2. The Course of Development

3. Time Events

4. The Course of Development Time Events

5. The Course of Development Time Events in time

6. The Course of Development Time Events in time

7. The Course of Development Time Events in time and space...

8. The Course of Development Time Events in time and space...

9. The Course of Development Time Events in time and space...

10. The Course of Development Events in time and space...... driven by patterned gene expression

11. The Course of Development Events in time and space...... driven by patterned gene expression Understanding Human Development

12. The Course of Development Understanding Human Development

13. The Course of Development Understanding Human Development

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

15. Why so difficult? DevelopmentComplex Process9 mo – 20 yrs Generation20 yrs Genetic recombinationUncontrolled Genetic manipulationDifficult / Impossible Genome size ~3 billion nucleotides How to attack a problem that’s too complex?

16. How to Attack a Complex Problem Probability of getting a full house?

17. How to Attack a Complex Problem Probability of getting a pair?

18. How to Attack a Complex Problem Probability of getting a pair in 2 cards? 1· 3/51

19. Simplification can help in understanding complexity

20. Understanding Human Development Why so difficult? DevelopmentComplex Process9 mo – 20 yrs Generation20 yrs Genetic recombinationUncontrolled Genetic manipulationDifficult / Impossible Genome size ~3 billion nucleotides

21. Process9 mo – 20 yrs Generation20 yrs Genetic recombinationUncontrolled Genetic manipulationDifficult / Impossible Genome size ~3 billion nucleotides Understanding Fly Development ~8 days ~14 days Controlled Difficult Still difficult How to simplify further? ~170 million nucleotides DevelopmentComplexComplex

22. Process Generation Genetic recombination Genetic manipulation Genome size ~8 days ~14 days Controlled Difficult Does such an organism exist? DevelopmentComplex ~170 million nucleotides Hours Easy Few million nucleotides Single phenomenon What do we want in a model organism? Understanding Any Development

23. Bacteria... but no development

24. Bacillus subtilis Temporally regulated differentiation Sporulation by Bacillus subtilis

25. Bacillus subtilis Temporally regulated differentiation Sporulation by Bacillus subtilis

26. Bacillus subtilis Temporally regulated differentiation Sporulation by Bacillus subtilis

27. Bacillus subtilis Temporally regulated differentiation Sporulation by Bacillus subtilis

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

29. Free-living Nostoc heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 O2O2 Heterocyst differentiation by Anabaena

30. Free-living Nostoc heterocysts Matveyev and Elhai (unpublished) CO 2 sucrose N2N2 NH 3 O2O2 Heterocyst differentiation by Anabaena

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

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

33. Anabaena Spatially 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 N 2 fixation Development of pattern

34. Fruiting body formation by Myxococcus Herd motility

35. Fruiting body formation by Myxococcus Herd development

36. Fruiting body formation by Myxococcus Extrinsic control over development

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

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

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

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

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

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

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

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

45. DNA binding protein RNA Pol Binding site P DNA No RNA No stimulusStimulus signal Bacterial regulation of gene expression Transcriptional factors

46. DNA RNA Pol Spo0A Binding site P RNA protein No stimulusStimulus signal Bacterial regulation of gene expression Transcriptional factors

47. Sporulation by Bacillus subtilis Control of initiation selective gene expression P ADP ATP P Spo genes Spores spo0A A P A P spo0B B B spo0F P F F kinA P K K Why???

48. PP Sporulation by Bacillus subtilis Phosphorelay as an integration processing device P ADP ATP P Spo genes Spores spo0A A P A P spo0B B B spo0F P F F kinA P K K Cell density Control by phosphatases ? - Cell cycle - DNA damage - Nutrient status

49. 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

50. Bacillus subtilis Temporally regulated differentiation Sporulation by Bacillus subtilis Control of timing by selective gene expression Set 0 Set II Set III Set IVSet V Fore-sporeMother cell

