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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).
6 Sarah L. Crittenden et al.

23. Plim-7::GFP (Caenorhabditis Genetics Center (CGC), http://

cbs.umn.edu/cgc, C. elegans strain #DG1575).
24. VALAP (equal parts petrolatum, lanolin, and paraffin wax,
melted to combine).

2.2 Web Resources 1. Reinke microarray data: http://cmgm.stanford.edu/
kimlab/

germline/.
2. In situ RNA expression database: http://nematode.lab.nig.ac.
jp/.
3. WormBase: http://www.wormbase.org/.
4. modENCODE: http://www.modencode.org/.
5. C. elegans strain collection (CGC): http://www.cbs.umn.edu/
CGC/.
6. WormBook: http://www.wormbook.org/.
7. WormMethods: http://www.wormbook.org/toc_wormmethods.
html.
8. WormAtlas: http://www.wormatlas.org.

3 Methods

3.1 Identification The C. elegans germline is composed of two U-shaped tubes con-
of the Progenitor Zone taining approximately 2000 germ cells in different states of differ-
in Wild-Type entiation, which can be observed in living animals (see Fig. 2a). The
C. elegans germline is syncytial, although cells in the progenitor zone are
Hermaphrodites nearly completely enclosed by membrane and are referred to as
germ cells. The undifferentiated germ cells, including GSCs, reside
at one end, adjacent to the somatic distal tip cell. The organization
of progenitor and meiotic cells is similar in hermaphrodites and
males. C. elegans hermaphrodites are self-fertile XX animals which
first make sperm, then oocytes, whereas males are XO animals that
make only sperm.
In wild-type young adult hermaphrodites (see Note 1), the
progenitor zone is approximately 20 cell diameters in length and
contains approximately 225–250 germ cells. The length of the
progenitor zone is defined as the number of cell diameters between
the distal tip cell and the transition zone (TZ) (Subheading 3.5,
Fig. 8, and refs [14, 41]). The TZ contains cells in early meiotic
prophase; when stained with DAPI, the DNA in these nuclei has a
distinctive crescent shape [53, 54] (crescents, Fig. 3, Subhead-
ings 3.3 and 3.5). Most cells between the DTC and the transition
zone are proliferating; however, approximately 50–100 germ cells
in the most proximal rows are premeiotic [14, 34]. In wild-type
germlines, apoptosis occurs in some oogenic cells in the late pachy-
tene stage of meiotic prophase (Subheading 3.4, ref. [55]). Apo-
ptosis does not occur in the male germline [55].
 C. elegans Germline Stem Cells 7

A undifferentiated
germ cells pachytene
germ cells

oocytes
sp

vulva uterus/spermatheca
wild-type
B
sp
intestine

sp

vulva
fbf-1 fbf-2
C undifferentiated germ cells throughout gld-2 gld-1; fbf-1 fbf-2
intestine

vulva

Fig. 2 Microscopy of wild-type and mutant germlines. (a) Differential interference contrast (DIC) micrograph of
a wild-type adult hermaphrodite. (b) DIC micrograph of an fbf-1 fbf-2 double mutant germline. Mature sperm
are seen at the distal end. (c) DIC micrograph of a gld-2 gld-1; fbf-1 fbf-2 mutant germline containing only
undifferentiated germ cells. Arrowheads indicate distal end of germline

3.2 Mounting Live 1. Make an agarose pad on slide: drop melted 4 % agar on glass
Animals for Light slide, quickly put another slide on top at a 90 angle and press
Microscopy (see to create a thin layer of agarose (about 0.4 mm thick) (see ref
Notes 2 and 3) [56], p. 596). Slides with lab tape can be used as spacers for the
top slide [57, 58].
2. Pick animals onto agarose pad next to a drop of M9 containing
0.25 mM levamisole (see Note 4). Levamisole will prevent ani-
mals from moving. Tricaine and tetramisole are also used [59].
Animals can be rescued from agarose pads by carefully sliding
coverslip to the side and then picking the animals from the pad
to a plate.
3. Cover with coverslip, avoiding bubbles. For short-term obser-
vation, it is not necessary to seal the edges of the coverslip. For
longer term imaging, coverslips can be sealed with VALAP [50].
4. Observe germline using differential interference contrast
(DIC) and/or fluorescence microscopy.
8 Sarah L. Crittenden et al.

Progenitor Transition
zone zone (TZ) Pachytene region

Wild-type

Progenitor TZ nuclei Pachytene

nuclei nuclei

Fig. 3 Identification of progenitor zone cells and meiotic germ cells. DAPI-stained distal arm of a wild-type
adult hermaphrodite. Progenitor zone, transition zone (TZ), and pachytene region nuclei are enlarged below.
Dashed and dotted lines indicate the boundaries between the zones. Note the crescents of DNA in the TZ
nuclei and the condensed strands of chromatin in the pachytene nuclei. Arrowhead points to DTC nucleus.
Modified from Eckmann et al., 2014 [63]

3.3 Immuno- We use two methods for extruding and processing gonads (somatic
histochemistry tissues remain associated with the germline) for immunohisto-
of Extruded Gonads chemistry: extrusion on slides and extrusion in solution ([41, 60];
Schedl lab website—protocols http://genetics.wustl.edu/tslab/).
One benefit of extruding in solution is the increased efficiency for
preparing large numbers of gonads, although there are anecdotal
reports of improved staining using the slide method. There are
several detailed protocols for staining C. elegans gonads that
include alternate fixation protocols [60, 61] (see Notes 5 and 6).
If only DAPI staining is required, skip the primary antibody step
and incubate the fixed germlines with DAPI. Continue on from
there (see Note 7).
1. Extrude gonads. To extrude on a slide, pick 10–20 animals into
7 μl PBS containing 0.25 mM levamisole on subbed slide. Cut
the animals with a syringe needle or scalpel behind the pharynx
or at the tail. The germline and intestine will extrude from the
cuticle (see Note 8). Make a sample area with a PAP pen to keep
the liquid from spreading across the slide (see Note 9). To
extrude in solution, pick or wash 10–100s of animals off plate
into a glass or plastic dish containing PBS, 0.25 mM levamisole
plus 0.1 % Tween 20 to prevent sticking. The total volume can
be anywhere from 200 μl to a couple milliliters depending on
your personal preference and size of the dish. Use a micropi-
pettor to transfer samples into a microfuge tube. For the fol-
lowing steps, the volumes should be matched to the technique
 C. elegans Germline Stem Cells 9

you use to extrude gonads. For extrusion on slides, generally

25–50 μl is sufficient to cover the sample area. For the solution
method, volumes for fixation, permeabilization, and washes are
generally 200–1000 μl, and volumes for antibody incubation
are 50–100 μl; we usually place tubes on a rotator or rocker
during all steps.
2. Fix. The fixation method will depend on the antibody antigen
combination you are using (see Note 5) [60]. A good place to
start is 1–3 % paraformaldehyde for 10–20 min. If you
extruded on a slide, keep the slides in a humidified chamber
(e.g., a box with a wet paper towel taped to the lid) throughout
the procedure.
3. Permeabilize. Remove paraformaldehyde and replace with
0.1 % Triton X100 in PBSB for 5 min at room temperature.
4. Block. In PBSBTw for 30 min at room temperature.
5. Incubate with primary antibody. Dilution, incubation time, and
temperature should be determined for each antibody.
6. Wash three times with PBSBTw for 15 min each.
7. Incubate with secondary antibody. Dilution, incubation time,
and temperature should be determined for each antibody in
PBSBTw (see Note 10). Include 0.1 μg/ml DAPI to stain DNA
(see Note 11).
8. Wash three times with PBSBTw for 15 min each.
9. Add 7–10 μl Vectashield or other mounting medium; if samples
are in a tube, pipet onto a slide, place coverslip over gonads, and
seal with nail polish. The volume of mounting medium will
depend on the size of the coverslip. The volume should be large
enough that gonads are not squashed, but small enough that
they are not floating around. Excess liquid can be carefully
wicked away using a kimwipe.
10. Observe using fluorescence microscopy.

3.4 Visualization 1. Incubate animals in 33 μM SYTO-12 (taken up by dying cells)

of DNA and Germline and/or 50 μg/ml Hoechst 33342 (DNA dye) in M9 with some
Apoptosis in Live E. coli in a microfuge tube for 4–5 h (see Notes 12 and 13).
Animals [55, 62] 2. Transfer to seeded plate for 30–60 min.
3. Mount on agarose pad for DIC microscopy.
4. Observe live animals on agarose pads (Subheading 3.2) using
fluorescence microscopy and DIC. Hoechst can be observed
using DAPI filter sets. SYTO-12 emits green fluorescence and
can be observed using FITC filter sets.
10 Sarah L. Crittenden et al.

