09.30 Nicolas Plachta Plenary - ASRM...Microsoft PowerPoint - 09.30 Nicolas Plachta Plenary Author:...
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Using Advanced Live Imaging to Visualize How the Embryo
Forms in Real Time
Nicolas PlachtaA*STAR, Singapore
LEARNING OBJECTIVES
● Describe how imaging techniques can be used to reveal mechanism controlling embryonic development.
● Identify key dynamic processes and morphogenetic events patterning the early mammalian embryo.
At the conclusion of this presentation, participants should be able to:
DISCLOSURE
● I have no commercial and financial relationships with commercial or financial interests and/or other relationships with manufacturers of pharmaceuticals, laboratory supplies, and/or medical devices.
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Imaging the Dynamics that Formthe Preimplantation Embryo
A ★S T A R
Institute of
Molecular &
Cell Biology
Nicolas Plachta
The Challenge
1-cell 16-cell8-cell >32-cell
Early Mouse Embryo
Human embryo
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1-cell 16-cell8-cell
Post-implantation
Fate
Shape
Position
Extra-embryonicPluripotent
>32-cell
Early Mouse Embryo
Mouse
?
C elegans
Cell Fate
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Oct4 Sox2 Nanog
Cdx2 Eomes Tead4 Gata4/6
Transcription Factors & Cell Fate
Oct4 protein levels
Levels & Lineages Uncorrelated
TF Function
Levels
Dynamics
Plachta et al.Nat Cell Biol (2011)
TF–DNA Binding Model
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Oct4-paGFP
Photo-Activatable FCS
Time
Flu
ctu
atio
ns
Correlation time
Co
rrel
ati
on
Diffusion
Fractions
Brownian
ACF curve
volume < 1 fL
Kaur et al. Nature Communications (2013)Zhao et al. Nature Protocols (2017)
TF–DNA Dynamics In Vivo
White et al.Cell (2016)
Fit
3D diffusion
Non-specific
Specific
Sox2
White et al.Cell (2016)
First Differences at 4-Cell Stage
Oct4
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Sox2–DNA Binding Predicts Fate
More Sox2–DNALess Sox2–DNA
4-cell 16-cell
White et al.Cell (2016)
New ways to reveal TF–DNA binding in vivo
Histone methylation controlsTF–DNA binding
TF–DNA binding predicts fate
summary
Embryo Compaction
Cell Shape
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E-cad–GFP
10 μm
Filopodia in Living Embryo
Fierro-Gonzalez et al.Nat. Cell Biol. (2013)
45 min
5 μm10 μm
Filopodia in Living Embryo
E-cad–GFP
Fierro-Gonzalez et al.Nat. Cell Biol. (2013)
SEM
Filopodia in Living Embryo
Fierro-Gonzalez et al.Nat. Cell Biol. (2013)
F-actin Membrane
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filopodia
compaction
Filopodia Grow when Cells Elongate
MembraneChromatin
Filopodia Retract when Cells Divide
RetractExtend Division
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Pre
5 μm
Filopodia Ablation Releases Tension
Post
2D
live
em
bry
o
E-cad Controls Filopodia, Shape & Compaction
Control E-cad siRNA
Inje
cted
ce
lls
Filopodia control compaction
In vivo system to study filopodia
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Cancer Cell Migrating CellNeuron
Pluripotent Inner Mass
Cell Position
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Spatially Orientated Divisions
inside
outside
DIC memb-mCherry
Filtered
Membrane Segmentation
Apical Constriction Forms Inner Mass
AsymmetricDivision
Apical Constriction
Samarage et al. Dev. Cell (2015)
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E-cad
E-cad
Actin-myosin
Mechanical Forces?
GFP-Myosin II
division
How to Measure Tension?
Tension
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x
XXXXx
Pre ablation / Post ablationUtrophin-GFP
XXXXx
Pre ablation / Post ablationUtrophin-GFP
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Out
In
OutOut
Cell Polarity Model‘…polarity is asymmetric
inherited…’
Apical capF-actin
Tension
0 min 10 min 15 min 30 min
Apical cap
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Cortical Flows Form Actin Rings De Novo
F-actin
F-actin rings
F-actin
Outside ringAt ring
Inside ring
Actin Rings Expand
Cell (2018)
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t = 0 hs
t = 24 hs
Actin Rings Zipper Along Junctions
F-actin
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Zippering Forms Tight Junctions
GFP-ZO1 / RFP-Utrophin
Cell (2018)
Cell (2018)
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How Microtubules Organize In Vivo?
Centrosome
MTOC
Many cells Mouse embryo
DNAa-tubulin Microtubule bridge
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DNAa-tubulin
Zenker et al. Science (2017)
Microtubules
Microtubules / Chromatin
Prophase Metaphase Telophase Cytokinesis Interphase
Microtubules
2–cells 4–cells 8–cells 16–cells
Zenker et al. Science (2017)
EB3-GFP
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CAMSAP3-GFP
t = 0 min
t = 30 min
t = 120 min
Microtubules
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Vescicles
Microtubule bridge
E-cad-GFP
Bridge microtubules Cell
membrane
E-cad-GFP
Control
CAMSAP3 KD
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Sister cells connected by bridge
Bridge is a non-centrosomalMTOC
MTOC directs transport
summary
Shape
Nat Cell Biol (2013)
Science (2017)
Filopodia Compact the Embryo
MTOC Polarizes Cells
Fate
Cell (2016)Biophys J (2015)
TF–DNA Dynamics
No
rma
lize
d G
(t)
t (ms)
3D diffusion
SpecificNon-specific
Fit
Specific binding
Non-specific binding
Nat Commun (2013)Nat Cell Biol (2011)
Position
Dev Cell (2015)
Mechanical Regulation
Actin Zippers Embryo
Cell (2018)
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Gastrulation Live
100 μm
Stephanie Bissiere Oz PompGrace Lim
Yanina Alvarez
Maxime Gasnier
Piotr Tetlak Melanie White
A ★S T A RSingapore
Institute of
Molecular &
Cell Biology