the course of development time events the course of development time events

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?