3.5 Scoring the The progenitor zone extends from the distal-most germ cell to the
Length and Cell start of the transition zone.
Number in the
1. Using DAPI-stained germlines (Subheading 3.3), identify the
Progenitor Zone distal end by locating the DTC nucleus (oval with diffuse DAPI
staining, Figs. 3 and 4) (see Note 14).
2. The start of the transition zone has been defined as either the
position of the first crescent [41] or the position at which there
are multiple crescents (to reduce the chance of scoring occa-
sional anaphase chromosomes that are curved similar to
crescent-shaped meiotic nuclei [8, 14, 63]). These two posi-
tions are usually within 1–3 rows of each other (see Note 15)
(Fig. 3).
3. Count germ cell rows and total number of cells (see Note 16)
starting with the row immediately adjacent to the DTC nucleus
and continuing to the transition zone (Fig. 4). Counting can
be done visually either through microscope eyepieces or on a
computer monitor. For training yourself to count with reason-
able accuracy, count the germline multiple times until your
numbers are within about 5 % of each other. A software tool
for counting germ cells (IRISES) is available and facilitates the
counting process with minimal manual input [64].
4. Progenitor zones in young adult wild-type hermaphrodite
germlines average 19–20 cell diameters from the distal end
(range 15–24) and contain 225–250 germ cells (range
200
to
300) (see Note 1). These numbers change with age (e.g.,
[27, 28]), so comparing staged animals is important. In addi-
tion, the number of germ cells in the progenitor zone does not
always correlate with the length (e.g., male progenitor zones
are longer than hermaphrodite, but contain the same number
of cells [23]).

Hoechst
DTC

1
2 3456
7 8 9 10 12 22 24
14 16 18 20

Fig. 4 Counting cell diameters from distal end of germline. Distal arm of a wild-
type adult hermaphrodite gonad stained with Hoechst. DTC nucleus is circled in
red, germ cell nuclei are circled in white. Numbers begin with 1 for the nucleus
closest to the DTC and increase moving proximally. Dashed line indicates TZ
boundary. Modified from Byrd et al., 2014 [62]
 C. elegans Germline Stem Cells 11

3.6 Characterization 1. sygl-1 and lst-1 expression. Two GLP-1/Notch targets, sygl-1
of the GSC Pool and and lst-1, are expressed in a subset of germ cells immediately
GSC Number adjacent to the DTC in the progenitor zone. The expression of
these genes is dependent on GLP-1/Notch signaling, making
their expression a useful marker for GLP-1/Notch responsive
germ cells [65] within the distal pool. Expression patterns for
endogenous sygl-1 and lst-1 can be examined by in situ hybri-
dization; detailed methods can be found in Kershner et al.,
2014 [65] (see Note 17). sygl-1 and lst-1 are also expressed in
maturing oocytes, so mutants that cause distal germ cells to
differentiate may express sygl-1 and lst-1 distally due to differ-
entiation rather than maintenance of an undifferentiated state.
sygl-1 expressing cells can also be visualized using animals car-
rying a single copy chromosomal insertion of a transgene con-
taining the sygl-1 promoter driving GFP::H2B [65]. Note that
some germlines expressing this transgene are not completely
normal and that expression is partially silenced. Germline
silencing is common in C. elegans and can be alleviated by
maintaining transgenic animals in a licensing background
[66–68] and/or by growing them at 25  C. See Kershner
et al., 2014 (Ref. [65] Materials and methods) for further
details.
2. emb-30 assay. In this assay, germ cell movement is blocked and
germ cells are scored for differentiation with time [15]. The
emb-30(ts) mutant at restrictive temperature blocks the meta-
phase to anaphase transition, arresting cell division and cell
movement [15]. After shift to restrictive temperature, the
distal-most 6–8 cell rows, containing about 50 germ cells,
arrest in M phase and remain undifferentiated, indicating that
this pool of cells is maintained in an immature state. More
proximal germ cells enter the meiotic cell cycle, indicating
that cells in that position in wild-type gonads have been trig-
gered to differentiate and would normally continue into meio-
sis as they move proximally (Fig. 5, compare panels (a) and (b),
Note 18).
(a) Maintain emb-30(tn377ts) at 15  C. Animals are fertile;
however, progenitor zones are slightly smaller than in wild
type. Maintain strains on plates that are equilibrated to
15  C. If you are determining distal pool size in mutants,
compare mutant; emb-30 to þ; emb-30 in parallel.
(b) Stage animals. For animals comparable to hermaphrodites
grown for 24 h after L4 at 20  C (see Note 1), pick L4s
and let them grow at 15  C for 36 h.
(c) Shift staged animals to restrictive temperature (25  C) by
picking adults to plates that have been equilibrated at
25  C.
12 Sarah L. Crittenden et al.

0 hours
B

15 hours anti-PH3 anti-GLD-1 DAPI

Fig. 5 emb-30 assay. Gonads were shifted from permissive (15  C) to restrictive (25  C) temperatures for
times shown in white lettering (Panel (a), 0 h at restrictive temperature; Panel (b), 15 h at restrictive
temperature). Gonads were extruded and stained with anti-PH3 (green), anti-GLD-1 (red), and DAPI (cyan).
Distal end is marked with white triangle, GLD-1 boundaries with red carats, most proximal PH3 with green
arrow. GLD-1 often has two stepwise increases in intensity—each is marked with a caret in the 0 h panels (a).
These steps can be absent or look graded in some gonads, making it hard to score. Also note the variable
nuclear morphology in distal end of the 15 h DAPI-stained gonad (b). Dashed line indicates TZ boundary.
Modified from Cinquin et al., 2010 [15]

(d) Every hour, or at your chosen time points, fix gonads

(Subheading 3.3) and stain with DAPI, anti-PH3 and
anti-GLD-1 or anti-HIM-3 (Fig. 5a, b). Score the posi-
tion of the boundary between metaphase-arrested nuclei
(visualized with DAPI and anti-PH3) and transition zone
nuclei (visualized with DAPI). Also score the position of
the boundary between low and high levels of meiotic
proteins such as GLD-1 and/or HIM-3. Positions can
be scored in germ cell diameters and/or microns (see
[15, 28]; Note 18).

3.7 Markers A number of markers are helpful for marking the progenitor zone,
for Progenitor Zone the distal pool of progenitor cells, differentiating progenitors, germ
and Early Stages cells in the meiotic cell cycle, and somatic gonad cells that affect
of Differentiation proliferation (Table 1, Fig. 8).
Progenitor zone markers include the Notch receptor, GLP-1,
which is abundant in membranes of progenitor zone cells [69];
endogenous sygl-1 and lst-1 transcripts ([65]; see Subheading 3.6)
and a transgene containing the sygl-1 promoter driving H2B::GFP
([65]; see Subheading 3.6), which are abundant in the distal-most
germ cells; the PUF family RNA regulators, FBF-1 and FBF-2,
which are abundant in the cytoplasm of progenitor zone cells
[70–72]; the p53 homolog, CEP-1, which is present in progenitor
zone nuclei [73]; and the meiotic cohesin, REC-8, which is
enriched in the nuclei of progenitor zone cells under certain fixa-
tion conditions [8, 41]. All of these markers are also expressed
either at lower levels in early meiotic prophase or in other parts of
the germline (Table 1, Fig. 8). Meiotic markers include the RNA
regulator, GLD-1, which increases in the cytoplasm of germ cells in
 C. elegans Germline Stem Cells 13

Table 1
Useful markers

Marker Molecular identity Pattern

lst-1 Novel GLP-1/Notch target Transcript is present in a distal subset of germ
cells [65]
sygl-1 Novel GLP-1/Notch target Transcript is present in a distal subset of germ
cells [65]
Psygl-1::H2B:: sygl-1 promoter driving GFP is present in a distal subset of germ cell
GFP::sygl-1 histone fused to GFP nuclei [65].
GLP-1 Notch receptor Membrane-associated, strong in mitotic germ
cells, lower levels in early meiotic region
[69]
FBF-1 and PUF family RNA regulators Cytoplasmic, strong in progenitor zone.
FBF-2 Levels decrease as germ cells progress
through meiosis. See Lamont et al., 2004
for details of expression patterns [70, 71]
REC-8 Cohesin On chromosomes in meiotic prophase (except
when used as mitotic marker) (see ref [41,
142])
CEP-1 C. elegans p53 Nuclear in progenitor zone and proximal
cells [73]
PH3 Histone H3 phosphorylated Chromosomes in late prophase (mitotic and
on serine 10 meiotic) and M phase [14, 41, 80]
GLD-1 maxi-KH/STAR domain Cytoplasmic, increasing in proximal mitotic
RNA binding protein cells, strong in meiotic germ cells [40, 47]
HIM-3 Component of central Cytoplasmic in proximal progenitor zone, on
element of synaptonemal chromosomes in meiotic prophase [42]
complex
PGL-1 P-granule component Only in germ cells, not in somatic cells [137]
SP56 Sperm-specific protein Spermatocytes and mature sperm [138]
RME-2 C. elegans yolk receptor Oocyte membranes [141]
Plag-2::GFP lag-2 promoter driving GFP Somatic DTC [14, 74]
Plim-7::GFP lim-7 promoter driving GFP Somatic sheath cells 1–4 [75, 121]

the proximal pool and peaks in the meiotic region [40, 47], and the
meiotic protein HIM-3, which is present in proximal progenitor
zone cells and becomes localized to chromosomes in early meiotic
prophase [41, 42]. Hansen and colleagues [41] used a combination
of REC-8 and HIM-3 staining to identify a region of overlap called
the meiotic entry region. The meiotic entry region has substantial
overlap with the premeiotic S region identified by cell cycle analysis
[14, 34, 41] (Fig. 8).
14 Sarah L. Crittenden et al.

