The Course of Development

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The Course of Development
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The Course of Development
Time
Events
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The Course of Development
Time
Events
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The Course of Development
Time
Events in time
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The Course of Development
Time
Events in time
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The Course of Development
Time
Events in time and space . . .
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The Course of Development
Time
Events in time and space . . .
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The Course of Development
Time
Events in time and space . . .
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The Course of Development
Events in time and space . . .
. . . driven by patterned gene expression
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The Course of Development
Understanding Human Development
Events in time and space . . . . . . driven
by patterned gene expression
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The Course of Development
Understanding Human Development
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The Course of Development
Understanding Human Development
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The Course of Development
Understanding Human Development
The fate of cells
Intrinsic control?
patterned in time and space
Extrinsic control?
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Understanding Human Development
Why so difficult?
Process 9 mo 20 yrs
Generation 20 yrs
Genetic recombination Uncontrolled
Genetic manipulation Difficult / Impossible
Genome size 3 billion nucleotides
Development Complex
How to attack a problem thats too complex?
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How to Attack a Complex Problem
Probability of getting a full house?
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How to Attack a Complex Problem
Probability of getting a pair?
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How to Attack a Complex Problem
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3/51
Probability of getting a pair in 2 cards?
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Simplification can help in understanding complexit
y
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Understanding Human Development
Why so difficult?
Process 9 mo 20 yrs
Generation 20 yrs
Genetic recombination Uncontrolled
Genetic manipulation Difficult / Impossible
Genome size 3 billion nucleotides
Development Complex
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Understanding Fly Development
Still difficult
Process 9 mo 20 yrs
8 days
Generation 20 yrs
14 days
Genetic recombination Uncontrolled
Controlled
Genetic manipulation Difficult / Impossible
Difficult
Genome size 3 billion nucleotides
170 million nucleotides
Development Complex
Complex
How to simplify further?
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Understanding Any Development
What do we want in a model organism?
Process
8 days
Hours
Generation
14 days
Hours
Genetic recombination
Controlled
Genetic manipulation
Difficult
Easy
Genome size
170 million nucleotides
Few million nucleotides
Development
Complex
Single phenomenon
Does such an organism exist?
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Bacteria
E. coli
. . . but no development
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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
Development in time and space
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Free-living Nostoc
Heterocyst differentiation by Anabaena
N2
CO2
O2
Matveyev and Elhai (unpublished)
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Free-living Nostoc
Heterocyst differentiation by Anabaena
NH3
N2
O2
CO2
Matveyev and Elhai (unpublished)
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AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
N2 fixation
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AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
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AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
Development of pattern
Mark Hill, University of New South
Wales http//anatomy.med.unsw.edu.au/cbl/embryo/No
tes/skmus7.htm
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Fruiting body formation by Myxococcus
Herd motility
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Fruiting body formation by Myxococcus
Herd development
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Fruiting body formation by Myxococcus
Extrinsic control over development
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Caulobacter crescentusCell cycle-regulated
differentiation
Cell cycle of Caulobacter
swarmercell
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Caulobacter crescentusCell cycle-regulated
differentiation
Cell cycle of Caulobacter
swarmercell
stalkcell
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Caulobacter crescentusCell cycle-regulated
differentiation
Cell cycle of Caulobacter
swarmercell
stalkcell
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Caulobacter crescentusCell cycle-regulated
differentiation
Cell cycle of Caulobacter
Intrinsic control over development
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Bacterial Development
End result...
much simpler
Anabaena heterocysts
Bacillus sporulation
Caulobacter cell cycle
Myxobacteria fruiting
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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
Control of initiation selective gene expression
?
How to makethe decision?
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Bacterial regulation of gene expression
Transcriptional factors
RNAPol
DNA
P
RNA
protein
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Bacterial regulation of gene expression
Transcriptional factors
DNA binding protein
RNAPol
No stimulus
Stimulus
DNA
Binding site
P
No RNA
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Bacterial regulation of gene expression
Transcriptional factors
DNA binding protein
RNAPol
No stimulus
Stimulus
DNA
Binding site
P
No RNA
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Bacterial regulation of gene expression
Transcriptional factors
RNAPol
Spo0A
No stimulus
Stimulus
DNA
Binding site
P
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Sporulation by Bacillus subtilis
Control of initiation selective gene expression
Why???
Spores
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Sporulation by Bacillus subtilis
Phosphorelay as an integration processing device
?
Spores
- Cell cycle- DNA damage- Nutrient status
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Sporulation by Bacillus subtilis
Control of initiation of development

