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Methods in
Molecular Biology 1463

Michael Buszczak Editor

Germline
Stem Cells
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
Germline Stem Cells

Second Edition

Edited by

Michael Buszczak
Department of Molecular Biology
University of Texas Southwestern Medical Center
Dallas, TX, USA
Editor
Michael Buszczak
Department of Molecular Biology
University of Texas Southwestern Medical Center
Dallas, TX, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-4015-8 ISBN 978-1-4939-4017-2 (eBook)
DOI 10.1007/978-1-4939-4017-2
Library of Congress Control Number: 2016945156

© Springer Science+Business Media New York 2008, 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

The registered company is Springer Science+Business Media LLC New York
Preface

Adult stem cells maintain tissue homeostasis by producing progeny that replace cells lost
through the course of normal cellular turnover or because of injury. Germline stem cells are
unique amongst stem cells because they produce daughter cells that develop into gametes,
which ultimately serve to give rise to the next generation. Thus, germline stem cells are
essential for the continued propagation of many sexually reproducing organisms.
Germ cells are imbued with several unique properties. They are the only cells to undergo
meiosis. Germ cells across species express many of the same markers and experience exten-
sive epigenetic reprogramming. These cells have specialized mechanisms that help maintain
the integrity of the genome and prevent the integration of selfish DNA elements. Several
somatic tumors express genes normally specific to germ cells and germ cells themselves can
give rise to specific types of cancer. Therefore, a better understanding of germ cells will have
a broad impact across many fields of biology.
Germline stem cell biology has witnessed an explosion of new findings and techniques
since that last edition of Germline Stem Cells in the Methods in Molecular Biology series.
The optimization of live-cell imaging techniques, improved cell purification protocols and
the identification of germ cells in a number of genetically tractable organisms now allows for
the characterization of germ cells at a greater depth and higher resolution than previously
attainable. This new edition of Germline Stem Cells is intended to provide researchers with
selected genetic, molecular, biochemical, and cell biological techniques used in germ cell
research. While the focus here is on primordial germ cells and germline stem cells, many of
the techniques and principles presented in the chapters of this issue may be applicable to
many different types of adult stem cells.
I would like to thank Prof. John M. Walker and the staff at Springer for their invitation,
assistance, and patience during the preparation of this book. I would like to thank Nevine
Shalaby for her help throughout this entire process. I would also like to express my sincere
appreciation and gratitude to the various contributors for sharing their insights and expertise
with the germline stem cell research community.

Dallas, TX, USA Michael Buszczak

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Analysis of the C. elegans Germline Stem Cell Pool. . . . . . . . . . . . . . . . . . . . . . . . . . 1

Sarah L. Crittenden, Hannah S. Seidel, and Judith Kimble
2 Methods for Studying the Germline of the Human Parasite
Schistosoma mansoni . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Julie N.R. Collins and James J. Collins III
3 Evaluation of the Asymmetric Division of Drosophila
Male Germline Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Mayu Inaba and Yukiko M. Yamashita
4 Live Imaging of the Drosophila Testis Stem Cell Niche . . . . . . . . . . . . . . . . . . . . . . 63
Leah J. Greenspan and Erika L. Matunis
5 RNA Isolation from Early Drosophila Larval Ovaries . . . . . . . . . . . . . . . . . . . . . . . . 75
Dana Gancz and Lilach Gilboa
6 Live-Cell Imaging of the Adult Drosophila Ovary Using
Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Nevine A. Shalaby and Michael Buszczak
7 Measurement of mRNA Poly(A) Tail Lengths in Drosophila
Female Germ Cells and Germ-Line Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Aymeric Chartier, Willy Joly, and Martine Simonelig
8 Identification of Germ-Line Stem Cells in Zebrafish. . . . . . . . . . . . . . . . . . . . . . . . . 103
Bruce W. Draper
9 Primordial Germ Cell Isolation from Xenopus laevis Embryos . . . . . . . . . . . . . . . . 115
Amanda M. Butler, Tristan Aguero, Karen M. Newman,
and Mary Lou King
10 Single-Cell Lineage Analysis of Oogenesis in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Lei Lei and Allan C. Spradling
11 Visualization and Lineage Tracing of Pax7+ Spermatogonial
Stem Cells in the Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Gina M. Aloisio, Ileana Cuevas, Yuji Nakada, Christopher G. Peña,
and Diego H. Castrillon
12 Transplantation as a Quantitative Assay to Study Mammalian
Male Germline Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Aileen R. Helsel and Jon M. Oatley
13 Mouse Fetal Germ Cell Isolation and Culture Techniques . . . . . . . . . . . . . . . . . . . 173
Cassy M. Spiller, Guillaume Burnet, and Josephine Bowles

