specificity
during synapse formation. Curr Opin Neurobiol 14, 83-8
Prior to synaptogenesis: transient rise in calcium
Morphological transition from growth cone to synaptic bouton
Importance of transport ‘packets’
e.g. PTV packets (Piccolo-Bassoon transport vesicle)
immaculate connections (imac): Pack-Chung et al (2007)
Nat. Neurosci. 10, 980-989
Signals for synaptogenesis? Agrin?
The mammalian
neuromuscular
synapse
Acetylcholine receptors
are diffusely distributed
across the muscle fibre
until the arrival of a neuron
Acetylcholine receptors cluster in response to the arrival
of a neuron: does the neuron promote synapse maturation
Purification of ‘Agrin’, a proteoglycan normally secreted
by the neuron, suggested Agrin induced synapse
maturation (Sanes et al., (1978) J.Cell Biol 78:176-198)
Agrin deficient neurons fail to
Induce neuromuscular synapse
maturation
1. Agrin recruits AchRs
2. Agrin induces transcription
Of AchRs from ‘synaptic nuclei’
3. Transcription of AchRs from
extra-synaptic nuclei is
downregulated
4. Rearrangement of muscle
cytoskeleton
5. Retrograde signal from the muscle
to the nerve to stabilise the synapse
Neural induction, migration, determination and differentiation
(lectures in module 301)
Axon outgrowth (301)
Axon guidance (301)
Target selection
Synaptogenesis (formation and function)
Synapse refinement (addition and subtraction)
Behavioural development
Marking synapses:
Live synapse elimination
Walsh and Lichtman
(2003) Neuron 37:
67-73
Live synapse growth:
Zito et al., (1999) Neuron
22: 719-729
Zito et al, 1999
Synaptic growth is regulated by a TGF-ß type-II
receptor wishful thinking (wit)
witA12/witB11
wt
Aberle et al., (2002) Neuron 33, 545-558
Marques et al.,(2002) Neuron 33, 529-543
Neural induction, migration, determination and differentiation
(lectures in module 301)
Axon outgrowth (301)
Axon guidance (301)
Target selection
Synaptogenesis (formation and function)
Synapse refinement (addition and subtraction)
Behavioural development:
Bate, M. (1999) Current Opinion in Neurobiology 9:670-5
Bate, M. (1998) International Journal of Developmental
Biology 42: 507-9
Reading Material:
Purves et al, 3rd Edition, Chapter 22.
Sanes, Reh and Harris., Development of the Nervous System.
2nd edition. Academic Press 2006
Bentley and Caudy (1983) Nature 304:62-65
Sanes et al., (1978) J. Cell Biol 78:176-198
Tessier-Lavigne and Goodman (2001) Science 274: 1123
Sanes and Lichtman (2001) Nature Reviews Neuroscience
2:791-805
Sanes and Lichtman (1999) Annual Reviews in Neuroscience
22:389-442
Jan and Jan (2001) Genes and Development., 15; 2627-2641