1. The Parts and the Whole: How Genes Allow an Animal to Build Itself Itai Yanai Biology, Technion

2. "The ship wherein Theseus and the youth of Athens returned had thirty oars, and was preserved by the Athenians down even to the time of Demetrius Phalereus, for they took away the old planks as they decayed, putting in new and stronger timber in their place, insomuch that this ship became a standing example among the philosophers, for the logical question of things that grow; one side holding that the ship remained the same, and the other contending that it was not the same." —Plutarch, Theseus The parts relative to the whole: The ship of Theseus

3. The Development of an Animal http://www.wormclassroom.org/files/worm/EmbryoHatch.swf

4. Introducing C. elegans C. elegans is studied as a model organism for a variety of reasons. It is a a multicellular eukaryotic organism that is simple enough to be studied in great detail. Strains are cheap to breed and can be frozen. When subsequently thawed they remain viable, allowing long-term storage.

5. Research from the humble worm has proved fruitful 3 Nobel Prizes in the past 10 years

6. The development of the nematode C. elegans is comprised of five somatic founder lineages

7. C MS E P3 ABs Expresses the genes expected from the gut Evidence for mosaic development: segregation of developmental potential in the embryo Dissociate cells Laufer, Bazzicalupo and Wood. Cell 1980

8. C MS E P3 ABs Expresses the genes expected from the gut Evidence for mosaic development: segregation of developmental potential in the embryo Dissociate cells med-1,2 end-1,3 elt-2,7 Maduro & Rothman. Dev. Bio. 2002

9. ABa ABp Makes half of the pharynx ABp ABa Evidence for regulative development: blastomere swapping experiment Priess and Thomson. Cell 1987

10. Cost per megabase of DNA sequence

11. The reason for the drop in cost: sequencing occurs in a massively parallel fashion

12. Illumina HiSeq 2000 Image: www.illumina.com

13. Rachelly Normand Bioinformatician Dr. Tal Katz-Ezov Head of Center Dr. Maayan Duvshani-Eshet Head of LS&E Infrastructure Unit Yvgenia Lapis Lab technician Maya Abraham Bioinformatician Karen Chait Bioinformatician Anastasia Diviatis Lab technician Noa Henig Bioinformatician With generous support from:

14. Now possible to gauge gene expression levels for all genes by sequencing the transcriptome. Reads genome sequence lowly expressed gene highly expressed gene

15. Which genes are expressed at which time in development? Levin, Hashimshony, Wagner and Yanai. Developmental Cell (2012)

16. Time Which genes are expressed at which time in development? Gene expression levels Different genes

17. Which gene expression is conserved?

18. Stage 7 in mid-development that is uniquely conserved across different nematodes.

19. Hashimshony, Wagner, Sher and Yanai. Cell Reports (2012) Our lab developed a method for single cell RNA-Seq

20. .... Gene 1 Gene 2 Gene 3 Gene 20,000 Cell 1 Cell 2 Cell 3 Our lab developed a method for single cell RNA-Seq high low Expression n level

21. Using the method we can identify differential expression between the sister cells mex-3 Bowerman et al. Cell 1996

22. Hashimshony, Wagner, Sher and Yanai. Cell Reports (2012) CEL-Seq for multiplexed single cell RNA-Seq CEL-Seq is a single cell transcriptomics method using in vitro transcription. CEL-Seq is linear, sensitive and reproducible. CEL-Seq is highly multiplexed allowing for the parallel examination of hundreds of cells.

23. We are able to isolate and culture blastomeres

24. Which developmental pathways are dependent upon interaction among the lineages? Whole embryoSeparated embryo time ? C MS E P3 ABs

25. Gene expression analysis on the C. elegans blastomere lineages

26. The developmental expression profiles distinguish the contribution of the founder lineages Time Expression level (log10) Whole embryo unc-120

27. The expression of many genes is lineage specific Whole embryo Developmental stages Exp. level

28. Developmental Stages Exp. Level The expression of most genes is not specific to particular lineages and adds up to the whole-embryo expression Whole embryo Developmental stages Exp. level