A cap

L4

B long external processes

cap

plexus Adult

Fig. 6 DTC morphology. Gonad from hermaphrodite expressing myristoylated GFP driven by the lag-2
promoter. Hermaphrodites have a single DTC/gonad arm. The DTC has a membranous cap that covers the
distal-most germ cells during larval development (a) and adulthood (b). During adulthood, the DTC extends a
complex network of processes (plexus) as well as long external processes. Male gonads have two DTCs per gonad
arm. Their morphology is less complex, but similar to the hermaphrodite. Modified from Byrd et al., 2014 [62]

3.8 Markers for the The somatic niche, the DTC, can be visualized using a transgene
Somatic Gonad with the lag-2 promoter driving GFP [14, 74]. Finer details of
membrane structure can be seen using a transgene with myristoy-
lated GFP [62]. In hermaphrodites, the DTC has a membranous
cap that surrounds the distal-most germ cells. The DTC produces
extensive processes in early adulthood; these processes surround
and intercalate into the membranes between the germ cells in the
distal pool (plexus, Fig. 6). During adulthood, the DTC also
extends long, external processes [14, 62, 75, 76]. There is a similar
DTC architecture in males; however, the lag-2 promoter is much
weaker in the male DTCs, making it harder to visualize processes
[23]. Other ligands such as APX-1 are also expressed in the niche
and play a role in stem cell maintenance [77] (see Note 19).
The somatic sheath cells also play a role in germline patterning,
especially during larval stages [78 and refs. therein]. Sheath cells can
be visualized using a transgene with the lim-7 promoter driving GFP.
The sheath cells also have extensive contact with the surface of the
germ cells and extensive intercalation between germ cells [75, 78].

3.9 Cell Cycle Cell cycle analysis allows estimation of cell cycle lengths, identifica-
Analysis tion of quiescent or slow-cycling cells, and identification of cells
entering meiotic S phase [79]. Anti-phosphohistone H3 (PH3)
staining, which labels condensed chromosomes in late G2 and M-
phase nuclei [80], is used to determine mitotic index (% of cells in
M phase) (Subheading 3.10) [14, 34, 41]. Alternatively, mitotic
figures can be scored by DAPI staining [44]. EdU labeling is used
to determine the S-phase labeling index (% of cells in S phase) and
to estimate total cell cycle length (Subheadings 3.11–3.18). Under
fixation conditions that preserve morphology well, EdU labeling
combined with nuclear size can be used to identify G1 vs. G2 cells
 C. elegans Germline Stem Cells 15

(Subheading 3.14). The proximal region of the progenitor zone,

where mitotic index is low and labeling index is the same as the rest
of the progenitor zone, is likely composed largely of premeiotic
germ cells (Subheading 3.18). Some premeiotic S-phase cells are
also found in the transition zone.

3.10 Mitotic Index 1. Prepare animals for antibody staining (see Subheading 3.3).
with Anti-PH3 2. Stain with anti-PH3. In our hands, mouse anti-PH3 (Cell
Signaling) reliably stains prophase and metaphase nuclei. Ana-
phase and telophase nuclei stain weakly and more variably.
3. Score position and number of PH3-positive nuclei in progeni-
tor zone (Fig. 5, see Note 16).
4. Count number of nuclei/row for normalizing.
5. Calculate mitotic index by dividing number of PH3-positive
cells by the total number of cells in the region of interest.

3.11 EdU Labeling EdU labeling can be performed by feeding or by soaking [23, 28,
34, 38, 52]. Both methods are appropriate for pulse labeling
(
15–30 min), but only feeding is appropriate for pulse-chase
labeling or for labeling over longer time periods, because the stress
of soaking can affect cell cycle dynamics (see Note 21). Germ cells
can also be labeled with BrdU [14, 81] and Cy3dUTP [33, 82, 83];
however, BrdU is more difficult than EdU to visualize well and
Cy3dUTP has to be injected.

3.12 Label with EdU 1. Grow an overnight culture of E. coli MG1693 in LB or in M9

by Feeding minimal media supplemented with 5 μg/ml thymine.
2. To 100 ml of E. coli M9 minimal media, add:
3 ml of MG1693 overnight culture.
100 μl 0.5 mM thymidine (0.5 μM).
200 μl 10 mM EdU (Invitrogen) (20 μM) (see Note 20).
Grow at least 24 h at 37  C. Spin to pellet E. coli. Resuspend in
about 1 ml M9.
3. Seed plates with 200–400 μl labeled MG1693. Try to cover
most of the plate so that animals are continuously feeding on
labeled bacteria. To prevent growth of bacteria after seeding
(which should be avoided because plates do not contain EdU),
use nematode growth media lacking peptone and containing
60 μg/ml carbenicillin. Alternatively, plates containing E. coli
M9 agar þ 60 μg/ml carbenicillin can be used, although we
have found that the ammonia in E. coli M9 minimal media can
affect the cell cycle of C. elegans germ cells. Plates can be stored
at 4  C for a couple of weeks.
4. Put animals on plates for desired time period.
16 Sarah L. Crittenden et al.

5. To chase, wash animals off plate with M9, wash once with M9,
and place animals on plate containing unlabeled bacteria.
6. Fix with 3 % PFA 30 min at room temperature. Weaker fixation
conditions can also be used (e.g., 1 % PFA for 10 min), but
they do not preserve chromosome morphology well and there-
fore make it more difficult to distinguish mitotic figures and the
nuclear size difference between G1 and G2 cells (see
Subheading 3.14).
7. Permeabilize with 0.1 % Triton X-100 5 min at room tempera-
ture or with 20  C methanol for 5–15 min.
8. Do the Click reaction according to the instructions. We do
2  30 min half reactions (suggested by [84]).

3.13 Label with EdU 1. Transfer animals by picking or washing into M9 þ 0.1 %
by Soaking Tween-20 þ 0.05–1 mM EdU. Incubate with rotation for
15 min.
2. Dissect gonads immediately (with no recovery period) and fix
according to the protocol for labeling with EdU by feeding,
above.

3.14 Calculating Cell cycle stage in mitotically dividing germ cells correlates with
Labeling Index and nuclear size: G1 and early S-phase nuclei are small; G2 and late S-
Identifying G1 and G2 phase nuclei are large; and mid S-phase nuclei are intermediate in size
Cells (Fig. 7, Movie 1). The size difference between G1 and G2 cells is
large enough that after pulse labeling with EdU to mark S-phase cells,
G1 and G2 cells can be distinguished by eye. This correspondence
between cell cycle stage and nuclear size has been vetted in wild-type
germlines of both sexes, but it may not hold true under conditions
that alter nuclear size, such as after knockdown of certain cell cycle
regulators [34, 85]. Keep in mind that the fraction of cells detected as
EdU-positive will be affected by the sensitivity of the detection sys-
tem. Early S-phase nuclei label weakly with EdU and may be detected
as G1 cells by image acquisition systems that are less sensitive.
1. Pulse animals with EdU for a short time, e.g., 15–30 min (see
Subheadings 3.11–3.13).
2. Fix and stain as described in Subheadings 3.11–3.13. Co-stain
with DAPI.
3. Examine germline at high magnification (e.g., 63), and iden-
tify G1, S-phase, and G2 cells using the following criteria. We
find it convenient to acquire z-stack images at 0.5–1 μm inter-
vals and to identify cell cycle stages on a computer monitor.
Cells can be easily marked and categorized using the Cell
Counter plug-in for ImageJ (http://fiji.sc/Cell_Counter).
(a) G1 cells are EdU-negative and have a nuclear size equal to
or smaller than the smallest EdU-positive cells (Fig. 7c,
Movie 1). Additional characteristics of G1 cells are that
 C. elegans Germline Stem Cells 17

