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Methods in
Molecular Biology 2047

Simon G. Sprecher Editor

Brain
Development
Methods and Protocols
Second Edition
Methods in M o l e c u l a r B i o lo g y

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

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in
all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-
by-step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
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constitute the key ingredient in each and every volume of theMethods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Brain Development

Methods and Protocols

Second Edition

Edited by

Simon G. Sprecher
Department of Biology, University of Fribourg, Fribourg, Switzerland
Editor
Simon G. Sprecher
Department of Biology
University of Fribourg
Fribourg, Switzerland

ISSN 1064-3745     ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-9731-2    ISBN 978-1-4939-9732-9 (eBook)
https://doi.org/10.1007/978-1-4939-9732-9

© Springer Science+Business Media, LLC, part of Springer Nature 2020

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Preface

How the brain works remains one of the largest enigmatic questions in modern biology.
Particularly, its cellular diversity, connectivity among neurons, formation of neuronal net-
works, use of distinct neurotransmitter systems, and underlying function for behavior raise
the question of how this highly interconnected organ develops. It is therefore not surpris-
ing that the intersection between developmental biology and neuroscience provides an
exceptional field to address and investigate impacting biological questions. Complementing
findings of an array of distinct animal model systems provide the basis of brain development
research. Our current understanding is based on widely used genetic model systems, includ-
ing the fruit fly, zebra fish, chicken, and mouse. These animal models are particularly
impacting since they allow elaborate genetic manipulations including transgenic expression
systems and conditional knockout or knockdown of developmental genes. However, the
advent of genome editing techniques makes it possible to extend investigations towards
other animals that may thus be used to answer specific questions of molecular neuroscience.
These noncanonical experimental systems further substantiate general principles and mech-
anisms in brain development. Questions that can be investigated often depend on the
methodological accessibility. Therefore, progress and developments in constantly improv-
ing laboratory technologies provide an essential ground for the advancement in the field.
This book aims to provide a comprehensive overview and introduction to widely used
leading-edge techniques on a representative range of animals. A particular focus lies on
recent technical advances in molecular genetics.

Fribourg, Switzerland Simon G. Sprecher

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Part I Invertebrate Models

1 Combining BrdU-Labeling to Detection of Neuronal Markers

to Monitor Adult Neurogenesis in Hydra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .   3
Wanda Buzgariu, Marie-Laure Curchod, Chrystelle Perruchoud,
and Brigitte Galliot
2 Reverse Genetic Approaches to Investigate the Neurobiology
of the Cnidarian Sea Anemone Nematostella vectensis . . . . . . . . . . . . . . . . . . . . . . .  25
Jamie A. Havrilak and Michael J. Layden
3 Generating Transgenic Reporter Lines for Studying Nervous System
Development in the Cnidarian Nematostella vectensis . . . . . . . . . . . . . . . . . . . . . . .  45
Fabian Rentzsch, Eduard Renfer, and Ulrich Technau
4 Immunostaining and In Situ Hybridization of the Developing
Acoel Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  59
Elena Perea-Atienza, Brenda Gavilán, Simon G. Sprecher,
and Pedro Martinez
5 Immunostaining of the Embryonic and Larval Drosophila Brain . . . . . . . . . . . . . . .  81
Frank Hirth and Danielle C. Diaper
6 Nonfluorescent RNA In Situ Hybridization Combined with Antibody
Staining to Visualize Multiple Gene Expression Patterns in the Embryonic
Brain of Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  97
David Jussen and Rolf Urbach
7 Analysis of Complete Neuroblast Cell Lineages in the Drosophila
Embryonic Brain via DiI Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Karoline F. Kraft and Rolf Urbach
8 Flybow to Dissect Circuit Assembly in the Drosophila Brain: An Update . . . . . . . . . 137
Emma L. Powell and Iris Salecker
9 Live Cell Imaging of Neural Stem Cells in the Drosophila Larval Brain . . . . . . . . . 153
Karolina Miszczak and Boris Egger
10 CRISPR/Cas9 Genome Editing to Study Nervous System
Development in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Cornelia Fritsch and Simon G. Sprecher
11 The Red Flour Beetle as Model for Comparative Neural Development:
Genome Editing to Mark Neural Cells in Tribolium Brain Development . . . . . . . . 191
Max S. Farnworth, Kolja N. Eckermann, Hassan M. M. Ahmed,
Dominik S. Mühlen, Bicheng He, and Gregor Bucher

vii
viii Contents

12 A Protocol for Double Fluorescent In Situ Hybridization

and Immunohistochemistry for the Study of Embryonic Brain Development
in Tribolium castaneum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Marita Buescher, Georg Oberhofer, Natalia Carolina Garcia-Perez,
and Gregor Bucher
13 Immunohistochemistry and Fluorescent Whole Mount RNA In Situ
Hybridization in Larval and Adult Brains of Tribolium . . . . . . . . . . . . . . . . . . . . . . 233
Vera S. Hunnekuhl, Janna Siemanowski, Max S. Farnworth, Bicheng He,
and Gregor Bucher
14 X-Ray Microscopy of the Larval Crustacean Brain . . . . . . . . . . . . . . . . . . . . . . . . . 253
Jakob Krieger and Franziska Spitzner
15 Immunolocalization of Neurotransmitters and Neuromodulators
in the Developing Crayfish Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Steffen Harzsch and Caroline Viertel
16 Immunostainings in Nervous System Development
of the Nematode C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Janet S. Duerr
17 Methods in Brain Development of Molluscs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Andreas Wanninger and Tim Wollesen
18 A Simple Method to Identify Ascidian Brain Lineage Cells at Neural Plate
Stages Following In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Clare Hudson
19 Spawning Induction and Embryo Micromanipulation Protocols
in the Amphioxus Branchiostoma lanceolatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Yann Le Petillon, Stéphanie Bertrand, and Héctor Escrivà

Part II Vertebrate Models

20 In Situ Hybridization and Immunostaining of Xenopus Brain . . . . . . . . . . . . . . . . . 363

Kai-li Liu, Xiu-mei Wang, Zi-long Li, Ying Liu, and Rong-qiao He
21 Morpholino Studies in Xenopus Brain Development . . . . . . . . . . . . . . . . . . . . . . . . 377
Jennifer E. Bestman and Hollis T. Cline
22 Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced
Tyramide Signal Amplification and Its Combination with Immunofluorescent
Protein Visualization in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Gilbert Lauter, Iris Söll, and Giselbert Hauptmann
23 Live Morphometric Classification of Sensory Neurons in Larval Zebrafish . . . . . . . 411
Gema Valera and Hernán López-Schier
24 Immunohistochemistry and In Situ Hybridization
in the Developing Chicken Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Richard P. Tucker, Tatsuto Ishimaru, and Qizhi Gong
25 Gene Silencing in Chicken Brain Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Georgia Tsapara, Irwin Andermatt, and Esther T. Stoeckli
 Contents ix

26 Transplantation of Neural Tissue: Quail–Chick Chimeras . . . . . . . . . . . . . . . . . . . . 457

Andrea Streit and Claudio D. Stern
27 Immunohistochemistry and RNA In Situ Hybridization
in Mouse Brain Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
Jinling Liu and Aimin Liu
28 The Cre/Lox System to Assess the Development of the Mouse Brain . . . . . . . . . . . 491
Claudius F. Kratochwil and Filippo M. Rijli
29 In Utero Electroporation to Study Mouse Brain Development . . . . . . . . . . . . . . . . 513
Emilie Pacary and François Guillemot

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
Contributors

Hassan M. M. Ahmed • Department of Developmental Biology, Johann-Friedrich-­

Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Department of Crop Protection, Faculty of Agriculture, University of Khartoum,
Khartoum-North, Khartoum, Sudan
Irwin Andermatt • Neuroscience Center Zurich, Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland
Stéphanie Bertrand • Sorbonne Université, CNRS, Biologie Intégrative des Organismes
Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France
Jennifer E. Bestman • Biology Department, William and Mary, Williamsburg, VA, USA
Gregor Bucher • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Department of Evolutionary Developmental Genetics, Georg-August-University
Göttingen, Göttingen, Germany
Marita Buescher • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany
Wanda Buzgariu • Department of Genetics and Evolution, iGE3, Faculty of Sciences,
University of Geneva, Geneva, Switzerland
Hollis T. Cline • The Dorris Neuroscience Center, The Scripps Research Institute, La
Jolla, CA, USA
Marie-Laure Curchod • Department of Genetics and Evolution, iGE3, Faculty of
Sciences, University of Geneva, Geneva, Switzerland
Danielle C. Diaper • Department of Basic and Clinical Neuroscience, Maurice Wohl
Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience,
King’s College London, London, UK
Janet S. Duerr • Department of Biological Sciences, Ohio University, Athens, OH, USA
Kolja N. Eckermann • Göttingen Graduate Center for Molecular Biosciences,
Neurosciences and Biophysics, Göttingen, Germany; Department of Developmental Biology
Johann-Friedrich-Blumenbach Institute, GZMB, University of Göttingen, Göttingen,
Germany
Boris Egger • Department of Biology, University of Fribourg, Fribourg, Switzerland
Héctor Escrivà • Sorbonne Université, CNRS, Biologie Intégrative des Organismes
Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, France
Max S. Farnworth • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Göttingen Graduate Center for Molecular Biosciences, Neurosciences and Biophysics,
Göttingen, Germany; Department of Evolutionary, Developmental Genetics, Georg-
August-University Göttingen, Göttingen, Germany
Cornelia Fritsch • Department of Biology, University of Fribourg, Fribourg, Switzerland
Brigitte Galliot • Department of Genetics and Evolution, iGE3, Faculty of Sciences,
University of Geneva, Geneva, Switzerland

xi
xii Contributors

Natalia Carolina Garcia-Perez • Department of Evolutionary Developmental Genetics,

