Discussion paper 6 Flashcards
Which cells of the retina project to the brain?
Ganglion cells
Why are topographical maps important?
Simplify processing, necessary for organized and normal brain processing
What is the question being addressed by this figure?
Where are EphA receptors being expressed?
What is the experimental technique being used in this figure?
In situ hybridization
What is the key takeaway from this figure?
EphA5 and EphA6 are expressed in RGCs, EphA3 is not
Areas with low levels of EphAs will target regions containing (low/high) levels of ligand, why?
High, because EphA interactions are repulsive: the less receptor, the less repulsion = more connections to cells with the ligand bound
What is the broad underlying question being addressed by this paper?
How do gradients mediate sensory map formation?
What is meant by absolute and relative levels of ephrin with respect to signaling?
Absolute: X concentration of ligand
Relative: Measuring differences in concentration of ligand regardless of the absolute concentration
What specific question is this discussion paper addressing?
Is mapping to the superior colliculus controlled by absolute or relative amounts of Eph?
What are the key features of Isl2 that make it a useful gene to piggyback EphA3 expression? (3)
- It is expressed broadly across the nasal-temporal axis: will increase the concentration evenly across the retina
- It is only expressed in ~50% of RGCs, some little gaps to serve as internal controls
- Expressed exclusively in ganglion cells
How is EphA3 gain of function achieved in this paper?
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!
What methods are being used in this figure?
Immunostaining
Which of these figures are the wild type and which are the mutant mouse?
CDEG = WT
FH = Mutant
What is the key takeaway of panel F in this figure?
When Isl2-EphA3 construct is expressed in mice, expression of Isl2 is unaffected – similar to the wildtype
What is the key takeaway of panel H in this figure?
In mutant mice containing the Isl2-EphA3 construct, EphA3 is expressed in a similar manner to Isl2
What is the key takeaway of this figure?
Expression of the EphA3-Isl2 construct does not affect normal expression of EphA5 and EphA6
What is being shown in panels A and D in this figure?
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)
In this figure, why do the authors use both heterozygous and homozygous knock-in mice?
They do this to check for gene dosage effects
Based on this figure, are EphA’s subject to gene dosage effects?
Yes – function in a dosage-dependent manner
Explain this figure
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
What is the assay being used in this figure? Briefly explain
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
Why is Dil’s lipophilic property important?
Because this allows it to diffuse across membranes and allows entire cells to fluoresce
Describe the excitation and emission spectra of Dil
Excited by green light and emits red light
What is DiAsp?
Similar to Dil but excited by blue light and emits green light
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?
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
Is mapping to the SC controlled by relative or absolute amounts of Eph?
Relative
What are the key observations in this figure? (3)
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
Why do the results of this figure tell us that mapping to the SC is a non-cell autonomous process?
Because if it was cell autonomous, the cells which are EphA3- would map to the same location as the WT
Describe the main takeaway of this figure?
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
What is the underlying question addressed by this figure?
Can the results from Dil anteroretrograde labelling be validated using retrograde labelling from SC to the RGCs?
What is the assay being used in this figure?
Dil and DiAsp retrograde labelling
What are the most important findings of this figure?
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
Based on findings from this data, what curve represents the EphA3+ overexpressing cells?
Orange curve likely expresses more EphA3 because these cells project more temporally (but this is not direct evidence for this!)
What are the authors trying to determine in this figure?
Trying to determine which curve belongs to the EphA3+ cells
Describe the experiment being performed in this figure
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+
What is the key takeaway from this figure?
The nasal (predicted EphA3+ cells) cells are colocalized with the beads
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)?
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?
