Lecture 12 (6) - Mechanisms of Axonal Pathfinding Flashcards
Attractive signal
microfilaments and microtubules accumulate at the contact site
Growth cone at tip
webby
• different regions, filopodia stabilized by actin
–> growth cone to attractive
Mechanisms of axonal pathfinding
- stereotropism
- haptotaxis
- chemotropism
Stereotropism
physical barriers
• axons move around physical objects
• some artificial substrates
• repair tissues/nerves
Haptotaxis
movement along substrate bound molecules
• most common
• bound to cell surface
- molecules of the extracellular matrix and cell surface molecules
- selective fasciculation of axons : labelled pathways hypothesis
Chemotropism
diffusabe factors
- Stereotropism - physical barriers
- collagen fibers
- plastic barrier
- artificial substrate
- tunnel
- Haptotaxis - movement of growth cones along substrate bound molecules
ECM: glycoproteins and proteoglycans
Cell adhesion proteins control
mobility and guide toward movement
• cell-cell adhesion
• cell-matrix adhesion
Cell-cell adhesion
- classical cadherins (E, N, P, VE), homophilic binding, associated with cytoskeleton (actin filaments) via catenins
- Ig family members (N-CAM, ICAM), homo- and heterophilic binding
Cell-matrix adhesion
- Integrins (many types), heterophilic binding associated with cytoskeletion (actin filaments), via talin, paxillin, filamin, etc.
- transmembrane proteoglycans (syndecans), heterophilic binding
To find molecules that axons like, use
cell culture assays
Cell culture as guidance molecules - assays are used for identifying
1. cell-adhesion assay
• can the neuron stick to other cells or a given substrate
Cell culture assays for identifying guidance
2. stripe assay
• petri dish - different areas with different molecules
• neuron in, where its axon grows =
which stripe is attractive or repulsive
Haptotaxis I - ECM-molecules facilitate or inhibit axonal growth
CSPG - chondroitin-sulfatproteoglycan
• dorsal roof plate of spinal cord
• when growth cone reaches CSPG, is repulsed and turns away
laminin attracts
Haptotaxis II - cell adhesion molecules are involved in axonal outgrowth, fasciculation and pathfinding
most growth cones grow on the surface of other axons or cells
• only if 2 molecules recognize each other
• floor plate instructs axons to grow along (cross midline) or repulses
• the midline is transient in embryos
Midline - axons express
axonin1 on surface
Cells of the floor plate have
NRCAm - both adhesions and bind to each other
• axon guided to cross midline
Incubate floor plate with
antibodies = 1 or 2 can’t pass
• antibodies block sites where 1 or other cross = can’t bind
- Selective fasciculation
the “labeled pathways” hypothesis
• guidance on axon
• G - neurons grow where nothing else arond axon
• recognizes molecules on axon P
• grows to decision point and sees P missing
–> stalls
= absence of whole axon tracks
Fasciculation is affected in
fasciclin II mutant flies
• FasII is expressed on pioneer axons of longitudinal tract
- Chemotropism
guidance by diffusable factors
• repulsive factors lead to growth cone collapse
• collapsin = growth cone turns away and collapses
• growth cone collapses, grows, tries again
Collapsin
growth cone turns away and collapses
• collapses, grows, tries again
Pathfinding of the DRG-axons is based on
repulsive factors
• spinal cord
• DRG cells grow straight bc repulsed by secreted substance
- Chemotropism
- NGF - nerve growth facctor
- all axons like NGF
- follows this diffusible factor
Both in vertebrates and invertebrates the CNS midline plays an important role in
organizing the axonal scaffold
• vertebrates - floor plate = epithelial cells, transient
• in embryo, guides axons in midline (attracts/repulses)
• cross = turn ant/post
Commissural
crosses ventral midline
Ipsilateral
doesn’t cross ventral midline
Netrin is expressed in the
CNS midline of Drosophila
• CNS midline - explant of growth plate to Dci
• Dci attracted to plate over distance
–> shows diffusible factor (later called netrin)
Netrin exists in
vertebrates and invertebrates