Johann-Friedrich-Blumenbach Institute, GZMB, University of Göttingen, Göttingen,
Germany
Brenda Gavilán • Departament de Genètica, Universitat de Barcelona, Barcelona, Spain
Qizhi Gong • Department of Cell Biology and Human Anatomy, University of
California, Davis, Davis, CA, USA
François Guillemot • The Francis Crick Institute, London, UK
Steffen Harzsch • Department of Cytology and Evolutionary Biology, Zoological Institute
and Museum, University of Greifswald, Greifswald, Germany
Giselbert Hauptmann • Department of Molecular Biosciences, The Wenner-Gren
Institute, MBW, Stockholm University, Stockholm, Sweden
Jamie A. Havrilak • Department of Biological Sciences, Lehigh University, Bethlehem, PA,
USA
Bicheng He • Department of Evolutionary Developmental Genetics,
Johann-Friedrich-­Blumenbach Institute, GZMB, University of Göttingen,
Göttingen, Germany; Department of Evolutionary Developmental Genetics,
Georg-August-University Göttingen, Göttingen, Germany
Rong-qiao He • State Key Laboratory of Brain and Cognitive Science, Institute of
Biophysics, Chinese Academy of Sciences, Beijing, China
Frank Hirth • Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical
Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's
College London, London, UK
Clare Hudson • Laboratoire de Biologie du Développement de Villefranche-sur-mer
(LBDV), Sorbonne Université, CNRS, Villefranche-sur-mer, France
Vera S. Hunnekuhl • Department of Evolutionary Developmental Genetics, ,
Georg-August-University Göttingen, Göttingen, Germany
Tatsuto Ishimaru • Department of Cell Biology and Human Anatomy,
University of California, Davis, CA, USA
David Jussen • Institute of Genetics, University of Mainz, Mainz, Germany
Karoline F. Kraft • Institute of Genetics, University of Mainz, Mainz, Germany
Claudius F. Kratochwil • Department of Biology, Zoology and Evolutionary Biology,
University of Konstanz, Konstanz, Germany
Jakob Krieger • Department of Cytology and Evolutionary Biology, Zoological Institute
and Museum, University of Greifswald, Greifswald, Germany
Gilbert Lauter • Department of Biosciences and Nutrition, Neo, Karolinska Institutet,
Huddinge, Sweden
Michael J. Layden • Department of Biological Sciences, Lehigh University, Bethlehem, PA,
USA
Yann Le Petillon • Sorbonne Université, CNRS, Biologie Intégrative des Organismes
Marins (BIOM), Observatoire Océanologique, Banyuls-sur-Mer, Banyuls-sur-Mer, France
Zi-long Li • State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, Beijing, China
Aimin Liu • Department of Biology, Center for Cellular Dynamics, Eberly College of
Science, Huck Institute of Life Sciences, The Penn State University, Pennsylvania, PA,
USA
 Contributors xiii

Jinling Liu • Department of Biology, Center for Cellular Dynamics, Eberly College of
Science, Huck Institute of Life Sciences, The Penn State University, Pennsylvania, PA,
USA
Kai-li Liu • State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, Beijing, China
Ying Liu • State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics,
Chinese Academy of Sciences, Beijing, China
Hernán López-Schier • Sensory Biology and Organogenesis, Helmholtz Zentrum Munich,
Munich, Germany
Pedro Martinez • Departament de Genètica, Universitat de Barcelona, Barcelona,
Spain; Institut Català de Recerca i Estudis Avancats (ICREA), Barcelona, Spain
Karolina Miszczak • Department of Biology, University of Fribourg, Fribourg,
Switzerland
Dominik S. Mühlen • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany;
Göttingen Graduate Center for Molecular Biosciences, Neurosciences and Biophysics,
Göttingen, Germany
Georg Oberhofer • Department of Evolutionary Developmental Genetics, Johann-­
Friedrich-­Blumenbach Institute, GZMB, University of Göttingen, Göttingen, Germany
Emilie Pacary • INSERM U1215, Neurocentre Magendie, Bordeaux, France; Université
de Bordeaux, Bordeaux, France
Elena Perea-Atienza • Departament de Genètica, Universitat de Barcelona, Barcelona,
Spain
Chrystelle Perruchoud • Department of Genetics and Evolution, iGE3, Faculty of
Sciences, University of Geneva, Geneva, Switzerland
Emma L. Powell • Visual Circuit Assembly Laboratory, The Francis Crick Institute,
London, UK
Eduard Renfer • Department for Molecular Evolution and Development, Faculty of Life
Sciences, Center of Organismal Systems Biology, University of Vienna, Vienna, Austria
Fabian Rentzsch • Sars International Centre for Marine Molecular Biology, University of
Bergen, Bergen, Norway; Department of Biological Sciences, University of Bergen, Bergen,
Norway
Filippo M. Rijli • Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
Iris Salecker • Visual Circuit Assembly Laboratory, The Francis Crick Institute, London,
UK
Janna Siemanowski • Department of Evolutionary Developmental Genetics,
Georg-August-University Göttingen, Göttingen, Germany
Iris Söll • Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm
University, Stockholm, Sweden
Franziska Spitzner • Department of Cytology and Evolutionary Biology, Zoological
Institute and Museum, University of Greifswald, Greifswald, Germany
Simon G. Sprecher • Department of Biology, University of Fribourg, Fribourg,
Switzerland
Claudio D. Stern • Department of Cell and Developmental Biology, University College
London, London, UK
Esther T. Stoeckli • Neuroscience Center Zurich, Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland
xiv Contributors

Andrea Streit • Faculty of Dental, Oral and Craniofacial Sciences, Centre for
Craniofacial and Regenerative Biology, King’s College London, London, UK
Ulrich Technau • Department for Molecular Evolution and Development, Faculty of
Life Sciences, Center of Organismal Systems Biology, University of Vienna, Vienna,
Austria
Georgia Tsapara • Neuroscience Center Zurich, Institute of Molecular Life Sciences,
University of Zurich, Zurich, Switzerland
Richard P. Tucker • Department of Cell Biology and Human Anatomy, University of
California, Davis, CA, USA
Rolf Urbach • Institute of Genetics, University of Mainz, Mainz, Germany
Gema Valera • Sensory Biology and Organogenesis, Helmholtz Zentrum Munich, Munich,
Germany
Caroline Viertel • Department of Cytology and Evolutionary Biology, Zoological
Institute and Museum, University of Greifswald, Greifswald, Germany
Xiu-mei Wang • State Key Laboratory of Brain and Cognitive Science, Institute of
Biophysics, Chinese Academy of Sciences, Beijing, China
Andreas Wanninger • Department of Integrative Zoology, Faculty of Life Sciences,
University of Vienna, Vienna, Austria
Tim Wollesen • European Molecular Biology Laboratory, Heidelberg, Germany
 Part I

Invertebrate Models
 Chapter 1

Combining BrdU-Labeling to Detection of Neuronal

Markers to Monitor Adult Neurogenesis in Hydra
Wanda Buzgariu, Marie-Laure Curchod, Chrystelle Perruchoud,
and Brigitte Galliot

Abstract
The nervous system is produced and maintained in adult Hydra through the continuous production of
nerve cells and mechanosensory cells (nematocytes or cnidocytes). De novo neurogenesis occurs slowly in
intact animals that replace their dying nerve cells, at a faster rate in animals regenerating their head as a
complete apical nervous system is built in few days. To dissect the molecular mechanisms that underlie
these properties, a precise monitoring of the markers of neurogenesis and nematogenesis is required. Here
we describe the conditions for an efficient BrdU-labeling coupled to an immunodetection of neuronal
markers, either regulators of neurogenesis, here the homeoprotein prdl-a, or neuropeptides such as
RFamide or Hym-355. This method can be performed on whole-mount animals as well as on macerated
tissues when cells retain their morphology. Moreover, when antibodies are not available, BrdU-labeling
can be combined with the analysis of gene expression by whole-mount in situ hybridization. This co-­
immunodetection procedure is well adapted to visualize and quantify the dynamics of de novo neurogen-
esis. Upon continuous BrdU labeling, the repeated measurements of BrdU-labeling indexes in specific
cellular populations provide a precise monitoring of nematogenesis as well as neurogenesis, in homeostatic
or developmental conditions.

Key words Hydra nervous system, Interstitial stem cells, Neurogenesis, Nematogenesis, In situ
hybridization, Immunofluorescence, Hydroxyurea, BrdU, prdl-a, Hym-355, RFamide

1 Introduction

1.1 Anatomy The freshwater hydrozoan Hydra belongs to Cnidaria, an early-­

of the Nervous branched eumetazoan phylum, sister group to bilaterians. Hydra is
System, Neurogenesis, an attractive model for biologists not only for its outstanding
and Nematogenesis regenerative properties but also for its highly dynamic neurogene-
in Hydra sis. Indeed the nervous system is continuously renewed with nerve
precursors produced in the body column, then displaced towards
the extremities where they terminally differentiate to replace the
nerve cells that die [1] (Fig. 1a). In addition, animals bisected at
any level along the body column replace the missing part in few

Simon G. Sprecher (ed.), Brain Development: Methods and Protocols, Methods in Molecular Biology, vol. 2047,
https://doi.org/10.1007/978-1-4939-9732-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020

3
4 Wanda Buzgariu et al.

A INTACT HYDRA POLYP C

apical differentiated
nerve cells
interstitial stem cells
sensory neurons

neurogenesis
nerve cell precursors

migration
interstitial stem cells 4x
nerve cell precursors

basal differentiated 8x
nerve cells
sensory-motor bipolar neurons

B HEAD-REGENERATING HYDRA
16

a.p a.p

nematoblasts

4 hpa 24 hpa 36 hpa

a.p=amputation plane ganglia neurons
nematocytes

Fig. 1 Adult neurogenesis in Hydra and cell types that form its nervous system. (a, b) Neurogenesis in adult
intact (a) and in head-regenerating (b) Hydra polyp (modified from ref. 2). Only nerve cells are shown, denser
in the apical (top) and basal (bottom) regions than in the body column where neurogenesis takes place. The
interstitial stem cells (ISCs) provide precursors for all nerve cells and nematocytes, which migrate towards the
extremities [3]. Terminal differentiation of nerve cells predominantly takes place at the extremities. In head-­
regenerating Hydra (b) interstitial cells and derivatives are first eliminated upon injury-induced cell death after
mid-gastric bisection [4] and de novo neurogenesis takes place at the tip where the apical nervous system get
built within 2 days, with precursors detected after 24 h [5, 6]. (c) ISCs divide every 24–30 h and frequently
appear as pairs (top-left panel). Nematogenesis starts with a series of synchronous syncytial divisions that
lead to the formation of nematoblast clusters that contain 4×, 8×, 16× or even 32× cells. At any stage, a
cluster can stop proliferating to enter differentiation, i.e. each nematoblast forms a venom capsule named
nematocyst (arrows) and a cnidocil (not visible), both fully mature in nematocytes [7, 8]. Note the moon-shaped
nucleus in nematocytes, compressed by the nematocyst. Hydra differentiates different classes of neurons:
sensory, sensory-motor bipolar, and multipolar ganglia neurons (right panels). Cells obtained after tissue mac-
eration [9], were immunodetected with an anti α-tubulin antibody followed by an anti-mouse secondary anti-
body coupled with Alexa-488 (green). The nuclei were counterstained with DAPI (light blue). Pictures were
taken on a SP8 Leica confocal microscope and optimized on Photoshop with channelmixer to lighten the dark
blue color and M-curves to increase the contrast. Scale bar: 10 μm

