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BIOL 309 > Early Vert Development (5) > Flashcards

Early Vert Development (5) Flashcards

1
Q

What does WE stand for?

A
  • whole embryo
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2
Q

What are the controls in this experiment?

A
  • WE-RT: whole embryo minus RT

- co: animal cap without dissociation

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3
Q

How is cDNA made and used?

A
    1. isolate RNA
    1. make cDNA
    1. then take cDNA and do PcR with different primers
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4
Q

What exactly were the procedures in the experiment testing if wnt functions as a morphogen?

A
  • cDNA created and exposed to different levels of wnt and PCR primers
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5
Q

Why is the primer H4 a control?

A
  • it should be present in all
  • if a band does not show up, this tells you that the cDNA is not good
  • positive control
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6
Q

Why is the primer actin a control?

A
  • it is present in mesoderm and so should not be present here (except in we)
  • negative control
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7
Q

Why is the primer NCAM2 a control?

A
  • it is present in neural tissue and so should be present here except in co
  • positive control
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8
Q

What results do we see about wnt as a morphogen?

A
  • wnt does act as a morphogen because the neural tissue is responding to different levels of wnt
  • as levels of wnt increase, the primers expressed change from Bf1, Otx2, En2 to Krox20
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9
Q

What do morphogen gradients do in Xenopus?

A
  • pattern Xenopus dorsal/ventral and anterior/posterior axes development
  • A-P neural ectoderm: wnt gradient
  • D-V mesoderm: BMP gradient
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10
Q

What gradient doe nodal form?

A
  • high in future dorsal and low in future ventral
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11
Q

What are microRNA?

A
  • short non-coding RNA that regulate gene transcription
  • not just translational OFF switches, they also fine-tune and stabilize gene expression
  • keeps genes expressed at an appropriate level
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12
Q

What approach can be used when examining if microRNA have a role in gradients?

A
  • in silico search (i.e. bioinformatics) to identify putative miRNA binding sites on all known components of the Nodal signaling pathway
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13
Q

What was found with the in silico search?

A
  • miR15c/16c as potential regulators of type II receptor for Nodal and activin ligands: Acvr2a
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14
Q

What do we see with correlative data about miR-15/16?

A
  • all are enriched on the ventral side
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15
Q

What experiment was done to test if MiR-15/16 function? The result?

A
  • loss-of-function/knockout by injecting antisense

- resulted in an expanded region of chordin expression (which is a spemann organizer molecule) (and too much Xnr)

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16
Q

How is miR-15/16 functioning based on the results?

A
  • indicates that the miR-15/16 may be fine tuning the boundary of the organizer by limiting a receptor for TGF-B like factors
  • inhibits some Acvr2a receptors
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17
Q

What is the chicken and egg question here?

A
  • so miR-15/16 acts to fine tune the spemann organizer gradient
  • but what acts to establish the miR-15/16 gradient?
  • could be the wnt pathway since wnt signalling and B catenin is highest on the dorsal side (opposite)
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18
Q

What experiment was done to test if the wnt pathway establishes the miR-15/16 gradient? What does this indicate?

A
  • knockout of B-catenin results in overexpression of miR-15/16 and no expression of chordin
  • however, if we add anti-miR15/16 we get a rescue and see expression of chordin again
  • indicates that b-catenin inhibits miR-15/16
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19
Q

What is MO?

A
  • morpholino oligonucleotide

- used in knockdown experiments

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20
Q

What is a blastula in frogs similar to in mammals?

A
  • frog blastula similar to mammal embryonic epiblast

- when germ layers are starting to be defined

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21
Q

What experiment was conducted to examine if d/v and a/p axis formation is conserved in mammals?

A
  • when a mouse egg is fertilized, there is a fertilization cone
  • put a fluorescent bead in fertilization cone and track the position of the bead throughout early preimplantation development
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22
Q

What results are seen in the fluorescent bead experiment?

A
  • known that mammals have an asymmetric division starting at the 2nd cleavage meaning that one cell divides and then the next
  • with bead, can see that blastomere associated with sperm entry point tends to divide first
  • first evidence of “stereotypical” asymmetry
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23
Q

What is the embryonic epiblast?

A
  • a flat sheet of cells

- part of the epiblast embryo

24
Q

At 7 days, what components does the mammalian embryo have?

A
  • called a blastocyst
  • contains trophoblast
  • contains inner cell mass (hypoblast and epiblast)
  • has a cavity called blastocoel
25
Q

At 9 days, what components does the mammalian embryo have?

A
  • embryonic epiblast!
  • amnionic ectoderm
  • cytotrophoblast
  • syncytiotrophoblast
26
Q

What are extraembryonic tissues?

A
  • tissues that the embryo produces to survive but is not ultimately part of the embryo
27
Q

What fate mapping was done with chicks?

A
  • fate mapping of embryonic epiblast
28
Q

Where does gastrulation in amniotes intiate?