vii
viii Contents

14 Rattus norvegicus Spermatogenesis Colony-Forming Assays. . . . . . . . . . . . . . . . . . 185

F. Kent Hamra
15 Identification of Mouse piRNA Pathway Components
Using Anti-MIWI2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Takamasa Hirano, Hidetoshi Hasuwa, and Haruhiko Siomi
16 Efficient Induction and Isolation of Human Primordial
Germ Cell-Like Cells from Competent Human
Pluripotent Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Naoko Irie and M. Azim Surani

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Contributors

TRISTAN AGUERO  Department of Cell Biology, University of Miami Miller School of

Medicine, Miami, FL, USA
GINA M. ALOISIO  Department of Pathology and Cecil H. and Ida Green Center for
Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas,
TX, USA
JOSEPHINE BOWLES  School of Biomedical Sciences, The University of Queensland, Brisbane,
QLD, Australia
GUILLAUME BURNET  School of Biomedical Sciences, The University of Queensland, Brisbane,
QLD, Australia
MICHAEL BUSZCZAK  Department of Molecular Biology, University of Texas Southwestern
Medical Center, Dallas, TX, USA
AMANDA M. BUTLER  Department of Cell Biology, University of Miami Miller School of
Medicine, Miami, FL, USA
DIEGO H. CASTRILLON  Department of Pathology and Cecil H. and Ida Green Center for
Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas,
TX, USA
AYMERIC CHARTIER  mRNA Regulation and Development, Institut de Génétique Humaine,
CNRS UPR1142 and University of Montpellier, Montpellier, Cedex 5, France
JULIE N.R. COLLINS  Department of Pharmacology, University of Texas Southwestern
Medical Centerr, Dallas, TX, USA
JAMES J. COLLINS III  Department of Pharmacology, University of Texas Southwestern
Medical Center, Dallas, TX, USA
SARAH L. CRITTENDEN  Department of Biochemistry, Howard Hughes Medical Institute,
University of Wisconsin-Madison, Madison, WI, USA
ILEANA CUEVAS  Department of Pathology and Cecil H. and Ida Green Center for
Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas,
TX, USA
BRUCE W. DRAPER  Department of Molecular and Cellular Biology, University of
California, Davis, Davis, CA, USA
DANA GANCZ  Department of Biological Regulation, Weizmann Institute of Science,
Rehovot, Israel
LILACH GILBOA  Department of Biological Regulation, Weizmann Institute of Science,
Rehovot, Israel
LEAH J. GREENSPAN  Department of Cell Biology, Johns Hopkins University School of
Medicine, Baltimore, MD, USA
F. KENT HAMRA  Department of Pharmacology, Cecil H. & Ida Green Center for
Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas,
TX, USA
HIDETOSHI HASUWA  Department of Molecular Biology, Keio University School of Medicine,
Tokyo, Japan
AILEEN R. HELSEL  Center for Reproductive Biology, School of Molecular Biosciences,
College of Veterinary Medicine, Washington State University, Pullman, WA, USA

ix
x Contributors

TAKAMASA HIRANO  Department of Molecular Biology, Keio University School of Medicine,

