1. Caenorhabditis elegans (C. elegans) An elegant worm

2. Why study worms? Sydney Brenner “Thus we want a multicellular organism which has a short life cycle, can be easily cultivated, and is small enough to be handled in large numbers, like a micro-organism. It should have relatively few cells, so that exhaustive studies of lineage and patterns can be made, and should be amenable to genetic analysis.” -- Excerpts from Proposal to the Medical Research Council, 1963

3. C. elegans: the chosen one! Photo credit: Ian D. Chin-Sang (Queen's University, Kingston, ON, Canada). Short generation time: 3 days Easily cultivated: can grow thousands on a petri dish, feed on non-hazardous bacteria, and cheap to maintain Small: 1 mm (about the size of a pinhead) Few cells: The adult has 959 hermaphrodrodite (XX) or 1031 (XO) cells Amenable to genetic analysis: maintained as hermaphrodites, but males exist for genetic studies, The genome is small- 100 Mb Transparency: allows for development to be analyzed from a single cell and all cells to be lineage

4. Life cycle of C. elegans Photo credit: http://www.scq.ubc.ca/genetic-studies-of-aging-and-longevity-in-model-organisms/

5. Anatomy of C. elegans Pharynx Intestine (yellow) Gonad (pink)Vulva Rectum Anus Epidermis head tail anterior posterior ~1 mm Fig. 8.43

6. Hermaphrodite (XX) Males (X0) Hermaphrodites do it by themselves Photo credit: http://homepages.ucalgary.ca/~dhansen/worms.gif

7. The C. elegans gonad: an extremely efficient reproductive system Fig. 8.42

8. Movie of C. elegans development

9. Within this lineage is the secret of embryonic development John Sulston

10. All neural synapses have been mapped

11. Learn to read a lineage diagram! = Cell death Line ending = differentiated cell Branching = cell division Increasing age of worm 1st stage larva 2nd stage larva embryo An entire C. elegans hermaphrodite worm consists of exactly 959 cells EVERY SINGLE TIME, allowing one to follow the cell lineage.

12. Cleavage Events Lineage P0 zygote2 cell stage4 cell stage8 cell stage

13. Most lineages consist of multiple tissue types but the P4, E and D cells gives rise to a single tissue type Fig. 8.43

14. Mutations can alter lineages in many ways

15. Question: 1.) How many cell divisions took place in the wildtype lineage? ____ 2.) In wild-type, how many total descendants will cell A have? ____ 3.) How many differentiated cells from the wild-type lineage will be a part of the adult worm? ____ 4.) What is the best description of the defect in mutant 1?

16. How are the invariant lineages established? ie. How do cells know who they are and what they are doing? Control of apoptosis Partitioning of cytoplasmic determinants Timing of developmental events Cell-Cell interactions

17. Even cell death is programmed into the lineage C. elegans was used to identify the machinery that regulates programmed cell death in vertebrates

18. The Nobel Prize in Physiology or Medicine 2002 "for their discoveries concerning ’ genetic regulation of organ development and programmed cell death'" Sidney BrennerH. Robert HorvitzJohn Sulston

19. Partitioning of cytoplasmic determinants P-granules (green) are cytoplasmic determinants that are formed from ribonucleoprotein complexes that specify the germ cells P0 AB P1 P3 P4 blue nucleigreen P-granules P granules are asymmetrically segregated into one cell, the P4 cell, which will give rise to the germline

20. Movie of P-granule movement

21. Photo credit: http://mbg.cornell.edu/cals/mbg/research/kemphues-lab/images/par_phenotypes.gif PARtition mutants (PAR) disrupt the asymmetric distribution of p-granlues

22. Moss E. 2007. Current Biology, R425. wildtype lin-14 (lof) lin-4 (lof) Lof= loss of function, gene function is disrupted Timing of developmental events Lin-14 is required for the timing of cell division in the L1 stage. Lin-4 regulates transition from L1 to L2 stage..

