Gastrulation occurs in an egg cylinder in mice. The ectoderm is separated in the proximal-posterior region of the embryo through the primitive streak and two wings of mesoderm. There is a node at one end where gastrulation will occur.
Summarise gastrulation in the mouse.
The definitive endoderm is formed through gastrulation. The mesoderm ingresses through the primitive streak.
The definitive endoderm is specified within the mesoderm population. This passes through the basement membrane to intercalate with visceral endode
How are the three germ layers specified in gastrulation?
Viotti et al., 2014 show FoxA2 marks mesoderm
Sox17 markst hose cells that will make definitive endoderm. This is done with immunohistochemistry.
When cells hit the ECM they acquire the definitive endoderm layer.
What markers are there for mesoderm and endoderm? Who shows this?
This is the combination of mesoderm and endoderm before placement. Some scientists don't like it.
Hadjantonakis -- Sox17 is required to produce definitive endoderm from bipotent mesoderm.
How this is done is unknown
What is known is that leaving streak ex
What is the mesendoderm? Is it a useful term?
Neurectoderm does not transit through the streak.
Neural specification occurs differently. Anterior neural tissue forms from the epiblast that does not transit through the streak.
The embryo then elongates to form posterior structures and the tail.
How does neural specification occur anteriorly and posteriorly?
There is a niche that appears to harbour stem/progenitor cells that populate extending posterior.
What happens at the node/streak border at the chordoneural hinge (CNH)?
Sox2 is a marker of early epiblast.
Brachyury is marker of primitive streak and nascent mesoderm.
In egg cylinder, these expression do not overlap.
What type of stem cells are in the neuroectoderm? What are
Wymeersch et al.
Position-dependent plasticity of distinct progenitor types in the primitive streak.
Bipotential cells -- some cells at the Node-streak border containing Brachyury AND Sox2 -- groups of cells after the non-overlap -- (E7 to E7.5).
These pe
What is special about markers in the nod streak border? Who shows this?
Cambray and Wilson, 2002
Axial progenitors with extensive potency in the chordoneural hinge.
Why?
Grafts cells from primitive streak or tail bud of mouse embryos results in various tissues.
Axial elongation probably requires stem cell population.
Transpla
How are mesoderm and neural lineages segregated?
Who shows this? What motivated this study? (the latter is the main question)
1) Grafting experiments from one to another -- but very technically demanding
2) Grafted embryos need to be ex utero -- can sustian development for a couple of days
3) Serial transpalntations required
How can potency and stem cell-ness be tested?
Cells labelled in the node contribute to notochord, not mesoderm. This tests for fate, but not for potency.
Grafted anterior streak contributes to posterior neurecotderm and somites.
Posterior neurectoderm cells can contribute to mesoderm even after poste
What are the results of neural and mesodermal tissue potency experiments?
Look for markers of host tissue not formally expressed in donor graft?
Look for evidence of cell division and lineage expansion
Functional test -- not possible in context of ex vivo
Grafted cells from CNH do end up having turning no markers of tissue they
How can we determine if incorporating donor cells are functional?
- single cell transplantations are inefficient and tricky/demanding
- serial transplantatiosn e.g. blood field = haemangioblasts
- repeatedly graft CNH tissue into node/streak border
- label donors with inert fluorescent marker
Serial grafting supports ex
How can bipotent cells be verified as stem cells?
Tzouanacou et al., 2009
Who redefined the lineage segregations by clonal analysis?
Though bipotential stem cells could be serially passaged, proof of origin was missing.
Generate labelled cells using spontaneous LacZ gene carrying inactivating sequence duplication to active LacZ -- rare inorganic homologous recombination. ROSA26 inactiv
Describe further experiments to look at bipotential stem cells.
- overrepresentation of 2, 4, 8, 16 multiples suggest regular cycling -- progenitors have self-renewing characteristics
- neurectoderm and mesoderm share common precursor that persists through axial elongation
- challenge paradigm that three germ layers v
What were the results of clonal analysis using rare LacZ staining?
Gouti et al.
Wnt signalling may specify -- with CHIR (Wnt agonist) more Brachyury and Sox2, whereas lack of Wnt agonism causes no Brachyury -- thus for neurectoderm specification
What signalling might define neuromesodermal progenitors?
Presence of labelled cells in node/streak in E9.5
Extension of proportion of clones to posterior neuropore
Increase in proportion of clones with N/M increased in 10.5 -- persistence
Small N/M clones found supporting bipotent activity
Loss of posterior lab
What evidence is there that there is stem cell like behaviour in node/streak region?