“Crowd Control” Turning Cells into Organs via Collective Migration
“Crowd Control” Turning Cells into Organs via Collective Migration
“Crowd Control” Turning Cells into Organs via Collective Migration
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<strong>“Crowd</strong> <strong>Control”</strong><br />
<strong>Turning</strong> <strong>Cells</strong> <strong>into</strong> <strong>Organs</strong> <strong>via</strong> <strong>Collective</strong> <strong>Migration</strong>
What is developmental biology?<br />
Developmental Biology tries to answer the first scientific question that many<br />
children ask……..<br />
Where do BABIES come from?
Morphogenesis: “the origin of shape”<br />
Embryos are not made of<br />
homogeneous matter that is<br />
‘sculpted’ <strong>into</strong> shape
We are made out of cells<br />
Number of people on earth = 6 000 000 000<br />
Number of cells in a person = 10 000 000 000 000 000<br />
<strong>Cells</strong> come in many shapes and sizes for different func7ons<br />
Nerve <strong>Cells</strong>:<br />
transmit electrical signals<br />
Red Blood <strong>Cells</strong>:<br />
Carry oxygen<br />
Macrophages:<br />
Eat invaders/dying cells
http://multimedia.mcb.harvard.edu/
Subcellular-scale<br />
Morphogenesis<br />
Morphogenesis is process by which cells/tissues/organs are shaped.<br />
Shape depends on dynamic interactions at different scales.<br />
Cell-Scale<br />
Tissue-Scale<br />
Many tissues are shaped through the directed migration of constituent cells.<br />
Cell migration is a ‘shape’ problem that spans all three levels of scale.
Dicty movie from Firtel<br />
lab (UCSD)<br />
Single cells can navigate using graded chemical cues<br />
needle emitting<br />
chemoattractant<br />
(cAMP)<br />
How are migrating cells guided in vivo?<br />
Motile Cell<br />
(D.discoideum)
<strong>Cells</strong> in their natural habitat prefer to migrate in groups<br />
Migrating collectives come in different forms - sheets, chains, clusters, branched networks.<br />
Most are considered to be chemotactic.<br />
Challenge: Demands visualisation and manipulation of dynamic cell behaviours in vivo.
Zebrafish allow the study of dynamic cell interactions in intact embryos<br />
Zebrafish<br />
4cm long, grown at high density.<br />
200-400 embryos/female/week (=1000s of embryos/day).<br />
Embryos completely transparent.<br />
Genetic, pharmacological and light-based manipulation.<br />
Kane & Karlstrom
Imaging cellular and molecular dynamics in living tissues<br />
GFP: Green Fluorescent Protein
What is it?<br />
The zebrafish lateral line: an in vivo model for collective migration<br />
Mechanosensory hair cell organs deposited by migrating epithelium (RG Harrison, 1906).<br />
lateral line in action (BBC Blue Planet)
What is it?<br />
The zebrafish lateral line: an in vivo model for collective migration<br />
Transgenic : mGFP fused to claudin B promoter (cldnb:gfp)<br />
Mechanosensory hair cell organs deposited by migrating epithelium (RG Harrison, 1906).<br />
Top view, 10x/0.25 NA<br />
frame every 4 mins<br />
multi-tasking - cells migrate, grow, divide, differentiate and change shape simultaneously.<br />
“Embryogenesis in a nutshell” or “An animal inside an animal”
<strong>Collective</strong> migration :<br />
a(nother) link between morphogenesis and cancer<br />
Zebrafish lateral line primordium<br />
- Migrating epithelium with invasive ‘tip’<br />
CXCR4b mRNA SDF1a mRNA<br />
Human mammary carcinoma (MCF-7)<br />
- Migrating epithelium with invasive ‘tip’<br />
(Friedl and Gilmour, Nature Reviews MCB 2009).<br />
Primordium guided by same chemokine-receptor (CXCR4-SDF1)* as many human tumors.<br />
*David et al 2002
Wild Wild Type Type<br />
(WT) (WT)<br />
How do signals control tissue migration?<br />
CXCR4/SDF1 signaling guides tissue migration<br />
Cxcr4/SDF1-deficient<br />
Cxcr4 Cxcr4<br />
mutant mutant<br />
Key Unkowns That Can Be Addressed<br />
How do guidance signals guide tissues?<br />
How is the response to cues regulated collectively?<br />
Do cells within groups guide each other? If so, how?<br />
Tissue guidance can be mediated non-autonomously by a few leader cells.<br />
wt cxcr4 mutant
Tissue Polarity/Directionality Requires Constant Cxcr4/SDF1 Signaling<br />
2 wt cells<br />
Ablate guiding tip<br />
change text to something<br />
more focussed on<br />
polarity/migration<br />
Tissue is unstable, quickly reverting to Cxcr4b mutant phenotype upon leader ablation.<br />
One Cxcr4b expressing cell is able to reverse tissue assembly and rescue migration.<br />
How does the chemokine path select and control leader cells?<br />
wt cxcr4 mutant
What determines the directionality of LL migration?<br />
SDF-1a mRNA<br />
SDF1a mRNA<br />
mcherrySDF1a<br />
SDF1a<br />
mcherrySDF1a
The primordium treats the stripe of SDF-1a as a “two way street”<br />
fused somites (fss)<br />
SDF1a<br />
SDF1a mRNA<br />
12% (27/226)<br />
Finding: SDF1 is unlikely to be present in a pre-patterned head-to-tail concentration gradient.<br />
Question: How do tissues ensure directional persistence in absence of gradients?<br />
Challenge: How can we see what the tissue ‘sees’?
