Discussion paper 6

Question 1

Which cells of the retina project to the brain?

Answer 1

Ganglion cells

Question 2

Why are topographical maps important?

Answer 2

Simplify processing, necessary for organized and normal brain processing

Question 3

What is the question being addressed by this figure?

Answer 3

Where are EphA receptors being expressed?

Question 4

What is the experimental technique being used in this figure?

Answer 4

In situ hybridization

Question 5

What is the key takeaway from this figure?

Answer 5

EphA5 and EphA6 are expressed in RGCs, EphA3 is not

Question 6

Areas with low levels of EphAs will target regions containing (low/high) levels of ligand, why?

Answer 6

High, because EphA interactions are repulsive: the less receptor, the less repulsion = more connections to cells with the ligand bound

Question 7

What is the broad underlying question being addressed by this paper?

Answer 7

How do gradients mediate sensory map formation?

Question 8

What is meant by absolute and relative levels of ephrin with respect to signaling?

Answer 8

Absolute: X concentration of ligand

Relative: Measuring differences in concentration of ligand regardless of the absolute concentration

Question 9

What specific question is this discussion paper addressing?

Answer 9

Is mapping to the superior colliculus controlled by absolute or relative amounts of Eph?

Question 10

What are the key features of Isl2 that make it a useful gene to piggyback EphA3 expression? (3)

Answer 10

1. It is expressed broadly across the nasal-temporal axis: will increase the concentration evenly across the retina
2. It is only expressed in ~50% of RGCs, some little gaps to serve as internal controls
3. Expressed exclusively in ganglion cells

Question 11

How is EphA3 gain of function achieved in this paper?

Answer 11

Insertion of EphA3 into the Isl2 gene 3’ UTR with an IRES site resulting in bicistronic mRNA (mRNA codes for Isl2 and EphA3)

Great because it leaves the Isl2 gene completely intact and is not a fusion protein!

Question 12

What methods are being used in this figure?

Answer 12

Immunostaining

Question 13

Which of these figures are the wild type and which are the mutant mouse?

Answer 13

CDEG = WT
FH = Mutant

Question 14

What is the key takeaway of panel F in this figure?

Answer 14

When Isl2-EphA3 construct is expressed in mice, expression of Isl2 is unaffected – similar to the wildtype

Question 15

What is the key takeaway of panel H in this figure?

Answer 15

In mutant mice containing the Isl2-EphA3 construct, EphA3 is expressed in a similar manner to Isl2

Question 16

What is the key takeaway of this figure?

Answer 16

Expression of the EphA3-Isl2 construct does not affect normal expression of EphA5 and EphA6

Question 17

What is being shown in panels A and D in this figure?

Answer 17

Confirming that EphA3 is being expressed in the ganglion cell layer of the retina (in the WT, it is normally not expressed there, but it is here which is what we want to see)

Question 18

In this figure, why do the authors use both heterozygous and homozygous knock-in mice?

Answer 18

They do this to check for gene dosage effects

Question 19

Based on this figure, are EphA’s subject to gene dosage effects?

Answer 19

Yes – function in a dosage-dependent manner

Question 20

Explain this figure

Answer 20

The figure is showing the spatial gradient on the nasal-temporal axis of the retina and is showing the EphA receptor concentration. On the temporal axis, there is the most EphA, and there is the least towards the nasal axis. Interspersed are Isl2-EphA3+ cells which express the same amount of A3 in each affected cell, but have differing amounts of A5 and A6 depending on their n-t positioning

Key: greater gene dosage of EphA’s in homo as opposed to hetero

Question 21

What is the assay being used in this figure? Briefly explain

Answer 21

Dil anterograde labelling – inject fluorescent label Dil in the retina and the dye will migrate to the superior colliculus, then you can measure where in the superior colliculus that area of the retina projects to

Question 22

Why is Dil’s lipophilic property important?

Answer 22

Because this allows it to diffuse across membranes and allows entire cells to fluoresce

Question 23

Describe the excitation and emission spectra of Dil

Answer 23

Excited by green light and emits red light

Question 24

What is DiAsp?

Answer 24

Similar to Dil but excited by blue light and emits green light

Question 25

Based on the findings from figures 1-4, how could the authors of this paper test whether mapping to the SC is controlled by relative or absolute amounts of Eph?

Answer 25

Use the Dil method of staining in WT and knockin mice, compare where they map to: In an absolute model, the knockins would have overlap with the wildtype mapping curve, in a relative model, they would probably not map to areas on this curve

Question 26

Is mapping to the SC controlled by relative or absolute amounts of Eph?

Answer 26

Relative

Question 27

What are the key observations in this figure? (3)

Answer 27

Projections are split in the knock-in retina Note that in the split cells, NEITHER OF THE DOTS PROJECTS TO THE SPOT WHERE THE FIRST DOT IS The homozygous mouse moves ~2x further than the heterozyogus mouse

Question 28

Why do the results of this figure tell us that mapping to the SC is a non-cell autonomous process?

Answer 28

Because if it was cell autonomous, the cells which are EphA3- would map to the same location as the WT

Question 29

Describe the main takeaway of this figure?

Answer 29

Mapping WT, heterozygote, and homozygote projections - dotted line in B and C is where the WT curve would be Notice that the observed data in B and C diverges from the WT curve Forming separate and independent retino-collicular maps in the homozygote esp EVIDENCE FOR THE RELATIVE MODEL

Question 30

What is the underlying question addressed by this figure?

Answer 30

Can the results from Dil anteroretrograde labelling be validated using retrograde labelling from SC to the RGCs?

Question 31

What is the assay being used in this figure?

Answer 31

Dil and DiAsp retrograde labelling

Question 32

What are the most important findings of this figure?

Answer 32

When injecting into different spots on the SC (on the Y axis), they map to the same region of the retina (X axis) -- evidence for the 2 curve/2 separate maps forming in the SC Validates the findings from the previous retrograde labelling model Notice the secondary site in green in the nasal region

Question 33

Based on findings from this data, what curve represents the EphA3+ overexpressing cells?

Answer 33

Orange curve likely expresses more EphA3 because these cells project more temporally (but this is not direct evidence for this!)

Question 34

What are the authors trying to determine in this figure?

Answer 34

Trying to determine which curve belongs to the EphA3+ cells

Question 35

Describe the experiment being performed in this figure

Answer 35

Doing in situ hybridization on cells from panel B to see if the cells are wild-type or EphA3+ Hoping that the more nasal cells will be EphA3+

Question 36

What is the key takeaway from this figure?

Answer 36

The nasal (predicted EphA3+ cells) cells are colocalized with the beads

Question 37

Given that ganglion cells appear to map "relative" to their Eph levels, what is the common hypothesis to describe how cells have "low middle and high" levels of Eph (how do they measure/know these relative amounts)?

Answer 37

There might be relative competition between ganglion cells -- the cells may be ordered to some degree by the time they reach the SC -- "first come first serve" potentially?