• Integration of signals through signal
  transduction

• Centers on phosphorylation of master protein

• DNA binding protein regulates transcription

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Bacillus subtilisTemporally regulated
differentiation
Sporulation by Bacillus subtilis
Control of timing by selective gene expression
Set 0
Set II
Set IV
Set V
Set III
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Promoter recognition by sigma factors
Sigma factor
RNA polymerase core enzyme
Figure from Griffiths et al (1996) Introduction
to Genetic Analysis, 6th ed., WH Freeman and Co.
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Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction
to Genetic Analysis, 6th ed., WH Freeman and Co.
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Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction
to Genetic Analysis, 6th ed., WH Freeman and Co.
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Promoter recognition by sigma factors
uvrB Repair DNA damage TTGTTGGCATAATTAAGTACGACGAG
TAAAATTAC ATACCT recA DNArecombination CACTTGAT
ACTGTA.TGAGCATACAGTATAATTGC TTCAACA rrnAB Ribosom
alRNA CTCTTGTCAGGCCG.GAATAACTCCCTATAATGCGCCACCACTG
str Ribosomal protein TTCTTGACACCTT.TCGGCATCGCCCT
AAAATTCG GCGTCG rpoA RNA polymerase TTCTTGCAAAGT
TGGGTTGAGCTGGCTAGATTAGC CAGCCA
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Promoter recognition by sigma factors
TTGaca
TAtAaT R
uvrB Repair DNA damage TTGTTGGCATAATTAAGTACGACGAG
TAAAATTAC ATACCT recA DNArecombination CACTTGAT
ACTGTA.TGAGCATACAGTATAATTGC TTCAACA rrnAB Ribosom
alRNA CTCTTGTCAGGCCG.GAATAACTCCCTATAATGCGCCACCACTG
str Ribosomal protein TTCTTGACACCTT.TCGGCATCGCCCT
AAAATTCG GCGTCG rpoA RNA polymerase TTCTTGCAAAGT
TGGGTTGAGCTGGCTAGATTAGC CAGCCA
Kp nifE nitrogenase accessory CTTCTGGAGCGCGAATTGCA
TCTTCCCCCT Kp nifU nitrogenase accessory
TCTCTGGTATCGCAATTGCT AGTTCGTTAT Kp
nifB nitrogenase accessory CCTCTGGTACAGCATTTGCA
GCAGGAAGGT Kp nifH nitrogenase CGGCTGGTATGTTCCCTGC
ACTTCTCTGCTG Kp nifM nitrogenase accessory
TGGCTGGCCGGAAATTTGCA ATACAGGGAT Kp
nifF nitrogenase accessory AACCTGGCACAGCCTTCGCA
ATACCCCTGC Kp nifL nitrogenase regulatn ATAAGGGCG
CACGGTTTGCATGGTTATCACC glnA P2 glutamine
synthetase AAGTTGGCACAGATTTCGCTTTATCTTTTTT
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Sigma factors in sporulation
HousekeepingSigma-A
Starvation-specificSigma-H
Starvation (and other signals)Stage 0
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Sigma factors in sporulation
Mother cell
Forespore
Mother-specificSigma-E
H
Forespore-specificSigma-F
H
Stage II/III
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Sigma factors in sporulation
Uniform presence of inactive sigma precursors
Starvation (and other signals)Stage 0
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Sigma factors in sporulation
Selective activation of sigma precursors
Active mother-specificSigma-E
H
Active forespore-specificSigma-F
H
Stage II/III
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Sigma factors in sporulation
Cascade of sigma factors
Late mother-specificSigma-K
Late forespore-specificSigma-G
Starvation (and other signals)Stage III
Stage IV
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Sporulation by Bacillus subtilis
Control of timing by selective gene expression

• Determined by specific, active sigma factors

• Presence and activation important

• Activation linked to morphological events

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AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
How to find regulation of pattern?
N2 fixation
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
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Genetic approach to Cell Biology
Isolation of Defective Gene
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AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
How to find regulation of pattern?
N2 fixation
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AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
How to find regulation of pattern?
Gene expression?
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Gene fusions to monitor expression
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Gene fusions to monitor expression
hetRRegulation
Reporter gene


NNNNNNNNNNNNNNNNNNATGNNNNNNNNNN
NNNNNN

NNNNNNNNNNNNNNNNNNTACNNN
NNNNNNNNNNNNN
5-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNN
NN3-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNN
NNNNN
RNA Polymerase
GTA ..(8).. TAC
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Detection of hetR gene expression through Green
Fluorescent Protein
The hydromedusa Aequoria victoria Source of
GreenFluorescent Protein
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Expression of hetR during differentiation
Weak and patchy
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Expression of hetR after differentiation
Strong and focused
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Expression of hetR after differentiation
HetR is required for its own induction!
Feedback Induction
Other examples
spo0A
eve
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Feedback Regulation
Stability
All-or-none
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Feedback Regulation
Alan Turings Reaction-Diffusion Model
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Feedback Regulation
Alan Turings Reaction-Diffusion Model
Initiation
Marcelo Walter, U Br Columbia
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Feedback Regulation
Alan Turings Reaction-Diffusion Model
Pattern emerging from random initiation
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Feedback Regulation
Alan Turings Reaction-Diffusion Model
Pattern emerging from random initiation
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Feedback Regulation
Alan Turings Reaction-Diffusion Model
What is the diffusible inhibitor?
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Heterocyst differentiation by Anabaena
How to find the hypothetical diffusible inhibitor?
Encodes diffusible inhibitor?
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Heterocyst differentiation by Anabaena
The nature of the hypothetical inhibitor
PatS
MLVNFCDERGSGR
Is PatS the predicted diffusible inhibitor?
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Heterocyst differentiation by Anabaena
The nature of the hypothetical inhibitor
RGSGR
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Heterocyst differentiation by Anabaena
The nature of the hypothetical inhibitor
Multiple heterocysts
But not ALL heterocysts
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Heterocyst differentiation by Anabaena
The nature of the hypothetical inhibitor
Nonrandomspacing
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Heterocyst differentiation by Anabaena
The nature of the hypothetical inhibitor
Heterocyst distributionis affected
But its not RANDOM
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Heterocyst differentiation by Anabaena
A natural example of the Turing model?

• Differentiation regulated by R-like protein, HetR

• Differentiation regulated by D-like protein, PatS

• Pattern is not completely determined by HetR
  and PatS

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Bacterial Development
End result...
much simpler
Anabaena heterocysts
Bacillus sporulation
Caulobacter cell cycle
Myxobacteria fruiting
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How to understand complexity?
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How to understand complexity?
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