days, e.g. a new head equipped with a complete nervous system

(Fig. 1b). The Hydra nervous system is made of nerve cells and
mechanosensory cells (named nematocytes or cnidocytes) that
form a nerve net much denser at the apical and basal extremities of
the animal. There it controls a series of more or less complex
behaviors such as peristaltic movements, walking, prey capture,
food ingestion, and reaction to light.
Cnidarian neurons are equipped with neurites but no axons,
therefore not polarized and often named “nerve cells.” Every
nerve cell is able to function as sensory, sensory-motor, or inter-
 Methods to Monitor Adult Neurogenesis in Hydra 5

neuron. Nevertheless, nerve cells show diverse anatomies, sensory,

bipolar, or multipolar as ganglia neurons (Fig. 1c), and the neuro-
nal phenotype of a given cell can change according to its position
along the body axis [10]. At the base of the apex, ganglia neurons
can form a nerve ring that allows coordinated behaviors [11].
Although cnidarian nervous systems are highly peptidergic with
peptide-gated ion channels as receptors [12, 13], they share with
bilaterians all the basic properties of synaptic conduction and
chemical neurotransmission [14, 15], even though the neuromus-
cular junction might have evolved independently in cnidarians and
bilaterians [16]. Still, the transcriptional factors that control neu-
rogenesis are largely evolutionarily conserved [2, 5, 9, 17–21] and
cnidarian nervous systems might represent the first evolutionary
attempt of centralized nervous system in eumetazoans [11].
The two myoepithelial layers of the body wall, the epidermis
and the gastrodermis, are made of cells that derive from three not
interchangeable stem cell populations: the two unipotent ectoder-
mal and endodermal epithelial stem cells (ESCs) and the multipo-
tent interstitial stem cells (ISCs). ISCs, predominantly located in
the central body column, give rise to a variety of cell types, includ-
ing the two cell lineages that build up the nervous system (Figs. 1a
and 2): the rare nerve cells and the abundant nematocytes, which
represent 3% and 35% of the total cell number, respectively [41,
42]. The nerve cells directly differentiate from migrating precur-
sors and then get displaced towards the extremities where they
form a dense diffuse nerve net [6, 42–44]. In intact animals
(homeostasis), neurogenesis is a slow process, leading to the
replacement of the nerve cells that get sloughed off at the extremi-
ties, while after bisection a faster de novo neurogenesis process
takes place in the regenerating structure (Fig. 3a).
In contrast to neurogenesis, nematogenesis is a multiple step
process where interstitial progenitors synchronously divide up to
five times, providing clusters that contain 4, 8, 16, or 32 nemato-
blasts (Figs. 1b and 2) [7]. Cells from a given cluster can exit the
cell cycle at any time from the 4-cell stage to differentiate as nema-
tocytes equipped with a sensory cnidocil and a vacuole named cni-
docyst that contains a paralyzing venom, the latter structure being
the hallmark of cnidarians [8]. The turnover of nematocytes is
highly dynamic as once the venom capsule is discharged, the
­nematocyte is eliminated and replaced. Interestingly, ISCs cycle
three to four times faster than ESCs and can thus be easily elimi-
nated upon pulse treatments of antimitotic drugs [45–47].
Elimination of the cycling interstitial cells leads to the loss of the
nervous system, after several weeks in intact animals, within few
days in regenerating ones, as such animals regenerate a missing
part that lacks a nervous system (Figs. 3b and 4). Nerve-free ani-
mals actually maintain their developmental properties if they are
6 Wanda Buzgariu et al.

Fig. 2 Molecular markers of neurogenesis and nematogenesis in Hydra. A partial list of genes expressed during
neurogenesis (upper) and/or nematogenesis (lower). Gene products detected with antibodies are written plain.
ISC interstitial stem cells, pc precursor cell, nb nematoblasts. The spatial and cell-type expression pattern of
genes presumably involved in neurogenesis and neurotransmission (103 and 156, respectively) was identified
by RNAseq transcriptomics [22], and for any Hydra gene of interest the spatial and cell-type expression pat-
terns are now available on the HydrAtlas server (https://hydratlas.unige.ch/blast/blast_link.cgi) [23]. Specific
signatures were obtained by analyzing the expression patterns of (1) neuropeptides such as RGamide,
RFamide, KVamide, or LWamide [24–26], the neuropeptide Hym-355 enhancing neuronal differentiation [3], (2)
86 genes identified in a microarray screened with cRNAs from nerve-less animals [27], (3) neurogenic genes
that regulate the proliferation and/or the differentiation of progenitors such as CnASH [17, 28], prdl-a [23],
COUP-TF, prdl-b [29], hyZic [30], cnox-2 [22], CREB [29], Myc1 [31, 32], FoxN1, PaxA, PaxB, Pou4F2 [22], (4)
the two Piwi proteins (HyWi, HyLi) strongly expressed in ISCs and nematoblasts [33], (5) some gap junction
proteins, innexin-2 being involved in neurotransmission [34]. No pan-neuronal marker was identified in Hydra
yet. As early markers of nematocyst differentiation, the minicollagens N-COL1 and NOWA [35, 36] are compo-
nents of the wall, spinalin of the spines [37], N-COL15 of the tubule [38], nematogalectins A and B of the tubule
from distinct types of nematocysts [39]. Finally, a proteomic analysis of the venom identified 410 secreted
proteins in nematocysts [40] (not shown here)
Fig. 3 The homeoprotein prdl-a as a marker of apical neurogenesis. (a) Double immunostaining of BrdU (green) and
prdl-a (red) expressed in apical neuronal progenitors or nerve cells of homeostatic animals (upper panel) or animals
having regenerated their head (lower panel). All animals were incubated with BrdU for 4 h and then washed out to
remain intact or to undergo mid-gastric bisection (red arrow). White arrow heads indicate the prdl-a+/BrdU+ cells,
which are more numerous in the regenerated than in the homeostatic head. (b) De novo neurogenesis evidenced
by prdl-a (red) immunostaining in the presence (top panel) or the absence (lowerpanel) of interstitial progenitors
after hydroxyurea (HU) treatment. The animals were exposed or not to 10 mM HU in three rounds (2× 24 h and
1× 32 h). After the third HU treatment, animals were transferred to HM, bisected (red arrow) and let to regenerate
for seven days. Note the absence of prdl-a+ cells in HU-treated animals. Nuclei were counterstained with DAPI
(blue). Image acquisition was done on a Leica LSM700 confocal microscope. Scale bar: 100 μm
8 Wanda Buzgariu et al.

Fig. 4 Loss of terminal neuronal differentiation in Hydra undergoing apical or basal regeneration in the absence
of ISCs or interstitial progenitors. HU treatment and regeneration were performed as in Fig. 3. The apical (a)
and basal (b) nerve nets were identified after RFamide immunostaining (green) and nuclear DAPI counterstain-
ing (purple). Note the sparse RFamide+ nerve cells in apical (a) and basal (b) regions regenerated after HU
treatment. Image acquisition was done on a Leica LSM700 confocal microscope. Scale bars: 100 μm

force-fed, pointing to an important morphogenetic role played by

the epithelial cells [46].
The Hydra Peptide Project identified about 500 peptides,
half neuropeptides, half epitheliopeptides, with specific roles in
neurotransmission, morphogenesis, and differentiation [48, 49].
However, not much is known concerning the role and the regu-
lation of the neuronal-epithelial cross talk on the slow and fast
modes of neurogenesis in intact and regenerating animals, respec-
tively. These questions require the monitoring of the expression of
neuronal markers in proliferating and differentiating progenitors as
well as in fully mature nerve cells (Fig. 5).

1.2 Principles The Hydra nervous system is well visualized with the immunos-
of Immunodetection taining procedures applied either on whole animals or on cells that
on Whole-Mount keep their morphology after tissue maceration [41]. In both con-
and Macerated texts, the procedure is based on the specific antigen–antibody rec-
Tissues in Hydra ognition followed by different detection techniques to visualize
the cells that express or overexpress a known antigen. The immu-
nodetection procedure follows the classical steps: fixation and per-
meabilization of the tissue, saturation of unspecific sites, and
antigen recognition by a specific antibody followed by detection
and visualization. The expression patterns obtained in intact or
regenerating Hydra as well as in specific cell types can also be veri-
fied at the transcriptomic level on the HydrAtlas server (https://
hydratlas.unige.ch) [23].
 Methods to Monitor Adult Neurogenesis in Hydra 9

Fig. 5 BrdU immunostaining combined with Hym-355 detection in homeostatic animals. The neuropeptide
Hym-355 is expressed in a subset of apical and basal neurons. The animals were exposed to BrdU for 4 h,
washed and subsequently maintained in HM for 2 days and then fixed. After the in situ hybridization procedure
to detect Hym-355 expressing nerve cells (a, purple), the animals were immunolabeled for BrdU (b, c, green).
In homeostatic condition, only few apical neurons do express Hym-355 and are positive for BrdU (arrows). The
bright field (a) and fluorescence (b) images were acquired with a Leica DM5500 fluorescence microscope. Box
in (b) corresponds to panel (c). Scale bars: 100 μm (a, b) and 20 μm (c)

1.2.1 Immunodetection The whole-mount immunodetection procedure is a robust method

on Whole-Mounts to detect with specific antibodies the spatial or temporal expression
pattern of neurogenic proteins or neuropeptides. In complement
the same procedure can be used to detect with antibodies raised
against tagged material incorporated into macromolecules such as
Bromodeoxyuridine (BrdU) incorporated into newly synthesized
DNA [50] or digoxygenin (DIG)-labeled riboprobes that bind
transcripts [51] (Fig. 5). As a consequence, immunodetection
allows the visualization of proteins, DNA, and transcripts in tissues
that show a preserved architecture.

1.2.2 Immunodetection To get a more accurate analysis at the cellular level, the immuno-
on Cells Obtained detection procedure can be performed on histological sections
After Tissue Maceration [18, 52] or on cells directly fixed on slides after tissue maceration
[41]: In the presence of acetic acid and glycerol, the tissues are dis-
sociated into single cells or small clusters, which are subsequently
fixed with paraformaldehyde (pFA) that preserves their typical cel-
lular architecture. Hydra-specific or exogenous epitopes as BrdU
can next be immunodetected. The maceration technique can be
adapted for small amount of tissue such as head- or foot-­
regenerating tips [4]. The strengths of this method are multiple: it
identifies cell types and specific subcellular localization, it allows
the precise quantification of each expressing cell type, including
neurons, interstitial cells or clusters of nematoblasts (Fig. 1b).