A
DAPI Progenitor zone Transition zone

EdU (S-phase)

10 µm

B C
Cell-cycle Nuclear EdU
phase size labeling? Example

G1 Small —

Early S Weak

Mid S Strong

Strong &
Late S
punctate

G2 Large —

Prophase n/a —

Metaphase n/a —
DAPI
EdU (S-phase) Anaphase n/a —
G1, EdU- small nuclei 10 µm
G2, EdU- large nuclei
Telophase n/a —
Paired occurence of G1 cells

Fig. 7 EdU labeling. (a) Image of an adult hermaphrodite germline stained with DAPI to visualize DNA (top) and
labeled with a 15-min pulse of EdU to mark S-phase cells (bottom). Images are maximum-intensity z-
projections. (b) Two-color images of adult hermaphrodite germlines treated as in (a). Magenta, DAPI staining.
Green, EdU. Yellow circles, G1 cells. Blue circles, G2 cells. Caret, occurrence of G1 cells in pairs. (c) EdU
labeling and nuclear size characteristics for cells in different stages of the cell cycle. Scale bar, 2 μm. n/a,
nuclear size not applicable during M phase

they are infrequent (
5–10 %) and nearly always occur in

pairs (Fig. 7b, Movie 1), a pattern consistent with G1
being very short [28, 34]. Chromatin in G1 cells tends
to be rather condensed (Fig. 7c), such that the 12 chro-
mosomes are often distinguishable as 12 DAPI-stained
puncta (not shown).
(b) S-phase nuclei are EdU-positive. S-phase nuclei can be
further categorized as early, mid, or late according to
nuclear size and pattern of EdU labeling (Fig. 7c,
Movie 1). Early S-phase nuclei are small and label weakly
Exploring the Variety of Random
Documents with Different Content
 220 itants, masters of families incorporated together into a
town fellowship, and such others whom they shall admit unto them,
only in civil things. ' The great- grandfather of Charles W., William W.
Reynolds, came from Westerly, R. I., and settled in Petersburgh in
1780. Prior to this, in 1777, he served in the defense of his country
against the English, at the battle of Bennington. He spent his
remaining days in Petersburgh, being supervisor in 1801, 1802 and
1803, and magistrate for many years. The grandfather of this
subject was Parley Reynolds, who was born in Petersburgh in 1780.
He became a merchant and for many years, in partnership with his
brother Thomas, conducted an extensive and profitable business in
Petersburgh, and was supervi,sor in 1837 and 1838. William W.
Reynolds, the father of Charles W., was born September 25, 1816,
and died June 4, 1876, and was supervisor in 1847, 1848, 1856 and
1857. He was married to Mary (born January 14, 1825), daughter of
Braddock Peckham, jr. (born June 4, 1781. died January 7, 1834),
and granddaughter of Bfaddock Peckham, sr. (born May 4, 1757,
died January 9, 1830), who was a soldier in a Rhode Island regiment
during the Revolutionary war. Previous to this service he was .second
in command in an expedition composed of patriotic citizens of
Wickford, R. I., that made a prisoner of the British General Prescott,
July 10, 1777, at Newport, R. I. ; the prisoner was delivered to
General Washington at Newburgh by the same party, and on July 18,
1777, was exchanged for Major-General Harry Lightfoot Lee. At the
close of his connection with this duty, he came to the valley of the
Little Hoosick, looking for a future home. He had but just arrived
when Captain Hull's company was being formed to go to the relief of
General Stark at Bennington; he joined this company, was made
lieutenant and served in that capacity at the battle of Bennington
and continued with the company until after the battle of Bemis
Heights and the surrender of Burgoyne, when the company was
disbanded ; he then joined the command of General Gates and with
that little army of 1,500 marched away to New Jersey. He was at the
defeat of Brandywine and on the bloody field of Monmouth. He
remained with General Gates's command until the latter was
superseded by Gen. Nathaniel Greene, and with him saw the
surrender of Cornwallis at Yorktown. At the termination of the war
he returned to his home in Rhode Island, and in 1786, accompanied
by his brother Abel, came to the beautiful valley of the Little Hoosick
and there reared a family of thirteen children and where many of his
descendants still reside. The first ancestor in this county of Braddock
Peckham was John Peckham of Newport, R. I., who was admitted an
inhabitant May 20, 1638; he married Mary Clarke, who was a sister
of the Rev. John Clarke from Bradfordshire, England, " one of the
ablest men of the seventeenth century and a founder of Rhode
Island." In 1648 John Peckham was one of the ten male members in
full communion of the First Baptist church. Charles W. Reynolds grew
to manhood on his father's farm, and obtained his education in the
common schools, at Fort Edward Institute and Alfred University.
When twenty-one years of age his father assisted him in purchasing
an interest in a general 1 "The government established by these
primitive settlers of Providence was an anomaly in the history of the
world. At the outset it was a pure democracy, which for the first time
guarded jealously the rights of conscience by ignoring any power in
the body politic to interfere with those matters that concern man
and his Maker. Principle, not precedent, formed their only standard
of judgment. Could the record of their proceedings have been
preserved (meetings were held monthly), with what interest should
we now pursue the debates of this earliest of modern
democracies!"— Arnold's History of Rhode Island.
 231 store in the village of Petersburgh in partnership with
the late David H. Kellyer where they soon after, in connection with
their mercantile interests, began the manufacture of shirts by
contract, and with such encouraging success that in 1874 they sold
their store and engaged exclusively in the fnanufacture of shirts on
their own account, in which undertaking they have been successful
as well as furnishing employment to a large number of people. Mr.
Reynolds makes the village of Petersburgh his home, but spends the
winters at his Albany residence where his children enjoy greater
educational advantages. In 1874 he married Lucy M. Gifford, born
December 7, 1856, a native of Albany and daughter of Alonzo (born
March 9. 1832) and Mary J. (Hakes) Gifford (born August 4. 1835),
who has borne him five children, as follows. William G., born August
13, 1875; George T., born September 21, 1878; Grace born
December 31, 1880; Alonzo P., born January 21, 1886: and Noyes,
born April 8, 1891. Mr. Reynolds has traveled extensively over the
United States, and in 1891, accompanied by his son William G., was
of the party of over two hundred Knights Templar who visited
Europe. Mr. Reynolds has never sought office, but in the spring of
1896 was elected supervisor of Petersburgh without opposition- and
at a considerable personal sacrifice consented to serve in that
capacity. Thacher, Ralph W., was born in Brockport, N. Y., April 24,
1839. He is a son of Dr. Ralph Thacher, who was born in Lebanon,
Conn., where five generations of Thachers have lived or were born.
Mr. Thacher's mother was Jerusha B. Harrison of Williamstown,
Mass. The first member of the Thacher family in America was the
Rev. Thomas Thacher, first pastor of the Old South church in
Bo.ston, Mass., from whom is also descended John Boyd Thacher,
mayor of Albany. Rev. Thomas Thacher landed at Boston in the ship
James in August, 1635, in charge of his uncle, Anthony Thacher, who
had been a curate of his father's church in Salisbury, England. Rev.
Peter Thacher, the father of Rev. Thomas, was rector of St.
Edmund's church at Salisbury, England, and lies buried in the
churchyard under the shadow of Salisbury cathedral. Ralph W.
Thacher, the subject of this sketch, and seventh in descent from Rev.
Thomas Thacher, spent the years of 1855 and 1856 at Williams
College and was graduated from Hamilton College in 1859. While at
Hamilton he was a member of the Phi Upsilon fraternity. After
leaving college Mr. Thacher removed to Albany, N. Y. , in 1860 and
engaged in the grain business with David N. Glazier and Harvey D.
Leonard. After three years Mr. Thacher was taken into partnership
and the firm became Glazier, Leonard & Co., which existed five
years. Mr. Leonard then retired and the firm became for two years
Glazier & Thacher. In 1870 Mr. Thacher withdrew and went to
Kansas, where he established the First National Bank of Ottawa, of
which he was cashier five years and vicepresident four years,
including two years after he returned to Albany, in 1877. When Mr.
Thacher returned to Albany he bought of David N. Glazier the
business that he was originally interested in. Mr. Glazier was then in
failing health and shortly after died. Mr. Thacher continued in this
business until July, 1891, coupling with it a mill and elevator at
Schenectady, N. Y., a mill and elevator at Kenwood, near Albany, two
malt houses in Albany and a coal yard in Schenectady, having in all
ninety employees. He retired from that business to go into the
export trade in New York in 1891, that being the year when there
was a shortage in all the wheat producing countries in the world
save America. Mr. Thacher was very successful
 222 in New York and in the fall of 1892 he retired from
active business on account of impaired health. In November, 1896,
he took the presidency of the Albany Art Union as a pastime,
growing out of his liking for amateur photography and to somewhat
satisfy his love of the beautiful in art. Mr. Thacher is a member of
Masters Lodge No. 4, F. & A. M., and a demitted member of Temple