In Drosophila, the ventral midline derives from the
mesectoderm
3 types of midline precursors give rise to the
unpaired midline neurons and glia
Axon scaffold
commissural axons cross betwen midline glial cells and neural cells
• 8 midline precursors per segment (gastrulation –> germ band elongation)
• 16 during division
• midline glial cells separate fasic. into ant and post commissural
Midline cells contribute to the
formation of the axonal scaffold
The ventral midline of crustaceans derive from
a medial column of cells that separates the neuroectoderm
• crustaceans - netrin in midline for axonal patterning
• no bilateral midline, have unpaired medial midline bc no welerm (?) band forms
Despite the different origin, individual midline progenitorsmight be
homologous in malocostraccans and insects
• similar position of midline precursors and cells
The ventral midline epithelium is
a transient structure that does not express any neural genes
• midline in centi and milli
• transient midline in spiders (like vertebrates)
• spiders split into 2 –> dorsal, come together when yolk into abdomen
= transient midline
Commissural axons cross the ventral midline despite
the gap between the 2 halves
• the axonal guidance molecule Netrin is expressed in the epithelial ventral midline
• commissural crosses on Netrin-positive cells
Netrin is required for
axonal guidance at the ventral midline
• same phenotype in Drosophila, spider, and in vertebrates - reduction of commissural axons
Pattern of axons at the ventral midline of Drosophila
• anterior and posterior commissures connected by longitudinal tracts
A large-scale screen for axonal pathfinding mutants resulted in the discovery of the
midline guidance system in Drosophila
• blue = midline glial cells
• slit = collapsed at midline
• Robo = roundabout, axons to midline, cross, go back
Robo
roundabout
• axons to midline, cross, go back
The commissures are absent in
commissureless mutants
• in axons meant to cross midline, commisureless need to sort Robo to lysosomes
- not on surface growth, can’t sense repulsive (slit)
- Robo on surface of growth cone and repelled by slit = doesn’t cross
High robo
strongly repelled by Slit
Low Robo
weakly repelled by Slit
comm ON
Robo sorted to lysosomes
comm OFF
Robo delivered to growth cone
Commissureless is expressed in axons that
cross the midline
• comm sort Robo (receives repulsive Slit)
• comm expressed = no Robo in growth cone
= can’t tell repulsive slit (repulse midline) –> cross midline
• comm off = high Robo expression
Commissureless sorts Robo to
allow midline crossing
In vertebrates, Robo3 functions
like comm in Drosophila
• no comm
• Robo3 represses Robo
• Robo3 expressed = Robo low = cross midline
• growth cone doesn’t recross, recognizes midline as repulsive
• Netrin attracts to CNS midline - doesn’t find Netrin attractive on other side
The human syndrome “horizontal gaze palsy and progressive scoliosis) is associated with mutations in
the Robo3 gene
• aberrant psilateral projections of major ascending and descending axons pathways
• failure of these axons to cross the midline in the hindbrain
• no horizontal eye movement
(can’t see sideways, only up and down)
Ips = on same side
(axons don’t cross midline in hindbrain)
Slit activates Robo in the midline resulting in
silencing of the netrin receptro DCC
In vertebrates, Robo3 functions like comm in Drosophila
• attracted to midline by Netrin
- have growth cone receptor for Netrin (dcc)
- bind Netrin and guided
• slit (repressor) in midline affects Robo in crossing
- Robo switches on and binds dcc receptor –> can’t bind attractive signal
(Robo prevents Netrin binding)
- growth cone crossed, dcc receptor no longer functional
• Robo mutant = recross
- plays role in silencing attractive signal
Growth cones guide axons through
the developing embryo to the target
Soluble ans substrate bound factors guide growth cones by
attraction and repulsion
A complex midline guidance system leads
commissural axons across the midline and prevents ipsilateral axons from crossing
