Early Vert Development (5) Flashcards
What does WE stand for?
- whole embryo
What are the controls in this experiment?
- WE-RT: whole embryo minus RT
- co: animal cap without dissociation
How is cDNA made and used?
- isolate RNA
- make cDNA
- then take cDNA and do PcR with different primers
What exactly were the procedures in the experiment testing if wnt functions as a morphogen?
- cDNA created and exposed to different levels of wnt and PCR primers
Why is the primer H4 a control?
- it should be present in all
- if a band does not show up, this tells you that the cDNA is not good
- positive control
Why is the primer actin a control?
- it is present in mesoderm and so should not be present here (except in we)
- negative control
Why is the primer NCAM2 a control?
- it is present in neural tissue and so should be present here except in co
- positive control
What results do we see about wnt as a morphogen?
- 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
What do morphogen gradients do in Xenopus?
- pattern Xenopus dorsal/ventral and anterior/posterior axes development
- A-P neural ectoderm: wnt gradient
- D-V mesoderm: BMP gradient
What gradient doe nodal form?
- high in future dorsal and low in future ventral
What are microRNA?
- 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
What approach can be used when examining if microRNA have a role in gradients?
- in silico search (i.e. bioinformatics) to identify putative miRNA binding sites on all known components of the Nodal signaling pathway
What was found with the in silico search?
- miR15c/16c as potential regulators of type II receptor for Nodal and activin ligands: Acvr2a
What do we see with correlative data about miR-15/16?
- all are enriched on the ventral side
What experiment was done to test if MiR-15/16 function? The result?
- 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)
How is miR-15/16 functioning based on the results?
- 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
What is the chicken and egg question here?
- 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)
What experiment was done to test if the wnt pathway establishes the miR-15/16 gradient? What does this indicate?
- 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
What is MO?
- morpholino oligonucleotide
- used in knockdown experiments
What is a blastula in frogs similar to in mammals?
- frog blastula similar to mammal embryonic epiblast
- when germ layers are starting to be defined
What experiment was conducted to examine if d/v and a/p axis formation is conserved in mammals?
- 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
What results are seen in the fluorescent bead experiment?
- 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
What is the embryonic epiblast?
- a flat sheet of cells
- part of the epiblast embryo
At 7 days, what components does the mammalian embryo have?
- called a blastocyst
- contains trophoblast
- contains inner cell mass (hypoblast and epiblast)
- has a cavity called blastocoel
At 9 days, what components does the mammalian embryo have?
- embryonic epiblast!
- amnionic ectoderm
- cytotrophoblast
- syncytiotrophoblast
What are extraembryonic tissues?
- tissues that the embryo produces to survive but is not ultimately part of the embryo
What fate mapping was done with chicks?
- fate mapping of embryonic epiblast
Where does gastrulation in amniotes intiate?
- initiates in the posterior region of the epiblast
(before gastrulation) - thickening area of blastoderm
- primitive streak taking shape
What is cell intercalation?
- cell movement that underlies primitive streak formation prior to gastrulation
- cells move up and outwards
Where is the site of gastrulation for frogs? For mice?
- frogs: dorsal lip
- mice: node and primitive groove
What does the primitive streak form?
- extends anteriorly forming primitive groove and node
What are the mechanisms of cell movements?
- ingression
- invagination
- involution
- epiboly
What kind of movement forms the primitive groove and node?
- involution
- cells crawling along the inside walls away from each other
Which cell types migrate in gastrulation (humans)?
- endoderm and then mesoderm migrate during gastrulation to displace the hypoblast
How can we observe the movement of the cells?
- electroporate with GFP expressing plasmid
inject DNA to a very specific region to get a subpopulation of cells to track
What does the amphibian dorsal blastopore lip do?
- first tissue that envelopes and comes to underlie ectoderm and induces secondary axis
How can we test if the amniote node is the anatomical equivalent of the amphibian dorsal blastopore lip? What does this indicate?
- 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
What are the two distinct signaling centres of the mouse early gastrula?
- node: equivalent of frog organizer
- AVE (anterior visceral endoderm): functional equivalent of frog anterior endomesoderm; extraembryonic
Is the mechanism underlying the induction of the node similar to that of amphibians (wnt pathway)? (correlative data)
- 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
What happens when wnt is knocked out in mice?
- 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
What is AVE equivalent to in frogs?
- anterior visceral endoderm equivalent to frog anterior endomesoderm (where cerberus, Dickkopf and Frzb function)
- in mouse, this is extraembryonic
What does AVE form from?
- AVE forms from migration of cells from medial regions
- this can be seen using a transgenic mouse and AVE-specific promotor: GFP
What was found to be expressed in AVE?
- cerberus-like (cerl) is expressed in the AVE in graded manner but not in the epiblast
- cerl: a cereberus homologue
Why are knock-out studies more difficult in mice?
- temperature shift experiment will affect the mother
- but still useful to do knockout studies
What is the procedure for doing a knock-out study in mice?
- 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
What results from cerk ko mutants?
- no overt defects
- because of redundancy
What factors does cerl effect?
- inhibits nodal and BMP4 but not wnt
- this makes it different from cerberus
What is another factor found to be expressed in AVE?
- Lefty
- could account for redundancy found for cerl
What results from lefty ko mutants?
- no overt defects
- again redundancy
What factors does lefty effect?
- lefty inhibits nodal
How would we knock out both cerl and lefty?
- knock out each separately then breed the mice together to get a double knock out
What do we see expressed in the wild type just prior to gastrulation compare to the double mutant mice?
- 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
What results with the double mutants?
- have an ectopic secondary axis
- (Otx2 expressed anteriorly, T expressed posteriorly in both tails)
What hypothesis do the double mutants support?
- nodal signaling specifies primitive streak formation
- 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
What are the two distinct signalling centres molecular paths?
- node: nodal and wnt produce primitive streak
- 2. AVE: cerl/lefty inhibit nodal leading to no primitive streak
