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BIOL 309 > Early Invertebrate Development (2) > Flashcards

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?
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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
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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)
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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
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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
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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

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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
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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
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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
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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

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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
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12
Q

What is cell autonomous development?

A
  • any cellular response or phenotype to a cellular or molecular mechanism occurring within that same cell
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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
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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
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15
Q

What does “determined” mean?

A
  • fate of cell is fixed
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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
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17
Q

What are “operational definitions”?

A
  • defined experimentally
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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
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19
Q

What is induction?

A
  • non-autonomous interaction

- when one cell type is influenced by another

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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
Q

What is seen by observing the GFP micromeres?

A
  • over time they separate into the centre
26
Q

What kind of experiments could you design with this approach? (GFP micromeres)

A
  • i.e. functional

- loss or gain of function (e.g. dominant neg, gene knockdown, overexpression)

27
Q

What occurs during the 2nd stage of gastrulation in the sea urchin?

A
  • 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
Q

Where does gastrulation start for protostomes? For deuterostomes?

A
  • starts at the mouth

- starts at the anus

29
Q

What does gut formation require?

A
  • requires narrowing and lengthening of archenteron

- driven by “convergent extension”

30
Q

What happens during the 3rd stage of gastrulation for sea urchins?

A
  • the secondary mesenchyme eventually fuses to blastocoel roof and forms mouth
  • the archenteron lengthens
31
Q

What is the pluteus to the adult sea urchin?

A
  • 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
Q

What is a starting point for an experimental set-up to study the molecular mechanisms of micromeres?

A
  • 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
Q

What is the wnt signalling pathway?

A
  • 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
Q

Where is activated disheveled found?

A
  • activated form of disheveled present in cytoplasm of micromeres
35
Q

What else affects the wnt pathway?

A
  • 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
Q

What happens in the absence of wnt?

A
  • ubiquitin-mediated proteasome degradation

- (B-catenin is degraded)

37
Q

What does LiCl treatment result in?

A
  • increase in nuc-beta-catenin

- causing more endomesoderm

38
Q

What does dnNCad treatment result in?

A
  • decrease in nuc-beta-catenin

- causing no endomesoderm

39
Q

What is the genomic regulatory network for development? How was it derived?

A
  • 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
Q

In the micromeres-PMC pathway, what is Pmar1?

A
  • 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
Q

In the micromeres-PMC pathway, what is Hes1?

A
  • bHLH transcription factor
  • it is inhibited by Pmar1
  • it inhibits Tbr, Ets, Delta, and Dri
42
Q

In the micromeres-PMC pathway, what is Delta?

A
  • sends secondary signal to veg2 (non-cell autonomous signalling)
  • is inhibited by HesC
43
Q

In the micromeres-PMC pathway, what are the 3 TF’s that regulate what?

A
  • Tbr, Ets, Dri
  • regulate expression of skeletogenic differentiation genes activated
  • can be inhibited by HesC
44
Q

Is the role of B-catenin in PMC specification autonomous or non-autonomous?

A
  • autonomous because it is functioning within the cells
45
Q

What happens in the rest of the embryo that does not normally express PMC?

A
  • no maternal nuclear b-catenin
  • no inductive signalling from pmar1 or delta because HesC and Hnf6 are activated
  • no skeletogenic differentiation genes activated
46
Q

What is the notch signalling pathway?

A
  • 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
Q

What led to identification of cell/tissue specific genes?

A
  • differential gene expression analysis
48
Q

What is the endo16 gene?

A
  • an endoderm specific gene

- (helps to form gut)

BIOL 309 (14 decks)

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