A
  • initiates in the posterior region of the epiblast
    (before gastrulation)
  • thickening area of blastoderm
  • primitive streak taking shape
29
Q

What is cell intercalation?

A
  • cell movement that underlies primitive streak formation prior to gastrulation
  • cells move up and outwards
30
Q

Where is the site of gastrulation for frogs? For mice?

A
  • frogs: dorsal lip

- mice: node and primitive groove

31
Q

What does the primitive streak form?

A
  • extends anteriorly forming primitive groove and node
32
Q

What are the mechanisms of cell movements?

A
  • ingression
  • invagination
  • involution
  • epiboly
33
Q

What kind of movement forms the primitive groove and node?

A
  • involution

- cells crawling along the inside walls away from each other

34
Q

Which cell types migrate in gastrulation (humans)?

A
  • endoderm and then mesoderm migrate during gastrulation to displace the hypoblast
35
Q

How can we observe the movement of the cells?

A
  • electroporate with GFP expressing plasmid

inject DNA to a very specific region to get a subpopulation of cells to track

36
Q

What does the amphibian dorsal blastopore lip do?

A
  • first tissue that envelopes and comes to underlie ectoderm and induces secondary axis
37
Q

How can we test if the amniote node is the anatomical equivalent of the amphibian dorsal blastopore lip? What does this indicate?

A
  • transplant the node in a chick to see if it makes a secondary axis
  • transplanted a duck Henson’s node (to be able to distinguish) and it induced a neural tube
  • indicates that Henson’s node is functionally equivalent to Spemann’s organizer
38
Q

What are the two distinct signaling centres of the mouse early gastrula?

A
  • node: equivalent of frog organizer

- AVE (anterior visceral endoderm): functional equivalent of frog anterior endomesoderm; extraembryonic

39
Q

Is the mechanism underlying the induction of the node similar to that of amphibians (wnt pathway)? (correlative data)

A
  • wnt3 is expressed in posterior visceral endodermal (PVE) and posterior epiblast region prior to gastrulation
  • the expression is consistent with the hypothesis that wnt induces organizer in mouse
40
Q

What happens when wnt is knocked out in mice?

A
  • used a conditional KO using epiblast specific promotor
  • wnt3-null mice undergo no gastrulation
  • means wnt3 is required for primitive streak formation
  • wnt3 function is required in epiblast but not in PVE
41
Q

What is AVE equivalent to in frogs?

A
  • anterior visceral endoderm equivalent to frog anterior endomesoderm (where cerberus, Dickkopf and Frzb function)
  • in mouse, this is extraembryonic
42
Q

What does AVE form from?

A
  • AVE forms from migration of cells from medial regions

- this can be seen using a transgenic mouse and AVE-specific promotor: GFP

43
Q

What was found to be expressed in AVE?

A
  • cerberus-like (cerl) is expressed in the AVE in graded manner but not in the epiblast
  • cerl: a cereberus homologue
44
Q

Why are knock-out studies more difficult in mice?

A
  • temperature shift experiment will affect the mother

- but still useful to do knockout studies

45
Q

What is the procedure for doing a knock-out study in mice?

A
  • start with embryonic stem cells in a dish
  • have gene of interest to target
  • use targeting construct that has homologous (left and right arm) called targeting vector to acquire plasmid
  • crossing over of dna at left arm and right arm with target dna resulting in deletion
  • implanted into female uterus
  • resulting mouse is chimera
  • mutation in egg or sperm and mutation can be carried on
46
Q

What results from cerk ko mutants?

A
  • no overt defects

- because of redundancy

47
Q

What factors does cerl effect?

A
  • inhibits nodal and BMP4 but not wnt

- this makes it different from cerberus

48
Q

What is another factor found to be expressed in AVE?

A
  • Lefty

- could account for redundancy found for cerl

49
Q

What results from lefty ko mutants?

A
  • no overt defects

- again redundancy

50
Q

What factors does lefty effect?

A
  • lefty inhibits nodal
51
Q

How would we knock out both cerl and lefty?

A
  • knock out each separately then breed the mice together to get a double knock out
52
Q

What do we see expressed in the wild type just prior to gastrulation compare to the double mutant mice?

A
  • goosecoid expression in posterior and slight in anterior
  • ectopic goosecoid expressing centre in the anterior region (and normal in posterior)
  • goosecoid specifies spemann organizer fate so we may see two axes
53
Q

What results with the double mutants?

A
  • have an ectopic secondary axis

- (Otx2 expressed anteriorly, T expressed posteriorly in both tails)

54
Q

What hypothesis do the double mutants support?

A
    1. nodal signaling specifies primitive streak formation
    1. AVE: crucial for correct A-P positioning by functioning as an anterior source of Nodal antagonists
  • cerl/lefty inhibit nodal which leads to no primitive streak
55
Q

What are the two distinct signalling centres molecular paths?

A
    1. node: nodal and wnt produce primitive streak

- 2. AVE: cerl/lefty inhibit nodal leading to no primitive streak

BIOL 309 (14 decks)

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