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:

The Authors 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, Postdoctoral fellow, Genomics Institute of the Novartis Research Foundation

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

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

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

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

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

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

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

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 % FBS

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

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 ?

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

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

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

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

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 of UNC-54 protein after 4 days’ incubation with anti- unc-54 dsRNA

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

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?