1.2.3 Antibodies As mentioned above, the Hydra nervous system produces

numerous neuropeptides. Since 1982, their immunodetection
on intact animals proved to be extremely useful to identify the
anatomical organization and localization of specific subsets of
neurons in Hydra [53, 54]. The vasopressin-like or RFamide
sera were critical to monitor the phenotypic conversion of neu-
rons as they get displaced along the body axis [10]. With the
10 Wanda Buzgariu et al.

development of bioinformatic tools and genomics, evolution-

arily conserved regions can be easily mapped and commercially
available antibodies raised against such mammalian epitopes can
be used to cross-react against Hydra proteins. Alternatively
monoclonal [55] or polyclonal antibodies can be raised against
Hydra proteins, either lab-made such as the sera against the
RFamide neuropeptide [53], the transcription factor CREB
[56], and the homeoprotein prdl-a [18] or commercially pro-
duced. The most efficient way to validate the specificity of a
given antibody is to perform gene silencing, usually through
RNA interference in Hydra, to assess the decrease in protein
abundancy by Western analysis or immunofluorescence [57].

1.2.4 Tissue Fixation Fixation and permeabilization are crucial steps as they allow the
and Tissue preservation of cells and tissue architecture. Paraformaldehyde (pFA)
Permeabilization and formaldehyde (FA) are two cross-linking agents commonly used
as fixatives; they ensure a good penetrability and preserve the anti-
gens by forming chemical bonds between the proteins. In contrast,
alcohols such as ethanol and methanol, or acetic acid are precipitat-
ing fixatives that disrupt the hydrophobic bonds and thus denature
the tertiary structure of proteins. In fact, ethanol or methanol allow
a good permeabilization by precipitating membrane proteins, lead-
ing to the formation of pores in the membrane, thus contributing to
the leakage of RNA or cytoplasmic proteins and to a poor cellular
preservation. Therefore, the commonly used method to fix Hydra
polyps for whole-mount immunodetection or in situ hybridization
(ISH) combines pFA and alcohol fixatives. As an alternative the
Lavdowsky fixative that combines ethanol, FA, acetic acid and water
(50/3.7/4/42), also takes advantage of the properties of cross-link-
ing and precipitating agents [55]. A variant of the Lavdowsky fixa-
tive that does not contain acetic acid is often used to detect nuclear
antigens. Finally, the mercury-containing “Helly” fixative or the
Zinc–FA fixative used on insect brains to preserve morphology and
improves immunodetection at synapses [58], can be used efficiently
when the other fixatives fail. For each new epitope/antibody several
fixatives need to be tested together with the fixing conditions such
as temperature (4 °C, 18 °C, 37 °C) or duration of fixation. Tissue
permeabilization is usually completed by adding a detergent such as
Triton X-100 or Tween 20, again applied for a period of time that
needs to be adapted to each antigen of interest.

1.2.5 Antigen Detection The detection of an antigen can be made directly with the primary
antibody or indirectly through a secondary antibody that binds to
the primary one. The indirect method is preferred as Hydra spe-
cific antibodies conjugated with fluorochrome are rare and immu-
nodetection is more sensitive when indirect as the signal is
amplified. A large choice of secondary antibodies coupled to
fluorochromes or enzymes are available. Amplification is optimal
 Methods to Monitor Adult Neurogenesis in Hydra 11

when a Tyramide detection system is used with conditions adapted

for each antibody (see supplemental figure 1 in ref. 21). Unspecific
binding of antibodies to reactive sites of proteins is blocked by BSA
(bovine serum albumin) or normal serum from the species used to
raise the secondary antibody. A common counterstaining is made
by nuclear staining, which allows a better cell-type and tissue-layer
identification through the shape and size of nuclei.

2 Materials

All stock solutions are prepared with MilliQ water in sterilized bot-
tles with screw-cup, then autoclaved or filtered through a 0.22 μm
Steritop filter, and finally stored at 4 °C or at room temperature
(RT) depending on the buffers.
After opening, the stock solution should be checked regularly
to be discarded before they become viscous or turbid, with visible
signs of contamination. Experiments are always performed with
fresh solutions, i.e. 10× stock solutions freshly diluted in sterile
dishware with MilliQ water to prepare the requested amount of 1×
solution for a single experiment.

2.1 Hydra 1. Hydra medium (HM): 1 mM NaCl, 1 mM CaCl2, 0.1 mM

Maceration KCl, 0.1 mM MgSO4, 1 mM Tris pH 7.6 [59]. HM is pre-
pared from three stock solutions that can be stored several
weeks at RT once autoclaved:
For the stock solution A (0.5 M Tris, 500×) dissolve 60.57 g
Tris-base in 900 ml H2O, adjust the pH to 7.7 and complete
the volume to 1000 ml. For the stock solution B (1 M MgSO4,
10,000×) dissolve 61.6 g MgSO4⋅7H2O in 250 ml H2O. For
the stock solution C (0.5 M CaCl2, 0.5 M NaCl, 0.05 M KCl,
500×) dissolve 54.77 g CaCl2⋅6H2O, 14.6 g NaCl, 1.85 g KCl
in 500 ml H2O. Autoclave the solutions A, B, and C. To pre-
pare 1 l HM solution, dilute 2 ml solution A, 100 μl solution
B and 2 ml solution C in 1000 ml H2O.
2. Macerating solution (MS): 7% acetic acid, 7% glycerol in
H2O. Add 0.7 ml glycerol and 0.7 ml glacial acetic acid to
8.6 ml H2O. Use the fume hood for preparation. Do not auto-
clave. Store at RT no longer than 2 weeks.
3. 10% Tween 80: Add 1 ml Tween 80 to 9 ml H2O (see Note 1).
Do not autoclave. Keep the solution for 1 month at RT.

2.2 Fixation 1. 4% Urethane: Dissolve 1 g urethane in 25 ml HM. Store at

4 °C. Always wear gloves as urethane is carcinogenic.
2. Lavdowsky fixative: 50% ethanol, 3.7% FA, 4% acetic acid. Add
1 ml 37% FA to 5 ml ethanol, 0.4 ml acetic acid and 4 ml
H2O. Prepare it fresh, do not store.
12 Wanda Buzgariu et al.

(a) Lavdowsky fixative without acetic acid: 50% ethanol, 3.7%

FA. Add 1 ml 37% FA to 5 ml ethanol and 4 ml
H2O. Prepare it fresh and do not store.
(b) 8% Paraformaldehyde (PFA): Dissolve 8 g pFA in 80 ml
HM preheated at 65–70 °C in a glass bottle and stir the
mixture on a water bath maintained at 70 °C under the
hood. Add 100 μl 1 N NAOH and stir the solution until
the solution becomes clear. After cooling to RT, adjust the
volume to 100 ml with HM and adjust the pH to 7.5 with
0.1 N NaOH. Filter the solution using a steriflip filter.
Store at 4 °C for 2 weeks maximum (see Note 2).
(c) 4% PFA: Dilute 50 ml 8% pFA with 50 ml H2O and read-
just the pH to 7.5 with 0.1 N NaOH.
(d) 3.7% Formaldehyde (FA): Add 1 ml 37% FA to 9 ml H2O.

2.3 BrdU BrdU (5-Bromo-2'deoxyuridine is an analog of thymidine that is

Immunodetection rapidly incorporated into DNA during the replication phase of the
cell cycle.
1. BrdU solution (10 mM): Add 154 mg BrdU to 50 ml HM in a
50 ml centrifugation tube, protect the tube from light and stir
the solution that can be stored at 4 °C for a few days (see Note
3).
2. 10× PBS buffer: 1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4,
11 mM KH2PO4. Dissolve 80 g NaCl, 2 g KCl, 29 g Na2HPO4
12H2O and 2 g KH2PO4 in 1000 ml H2O. Verify that pH is
6.8. Autoclave and keep at RT (see Note 4).
3. PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.1 mM
KH2PO4. Dilute 50 ml 10× PBS to 500 ml (see Note 4).
4. PBST: 0.5% (v/v) Triton X-100 in PBS. Add 0.5 ml Triton
X-100 to 100 ml PBS (see Note 1).
5. Blocking solution: 2% BSA. Dissolve 1 g BSA in 50 ml PBS and
stir until the solution becomes clear. Filter through a Steriflip
and store at 4 °C (see Note 5).
6. 2 N HCl: Add 2 ml 37% HCl to 10 ml of H2O (see Note 6).
7. DAPI (4,6-diamidino-2-phenylindole) stock solution (1 mg/
ml): Dissolve 10 mg DAPI in 10 ml H2O. Aliquot and store at
−20 °C.
8. DAPI working solution (1 μg/ml): Dilute 1 μl DAPI 1 mg/ml
into 1 ml PBS.

2.4 Antibodies 1. The polyclonal anti-prdla antibody was produced in rabbit

after immunization with the N-terminal fragment of prdl-a
fused with 6HIS [18]. The polyclonal anti-RFamide antibody
was produced in rabbits and kindly provided by Cok
 Methods to Monitor Adult Neurogenesis in Hydra 13

Grimmelikhuijzen [53]. For BrdU immunodetection the

BrdU labeling and detection kit from Roche (ref 11444611001)
provides the most reliable results. Dilute the antibody 1:20 in
the labeling solution found in the kit (see Note 7).
2. The optimal titer for each primary antibody should be estab-
lished after testing a series of dilutions; too high concentra-
tions increase the background staining as the antibody
accumulate at the cell or tissue surface, too low concentrations
provide low-­intensity signals due to a reduced antigen–anti-
body interaction.
3. In the indirect immunostaining procedure, the secondary anti-
body needs to be chosen according to the available filters and
lasers that equip the fluorescent/confocal microscopes. If a
double antigen detection is required, select the secondary anti-
bodies considering the possible cross talk between the excita-
tion and emission wavelengths of each fluorochrome.

2.5 Mounting The samples should be mounted in a medium adapted for fluores-
Medium cence, i.e. characterized by a high refractory index, no autofluores-
cence and protecting against photobleaching. The MOWIOL
mounting medium (polyvinyl alcohol) fulfills these criteria. It is a
widely used lab-made preparation, less expensive than the com-
mercially available ones.
1. 0.2 M Tris pH 8.5: Dissolve 2.42 g Tris base in 100 ml of
H2O. Adjust in a fume hood the pH to 8.5 with approximately
710 μl of 37% HCl solution.
2. Add 24 g MOWIOL 4–88 to 60 g glycerol and stir well.
Subsequently add 60 g H2O and mix well for another 2 h at
RT.
3. Add 100 ml 0.2 M Tris pH 8.5 and continuously stir at 52 °C
on a heating plate until the powder is completely dissolved.
This might take 4–5 h.
4. Dispatch the mix in 50 ml centrifugation tubes and centrifuge
at 5000 × g for 15 min.
5. Carefully collect the supernatant and aliquot into 15 ml coni-
cal tubes.
6. Store the Mowiol mounting medium at −20 °C up to 1 year.
For current use, keep aliquots at 4 °C.

