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

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.......

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.......

Materials

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