Axon guidance

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Axon guidance Herwig Baier Email
herwig.baier_at_ucsf.edu Phone x2-4301 Office Rock
Hall 348F
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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
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Growth 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)
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The cytoskeleton of the growth cone continuously
changes during outgrowth and navigation.
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Actin Tubulin
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Actin-GFP
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Growth cones are highly dynamic structures.
Mauthner cell axon labeled with DiI in the spinal
cord of a zebrafish embryo contacting a
motoneuron (left) and forming an en passant
synapse (right) Jontes et al., 2000
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How does the growth cone get from A to
B? Consider Enormous distances. Neuronal
diversity. 3 possibilities (discuss on white
board)
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Growth cones turn in response to gradients of
axon guidance molecules
Dickson, 2002
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Axon guidance cues can be either attractive or
repulsive
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Four families of axon guidance molecules and
their receptors Netrins (DCC, Unc5) Slits
(Robo) Semaphorins (plexin, neuropilin)
Ephrins/Eph (Eph/ephrin)
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Gradient reading requires detection of small
concentration changes (a few percent over the
length of the growth cone)
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Gradient reading can be achieved by two mechanisms
Netrin gradient
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1) Local autocatalysis (plus lateral inhibition)
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2) Adaptation
Shallow, unreadable gradient
Intracellularly enhanced gradient
No gradient
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Growth cone "sensory physiology"
1) Local autocatalysis amplifies a small
concentration difference to generate a larger
absolute difference. Lateral inhibition
prevents the autocatalysis to spread and
suppresses competing activation foci. 2)
Adaptation shifts the baseline down to generate a
larger relative concentration difference.
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Growth cones are sensitive to external
concentration differences of ca. 2
1) Local autocatalysis (plus lateral inhibition)
300100
102100
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2) Adaptation
Shallow gradient
Enhanced gradient
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Calcium imaging with indicator dyes
Fluo-3
Excitation wavelength (confocal Argon laser)
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Growth cone guidance by local calcium increase
and decrease
Hi Cae
Lo Cae
Zheng, 2000
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Caged calcium Released by UV spot
illumination of a growth cone loaded with NP-EGTA
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Growth cone behavior depends on resting CaI
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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).
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Guidance across the midline Conservation of
mechanisms
Dickson, 2002
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Crossing the midline Molecules and mechanisms
Stein Tessier-Lavigne, 2001
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Crossing the midlineA smooth journey controlled
by dynamic receptor interactions
Stein Tessier-Lavigne, 2001
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Local protein synthesis is required for axon
guidance beyond the midline
Brittis et al., 2002
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Morphogens BMP and Hedgehog in commissural axon
guidance
Charron et al., 2003
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Morphogens Wnt and Shh in caudo-rostral axon
guidance
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Discussion paper Retinal axon pathfinding
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Genetic analysis of the retinotectal projection
Optic nerve
DiI injection DiO injection
Chiasm
Retina
Optic tract
Tectum
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A screen for mutations disrupting axon
pathfinding and retinotopyin zebrafish
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Lipophilic axon tracers
DiI (di-C18-...indo-carbocyanine)
DiO (di-C18-oxa-carbocyanine)
DiD (di-C18...-indo-di-carbocyanine)
DiA (di-C16-aminostyrylpyrimidinium)
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Axon pathfinding phenotypes discovered in the
retinotectal screen
Baier et al., 1996 Trowe et al., 1996 Karlstrom
et al., 1996
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Somatotopic mapping Body surface map in the
cortex
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The retinotectal projection creates a faithful
map of the visual space in the brain
D V (L) V D (M) N P (C) T A (R)
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Sperry's chemoaffinity theory Connections
between retinal and tectal neurons are specified
by "key-and-lock" interactions of cell-surface
molecules specific to these cells.
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Positional information is graded and is being
"read" by retinal axons
1. Growth cone guidance
2. Axon branching (not in all systems)
3. Refinement of axonal arbors
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Axon guidance by gradients of attractive and
repulsive cues in a two-dimensional field
Branching
D V
Normal route
Guidance from ectopic position
A P
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In vitro retinotectal guidance The stripe assay
Stripe assay was first carried out with crude
membrane preparations from different parts of the
tectum.
ant
post
Walter et al., 1987
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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
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Stripe assay results
Ephrin-A2/mock
Ephrin-A5 (12)/mock
Ephrin-A5 (14)/mock
Monschau et al., 1997
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Ephrin-A2 and A5 both specify A/P position in the
tectum
assuming crowding results in a countergradient
and/or more competition in anterior tectum
Feldheim et al., 2000
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Basic model of retinotectal mapping (along the
A/P axis)
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Axon competition for tectal territory? Evidence
from surgical manipulations
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Map regulation in genetically manipulated mice
wt het hom
Brown et al., 2001
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Knock-in cells (EphA-OE) and wild-type cells form
two overlapping maps
Brown et al., 2001
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Summary of findings in the Isl2-EphA3 knock-in
mice Two maps neither map is normal.
Wild-type axons are "pushed" more anteriorly by
EphA overexpressors. EphA level determines
target position, regardless where the RGC
resides. Relative, not absolute, EphA levels
determine final positions.
Additional mechanisms Competition for target
space? Additional gradients?
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Topographic organization of the olfactory
system Not a smooth map as in other sensory
systems
Mombaerts, 2001
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Molecular basis of olfactory reception
1000 receptor genes clustered in the genome.
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One-neuron-one receptor choice Negative feedback
from functional odorant receptor (OR) ensures
monoallelic expression of receptor genes
Serizawa et al. (2003)
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OR gene swapping reveals a role for the OR
protein in axon pathfinding
Mombaerts et al., 1996 Wang et al., 1998
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Olfactory neuron targeting experiments A single
OR gene (in fact only one allele of the gene) is
selected from a repertoire of 2 alleles x 1000
genes. This monoallelic choice is regulated by
transcriptional control, using negative feedback
from functional OR protein (not by gene
rearrangements). The OR confers a narrow
odorant specificity on the sensory neuron. OR
protein is also present on the surface of axons
and is required for correct glomerulus
targeting. The receptor-swap experiment shows
that the receptor is not sufficient for
glomerulus targeting. Other guidance systems
(ephrin-A/EphA and Sema3a) also play a role.
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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). 6) The
molecular organization of the olfactory system
poses a challenge to axon guidance mechanisms.
Complementary gradients cannot account for
pathfinding decisions and glomerulus choice.
Somehow, stochastic decisions are linked to
target specificity.