Chapter, R. A. M. ; he was also a charter member of the Fort Orange
and Albany Clubs. He is now a member of the University Club of
New York and of the New York Produce Exchange. He was formerly
a member of the Boston Chamber of Commerce and the Chicago
Board of Trade. His first wife was Anna Elizabeth Glazier, of
Brockport, N.Y., by whom he has one daughter. Mrs. F. W. Stedman,
of Albany. His present wife was Louisa C. Huntington, of Albany, by
whom he has a son, Ralph Huntington Thacher. Lawson, Stephen,
was born in 1830, and is a son of Levi, and grandson of Lawrence
Lawson, who first settled at Bethlehem and later at Rufus Corners,
where he died and left two sons, James and Levi. Levi came to
Coeymans in 1830 and bought the farm where Stephen now lives.
He was a farmer and died in 1860. He had four sons: Henry, William,
Isaac, and Stephen, who remained on the homestead, and has two
sons: Frederick and Howard. Griffen, Edward C, son of Edward and
Harriett (Perkins) Griffen, was born in Newark, N. J., September 5,
1868. In 1875 he moved with his parents to Schuylerville, N. Y.,
where he attended the high school at that place. Subsequently he
attended the Albany Business College and graduated from that
institution June 6, 1887, when he entered the employ of Henry
Russell, flour merchant, and remained with him seven years, rising
to the position of bookkeeper. In January, 1894, Mr. Griffen resigned
his position with Mr. Russell and opened a store at No. 43 Hudson
avenue, where he deals in flour, feed, hay and grain. He is one of
Albany's youngest merchants and is respected for his integrity,
perseverance and fair dealing. February 10, 1892, he married
Harietta Meader of (Juaker Springs, N. Y., and they have one son,
Chauncey Rider. Miller, S. Edward, jr., was born in Albany, N. Y., in
1855. His father for many years was a prominent merchant on
Broadway. His mother's maiden name was Sarah Frances Silsby. On
the paternal side, Mr. Miller is descended from Elizabeth Staats
(great- grandmother) who was born just below Albany in the old
Staats homestead, the oldest inhabited house in America, bearing
date of erection of 1630. Mr. Miller received his education in the
public and high schools and was bookkeeper for Coming & Co. until
1882, when he opened a men's furnishing store at No. 36 Maiden
Lane. His business rapidly increased so that in 1891 he took
premises at No. 34 Maiden Lane; now he occupies Nos. 34 and 36.
He began this business in a small way and owing to his pleasant
manner and fair dealings, was not long in having it very well
established. He now has a plant outside used solely for the
manufacture of shirts giving employment to a large number of
hands. Mr. Miller has a large double store and does the largest
strictly furnishing goods business in the State, outside of New York
and Buffalo. He has a very large custom shirt trade extending to all
parts of the United States, and the Hanan shoe agency which is
developing into a large business. He is a member of the Albany Club,
Old Guard, Albany Zouave Cadets and the Empire and Capital City
Curling Clubs. Mr. Miller is also
 a life member, ex-vice-president and director of the Young
Men's Association and a member of the Y. M. C. A. In 1880 he
marriedSarah Louise Nash, daughter of John II. Nash and sister of
Willis G. Nash, cashier of the New York State Bank. They have two
children: Louise Adele and Edgar Nash. Danaher, John E.. son of
Francis M. and Mary E. (Hillenbrant) Danaher, was born in Albany, N.
Y., March 4, 1861. He attended the publicschools and Christian
Brothers' Academy and graduated from the Albany High School in
1878. After leaving the high school he obtained a situation as
bookkeeper for Tallmadge & Carter, commission merchants, and
remained with this firm a year and a half. Subsequently he was
bookkeeper and afterward traveling salesman for William H.
Livingston, wholesale liquor dealer, with whom he remained seven
years, when in 1886, he started in the wholesale liquor business for
himself at No. 34 Green street. He remained at that location for one
year and then owing to increased business he moved to Nos. 304
and 396 Broadway, where he was located five years, when his
business became so large that he was compelled to find more
suitable quarters and moved to his present location No. 97 Hudson
avenue, corner of Grand street, with storehouse in the rear at No. 14
Grand street. Mr. Danaher is a member of the Catholic Union, the
Commercial Traveler's Club, and is a member of the Board of Control
of the National Wholesale Liquor Dealers Association of America. He
married Elizabeth B., daughter of Patrick McCarthy, for many years a
builder and alderman of Albany. They have one daughter, Hortense
E. Mr. Danaher's success may be accounted for so.-newhat by the
fact that he was born of that good stock, Irish and German. His
maternal grandparents were of the first German immigrants to locate
in Albany, where they came in 1830. Mr. Danaher if a self made man
and great praise is due to his efforts. He does a strictly wholesale
business, being a large direct importer of wines and brandies and
has sole control of the "Optimus" brand of whiskey. He has a large
busmess equal to and as important as any in Albany. Ertz Berger,
Edmund J., son of William G. and Mary L. (Sheridan) Ertz Berger,
was born in Albany, N. Y., Septembers, 1856. About 1765, Daniel
Ertz Berger came to America from Basil, Switzerland, and settled in
Albany and engaged in trading skins and furs with the Indians, and
was in many a bloody encounter with them. Daniel, his son, the
grandfather of Edmund J., vyas born in Albany in 1788, and
Charlotte Dunlap, his wife, was born in Albany in 1794. William G.,
the father of Edmund J., was a manufacturer of cigars and candies in
Albany and did an extensive business during the war. He died in
1885, aged seventy-five. Edmund J.'s mother died when he was two
years old and he went to live with an uncle who put him through the
public schools and high school, from which he was graduated in the
English and mathematical course in 1874. He then entered the
employ of S. L. Munson, shirt and collar manufacturer, where he
learned the business and with whom he remained twenty years,
rising rapidly until he had entire charge of the shirt department. In
1881 Mr. Ertz Berger went West on an extensive trip for his health.
In 1894 he entered the Hudson River Garment Company in
partnership with William R. McGraw, and is now junior partner and
financial manager. Mr. Ertz Berger is a member of the Unconditional
Republican Club, the Ancient Essenic Order and treasurer of the
Albany Bicycle Club. In 1883 he married Eloise Ross of Albany, and
they have one daughter. Edna D.
 224 Cass, Lewis.— This citizen of Albany, for many years
]5rominent aniony those interested in the welfare of the city, was
born at Decatur, Otsego county, N. Y., December 30, 1853. His father
was a farmer, and his early life was passed upon his father's farm. At
the age of twelve, he was left an orphan. At the age of sixteen, he
began to teach in the district schools in Otsego county, at "a dollar a
day and boarded around." Afterwards he passed successfully
through the State Normal School, Colgate Academy at Hamilton, N.
Y , graduating from the former in 1872 and the latter in 1874. He
pursued a collegiate course at Union College, and graduated from
that institution in 1878. In the summer of 1878, he began to study
law with the celebrated firm of Smith, Bancroft & Moak, where he
remained for three years, when he opened an office of his own for
the transaction of business. In 1886 he married Miss Kate Landon,
eldest daughter of Judge Landon of Schenectady, N. Y. Mr. Cass early
took a high rank as a lawyer, and especially as an advocate, being
connected with many important litigations, notably, the case of "
McDonald against the Village of Gloversville," and " The Trumbell will
case " in Albany county, and many other important litigations in
Circuit, Probate and Criminal Courts. He was attorney for the New
York State Dairy Commissioner, and afterwards for the Commissioner
of Agriculture of the State of New York for seven years, and for the
past two years attorney for the New York State Veterinary Medical
Society. Mr. Cass is well known as an ardent, fearless advocate of
progress, and has been a potent factor in various reforms and
improvements in the city, notably, the project of the construction of
Beaver Park in the south portion of the city. To no one man is there
more credit due for this much needed improvement than to Mr. Cass.
Being a forcible and fluent public speaker, his services are eagerly
sought in political campaigns. Although deeply interested in politics
and political affairs, he has never sought nor held a political office,
preferring to remain a private citizen. He has a well selected library
of classic and historic literature and fiction, with which he is
exceedingly familiar. He was selected in 1888, to deliver the annual
address before the Adelphic Society of Union College, and chose for
his subject "The Duty of the Educated Man to Business and Society."
Another topic upon which he has been heard with interest and
propriety is "The Puritans," which perhaps is his best known lecture.
Love for his early occupation abides with him, as shown by the fact
that he is one of the most successful amateur florists in the city,
turning his special attention to roses, having ,a collection
unsurpassed by any in the city. Gilbert, Henry S., is one of the
