The Course of Development The Course of

The Course of Development

The Course of Development Time Events

The Course of Development Time Events in time

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 Understanding Human Development Events in time and space. . . driven by patterned gene expression

The Course of Development Understanding Human Development

The Course of Development Understanding Human Development The fate of cells patterned in time and space Intrinsic control? Extrinsic control?

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 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 1 · 3/51 Probability of getting a pair in 2 cards?

Simplification can help in understanding complexity

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

Understanding Fly Development Still difficult Process ~8 mo – 20 yrs 9 days Generation 20 days ~14 yrs Genetic recombination Uncontrolled Controlled Genetic manipulation Difficult / Impossible Genome size ~3 billion nucleotides ~170 million nucleotides Development Complex How to simplify further?

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?

Bacteria . . . but no development

Bacillus subtilis Sporulation by Bacillus subtilis Temporally regulated differentiation

Bacillus subtilis Sporulation by Bacillus subtilis Temporally regulated differentiation Development in time and space

Heterocyst differentiation by Anabaena Free-living Nostoc heterocysts N 2 O 2 Matveyev and Elhai (unpublished) rose suc CO 2

Heterocyst differentiation by Anabaena Free-living Nostoc NH 3 heterocysts N 2 O 2 NH 3 Matveyev and Elhai (unpublished) rose suc CO 2

Anabaena Heterocyst differentiation by Anabaena Spatially regulated differentiation Time after nitrogen removal 0 h 3 h 6 h 9 h 12 h 18 h N 2 fixation

Anabaena Heterocyst differentiation by Anabaena Spatially regulatedof pattern differentiation Development Time after nitrogen removal 0 h 3 h 6 h 9 h 12 h 18 h N 2 fixation Mark Hill, University of New South Wales http: //anatomy. med. unsw. edu. au/cbl/embryo/Notes/skmus 7. htm

Fruiting body formation by Myxococcus Herd motility

Fruiting body formation by Myxococcus Herd development

Fruiting body formation by Myxococcus Extrinsic control over development

Caulobacter Cell cycle of crescentus Cell cycle-regulated differentiation swarmer cell

Caulobacter Cell cycle of crescentus Cell cycle-regulated differentiation swarmer cell stalk cell

Caulobacter Cell cycle of crescentus Cell cycle-regulated differentiation Intrinsic control over development

Bacterial Development End result. . . much simpler Bacillus sporulation Anabaena heterocysts Myxobacteria fruiting Caulobacter cell cycle

Bacillus subtilis Sporulation by Bacillus subtilis Temporally regulated differentiation Control of initiation selective gene expression How to make the decision? ?

Bacterial regulation of gene expression Transcriptional factors RNA Pol DNA P RNA protein

Bacterial regulation of gene expression Transcriptional factors signal No stimulus Stimulus DNA binding protein RNA Pol DNA Binding P site No RNA

Bacterial regulation of gene expression Transcriptional factors signal No stimulus Stimulus RNA Spo 0 A Pol DNA Binding P site RNA protein

Sporulation by Bacillus subtilis Control of initiation selective gene expression Why? ? ? P K F P B P A ATP Spores P Spo genes KP F B P A ADP Spo genes kin. A spo 0 F spo 0 B spo 0 A

Sporulation by Bacillus subtilis Phosphorelay as an integration processing device Cell density P K P F P ? B Control by phosphatases P P A ATP Spores P Spo genes KP F B P A ADP Spo genes kin. A spo 0 F spo 0 B spo 0 A - Cell cycle - DNA damage - Nutrient status

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

Bacillus subtilis Sporulation by Bacillus subtilis Temporally regulated differentiation Control of timing by selective gene expression Set 0 Set II Set V Set III Fore-spore Mother cell

Promoter recognition by sigma factors Sigma factor RNA polymerase core enzyme ' Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6 th ed. , WH Freeman and Co.

Promoter recognition by sigma factors Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6 th ed. , WH Freeman and Co.

Promoter recognition by sigma factors uvr. B rec. A rrn. AB str rpo. A Repair DNA damage DNArecombination Ribosomal. RNA Ribosomal protein RNA polymerase A TTGTTGGCATAATTAAGTACGACGAGTAAAATTAC ATACCT CACTTGATACTGTA. TGAGCATACAGTATAATTGC TTCAACA CTCTTGTCAGGCCG. GAATAACTCCCTATAATGCGCCACCACTG TTCTTGACACCTT. TCGGCATCGCCCTAAAATTCG GCGTCG TTCTTGCAAAGTTGGGTTGAGCTGGCTAGATTAGC CAGCCA TTGaca TAt. Aa. T R

Promoter recognition by sigma factors TTGaca uvr. B rec. A rrn. AB str rpo. A Kp nif. E Kp nif. U Kp nif. B Kp nif. H Kp nif. M Kp nif. F Kp nif. L gln. A P 2 Repair DNA damage DNArecombination Ribosomal. RNA Ribosomal protein RNA polymerase nitrogenase accessory nitrogenase accessory nitrogenase regulat’n glutamine synthetase TAt. Aa. T R TTGTTGGCATAATTAAGTACGACGAGTAAAATTAC ATACCT CACTTGATACTGTA. TGAGCATACAGTATAATTGC TTCAACA CTCTTGTCAGGCCG. GAATAACTCCCTATAATGCGCCACCACTG TTCTTGACACCTT. TCGGCATCGCCCTAAAATTCG GCGTCG TTCTTGCAAAGTTGGGTTGAGCTGGCTAGATTAGC CAGCCA N CTTCTGGAGCGCGAATTGCA TCTTCCCCCT TCTCTGGTATCGCAATTGCT AGTTCGTTAT CCTCTGGTACAGCATTTGCA GCAGGAAGGT CGGCTGGTATGTTCCCTGCACTTCTCTGCTG TGGCCGGAAATTTGCA ATACAGGGAT AACCTGGCACAGCCTTCGCA ATACCCCTGC ATAAGGGCGCACGGTTTGCATGGTTATCACC AAGTTGGCACAGATTTCGCTTTATCTTTTTT CTGG-A TTGCA

