Early Invertebrate Development (2) Flashcards
1
Q
What are the main questions surrounding cell fate determination?
A
- what cell types are these undifferentiated embryonic cells fated to become?
- when are these cell types determined?
- how are these cell types determined?
2
Q
What study might we do to examine cell fate determination?
A
- “lineage tracing”
- identify cell/tissue type by virtue of morphology and location
- inject fluorescent dyes at 60 cell stage and then use fluorescence microscopy at pluteus stage
- label DiI and DiO
3
Q
What may become difficult if a sea urchin 64-cell embryo looked completely symmetrical?
A
- it would be very difficult to replicate the experiment
- this is not an issue however because sea urchins are asymmetrical at the 6th cleavage (60 cell stage) (micromeres at vegetal pole)
4
Q
What was found when veg1 cells were lineage traced?
A
- injected with DiI
- became endoderm (foregut, midgut, and hindgut)
- became ectoderm
- veg1 cells are uncommitted at this stage
5
Q
What was done after veg1 cells were lineage traced?
A
- all cells were lineage traced to create a “fate map” at the 60-cell stage
6
Q
What study can be done to see when veg1 ectoderm and endoderm lineages separate?
A
- inject at the 8th cleavage and trace lineage
- even at the 8th cleavage stage, ectoderm and endoderm lineages have not segregated
7
Q
What does a fate map tell us?
A
- what a cell will become based on its position in the embryo
- this does not mean that at the 60 cell stage the fates of the cells are determined
8
Q
What experiment can address the question, at what developmental time point do cell fates become committed?
A
- move a cell to another location and see if it changes it’s fate
- inject cytoplasm into a cell
- remove cells and see if certain tissues develop
9
Q
What experiment was done to look at when cell fates become committed?
A
- Okazaki
- dissociated embryos into single cells with agitation in calcium free sea water
- to look at if micromeres at the 16-cell stage are specified to become spicules
10
Q
Why does Ca++ free sea water dissociate cells?
A
- cell-cell adhesion is mediated by cadherin proteins
- the removal of calcium prevents cadherin-cadherin binding
11
Q
What is cell non-autonomous development?
A
- any cellular response or phenotype that occurs due to the influence of another cell(s) or external factor
12
Q
What is cell autonomous development?
A
- any cellular response or phenotype to a cellular or molecular mechanism occurring within that same cell
13
Q
What was observed when the micromeres were isolated at the 16-cell stage?
A
- they still formed skeletogenic mesenchyme/”spicules”
- this means they were already specified
- autonomous development
14
Q
What does “specified” mean?
A
- fate of a cell is established but not fixed (can still be altered by external factors)
- if cultured in isolation, it will follow its fate
15
Q
What does “determined” mean?
A
- fate of cell is fixed
16
Q
At the pluteus stage, are all cell fates determined and irreversible?
A
- if you expose pluteus to fish mucus, it causes budding that falls off and forms mini-pluteus
- could be adaptation for when they are in environment with lots of fish
17
Q
What are “operational definitions”?
A
- defined experimentally
18
Q
What experimental approach was used to determine if cell-cell interactions are necessary for urchin development?
A
- culture embryo in absence of different layers
- found that animal hemisphere alone results in complete animalization (ring)
- animal hemisphere and micromeres results in a recognizable larva (endoderm structure formed by animal layers)
- conclusion: micromeres play an instructive role in endoderm formation
19
Q
What is induction?
A
- non-autonomous interaction
- when one cell type is influenced by another
20
Q
To what extent can the micromeres induce endoderm? (What other experiments and results?)
A
- transplanted additional micromeres to top of animal pole: micromeres induced a secondary “vegetal” axis
21
Q
What is gastrulation?
A
- a coordinated movement of cells that underlies germ layer formation
22
Q
What happens during gastrulation (1st stage) in sea urchins?
A
- starts from blastula
- primary mesenchyme cells are within internal cavity
- blastomeres undergo movements and change in their positions relative to one another
- embryo contains three “germ” layers: ectoderm, mesoderm and endoderm
23
Q
What experiment would be used to determine where the internal primary mesenchyme cells come from?
A
- inject GFP into a fertilized sea urchin egg, all cells will express GFP
- remove the GFP micromeres and add them to a normal 60-cell stage host embryo (transplantation)
24
Q
What does galtase-GFP mRNA allow for?