leading citizens of Guilderland. He was born in the town of New
Scotland, March 5, 1846. His father was Williams Gilbert, born in the
town of Bethlehem, April 18, 1823. His paternal grandfather was
also Williams, who married first Ora Hart, who bore him eleven
children: Glazier, Noah, Elkanah, Maria, Laura, Ann, Bradley, Alvin
and Calvin (twins) and Prudence: his second wife was Charity
Barber, by whom he had four children: Eliza, Rachel Ann, Joseph and
Elisha. WilHams. father of Henry S., married Hannah Houghton (born
in NewScotland, April 4, 1831) in December, 1843; she was one of a
family of ten children born to David ( born January 24, 1878) and
4nna (Bryant) Houghton (born February 2, 1777), and
granddaughter of John and Dorcas (Lawrence) Bryant ; her brothers
and sisters were Polly, Lucy, John, Silas, Eli, Catharine, Smith, Sally
and Jane Ann ; she was the last survivor of her family. Williams
followed farming all his life, living
 325 some years in New Scotland and in 1856 removing to
Guilderland where he bought a farm and resided until 1865, when
he sold his farm and removed to Glenville, Schenectady county;
there he bought a farm on which he resided until his death, which
occurred in September, 1873. The only child of Williams and Hannan
(Houghton) Gilbert was Henry S., the subject of this sketch. Mrs.
Gilbert survived her husband many years, cared for by her son till
the time of her death, January 14, 1895. Henry S. Gilbert attended
the district school and remained with his father until the latter's
death, when he sold the farm and bought his present one of 100
acres near Fuller's Station, to which he moved in 1874. He has been
successfully engaged in dairying, keeping a fine lot of choice cows;
he also takes much pride in keeping fine horses. In 1890-91 he
engaged in mercantile business at Fuller's Station, where he owned
a store, and where he was also postmaster under Harrison's
administration, but not liking the business he sold out and returned
to his farm, on which he has since resided. He deals in agricultural
implements, handling the Johnson harvesting machines; he is a
director and stockholder in the Altamont Driving Park and Fair
Associations, and was chairman of the committees on fruit and
vegetables, and on stock and poultry, also horses. In January, 1867,
he married Helen C. Weaver, a native of Glenville, Schenectady
county, daughter of Benjamin aad Hannah (Clossen) Weaver. They
have two children, William W., born January 14, 1868, and Burton
H., born April 29, 1876. William W. married Hattie, daughter of Leroy
Main, and has one child, Ethel ; he remained on the farm with his
father until April, 1896, when he removed to Voorheesville where he
now resides. Burton A. is at home with his parents. Frederick,
Charles F., son of Philip and Catharine (Gomph) Frederick, was born
in Albany, N. Y., August 21, 1865. He is a grandson of Philip
Frederick, who was born in Germany, and who came to Albany in
1830, where he engaged in the furniture business and was one of
the best known and most highly respected citizens of Albany. His
son, the father of the subject of this sketch, followed his father's
business with the addition of the undertaker's business, and gave
promise of building up a remarkable business, but was cut off in
early manhood. He died in 1874, aged thirty-seven, leaving a family
of eight children, all of whom are now living. He was prominent in
fraternal and social circles, being a Mason, an Odd Fellow, and
Knight of Pythias; he was also an ex-niember of the 25th Regiment,
and in 1870 represented the then Tenth ward in the Board of
Supervisors. Charles F. Frederick, the subject of this sketch, was
educated in the public schools and learned the trade of bookbinder
with R. G. Hendrie, with whom he remained eight years; at the end
of five years he was promoted to the position of foreman of Mr.
Hendrie's establishment and held that position when he left Mr.
Hendrie's employ. In 1886 Mr. Frederick removed to Washington, D.
C, where he obtained an appointment as l30okbinder in the
government printing office and remained there si.x years, resigning
to go into the grocery business in Washington. He was compelled to
abandon this business after three years owing to ill health, and in
September, 1895, returned to Albany. In January, 1896, he took a
course in the United States Embalming College in New York city,
from which he received a diploma. In March of the same year he
started his present business, that of undertaker and embalmer, at
No. 118 Washington avenue. Mr. Frederick is a member of the
American Legion of
 Honor, the International Brotherhood of Bookbinders and
Clinton Lodge No. T, I. O. O. F. November 16, 1887, he married
Sarah Furman of Albany, and they have one son, Charles F., jr. Van
Valken burgh, Hon. John W., was born in the village of Chatham,
Columbia county, N. Y., June 23, 1826, and is a son of James B. Van
Valkenburgh, also of Chatham, who fought gallantly at Plattsburgh
during the war of 1812. He lived until he was eighty-one years of
age, dying August 15, 1868. The maiden name of Mr. Van
Valkenburg's mother was Clarinda Pitts, an aunt of Hon. Edmund
Pitts, ex-speaker of the Assembly. She died July 3, 1871, at the age
of eighty-one. His grandfather, Bartholomew Van Valkenburgh, was a
native of Holland and came to America at an early date, settling at
Chatham, N. Y. He served with distinction in the Revolutionary war.
In his early youth, J. W. Van Valkenburgh, the subject of this sketch,
attended the common schools in Chatham and worked on his
father's farm. When he became of age he joined a military company
and on November 16, 1849, was comtnissioned first lieutenant in the
old 23d Regiment, N. Y. Militia. This commission he held thirty-six
years, until the regiment went out of existence. In 1853 Mr. Van
Valkenburgh's services were secured to push forward the work of the
Lebanon Springs Railroad, and he is said to have thrown out the first
shovel of earth and hired the first man on the work. He displayed
great energy and ability in this enterprise. He took a deep interest in
politics and early joined the Democratic party. In 1853 he was
appointed deputy sheriff of Columbia county and served for three
years. In 1856 he was made route agent for the general post-office
department and ran the first night express train on the Harlem
Railroad from Albany to New York. When the Civil war broke out Mr.
Van Valkenburgh offered his services and was commissioned first
lieutenant of Co. E, 128th Regiment, N. Y. Vols. August 22, 1862, he
was duly mustered into the service. His career was a most creditable
one. In January, 1863, he served as a member of a court martial in
New Orleans, and continued in the service until April 18, 1864, when
on advice of a surgeon he tendered his resignation and was
honorably discharged. In 1865 he accepted a position as conductor
on the Harlem Railroad. The following year he was elected member
of assembly from Columbia County. In 1867 Mr. Van Valkenburgh
removed to Albany and has since been an active and esteemed
citizen of that city. In 1868 he accepted the superintendency of the
Albany and Susquehanna Railroad and in 1872 became interested in
the New York and Albany Railroad, now known as the New York
Railway. When the Lebanon Springs Railroad became involved Mr.
Van Valkenburgh was appointed receiver and held that position for
three years. In 1873 he was elected a member of assembly from
Albany county and has thus had the honor to represent both Albany
and Columbia counties. Hennessy, John V., M. D., son of Thomas and
Margaret (McKinley) Hennessy, was born in New York city in 1854.
When he was a boy his parents removed to Bath-on-the-Hudson;
here young Mr. Hennessy attended the public schools. After leaving
school he obtained a situation as clerk in the office of his father, who
was a well known and prosperous builder in Albany. He remained
with his father until 1880, when he entered the Albany Medical
College and in 1884 was graduated from that in.stitution, receiving
the degree of M. D. Dr. Hennessy has practiced in Albany since his
crraduation. He is a surgeon on the staff of St. Peter's Hospital, at 
 tending physician at the Boys' Orphan Asylum, lecturer on
materia medica at the Albany Medical College and a member of the
Albany County Medical Society. In 1878 he married Sarah Elizabeth
Kane of Amsterdam, N. Y. Williams, C. Franlc, son of Isaac A. and
Sarah M. (Carpenter) Williams, was born in Brattleboro, Vt., October
17, 1859, and attended the public schools of Brattleboro, and
Worcester, Mass., after which he learned the printer's trade in
Brattleboro. In 1878 Mr. Williams removed to Albany, N. Y., where he
followed his trade until 1880, when he opened a printing office in S.
R. Gray's building in partnership with J. H. Prouty. This partnership
lasted for four years, when Mr. Williams organized the C. F. Williams
Printing Company, which existed until 1892, when it was completely
burned out at No. 36 Beaver street. Immediately after this fire the
company was dissolved and Mr. Williams resumed alone at his
present location. No. 9-11 Green street. Mr. Williams is a member of
Ancient City Lodge No. 453, F. & A. M., Albany Lodge No. 641, K. A.
E. O. , Unconditional and Capital City Clubs and Albany Republican
League. June 13, 1884, he married Frances E. A. Pangburn of
Albany, and they have three children. Grogan, Michael, was born in
Ireland and was brought to America when an infant, John Grogan,
