A Primary Culture System for A Primary Culture System for Functional Analysis of Functional Analysis of C. elegans C. elegans

Neurons & Muscle CellsNeurons & 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

The AuthorsThe 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,

1997-2002 Postdoctoral fellow, Genomics

Institute of the Novartis Research Foundation

Current ResearchCurrent 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 PaperAims 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)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 C.elegans cell culturecell culture

Large-scale cultures are in their infancyProblems include: - cell survival

- adherence to growth substrate- lack of cellular differentiation- poor reproductibility of results

Large-scale cell culturesLarge-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

MethodsMethods 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?Why are coating agents important?

• Provides an anchor for cell-surface proteins• Prevents cell clumping and uneven growth

Methods, continuedMethods, 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

Major FindingsMajor 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 contactCell-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 cellsMorphology 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 Expression patterns in vitroin 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 cellsIsolation 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 applicationsMechanical 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 RNAi feasibility in vitroin 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

ConclusionsConclusions

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

QuestionsQuestions

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?