1. No category

If you can do anything,
how to make sure you do
the right thing at the right time?
Stem cells, pluripotency, and
developmental timing in the embryo
Wolfgang Driever
FRIAS - Dec 1, 2010
Tuesday, January 25, 2011
http://www.decision-making-confidence.com/how-do-i-make-good-decisions.html
The act of making up your mind about something, or
a position or opinion or judgment reached after consideration
The process of selecting from several choices, products or ideas,
and taking action.
Tuesday, January 25, 2011
A simple decision making system
in 2D space and time
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Tuesday, January 25, 2011
Decision making in time
state
naive
reversible
decision making
time
Tuesday, January 25, 2011
Information input into decision making
- generate structured decision patterns
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Tuesday, January 25, 2011
Decision making in time
state
naive
reversible
decision making
with
spatial structure
information input
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time
Problems
No stable decisions
Fuzzy pattern
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Well defined
pattern
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Stable and well defined pattern maintained
even in absence of continued information
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What additional regulatory input
may be needed to achieve this
goal?
Tuesday, January 25, 2011
Decision making in time
state
open
state
pluripotent
reversible
decision
making
sharpening
decision
(integrate
info
over
time)
fixed
final
states
time
promote undecided state
repress decison
promote and fix decision
spatial information input
Tuesday, January 25, 2011
How do cells make such decisions
in real life?
The earliest cells in the embryo can
do anything.
They are pluripotent.
Mouse
Tuesday, January 25, 2011
Zebrafish
Human
Pluripotency and decision making in the embryo
3h
pluripotent
5h
deciding
12h
head
fate trunk
determined
tail
Tuesday, January 25, 2011
Decision making
time window
Pluripotency and decision making in the embryo
3h
pluripotent
spatial information
5h
deciding
12h
head
fate trunk
determined
tail
Tuesday, January 25, 2011
Brain - Neuroectoderm
Skin - Ektoderm
Muscle, Organs
Endo-Mesoderm
germ layers
Decision making in time
state
3h
open
state
pluripotent
12h
5h
reversible
decision
making
sharpening
decision
promote undecided state
Pou5f1/Oct4 “stem cell factor”
controls gene expression
spatial information input
Tuesday, January 25, 2011
fixed
final
states
time
Pou5f1 / Oct4
is a pluripotency control gene in mammals
embryos
Oct4
Oct4
Oct4 expressing
inner cells are
source of
embryonic stem
(ES) cells
Tuesday, January 25, 2011
Pou5f1 / Oct4
is a pluripotency control gene in mammals
Oct4 transcription factor controls
expression of other genes
embryos
Oct4
Oct4
Oct4 expressing
inner cells are
source of
embryonic stem
(ES) cells
Tuesday, January 25, 2011
Mammalian core pluripotency network
Niwa, Development 2007
How to investigate the temporal
sequential states during the
decision making process?
Tuesday, January 25, 2011
Determine temporal profiles of genes regulated by
Pou5f1:
Normal wildtypes compared to Pou5f1 mutants
Compare + Pou5f1 (wildtype) against - Pou5f1 (MZspg mutants)
pluripotency
lineage restriction
Tuesday, January 25, 2011
A large number of genes are not activated or
activated to abnormal levels in Pou5f1 mutants
1005 genes
reduced or absent
without Pou5f1
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844 genes
increased in
without Pou5f1
An even larger number of genes is activated
prematurely or with delay: 7677 genes
delayed
prematurely
activated
hr
Tuesday, January 25, 2011
Pou5f1 targets - developmental regulators
precisely timed: downregulated in MZspg
prematurely expressed in MZspg
downregulated in absence of Pou5f1:
Sox genes, many transcriptional repressors
prematurely expressed in absence of Pou5f1:
differentiation genes, promoters of differentiation
Tuesday, January 25, 2011
Effect of loss of Pou5f1 on
Decision making in time
state
3h
open
state
pluripotent
12h
5h
premature activation