2.6 Equipment 1. Stereomicroscope.

2. Pasteur glass pipette.
3. 360° vertical rotator.
4. Shaker.
5. Superfrost Plus microscope slides (Thermo Scientific, Gerhard
Menzel).
14 Wanda Buzgariu et al.

6. Coverslips: 22 × 40 mm, 0.13–0.17 thickness.

7. Surgical scalpel N°3.
8. Surgical blades N°10 (Ruttgers Solingen).
9. Plastic Petri dishes (6 and 9 cm diameter).
10. Silicon bulbs, 5 mm diameter for Pasteur pipette.
11. Steriflip and steritope for filtration (Millipore).
12. Hydrophobic pen (DAKO Pen).
13. Slide staining tray, Simport Scientific.

3 Methods

3.1 Hydra Tissue 1. Collect five intact animals in a 1.5 ml Eppendorf tube with the
Maceration Protocol help of a Pasteur pipette. Remove the medium and replace  it
and Fixation with 400 μl of fresh HM, repeat the washing 2–3× times (see
Note 8).
2. After the last wash, eliminate carefully all the liquid and add
100 μl MS. Let the tube on a rack and mix gently, from time
to time, until the tissue dissociates into a homogenous cell sus-
pension (see Note 9).
3. After 30–60 min, fix the cell suspension by adding 100 μl 8%
pFA, mix gently and let incubate for 1 h at RT.
4. Add 10 μl of 10% Tween 80 and mix gently.
5. With the DAKO PEN, label a square of about 25 × 20 mm on
a Superfrost Plus slide. Let it dry for about 10 min.
6. Add the cell suspension drop by drop on the surface delimi-
tated by the hydrophobic marker lines.
7. Let the slides dry for about 2 days at RT.
8. The slides are ready to be processed or can be stored into a box
at −20 °C.

3.2 Hydra Fixation 1. Collect 15–20 intact animals with a glass Pasteur pipette in a
for ISH and Whole-­ graded 2 ml Eppendorf tube and adjust the final volume to
Mount 1 ml HM.
Immunostaining 2. Add 1 ml 10 mM BrdU solution and transfer the animals in a
plastic Petri dish (6 cm diameter) that contains 10 ml 5 mM
BrdU solution. Protect from light and incubate for the desired
period of time (see Note 3).
3. To wash out BrdU, collect the animals in 2 ml tube and wash
them several times in HM by gently aspirating the liquid and
replacing it with fresh medium.
 Methods to Monitor Adult Neurogenesis in Hydra 15

4. To initiate regeneration, transfer the animals in a plastic Petri

dish (9 cm diameter) in 50 ml HM and place the dish under
the stereomicroscope. Let the animals relax for a few minutes.
5. Bisect the animals with a scalpel at half of the body column
length and transfer the head regenerating halves to a new Petri
dish filled with 50 ml HM. Let the animals to regenerate (see
Note 10).
6. Collect 15–20 intact or regenerated animals in a graded 2 ml
Eppendorf tube and wash them several times with HM as
described at step 3.
7. Adjust the final volume to 1 ml. Let the animals to relax a few
minutes.
8. Immediately before fixation, add 1 ml 4% urethane to reach a
2% final concentration. Mix gently and wait for 60 s until the
animals are completely relaxed (see Note 11).
9. Remove 1 ml urethane solution and immediately add 1 ml 8%
pFA to reach a 4% final concentration. Mix well and let the
animals fall to the bottom of the tube and aspirate the maxi-
mum amount of the liquid.
10. Wash 3–4 times with 4% PFA by eliminating each time the
liquid and replacing it with fresh fixative.
11. Place the tubes on a shaker/rotator and fix the animals for 4 h
at RT (see Note 12).
12. Aspirate the PFA and replace it with ethanol 100%; replace
several times the ethanol until the brown pigment is
solubilized.
13. Store the fixed samples at −20 °C in ethanol until the ISH
procedure.

3.3 Hydra Fixation 1. For BrdU incubation and animal fixation, proceed as in
for BrdU Subheading 3.2 from steps 1 to 8.
Immunostaining 2. Remove the maximum amount of the urethane solution and
on Whole-Mount replace it with Lavdowsky fixative without acetic acid. Wash
several times with the fixative.
3. Place the tubes on a shaker or rotator and let the animals fix
either for 1 h at 37 °C or overnight at 4 °C (see Note 12).
Proceed immediately after fixation with the immunodetection.

3.4 Immuno­ The immunodetection procedure is performed in a dark humidity

detection of Hydra chamber (see for example the StainTray slide staining system pro-
Cells After Tissue posed by Sigma). During antibody incubation, the atmosphere is
Maceration kept humid by adding PBS in the bottom of the chamber.
16 Wanda Buzgariu et al.

1. Rehydrate the cells by washing 3× 10 min with PBS (see

Note 13).
2. Incubate the cells with 0.1% PBST for 30 min (see Note 14).
3. Add the blocking solution for 1 h at RT (see Note 15).
4. Dilute the primary antibody as desired in blocking solution.
5. Gently remove the blocking solution and add 100 μl of ­primary
antibody solution. Incubate overnight at 4 °C or alternatively
3–4 h at RT (see Note 16).
6. Wash the slides 4× 10 min with PBS.
7. Dilute the secondary antibody in PBS as recommended by the
supplier.
8. Remove PBS, add the secondary antibody solution and incu-
bate for 2–4 h protected from light.
9. Wash the slides 4× 10 min with PBS.
10. Incubate with 1 μg/ml DAPI for 10 min (see Note 17).
11. Wash the slides 2× 5 min with PBS.
12. Wash the slides fast with H2O and let them dry at RT
for 15–20 min protected from light.
13. Once dried, add one drop of mounting medium and apply the
coverslip by holding it at 45 °C. Gently descend the cover-
slip allowing the mounting medium to cover the surface
delimitated by the square. Avoid producing air bubbles (see
Note 18).

3.5 Immunostaining The procedure presented below is a general protocol, which has to
on Whole-Mount be adapted for each antibody. Unless specified, the incubation
Hydra steps are performed at RT in 1.5 ml Eppendorf tubes. The proto-
col should be adjusted according to the fixative. If samples are
freshly fixed with Lavdowsky fixative, with or without acetic acid,
start with step 1. If samples are stored in ethanol after pFA fixa-
tion, directly proceed to step 2.
1. Remove the Lavdowsky fixative and replace it with PBS. Repeat
quickly the washing step twice. Proceed to step 3.
2. Rehydrate the pFA-fixed animals stored in ethanol by washing
successively in 75%, 50% and 25% ethanol, each step for
5–10 min (see Note 8).
3. Wash 4× 10 min with PBS.
4. Remove PBS, add PBST to permeabilize for 30 min (see Note
14).
5. Optional for nuclear antigen detection. Otherwise proceed to
step 7. Remove PBST and incubate in 2 N HCl for 30 min (see
Note 19).
 Methods to Monitor Adult Neurogenesis in Hydra 17

6. Remove HCl and wash quickly 6× in PBS to eliminate all HCl

traces. Complete with 2× 5 min washes in PBS (see Note 20).
7. Add the blocking solution for 60 min at RT (see Note 15).
8. Dilute the primary antibody as desired in blocking solution.
9. Remove the blocking solution, add at least 100 μl primary
antibody solution. Incubate overnight at 4 °C or alternatively
3–4 h at RT (see Note 21).
10. Remove the primary antibody, wash quickly twice in PBS, then
4× 10 min.
11. Dilute the secondary antibody in PBS as recommended by the
supplier.
12. Remove PBS, add the secondary antibody solution and incu-
bate for 2–4 h protected from light (see Note 22).
13. Remove the secondary antibody, wash quickly twice in PBS,
then 4× 10 min.
14. Remove PBS and add 300 μl 1 μg/ml DAPI for 10 min (see
Note 23).
15. Wash quickly twice with PBS, then 2× 5 min.
16. Wash quickly with H2O.
17. Mount the animals on glass slide with the mounting medium.
Gently descend the coverslip allowing the mounting medium
to cover the surface. Avoid producing air bubbles (see
Note 24).

3.6 Immunostaining The immunodetection protocol described above can be applied to

on Whole-Mount samples previously processed for in situ hybridization (ISH) to
Hydra After In Situ combine the detection of both protein and gene expression. Since
Hybridization the 1990s, gene expression patterns can be investigated in whole-­
mount Hydra. The classical procedure comprises fixation of the
samples, hybridization of Digoxygenin (DIG) or Fluorescein-­
labeled riboprobes to the targeted transcript, and immunodetec-
tion of these hybridized riboprobes with an anti-DIG or an
anti-Fluorescein antibody coupled to alkaline phosphatase, fol-
lowed by a colorimetric detection of the coupled enzyme with
NBT/BCIP substrate (for a detailed protocol, see ref. 53). The
presentation of the complete ISH procedure is omitted here and
only the steps that link the two methods are detailed.
1. At the end of the ISH procedure, stain the samples with NBT/
BCIP as in [51] and follow carefully the development of the
staining. Once the pattern is obtained, wash out as indicated
the NBT/BCIP substrate to block the staining.
2. Post-fix the samples in 3.7% FA for 30 min at RT.
3. Wash out FA by rinsing two to three times with methanol.
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 GENIUS
IN
SUNSHINE AND SHADOW

By the Same Author

EDGE-TOOLS OF SPEECH.

One fine octavo volume. $3.50.

"A vast storehouse of the best thought of the world."—Boston:

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It is indispensable in the library and at the office."—Gazette.

"He has classified his quotations alphabetically under the head of

subjects
('Ability,' 'Absence,' etc.), and has collected the most famous literary
or historical
sayings bearing on each subject. Thus the word 'Ability' is made the
text of wise utterances from Napoleon I., Dr. Johnson, Wendell
Phillips, Longfellow,
Maclaren, Gail Hamilton, Froude, Beaconsfield, Zoroaster,
Schopenhauer,
La Rochefoucauld, Matthew Wren, Gibbon, and Aristotle. It has no
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GENIUS

IN SUNSHINE AND SHADOW

 BY

MATURIN M. BALLOU
AUTHOR OF "EDGE-TOOLS OF SPEECH," ETC.

'Tis in books the chief

Of all perfection to be plain and brief.
Butler

BOSTON
TICKNOR AND COMPANY
1887

Copyright, 1886,
By Maturin M. Ballou.

All rights reserved.

University Press:
John Wilson and Son, Cambridge.

