Regulation of Endogenous SC Population Flashcards
Purpose of Pre-implantation Development
Produce an implantation competent blastocyst containing:
- Differentiated outer trophectoderm capable of pumping fluid into the blastocyst cavity, interacting with the uterus for implantation and producing the placenta.
- Aggregate of ESCs in the ICM
Blastocyst Formation
- Compaction of the 8 cell embryo to form the morula, where individual cells are no longer evident.
- Increase in cell adhesion dependent on Ca2+ and polarisation - outer cells become polarised, inner surface has contact. Becomes trophectoderm. Inner cells are not polarised. Have contact all round. Becomes ICM.
- Cavitation occurs at the 30 cell stage to form the blastocyst.
What are the 2 inhibitors used to maintain the naive mESC state?
- ERK pathway inhibitors.
- Glycogen Synthase Kinase inhibitors - demonstrates that activation of the Wnt pathway sustains the expression of pluripotent TFs Oct-4, REX1 and Nanog in the absence of supplemented LIF.
Epiblast Lineage Markers
Nanog, Sox-2, Oct-4
Primitive Endoderm Lineage Markers
Gata4, Gata6, Sox17, Sox7
Trophoblast Lineage Markers
CDX2, Eomes, Gata3
What does ‘salt and pepper’ describe
The fact that, after the first cell fate and prior to the second cell fate, cells of the ICM are not sorted, and express epiblast and primitive endoderm markers in a random ‘salt and pepper’ fashion.
In the first cell fate decision - how do the inside cells become ICM?
− AMOT binds to apical junctions via Nf2, activating the Lats 1/2 kinases
− Active Lats1/2 phosphorylate Yap, resulting in exclusion of Yap from the nucleus.
− Without Yap, the Hippo TF, TEAD4 cannot induce expression of Cdx2.
− In the absence of Cdx2, Oct-4 is upregulated
In the first cell fate decision - how do the outside cells become trophectoderm?
− Cell polarity sequesters AMOT to the apical protein complex, it cannot bind to the apical junction via Nf2, so does not activate the Lats1/2 kinase
− Yap is not phosphorylated, so Yap is transported into the nucleus where it acts as a co-factor for TEAD4.
− TEAD4 drives the expression of Cdx2
− Cdx2 suppresses the expression of Oct-4
Outside cells are thought to additionally be exposed to polarity signals – keeps them polar to maintain apical and basolateral domain!! O
How is the second cell fate decision regulated?
Prior to the 32 cell stage, the Epi marker Nanog and the PreE marker Gata6 are coexpressed Gata6 expression is FGF4 independent but Mek-dependent at this stage. Segregation of the cells occurs from the 32 cell stage onwards:
− In Epi cells, Nanog inhibits Gata6 expression
− In FGFR-2 expressing PreE cells, FGF4 (supplied by the Epi cells) inhibits Nanog via Mek, therefore strengthening Gata6 expression.
− Oct-4 is activated in a Mek dependent manner in PE cells
− Oct-4 and Gata6 are needed for expression of downstream PE specific TFs – Pdgfra, Sox17 and Gata4.
− PE cells are then sorted into a layer lining the blastocyst cavity. They are no longer sensitive to FGF signaling, and express Sox7 in a Gata-6 and Oct-4 dependent manner.
Explain how stochastic gene expression helps with the second cell fate decision?
All cells will be supplied with equal amounts of FGF4. However, there are small differences in the expression of FGFR2 - this is random. Those cells with higher FGFR2 will respond to FGF4, and commence the signalling that results in them becoming PrE cells (inhibition of Nanog and strengthening of Gata6).
What results in transformation from the early to the late epiblast stage?
Nanog essential for formation of naïve epiblast is downregulated via ERK signaling. This shifts it to late epiblast.
Also speculated that in the absence of ERK activation, naïve epiblast can pass through a trophoblast precursor state, that becomes reinforced by positional signals
What results in transformation from the PrE to the parietal and visceral endoderm?
ERK signaling results in downregulation of Oct-4.
