Discussion paper 2 Flashcards
What are the 2 key criteria which make up a morphogen?
- forms a concentration gradient
- different concentrations of morphogens induce different responses in cells
Describe the French Flag model of patterning
Cell fates are generated according to position along a concentration gradient, and different cell fates are specified at different concentration thresholds
Why are sharp boundaries between cell types important in the developing neural tube?
Nervous system requires the formation of distinct circuits - formation of domains may simplify the development process
Describe the key result (ie. what TF activity) of Shh signaling
Activation and nuclear import of GliA
What 2 molecules does smoothened inhibit?
PKA and Slimb
What inhibits Patched?
Hedgehog
What inhibits smoothened?
Patched
Why might the french flag model of patterning be an oversimplification of what is actually happening?
Cells position relative to the source of a morphogen is constantly changing during the time of exposure
What is the model organism of this paper?
Danio rerio (zebrafish)
Describe discoidal cleavage
Faster cleavage around the animal pole of the embryo
Describe the methods of this study and how zebrafish made this a good model to use these methods
In toto live cell imaging: watching the cells as they divide when using different fluorescent markers
Zebrafish is a good model for this because it is clear
Gastrulation in zebrafish happens around this time (hours)
8h
Neurulation in zebrafish happens around this time (hours)
16h
By what point (hour) are zebrafish embryos capable of swimming around (in their larval form)
72h - short generation time and rapid development
When the zebrafish neural plate starts condensing in on itself, what structure is formed?
Neural keel
The main source of Shh in zebrafish comes from this area
Notochord
The in toto imaging setup of this paper combines these 2 types of microscopy
Confocal and fluorescent
Describe (very basically) how fluorescent imaging works
Shine light at a fluor, it will be excited by this wavelength of light and emit light at a lower wavelength (e.g. excite at blue, emit at green for GFP)
When can multiple fluorescent markers be used at the same time?
When they have non-overlapping excitation and emission spectra
Confocal microscopy allows acquisition of a high resolution (1) of images
Z-stack: allows for 3D imaging (gain sections in the x and y plane, then overlap these to get information about the z plane)
What is the “4th dimension” in this paper (where 1-3rd dimensions are x, y, and z)
Time - see changes in all three planes over time
mem-citrine is a fluorescent marker for (1). What colour is it?
Red: membrane localized cells
What is H2B-cherry a marker of?
Membrane localization (H2B = “histone 2b”)
What is the key advantage of in toto cell imaging?
You can track individual cell movements
Describe what the LM/DV distance is in this study
The distance from the middle top of the mesoderm/notochord to the cell of interest
What is the objective of Figure 2A?
Wanted to look at the dynamics of Shh signaling, is the source of Shh changing over time?
Describe the utility of the Shh:gfp reporter
Indicates where Shh-producing cells are located
What is the main takeaway from figure 2A?
The source of Shh morphogen is changing shape over time, and therefore the level of Shh that a cell is exposed to is not uniform
In the last panel at 15h, Shh is mainly located…
In the notochord and along the medial floor plate
What is the objective of figure 2B?
Visualize the levels of Shh over time, but instead of looking at the source of Shh, they look at patched activation indirectly, through the presence of kaede (which is downstream)
What are the key takeaways from fig 2B?
Not all neighbouring cells express equal amounts of kaede, but they later become more concentrated and organized which could be an indicator of cell sorting
What is the key difference btw the green dots (10 hours post-fertilization) and the blue triangles (14.5 hpf)? What does this signify?
There is a broader distribution of kaede+ cells earlier in development, and this distribution “tightens up” later in development - this could be indicative of cell sorting
What is the significance of the data in figure 2 with respect to the underlying hypothesis of the paper?
The results challenge the FF model, there are temporal and positional differences in Shh+ cells and in Shh secreting cells. Local heterogeneity in response contradicts the french flag model!
What is the objective of figure 3A?
Wanted to characterize the spatial distribution and timing of specific motor neuron progenitors using the mnx1 promoter (marks motor neuron progenitors)
What is indicated by the red arrows in figure 3A?
mnx1-negative cells are intermingled with mnx1-positive cells
What is the key takeaway from figure 3A?
Earlier in development (e.g. 12h), there is a “salt and pepper” distribution of mnx1+/- cells, and by 14 (for example), there is a lot less mixing
What is the objective of figure 3D?
To characterize the spatial distribution and timing of specification of dorsal motor neuron progenitors using the olig2 promoter
Why did the authors look at the mnx1 promoter in figure 3A? (i.e. what is mnx1 marking?)
mnx1 marks ventral motor neuron progenitors
Why did the authors look at the olig2 promoter in figure 3D? (i.e. what is olig2 marking?)
olig2 marks dorsal motor neuron progenitors
What are the key observations of figure 3D?
Early on there are scattered GFP+ cells intermingled with GFP- cells under the olig2 promoter - “salt and pepper” pattern
What are the important parts of Figure 3E?
The olig2 “stripe” characteristic of later observations (more in line with the FF model) arises from a mixed population of cells, and later gets sorted into the clean line we see later
What is cyclopamine? How and why is it used in Figure 3C?
Cyclopamine is an inhibitor of Shh - it is being used here to determine at which stage application of cyclopamine will have the largest effect on pMN specification
What do the results of using cyclopamine on developing neural tubes show in figure 3C?
Early inhibition of Shh reduces the population of pMNs, indicating that this fate specification occurs early on (only mild effects when application is later)
Why is the result of figure 3C important for the paper?
It suggests that Shh is required for fate specification, but that cells are not “sorted” early on
What is the rationale underlying the experiments for figure 4A?
To determine whether cell fates are specified early or late, they are looking at individual cell divisions and the fate of both the daughter cells and granddaughter cells - if the clones share the same fate, fate must be specified early
Describe why the cell-fate mapping experiments of figure 4 were necessary to prove that cell sorting occurs in the neural tube
If cell sorting occurs, cells would need to maintain their early specified fate, if the cells change fate later, the french flag model could be correct
What are the key observations of Figure 4A? What conclusions do these observations support?
A disproportionately high number of clones have the same cell fate. Cells therefore, must be specified at an early time point
Describe how the experiment in figure 5A was performed
Embryos were labelled with H2B-cherry and mem-citrine (to visualize membranes and nuclei) and then cell fates were marked with different labels. Using their imaging software, they went back in time to see where the cells came from to track their relative position
Point out the most important findings of Figure 5A on the graph for Figure 5B
There are dispersed cell fates at 10hpf that reorganize by 16 hpf
What conclusion can be made from figures 5A and 5B? (2)
- The french flag model can be ruled out
- progenitor domains are formed by a cell sorting process
What is the underlying hypothesis in Figure 5F, and what was the authors rationale for examining cdh2?
Hypothesis: cell adhesion is required for cell sorting
Chose cdh2 because this is a neural cell adhesion protein which is required for movement of all neural progenitors
What are the experimental approaches of figure 5F?
- dominant-negative cdh2
- cadherin MO knockdown
Both of these are loss of function experiments
What are the important findings of figure 5F?
when cdh2 is knocked down, some mnx1:gfp+ cells are misplaced in a wider and more mixed pattern (both the right panels are the knockdowns)
What is being demonstrated by Figure 5G?
In the knockdown (red), there is a wider dispersal of mnx1:gfp+ cells when cadherin is perturbed
What are the overall results of Figure 5?
Cell sorting occurs in the developing neural tube, and is likely mediated by cadherin-mediated interactions
What is a weakness of the paper discussed in class?
Cdh2-mediated sorting is not the whole story: cdh2 is expressed by all neural progenitors, so does not explain why or how specificity of domains is determined
