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