2Wiring of the nervous system Axon guidance is part of a genetic program that controls neuronal connections.• Patterning of the brain• Neuronal cell fate determination• Neuronal differentiation• Axon pathfinding• Dendrite development• Map formation• Layer formation• Synaptogenesis• Synaptic competition, homeostasis, and plasticity
3Steps during neural development: NeurogenesisCompartmentalizationNeural differentiationNeural migrationAxonal pathfindingSynaptogenesis
4Axonal growth coneKarl H. PfenningerMovement of the growth cone is mediated by a cytoskeletal lattice containing the motor proteins actin and myosin. As the neurite extends behind the moving growth cone, the microtubule backbone of the neurite is constructed from molecules of tubulin.
5Guidance of Axons by short- and long-range cues: Attractive or repulsive Four types of mechanisms contribute to the guidance of the growth cone:Contact attraction, chemoattractioncontact repulsion, chemorepulsion.Individual growth cones might be "pushed" from behind by a chemorepellent, "pulled" from in front by a chemoattractant, and "hemmed in" by attractive and repulsive local cues (cell surface or extracellular matrix molecules).Adapted from Tessier-Lavigne and Goodman (1996).
6Molecular guidance molecules Conserved families of guidance molecules (A) and their receptors (B). Examples:● SLIT secreted proteins, control midline repulsion, dual role, signaling through roundabout receptors (Robo)● Ephrins (A +B) membrane anchored, repellent and attractive functions, receptors: EphA, EphB●Netrins and their receptors●Semaphorins 5 different subfamilies characterized by a 500 aa semaphorin domain, secreted and anchored.Cell Adhesion Molecules ( N-CAM, L1 or Fasciclins
7Semaphorins example for dual function: D) In the presence of NGF Sema III has a repellent effect on neurite growthE) In the presence of NT3, Sema III elicits outgrowth of neurites (NT-3).Secreted (subclass 2 + 3) or membrane bound ligands (GPI anchored or transmembrane domain)have Chemorepellent or chemoattractive functions.
8Linkage of the actin cytoskeleton to a permissive surface is required for forward advance. Actin is polymerized at the leading edge of the growth cone (right) and is swept toward the rear.If the actin meshwork is not linked to cell surface receptors that bind permissive molecules on adjacent cell surfaces, the actin cycles from front to rear but does not advance the growth cone.If the actin meshwork is attached to these receptors, the meshwork remains in place and newly polymerized actin helps advance the leading edge.Modified from Lin et al.(1994).
10Projections from preplate guide thalamocortical fibers Top: Preplate cells send out their axons towards the internal capsule (red). Thalamic axons project through the IC and meet cortical axons.Right: Handshake between thalamic and preplate axons and precise topography of early thalamicortical projectionsNote:Axons travel together (fasciculation)Axons use preexisting projectionsGuidepost cells show the way
11Growth cones are sensory-motile organelles at the tip of growing axons and dendrites.Golgi-stained section of the spinal cord(specimen prepared by Ramon y Cajal, 1892, photographed 100 years later)
12The cytoskeleton of the growth cone continuously changes during outgrowth and navigation.
14Growth cones are highly dynamic structures. Mauthner cell axon labeled with DiI in the spinal cord of a zebrafish embryocontacting a motoneuron (left) and forming an en passant synapse (right)Jontes et al., 2000
15How does the growth cone get from A to B? Consider• Enormous distances.• Neuronal diversity.
16Growth cones turn in response to gradients of axon guidance molecules Dickson, 2002
17Axon guidance cues can be either attractive or repulsive
18Four families of axonguidance moleculesand their receptors• Netrins (DCC, Unc5)• Slits (Robo)• Semaphorins (plexin, neuropilin)• Ephrins/Eph (Eph/ephrin)
19Gradient reading• requires detection of small concentration changes (a few percent over the length of the growth cone)
20Gradient reading can be achieved by two mechanisms Netrin gradientnnnnnnnnnnnnnnnnn1) Local autocatalysis(plus lateral inhibition)nnnnn2) AdaptationNo gradientShallow, unreadablegradientIntracellularlyenhanced gradient
21Growth cone "sensory physiology" 1) Local autocatalysis amplifies a small concentration differenceto generate a larger absolute difference. Lateral inhibition preventsthe autocatalysis to spread and suppresses competing activation foci.2) Adaptation shifts the baseline down to generate a larger relativeconcentration difference.
22Growth cones are sensitive to external concentration differences of ca 1) Local autocatalysis(plus lateral inhibition)300:100102:1003 : 12) AdaptationShallow gradientEnhanced gradient
23Calcium imaging with indicator dyes Fluo-3Excitation wavelength(confocal Argon laser)
40Axon pathfinding phenotypes discovered in the retinotectal screen Baier et al., 1996; Trowe et al., 1996; Karlstrom et al., 1996
41Somatotopic mapping: Body surface map in the cortex
42The retinotectal projection creates a faithful map of the visual space in the brainD V (L)V D (M)N P (C)T A (R)
43Sperry's chemoaffinity theory Connections between retinal and tectal neurons are specified by"key-and-lock" interactions of cell-surface molecules specificto these cells.
44Positional information is graded and is being "read" by retinal axons 1. Growth cone guidance2. Axon branching (not in all systems)3. Refinement of axonal arbors
45Axon guidance by gradients of attractive and repulsive cues in a two-dimensional fieldBranchingDVNormalrouteAttraction=RepulsionGuidancefrom ectopic positionA P
46In vitro retinotectal guidance: The stripe assay Stripe assay was first carried out with crude membrane preparationsfrom different parts of the tectum.antpostWalter et al., 1987
47Stripe assay......was used to test molecules that were differentially expressed between anterior and posterior tectum.In 1995, the Bonhoeffer and Flanagan labs independently discovered the ephrins (under different names).Ephrin-A2 and ephrin-A5 are expressed as gradients in the tectum. Their receptors are expressed as gradients in the retina.ant: low ephrin-Apost: high ephrin-A
48Stripe assay results Ephrin-A2/mock Ephrin-A5 (1:2)/mock Monschau et al., 1997
49Ephrin-A2 and A5 both specify A/P position in the tectum assuming crowding results in a countergradient and/ormore competition in anterior tectumFeldheim et al., 2000
50Basic model of retinotectal mapping (along the A/P axis)
51Axon competition for tectal territory? Evidence from surgical manipulations
52Summary1) Growth cones are sensory and motile organelles at the tip of axons2) families of axon guidance molecules are responsible for most of the pathfinding decisions observed so far in the nervous system.3) Axon guidance depends on gradient sensing by the growth cone (or entire axon).4) Growth cone responses are not static, but are dynamically regulated by the local environment and the intracellular state.5) Most sensory projections are topographically organized (neighborhood is preserved). This is achieved by axon guidance (plus other mechanisms).