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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Published: October 22nd, 2018

DOI:

10.3791/58339

1Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, 2Department of Genetics, Washington University School of Medicine, St. Louis, Missouri

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.

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.

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....

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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.......

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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.......

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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.......

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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-....

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Name Company Catalog Number Comments
E. coli MG1693 Coli Genetic Stock Center 6411 grows fine in standard unsupplemented LB
E. coli OP50 Caenorhabditis Genetics Center OP50
Click-iT EdU Alexa Fluor 488 Imaging Kit Thermo Fisher Scientific C10337
5-Ethynyl-2′-deoxyuridine Sigma 900584-50MG or use EdU provided in kit
Glucose Sigma D9434-500G D-(+)-Dextrose
Thiamine (Vitamin B1) Sigma T4625-5G Reagent Grade
Thymidine Sigma T1895-1G BioReagent
Magnesium sulfate heptahydrate Sigma M1880-1KG MgSO4, Reagent Grade
Sodium Phosphate, dibasic, anhydrous Fisher BP332-500G Na2HPO4
Potassium Phosphate, monobasic Sigma P5379-500G KH2PO4
Ammonium Chloride Sigma A4514-500G NH4Cl, Reagent Plus
Bacteriological Agar US Biological C13071058
Calcium Chloride dihydrate Sigma C3881-500G CaCl
LB Broth (Miller) Sigma L3522-1KG Used at 25g/L
Levamisole Sigma L9756-5G 0.241g/10ml
Phosphate buffered saline Calbiochem Omnipur 6506 homemade PBS works just as well
Tween-20 Sigma P1379-500ML
16% Paraformaldehyde, EM-grade ampules Electron Microscopy Sciences 15710 10ml ampules
100% methanol Thermo Fisher Scientific A454-1L Gold-label methanol is critical for proper morphology with certain antibodies
Goat Serum Gibco 16210-072 Lot 1671330
rabbit-anti-WAPL-1 Novus biologicals 49300002 Lot G3048-179A02, used at 1:2000
mouse-anti-pH3 clone 3H10 Millipore 05-806 Lot#2680533, used at 1:500
goat-anti-rabbit IgG-conjugated Alexa Fluor 594 Invitrogen A11012 Lot 1256147, used at 1:400
goat-anti-mouse IgG-conjugated Alexa Fluor 647 Invitrogen A21236 Lot 1511347, used at 1:400
Vectashield antifade mounting medium containing 4',6-Diamidino-2-Phenylindole Dihydrochloride (DAPI) Vector Laboratories H-1200 mounting medium without DAPI can be used instead, following a separate DAPI incubation
nail polish Wet n Wild DTC450B any clear nail polish should work
S-medium various see wormbook.org for protocol
M9 buffer various see wormbook.org for protocol
M9 agar various same recipe as M9 buffer, but add 1.7% agar
Nematode Growth Medium various see wormbook.org for protocol
dissecting watch glass Carolina Biological 42300
Parafilm laboratory film Pechiney Plastic Packaging PM-996 4 inch wide laboratory film
petri dishes 60 mm diameter
Long glass Pasteur pipettes
1ml centrifuge tubes MidSci Avant 2926
Tips
Serological pipettes
500 mL Erlenmyer flask
Aluminum foil
25G 5/8” needles BD PrecisionGlide  305122
5ml glass centrifuge tube Pyrex 
Borosilicate glass tubes 1ml
glass slides
no 1 coverslips 22 x 40 mm no 1.5 may work, also
37 °C Shaker incubator
Tabletop Centrifuge
Clinical Centrifuge IEC 428 with 6 swinging bucket rotor
Mini Centrifuge
20 °C incubator
4 °C refrigerator
-20 °C freezer
Observer Z1 microscope Zeiss
Plan Apo 63X 1.4 oil-immersion objective lens Zeiss
Ultraview Vox spinning disc confocal system PerkinElmer  Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy 

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