A
- allows GFP to be tethered to the golgi apparatus
25
What is seen by observing the GFP micromeres?
- over time they separate into the centre
26
What kind of experiments could you design with this approach? (GFP micromeres)
- i.e. functional
| - loss or gain of function (e.g. dominant neg, gene knockdown, overexpression)
27
What occurs during the 2nd stage of gastrulation in the sea urchin?
- cells at bottom (primary mesenchyme cells) become thickened forming the vegetal plate
- invagination occurs
- forms the blastopore (outside "hole"), and the archenteron (invaginated cavity)
- primary mesenchyme cells and secondary mesenchyme cells present
28
Where does gastrulation start for protostomes? For deuterostomes?
- starts at the mouth
| - starts at the anus
29
What does gut formation require?
- requires narrowing and lengthening of archenteron
| - driven by "convergent extension"
30
What happens during the 3rd stage of gastrulation for sea urchins?
- the secondary mesenchyme eventually fuses to blastocoel roof and forms mouth
- the archenteron lengthens
31
What is the pluteus to the adult sea urchin?
- pluteus is the temporary vessel within which the actual sea urchin "dormantly" resides
- sea urchin is called imaginal rudiment, formed from invaginations of ectoderm and secondary mesenchyme
- surrounding pluteus degenerates
32
What is a starting point for an experimental set-up to study the molecular mechanisms of micromeres?
- start with correlative data
- beta-catenin protein localization in the 60-cell sea urchin embryo
- correlative data: expression of beta-catenin marks cells fated to become 'mesoderm and endoderm' (endomesoderm)
33
What is the wnt signalling pathway?
- wnt binds to frizzled protein receptor
- disheveled is activated
- disheveled inhibits GSK-3
- this prevents GSK-3 from inhibiting B-catenin
- B-catenin is active and allows for transcription
34
Where is activated disheveled found?
- activated form of disheveled present in cytoplasm of micromeres
35
What else affects the wnt pathway?
- many molecules can have an affect to turn pathway on or off
- lithium chloride can inhibit GSK-3 and activate the pathway
- dnNCad can inhibit B-catenin
36
What happens in the absence of wnt?
- ubiquitin-mediated proteasome degradation
| - (B-catenin is degraded)
37
What does LiCl treatment result in?
- increase in nuc-beta-catenin
| - causing more endomesoderm
38
What does dnNCad treatment result in?
- decrease in nuc-beta-catenin
| - causing no endomesoderm
39
What is the genomic regulatory network for development? How was it derived?
- map of all genes/signalling/interactions required for development
- derived from large-scale perturbation analyses (computational methodologies, genomic data, cis-regulatory analysis, molecular embryology)
40
In the micromeres-PMC pathway, what is Pmar1?
- homeodomain transcription factor
- is activated by otx and B-catenin
- activates early signal to veg2 (non cell autonomous signalling)
- inhibits Hnf6 and HesC (doulble negative gate)
41
In the micromeres-PMC pathway, what is Hes1?
- bHLH transcription factor
- it is inhibited by Pmar1
- it inhibits Tbr, Ets, Delta, and Dri
42
In the micromeres-PMC pathway, what is Delta?
- sends secondary signal to veg2 (non-cell autonomous signalling)
- is inhibited by HesC
43
In the micromeres-PMC pathway, what are the 3 TF's that regulate what?
- Tbr, Ets, Dri
- regulate expression of skeletogenic differentiation genes activated
- can be inhibited by HesC
44
Is the role of B-catenin in PMC specification autonomous or non-autonomous?
- autonomous because it is functioning within the cells
45
What happens in the rest of the embryo that does not normally express PMC?
- no maternal nuclear b-catenin
- no inductive signalling from pmar1 or delta because HesC and Hnf6 are activated
- no skeletogenic differentiation genes activated
46
What is the notch signalling pathway?
- notch is a transmembrane protein
- the ligand it binds to is delta (another transmembrane protein in a signalling cell)
- when they bind, a protease is activated and allows activation of gene transcription
47
What led to identification of cell/tissue specific genes?
- differential gene expression analysis
48
What is the endo16 gene?
- an endoderm specific gene
| - (helps to form gut)
