9. In vitro models of patterning Flashcards
What are the stem cell lines derived from a blastocsyst?
ESCs - from epiblast
TSCs - from TE
XEN (Extraembryonic endoderm) - from PrE
Questions answered in this lecture
- What are the mechanisms leading sorting of different cell populations upon aggregation in vtiro?
- What are the mechanisms enabling formation of cavity?
- Signalling dynamics and tissue mechanis in developmental systems
Explain what are micropatterns
Micropatterns - self-organised dveelopmental models in 2D - deposition of EXM in controlled manner - studying stem cell patterning in vitro in chosen conditinos
What are the advantages of 3D in vitro culture method in comparison to 2D?
Cells gain 3D interactions with EXM - can model developmet rather than only cell interactions - more potent for studying larger scale development
What lead researchers to believe that embyronic cell lines know how to organise them to form embyro like structures in vitro?
When ESCs + TSCs + XEN aggregated - they self-organised like in embryos - TSCs arranged outside - ES inside w/o any external signal to guide them
Lead to questions: what are the mechanisms leading to cell sorting and formation of boundries?
What were the first experiments done to study embryo dissociation and reaggregation?
Townes and Holtfreter 1950s dissociated amphibian embryos into single cells with high pH / trypsin- reaggregated in vitro -> cells were able to distinguish between self and non-self => proposed the hypothesis of different cell affinities for each other
What were the two proposed cell adhesion hypotheses for cell sorting in embryo cell reaggregation experiments?
Cell sorting hypotheses:
- Differential cell adhesion hypothesis (DAH) wrong
Steinberg 1970 proposed that depending on cell surface molecules cells have different adhesion abilities which leads to sorting
- Differential interfacial tension hypothesis (DITH) correct
Harris 1976 proposed that different affinities between cells arise due to cortical tensions within cells
Mina idea - cell sorting is the result of energy minimisation process - if cells come together free E released - more stable
Explain how adhesion strength and contact tension cooperate in cell adhesion
In cell adhesion: adhesion strength = contact tension - cadherins on cell surface interact like hooks - if tension applied - adhesion becomes stronger - can withstand more
Notes from the slide:
- Adhesiveness is largely due to repression of cortical contractility at cell contact - the contribution of adhesive E is relatively marginal (207% in Zebrafish gastrula cells)
- Cadherins are adhesive molecules that recruit cytoplasmic components responsible for local respression or cortical tension. Other molecules like Ephrins can elicit an increase in local tension and therefore induce repulsion
How differential interfacial tensions hypothesis (DITH) was tested?
Krieg et al. determined that cell sorting was dependent on cortical tensions - not cadherin levels (disproved DAH):
- cadherins were quantified in Zebrafish gastrulation embryos
- force microscopy performed on isolated germ layers - press on cells with lever - see how stiff (calculate using equations)
=> found Ect highest cortical tension, Meso lower, End lowest cortical tension
-germ layer lineages labelled - Meso + Ect cells mixed - self-organised - lower cortical tension Meso surrounded higher tension Ecto (inside) - more stretchy surrounded more stiff cells
What were the results of Krieg et al. investigating DITH theory?
Results were in line with DITH theory: cortical tension determines cell sorting (not adhesion molecules), germ layer cells have different cortical tensions, Ecto>Meso>Endo cortical tension
What is the fault in the logic between DITH theory and embryo development?
Arrangement of cortical tension based germ layer cell arrangement is reversed in embryo development
Epithelial embryo coating
Explain how epithelial coating controls tissue position in embryos
Epithelial layer - the most outer in embryos - creates asymmetry by polarization of epithelial layer and non-adhesive apical surface
Epithelial coating controls tissue positioning and permits elongation - tested in experiment - w/o epithelium A/P mesoderm engulf and tissue becomes spherical instead of elongation - with ectodermal layer cells maintain serial arrangements and tissue elongates -> same found out when modelled in silico
What were the two considered mechanisms for cavity formation in blastocysts?
Mechanisms for cavitation:
- apoptosis driven cavitation: cell divivions - induction of apoptosis in inetrnal cells - clearance of apoptotic cells -> cavity
- polarization driven hollowing: polar transport of cellular organelles -> fluid filled lumen -> tight junctions
In which culture method cavity can form in stem cell models?
Only in 3D - need spatial organisation in EXM - mimics organisation and maturation of epithelail cells - formation via hollowing observed
What other developmental events coincide with cavity formation in vivo?
Cavity formation in vivo coincides with:
- basement membrane formation
- epithelial maturation
How does cavity form in peri-implantation mouse embryo?
Through hollowing, forming a rossette in epiblast - forming a fluid filled cavity within the rossette - apical-basal domain polarization - dusion with ExE forming lumen
How does the cavity form from morula to blastocyst stage?
- No basal lamina to use as starting point
- Cavity always forms at the interface between ICM and TE
Mechanism: little cavities form all over -> TE expresses tight junctions -> little cavities merge at ICM-TE interface
Experiment to prove small lumens fusing into big one in morula-blastocyst cavity formation
Dumortier et al. 2019 observed embryos under light sheet microscopy - observed microfractures in between cells due to hydraulic pressure -> microlumens -> which later merge
Explain what is the theory behind how micro-lumens fuse into one lumen
Direction of fluid flow of micro-lumens is dictated by tensions asymmetry and area asymmetry
Otswald ripening effect - 2 lumen will fuse when:
- one with higher tension will move to fuse with another with lower tension
- one with smaller area will move to fuse into higher area
=> final merged lumen in between higher-lower cortical tension cells (TE-ICM)
What experiment was done to determine if cell tension or adhesion drives cavity formation?
