Discussion Paper 3 Flashcards
What is the underlying question of the discussion paper?
How does morphogen signaling lead to distinct boundary formation?
What kind of motor neurons are being examined in this paper?
Lateral motor column neurons, and neurons of the column of terni
Hoxc6 is associated with the (brachial/thoracic) region of the LMC
Brachial
Hoxc9 is associated with the (brachial/thoracic) region of the LMC
Thoracic
What hox genes represent the boundary between brachial and thoracic LMC/CT neurons?
Hoxc6 and Hoxc9
Which other hox genes form a boundary just above the brachial and thoracic boundary? Which one is more brachial?
Hoxc5 and Hoxc8, where Hoxc5 is more brachial
What neural marker is coexpressed with Hoxc6?
RALDH2
What neural marker is coexpressed with Hoxc9?
BMP5
What is the take home message of Figure 1?
Hoxc6 and Hoxc9 define motor neuron columnar regions which correspond to LMC and CT neurons respectively. These can be assessed by the markers RALDH2 and BMP5 respectively
What is the underlying question of Figure 2
Is the positional identity of MNs at brachial and thoracic levels established by graded FGF signaling?
What is the utility of counting somites in this paper?
This is a method used to age the embryo
Why did the authors decided to examine FGF in this paper?
Because this was a candidate morphogen which was expressed in a gradient which differed in the anterior-posterior axis. BMP and WNT and Shh differ in a D-V manner, so would not be likely to have had much effect
What is the experimental approach used in Figure 2?
Overexpress FGF8 or FGFR in the neural tube using in ovo electroporation
Describe the technique to perform in ovo electroporation
Insert electrodes into a live egg with plasmid DNA, when current is applied the DNA will move to the positive electrode because it is negatively charged. Then, the plasmid DNA will be expressed by the positive side, and the other side will function as a sort of control
Why is only one side of the chick neural tube electroporated?
That is just a consequence of how in ovo electroporation works, only the positive side gets the plasmid DNA electroporated into it
What are the most important findings of figure 2?
When FGF is overexpressed in the brachial region, there is a loss of Hoxc6 and an expansion of Hoxc9
What is being demonstrated in this figure?
In the brachial region, when FGF8 is overexpressed, there is a reduction of Hoxc6 and an expansion of Hoxc9 where Hoxc6 was originally
What are the key findings in this figure?
When FGF8 is overexpressed in the brachial region, there is loss of RALDH2 (the brachial marker) and there is gain of BMP5 – this indicates that you are losing brachial identity and gaining thoracic identity
What does the data in this figure tell us about the role of Hoxc9 with respect to Hoxc6 and RALDH2 expression?
Trick question: nothing. This is correlative data only
What is the underlying question of figure 3?
Can the positional identity of MNs at the brachial and thoracic levels be specified by Hoxc6/Hoxc9 expression?
What is the key experimental approach being used in figure 3?
Overexpression of hoxc6 and hoxc9 in the neural tube using in ovo electroporation
What are the most important findings in this figure?
When hoxc9 is overexpressed brachially, there is a reduction of hoxc6 and RALDH2, and a gain of BMP5 expression
What do the results of figure 3 here tell us about the relationship between hoxc6 and hoxc9?
Hoxc9 likely inhibits hoxc6
Based on the results in this figure, describe what we know about the relationships between Hoxc6, Hoxc9, RALDH2, and BMP5
When Hoxc6 is expressed in the thoracic region, what occurs?
Hoxc9 expression is lost, there is ectopic expression of RALDH2, and there is a loss of BMP5
What do these results say about the relationship between Hoxc6 and Hoxc9?
Hoxc6 likely inhibits Hoxc9
What is the underlying question of figure 4?
Is the patterning of pMNs at brachial and thoracic levels affected by FGF - looking at this with respect to timing
What is the experimental approach used in figure 4?
Electroporate the embryos at stage 12 to get ectopic FGF expression and then do in situ hybridization/immunostaining at a later time
Highlight the most important findings from this figure
Note that this figure is indicating mRNA, NOT protein!
When FGF8 is overexpressed in brachial regions, there is an expansion of all Hox9 paralogues and Hoxc6 mRNA in the brachial regions
What assay is being used in this figure?
Immunolabelling
What is the key takeaway of this figure?
Previously it was shown that Hoxc6 mRNA was present in the brachial regions even after ectopic FGF8 expression, but there is an expansion of Hoxc9 protein and a loss of Hoxc6 protein
What is the underlying question being addressed by figure 5?
Do Hoxc6 and Hoxc9 function in post-mitotic MNs to direct cell fate?
What is the experimental approach being used in figure 5?
Electroporate ectopic hox genes under the Hb9 promoter
What is the key takeaway from this figure?
When Hoxc9 is expressed in post-mitotic neurons in the brachial region, there is a reduction of Hoxc6 and a loss of RALDH2, but no gain of BMP5
Sufficient to suppress brachial traits, but insufficient to induce CT identity post-mitotically
What are the key takeaways of this figure?
When Hoxc6 is expressed thoracically, Hoxc9 and BMP5 are suppressed, and RALDH2 expression is induced
Ectopic Hoxc6 expression is capable of inducing LMC fate post-mitotically
Why might it be important that there are many Hox genes overlapping in the brachial region of the motor column?
Repressive interactions between the different hox genes might determine motor pool fates - different areas of projection maybe?
