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: 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 ) 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: ‘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 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) wild type (untransfected) Cdc42RacRho

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 Ti1 pioneer axons in grasshopper 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 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 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 Drosophila embryo side view anteriorposterior ventral view dorsal ventral

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 Gradient of Nerve Growth Factor Competing growth cones Target cell Secreting 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 process of programmed cell death (aka apoptosis) Target cell Secreting 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 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; )

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, ) 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,

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, 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: )

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

Zito et al, 1999 Marking synapses: Live synapse elimination Walsh and Lichtman (2003) Neuron 37: Live synapse growth: Zito et al., (1999) Neuron 22:

Aberle et al., (2002) Neuron 33, Marques et al.,(2002) Neuron 33, wit A12 /wit B11 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 9:670-5 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: Tessier-Lavigne and Goodman (2001) Science 274: 1123 Sanes and Lichtman (2001) Nature Reviews Neuroscience 2: Sanes and Lichtman (1999) Annual Reviews in Neuroscience 22: Jan and Jan (2001) Genes and Development., 15;