reversible
decision
making
sharpening
decision
fixed
final
states
delay
X
promote undecided state
Pou5f1/Oct4 “stem cell factor”
controls gene expression
spatial information input
Tuesday, January 25, 2011
time
Directly activated by Pou5f1: Sox genes
Pou5f1
no Pou5f1
Sox transcription factors
cooperate with Pou5f1 to induce
downstream target genes
Tuesday, January 25, 2011
1
Spatial information: several germ layer specific Pou5f1
targets are transcriptional repressors:
Sox-dependent
Pou5f1
Sox-dependent
no Pou5f1
Pou5f1
klf2b
her3
Sox-independent
Pou5f1
no Pou5f1
foxD3
Tuesday, January 25, 2011
no Pou5f1
Decision making in time
state
open
state
pluripotent
reversible
decision
making
sharpening
decision
fixed
final
states
time
Pou5f1
Sox act with Pou5f1
repress
differentiation (ROD)
promote decision promote
repress decison
differentiation (POD)
spatial information input
Tuesday, January 25, 2011
Tuesday, January 25, 2011
Temporal behaviour of Sox, RODs and POD
Simulation versus measurements
with Pou5f1
without Pou5f1
“half” amount of Pou5f1
prediction
“half” amount of Pou5f1
measurements
Pou5f1 targets:
early “monophasic”
activation
(ROD)
Tuesday, January 25, 2011
Pou5f1 + Sox targets:
delayed “biphasic”
activation
(ROD)
differentiation genes
prematurely activated in
absence of Pou5f1
Explanation for the Dual Role of Pou5f1
The dual role of Pou5f1 in activating promotors of differentiation
(PoDs) and also repressing them indirectly by RODs allows to
control the time of differentiation
Tuesday, January 25, 2011
Decision making in time
state
open
state
pluripotent
reversible
decision
making
sharpening
decision
fixed
final
states
time
Pou5f1
Sox acts with Pou5f1
repress
repressdecison
PODs
Her3 ROD Type II
FoxD3 ROD Type I
repress
repressdecison
PODs
promote decision
promote decision
spatial information input
Tuesday, January 25, 2011
Decision making in time I: early Pou5f1
and late Pou5f1/Sox dependent activation
state
open
state
pluripotent
reversible
decision
making
sharpening
decision
fixed
final
states
time
Pou5f1
Sox acts with Pou5f1
promote decision
promote decision
spatial information input
Tuesday, January 25, 2011
Decision making in time II: balance of repression
and activation times developmental progress
state
open
state
pluripotent
reversible
decision
making
sharpening
decision
fixed
final
states
time
Pou5f1
Sox acts with Pou5f1
repress
repressdecison
PODs
Her3 ROD Type II
FoxD3 ROD Type I
repress
repressdecison
PODs
promote decision
promote decision
spatial information input
Tuesday, January 25, 2011
Is this specific for zebrafish?
Or can we learn how to drive stem
cells into specific cell types in
mammals?
Tuesday, January 25, 2011
Overexpression of mouse Pou5f1 rescues zebrafish
embryos mutant for Pou5f1
Evolutionary conservation of function
Tuesday, January 25, 2011
Overexpression of mouse Pou5f1 rescues zebrafish
embryos mutant for Pou5f1
Evolutionary conservation of function
Tuesday, January 25, 2011
Overexpression of mouse Pou5f1 rescues zebrafish
embryos mutant for Pou5f1
Evolutionary conservation of function
Mouse and zebrafish Pou5f1 share targets:
453
49
zebrafish
Tuesday, January 25, 2011
406
mouse
Comparison with mouse Oct4 target sets
(genes with homology info only):
Sharov et al, BMC Genomics 2008
(Matoba et al., 2006 and Loh et al., 2006)
Hypothesis: The stem cell regulatory network
evolved from a regulatory network
controlling developmental timing
Tuesday, January 25, 2011
Relevance?
Tuesday, January 25, 2011
Insight into controlled differentiation of
stem cells to replace lost tissues:
Regenerative medicine
Use knowledge of combination of tissue
specific repressors of differentiation to
generate defined cell types
Tuesday, January 25, 2011
Acknowledgements
Daria Onichtchouk
Florian Geier
Jens Timmer (FRIAS)
& Rebecca Mössner, Kay Kotkamp, Bozena Polock, Björn Wendik, Esther Kur, Bernadette Lippok, Sungmin
Song, Heinz-Georg-Belting, Martina Lachnit
Collaborations: Thorsten Kurz (ZBSA Genomics Core)
Onichtchouk et al., Molecular Systems Biology, 6:354 Mar 9 - 2010
[email protected]
Tuesday, January 25, 2011
Tuesday, January 25, 2011
Tuesday, January 25, 2011