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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Abstract

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Introduction

In animal development, hundreds, thousands, millions, billions, or even trillions of cell divisions are required to form the adult organism. The cell cycle, the set of cellular events composed of G1 (gap), S (synthesis), G2 (gap), and M (mitosis) define the series of events that are executed each cell division. The cell cycle is dynamic and best appreciated in real time, which can be technically difficult. The techniques presented in this protocol allow one to make the measurements of the phases and timing of the cell cycle from still images.

Labeling with nucleoside analogs such as 5-ethynyl-2'-deoxyuridine (EdU) or 5-bromo-2'-deox....

Protocol

1. Preparation of EdU-labeled Bacteria

  1. Grow a starter culture of MG1693. Escherichia coli (E. coli) MG1693 carries a mutation in thyA.
    1. Streak out E. coli MG1693 from a frozen glycerol stock onto a 120 mm lysogeny broth (LB) agar Petri dish. Culture at 37 °C overnight.
    2. Inoculate from two individual E. coli MG1693 colonies into two duplicate 4 mL tubes of liquid LB. Culture at 37 °C for ~16 h.
      NOTE: MG1693 grows fine i.......

Representative Results

Since DNA synthesis is required to incorporate EdU, one can conclude that EdU-labeled nuclei underwent S-phase during the EdU-labeling time window. One may interpret the nuclei that label in a 30 min feeding with EdU labeled bacteria as nuclei in S-phase at the time of dissection. Nuclei that label in a longer continuous EdU feeding experiment may have labeled early in the time window and since left S-phase, or may have labeled in the late part of the EdU time window. EdU signal co-locali.......

Discussion

Preparation of EdU-labeled bacteria (step 1) is critical for this protocol, and the first point for troubleshooting. Wild-type young adult hermaphrodites label very reliably in a 4 h EdU-pulse, making this a useful control for every new batch of EdU-labeled bacteria. Additionally, intact EdU-labeled bacteria that enter the intestine (in older animals or certain pharynx/grinder defective mutants) will label with click chemistry and appear as bright oblong puncta in the gut. An alternative technique for labeling hermaphrod.......

Acknowledgements

We are grateful to the E. coli stock center for MG1693; Wormbase; the Caenorhabditis Genetics Center which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40OD010440) for strains; Zach Pincus for statistical advice; Aiping Feng for reagents; Luke Schneider, Andrea Scharf, Sandeep Kumar, and John Brenner for training, advice, support, and helpful discussion; and the Kornfeld and Schedl labs for feedback on this manuscript. This work was supported in part by National Institutes of Health [R01 AG02656106A1 to KK, R01 GM100756 to TS] and a National Science Foundation predoctoral fellowship [DGE-1143954 and DGE-....

Materials

NameCompanyCatalog NumberComments
E. coli MG1693Coli Genetic Stock Center6411grows fine in standard unsupplemented LB
E. coli OP50Caenorhabditis Genetics CenterOP50
Click-iT EdU Alexa Fluor 488 Imaging KitThermo Fisher ScientificC10337
5-Ethynyl-2′-deoxyuridineSigma900584-50MGor use EdU provided in kit
GlucoseSigmaD9434-500GD-(+)-Dextrose
Thiamine (Vitamin B1)SigmaT4625-5GReagent Grade
ThymidineSigmaT1895-1GBioReagent
Magnesium sulfate heptahydrateSigmaM1880-1KGMgSO4, Reagent Grade
Sodium Phosphate, dibasic, anhydrousFisherBP332-500GNa2HPO4
Potassium Phosphate, monobasicSigmaP5379-500GKH2PO4
Ammonium ChlorideSigmaA4514-500GNH4Cl, Reagent Plus
Bacteriological AgarUS BiologicalC13071058
Calcium Chloride dihydrateSigmaC3881-500GCaCl
LB Broth (Miller)SigmaL3522-1KGUsed at 25g/L
LevamisoleSigmaL9756-5G0.241g/10ml
Phosphate buffered salineCalbiochem Omnipur6506homemade PBS works just as well
Tween-20SigmaP1379-500ML
16% Paraformaldehyde, EM-grade ampulesElectron Microscopy Sciences1571010ml ampules
100% methanolThermo Fisher ScientificA454-1LGold-label methanol is critical for proper morphology with certain antibodies
Goat SerumGibco16210-072Lot 1671330
rabbit-anti-WAPL-1Novus biologicals49300002Lot G3048-179A02, used at 1:2000
mouse-anti-pH3 clone 3H10Millipore05-806Lot#2680533, used at 1:500
goat-anti-rabbit IgG-conjugated Alexa Fluor 594InvitrogenA11012Lot 1256147, used at 1:400
goat-anti-mouse IgG-conjugated Alexa Fluor 647InvitrogenA21236Lot 1511347, used at 1:400
Vectashield antifade mounting medium containing 4',6-Diamidino-2-Phenylindole Dihydrochloride (DAPI)Vector LaboratoriesH-1200mounting medium without DAPI can be used instead, following a separate DAPI incubation
nail polishWet n WildDTC450Bany clear nail polish should work
S-mediumvarioussee wormbook.org for protocol
M9 buffervarioussee wormbook.org for protocol
M9 agarvarioussame recipe as M9 buffer, but add 1.7% agar
Nematode Growth Mediumvarioussee wormbook.org for protocol
dissecting watch glassCarolina Biological42300
Parafilm laboratory filmPechiney Plastic PackagingPM-9964 inch wide laboratory film
petri dishes60 mm diameter
Long glass Pasteur pipettes
1ml centrifuge tubesMidSci Avant2926
Tips
Serological pipettes
500 mL Erlenmyer flask
Aluminum foil
25G 5/8” needlesBD PrecisionGlide 305122
5ml glass centrifuge tubePyrex 
Borosilicate glass tubes 1ml
glass slides
no 1 coverslips 22 x 40 mmno 1.5 may work, also
37 °C Shaker incubator
Tabletop Centrifuge
Clinical CentrifugeIEC428with 6 swinging bucket rotor
Mini Centrifuge
20 °C incubator
4 °C refrigerator
-20 °C freezer
Observer Z1 microscopeZeiss
Plan Apo 63X 1.4 oil-immersion objective lensZeiss
Ultraview Vox spinning disc confocal systemPerkinElmer Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy 

References

  1. Fox, P. M., Vought, V. E., Hanazawa, M., Lee, M. -. H., Maine, E. M., Schedl, T. Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline. Development. 138 (11), 2223-2234 (2011).
  2. Crittenden, S. L., Leonhard, K. A., Byrd, D. T., Kimble, J.

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