Axon guidance 2

 What surgical experiments suggested that retinotopic connections between ganglion axons and the optic tectum
was based on molecular cues?
First, you can remove half of the retina, say the anterior retinal ganglion cells. When you inject dye that is
picked up by the remaining posterior retinal ganglion cells, these neurons project to the anterior tectum. The
opposite is true when you remove the posterior retinal ganglion cells; dye gets picked up by the remaining
anterior retinal ganglion cells and get carried to the posterior tectum. This means that there is preservation of a
retinotopic map.

You can then take this one step further and not only remove half of the retinal ganglion cells but cut the optic
nerve as well. Cutting the optic nerve causes degeneration of the axons that project to the tectum, making it
devoid of synapses. When you do this, the remaining anterior or posterior retinal ganglion cells will pick up the
dye and regenerate back to the posterior or anterior tectum, respectively, even though there were no more
synapses in the tectum. This means that there is some molecular cue that guides newly formed or regenerating
axons from the retina to the proper location in the tectum. At the time, this was coined “chemospecificity.”

Later on, you could do more detailed experiments where you surgically swap one area of the tectum with
another, then cut the optic nerve and allow the retinal ganglion axons to regenerate. What you find is that if
they originally projected to the anterior tectum, but then you swap the anterior and posterior tectum and cut
the optic nerve, the regenerating ganglion cells would project to the posterior tectum now; that is there is
some molecular cue that is produced by the original tissue that guides axons even after it has been moved:

 What are some ways in which one can trace where ganglion cell axons project in the optic tectum?
One way is to use electrophysiology: you can put an electrode in a certain region of the optic tectum, then shine
light into different regions of the visual field and see where in the visual field when light is present do you see
action potentials in the optic tectum. Doing this for different regions can generate a map showing where ganglion
cell axons in the retina project to in the optic tectum.

The other way is to look at this directly using dyes into retinal ganglion cells and see where in the optic tectum
they project to. We talked about some of these dyes in earlier lectures.

Generation of a retinotopic map is important to determine molecular cues that guide axons. You need to
know which retinal ganglion cells project to where in the optic tectum first; you can then manipulate this and
see if you can interfere with this process to determine which molecular cues are important.

 What in vitro bioassay experiment was used to show that some retinal axons were responding to a repulsive cue,
instead of an attractive one? Which retinal cells respond to the repulsive cue? What variation on this bioassay
was used to isolate EphrinA as the cue?
A stripe assay was used to show retinal axons were responding to a repulsive one. You can take cells from the
tectum and extract cell membranes, and plate this membrane extract on a dish in alternating stripes:
membranes from the anterior in one stripe, membranes from the posterior in the next, anterior in the next,
posterior in the next, etc.:
 As shown above, temporal retinal ganglion axons only grow along stripes containing anterior tectum
membrane extracts. However, this can be due to either something on the anterior membrane extract that the
temporal retinal ganglion axon likes, or something on the posterior membrane extract that the temporal retinal
ganglion axons do not like. To test this, you can heat treat either the anterior or posterior membrane extracts to
denature the proteins. If you denature the anterior membrane extracts, you get the same as above suggesting that
the axon guidance probably is done through a repulsive cue from the posterior membrane extracts. If you heat
treat and denature the posterior membrane extracts, you get the following:

This means that it is an inhibitory cue from the posterior membranes of cells in the tectum that guide
temporal retinal axon growth.
*Important to know is that the stripe assay does not work for the nasal retinal ganglion cells, as these cells
grow equally well in either anterior or posterior membrane extracts of the tectum. So only the temporal retinal
ganglion cells respond to a repulsive cue from membrane extracts of the posterior tectum.

The same stripe assay was used to show that if you treat the posterior membrane tectum extracts with something
that breaks GPI links, a covalent bond that attaches extracellular proteins to the membrane surface, you lose the
ability of the temporal retinal ganglion axon to properly stay within the posterior membrane extract stripe. At the
same time, another lab Ephrin receptor and Ephrins on the growing axon as repulsive cue responsible for
this: EphA receptor is a transmembrane receptor expressed on the membrane of the temporal retinal axons,
and EphrinA ligand is a GPI-linked ligand expressed on the cell membranes of cells in the tectum.

 Where is EphrinA made in the optic tectum, and why do only some retinal axons respond to EphrinA? What is
the receptor for EphrinA, and how is the receptor expressed?
EphrinA is made in the posterior region of the optic tectum, and the receptor for Ephrin A, EphA receptor, is
primarily expressed by temporal retinal ganglion axons. For this reason, only the temporal retinal ganglion
axons can respond to EphrinA which is more highly expressed in the posterior tectum and avoid the tectum
(remember this is a repulsive cue). Therefore, the temporal retinal ganglion axons will migrate to the anterior
tectum, and avoid the posterior tectum where there is a lot of the repulsive EphrinA cue:
 When EphrinA binds to the EphA receptor on the growth cone, it causes growth cone collapse.

 What are some possible reasons why the nasal axons grow into the posterior instead of the anterior of the tectum?
One reason is that the nasal axons grow into the posterior tectum by default; they lack the response to the
repulsive cue Ephrin A because they have low expression of the EphA receptor.
Genetically, you can have the tectum express less EphrinA’s which causes the temporal retinal ganglion
axons to now go into the posterior tectum; you can also cause the nasal retinal ganglion cells to express more
EphA receptor, which causes them to go anterior as they are repulsed by the posterior tectum.

 What is meant by reverse Ephrin-Eph signaling? Give an example.

So you need to know forward versus reverse signaling.
Forward signaling is the normal or what you would expect: The ligand EphrinA from the posterior tectum
binds to the EphA receptor on the temporal retinal ganglion axons, which causes them to undergo growth cone
collapse.
Reverse signaling is the opposite: The receptor EphA expressed on the anterior tectum can bind to the
“ligand” EphrinA on the nasal retinal ganglion axons to cause the retinal ganglion axons to undergo repulsion;
in this case the EphrinA ligand on the membrane of the nasal retinal ganglion axon can behave like a receptor and
cause growth cone collapse when bound to the EphA receptor.

 Give an example where a member of Ephrin protein family is used as an attractive guidance cue
EphB receptor and Ephrin B ligands are attractive, which has both forward and reverse signaling in dorsal-
ventral cues from the retinal ganglion cells to the tectum. The ventral retinal ganglion axons have the EphB
receptor, and the dorsal tectum contains high levels of the Ephrin B ligand. When a ventral retinal ganglion
axon sees the Ephrin B ligand on the dorsal tectum, it binds the EphB receptor and recruits these axons there:
hence forward signaling of an attractive guidance cue.

The dorsal retinal ganglion axons and ventral tectum have reverse signaling: there is high expression of the
Ephrin B ligand on the dorsal retinal ganglion axons and high levels of the EphB receptor on the ventral
tectum. When the dorsal retinal ganglion axons containing EphrinB ligand bind to the EphB receptor on the
ventral tectum, it recruits the dorsal retinal ganglion axons as an attractive cue; it is a reverse signaling because
the ligand EphrinB is on the axon and acts as a “receptor” when bound to the EphB receptor on the ventral
tectum.