1. ESTABLISHMENT OF NEURONAL IDENTITY
LECTURE 22: AXONAL GUIDANCE
REQUIRED READING: Kandel text, Chapter 54
ESTABLISHMENT OF NEURONAL IDENTITY
AXONAL GUIDANCE TO TARGETS
NEUROTROPHIC SUPPORT FROM TARGET TISSUES
SYNAPTOGENESIS

2. HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?
THEORY 1: AXON PROJECTIONS ARE IMPRECISE…PROPER CONNECTIONS ARE WORKED
OUT BY ELECTRICAL ACTIVITIES DURING EARLY EXPERIENCE
THEORY 2: CHEMOSPECIFICITY MODEL….AXONS ARE DIRECTED TO THEIR PRECISE TARGETS
BY CHEMICAL SIGNALS THAT EXIST INDEPENDENT OF EXPERIENCE
Roger Sperry’s retinal axon
regeneration experiment
supported the
chemospecificity model
When optic nerve is cut, axons
regenerate and restore
normal sight and behavior
When optic nerve is cut and
the eyeball is rotated 180 deg
in socket, axons regenerate
and restore sight, but behavior
is maladaptive
FROG THINKS “UP” IS “DOWN”
And “LEFT” is “RIGHT”

3. AXON GUIDANCE IS A MULTI-STEP PROCESS
Temporal
Retina
Nasal
Migration towards retinal exit point (axon convergence)
Turning to exit retina and fasiculation
Fasiculated extension towards chiasm
Ipsilateral vs. contralateral migration at optic chiasm (divergence point)
Fasiculated extension into brain
Projection into lateral geniculate or into optic tectum (divergence point)
Projection a certain distance along surface of tectum (divergence step)
Diving into the tectum
Stopping at specific depths into tectum (divergence step)
Synaptogenesis

4. AXON DIVERGENCES AT OPTIC CHIASM ALLOW CONVERGENCE OF
CONGRUENT INFORMATION FROM BOTH RETINAS
Nasal hemiretinal axons cross over at chiasm
(contralateral projection)
Temporal hemiretinal axons do not cross over
(ipsilateral projection)

5. ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE
DURING ATTRACTION AND REPULSION:
GROWTH CONE STRUCTURE
ACTIN FILAMENTS
IN FILOPODIA
AND LAMELLIPODIA
MICROTUBULES
ALONG AXON AND
IN CORE OF
GROWTH CONE

6. ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE
DURING ATTRACTION AND REPULSION:
LAMELLIPODIA/FILOPODIA DRIVE AXON EXTENSION

7. ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE
DURING ATTRACTION AND REPULSION:
REPULSIVE CUES CAUSE GROWTH CONE COLLAPSE
GROWTH CONE
BEFORE
SEMAPHORIN
EXPOSURE
SAME GROWTH
CONE AFTER

8. ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS
Gas, Gai, Gaq
~45,000 MW
HETEROTRIMERIC
H-Ras, K-Ras, N-Ras
~21,000 MW
RAS FAMILY
SMALL
GTPases
RhoA, Rac, Cdc42
~21,000 MW
RHO FAMILY
RalA, RalB
~21,000 MW
RAL FAMILY
GDP
GTP
INACTIVE
G : GDP
G : GTP
ACTIVE Pi

9. ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS
Common structure of small G proteins allows design of
constitutively active (CA) and dominant negative (DN) mutant proteins
CA-RhoA , CA-Rac , CA-Cdc42 expressed in fibroblasts induce specific architectural structures
FOCAL ADHESIONS
&
STRESS FIBERS
LAMELLIPODIA
FILOPODIA

10. ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS

11. TO MODIFY GROWTH CONE ARCHITECTURE AND STEER DIRECTION OF OUTGROWTH
EXTRACELLULAR AXON GUIDANCE MOLECULES ACT THROUGH RHO-FAMILY G PROTEINS
TO MODIFY GROWTH CONE ARCHITECTURE AND STEER DIRECTION OF OUTGROWTH
ATTRACTANT GUIDANCE MOLECULES
LOCAL ACTIVATION OF RAC and CDC42
LOCAL EXTENSION OF FILOPODIA and
SPREAD OF LAMELLIPEDIA
GROWTH CONE EXTENDS TOWARD
ATTRACTANT
REPULSIVE GUIDANCE MOLECULES
LOCAL ACTIVATION OF RHO
LOCAL ASSEMBLY OF ACTOMYOSIN
BRIDGES
GROWTH CONE CONTRACTS AWAY
FROM REPELLANT

12. DIFFERENT TYPES OF AXON GUIDANCE CUES

13. TOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUM
THE SURFACE OF TECTUM AS A TOPOLOGICALLY PRESERVED REPRESENTATION
OF THE VISUAL FIELD
AXONS FROM MOST ANTERIOR OF RETINA PROJECT TO MOST POSTERIOR TECTUM
AND VISA VERSA
WHAT IS NATURE OF THE GUIDANCE SYSTEM?

