ppt slides - MCD Biology

MCDB 4650 Class 23
Patterning of the nervous system
Learning goals:
• Describe the general mechanisms by which axons find their targets,
and give examples of kinds of molecules involved.
• Explain how both inhibitory and excitatory cues can determine axonal
pathfinding.
• Interpret and design experiments that would demonstrate the
importance of certain molecules in pathfinding.
Reminder: Exam next Monday 6-7:30 pm
Review session this Friday at 2 pm, B121

Defects in neuronal patterning:
real examples of problems in the signaling pathways
Lack of Shh
Holoprosencephaly (fusion of the two sides of the brain into one)

Axons are extremely
active!
Anatomy of a neuron

Do neurons follow a trail of breadcrumbs?
A series of signposts? A beacon?

Different groups of motor neurons
innervate different classes of muscles
Why?
Cells in
different areas
of spinal cord
could be
committed OR
Local
molecules
could be
attracting the
axons
differently

Expression of different transcription factors in different
regions of spinal cord

Known Mechanisms of Axon Pathfinding
• Non-specific, generalized
outgrowth cues
(permissive):
Fibronectin, laminin
• Previously pioneered
pathway (adhesive)
N-CAM, cadherins,
fasciclins
• Target-Specific pathfinding:
• adhesive or diffusible, repulsive or attractive

In amphibians, axons from the retina project to a brain
region called the tectum
Visual processing center of frog brain
If the connections
between retina and
tectum are cut, they
re-grow, providing a
model for studying
how axons know where
to grow

Experiments that led to the hunt for guidance cues
• Shine a light on retina, record electrical activity from tectum
• a cell in one part of the retina always produced activity in a
particular part of the tectum
• Inject a visible label into neurons
• an injected cell from the retina always projected to a
particular place in the tectum
Conclusion: Cells
in retina have
specific targets
in the tectum

But do these cells “know” their fate
(ie, are they committed to project to a certain location?
nasal
temporal
Nasal axons: posterior tectum
Temporal axons: anterior tectum
Dorsal axons: lateral tectum
Ventral axons: medial tectum
To test commitment (1960s):
Sperry and colleagues removed the eyes of frogs, severing the axonal
connections.
Replace eyes, rotated 180 degrees relative to their normal position

Sperry’s experiment and result:

What does this experiment demonstrate about
retinal-tectal projection?
a. The retinal neurons are committed to target
a particular region of tectum
b. The retinal neurons are not committed to
target a particular region of the tectum.
c. The location of the retinal neurons, not
their identity, determines their path
d. The neurons target the tectum randomly

Why do the axons know where to grow
despite the fact that they are now in a
different starting location?
They have receptors that bind ligands in the environment;
this information system allows them to navigate.
If a neuron is committed to its fate, will its axon always go to
the correct location?
Not necessarily: We have to consider the molecular composition
within the axon, as well as the molecular composition of the
environment, and of the target!

Beacon or bread crumbs?
• Chemotaxis (Beacon)
long range response to a gradient of a molecule
• Haptotaxis or contact-mediated guidance (bread crumbs)
short range response to contact with cell/substrate (can also be
a gradient)

A sampling of axon guidance molecules (do not memorize!)
Ligands:
always in
the environment
Receptors: always on
the surface of the axon
From B. Dickson, Science V 298, pp. 1959-1964.
Domain names can be found at http://smart.emblheidelberg.de/browse.shtml

CHEMOTAXIS: growth of neurons towards a specific target based on
the concnetration of a diffusible ligand
How did scientists identify these molecules and figure out how they
worked?

After purification of possible molecules, express these molecules in fibroblasts
(normally do not generate any axon outgrowth on their own)
Floor plate Fibros +
Vector
(control)
Fibros +
Netrin-1
Fibros +
Netrin-2

Floor
plate
Fibros +
Vector
(control)
Fibros +
Netrin-1
Fibros +
Netrin-2
What else would you want to do?
What can you conclude?
a. Only netrin 1 is a specific
chemoattractant for these
neurons
b. Only netrin 2 is a specific
chemoattractant for these
neurons
c. Both netrin 1 and 2 are
specific chemoattractants
for these neurons
d.Both netrin 1 and 2 are
chemoattractants, but not
necessarily specific

mRNA localization shows
• a gradual gradient of Netrin-2 and Shh
• a steep gradient of Netrin 1, in and directly adjacent to the floorplate

Summary of axon guidance molecules:
• They often exist in a gradient, either diffusible
or membrane bound
• They can be attractive to one set of neurons and
repulsive to another set dependent on the
combination of factors present in any given neuron
• Let’s return to the retino-tectal projection to
explore this model further

A model for axon targeting: the retino-tectal projection
in amphibians
• The information from one eye is sent exclusively to the opposite
side of the brain (right retina to left tectum
• Each RGC axon projects to a specific spot within the tectum
• Targeting within the tectum is due to haptotaxis (membrane bound
ligands)

From this experiment, we could conclude:
a. The nasal axons are attracted by
molecules in both anterior and posterior
tectum.
b. The nasal axons are not repelled by
molecules in the posterior tectum.
c. The temporal axons are repelled by
molecules in posterior tectum cells.
d. The temporal axons are attracted by
molecules in the anterior tectum cells.
e. Each set of axons is attracted to a
specific region in the tectum by a
different attractive molecule
To really be confident, you need more information

What to do next?
• Watch the axons as they encounter Ephrin ligand
• Purify proteins from different regions of the tectum.
• Identify proteins present
• Test the proteins to see if they act as guidance
molecules
• Discovered the Ephrin receptor and ligand—work through
contact-mediated guidance (haptotaxis)
• Ephrin Receptor was found in different concentrations
across the retina, and the ligand in different
concentrations across the tectum!

Ephrins and their receptors are present in
a gradient across both the tectum and the retina
Eph receptor
in the retina
Ephrin ligand
in the tectum

High EphR
(low to high Eph ligand)
Low EphR
Why do the axons from the nasal retina go to the
posterior tectum, rather than being stopped in the
anterior tectum?
a. The nasal axons are more sensitive to the Eph ligands
b. The nasal axons are less sensitive to the ligands
c. The nasal axons are unresponsive to the Eph ligand
d. None of the above

Watching neurons as they encountered Eph targets also showed
“repulsion”
this was the first example of repulsive activity working as
guidance
In this case, repulsion is based on both the density of the receptors,
and the concentration of the ligand
Axons with a low concentration of receptors are not repelled by a low
concentration of ligand, so they project to the posterior tectum, and
synapse there.

Cool cre-lox system for labeling neurons and their axonal projections