Tokyo, Japan; Division for Mammalian Development, National Institute of Genetics,
Mishima, Shizuoka, Japan
MAYU INABA  Department of Cell and Developmental Biology, Life Sciences Institute,
Howard Hughes Medical Institute, University of Michigan Medical School,
Ann Arbor, MI, USA; Department of Molecular Biology, University of Texas
Southwestern Medical Center, Dallas, TX, USA
NAOKO IRIE  Wellcome Trust Cancer Research UK Gurdon Institute, University of
Cambridge, Cambridge, UK; Department of Physiology, Development and Neuroscience,
University of Cambridge, Cambridge, UK
WILLY JOLY  mRNA Regulation and Development, Institut de Génétique Humaine, CNRS
UPR1142 and University of Montpellier, Montpellier, Cedex 5, France
JUDITH KIMBLE  Department of Biochemistry, Howard Hughes Medical Institute,
University of Wisconsin-Madison, Madison, WI, USA
MARY LOU KING  Department of Cell Biology, University of Miami Miller School of
Medicine, Miami, FL, USA
LEI LEI  Department of Cell and Developmental Biology, University of Michigan
Medical School, Ann Arbor, MI, USA
ERIKA L. MATUNIS  Department of Cell Biology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
YUJI NAKADA  Department of Pathology and Cecil H. and Ida Green Center for
Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas,
TX, USA
KAREN M. NEWMAN  Department of Cell Biology, University of Miami Miller School of
Medicine, Miami, FL, USA
JON M. OATLEY  Center for Reproductive Biology, School of Molecular Biosciences, College of
Veterinary Medicine, Washington State University, Pullman, WA, USA; Center for
Reproductive Biology, College of Veterinary Medicine, Washington State University,
Pullman, WA, USA
CHRISTOPHER G. PEÑA  Department of Pathology and Cecil H. and Ida Green Center for
Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas,
TX, USA
HANNAH S. SEIDEL  Department of Biochemistry, Howard Hughes Medical Institute,
University of Wisconsin-Madison, Madison, WI, USA
NEVINE A. SHALABY  Department of Molecular Biology, University of Texas Southwestern
Medical Center, Dallas, TX, USA; Institute for Biology, Freie Universit€
at, Berlin,
Germany
MARTINE SIMONELIG  mRNA Regulation and Development, Institut de Génétique
Humaine, CNRS UPR1142 and University of Montpellier, Montpellier, Cedex 5, France
HARUHIKO SIOMI  Department of Molecular Biology, Keio University School of Medicine,
Tokyo, Japan
CASSY M. SPILLER  School of Biomedical Sciences, The University of Queensland, Brisbane,
QLD, Australia
ALLAN C. SPRADLING  Department of Embryology, Howard Hughes Medical Institute,
Carnegie Institution for Science, Baltimore, MD, USA
 Contributors xi

M. AZIM SURANI  Wellcome Trust Cancer Research UK Gurdon Institute, University of

Cambridge, Cambridge, UK; Department of Physiology, Development and Neuroscience,
University of Cambridge, Cambridge, UK
YUKIKO M. YAMASHITA  Department of Cell and Developmental Biology, Life Sciences
Institute, Howard Hughes Medical Institute, University of Michigan Medical School, Ann
Arbor, MI, USA
 Chapter 1

Analysis of the C. elegans Germline Stem Cell Pool

Sarah L. Crittenden, Hannah S. Seidel, and Judith Kimble

Abstract
The Caenorhabditis elegans germline is an excellent model for studying the regulation of a pool of stem cells
and progression of cells from a stem cell state to a differentiated state. At the tissue level, the germline is
organized in an assembly line with the germline stem cell (GSC) pool at one end and differentiated cells at
the other. A simple mesenchymal niche caps the GSC region of the germline and maintains GSCs in an
undifferentiated state by signaling through the conserved Notch pathway. Downstream of Notch signaling,
key regulators include novel LST-1 and SYGL-1 proteins and a network of RNA regulatory proteins. In this
chapter we present methods for characterizing the C. elegans GSC pool and early germ cell differentiation.
The methods include examination of the germline in living and fixed worms, cell cycle analysis, and analysis
of markers. We also discuss assays to separate mutants that affect the stem cell vs. differentiation decision
from those that affect germ cell processes more generally.

Key words Stem cells, Progenitor cells, C. elegans, Germline, Proliferation, Meiosis, Mitosis, EdU,
Cell cycle

1 Introduction

Identification of stem cells and the pathways that regulate them are
important for both clinical research and more basic biomedical
science. The Caenorhabditis elegans germline is a simple and well-
studied model for understanding the genetic and molecular regu-
lation of stem cells [1–13]. Several qualities distinguish the
C. elegans gonad as a model for GSC regulation. First, in contrast
to other GSC models with asymmetrically dividing stem cells,
C. elegans GSCs are maintained as a pool (Fig. 1a) [14, 15].
Second, all stages of germ cell development, from stem cell to
differentiated gamete, are present in the adult gonad at one time
(Fig. 1a). Third, establishment and maintenance of C. elegans
germline stem cells is controlled by a simple, single-celled

Electronic supplementary material: The online version of this chapter (doi: 10.1007/978-1-4939-4017-2_1)
contains supplementary material, which is available to authorized users.

Michael Buszczak (ed.), Germline Stem Cells, Methods in Molecular Biology, vol. 1463,
DOI 10.1007/978-1-4939-4017-2_1, © Springer Science+Business Media New York 2017
1
2 Sarah L. Crittenden et al.

