1. Lecture 9 C. elegans cell biology C. elegans genetics C. elegans genome

2. Theoretically perfect model organism Well characterized life cycle-all stages easily accessible. Well characterized genetic system. Well characterized genome-basically sequenced and annotated. The ability to reintroduce DNA into the organism-transgenesis. Closely related to humans-funding purposes.

3. Caenorabditis elegans Life cycle: short 3 1/2 days, transparent organism, complete cell lineage known. Genetic system: both classical and RNAi Genome: first metazoan sequenced 1998 Transgenesis: injection of DNA Related to humans?

4. Caenorabditis elegans Hermaphrodite Horvitz and Sternberg Nature 351, 535

5. Hermaphrodite and male Wood 1998 The Nematode C. elegans

6. Cross section tube within a tube Wood 1998 The Nematode C. elegans

8. Life cycle Wood 1998 The Nematode C. elegans

9. Life cycle Hermaphrodite 558 nuclei Males 560 nuclei Wood 1998 The Nematode C. elegans

10. Hermaphrodite and male gonadogenesis Wood 1998 The Nematode C. elegans

11. Life cycle Hermaphrodite 959 somatic nuclei Male 1,031 somatic nuclei Wood 1998 The Nematode C. elegans

12. Worm’s brain White et al. Phil. Trans. Royal Soc. London 314, 1-340

13. All neuronal connections known White et al. Phil. Trans. Royal Soc. London 314, 1-340

14. Hermaphrodite and male Wood 1998 The Nematode C. elegans

15. Fertilization and the first divisions Kalthoff Analysis of Biological Development

16. Complete cell lineage Slack and Ruvkun Annu. Rev, Genet. 31, 611

17. Cell lineage Early divisions Lineage structure and nomenclature Cell death Repeated lineages

18. First four divisions and major blast cells Wood 1998 The Nematode C. elegans

19. First four divisions and major blast cells

20. Complete cell lineage Slack and Ruvkun Annu. Rev, Genet. 31, 611

21. Wormbase

22. Temporal and spatial information AB time AB.aAB.p

23. M.vlpaa Key blast cells are given upper case letters The progeny are named by adding lower case letters indicating the division axis: a-anterior p-posterior d-dorsal v-ventral l-left r-right

24. Mgreat great great grandmoth M.vgreat great grandmother M.vlgreat grandmother M.vlpgrandmother M.vlpamother M.vlpaadaughter Following the lineage

25. Cell death AB.alaaaala alar alal lr DEAD Neuron in ring ganglion Kalthoff Analysis of Biological Development

26. Repeated lineages Wormbase

27. Repeated lineages Wormbase

28. How is cell fate determined? English vs American view

29. Complete cell lineage Slack and Ruvkun Annu. Rev, Genet. 31, 611

30. Fertilization and the first divisions Kalthoff Analysis of Biological Development

31. How is cell fate determined? English vs American view Experimental approach: laser cell ablation

32. Nonautonomous determination Induction Equivalence groups

33. Induction 1 2 A cell or group of cells removed from a second cell that directs the developmental fate of a second cell or group of cells.

34. Example of induction Anchor cell-gonad signals Epidermis Vulva

35. Repeated lineages Wormbase

36. Equivalence groups: Group of cells that have equivalent pluripotent cell fates.

37. Z1.ppp Z4.aaa Individual AIndividual B Anchor cell/ Ventral uterine cell equivalence group AC VU

38. Z1.ppp Z4.aaa Experiment AExperiment B Anchor cell/ Ventral uterine cell equivalence group AC Cell ablation experiment

39. Z1.ppp Z4.aaa Experiment AExperiment B Anchor cell/ Ventral uterine cell equivalence group AC Cell ablation experiment The remaining cell always becomes an AC. The AC fate is the 1° (primary) cell fate.

40. Vulva equivalence group Wormbase

41. P3.p P8.p XX X Y YZ Vulva equivalence group

42. P3.p P8.p XX X Y YZ Vulva equivalence group X X Y YZ X X Y Y Z Z

43. Z is the 1° cell fate Y is the 2° cell fate X is the 3° cell fate

44. C. elegans genetics 1. Self-fertilization 2. Systematic approach with RNAi

45. Self-fertilization and homozygousity m/+ F 0 m/m m/+ +/+ F 1 m/m m/+ +/+ F 2 Self the population

46. Mutagenesis and screens P0P0 young hermaphrodite EMS +/+ +/+ +/+ +/+ +/+ +/m +/+ +/+ ….. F 1 self F2F2 All wild-type

47. Males X X hermaphrodite X O male At a frequency of 1/1000, males arise due to nondisjunction of the X chromosome.

48. Complementation analysis males m 1 /m 1 X hermaphordites m 2 /m 2 Look at males only?

49. Complementation analysis males m 1 /m 1 X hermaphordites m 2 /m 2 1. All males have mutant phenotype 2. All males are wild-type

50. Non complementation screen male a + m - /a + m - X hermaphrodite a - m + /a - m + EMS Most Wild-type

51. Non complementation screen male a + m - /a + m - X hermaphrodite a - m + /a - m + EMS Most Wild-type a - m + a + m -

52. Non complementation screen male a + m - /a + m - X hermaphrodite a - m + /a - m + EMS Most Wild-type Some a - a - m + a + m -

53. Non complementation screen male a + m - /a + m - X hermaphrodite a - m + /a - m + EMS Most Wild-type Some a - a - m + a + m - a - m +

54. Non complementation screen male a + m - /a + m - X hermaphrodite a - m + /a - m + EMS Most Wild-type Some a - a - m + a + m - a - m + Rare m -

55. Non complementation screen male a + m - /a + m - X hermaphrodite a - m + /a - m + EMS Most Wild-type Some a - a - m + a + m - a - m + Rare m - a - m -new a + m -

56. Transgenesis YFG roll D Look for rolling progeny F 1 Horvitz and Sternberg Nature 351, 535

57. Transgenesis YFG roll D Look for rolling progeny F 1 Look for rolling progeny in F 2 Horvitz and Sternberg Nature 351, 535

58. Transgenesis Nucleus of F2 rolling progeny YFGroll D YFG roll D Large concatenated arrays that are stablely maintained.

59. NCBI

60. RNAi inhibition of gene expression 1. RNAi discovered in C. elegans and plants. 2. Double stranded RNA results in the degradation of homologous mRNA. 3. Double stranded RNA can be fed to worms in the E. coli they eat. 4. Allows for the systematic inhibition of all 20,000 genes of C. elegans.

61. Systematic RNAi screens in C. elegans Tuschl Nature 421, 220