having preceded him two years before and who had directly located
in West Troy, was a pioneer settler and for years in the employ of
the Harrington planing mill. Here Mr. Grogan has spent most of his
life, first acquiring the cooper's trade, which he followed for thirteen
years. He served one year as clerk in the weighlock and then
entered the county clerk's office under John Larkin, acting as clerk
for four years. In 1884 he was appointed deputy sheriff', filling the
position for eleven years. Murray, Wilham H., M. D., son of Francis
and Sarah (Lockwood) Murray, was born in Poundridge, Westchester
county, N. Y., December 8, 1845. He attended Betts's Academy at
Stamford, Conn., and graduated from that institution in 1863. In the
fall of that year he entered Union College at Schenectady, N. Y. , and
graduated in 1877, receiving the degree of A. B. During the year
1867-68, he taught school at Bellefonte, Pa., with Governor
Hastings, present governor of Pennsylvania. In the fall of 1868 Dr.
Murray entered the Albany Medical College and received the degree
of M. D. from that institution in 1869. In 1868 he married Martha W.
Bouck, granddaughter of the late Governor Bouck; they have two
children living, Frank and Bessie. In 1870 Dr. Murray began the
practice of medicine in Albany and has since continued there,
making a specialty of obstetrics. He has been prominently identified
with the Democratic party and has sacrificed much time to further
the interests of the city of Albany; there is no man better known or
more highly respected in his ward, the Si.\teenth. He can call
everybody by name. His love for his profession and his devotion to
his fellows have contributed to his holding the following offices;
Supervisor of his ward for five terms, president of the Board of
Aldermen one term, district physician, police surgeon, county
physician, coroner's physician, penitentiary physician, and at present
city physician. Dr. Murray has been president of the Board of
Trustees of the Hospital for Incurables since its foundation. He has
also been prominently identified with social and fraternal
organizations; he has been through all the chairs in Odd Fellowship,
and is a member of all Masonic bodies, and has the thirty-second
degree; he has also been a
 228 member of the K. of P. and Red Men. He is now a
member of the Albany and Acacia Clubs and the Albany County
Medical Society. Hall. Charles Roswell, son of John Peck and Sarah
Hart (Purdy) Hall, was born September 17, 1853, in Guilford,
Chenango county, N. Y., where his father owned a farm and died in
1875. The family were early settlers of Connecticut, coming
originally from England in the seventeenth century, and held
commissions in the State troops of their State in the Colonial wars,
and in the Continental army during the war of the Revolution. Mr.
Hall after receiving a common school education, became a teacher in
his native town, and in the fall of 1870 entered the State Normal
School at Brockport, N. Y. After entering and before finishing at the
State Normal School he taught school several terms, in this State,
and in the States of Massachusetts, Connecticut and New Jersey. He
read law with Judge Alberto T. Roraback in Canaan, Conn., with Hon.
Horace Packer, in Oxford, and with Judge Albert F. Gladding in
Norwich, from whose office he was admitted to the bar at Saratoga
in September, 1880. He began the practice of his profession in
Norwich, where he was elected justice of the peace, clerk to the
Surrogate's Court, and in Ja.uua.Ty, 1884, he received an
appointment as assistant to Attorney-General O'Brien, with charge of
the Land Department of that office. In the fall of 1880 he was
offered and accepted the office of Deputy Comptroller, being the
youngest man to hold that important position, and occupied it until
the close of the term of the then comptroller. Later he formed a
copartnership for the practice of the law with Mr. Frederick E.
Wadhams, the special study of the law in reference to State lands
and the tax laws made while he was assistant attorney-general, and
deputy comptroller, being found to be of great advantage. April 16,
1889. he was appointed deputy superintendent of the Banking
Department, by the then superintendent, Willis S. Paine, and has
remained connected with that department since. He has filled every
position in it from deputy and acting superintendent to bookkeeper,
has made a special study of the laws affecting the organization,
conduct and supervision of financial institutions, both under the
State Banking Laws and the National Bank Act, and is the author of
Hall's Bank Laws, a recognized authority on such subjects. He has
written much for the press, has delivered lectures and read papers
on financial subjects, has won honors as an orator, has always been
a staunch Democrat, being delegate to local, State and National
conventions. He is a member of the Albany Clubs and other
organizations. Collins, Hon. Lorenzo D., was born in the town of
Whitehall, Washington county, July 13, 1821. He is of Puritan
ancestry and Revolutionary stock, both grandfathers having served
in the Revolutionary war. His father, Daniel Collins, fought in the war
of 1813. Mr. L. D. Collins received a district school education and
when nineteen years of age, left his father's farm and located in
West Troy, Albany county, where two years later, he opened a canal
barn and grocery and provision store. He was a member of the old
Whig party and when the Republican party was formed in 1856, he
became a member and has been very active ever since. Mr. Collins
was trustee of the village of West Troy in 1853 and the next year
was chosen village president; in 1859 and 1860, he was a member
of the Assembly and in 1866 was elected State senator. While in the
Senate, he was chairman of the committee on canals and in 1867
introduced in the Senate a bill for the erection of the New Capi 
 239 tol building, which he liad passed. Every bill he
introduced, while in the Legislature, was passed and became law. In
1865 he was a delegate to the International Convention at Detroit,
Mich. In 1895 when the town of Watervliet was divided and the town
of Colonie erected, Mr. Collins was chosen the first supervisor and
was reelected in the spring of 1896. He was named by Governor
Morton as one of the delegate.s from New York to the National
Farmers' Congress and Good Roads Parliament, which were held at
Atlanta, Ga., during the Cotton States and International Expcisition in
1895. He is president of the State Farmers' League and chairman of
the executive committee of the New York State Farmers' Congress,
both of which were organized largely through his individual efforts.
Mr. Collins was a director of the Union National Bank of Troy, for
twenty years, and was for six years captain of the Light Guards, a
military company of West Troy, Albany county. He is a charter
member of Evening Star Lodge, No. 75, F. & A. M., of West Troy.
Anlemann, Herman W., son of Gottlieb and Augusta (Scherff)
Antemann, was born in Saxony, Germany, April 21, 1847. He came to
America with his parents when he was five years old and settled in
Albany, N. Y., where he was educated in a private German school
and the public schools. He obtained his first employment with
Thomas R. Van Loon at No. 480 Broadway, where he learned the
jewelry business. In 1870 Mr. Antemann and Mr. Van Loon formed a
partnership. Six months later Mr. Van Loon sold out to Mr. Antemann
and for the past twenty-four years Mr. Antemann has been in
business at his present location, No. 14 James street, where he now
does a large business as a manufacturing jeweler. He is a member of
the Evangelical Lutheran Church of the Redeem.er and a member
and dii-ector of the Albany Musical Association. February 10, 1870,
he married Elizabeth Huber of Albany, by whom he has four children,
Elizabeth, Kathryn, Millie and Augusta Elsie. Winne, Charles Visscher,
is descended from Pieter Winne, born in Ghent, Flanders, and
Tannatje Adams, his wife, born in Leeuwaerden, ^'rieslandt, who
came to America and settled in what is now Bethlehem, Albany
county, July 6, 1684. The line of descent is (1) Pieter Winne; (2)
Livinus, 1647-1706, of Albany, married first Teuntje Martense and
second Mrs. Williamje Viele Schermerhorn ; (3) Benjamin (by second
wife), 1705-1797, married Rachel Van Arnam ; (4) Livinus, 1745-
1825, married Marytje Lansing; (5) Livinus Lansing, 1783-1816,
married Ann Visscher, attorney, graduated from Union College in
1804, captain U. S. Army 1812, and served in that war; and (6)
Nanning Visscher, 1807-1858, a physician, graduated from Union
College in 1824 and from Yale in 1826, commissioned surgeon with
rank of lieutenant-colonel on Maj.-Gen. Stephen Van Rensselaer's
staff, and married Rachel, daughter of Garrett Van Zandt Bleecker.
All these spent their active careers in Albany. Charles V. Winne, son
of Dr. N. V., was born January 27, 1848, was educated at the Albany
Boys' Academy and in 1871 entered the employ of the D. & H. C.
Co., where he has since remained. He was first attached to the
engineering corps and since 1873 has been in the paymaster's
office, becoming paymaster in June, 1891. He is a member of
Temple Lodge No. 14, F. & A. M., Temple Chapter No. 5, R. A. M., the
Fort Orange Club, the Old Guard Albany Zouave Cadets, and the
Ridgefield Athletic and Albany Camera Clubs; has been president of
the Young Men's Association since 1894; was commodore of the
American Canoe Association