14. AN AXONAL REPELLENT PRODUCED BY TECTUM
POSTERIOR RETINA AXONS
WON’T GROW ALONG
POSTERIOR TECTAL MEMBRANES;
THEY GROW ALONG
ANTERIOR TECTAL MEMBRANES
OR NO MEMBRANES
ANTERIOR RETINA AXONS
SHOW NO SUBSTRATE
PREFERENCE
CONCLUSION:
POSTERIOR TECTUM PRODUCES
A CHEMOREPELLENT SPECIFIC
FOR POSTERIOR RETINA AXONS
THE CHEMOREPELLENT WAS
PURIFIED FROM TECTUM
TISSUE HOMOGENATES
REPELLENT FOUND TO BE PROTEIN
NOW TERMED EPHRIN

15. EPHRINS AND THEIR RECEPTORS
EPHRINS ARE CELL-SURFACE
PROTEINS:
“A-TYPE” ARE GPI-ANCHORED
“B-TYPE” ARE TRANSMEMBRANE
EPHRIN RECEPTORS ARE
RECEPTOR TYROSINE KINASES
THE SIGNALING CAPACITIES
OF EPHRIN RECEPTORS
ARE VERY DIFFERENT FROM
OTHER RTKs,
SUCH AS THE
NEUROTROPHIN RECEPTORS

16. GRADIENT EXPRESSION OF EPHRINS ON TECTUM
AND RECEPTORS ON RETINAL AXONS
EPHRIN EXPRESSION IS HIGHEST ON POSTERIOR TECTUM
EPH EXPRESSION IS HIGHEST ON POSTERIOR RETINAL GANGLION CELLS
AND THEIR AXONS, WHICH STOP IN ANTERIOR TECTUM
LOW EPH EXPRESSION ON ANTERIOR GANGLION CELLS ALLOWS
THEIR AXONS TO ADVANCE TO POSTERIOR TECTUM

17. ECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL AXONS
PATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED BY INFECTING
TECTUM WITH REPLICATION-COMPETENT VIRAL VECTOR EXPRESSING EPH
POSTERIOR RETINAL AXONS WITH HIGH EPHRIN RECEPTOR LEVELS
AVOID EPHRIN OVEREXPRESSION PATCHES
ANTERIOR RETINAL AXONS ARE IMMUNE TO EPHRIN PATCHES
(A MUTATION ELIMINATING EPHRIN EXPRESSION IN BRAIN ALLOWS
POSTERIOR RETINAL AXONS TO PROJECT THROUGHOUT TECTUM)

18. EPHRIN INDUCES EPH RECEPTOR CLUSTERING AND ACTIVATES THE RECEPTOR
CYTOPLASMIC DOMAIN TO INITIATE SIGNALING LEADING TO REPULSION
EPHRIN
EPH
SIGNALING
&
REPULSION
NGF
TRK-EPH
SIGNALING
&
REPULSION

19. CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR COMMISURAL AXONS
COMMISURAL AXONS PROJECT VENTRALLY TO FLOOR PLATE
FLOOR PLATE EXPLANT ATTRACT COMMISURAL AXONS IN EXPLANT
OF DORSAL SPINAL CORD

20. IDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS RECEPTOR
COMMISURAL AXONS IN NETRIN AND DCC
MUTANT MICE FAIL TO PROJECT TO FLOOR PLATE

21. NETRIN/DCC INTERACTION CAN ALSO BE CHEMOREPULSIVE
ROLE OF CYCLIC AMP?
UNC-5 STATUS DETERMINES WHETHER DCC-POSITIVE
AXON IS ATTRACTED OR REPELLED BY NETRIN
TROCHLEAR MOTONEURONS
NORMALLY REPELLED BY NETRIN,
BUT ARTIFICIAL ACTIVATION OF
PKA IN CELLS CAUSES
ATTRACTION TO NETRIN SOURCE

22. CONSERVED FAMILIES OF GUIDANCE MOLECULES AND THEIR RECEPTORS GUIDANCE

23. AXON NAVIGATION THROUGH MULTIPLE GUIDANCE REGIONS REQUIRES
CHANGES IN GROWTH CONE SENSITIVITY TO INDIVIDUAL GUIDANCE CUES
SENSITIVITY TO ATTRACTANT IS LOST!!!

24. MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUES
I. CHANGE IN INTRACELLULAR CYCLIC NUCLEOTIDE LEVELS
Early in their outgrowth, retinal axons
travel into netrin-expressing region
of optic stalk
Netrin is attractive guidance cue
Axons express high cAMP
Later in their outgrowth, retinal axons
avoid netrin-expressing deep regions
of thalamus and tectum
Netrin is repulsive guidance cue
Axons express low cAMP
Netrin mRNA
(blue)
Retinal ganglion
axons (brown)
from Shewan et al., Nature Neuroscience 5:955 (2002)

25. MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUES
II. SILENCING A GUIDANCE CUE
COMMISURAL AXONS ARE
ATTRACTED TO MIDLINE FLOOR PLATE,
CROSS ONCE,
AND NEVER CROSS AGAIN
Midline floor plate produces
Netrin attractant and Slit repellent
Initially, axons are attracted by netrin and
are insensitive to slit repulsion
Upon reaching floor plate, Slit does two things:
SILENCES netrin attraction
INDUCES Slit repulsion
SLIT ACTS LOCALLY TO MODIFY
THE PROPERTIES OF THE
GROWTH CONE

26. SLIT SILENCES NETRIN BY GENERATING ROBO/DCC RECEPTOR COMPLEXES
AND SILENCING MUST PRECEDE SLIT-MEDIATED REPULSION
Slit induces DCC/Robo complexes,
and the cytoplasmic domain of Robo
binds the cytoplasmic domain of
DCC to block attractive signaling
The formation of DCC/Robo complexes
may also promote exocytosis of
Robo-containing vesicles,
thereby increasing surface Robo and
allowing for Slit-mediated repulsion
Only after axon passes through floor plate,
it is no longer sensitive to
floor plate attraction
it becomes sensitive to
Slit repulsion