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.
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
1-cell 16-cell8-cell
Post-implantation
Fate
Shape
Position
Extra-embryonicPluripotent
>32-cell
Early Mouse Embryo
Mouse
?
C elegans
Cell Fate
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
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
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
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
filopodia
compaction
Filopodia Grow when Cells Elongate
MembraneChromatin
Filopodia Retract when Cells Divide
RetractExtend Division
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
Cancer Cell Migrating CellNeuron
Pluripotent Inner Mass
Cell Position
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)
E-cad
E-cad
Actin-myosin
Mechanical Forces?
GFP-Myosin II
division
How to Measure Tension?
Tension
x
XXXXx
Pre ablation / Post ablationUtrophin-GFP
XXXXx
Pre ablation / Post ablationUtrophin-GFP
Out
In
OutOut
Cell Polarity Model‘…polarity is asymmetric
inherited…’
Apical capF-actin
Tension
0 min 10 min 15 min 30 min
Apical cap
Cortical Flows Form Actin Rings De Novo
F-actin
F-actin rings
F-actin
Outside ringAt ring
Inside ring
Actin Rings Expand
Cell (2018)
t = 0 hs
t = 24 hs
Actin Rings Zipper Along Junctions
F-actin
Zippering Forms Tight Junctions
GFP-ZO1 / RFP-Utrophin
Cell (2018)
Cell (2018)
How Microtubules Organize In Vivo?
Centrosome
MTOC
Many cells Mouse embryo
DNAa-tubulin Microtubule bridge
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
CAMSAP3-GFP
t = 0 min
t = 30 min
t = 120 min
Microtubules
Vescicles
Microtubule bridge
E-cad-GFP
Bridge microtubules Cell
membrane
E-cad-GFP
Control
CAMSAP3 KD
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)
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