 230 in 1892; was for six years captain of the Mohican
Canoe Club; and is secretary of the Albany Country Club; a trustee
and treasurer of the Albany City Homoeopathic Hospital, member of
the Holland Society of New York and recorder of the Board of
Governors of the American Cauoe Association, in which he is very
prominent. Young, William P., was born in the town of New Scotland,
August 7, 1834, Peter, his grandfather, being a native of the town of
Knox, where he was born about 1784, and where he spent his days
as a farmer. He was a prominent and active member of the State
militia, in which he took great pride and spent considerable money,
being an officer in a company of cavalry. His first wife was Miss
Toles, by whom he had six sons and four daughters, his second wife
being Miss Bundy, by whom three children were born. He died in
1864, at the age of eighty years. Peter, the father, was born in Knox,
June 6, 1806. He commenced at the age of sixteen to learn the
carpenter's trade and followed it about forty years, when, in 1851 he
bought a farm in Guilderland and in 1856 bought an adjoining farm.
In 1863 he engaged in farming in Guilderland, where he spent his
remaining days. He was also a member of and drummer in the State
militia. His wife was Rebecca (Williams) Austin, and their children
were John A., Charles W., Henry W., Sarah A., Margaret J., Lois R.,
Mary (who died at the age of twenty-five), Eliza O. and Gouvenier M.
He died August 15, 1881, at the age of seventy-five, and his wife
died April 28, 1892, at the age of seventy-seven. William P. remained
at home until twenty-one years of age, when he rented, in 1856, a
farm for one year for himself in the town of Coeymans. In 1857 he
returned to Guilderland and worked his father's farms for nine years,
and in 1866 purchased a farm in New Scotland which he still owns.
In 1883 he bought a second farm in New Scotland, where he now
resides. He has made a specialty of fruit culture and has several
varieties on his farms. The farm on which he now lives is known as
the Dr. Sager farm, and was originally owned by Dr. Day. Dr. Sager
lived with Dr. Day and later married his adopted daughter. Mr. Young
has erected new houses and barn buildings on both of his farms,
being his own architect. December 9, 1854, he married Mary S.
Koonz. born in New Scotland and daughter of Samuel and Elizabeth
(Folmsbee) Koonz, and granddaughter of Nathaniel and Catherine
(Cline) Koonz; the latter lived to be 106 years of age. To Mr. and
Mrs. Young were born four children: Mary, widow of Albei-t Relyea,
who died January 4, 1885, was married to him August 18, 1875,
leaving two children surviving her: Lizzie B., and Levi E. William H.
married Libbie Main of Guilderland, March 10, 1885, and have two
children: Olive and Lelah ; he is an extensive berry grower. Hannah
E. married Henry Goodfellow of Guilderland, October 7, 1880, and
have two children: Florence and Ernest. Elizabeth E., who still
resides at home. Of the brothers and sisters of William P. Young,
John A. Young resides at Brodhead, Wis., having married in 1852
Maria Groat of Guilderland, by whom he has had four children.
Charles W. Young lives at Whitewater, Wis., and married Mary Jane
Chism. and has no children. Sarah A. Young married Peter Van
Patten and now lives in Centralia, Kansas, having one daughter.
Margaret J. Young married Charles Gemlich and resides in
Guilderland and has one son. Henry W. Young was married to
Joanna Gates and lives in the city of Albany. They are the parents of
two children. Lois R. Young married David Van Patten, a brother of
Peter, and lives on an adjoining farm in Centralia, Kas. Thev have
two chil 
 231 dren. Eliza O. Young married Charles Severson and
resides in Guilderland, having borne him one child. Gouvenier M.
Young resides at Whitewater, Wis., having married Elva Martm of
Guilderland, by whom he has had two children, of whom one
survives. Niles, Nathaniel, son of John H. and Fannie (Mosher) Niles,
was born in Bethlehem, Albany county, September 1, 1856, and is a
grandson of Nathaniel Niles, who came from Connecticut to
Coeymans, Albany county, at an early day and died there in 1876.
The latter was prominent in town affairs, serving as supervisor, etc.
John H. Niles, a farmer by occupation, died in 1861. Nathaniel Niles
attended the public and private schools, was graduated from the
Albany Free Academy in 1874 and from Dartmouth College, with the
degree of A. B., in 1878, and read law in Albany with Judge Rufus W.
Peckham. He was admitted to the bar in 1880 and for a time acted
as clerk for Peckham, Rosendale & Hessberg, in whose offices and
the offices of their successors, he has successfully practiced his
profession ever since. In politics he is a Democrat. Mead, Charles W.,
son of Delois L., was born in Clymer, N. Y., December 3, 1843, and
pursued his education under private tutors and in the academies of
Chautauqua county, graduating in 1863. He completed his collegiate
studies at Painesville, Ohio, and for seven years was principal of
academies and union schools in his native county. In the fall of 1870
he came to Albany and entered the Albany Law School, from which
he was graduated and admitted to the bar in 1871. He immediately
began the practice of his profession and in 1877 formed a
copartnership with Samuel S. Hatt. which still continues, the present
firm being Mead, Hatt & Palmer. He is a staunch Republican and in
1882 was appointed a U. S. circuit court commissioner, which
position he has since held. He takes an active interest in the welfare
of the citv, was at one time a member of the legislative branch of its
government, and has given considerable attention and takes high
rank in the social and fraternal organizations of Albany. He is a
member of Grand Lodge, F. & A. M., is prominently identified with
the fraternal co-operative associations, and was the representative
of one of the leading orders of the State in the matter of State
legislation and one of the framers of the present law governing the
same. In 1874 he married M. Manila Burnap, one of the leading
contraltos of Albany, and they have one daughter, Edith M. Amyot,
Bruno E., D. D. S., is a leading member of the dental profession in
Cohoes, and is a son of Bruno Arayot, who has been a resident of
this place for nearly half a century. He came from Vercherer,
Province of Quebec. Doctor Amyot was born in 1869 in Cohoes and
was educated in the parochial schools. At the age of nineteen he
entered the New York College of Dentistry, and after two vears
graduated, in 1890, beginning practice here at once, where
he_enjoys a large patronage. He is a member of the Third District
Dental Society of New York State. September 30, 1896, he married
Miss Rosa de Lima Masson of Cohoes. Beras, James H., was born in
1863, a son of James Berns, an artist; his mother being a teacher,
made the home of his childhood a dwelling of culture and
refinement. Mr. Berns is a Democrat and is a member of the County
Committee. James H. is one of the leading young lawyers of Cohoes,
and came to the front because of his able handhng of the celebrated
case of Cahill, who was indicted for
 232 shooting his brother-in-law, Charles Scholield, at
Cohoes. In 1893 he entered the Albany Law School, after graduating
from the High School and the Albany business College. After his
admission to the bar in 1894, he opened an office and began
practice. Bullock, Joseph, came to Cohoes as early as 1846, and has
been a resident here since, with the exception of eight years in
Lockport, where he was engaged in the knitting business. He was of
Dutch ancestry, born in Guilderland, in 1835, and decidedly a self-
made man, adding to his limited education by close observation and
personal research. In 1872 he returned to Cohoes and in 1877
established a baking business, which he conducted with marked
success until it was purchased in 1894 by his son, John H. Bullock,
who still conducts it at No. 116 Remsen street. Mr. Bullock is a man
of great strength of character and convictions. He appreciates highly
the picture of the domicile of his youth where both father and
mother were l)orn ; it was built in 1704 and is yet intact; the Ijrick in
the fireplace and chimney were brought from Holland. Belanger,
Israel, justice of the peace, and a scholarly young man, had the
courage and perseverance to break the fetters of circumstances
which surrounded his youth, and gain his way to the front "amid the
maddening crowd's ignoble strife."' When nine years old he began
life in the mill where he remained until twenty years of age as a
weaver. He then returned to Joliette, Quebec, where he was born in
1863, and entered Joliette College. In 1890 he graduated with
degree of Bachelor of Letters from Laval University, QueV^ec, and
came to Cohoes. Here he studied law with Hon. George H. Fitts and
was admitted to the bar in 1892. Besides his law practice and office
duties, he is identified with an insurance and real estate agency. He
is now justice of peace of the city of Cohoes. Campbell, Hon.
George, a well known citizen, long identified with the interests of
Cohoes, is of Canadian birth, and first located at Cohoes in 1847,
and after sixteen years' residence at Waterford, where he learned
the machinist's trade and was for a time in partnership with George
Gage, he returned to this city in 1863, and established with John
Clute the present firm. In 1873 they erected a commodious modern
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