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 organisethe ‘wiring diagram’.
Undifferentiated neuronalcells grow to become morphologically distinctand 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 thiscomplexity?
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 andothers (ca. 1890-1910)
Coghill and others (1929) ‘individuation vs integration in the development of behaviour’ : Neurons, by their activityand ‘learning’, select the correct connections duringdevelopment. ‘primitive thrashings of developing organisms’.
The Chemoaffinity Hypothesis:
Sperry, R.W. (1943) J. Expl. Zool. 92: 263-279 ‘Effect of 180degree rotation of the retinal field on visuomotor coordination
The Chemoaffinity Hypothesis:
Severing the optic nerve, rotating the eye 180 degreesand allowing the nerve to regenerate results in visuomotorimpairments 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 opticnerve fibres followedby nerve regenerationleads tovisuomotor defectsin frogs
(importance of a ‘midline’ choicepointthe brain is bilaterallysymmetric)
Growth cones are active and dynamic projections rich inmicrotubules 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 theextra-cellular matrix)
Time dependent
Actin cytoskeletondynamics can be regulated by small monomericG-proteins
Rho - induces stressfibres
Cdc42 - inducesfilopodia
Rac - induceslamellipodia
Fibroblasts transfectedwith a small G-proteinand stained for actin(G-protein is engineered so that it cannot hydrolyseGTP and is thereforeconstitutively active)
wild type(untransfected)
Cdc42 Rac Rho
Summary
Axons can use manycues and combinations ofcues to guide them to their correct location.
These cues are interpretedby the growth cone as theperceived cues act to regulate the actin cytoskeleton and determine the directionof 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
Ti1 pioneer axons ingrasshopper embryo(Bentley and Caudy 1983)
Dictinct identifiable cells act as local routemarkers to give direction to growing pioneer axons
Contact mediated attraction
Growth cones adhere to substratecell upon detection of a positivecue (a cell surface molecule)
Mediated by:CAMs (IgG superfamily proteins)cadherinsephrins/Eph receptorsintegrins
Contact mediated repulsion
Growth cones retreat froma cell upon detection of a negative cue (a cell surface molecule)Mediated by:collapsins/semaphorins
growth
growth
The collapsins/semaphorins
Chemoattraction
Long distance cueSecretedMediated by:Nerve Growth FactorNetrin/DCC/unc5 interaction
Gradient of secreted cue
The Netrins/DCC/unc5
Chemorepulsion
Long distance cueSecretedMediated by:slit/roundabout interactionsemaphorins/collapsins
Gradient of secreted cue
Slit/roundabouts
The Drosophila embryonic ventral nerve cord
Drosophila embryo side view
anterior posterior
ventral view
dorsal
ventral
Fasciculation: pioneers vs followers
Followers can fasciculateand de-fasciculate and usecomplex combinations of cuesto do so
Trophic support, a mechanism for regulating numbers and direction of growth cones
growth
Gradient of Nerve Growth Factor
Competing growth cones
Target cellSecreting NGF
Trophic support, a mechanism for regulating numbers and direction of growth cones
growth
Gradient of Nerve Growth Factor
Competing growth cones
Growing nerves that receive insufficient NGF die by a processof programmed cell death (aka apoptosis)
Target cellSecreting NGF
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 predominatesand becomes the axon,others become the dendrites.Thereafter, guidance cues may be similar to thoseguiding axons, growth occursin similar timewindowDendrites may also utilise‘tiling’.
The Drosophilalarval body wallis innervatedby sensory dendritesof many differentclasses(Grueber et al., 2002Development, 129;2867-78)
Sensory dendritesoccupy territories that Exclude dendrites of the same sensory class. Ablation identifies a mutual inhibitionthat 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 toefficient tiling: isoneuronal recognition
DSCAM mediates isoneuronal recognition by an inhibitory mechanismregulated 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 synapseto form from a growth cone?
Many of the molecules regulating guidance are alsoinvolved in synaptogenesis: are these cues inductive?
Partner recognition (cessation in growth)?: adhesion moleculessidekicks, flamingo, DSCAM, SYG1, SYG2Shen (2004) Molecular mechanisms of target specificityduring synapse formation. Curr Opin Neurobiol 14, 83-8
Prior to synaptogenesis: transient rise in calcium
Morphological transition from growth cone to synaptic boutonImportance 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 mammalianneuromuscular synapse
Acetylcholine receptorsare diffusely distributedacross the muscle fibreuntil the arrival of a neuron
Acetylcholine receptors cluster in response to the arrivalof a neuron: does the neuron promote synapse maturation
Purification of ‘Agrin’, a proteoglycan normally secreted by the neuron, suggested Agrin induced synapsematuration (Sanes et al., (1978) J.Cell Biol 78:176-198)
Agrin deficient neurons fail to Induce neuromuscular synapsematuration
1. Agrin recruits AchRs2. 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
Zito et al, 1999
Marking synapses:Live synapse eliminationWalsh and Lichtman(2003) Neuron 37:67-73
Live synapse growth:Zito et al., (1999) Neuron22: 719-729
Aberle et al., (2002) Neuron 33, 545-558
Marques et al.,(2002) Neuron 33, 529-543
witA12/witB11 wt
Synaptic growth is regulated by a TGF-ß type-II receptor wishful thinking (wit)
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 9:670-5Bate, 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 304:62-65Sanes et al., (1978) J. Cell Biol 78:176-198Tessier-Lavigne and Goodman (2001) Science 274: 1123Sanes and Lichtman (2001) Nature Reviews Neuroscience2:791-805Sanes and Lichtman (1999) Annual Reviews in Neuroscience22:389-442Jan and Jan (2001) Genes and Development., 15; 2627-2641