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

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”

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

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)

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

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

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

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

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

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

DIFFERENT TYPES OF AXON GUIDANCE CUES

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?

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

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

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

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)

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

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

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

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

CONSERVED FAMILIES OF GUIDANCE MOLECULES AND THEIR RECEPTORS GUIDANCE

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

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)

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

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