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LECTURE 22: AXONAL GUIDANCE
REQUIRED READING: Kandel text, Chapter 54
ESTABLISHMENT OF NEURONAL IDENTITY
AXONAL GUIDANCE TO TARGETS
NEUROTROPHIC SUPPORT FROM TARGET TISSUES
SYNAPTOGENESIS
HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?HOW IS PROPER “WIRING” OF NERVOUS SYSTEM ACHIEVED?
THEORY 1: AXON PROJECTIONS ARE IMPRECISE…PROPER CONNECTIONS ARE WORKEDTHEORY 1: AXON PROJECTIONS ARE IMPRECISE…PROPER CONNECTIONS ARE WORKED OUT BY ELECTRICAL ACTIVITIES DURING EARLY EXPERIENCEOUT BY ELECTRICAL ACTIVITIES DURING EARLY EXPERIENCE
THEORY 2: CHEMOSPECIFICITY MODEL….AXONS ARE DIRECTED TO THEIR PRECISE TARGETSTHEORY 2: CHEMOSPECIFICITY MODEL….AXONS ARE DIRECTED TO THEIR PRECISE TARGETS BY CHEMICAL SIGNALS THAT EXIST INDEPENDENT OF EXPERIENCEBY CHEMICAL SIGNALS THAT EXIST INDEPENDENT OF EXPERIENCE
Roger Sperry’s retinal axonRoger Sperry’s retinal axonregeneration experimentregeneration experiment
supported the supported the chemospecificity modelchemospecificity model
When optic nerve is cut, axonsWhen optic nerve is cut, axonsregenerate and restoreregenerate and restore
normal sight and behaviornormal sight and behavior
When optic nerve is cut When optic nerve is cut andandthe eyeball is rotated 180 degthe eyeball is rotated 180 degin socketin socket, axons regenerate, axons regenerate
and restore sight, but behaviorand restore sight, but behavioris maladaptiveis maladaptive
FROG THINKS “UP” IS “DOWN”FROG THINKS “UP” IS “DOWN”And “LEFT” is “RIGHT”And “LEFT” is “RIGHT”
TemporalRetina
NasalRetina
AXON GUIDANCE IS A MULTI-STEP PROCESSAXON GUIDANCE IS A MULTI-STEP PROCESS
1.1. Migration towards retinal exit point (axon convergence)Migration towards retinal exit point (axon convergence)2.2. Turning to exit retina and fasiculationTurning to exit retina and fasiculation3.3. Fasiculated extension towards chiasmFasiculated extension towards chiasm4.4. Ipsilateral vs. contralateral migration at optic chiasm (divergence point)Ipsilateral vs. contralateral migration at optic chiasm (divergence point)5.5. Fasiculated extension into brainFasiculated extension into brain6.6. Projection into lateral geniculate or into optic tectum (divergence point)Projection into lateral geniculate or into optic tectum (divergence point)7.7. Projection a certain distance along surface of tectum (divergence step)Projection a certain distance along surface of tectum (divergence step)8.8. Diving into the tectumDiving into the tectum9.9. Stopping at specific depths into tectum (divergence step)Stopping at specific depths into tectum (divergence step)10.10. SynaptogenesisSynaptogenesis
AXON DIVERGENCES AT OPTIC CHIASM ALLOW CONVERGENCE OFAXON DIVERGENCES AT OPTIC CHIASM ALLOW CONVERGENCE OFCONGRUENT INFORMATION FROM BOTH RETINASCONGRUENT INFORMATION FROM BOTH RETINAS
Nasal hemiretinal axons cross over at chiasmNasal hemiretinal axons cross over at chiasm(contralateral projection)(contralateral projection)
Temporal hemiretinal axons do not cross overTemporal hemiretinal axons do not cross over(ipsilateral projection)(ipsilateral projection)
ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEDURING ATTRACTION AND REPULSION:DURING ATTRACTION AND REPULSION:
GROWTH CONE STRUCTUREGROWTH CONE STRUCTURE
ACTIN FILAMENTSACTIN FILAMENTSIN FILOPODIAIN FILOPODIA
AND LAMELLIPODIAAND LAMELLIPODIA
MICROTUBULESMICROTUBULESALONG AXON ANDALONG AXON AND
IN CORE OFIN CORE OFGROWTH CONEGROWTH CONE
ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEDURING ATTRACTION AND REPULSION:DURING ATTRACTION AND REPULSION:
LAMELLIPODIA/FILOPODIA DRIVE AXON EXTENSIONLAMELLIPODIA/FILOPODIA DRIVE AXON EXTENSION
ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEACTIN CYTOSKELETON DYNAMICS IN GROWTH CONEDURING ATTRACTION AND REPULSION:DURING ATTRACTION AND REPULSION:
REPULSIVE CUES CAUSE GROWTH CONE COLLAPSEREPULSIVE CUES CAUSE GROWTH CONE COLLAPSE
GROWTH CONEGROWTH CONEBEFOREBEFORE
SEMAPHORINSEMAPHORINEXPOSUREEXPOSURE
SAME GROWTHSAME GROWTHCONE AFTERCONE AFTERSEMAPHORINSEMAPHORIN
EXPOSUREEXPOSURE
ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS
Gs, Gi, Gq~45,000 MW HETEROTRIMERIC
H-Ras, K-Ras, N-Ras ~21,000 MW
RhoA, Rac, Cdc42 ~21,000 MW
RalA, RalB ~21,000 MW
RAS FAMILY
RHO FAMILY
RAL FAMILY
SMALL
GTPases
G : GTPG : GDP
GDP GTP
Pi
INA
CT
IVE
AC
TIV
E
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
ACTIN CYTOSKELETON DYNAMICS: THE ROLE OF RHO-FAMILY G PROTEINS
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 andSPREAD OF LAMELLIPEDIA
GROWTH CONE EXTENDS TOWARDATTRACTANT
REPULSIVE GUIDANCE MOLECULES
LOCAL ACTIVATION OF RHO
LOCAL ASSEMBLY OF ACTOMYOSINBRIDGES
GROWTH CONE CONTRACTS AWAYFROM REPELLANT
DIFFERENT TYPES OF AXON GUIDANCE CUESDIFFERENT TYPES OF AXON GUIDANCE CUES
TOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUMTOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUM
THE SURFACE OF TECTUM AS A TOPOLOGICALLY PRESERVED REPRESENTATIONTHE SURFACE OF TECTUM AS A TOPOLOGICALLY PRESERVED REPRESENTATIONOF THE VISUAL FIELDOF THE VISUAL FIELD
AXONS FROM MOST ANTERIOR OF RETINA PROJECT TO MOST POSTERIOR TECTUMAXONS FROM MOST ANTERIOR OF RETINA PROJECT TO MOST POSTERIOR TECTUMAND VISA VERSAAND VISA VERSA
WHAT IS NATURE OF THE GUIDANCE SYSTEM?WHAT IS NATURE OF THE GUIDANCE SYSTEM?
AN AXONAL REPELLENT PRODUCED BY TECTUMAN AXONAL REPELLENT PRODUCED BY TECTUM
POSTERIOR RETINA AXONSPOSTERIOR RETINA AXONSWON’T GROW ALONGWON’T GROW ALONG
POSTERIOR TECTAL MEMBRANES;POSTERIOR TECTAL MEMBRANES;THEY GROW ALONGTHEY GROW ALONG
ANTERIOR TECTAL MEMBRANESANTERIOR TECTAL MEMBRANESOR NO MEMBRANESOR NO MEMBRANES
ANTERIOR RETINA AXONSANTERIOR RETINA AXONSSHOW NO SUBSTRATESHOW NO SUBSTRATE
PREFERENCEPREFERENCE
CONCLUSION:CONCLUSION:POSTERIOR TECTUM PRODUCESPOSTERIOR TECTUM PRODUCESA CHEMOREPELLENT SPECIFICA CHEMOREPELLENT SPECIFIC
FOR POSTERIOR RETINA AXONSFOR POSTERIOR RETINA AXONS
THE CHEMOREPELLENT WASTHE CHEMOREPELLENT WASPURIFIED FROM TECTUMPURIFIED FROM TECTUMTISSUE HOMOGENATESTISSUE HOMOGENATES
REPELLENT FOUND TO BE PROTEINREPELLENT FOUND TO BE PROTEINNOW TERMED NOW TERMED EPHRINEPHRIN
EPHRINS AND THEIR RECEPTORSEPHRINS AND THEIR RECEPTORS
