A Primary Culture System for Functional Analysis of C. elegans Neurons & Muscle Cells

1. A Primary Culture System for Functional Analysis of C. elegans Neurons & Muscle Cells Christensen M., Estevez A., Yin X., Fox R., Morrison R., McDonnell M., Gleason C., Miller DM., and Strange K. (2002) Neuron 33: 503-514

2. Kevin Strange, Ph.D. Professor of Anesthesiology, Pharmacology, and Molecular Physiology and Biophysics Ph.D. in Zoology, University of British Columbia, 1983 Michael Christensen, Ph.D. Pharmacology graduate student, 1997-2002 Postdoctoral fellow, Genomics Institute of the Novartis Research Foundation The Authors

3. Current Research • Laboratory focuses on physiology of ion channels and cellular osmoregulation • Most work is carried out in C. elegans and Drosophila. • Primary tools of research are: - patch clamp electrophysiology - quantitative microscopy - protein chemistry - reverse and forward genetics screening

4. Aims of this Paper • Provide a means of primary cell culture of nematode cells • To generate enriched populations of differentated C. elegans cells in culture • To grow cells large enough for patch-clamp studies

5. The Problem(s) • Tough cuticle limits access to specific cells • Individual cells or organs cannot be readily isolated in significant quantities • Mechanistic studies are severely curtailed: readings of membrane potentials and ion channel activity are blocked by cuticle • On the plus side: Detailed imaging studies are easy to perform on C.elegans cells

6. C.elegans cell culture • Large-scale cultures are in their infancy • Problems include: - cell survival - adherence to growth substrate - lack of cellular differentiation - poor reproductibility of results

7. Large-scale cell cultures • Currently, only embryonic C. elegans cells are known to have been successfully cultivated • If cells adhere to growth substrate, then morphological differentiation can occur

8. Methods • Culture media: L-15 with 10% fetal bovine serum • Successful glass coating agents include: • Peanut lectin • poly-L-lysine • Mixture of poly-D-lysine and laminin

9. Why are coating agents important? • Provides an anchor for cell-surface proteins • Prevents cell clumping and uneven growth

10. Methods, continued • First, adults are lysed using 0.5M NaOH and 1% NaOCl (bleach) • Lysis is stopped by 2 washes w/egg buffer • Eggs are pelleted and washed 3 more times • Eggshells are digested using chitinase • Embryos are dissociated by gentle pipetting • Intact embryos and clumps are filtered out • Remaining cells are plated in L-15 + 10% FBS

11. Major Findings • Primary embryonic cells can be cultured • Limited cellular differentiation will occur • Cells can develop more specialized morphologies if there are signaling cues, such as cell-cell contact Figure 2F: Muscle cell forming a Y-shaped synapse upon contact with a motoneuron in vitro This is analogous to the neuromuscular junction in vivo

12. Cell-cell contact • Embryonic cells of the same type, when they touch, can differentiate into 2 types • This is called a “binary switch” Red – Neuron specific marker unc-54 Green – Myo-3 muscle marker Yellow - Coexpression of unc-54 + unc-119 = Head muscle cell ?

13. Morphology of cultured cells • Roughly ~30% are body-wall muscle type • ~70% express neural progenitor unc-119 • Neural cells differentiate only to the relative extent that they are found in L1 larvae

14. Expression patterns in vitro • Levels of reporter gene expression for A and B motoneurons are in agreement with levels found in vivo • Postembryonic expression patterns (e.g.motoneurons found in L2 larvae) are not observed in vitro

15. Isolation of GFP expressing cells • Can use FACS to enrich subpopulations • Sorted populations can show subsequent small variations in GFP expression levels • Mitosis continues for ~24 hours in culture, suggesting that precursor cells are present Roughly 30% of cells are in mitosis or S-phase

16. Mechanical applications • Authors show that patch-clamp studies are feasible on cultured embryonic cells • Previous attempts at patch-clamping cultured cells had failed because cells were too small

17. RNAi feasibility in vitro • Addition of double-stranded RNA is demonstrated to interfere with normal gene expression of cultured cells in vitro • Authors showed a 50%-90% reduction in GFP levels after targeted dsRNA exposure • Western blotting shows nearly undetectable levels ofUNC-54 protein after 4 days’ incubation with anti-unc-54 dsRNA

18. Conclusions • In vitro culturing of up to 2 weeks’ duration is possible on disrupted embryos • In vitro cell populations are roughly proportional to those found in embryos • Cell fates are altered by disrupting the embryo; this lack of normal cues for differentiation suggests that a non-autonomous developmental process is occuring at this stage

19. Questions • What are the developmental cues essential for further cellular specialization? • How to mimic the physical cues for development? • Could promoters / transcription factors be used to provide the necessary triggers for development? • Can RNAi be used to repress certain developmental pathways in vitro? • Can immortal cell lines be created by cultivating cells that divide continuously?