Progenitor Zone
Early meiotic
Distal pool Proximal pool prophase

DTC
niche

Stem cell-like Transition Differentiated

state between states state

B LST-1
SYGL-1
GLP-1/Notch Germline stem cell
signaling ? FBF-1 self-renewal
FBF-2

C Germline development #GC

L1
Proliferation phase

2
L2
16
L3
~60
L4
~400
Maintenance phase

Adult
(24 hour) ~2000
cell death
Adult embryos
(96 hour) ~2000

Fig. 1 Introduction to C. elegans germline development. (a) Diagram of adult

hermaphrodite. Gonad arms are color coded. Yellow, progenitor zone. Green,
meiosis. Pink, oocytes. Blue, sperm. Red, the somatic distal tip cell (DTC), which
is located at the distal end and provides a niche for proliferating germline stem
cells. Germ cells differentiate as they move away from the DTC, toward the
proximal end of the germline. (b) Pathway controlling the progenitor/
differentiation switch in the C. elegans germline. (c) Diagram showing the
stages of germline development from the larval proliferative phases through
the adult maintenance phase. Approximate germ cell numbers for each stage are
given on the right
 C. elegans Germline Stem Cells 3

mesenchymal niche, the distal tip cell (DTC) (Fig. 1a) [16]. Finally,
regulators of stem cell self-renewal have been identified and ana-
lyzed in depth (Fig. 1b). Both the Notch signaling pathway [1] and
the PUF family of RNA regulators maintain stem cells in C. elegans
[1, 7, 17, 18]. These regulators also control stem cells in other
systems. For example, Notch signaling has been suggested to play
roles in stem cell regulation in vertebrates ([4] and refs. therein).
PUF proteins are required for germline stem cell self-renewal in
flies ([1] and refs. therein) and have been found in human sperma-
togonia and embryonic stem cells [19].
The C. elegans GSCs generate the germline during larval devel-
opment, expanding it from 2 to 2000 cells (Fig. 1c); they maintain
the germline during adulthood, replenishing it as mature gametes
are lost to cell death and fertilization (Fig. 1c); and they regenerate
the germline after starvation [14, 20–22]. GSCs are pluripotent,
giving rise to both sperm and oocytes, which, after fertilization,
produce a totipotent embryo.
GSC behavior is influenced by sexual identity [23], food abun-
dance [21, 22, 24, 25], food quality [26], age [27, 28], and rate of
gamete production [23, 29]. In addition, a number of screens have
identified genes that modify the activity of the core pathway
controlling GSCs. Modifiers include splicing factors, components
of the proteasome, RNA binding proteins, and genes of unknown
function (see Refs. in [1, 2, 30–32]).
In this chapter we describe methods for studying C. elegans
germline stem and progenitor cells. Criteria to identify stem cells
can vary; thus it is crucial to define what is known in the system
being worked on and to define the criteria for identifying cell types.
In the C. elegans germline, immature stem and progenitor cells are
at the distal end, adjacent to the distal tip cell (Fig. 1a). The region
of the germline containing these cells has been referred to by
various names, including mitotic region, mitotic zone, and prolif-
eration zone. It has become clear that the undifferentiated state is
maintained independent of the cell cycle state, so a term such as
progenitor zone [27, 28] is more appropriate. The progenitor zone
contains the germline stem cells (GSCs) as well as their progeny in
various early states of differentiation. Lineage tracing is not yet
possible in the C. elegans germline, so GSCs cannot be unequivo-
cally identified. However, several experiments indicate that GSCs
reside within the distal part of the progenitor zone. There is a pool
of
35–70 undifferentiated cells found within the distal 5–8 rows
of the germline [15] that have similar cell cycle properties [14,
33–35], can regenerate the entire germline after starvation
[21, 22], and express high levels of the mitotic activators GLP-1,
lst-1, sygl-1, FBF-1 and FBF-2 and low levels of the meiotic activa-
tors GLD-1 and GLD-2 (Fig. 1b and Fig. 8) [1, 9, 15, 31, 36–38].
This group of cells comprises the GSC pool.
4 Sarah L. Crittenden et al.