Organ assembly is the biological function of the primordium<br />
Stable epithelium Migrating epithelium<br />
Mesenchyme<br />
Adhesion<br />
γTubulin-cherry cldnB-GFP<br />
How are organ assembly and migration coupled?<br />
Motility
FGF ‘foci’ nucleate epithelial organ formation<br />
FGF-10<br />
B<br />
CC<br />
FGF-deficient<br />
γTubulin-GFP membrane-cherry<br />
Polarity can be down and up regulated <strong>via</strong> manipulation of FGF-signaling<br />
D<br />
E<br />
+ FGF inhibitor<br />
F<br />
G<br />
fgfr1 gfp<br />
fgf3 gfp<br />
fgf10 fgf10 gfp gfp<br />
wt pea3 gfp fgf3-/-,fgf10-/- pea3 gfp<br />
wt pea3 fgf3-/-,fgf10-/-<br />
wt<br />
50µm<br />
H<br />
pea3 gfp<br />
200µm 200µm<br />
200µm 200µm<br />
pea3<br />
50µm<br />
pea3 gfp fgf3-/-,fgf10-/ pea3 gfp<br />
50µm<br />
50µm
FGF ‘foci’ nucleate epithelial organ formation<br />
FGF-10<br />
FGF-deficient<br />
Polarity can be tuned by genetic/chemical manipulation of FGF-signaling<br />
washout FGF inhibitor<br />
Preventing organ formation arrests directed migration.<br />
SU5402 ‘washout’<br />
side view
Testing role of tissue-scale polarity by targeted laser dissection<br />
epithelial<br />
Genetics reveals that subdivison of the tissue requires different signaling pathways.<br />
Pulsed “ laser knife”(Nd:YAGatl 1⁄4:355nm, 1.2 NA)<br />
mesechyme-like ‘leaders’<br />
EB1GFP labeled MTs
Testing role of tissue-scale polarity by targeted laser dissection<br />
epithelial<br />
Tissue is polarised in terms of migratory potential.<br />
mesechyme-like ‘leaders’<br />
70 70 uM uM<br />
Rosettes ‘melt’ after leader<br />
ablation.<br />
“leading edge+rosettes’<br />
configuration is highly robust.<br />
Enconsin-GFP
Mesenchymal Leaders + Epithelial Rosette = Directional Persistence<br />
Distance in microns<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Mean displacement of cells in fragment including rosette<br />
Std<br />
Mean Displacement<br />
20 40 60 80 100 120 140 160<br />
Time in minutes<br />
MSD in microns 2 /minutes<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
Mean square displacement of cells in fragment<br />
Std<br />
Mean square Displacement<br />
20 40 60 80 100 120 140 160<br />
Time in minutes<br />
SDF1
Gilmour Lab 2011<br />
Erika Donà<br />
Sevi Durdu<br />
Andreea Gruia<br />
Andreas Kunze<br />
Chrisoph Moehl<br />
Li-Kun Phng<br />
Céline Revenu<br />
Ulrike Schulze<br />
Sebastian Streichan<br />
Guillaume Valentin<br />
Former Lab<br />
Petra Haas*<br />
Ana Fernandez-Minan<br />
Gülcin Cakan<br />
Virginie Lecaudey<br />
Acknowledgements<br />
EMBL<br />
Lars Hufnagel*<br />
Ernst Stelzer<br />
Carsten Schultz<br />
EMBL ALMF<br />
Rainer Pepperkok<br />
Yury Belyaev<br />
Stefan Terjung<br />
Christian Tischer<br />
EMBL MACF<br />
Alan Sawyer<br />
Sabin Antony
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