29. The sum of the parts of the embryo generally matches the whole embryo. Time Number of molecules Whole embryo Parts + + +

30. An example of a gene with missing expression Developmental Stages Exp. Level pha-4 Whole embryo

31. Sum analysis Time Ratio (Sum of Parts / Whole) Over time, fewer genes are recapitulated completely

32. Genes involved in morphogenesis, such as the fusogen eff-1, are also missing in the parts. Developmental Stages Exp. Level eff-1

33. Spatial specificity Not time-specific time-specific Genes that are temporally restricted are also spatially restricted

34. C MS E P3 ABs Expresses the genes expected from the gut Known pathways can be recovered by detecting lineage specific expression med-1,2 end-1,3 elt-2,7

35. We are currently validating a predicted AB neurogenesis pathway Developmental Stages Exp. Level

36. GB Muller. Nature Reviews Genetics (2008) Extending the evolutionary synthesis

37. The evolution of pathways can be studied by comparing across distantly related species

38. Currently we are studying an entire phylum with Tardigrades as an outgroup.

39. Vlad GrishkevichShay Ben-ElazarAvital PolskyDavid SilverMartin Feder Our group at the Technion Gal AvitalNaftalie Senderovich Natalia Mostov Leon Anavy Michal Levin Sally Khair Dr. Tamar Hashimshony Assist. Prof. Itai Yanai

40. Thank you!

42. AB P 1 The vitellogenins are expressed in the whole embryo but not in the cells, even early on. Whole Parts No expressionHigh expression

43. GLP-1/Notch P0P0 P1P1 P2P2 EMS ABp ABa AB APX-1/Delta How is the pharynx programmed? A first Notch signal is received by ABp but not ABa glp-1 apx-1

44. GLP-1/Notch P0P0 P1P1 P2P2 EMS ABp ABa AB APX-1/Delta hlh-27 tbx-37,38 lag-2 How is the pharynx programmed? Without the signal, no hlh-27 and extra lag-2 and tbx-37,38 hlh-27 lag-2 tbx-37,38

45. GLP-1/Notch P0P0 P1P1 P2P2 EMS ABp ABa AB APX-1/Delta MSE ABa descendants GLP-1/Notch DSL-1??/Delta tbx-37,38lag-1 pha-4 ceh-22 Anterior Posterior lag-1 is present throughout though, another signal is missing? lag-1 ceh-22 pha-4

46. GLP-1/Notch P0P0 P1P1 P2P2 EMS ABp ABa AB APX-1/Delta MSE ABa descendants GLP-1/Notch DSL-1??/Delta tbx-37,38lag-1 pha-4 ceh-22 Anterior Posterior tbx-35 pha-4 med-1,2 MS descendants Why doesn’t the MS blastomere make pharynx? pha-4 tbx-35

47. How many genes overall are missing? Fraction covered (log2) Number of genes

48. Sulston et al. Dev. Bio. 1983 C. elegans development proceeds by an invariant cell lineage

49. C. elegans embryos develop according to an invariant cell lineage P0

50. 500 bp Chromosome II CEL-Seq reads have “sense” orientation

51. The expression of many genes is lineage specific Whole embryo Developmental stages Exp. level

52. CEL-Seq is highly reproducible

53. HOX gene expression across lineages

54. The five WNT ligands are not accounted for suggesting that this pathway is crucial for morphogenesis Developmental Stages Exp. Level

55. Tamar HashimshonyNoa SherFlorian Wagner The CEL-Seq team

56. “Theories come and go.. the frog remains” Jean Rostand

57. Dr. Tamar Hashimshony Martin Feder David Silver The Team Avital Polsky Michal Levin

58. The maternal expression is degraded asynchronously across the lineages Developmental Stages Exp. Level puf-5 oma-2 Developmental Stages

59. The maternal expression is degraded asynchronously across the lineages Developmental Stages Exp. Level puf-5

60. The maternal expression of many genes is degraded asynchronously

61. Zygotic expression is initiated asynchronously across the lineages

62. M03D4.4 may be a novel myogenic factor Developmental Stages Exp. Level Not known to be muscle-specific