Sigma factors in sporulation Housekeeping Sigma-A A H A A A H Starvation (and other signals) Stage 0 Starvationspecific Sigma-H

Sigma factors in sporulation Mother-specific Mother cell Sigma-E E Forespore A H F A E A A Stage II/III H F Forespecific Sigma-F

Sigma factors in sporulation Uniform presence of inactive sigma precursors E A A H E A F E H F Starvation (and other signals) Stage 0

Sigma factors in sporulation Selective activation of sigma precursors Active motherspecific Sigma-E E A H E A F F A A Stage II/III E H F Active forespecific Sigma-F

Sigma factors in sporulation Cascade of sigma factors Late motherspecific Sigma-K K G E F A K E G F A Starvation Stageother signals) (and IV Stage III Late forespore -specific Sigma-G

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

Anabaena Heterocyst differentiation by Anabaena Spatiallyfind regulation of pattern? regulated differentiation How to Time after nitrogen removal 0 h 3 h 6 h 9 h 12 h 18 h N 2 fixation

Genetic approach to Cell Biology

Genetic approach to Cell Biology Isolation of Defective Gene

Anabaena Heterocyst differentiation by Anabaena Spatiallyfind regulation of pattern? regulated differentiation How to Time after nitrogen removal 0 h Rare mutants 3 h het. R 6 h 9 h 12 h 18 h N 2 fixation Many mutants

Anabaena Heterocyst differentiation by Anabaena Spatiallyfind regulation of pattern? regulated differentiation How to het. R (wild-type) het. R- +N -N het. R +N -N Gene expression?

Gene fusions to monitor expression het. R Regulation het. R gene 5’-GTA 3’-CAT . . (8). . TACNNNNNTANNNTNNNNNNNNNNNNNNATGNNNNNNNN ATGNNNNNATNNNANNNNNNNNNNNNNNTACNNNNNNNN RNA Polymerase Reporter gene 5’-GTGAGTTAGCTCACNNNNNTANNNTNNNNNNNNNNNNNNATGNNNNNNNN 3’-CACTCAATCGAGTGNNNNNATNNNANNNNNNNNNNNNNNNTACNNNNNNNN GTA . . (8). . TAC

Gene fusions to monitor expression het. R Regulation Reporter gene 5’-GTA 3’-CAT . . (8). . TACNNNNNTANNNTNNNNNNNNNATGNNNNNNNN ATGNNNNNATNNNANNNNNNNNNTACNNNNNNNN RNA Polymerase GTA . . (8). . TAC

Detection of het. R gene expression through Green Fluorescent Protein The hydromedusa Aequoria victoria Source of Green Fluorescent Protein

Expression of het. R during differentiation Weak and patchy

Expression of het. R after differentiation Strong and focused

Expression of het. R after differentiation het. R+ het. R expression het. R- (wild-type) het. R 0 Het. R het. R (wild-type) Hrs after -N 18 Het. R is required for its own induction! Feedback Induction Other examples: spo 0 A eve

Feedback Regulation Temperature Feedback Inhibition Stability Temperature Feedback Induction All-or-none

Feedback Regulation Alan Turing’s Reaction-Diffusion Model color + R D Marcelo Walter, U Br Columbia

Feedback Regulation Alan Turing’s Reaction-Diffusion Model color + Initiation Giraffe Marcelo Walter, U Br Columbia R D Model

Feedback Regulation Alan Turing’s Reaction-Diffusion Model Pattern emerging from random initiation

Feedback Regulation Alan Turing’s Reaction-Diffusion Model color + R het. R D What is the diffusible inhibitor?

Heterocyst differentiation by Anabaena How to find the hypothetical diffusible inhibitor? genome (chopped) plasmid ? Encodes diffusible inhibitor?

Heterocyst differentiation by Anabaena The nature of the hypothetical inhibitor (typical size of gene) Pat. S Active part of sequence MLVNFCDERGSGR Is Pat. S the predicted diffusible inhibitor?

Heterocyst differentiation by Anabaena The nature of the hypothetical inhibitor color + RGSGR + het. R R D + RGSGR + pat. S- Het. R het. R

Heterocyst differentiation by Anabaena The nature of the hypothetical inhibitor +N -N pat. S+ (wild-type) pat. S - Multiple heterocysts But not ALL heterocysts

Heterocyst differentiation by Anabaena The nature of the hypothetical inhibitor Nonrandom spacing

Heterocyst differentiation by Anabaena The nature of the hypothetical inhibitor Heterocyst distribution is affected But it’s not RANDOM

Heterocyst differentiation by Anabaena A natural example of the Turing model? • Differentiation regulated by R-like protein, Het. R • Differentiation regulated by D-like protein, Pat. S • Pattern is not completely determined by Het. R and Pat. S

Bacterial Development End result. . . much simpler Bacillus sporulation Anabaena heterocysts vs Myxobacteria fruiting Caulobacter cell cycle

How to understand complexity?