Axon Outgrowth and Pathfinding

Wiring 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

Steps during neural development:

• Neurogenesis• Compartmentalization• Neural differentiation• Neural migration• Axonal pathfinding• Synaptogenesis

Axonal growth cone

Movement 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.

Karl H. Pfenninger

Guidance 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).

Molecular 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

Semaphorins example for dual function:

.

E) In the presence of NT3, Sema III elicits outgrowth of neurites (NT-3)

D) In the presence of NGF Sema III has a repellent effect on neurite growth

Dual function:

Secreted (subclass 2 + 3) or membrane bound ligands (GPI anchored or transmembrane domain)have Chemorepellent or chemoattractive functions.

Linkage 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).

Ephrins +Eph receptors

Ephrin-expressing cell (top) interacting with Eph-receptor expressing cell (bottom). Ligand–receptor interactions (green) are dimeric or oligomeric Eph–ephrin complexes. GPI, glycosylphosphatidylinositol; SAM, sterile alpha -motif. Functions:Vascular developmentBorder formationCell migrationAxon guidanceSynaptic plasticity

Klas Kullander1 & Rüdiger Klein, 2002

Projections 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 projections

Note: Axons travel together (fasciculation)Axons use preexisting projectionsGuidepost cells show the way

Growth cones are sensory-motile organelles at the tipof growing axons and dendrites.

Golgi-stained section of the spinal cord(specimen prepared by Ramon y Cajal, 1892, photographed 100 years later)

The cytoskeleton of the growth cone continuously changesduring outgrowth and navigation.

ActinTubulin

Growth 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

How does the growth cone get from A to B?

Consider• Enormous distances.• Neuronal diversity.

Growth cones turn in response to gradients of axon guidance molecules

Dickson, 2002

Axon guidance cues can be either attractive or repulsive

Four families of axonguidance moleculesand their receptors

• Netrins (DCC, Unc5)

• Slits (Robo)

• Semaphorins (plexin, neuropilin)

• Ephrins/Eph (Eph/ephrin)

Gradient reading

• requires detection of small concentration changes (a few percent over the length of the growth cone)

Gradient reading can be achieved by two mechanisms

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nn

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nn

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1) Local autocatalysis(plus lateral inhibition)

No gradient Shallow, unreadablegradient

2) Adaptation

Intracellularlyenhanced gradient

Netrin gradient

1) Local autocatalysis amplifies a small concentration difference to 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.

Growth cone "sensory physiology"

Growth cones are sensitive to external concentration differences of ca. 2%

1) Local autocatalysis(plus lateral inhibition)

Shallow gradient

2) Adaptation

Enhanced gradient

102:100

300:100

3 : 1

Calcium imaging with indicator dyes

Fluo-3

Excitation wavelength(confocal Argon laser)

Growth cone guidance by local calcium increase and decrease

Zheng, 2000

Hi [Ca++]e

Lo [Ca++]e

Caged calcium:Released by UV spot illumination

of a growth cone loaded with NP-EGTA

Growth cone behavior depends on resting [Ca++]I

Three examples of axon guidance in vivo

1) Navigation of commissural axons towards and across the midline.

2) Retinotectal map formation.

3) Olfactory system (if time allows).

Guidance across the midline

• Conservation of mechanisms

Dickson, 2002

Crossing the midline: Molecules and mechanisms

Stein & Tessier-Lavigne, 2001

Stein & Tessier-Lavigne, 2001

Crossing the midline:A smooth journey controlled by dynamic receptor interactions

Local protein synthesis is required for axon guidance beyond the midline

Brittis et al., 2002

Morphogens BMP and Hedgehog in commissural axon guidance

Charron et al., 2003

Morphogens Wnt and Shh in caudo-rostral axon guidance

Discussion paper: Retinal axon pathfinding

Genetic analysis of the retinotectal projection

DiI injection

DiO injection

Chiasm

Retina

Tectum

Optictract

Opticnerve

A screen for mutations disrupting axon pathfinding and retinotopyin zebrafish

Lipophilic axon tracers

DiI (di-C18-...indo-carbocyanine) DiO (di-C18-oxa-carbocyanine)

DiD (di-C18...-indo-di-carbocyanine) DiA (di-C16-…amino…styryl…pyrimidinium)

Axon pathfinding phenotypes discovered in the retinotectal screen

Baier et al., 1996; Trowe et al., 1996; Karlstrom et al., 1996

Somatotopic mapping: Body surface map in the cortex

The retinotectal projection creates a faithful mapof the visual space in the brain

D V (L)

V D (M)

N P (C)

T A (R)

Sperry's chemoaffinity theory

Connections between retinal and tectal neurons are specified by"key-and-lock" interactions of cell-surface molecules specificto these cells.

Positional information is gradedand is being "read" by retinal axons

1. Growth cone guidance

2. Axon branching (not in all systems)

3. Refinement of axonal arbors

Axon guidance by gradients of attractive and repulsive cuesin a two-dimensional field

Branching

Guidance

from ectopic position

Normalroute

D

V

A P

Attraction=Repulsion

In vitro retinotectal guidance: The stripe assay

Walter et al., 1987

Stripe assay was first carried out with crude membrane preparationsfrom different parts of the tectum.

ant post

Stripe 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-A post: high ephrin-A

Ephrin-A5 (1:2)/mock

Ephrin-A5 (1:4)/mock

Ephrin-A2/mock

Stripe assay results

Monschau et al., 1997

assuming crowding results in a countergradient and/ormore competition in anterior tectum

Ephrin-A2 and A5 both specify A/P position in the tectum

Feldheim et al., 2000

Basic model of retinotectal mapping (along the A/P axis)

Axon competition for tectal territory?Evidence from surgical manipulations

Summary

1) Growth cones are sensory and motile organelles at the tip of axons

2) 4 + 4 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).