Farther from the DTC, the proximal pool includes germ cells in
the early phases of differentiation. Two separate decisions, both of
which are required for gametogenesis, occur in this pool: the
decision to enter the meiotic cell cycle [15] and the decision to
become sperm or oocyte [39]. The proximal pool cells express
markers of both meiotic and sexual differentiation, such as GLD-
1, HIM-3, and FOG-1 (Fig. 8) [15, 38, 40–43]. Proximal pool
germ cells are similar to transit-amplifying cells in other systems in
that they have been triggered to differentiate but are still dividing
mitotically. Their amplification is not robust; they are estimated to
divide 1–2 times before entering meiosis [38]. In addition to
transit-amplifying cells, the proximal pool includes germ cells that
are premeiotic, defined as cells that will enter meiotic prophase
without passing through mitosis [14, 34, 41] (Fig. 1a and
Fig. 8). Since, at present, there are no good markers for premeiotic
S phase, we cannot reliably distinguish premeiotic S-phase cells
from mitotically dividing cells.
An outstanding question is whether all cells in the progenitor
zone have stem cell potential. It is technically difficult to test this
directly in the C. elegans germline; however, it is known that distal
and proximal pools have shared characteristics. They all express the
GLP-1/Notch receptor and depend on its activity to inhibit entry
into meiosis. They all cycle and do so at similar rates and they also
divide with a random orientation [8, 14, 23, 33, 34, 44]. They all
have sexual identity based on sex-specific differences in morphol-
ogy, gene expression, and cell cycle rate [23, 33, 43, 45–47].
Whether these shared characteristics indicate shared potential is
unclear.
We will present methods for identifying and characterizing
undifferentiated and proliferating germ cells, including identifica-
tion of the progenitor zone, putative stem cells, and the premeiotic
region. We will then discuss how we characterize new mutants.
There are excellent chapters about the germline and useful techni-
ques available on the WormBook website (http://www.wormbook.
org), the WormBook Methods website (http://www.wormbook.
org/toc_wormmethods.html), and in several books [48–51].

2 Materials

2.1 Reagents 1. 4 % agarose in dH2O for microscopy of live C. elegans.

2. M9: 3 g/L KH2PO4, 6 g/L Na2HPO4, 5 g/L NaCl, 1 mM
MgSO4.
3. E. coli M9 minimal media: 3 g/L KH2PO4, 6 g/L Na2HPO4,
0.5 g/L NaCl, 1 g/L NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2,
0.4 % glucose, 1.25 μg/ml thiamin.
4. M9 plus 0.25 mM levamisole.
 C. elegans Germline Stem Cells 5

5. Slides and coverslips.

6. Subbing solution:
(a) Bring 200 ml distilled water to 60  C.
(b) Add 0.4 g gelatin.
(c) Cool to 40  C.
(d) Add 0.04 g chrome alum.
(e) Add 1 mM sodium azide.
(f) Add poly-L-lysine (Sigma) to 1 mg/ml.
Store subbing solution at 4  C. To sub slides, put subbing
solution on slide for 10 min at room temperature. Wick off
excess liquid. Dry in 65  C oven for
30 min. Slides can be
stored in the oven or at room temperature.
7. Paraformaldehyde (16 % stock, Electron Microscopy Sciences).
8. PBSB: PBS (for 1 L: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4,
0.24 g KH2PO4, pH to 7.2 with NaOH) containing 0.5 %
BSA.
9. PBSBTw: PBSB containing 0.1 % Tween 20 to prevent
sticking.
10. DAPI (40 ,6-diamidino-2-phenylindole) (Molecular Probes,
Invitrogen).
11. Vectashield (Vector Labs).
12. Hoechst 33342 (Molecular Probes, Invitrogen).
13. SYTO-12 (Molecular Probes, Invitrogen).
14. Rabbit anti-PH3 polyclonal antibody (Upstate Biotechnology).
15. Mouse anti-PH3 monoclonal antibody (Cell Signaling
Technology).
16. M9-agar plates: 1.2 % agar, 0.6 % agarose in M9 salts contain-
ing 0.1 mg/ml carbenicillin [52].
17. Click-iT® EdU Alexa Fluor® 488 Imaging Kit (ThermoFisher).
18. TO-PRO-3 (Molecular Probes, Invitrogen).
19. Thymidine-deficient E. coli MG1693 (E. coli Genetic Stock
Center (CGSC), http://cgsc.biology.yale.edu/top.html,
CGSC #6411).
20. Psygl-1::H2B::GFP::sygl-1; Poma-1::OMA-1::GFP (Caenorhabditis
Genetics Center (CGC), http://cbs.umn.edu/cgc, C. elegans
strain #JK5018).
21. Plag-2::GFP (Caenorhabditis Genetics Center (CGC), http://
cbs.umn.edu/cgc, C. elegans strain #JK2868).
22. Plag-2::myr::GFP (Caenorhabditis Genetics Center (CGC),
http://cbs.umn.edu/cgc, C. elegans strain #JK4475).
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