PREFACE.
The volume in hand might perhaps better have been entitled
"Library Notes," as the pages are literally the gathered notes of the
author's library-hours. The reader will kindly peruse these pages
remembering that they assume only to be the gossip, as it were, of
the author with himself,—notes which have grown to these
proportions by casual accumulation in the course of other studies,
and without consecutive purpose. That these notes thus made have
been put into printed form, is owing to the publisher's chance
knowledge and hearty approval of them. These few lines are by way,
not of apology,—no sensible person ever made an apology,
according to Mr. Emerson,—but of introduction; so that the reader
may not fancy he is to encounter a labored essay upon the theme
suggested by the title of the volume.
These pages may not be without a certain wholesome influence, if,
fortunately, they shall incite others to analyze the character of
genius as exhibited by the masters of art and literature. The facts
alluded to, though familiar to many, are not so to all; wherefore the
volume may indirectly promote the knowledge of both history and
biography, at the same time leading the thoughtful reader to seek
further and more ample information concerning those individuals
who are here so briefly introduced.
M. M. B.

GENIUS
IN SUNSHINE AND SHADOW.
 CHAPTER I.
The ever-flowing tide of time rapidly obliterates the footprints of
those whom the world has delighted to honor. While it has caused
heroic names, like their possessors, to lapse into oblivion, it has also
shrouded many a historical page with the softened veil of distance,
like ivy-grown towers, rendering what was once terrible now only
picturesque. In glancing back through thousands of years, and
permitting the mind to rest on the earliest recorded epochs, one is
apt to forget how much human life then, in all its fundamental
characteristics, was like our own daily experience. There never was a
golden age; that is yet to come. The most assiduous antiquarian has
only corroborated the fact that human nature is unchanged.
Conventionalities, manners and customs, the fashions, may change,
but human nature does not. As an example of the mutability of
fame, we have only to ask ourselves what is actually known to-day
of Homer,[1] Aristophanes, and their renowned contemporaries, or
even of our more familiar Shakespeare?[2] Of the existence of the
first named we have evidence in his two great epics, the Iliad and
the Odyssey; but, though deemed the most famous poet that ever
lived, we do not even know his birthplace.

"Ten ancient towns contend for Homer dead,

Through which the living Homer begged his bread."

The cautious historian only tells us that he is supposed to have

flourished about nine hundred years before the time of Christ; while
there are also learned writers who contend that no such person as
Homer[3] ever lived, and who attribute the two most famous poems
of antiquity to various minstrels or ballad-mongers, who celebrated
the "tale of Troy divine" at various periods, and whose songs and
legends were fused into unity at the time of Pisistratus.

Over the personality of Aristophanes,[4] the great comic poet of

Greece, who is supposed to have flourished some five or six hundred
years later than Homer, there rests the same cloud of obscurity, and
he is clearly identified only by eleven authentic comedies which are
still extant, though he is believed to have written fifty. Of
Shakespeare, born some two thousand years later (1564), how little
is actually known beyond the fact of his birthplace! Even the
authorship of his plays, like that of Homer's poems, is a subject of
dispute. Perhaps, however, this loss of individuality but adds to the
influence of the poet's divine mission. The really great men of
history, benefactors of their race, are those who still live in the
undying thoughts which they have left behind them.

In this familiar gossip we propose to glance briefly at such names as

may suggest themselves, without observing any strict system of
classification. The theme is so fruitful, the pages of history so teem
with portraits which stand forth in groups to attract the eye, that one
hardly knows where to begin, what matter to exclude, what to
adduce; and therefore, closing the elaborate records of the past, we
will trust to momentary inspiration and the ready promptings of
memory.
The first thought which strikes us in this connection is, that the
origin of those whom the world has called great—men who have
written their names indelibly upon the pages of history—is often of
the humblest character. Such men have most frequently risen from
the ranks. In fact, genius ignores all social barriers and springs forth
wherever heaven has dropped the seed. The grandest characters
known in art, literature, and the useful inventions have illustrated
the axiom that "brave deeds are the ancestors of brave men;" and it
would almost appear that an element of hardship is necessary to the
effective development of true genius. Indeed, when we come to the
highest achievements of the greatest minds, it seems that they were
not limited by race, condition of life, or the circumstances of their
age. "It is," says Emerson, "the nature of poetry to spring, like the
rainbow daughter of Wonder, from the Invisible, to abolish the past
and refuse all history." But this of course refers only to poetry in its
loftiest and noblest conceptions and sentiments; and then only in
passages of a great work.
Æsop, the fabulist, who flourished six hundred years before Christ,
and whose fables are as familiar to us after the lapse of twenty-five
hundred years as household words; Publius Syrus,[5] the eminent
moralist, who lived in the time of Julius Cæsar, and whose wise
axioms are to be found in every library; Terence,[6] the Carthaginian
poet and dramatist; Epictetus, the stoic philosopher,—all were slaves
in early life,[7] but won freedom and lasting fame by force of their
native genius. No man is nobler than another unless he is born with
better abilities, a more amiable disposition, and a larger heart and
brain. The field is open to all; for it is fixedness of purpose and
perseverance that win the prizes of this world,—qualities that can be
exercised by the most humble.
Protagoras, the Greek sophist and orator, was in his youth a street
porter of Athens, carrying loads upon his back like a beast of
burden. He was a singularly independent genius, and was expelled
from his native city because he openly doubted the existence of the
gods. His countryman, Cleanthes the stoic, was also "a hewer of
stone and drawer of water," but rose among the Athenians to be
esteemed as a rival of the great philosopher Zeno. He wrote many
works in his day,—about three hundred years before the Christian
Era,—none of which have been preserved except a hymn to Jupiter,
which is remarkable for purity of thought and elevation of sentiment.
We need not confine ourselves, however, to so remote a period to
illustrate that genius is independent of circumstances. In our random
treatment of the subject there occurs to us the name of Bandoccin,
one of the most learned men of the sixteenth century, who was the
son of an itinerant shoemaker, and who was himself brought up to
the trade. Gelli, the prolific Italian author, and president of the
Florentine Academy, was a tailor by trade, and of very humble birth.
His moral dialogues entitled, I Capricci del Bottajo ("The Whims of
the Cooper"), have been pronounced by competent critics to be
extraordinary for originality and piquancy, while all his works are
remarkable for purity of diction. Canova, the sculptor of world-wide
fame, was the son of a day-laborer in the marble quarries. Opie, the
distinguished English painter, earned his bread at the carpenter's
trade until his majority, but before his death became professor of
painting in the Royal Academy. Amyot, the brilliant scholar, and
professor of Greek, Hebrew, and Latin, who is ranked among those
who have contributed most towards the perfection of the French
language, learned to write upon birch-bark with charcoal, while he
lived on a loaf of bread per day. This man rose to be grand almoner
of France, and proved that courage, perseverance, and genius need
no ancestors.[8]
Akenside, the English didactic poet, wit, essayist, and physician,
author of the "Pleasures of the Imagination," was a butcher's boy.
His developed genius caused him to be appointed to the Queen's
household. Sir Humphry Davy was an apothecary's apprentice in his
youth. Matthew Prior, the English poet and diplomatist, began life as
a charity scholar. Rollin, famous for his "Ancient History," was the
son of a poor Parisian cutler, and began life at an iron-forge. James
Barry, the eminent historical painter, was in his minority a foremast
hand on board an Irish coasting-vessel. D'Alembert, the remarkable
French mathematician, author, and academician, was at birth a poor
foundling in the streets of Paris, though it must be added that he
was the illegitimate and discarded son of Madame de Tencin, one of
the wickedest, most profligate, most cynical, and ablest of the high-
placed women of France. D'Alembert scorned her[9] proffered help
when she, learning that he was the offspring of one of her desultory
amours, attempted to assist him by her money and patronage. He
lived austerely poor, and his love was lavished, not on his natural, or
rather unnatural, mother, but on the indigent woman who had
picked him up in the street, and who by self-denial had enabled him
to obtain sustenance and education. As soon as he was old enough
to realize his true situation, he said, "I have no name, but with God's
help I will make one!" The time came when Catherine II. of Russia
offered him one hundred thousand francs per annum to become the
educator of her son, which he declined.
Béranger, the lyric poet of France, whose effectiveness and purity of
style defy criticism, was at one time a barefooted orphan on the
boulevards of the great city. His verses, bold, patriotic, and satirical,
were in every mouth among the masses of his countrymen,
contributing more than any other cause to produce the revolution of
1830.[10] He had the noble independence to refuse all official
recognition under government. Rachel, it will be remembered, was in
her childhood a street-ballad singer. A resident of the French capital
once pointed out to the writer a spot on the Champs Élysées where
at the age of twelve, so pale as to seem scarcely more than a
shadow, she used to appear daily, accompanied by her brother. A
rude cloth was spread on the ground, upon which she stood and
recited tragic scenes from Corneille and Racine, or sang patriotic
songs for pennies, accompanied upon the violin by her brother.
Her attitudes, gestures, and voice always captivated a crowd of
people. Rachel was a Jewish pedler's daughter, though she was born
in Switzerland; and in these youthful days she wore a Swiss costume
upon the boulevards.[11]
Boccaccio, the most famous of Italian novelists, was the illegitimate
son of a Florentine tradesman, and began life as a merchant's clerk.
It is well known that Shakespeare borrowed the plot of "All's Well
that Ends Well" from Boccaccio.[12] In fact, the "Decamerone"
furnished him with plots for several of his plays. Chaucer derived
from the same source his poem of the "Knight's Tale." We never
hear shallow people reflecting upon the Bard of Avon for taking
some of his plots from earlier writers, and weaving about them the
golden threads of his superb genius, without recalling Dryden's
remark relative to Ben Jonson's adaptations and translations from
the classics, in such plays as "Catiline" and "Sejanus." "He invades
authors," says Dryden, "like a monarch; and what would be theft in
other writers is but victory in him." Sterne's idea upon the same
subject also suggests itself. "As monarchs have a right," he says, "to
call in the specie of a State and raise its value by their own
impression, so are there certain prerogative geniuses who are above
plagiaries, who cannot be said to steal, but from their improvement
of a thought, rather to borrow it, and repay the commonwealth of
letters with interest again, and may more properly be said to adopt
than to kidnap a sentiment, by leaving it heir to their own fame."
Columbus, who gave a new world to the old, was a weaver's son,
and in his youth he earned his bread as a cabin-boy in a coasting-
vessel which sailed from Genoa. The story of the great Genoese pilot
possesses a more thrilling interest than any narrative which the
imagination of poet or romancer has ever conceived. His name
flashes a bright ray over the mental darkness of the period in which
he lived. In imagination one sees him wandering for years from
court to court, begging the necessary means wherewith to prosecute
his inspired purpose,[13] and finally, after successfully accomplishing
his mission, languishing in chains and in prison.
How naturally Halleck's invocation to Death, in "Marco Bozarris,"
occurs to us here, as the hero, when his object has been attained,
joyfully faces the grim monarch:

"Thy grasp is welcome as the hand

Of brother in a foreign land;
Thy summons welcome as the cry
That told the Indian isles were nigh,
To the world-seeking Genoese,
When the land wind from woods of palm
And orange-groves and fields of balm
Blew o'er the Haytian seas."

