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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 QuickTime™ and a ÉÇÅ[ÉVÉáÉì JPEG A decompressor are needed to see this picture. 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