Dumortier et al. 2019 E-Cad KO embryos: maternal Cdh1 KO -> embryo comapred to WT - changes organisation but lumen still forms => not cell adhesion drives cavity formation
Adhesion and cortical tension affect cell fate but not cavity formation
What experiment was done to evaluate if contractivity affects cavity formation?
Dumortier et al. 2019 produced maternal non-muscle myosin (Myh9) KO - embryos were less contractile than WT -> mechanical tension is necessary for embryo organisation
Idk what’s wrong in fluorescent image
What is the biggest factor in guiding patterning in human embryonic stem cells in 2D
Spatial confinement guides patterning - where cell is located - middle vs edge
How do cells in micropatterns respond to BMP signalling?
Over time BMP signalling activity organises in a radial gradient - lowest in the middle - highest at the outer edge
After 1h: all cells respond to BMP - active SMAD
After 24h: only outer edge cells respond to BMP - active SMAD
How does BMP signalling gradient activity organise itself in a radial gradient within cells ina micropattern?
A secondary signal is secreted by inner cells to antagonise BMP - Noggin and Chordin
Bra still observed when Noggin and Chrodin inhibited -> further looked into the infleucne of cell density on BMP inhibition
Idk how Bra and Cdx2 meant to interact with BMP
What is the main determinant in the cells ability to respond to BMP in micropatterns
Spatial organisation of the cell - where/how it is located in relation to other cells:
- in the middle BMP inhibitors NOggin and Chordin expressed - inibit BMP
- at high cell density not all cells respond to BMP but at low levels do
How come cells in the middle of the colony in micropatterns respond to BMP differently?
Etoc et al. 2016 study:
Due to higher cell density in the middle of the colony the surface receptos that respond to BMP are hidden - BMP can’t bind -> no signal
Cells at the outer edges - lower cell density - their sruface receptors more exposed -> BMP can bind and induce a response
What was the experiment done to show different cell basal vs apical sensitivities to ligands?
Cell were grown on filters - BMP4 introduced from top / bottom -> SMAD downstream of BMP4 activity measured
=> cells respond to BMP4 when provided from bottom - cell internal asymmetry makes response different
Read Etoc et al. 2016 paper
How was the importance of cell polarity in infleuncing cells competence to respond to BMP signalling tested in vivo?
In vivo test for improtance of cell polarity in cells competence to repsond to BMP siganlling - tested by apical basal staining in transverese sections (??) read the paper to understand
How is the BMP gradient within embryo translated into different cell fates?
French flag model - positional information model - based on gradient thresholds different fates arise
But when investigated response to BMP levels - observed On/Off phenotype instead of gradient dependent (french flag) -> BMP levels affect Sox2, Cdx2 On/Off at high adn low levels - but doesn’t induce anything at middle levels - Bra induction never emerged from middle BMP levels => What causes Bra expression? - need presence of secondary signal
What is the secondary signal beyond BMP which drives Bra expression?
BMP4 -> Nodal -> Bra
BMP doesn’t act as a morphogen (not french flag) - all three cell fates emerge at BMP yes (high BMP) -> Cdx2, no BMP (low BMP) -> Sox2, and BMP+Nodal -> Bra
Secondary Nodal signal is necessary for mesoderm fate emergence
How do signals from BMP and Nodal interact?
Nodal and BMP signals converge at Smad4 activity - but still achieve distinct outcomes becasue bind different second protein (Smad1/ Smad2) to Smad4
How do BMP and Nodal achieve distinct outcomes if they both work via Smad4?
How does Nodal change in self-organised patterning?
Rapid change in edogenous Nodal during patterning - low in the middle - higher on the outside => depending on cell localisation within the colony - BMP4 and Nodal experienced at different conc => signalling dynamics in patterning
How does neuroectoderm patterning form in micropatterned cell colonies?
Depending on BMP acitvity -> neural plate (low), neural plate border (NPB) (medium), epidermal ectoderm (NC cells) (high)
What has been proposed to induce mechanical force influencing cells response to signalling molecules?
Tissue scale morphogenesis infleunces cells abilities to respond to signalling molecules - different levels of stretch experienced by cells, for example, in folding of ectodermal epithelium - based on sretch experienced dteermines their ability to respond to BMP
Can be studeid by traction force microscopy - method to measure cell generated pulling force
Explain the experiment used to study traction forces within colonies
Traction forces of cells at different parts of the olony were studied by traction force microscopy - observed that traction forces are greater at the periphery than centre of the colony
KO of acto-myosin contractility abolishes gradient of traction forces
What is the link between acto-myosing contractility and cell fate?
pSmad1 activity (downstream of BMP) is conditional on:
- contractility (cell shape / stretching)
- cell density
Explain how pSMAD1 activity was tested if it was conditional on cell shape / stretching
Experiment: microfluidic cells were used - cells placed on top - control air pressure - stretch cells on the surface - increase in cell tension, cell shape changes, stretches -> pSMAD1 (downstream of BMP) becomes active
Key lecture messages
