The Course of Development
The Course of Development
Time
Events
The Course of Development
Time
Events
The Course of Development
Time
Events in time
The Course of Development
Time
Events in time
The Course of Development
Time
Events in time and space . . .
The Course of Development
Time
Events in time and space . . .
The Course of Development
Time
Events in time and space . . .
The Course of Development
Events in time and space . . .. . . driven by patterned gene expression
The Course of Development
Events in time and space . . . . . . driven by patterned gene expression
Understanding Human Development
The Course of DevelopmentUnderstanding Human Development
The Course of DevelopmentUnderstanding Human Development
The Course of Development
The fate of cellspatterned in time and space
Intrinsic control?Extrinsic control?
Understanding Human Development
Understanding Human DevelopmentWhy so difficult?
Development Complex
Process 9 mo – 20 yrs
Generation 20 yrs
Genetic recombination Uncontrolled
Genetic manipulation Difficult / Impossible
Genome size ~3 billion nucleotides
How to attack a problem that’s too complex?
How to Attack a Complex Problem
Probability of getting a full house?
How to Attack a Complex Problem
Probability of getting a pair?
How to Attack a Complex Problem
Probability of getting a pair in 2 cards?
1 · 3/51
Simplification can help
in understanding
complexity
Understanding Human DevelopmentWhy so difficult?
Development Complex
Process 9 mo – 20 yrs
Generation 20 yrs
Genetic recombination Uncontrolled
Genetic manipulation Difficult / Impossible
Genome size ~3 billion nucleotides
Process 9 mo – 20 yrs
Generation 20 yrs
Genetic recombination Uncontrolled
Genetic manipulation Difficult / Impossible
Genome size ~3 billion nucleotides
Understanding Fly Development
~8 days
~14 days
Controlled
Difficult
Still difficult
How to simplify further?
~170 million nucleotides
Development ComplexComplex
Process
Generation
Genetic recombination
Genetic manipulation
Genome size
~8 days
~14 days
Controlled
Difficult
Does such an organism exist?
Development Complex
~170 million nucleotides
Hours
Hours
Easy
Few million nucleotides
Single phenomenon
What do we want in a model organism?
Understanding Any Development
Bacteria
. . . but no development
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilis
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilis
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilis
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilis
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilis
Development in time and space
Free-living Nostoc
heterocysts
Matveyev and Elhai (unpublished)
CO2
sucroseN2
O2
Heterocyst differentiation by Anabaena
Free-living Nostoc
heterocysts
Matveyev and Elhai (unpublished)
CO2
sucroseN2
NH3
NH3
O2
Heterocyst differentiation by Anabaena
AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
Time after nitrogen removal
0 h
3 h
6 h
9 h
12 h
18 h N2 fixation
AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
Time after nitrogen removal
0 h
3 h
6 h
9 h
12 h
18 h N2 fixation
AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
Mark Hill, University of New South Wales
http://anatomy.med.unsw.edu.au/cbl/embryo/Notes/skmus7.htm
Time after nitrogen removal
0 h
3 h
6 h
9 h
12 h
18 h N2 fixation
Development of pattern
Fruiting body formation by MyxococcusHerd motility
Fruiting body formation by MyxococcusHerd development
Fruiting body formation by MyxococcusExtrinsic control over development
Caulobacter crescentusCell cycle-regulated differentiation
Cell cycle of Caulobacter
swarmercell
Caulobacter crescentusCell cycle-regulated differentiation
Cell cycle of Caulobacter
swarmercell
stalkcell
Caulobacter crescentusCell cycle-regulated differentiation
Cell cycle of Caulobacter
swarmercell
stalkcell
Caulobacter crescentusCell cycle-regulated differentiation
Cell cycle of CaulobacterIntrinsic control over development
End result... much simplerBacillus sporulation
Myxobacteria fruiting
Anabaena heterocysts
Caulobacter cell cycle
Bacterial Development
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilis
?How to makethe decision?