EPHRINS ARE CELL-SURFACEEPHRINS ARE CELL-SURFACEPROTEINS:PROTEINS:
““A-TYPE” ARE GPI-ANCHOREDA-TYPE” ARE GPI-ANCHORED““B-TYPE” ARE TRANSMEMBRANEB-TYPE” ARE TRANSMEMBRANE
EPHRIN RECEPTORS AREEPHRIN RECEPTORS ARERECEPTOR TYROSINE KINASESRECEPTOR TYROSINE KINASES
THE SIGNALING CAPACITIESTHE SIGNALING CAPACITIESOF EPHRIN RECEPTORSOF EPHRIN RECEPTORS
ARE VERY DIFFERENT FROMARE VERY DIFFERENT FROMOTHER RTKs,OTHER RTKs,SUCH AS THESUCH AS THE
NEUROTROPHIN RECEPTORSNEUROTROPHIN RECEPTORS
GRADIENT EXPRESSION OF EPHRINS ON TECTUMGRADIENT EXPRESSION OF EPHRINS ON TECTUMAND RECEPTORS ON RETINAL AXONSAND RECEPTORS ON RETINAL AXONS
EPHRIN EXPRESSION IS HIGHEST ON POSTERIOR TECTUMEPHRIN EXPRESSION IS HIGHEST ON POSTERIOR TECTUM
EPH EXPRESSION IS HIGHEST ON POSTERIOR RETINAL GANGLION CELLSEPH EXPRESSION IS HIGHEST ON POSTERIOR RETINAL GANGLION CELLSAND THEIR AXONS, WHICH STOP IN ANTERIOR TECTUMAND THEIR AXONS, WHICH STOP IN ANTERIOR TECTUM
LOW EPH EXPRESSION ON ANTERIOR GANGLION CELLS ALLOWSLOW EPH EXPRESSION ON ANTERIOR GANGLION CELLS ALLOWSTHEIR AXONS TO ADVANCE TO POSTERIOR TECTUMTHEIR AXONS TO ADVANCE TO POSTERIOR TECTUM
ECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL AXONSECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL AXONS
PATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED BY INFECTINGPATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED BY INFECTINGTECTUM WITH REPLICATION-COMPETENT VIRAL VECTOR EXPRESSING EPHTECTUM WITH REPLICATION-COMPETENT VIRAL VECTOR EXPRESSING EPH
POSTERIOR RETINAL AXONS WITH HIGH EPHRIN RECEPTOR LEVELSPOSTERIOR RETINAL AXONS WITH HIGH EPHRIN RECEPTOR LEVELSAVOID EPHRIN OVEREXPRESSION PATCHESAVOID EPHRIN OVEREXPRESSION PATCHES
ANTERIOR RETINAL AXONS ARE IMMUNE TO EPHRIN PATCHESANTERIOR RETINAL AXONS ARE IMMUNE TO EPHRIN PATCHES
(A MUTATION ELIMINATING EPHRIN EXPRESSION IN BRAIN ALLOWS(A MUTATION ELIMINATING EPHRIN EXPRESSION IN BRAIN ALLOWSPOSTERIOR RETINAL AXONS TO PROJECT THROUGHOUT TECTUM)POSTERIOR RETINAL AXONS TO PROJECT THROUGHOUT TECTUM)
EPHRIN INDUCES EPH RECEPTOR CLUSTERING AND ACTIVATES THE RECEPTORCYTOPLASMIC DOMAIN TO INITIATE SIGNALING LEADING TO REPULSION
SIGNALINGSIGNALING&&
REPULSIONREPULSION
SIGNALINGSIGNALING&&
REPULSIONREPULSION
EPH
EPHRINEPHRIN
TRKTRK-EPH
NGFNGF
CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR COMMISURAL AXONS CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR COMMISURAL AXONS
COMMISURAL AXONS PROJECT VENTRALLY TO FLOOR PLATECOMMISURAL AXONS PROJECT VENTRALLY TO FLOOR PLATE
FLOOR PLATE EXPLANT ATTRACT COMMISURAL AXONS IN EXPLANTFLOOR PLATE EXPLANT ATTRACT COMMISURAL AXONS IN EXPLANTOF DORSAL SPINAL CORDOF DORSAL SPINAL CORD
IDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS RECEPTORIDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS RECEPTOR
COMMISURAL AXONS IN NETRIN AND DCCCOMMISURAL AXONS IN NETRIN AND DCCMUTANT MICE FAIL TO PROJECT TO FLOOR PLATEMUTANT MICE FAIL TO PROJECT TO FLOOR PLATE
NETRIN/DCC INTERACTION CAN ALSO BE CHEMOREPULSIVENETRIN/DCC INTERACTION CAN ALSO BE CHEMOREPULSIVEROLE OF CYCLIC AMP?ROLE OF CYCLIC AMP?