51. 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

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

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

54. AA uvrB Repair DNA damage TTGTTGGCATAATTAAGTACGACGAGTAAAATTAC ATACCT recA DNArecombination CACTTGATACTGTA.TGAGCATACAGTATAATTGC TTCAACA rrnAB RibosomalRNA CTCTTGTCAGGCCG.GAATAACTCCCTATAATGCGCCACCACTG str Ribosomal protein TTCTTGACACCTT.TCGGCATCGCCCTAAAATTCG GCGTCG rpoA RNA polymerase TTCTTGCAAAGTTGGGTTGAGCTGGCTAGATTAGC CAGCCA TTGaca TAtAaT R Promoter recognition by sigma factors

55. NN uvrB Repair DNA damage TTGTTGGCATAATTAAGTACGACGAGTAAAATTAC ATACCT recA DNArecombination CACTTGATACTGTA.TGAGCATACAGTATAATTGC TTCAACA rrnAB RibosomalRNA CTCTTGTCAGGCCG.GAATAACTCCCTATAATGCGCCACCACTG str Ribosomal protein TTCTTGACACCTT.TCGGCATCGCCCTAAAATTCG GCGTCG rpoA RNA polymerase TTCTTGCAAAGTTGGGTTGAGCTGGCTAGATTAGC CAGCCA TTGacaTAtAaT R Kp nifE nitrogenase accessory CTTCTGGAGCGCGAATTGCA TCTTCCCCCT Kp nifU nitrogenase accessory TCTCTGGTATCGCAATTGCT AGTTCGTTAT Kp nifB nitrogenase accessory CCTCTGGTACAGCATTTGCA GCAGGAAGGT Kp nifH nitrogenase CGGCTGGTATGTTCCCTGCACTTCTCTGCTG Kp nifM nitrogenase accessory TGGCTGGCCGGAAATTTGCA ATACAGGGAT Kp nifF nitrogenase accessory AACCTGGCACAGCCTTCGCA ATACCCCTGC Kp nifL nitrogenase regulat’n ATAAGGGCGCACGGTTTGCATGGTTATCACC glnA P2glutamine synthetase AAGTTGGCACAGATTTCGCTTTATCTTTTTT CTGG-ATTGCA AA Promoter recognition by sigma factors

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

57. Sigma factors in sporulation A A A A H H E E F F Stage II/III Mother cellForespore Forespore- specific Sigma-F Mother-specific Sigma-E

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

59. F F F F E E Sigma factors in sporulation A A A A H H E E F F Stage II/III E F Selective activation of sigma precursors Active mother- specific Sigma-E Active forespore- specific Sigma-F

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

61. 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

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

63. Genetic approach to Cell Biology

71. Isolation of Defective Gene Genetic approach to Cell Biology

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

73. Anabaena Spatially regulated differentiation Heterocyst differentiation by Anabaena How to find regulation of pattern? hetR Gene expression? hetR - +N -N +N -N +N hetR (wild-type)

74. GTA..(8).. TAC 5’-GTGAGTTAGCTCACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CACTCAATCGAGTGNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN Reporter gene Gene fusions to monitor expression 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNNNNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN hetR gene RNA Polymerase hetR Regulation

75. GTA..(8).. TAC NNNNNNNNNNNNNNNNNNATGNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNTACNNNNNNNNNNNNNNNN Reporter gene Gene fusions to monitor expression 5’-GTA..(8).. TACNNNNNNNNNNTANNNTNNNNNNNNNN 3’-CAT..(8).. ATGNNNNNNNNNNATNNNANNNNNNNNNN RNA Polymerase hetR Regulation

76. Detection of hetR gene expression through Green Fluorescent Protein The hydromedusa Aequoria victoria Source of Green Fluorescent Protein

77. Expression of hetR during differentiation Weak and patchy

78. Expression of hetR after differentiation Strong and focused

79. hetR - hetR (wild-type) hetR expression 0 18 Hrs after -N HetR is required for its own induction! hetR + hetR - hetR (wild-type) HetR Feedback Induction Expression of hetR after differentiation Other examples: spo0Aeve

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

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

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

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

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

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

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

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

88. Heterocyst differentiation by Anabaena The nature of the hypothetical inhibitor + RGSGR hetR HetR R color D + hetR + RGSGR HetR + patS -

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

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

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

92. 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

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

94. How to understand complexity?