Control of initiation selective gene expression
Bacterial regulation of gene expression
Transcriptional factors
DNA
RNA
protein
RNAPol
P
No stimulus
DNA binding protein
RNA
Pol
Binding site
P
DNA
No RNA
Stimulus
signal
Bacterial regulation of gene expression
Transcriptional factors
DNA binding protein
RNA
Pol
Binding site
P
DNA
No RNA
No stimulusStimulus
signal
Bacterial regulation of gene expression
Transcriptional factors
DNA
RNAPolSpo0A
Binding site
P
RNA
protein
No stimulusStimulus
signal
Bacterial regulation of gene expression
Transcriptional factors
Sporulation by Bacillus subtilisControl of initiation selective gene expression
P
ADP
ATP
P
Spo genes
Spo genes
Spores
spo0A
A
PA
P
spo0B
B
B
spo0F
P
F
F
kinA
PK
K
Why???
PP
Sporulation by Bacillus subtilisPhosphorelay as an integration processing device
P
ADP
ATP
P
Spo genes
Spo genes
Spores
spo0A
A
PA
P
spo0B
B
B
spo0F
P
F
F
kinA
PK
K
Cell density
Control by phosphatases?
- Cell cycle- DNA damage- Nutrient status
Sporulation by Bacillus subtilisControl of initiation of development
• Integration of signals through signal transduction
• Centers on phosphorylation of master protein
• DNA binding protein regulates transcription
Bacillus subtilisTemporally regulated differentiation
Sporulation by Bacillus subtilisControl of timing by selective gene expression
Set 0
Set II
Set IIISet IVSet V
Fore-spore Mother cell
Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.
'
RNA polymerase core enzyme
Sigma factor
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.
Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.
Promoter recognition by sigma factors
AuvrB Repair DNA damage TTGTTGGCATAATTAAGTACGACGAGTAAAATTAC ATACCTrecA DNArecombination CACTTGATACTGTA.TGAGCATACAGTATAATTGC TTCAACArrnAB RibosomalRNA CTCTTGTCAGGCCG.GAATAACTCCCTATAATGCGCCACCACTGstr Ribosomal protein TTCTTGACACCTT.TCGGCATCGCCCTAAAATTCG GCGTCGrpoA RNA polymerase TTCTTGCAAAGTTGGGTTGAGCTGGCTAGATTAGC CAGCCA
TTGaca TAtAaT R
Promoter recognition by sigma factors
N
uvrB Repair DNA damage TTGTTGGCATAATTAAGTACGACGAGTAAAATTAC ATACCTrecA DNArecombination CACTTGATACTGTA.TGAGCATACAGTATAATTGC TTCAACArrnAB RibosomalRNA CTCTTGTCAGGCCG.GAATAACTCCCTATAATGCGCCACCACTGstr Ribosomal protein TTCTTGACACCTT.TCGGCATCGCCCTAAAATTCG GCGTCGrpoA RNA polymerase TTCTTGCAAAGTTGGGTTGAGCTGGCTAGATTAGC CAGCCA
TTGaca TAtAaT R
Kp nifE nitrogenase accessory CTTCTGGAGCGCGAATTGCA TCTTCCCCCTKp nifU nitrogenase accessory TCTCTGGTATCGCAATTGCT AGTTCGTTATKp nifB nitrogenase accessory CCTCTGGTACAGCATTTGCA GCAGGAAGGTKp nifH nitrogenase CGGCTGGTATGTTCCCTGCACTTCTCTGCTGKp nifM nitrogenase accessory TGGCTGGCCGGAAATTTGCA ATACAGGGATKp nifF nitrogenase accessory AACCTGGCACAGCCTTCGCA ATACCCCTGCKp nifL nitrogenase regulat’n ATAAGGGCGCACGGTTTGCATGGTTATCACCglnA P2 glutamine synthetase AAGTTGGCACAGATTTCGCTTTATCTTTTTT
CTGG-A TTGCA
Promoter recognition by sigma factors
Sigma factors in sporulation
A A
AA H
H
H
H
Starvation (and other signals)Stage 0
Starvation-specificSigma-H
HousekeepingSigma-A
Sigma factors in sporulation
A
A
AA
H
H
E
E F
F
Stage II/III
Mother cell Forespore
Forespore-specificSigma-F
Mother-specificSigma-E
Sigma factors in sporulation
A A
AA H
H
H
H
Starvation (and other signals)Stage 0
E
E E
F
FF
Uniform presence of inactive sigma precursors
F
F
F
F
E
E
Sigma factors in sporulation
A
A
AA
H
H
E
E F
F
Stage II/III
E
F
Selective activation of sigma precursorsActive mother-
specificSigma-E
Active forespore-specificSigma-F
Sigma factors in sporulation
A
A
E
E F
F
K
K
G
G
Starvation (and other signals)Stage IIIStage IV
Late forespore-specificSigma-G
Late mother-specificSigma-K
Cascade of sigma factors
Sporulation by Bacillus subtilisControl of timing by selective gene expression
• Determined by specific, active sigma factors
• Presence and activation important
• Activation linked to morphological events
AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
Time after nitrogen removal
0 h
3 h
6 h
9 h
12 h
18 h N2 fixation
How to find regulation of pattern?
