Discussion paper 1 Flashcards
In order to have a secondary body axis (as per the Spemann/Mangold experiment), there must first be a secondary site of…
Gastrulation
The organizer is fated to become this kind of tissue (dorsal/ventral, germ layer)
Dorsal mesoderm
How was noggin discovered as a neural inducer?
Was found in a cDNA scan to identify neural inducing candidate genes, and screening determined that the molecule was in the right place at the right time, supporting noggin’s role as a NIF
What is the main, broad underlying hypothesis of the paper?
Is noggin (and are other BMP antagonists) sufficient and necessary for the formation of dorsal structures?
Where is the main source of BMP inhibition?
The dorsal mesoderm AKA the organizer
“Dorsalization” of the ectoderm refers to…
The induction of neural tissues on the dorsal embryo
Why is xenopus tropicalis a good model organism for this study?
- Eggs are large and easy to manipulate
- Has a simple diploid genome which is good for genetic studies (laevis is pseudotetraploid)
- tropicalis has a shorter generation time, which makes it a more practical model
What molecule is being targeted by morpholino oligonucleotides?
mRNA
Why does this study act as a cautionary tale for loss-of-function studies?
Previous loss-of-function studies have knocked out noggin (and other BMP inhibitors) and seen little effect on neural development, but nobody had knocked out more than 2 BMP antagonists, therefore they did not get to examine the redundancy
What is one of the pitfalls of this paper?
They do not report number of trials, may be cherry-picking data, minimal to no statistical analysis
What is the underlying question addressed by Figure 1?
Are BMP inhibitors other than noggin expressed in the organizer, when and where are they expressed?
What experimental approach is used by Figure 1?
In situ hybridization
What BMP inhibitors are expressed by the organizer? (5)
noggin, chordin, follistatin, xnr3, cereberus
What is the take-home message of figure 1?
Other BMP antagonists are expressed in the organizer, and they are expressed at the correct time to function as neural inducing factors (consistent with other studies)
What is the underlying research question behind Figure 2?
Do single or double knockouts of BMP antagonists have an effect on neural plate development?
What experimental approach (2) is being used in figure 2?
Knockout of BMP antagonists using MOs, assay for sox2 expression (in situ hybridization)
Why is sox2 an appropriate assay for determining if neural plate structures are forming?
It is an early marker of neural induction
What are the two kinds of morpholino antisense knockdowns? Which is used in the paper?
- translational blocking “atgMO” (blocks the start codon) – used in the paper!
- splicing modification “gtMO” (blocks a splice modification)
How does MO knockdown eliminate a certain gene product?
Antisense RNA binds to a region of the mRNA for a gene, blocking its translation and resulting in a loss of the gene product
When are MOs injected into developing embryos?
At the one-cell stage
What is the take-home message of figure 2?
If 1 or 2 BMP antagonists are knocked down, the embryo will have relatively normal development – minimal to no alteration of neural induction
What is the underlying hypothesis of figure 3?
Do BMP inhibitors function in a redundant manner to induce neural tissue
What is the experimental approach used in figure 3?
MO knockdown and sox2 in situ hybridization, but this time MO knockdown is of noggin, chordin, and follistatin at the same time
What is the take-home message of figure 3?
The BMP antagonists secreted from the organizer function in a redundant manner to promote the development of neural tissue and dorsal mesoderm
In figure 3, why did the authors do in situ hybridization for sox3 in addition to sox2? What is sox3?
They did this as a confirmation experiment, sox3 is another marker of the neural plate
What is being examined in this part of figure 3? Why did the authors examine expression of myf5 and myoD?
Looking at other markers of the mesoderm: in this image, the markers are telling us that the dorsal mesoderm is prevented from being formed
What is the underlying research question in figure 4?
Are the MO knockdown phenotypes due to the specific targeting of BMP inhibitors and pathway? Is something else being targeted?
What approach is being used in figure 4?
Rescue experiments: injection of fugu noggin
What is the take-home message of figure 4?
The phenotype with loss of dorsal structures in the knockdown experiments is due to loss of BMP inhibition and is not an artifact of a nonspecific effect
What is the underlying question of figure 5?
If the dorsal ectoderm and mesoderm (neural) are lost, are they replaced by ventral ectoderm and ventral mesoderm?
What were the key methods used in figure 5?
FCN and B-catenin knockouts then in situ hybridization for ventral markers
What is the take-home message of figure 5?
BMP inhibition is required to inhibit the formation of ventral mesoderm, when tissue does not differentiate into dorsal tissue, it becomes ventralized (binary cell fate choice)
What is the take home message of figure 6?
In the triple knockdown experiments, the organizer is still formed, therefore phenotype is not due to loss of organizer, but loss of BMP antagonism
