Transcript Axon guidance

Axon Guidance and
Synaptogenesis
Module 632
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
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 Cont:
Uncrossing of optic
nerve fibres followed
by nerve regeneration
leads to
visuomotor defects
in frogs
(importance of a
‘midline’ choicepoint)
The Chemoaffinity Hypothesis of Sperry:
1. Axons have differential 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
Experimental Systems to Identify Axonal Guidance Cues:
The vertebrate retinotectal system
Explants
Vertebrate spinal cord/neural tube
Drosophila embryonic ventral nerve cord
C.elegans nervous system
Zebrafish oculomotor system
In vitro growth cone manipulation
Explants:
Tissue from growing neurons and target regions can be
Manipulated in vivo
Pain fibres of the DRG are repulsed by sema3a
The vertebrate spinal cord/neural tube
Advantages: in vivo/in vitro
Manipulation, vertebrate genetic
Models (mouse)
The Drosophila embryonic
ventral nerve cord
anterior
posterior
ventral view
dorsal
ventral
Drosophila embryo
side view
Drosophila and C.elegans: advantages as systems for
analysis of axon guidance:
Advanced genetics
Sequenced genomes
Small size
Small nervous systems with a well defined wiring diagram
Mutagenesis screens in Zebrafish (a vertebrate)have indentified
many molecules and mechanisms involved in regulating
retinotectal axon pathfinding
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
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Microtubules (provide rigidity and drive)
F-actin (motility and direction)
Membrane addition (new growth and sensing of environment)
Adhesion molecules and receptors (homophilic and heterophilic)
Guidance Cues:
Target derived (positive and negative cues)
Local vs long range (diffusable vs cell attached in the
extra-cellular matrix)
Time dependent
Responsiveness of growth cones to guidance cues can be
Exploited in vitro
Guideposts
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
Molecules mediating contact mediated attraction:
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
The Nerve Growth Factors/Trk receptors
Chemorepulsion
Long distance cue
Secreted
Mediated by:
slit/roundabout interaction
semaphorins/collapsins
Gradient of secreted cue
Slit/roundabouts
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)
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
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
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
(Chen et al.,(2006)Cell 125:607-620)
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?
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
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