UNC-5 STATUS DETERMINES WHETHER DCC-POSITIVEUNC-5 STATUS DETERMINES WHETHER DCC-POSITIVEAXON IS ATTRACTED OR REPELLED BY NETRINAXON IS ATTRACTED OR REPELLED BY NETRIN
TROCHLEAR MOTONEURONSTROCHLEAR MOTONEURONSNORMALLY REPELLED BY NETRIN,NORMALLY REPELLED BY NETRIN,BUT ARTIFICIAL ACTIVATION OFBUT ARTIFICIAL ACTIVATION OF
PKA IN CELLS CAUSESPKA IN CELLS CAUSESATTRACTION TO NETRIN SOURCEATTRACTION TO NETRIN SOURCE
GUIDANCEMOLECULES
GUIDANCERECEPTORS
CONSERVED FAMILIES OFGUIDANCE MOLECULES AND THEIR RECEPTORS
n
AXON NAVIGATION THROUGH MULTIPLE GUIDANCE REGIONS REQUIRESCHANGES IN GROWTH CONE SENSITIVITY TO INDIVIDUAL GUIDANCE CUES
SENSITIVITY TO ATTRACTANT IS LOST!!!
MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUESI. CHANGE IN INTRACELLULAR CYCLIC NUCLEOTIDE LEVELS
Netrin mRNANetrin mRNA(blue)(blue)
Retinal ganglionRetinal ganglionaxons (brown)axons (brown)
from Shewan et al., Nature Neuroscience 5:955 (2002)
Early in their outgrowth, retinal axonsEarly in their outgrowth, retinal axonstravel intotravel into netrin-expressing region netrin-expressing region
of optic stalkof optic stalk
Netrin is attractive guidance cueNetrin is attractive guidance cue
Axons express high cAMPAxons express high cAMP
Later in their outgrowth, retinal axonsLater in their outgrowth, retinal axonsavoidavoid netrin-expressing deep regions netrin-expressing deep regions
of thalamus and tectumof thalamus and tectum
Netrin is repulsive guidance cueNetrin is repulsive guidance cue
Axons express low cAMPAxons express low cAMP
MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON GUIDANCE CUESII. SILENCING A GUIDANCE CUE
COMMISURAL AXONS ARE COMMISURAL AXONS ARE ATTRACTED TO MIDLINE FLOOR PLATE, ATTRACTED TO MIDLINE FLOOR PLATE,
CROSS ONCE,CROSS ONCE,AND NEVER CROSS AGAINAND NEVER CROSS AGAIN
Midline floor plate producesMidline floor plate producesNetrin attractant and Slit repellentNetrin attractant and Slit repellent
Initially, axons are attracted by netrin andInitially, axons are attracted by netrin andare insensitive to slit repulsionare insensitive to slit repulsion
Upon reaching floor plate, Slit does two things:Upon reaching floor plate, Slit does two things:1)1) SILENCES netrin attractionSILENCES netrin attraction
2)2) INDUCES Slit repulsionINDUCES Slit repulsion
SLIT ACTS LOCALLY TO MODIFYSLIT ACTS LOCALLY TO MODIFYTHE PROPERTIES OF THETHE PROPERTIES OF THE
GROWTH CONEGROWTH CONE
SLIT SILENCES NETRIN BY GENERATING ROBO/DCC RECEPTOR COMPLEXESAND SILENCING MUST PRECEDE SLIT-MEDIATED REPULSION
Slit induces DCC/Robo complexes,and the cytoplasmic domain of Robo
binds the cytoplasmic domain ofDCC to block attractive signaling
The formation of DCC/Robo complexesmay also promote exocytosis of
Robo-containing vesicles,thereby increasing surface Robo andallowing for Slit-mediated repulsion
Only after axon passes through floor plate,Only after axon passes through floor plate,it is no longer sensitive toit is no longer sensitive to
floor plate attractionfloor plate attraction
Only after axon passes through floor plate,Only after axon passes through floor plate,it becomes sensitive toit becomes sensitive to
Slit repulsionSlit repulsion