Patterning Flashcards
What organization centre precedes the Spemann Organizer?
The Nieuwkoop centre
The formation of the Niewkoop centre is mediated by (1) signaling
B-catenin
What is secreted by the Nieuwkoop centre to induce formation of the organizer?
BMP
In the pictured experiment (similar to but not the same as the Spemann/Mangold transplantation experiment) when the gastrula dorsal lip is being transplanted into presumed epidermis, what is the key result?
When the dorsal gastrula lip is transplanted at different times, a secondary axis is established, but the dorsal structures that it becomes are different - don’t get an entire head, just get different parts of the head
What information does this experiment tell us? (2)
There must be molecules that act along the A-P axis of the dorsal mesodern in a graded manner, and signals from the underlying mesoderm (archenteron roof) instruct the neural plate in a graded manner
Define the endomesoderm and the utility of this name
It is the tissue that is first involved in the formation of the organizer, and it is called this because it is not fully endoderm, but it is fated to become the most anterior parts of the mesoderm / nervous tissues
What key BMP inhibitor is expressed in the endomesoderm?
Cerberus
What occurs when cerberus is injected into vegetal structures?
Results in ectopic head formation but no secondary axis
What 3 important molecules does cerberus inhibit?
Nodal, BMP4 and Wnt
Where are Nodal, BMP4 and Wnt expressed?
In the anterior mesoderm and endoderm (head-fated region)
Nodal and BMP4 are members of this family of molecules
TGF-B
What are the general steps in the Wnt signaling pathway?
Wnt > frizzled > disheveled > GSK-3 > B-catenin
When Wnt is absent, beta-catenin is…
Degraded
When Wnt is present, beta-catenin is…
Active
How does GSK-3 impact B-catenin functioning
GSK-3 is a kinase, it phosphorylates B-catenin when active (GSK-3 not phosphorylated) and then B-catenin is targeted and degraded by proteasomes
What happens if you knock down cerberus using morpholinos? Why?
Nothing - functionally redundant
What molecules are functionally redundant to cerberus? (2)
Frzb and Dickkopf
Cerberus, Frzb, and Dickkopf are all inhibitors of this molecule
Wnt
What kinds of molecules are cerberus, fzrb and dickkopf?
proteins
When a tissue receives Wnt signals, what kind of neural tissue does this induce?
Induces trunk-like (posterior) fates
Another name for Nodal is…
Xnr3
What is the name of the dorsal endomesoderm promoter used to explore the function of cerberus?
McerP
What happens to the development of head structures when BMP4 and Nodal are overexpressed?
Nothing - no substantial loss of head structures because cerberus is the key molecule for inducing head structures
What is key evidence that any morphogen is a secreted molecule?
If there is a concentration gradient present
Head structures are formed when Wnt is (active/inhibited)
Inhibited
Where is Wnt concentration highest on the anterior-posterior axis?
Posterior
Where is Wnt concentration lowest on the anterior-posterior axis?
Anterior
Where is cerberus concentration highest on the anterior-posterior axis?
Anterior
Describe the experiment used to determine if the effects of Wnt are concentration dependent?
Animal cap cells were removed and dissociated from each other (dissociation removes BMP singaling)
Cells were reaggregated, which causes neural induction
Once the cells were reaggregated, RT-PCR was used to assess the TF profile of the dissociated ectoderm
Next, Wnt was added in a graded manner to the neurally-induced tissue to see what would happen. Different neural markers of forebrain/hindbrain can be used to determine how anterior the structures which formed under different additions of Wnt were
Does Wnt signaling function in a concentration-dependent manner?
Yes
Why was RT-PCR done in this experiment?
Because the cells would have had mRNA for the genes of interest, and to amplify genes with PCR the target molecules need to be double stranded because the PCR enzymes only work on DNA, not RNA. The “RT” part of PCR involves making a probe and then synthesizing a complimentary strand
What kind of molecule is Wnt?
Morphogen
What are the 2 key criteria for a molecule to be a morphogen?
- forms a concentration gradient
- different concentrations of the morphogen induce different responses (e.g. cell fate) in responding cells
What is the morphogen responsible for the patterning of what will become the retinal-tectal projection (in xenopus)?
Vax2
In the spinal cord, the dorsal section is mostly devoted to this kind of information
Sensory input
In the spinal cord, the ventral section is mostly devoted to this kind of information
Motor output
What are the 3 major classes of neurons or receptors which make up majority of sensory input to the spinal cord
Nociceptors, mechanoreceptors, proprioceptors
Sensory neurons are derived from these cells early in development
Neural crest cells
Describe the depth at which nociceptors, proprioceptors, and mechanoreceptors reach within the spinal cord
Nociceptors - Most dorsal (region I, II)
Mechanoreceptors - Intermediate (region III, IV)
Proprioceptors - Most ventral (but still not actually on the ventral side bc that’s where motor neurons are, region V)
Define a central pattern generator
Underlying neural circuit which forms rhythmic movements
Projections from dorsal sensory axons usually synapse to these before synapsing to a motor neuron
Spinal interneurons
What would be a good way to design an experiment to explain how distinct neuronal cell types are specified along the D-V axis?
Look for anatomical correlates: transplant these anatomical correlates to a new region and see if similar patterning is generated
Describe the expression of Shh in the neural tube relative to the notochord
Secreted from the notochord, distributed ventrally (high) to dorsally (low) in a semi-graded manner (some discrepancies, not perfect
When Shh expression is high, these cells later go on to express…
GliA (activator) - this is a TF downstream of Shh (not cleaved)
Cells which express GliA will express these downstream TFs
Class-II
What molecule forms an inverse TF gradient to GliA?
GliR (Repressor, cleaved form of Gli)
Why is the formation of TF gradients significant?
This is the mechanistic basis for tissue patterning
Define combinatorial coding
A conserved mechanism which enables neuronal cell type diversity where a morphogen gradient directs differential TF expression
What is the advantage of combinatorial coding?
It is economical
When a large number of distinct TF expression domains exist, what is the significance of this?
It leads to distinct cell types
Describe a key difference in mammalian vs zebrafish neurulation
Mammals: get a flat sheet which bends
Zebrafish: get a flat sheet which smushes together - get the neural keel stage
End result is basically the same
What are the 2 key ways you can express exogenous genes in zebrafish
- make a transgenic fish (permanent genetic changes)
- Inject mRNA directly into a fertilized zebrafish egg (this method can be transient)
Define a promoter
A region of non-coding DNA which normally lies upstream of a gene and directs transcription of that gene
What is one potential setback of using transgenic animal models?
Insertion of a gene of interest into a genome is completely random, don’t know where that gene is going to end up - could be anywhere