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Genetic approach to Cell Biology
Isolation of Defective Gene
Genetic approach to Cell Biology
AnabaenaSpatially regulated differentiation
Heterocyst differentiation by Anabaena
Time after nitrogen removal
0 h
3 h
6 h
9 h
12 h
18 h N2 fixation
How to find regulation of pattern?
Many mutants
Rare mutants
hetR
AnabaenaSpatially regulated differentiation
Heterocyst differentiation by AnabaenaHow to find regulation of pattern?
hetR
Gene expression?
hetR-
+N-N
+N-N
-N+N
hetR
(wild-type)
GTA ..(8).. TAC
5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
Reporter gene
Gene fusions to monitor expression
5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
hetR gene
RNA Polymerase
hetRRegulation
GTA ..(8).. TAC
NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN
Reporter gene
Gene fusions to monitor expression
5’-GTA ..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNN3’-CAT ..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNN
RNA Polymerase
hetRRegulation
Detection of hetR gene expression
through Green Fluorescent Protein
The hydromedusa Aequoria victoria
Source of GreenFluorescent Protein
Expression of hetR during differentiation
Weak and patchy
Expression of hetR after differentiation
Strong and focused
hetR-
hetR
(wild-type)
hetRexpression
0 18Hrs after -N
HetR is required for its own induction!
hetR+
hetR-
hetR
(wild-type)
HetR
Feedback Induction
Expression of hetR after differentiation
Other examples:
spo0A eve
Tem
pera
ture
Feedback Induction
Tem
pera
ture
Feedback Inhibition
Feedback Regulation
Stability
All-or-none
Feedback Regulation
Alan Turing’s Reaction-Diffusion Model
R
color
D
+
Marcelo Walter, U Br Columbia
Feedback Regulation
Alan Turing’s Reaction-Diffusion Model
R
color
D
+
Giraffe Model
Initiation
Marcelo Walter, U Br Columbia
Feedback Regulation
Alan Turing’s Reaction-Diffusion Model
Pattern emerging from random initiation
Feedback Regulation
Alan Turing’s Reaction-Diffusion Model
Pattern emerging from random initiation
Feedback Regulation
Alan Turing’s Reaction-Diffusion Model
R
color
D
+ hetR
What is the diffusible inhibitor?
Heterocyst differentiation by AnabaenaHow to find the hypothetical diffusible inhibitor?
plasmid
(chopped)
Encodes diffusible inhibitor?
genome
?
Heterocyst differentiation by AnabaenaThe nature of the hypothetical inhibitor
(typical size of gene)
Active part of sequence MLVNFCDERGSGR PatS
Is PatS the predicted diffusible inhibitor?
Heterocyst differentiation by AnabaenaThe nature of the hypothetical inhibitor
+ RGSGR
hetR
HetR
R
color
D
+ hetR
+ RGSGR HetR+
patS-
Heterocyst differentiation by AnabaenaThe nature of the hypothetical inhibitor
+N -N
patS+
(wild-type)
+N -N
patS -
Multiple heterocysts
But not ALL heterocysts
Heterocyst differentiation by AnabaenaThe nature of the hypothetical inhibitor
Nonrandomspacing
Heterocyst distributionis affected
Heterocyst differentiation by AnabaenaThe nature of the hypothetical inhibitor
But it’s not RANDOM
Heterocyst differentiation by AnabaenaA 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
End result... much simplerBacillus sporulation
Myxobacteria fruiting
Anabaena heterocysts
Caulobacter cell cycle
Bacterial Development
vs
How to understand complexity?
How to understand complexity?