23. L1L2L3L4Adult LIN-4 LIN-14 Levels Time wildtype lin-14 (lof) lin-4 (lof) Graph of LIN-14 and LIN-4 levels in a wildtype embryo

24. L1L2L3L4Adult LIN-4 LIN-14 Levels Time wildtype lin-14 (lof) lin-4 (lof) Graph of LIN-14 and LIN-4 levels in a wildtype embryo If you have a mutation that results in an INCREASED level of LIN-14 (gain of function) which lineage would you expect

25. lin-4 does not encode a protein— what???? It encodes for a microRNA lin-4 lin-14 lin-4 Translation blocked! lin-14 lin-4

26. Cell-Cell Interactions: the P2 impact! Apx-1/Delta- like ligand Glp-1/Notch receptor mom-2/ Wnt ligand mom-5/ Wnt receptor Signal from P2 cell required to induce EMS cell to produce E cell which forms the gut (see p. 248)

27. How to cell interactions relate to the formation of an organ? Vulva formation!

28. Getting the terminology down: C. elegans Vulva Figure 6.27 Anchor cell (AC) Gonad VPCs Early larval stage P3.p-P8.p are the Vulva Precursor Cells (VPCs) AC Basement membrane Gonad Later larval stage P5.p,P6.p and P7.p lineages make the vulva P3.p,P4.p and P8.p lineages non-vulval 1° 2° 3° P6.pP7.pP5.pP3.pP4.pP8.p

29. Inductive and lateral signals induce the vulva gonad Anchor cell VPCs P3 P4 P5 P6 P7 P8 VPCs after induction 1° 3° 2° The primary and secondary cells form the vulva

30. How’d you know that? Cell ablation studies helped identify key players in vulva formation Lecture notes: experiment 1

31. Movie of cell ablation

32. gonad anchor cell 3° cell If anchor cell signaling is disrupted, all VPCs cells adopt a non-vulva fate 3° cell no vulva

33. The VPCs have multipotential gonad Anchor cell 1° 2° 3° What is causing the VPCs to be different? Early stage Later stage gonad Anchor cell VPCs P3 P4 P5 P6 P7 P8

34. Let’s do an experiment: what happens when the P6.p cell is ablated? gonad Anchor cell VPCs P3 P4 P5 P7 P8 1° 2° 3° 2° 3° A B 2° 1° 2°3° C P6 Lecture notes: experiment 2

35. What genes specify the VPC cell fate? Looked for mutants that disrupted vulva formation 1) No vulva: worms hatch inside (yuck!!) 1) Too many vulvas Lecture notes: experiment 3

36. Inductive and lateral signals induce the vulva gonad Anchor cell VPCs P3 P4 P5 P6 P7 P8 VPCs after induction 1° 3° 2° The primary and secondary cells form the vulva

37. Lin-3/Epidermal Growth Factor (EGF) Let-23/EGF Receptor Sem-5/GRB2 Let-60/RAS Lin-45/RAF P6.p becomes the primary cell! The vulvaless mutations helped define the Ras pathway

38. The Ras pathway is abnormally activated in many human tumors eg: pancreatic cancer, colorectal cancer, lung adenocarcinoma, gall bladder cancer, bile duct cancer and thyroid cancer signal (VPC cells) LIN-3 Another representation of the RAS pathway

39. The Ras mutation is so prevalent that kits are available to test of mutations that are linked to cancer

40. A signal from P6.p actives notch (lin-12) in P5.p and P7.p Figure 6.27

41. Both membrane and receptor are membrane bound! The transmembrane receptor is the Lin-12 protein, a receptor protein related to Notch “ Primary cell” “ Secondary cells”

42. Generation of Different Cell Types From Equivalent Cells in C. elegans: Initial specification of the Anchor Cell also requires Notch Figure 6.28 The signal: lag-2 (delta) The receptor: lin-12 (notch)

43. Does the Notch pathway remind you of anything you learned earlier? No notch=neural!

44. Nervous system Epidermis Extra nervous system No epidermis! Some cells become neuroblasts and signal their neighbors to remain epidermis If signal is missing... all cells eventually ingress and become neuroblasts The story of epidermal vs. neuronal fate in Drosophila