De Foe, the author of "Robinson Crusoe," and of over two hundred

other books, was a hosier by trade, the son of a London butcher
named James Foe. The particle De was added by the son without
other authority than the suggestion of his own fancy. Cardinal
Wolsey and Kirke White were also sons of butchers.
Claude Lorraine, the glorious colorist, whose very name has become
a synonym in art, was in youth employed as a pastry-cook. Molière,
the great French dramatist and actor, who presents one of the most
remarkable instances of literary success known to history, was the
son of a tapestry-maker, and was himself at one time apprenticed to
a tailor, and afterwards became a valet-de-chambre. When Molière
was valet to Louis XIII., he had already appeared upon the stage,
and was rather sneered at by the other members of the king's
household. The generous monarch observed this, and determined to
put a stop to it: "I am told you have short commons here, Molière,
and some of my people decline to serve you," said Louis, as he rose
from his breakfast one day. "Sit down here at my table. I warrant
you are hungry." And the king cut him a portion of chicken and put it
upon his plate just at the moment when a distinguished member of
the royal household entered. "You see me," said the king, "giving
Molière his breakfast, as some of my people do not think him good
enough company for themselves." From that hour the royal valet
was treated with due consideration. William Cobbett, the English
author and vigorous political writer, was a poor farmer's boy and
entirely self-educated. Izaak Walton, the delightful biographist and
miscellaneous author, whose "Complete Angler" would make any
man's name justly famous, was for years a linen-draper in London.
Pope and Southey were the sons of linen-drapers.
How rapidly instances of the triumphs of genius over circumstances
multiply upon us when the mind is permitted to roam at will through
the long vista of the past! Cervantes, the Spanish Shakespeare,
whose "Don Quixote" is as much a classic[14] as "Hamlet," was a
common foot-soldier in the army of Castile. In 1575 he was captured
by an Algerine corsair and carried as a slave to Algiers, where he
endured the most terrible sufferings. He was finally ransomed and
returned to Spain. Alexandre Dumas's grandmother was an African
slave. Hugh Miller, author, editor, poet, distinguished naturalist,
whose clear, choice Saxon-English caused the Edinburgh "Review" to
ask, "Where could this man have acquired his style?" was a stone-
mason, and his only college was a stone-quarry.[15]
Keats, the sweetest of English poets, whose delicacy of fancy and
beauty of versification are "a joy forever," was born in a stable.
Oliver Cromwell, one of the most extraordinary men in English
history, famous as a citizen, great as a general, and greatest as a
ruler, was the son of a malt-brewer. Howard, the philanthropist and
author, whose name stands a monument of Christian fame, was at
first a grocer's boy. Rossini, one of the greatest of modern
composers, was the son of an itinerant musician and a strolling
actress. Andrea del Sarto was the son of a tailor, and took his name
from his father's trade. Perino del Vaga was born in poverty and
nearly starved in his boyhood. Perugino, whose noble painting of the
"Infant Christ and the Virgin" adorns the Albani Palace at Rome,
grew up in want and misery. We all remember the story of the
shepherd-boy Giotto, who finally came to be so eminent a painter,
and the intimate friend of Dante; like Michael Angelo, he was an
architect and sculptor. Paganini, one of the greatest of instrumental
performers that ever lived, was born in poverty and was illegitimate.
He gained enormous sums of money by his wonderful exhibitions
and musical compositions, but was spoiled by adulation, becoming
reckless and dissipated. His performances in the cities of Europe
created a furore before unparalleled in the history of music, and
never since surpassed.
Wilson the unequalled ornithologist, Dr. Livingstone the heroic
missionary and African traveller, and Tannahill[16] the Scottish poet,
—author of that familiar and favorite song, "Jessie, the Flower of
Dumblane,"—earned their living in youth as journeymen weavers.
Joost van den Vondel, the national poet of Holland, was a hosier's
apprentice. Molière, already referred to, began his career as a
journeyman tailor, but occasionally his maternal grandfather took
him to the play, and thus were sown the seeds which led to his
greatness as a dramatic author and actor. Samuel Woodworth,
author of the "Old Oaken Bucket," one of the sweetest lyrics in our
language, was a journeyman printer. Richard Cobden, statesman,
economist, and author, was a poor Sussex farmer's son, whose
youthful occupation was that of tending sheep. John Bright, the
intimate friend and coadjutor of Cobden, one of the greatest, most
eloquent, and most successful of English reformers, was the son of a
cotton-spinner. Lord Clyde, the successful general who crushed the
rebellion in India, and who was made a peer of England, was the
son of a carpenter. The motto of his life, always inscribed upon the
fly-leaf of his pocket memorandum-book, was: "By means of
patience, common-sense, and time, impossibilities become possible."

John Bunyan,[17] the author of "Pilgrim's Progress," the solace and

delight of millions, and a text-book for all future time, was a tinker.
His great work is said to have obtained a larger circulation than any
other English book except the translation of the Bible. Benjamin
Franklin, statesman, philosopher, epigrammatist, was a tallow-
chandler.[18] Nathaniel Bowditch, the eminent mathematician, was a
cooper's apprentice. He was twenty-one years of age before he may
be said to have begun his education, but in his prime was a Fellow
of the Royal Society of London, and was offered the chair of
mathematics in Harvard College. Hiram Powers, the first sculptor
from this country to win European fame, was brought up a
ploughboy on a Vermont farm; his "Greek Slave" gave him high rank
among modern sculptors. Elihu Burritt, the remarkable linguist, was
a Connecticut horse-shoer. Whitefield, the eloquent English preacher
and father of the sect of Calvinistic Methodists, was in youth the
stable-boy of an English inn. Cardinal Wolsey, chief minister of Henry
VIII., was brought up to follow his father's humble calling of a
butcher. Horne Tooke, the English wit, priest, lawyer, and genius,
was the son of a poulterer.[19] Correra, afterwards president of
Guatemala, was born in poverty, and for years was a drummer-boy
in the army, where he was laughed at for saying that the world
should some day hear from him, being reminded that his present
business was to make a noise in the world. But he meant what he
said, and acted under Lord Clyde's motto. He rose by degrees to the
highest position in the gift of his countrymen. "To the persevering
mortal the blessed immortals are swift," says Zoroaster.

Ebenezer Elliott, the English "Corn-Law Rhymer,"[20] was a

blacksmith, but a poet by nature, and his songs created a political
revolution in his native land, though unlike Béranger's, in France, it
was a peaceful revolution. He was ever a true champion of the poor
and oppressed. In the latter portion of his life he was in easy
pecuniary circumstances. William Lloyd Garrison,[21] the beloved
philanthropist, orator, and writer, was born in poverty, and was early
apprenticed to a shoemaker, but became a journeyman printer
before his majority. He suffered imprisonment for his opinions' sake,
and may be said to have been the father of Abolitionism in America,
fortunately living long enough to see the grand effort of his life
crowned with success, in the emancipation of the blacks and the
abolishment of slavery throughout the length and breadth of his
native land. Kepler, the famous German astronomer, was the son of
a poor innkeeper, and though enjoying royal patronage, often felt
the pressure of poverty. Coleridge said: "Galileo was a great genius
and so was Newton; but it would take two or three Galileos and
Newtons to make one Kepler." We owe our knowledge of the laws of
the planetary system to him.
Sir Richard Arkwright, inventor of the spinning-jenny, and founder of
the great cotton industries of England, never saw the inside of a
schoolhouse until after he was twenty years of age, having long
served as a barber's assistant. Justice Tenterden, and Turner,
greatest among landscape-painters, were also brought up to the
same trade. James Brindley, the English engineer and mechanician,
and Cook, the famed navigator, were day-laborers in early life.
Romney, the artist, John Hunter, the physiologist, Professor Lee, the
Orientalist, and John Gibson, the sculptor, were carpenters by trade.
Shakespeare was a wool-comber in his youth. These low estates, the
workshop and the mine, have often contributed liberally to recruit
the ranks of those whom the world has recognized as men of
genius.
Horace Mann declared that education is our only political safety. He
might have gone further, and said our only moral safety also. It is
not, however, the school and the college alone that bring about this
grand object, though they are natural adjuncts. Real education is the
apprenticeship of life, and that is always the best which we realize in
our struggle to obtain a livelihood. Genius, as a rule, owes little to
scholastic training,—within these pages there will be found proof
sufficient of this. Sir T. F. Buxton says he owed more to his father's
gamekeeper, who could neither read nor write, than to any other
source of knowledge. He said this man was truly his "guide,
philosopher, and friend," whose memory was stored with more
varied rustic knowledge, good sense, and mother wit, than his young
master ever met with afterwards. He adds that he was his first
instructor, and that he profited far more by his remarks and
admonitions than by those of his more learned tutors.[22]
Perhaps at first thought it may seem singular that so many
unschooled geniuses should have risen to be famous in their several
departments, but it is because they were geniuses. They saw and
understood nature and art by intuition, while those of us who can
claim no such distinction have been compelled to acquire knowledge
by plummet and line, so to speak. "The ambition of a man of parts,"
says Sydney Smith, "should be not to know books, but things; not to
show other men that he has read Locke, and Montesquieu, and
Beccaria, and Dumont, but to show that he knows the subjects upon
which they have written." Let us pursue our examples still further,
for they are both interesting and remarkable when brought thus
together.

Benjamin West[23] was born in Pennsylvania, a poor farmer's boy;

but the genius of art was in him, and after patient study he became
an eminent painter, finally succeeding Sir Joshua Reynolds as
president of the Royal Academy in 1792. George III. was his
personal friend and patron. He was so thoroughly appreciated there
that he made England his home, where he died in 1820. John
Britton, author of the "Beauties of England and Wales," as well as of
several valuable works on architecture, was born in a mud cabin in
Wiltshire, and was for years engaged as a bar-tender. He was finally
turned adrift by his employer with two guineas in his pocket, but
before his death his list of published books exceeded eighty
volumes! Sir Francis Chantrey, the eminent sculptor, was in his
minority a journeyman carver in wood. Talma, the great tragic actor
of France, and favorite of the first Napoleon, was a dentist by trade.
Gifford, the eminent English critic and essayist, was "graduated"
from a cobbler's bench. When Cicero was asked concerning his
lineage, he replied, "I commence an ancestry." Beaumarchais, the
successful French dramatist, author of the "Barber of Seville" and
the "Marriage of Figaro," was a watchmaker by trade, but developed
such versatile genius as finally to excite the jealousy of the
unscrupulous Voltaire.
Thomas Ball, the sculptor, who has done so much to ornament the
parks and squares of Boston, used as a lad to sweep out the halls of
the Boston Museum.[24] The author has often been within the walls
of his pleasant studio in the environs of Florence, adjoining his
charming domestic establishment. It is near to the spot where
Powers produced his "Greek Slave," and overlooks the lovely city of
Florence, divided by the Arno. Andrew Jackson, who became
President of the United States, was the son of a poor Irish emigrant,
and so was John C. Calhoun, the great Southern statesman and
Vice-President. Abraham Lincoln and the late President Garfield were
both sons of toil, the former being commonly designated as "the rail-
splitter," the latter as "the canal-boy." Andrew Johnson was a
journeyman tailor. Henry Wilson was a cobbler at the bench until he
was nearly twenty-one. So also was Andersen,[25] the Danish
novelist. Jasmin, who has been called the Burns of France, was the
son of a street beggar. Allan Cunningham, poet, novelist, and
miscellaneous writer, began life as a stone-mason; he became the
father of four sons, all of whom won distinction in literature. Among
the father's novels was that of "Paul Jones," which was remarkably
successful. Dr. Isaac Miller, Dean of Carlisle, began life as a weaver,
and Dr. Prideaux, Bishop of Worcester, earned his living in youth as a
kitchen-boy at Oxford. Watt, the great Scotch inventor, whose
steam-engine has revolutionized modern industry, and Whitney,
inventor of the cotton-gin, were street gamins in childhood. Both
these inventors were thought by their associates to be "beside
themselves" as they grew towards maturity. "No man is quite sane,"
says Emerson; "each has a vein of folly in his composition, a slight
determination of blood to the head, to make sure of holding him
hard to some one point which nature has taken to heart."
The world's great men, according to the acceptation of the term,
have not always been great scholars. General Nathaniel Greene, the
successful Revolutionary commander, second only in military skill to
Washington, was brought up at a blacksmith's forge. Horace Greeley,
orator and journalist, was the son of a poor New Hampshire farmer
and earned his living for years by setting type. William Sturgeon the
able and famous electrician, Samuel Drew the English essayist, and
Bloomfield the poet, all rose from the cobbler's bench; and so did
Thomas Edwards, the profound naturalist. Robert Dodsley, the poet,
dramatist, and friend of Pope began life as a London footman in
livery. His tragedy of "Cleone" was so successful and well
constructed, that Dr. Johnson said, "If Otway had written it, no other
of his pieces would have been remembered," which was certainly
extravagant praise.[26] Douglas Jerrold was born in a garret at
Sheerness. Hobson, one of England's admirals, was a tailor's
apprentice in early life. Huntington, the remarkable preacher and
revivalist, was originally a coal-heaver, and Bewick, the father of
wood-engraving, was a laborer in a coal mine for many years.
John Gay, the English poet, was not "born with a silver spoon in his
mouth," but in youth he came up to London, where he served as a
clerk to a silk-mercer. "How long he continued behind the counter,"
says Dr. Johnson, "or with what degree of softness and dexterity he
received and accommodated the ladies, as he probably took no
delight in telling it, is not known." He wrote comedies, fables, farces,
and ballads, and wrote well, and was vastly popular. Gay was a great
gourmand, very lazy, and fond of society.[27] The silk-mercer's clerk
attained the much-coveted honor of resting at last in Westminster
Abbey. Boffin, the great navigator, served at first before the mast as
a common sailor. Robert Dick, the geologist and botanist, followed
his trade as a baker through his whole life.
Would it not seem, in the light of these many instances, that
practical labor forms the best training even for genius?
Linnæus (Karl von Linné), the great Swedish botanist, the most
influential naturalist of the eighteenth century, was a shoemaker's
apprentice. His works upon his favorite study are authority with
students of science all over the world. He became physician to the
king and made his home at Stockholm, but roamed over all
Scandinavia in pursuing his special science of botany and also that of
zoölogy. He will always be remembered as having been the first to
perfect a systematic and scientific classification of plants and
animals. He lies buried in the Upsala Cathedral.
Thorwaldsen, the great Danish sculptor, was the son of an humble
Icelandic fisherman, but by reason of native genius he rose to bear
the name of the greatest of modern sculptors. He left in the
Copenhagen museum alone six hundred grand examples of the art
he adorned. Many of our readers will remember having seen near
Lucerne, Switzerland, one of his most remarkable pieces of
sculpture, representing a wounded and dying lion of colossal size,
designed to commemorate the heroic fidelity of the Swiss guards
who fell Aug. 10, 1792. Thorwaldsen was passionately fond of
children, so that the moment he entered a house he gathered all the
juveniles about him; and in most of his marble groups he introduces
children. He never married, but made his beautiful mistress, the
Roman Fortunata, celebrated by repeating her face in many of his
ideal groups. Thorwaldsen gave an impulse to art in his native
country which has no like example in history; indeed, art is to-day
the religion of Copenhagen, and Thorwaldsen is its prophet.
George Stephenson, the English engineer and inventor, was in his
youth a stoker in a colliery, learning to read and write at a laborers'
evening school. John Jacob Astor began life as a pedler in the
streets of New York, where his descendants own a hundred million
dollars worth of real estate.[28] The elder Vanderbilt, famous not
alone for his millions but also for his vast enterprise in the
development of commerce and railroads, served as a cabin-boy on a
North River sloop during several years of his youth. George Peabody,
the great American philanthropist and millionnaire, was born in
poverty. Fisher Ames, the eminent statesman and orator, eked out a
precarious living for years as a country pedagogue. Greatness lies
not alone in the possession of genius, but in the right and effective
use of it.
We have given examples sufficient to illustrate this branch of our
subject, though they might be almost indefinitely extended. It was
Daniel Webster[29] who said that "a man not ashamed of himself
need not be ashamed of his early condition in life." Titles are
vendible, but genius is the gift of Heaven.
Enthusiasm is the heritage of youth; it plans with audacity and
executes with vigor: "It is the leaping lightning," according to
Emerson, "not to be measured by the horse-power of the
understanding." In the accomplishment of great deeds it is
undoubtedly the keenest spur, and consequently those who have
become eminent in the history of the world have mostly achieved
their greatness before gray hairs have woven themselves about their
brows. Unless the tree has borne ample blossoms in the spring, we
shall look in vain for a generous crop in the fall. Notwithstanding the
abundance of axioms as to youth and rashness dwelling together, we
have ample evidence that it is the period of deeds, when the senses
are unworn and the whole man is in the vigor of strength and
earnestness. Goethe tells us that the destiny of any nation depends
upon the opinions of its young men. Let us recall a few examples, in
corroboration of this view, among those who have made their mark
upon the times in which they lived.
Alexander the Great reigned over the Macedonians at sixteen; Scipio
was but twenty-nine at the zenith of his military glory; Charles XII.
[30] was only nineteen when, as commander-in-chief, he won the
famous battle of Narva; Condé was twenty-two when he gained the
battle of Rocroi; Scipio the Younger conquered Carthage at thirty-six,
and Cortes subdued Mexico at the same age. At thirty Charlemagne
was master of France and Germany; at thirty-two Clive had
established the British power in India. Hannibal won his greatest
victories before he was thirty, and Napoleon was but twenty-seven
when he outgeneralled the veteran marshals of Austria on the plains
of Italy. George Washington won his first battle as a colonel at
twenty-two; Lafayette was a major-general in our army at the age of
twenty. Nor are we to look only for youthful greatness among those
who have won laurels in war. William Pitt was prime minister of
England at twenty-four; Calhoun had achieved national greatness
before he was thirty; while the names of John Adams, Alexander
Hamilton, and the elder Pitt in England also suggest themselves in
this connection.[31]
Handel composed sonatas at ten years of age; Mozart was equally
precocious, and died at thirty-six, at which age Shakespeare had
written "Hamlet." Bellini, the composer, had produced "II Pirata," "La
Sonnambula," and "La Norma," before his thirtieth year; "I Puritani"
was finished at thirty, and he died two years later. Charles Matthews
the elder began to write for the press at fourteen, and Moore wrote
verses for print at the same age; undoubtedly both were open to
cool and judicious criticism.[32] Henry Kirke White published a
volume of poems at seventeen. Bryant, the first American poet of
celebrity, began to write verses at the age of ten, and his most
celebrated poem, "Thanatopsis," was written before he was twenty.
Fitz-Greene Halleck, author of "Marco Bozzaris," wrote verses for the
magazines at fourteen. Congreve was at the height of his literary
fame at four-and-twenty,—he to whom Dryden said Shakespeare
had bequeathed his poetical crown, and to whom Pope dedicated his
version of the Iliad. Watt invented the steam-engine before he was
thirty. The reproof administered by his grandmother for his idleness
in taking off and replacing the cover of the teakettle, and "playing
with the steam to no purpose," will occur to the reader. Joan of
Arc[33] was but eighteen when she raised the siege of Orléans and
conquered city after city, until Charles VII. was crowned king at
Rheims.
Guizot, the distinguished French statesman and historian, seems to
have been "a child who had no childhood." At eleven years of age he
was able to read in their respective languages Thucydides,
Demosthenes, Dante, Schiller, Gibbon, and Shakespeare.
Robert Hall, the eloquent English clergyman, was a remarkable
instance of early mental development. It is said that before he was
ten years of age he perused with interest and understanding
Edwards's treatises on the "Affections" and on the "Will." His
sermons, essays, and writings generally were eagerly read and
admired by the public; but excessive application at last brought on
insanity. It was gracefully said of him that his imperial fancy laid all
nature under tribute. Even in madness he did not lose his power of
retort. A hypocritical condoler visited him in the mad-house, and
asked in a servile tone: "Pray, what brought you here, Mr. Hall?" Hall
touched his brow significantly with his finger, and replied, "What'll
never bring you, sir,—too much brains!"[34]
Macaulay had already won an exalted reputation for prose and
poetry before he was twenty-three, and N. P. Willis, before he left
college, had achieved enduring fame by his sacred poems,[35]
which, in fact, he never afterwards excelled in a long and successful
literary career. Schiller wrote and published in his fourteenth year a
poem on Moses. Klopstock began his "Messiah" at seventeen, and
Tasso had produced his "Rinaldo," and completed the first three
cantos of "Jerusalem Delivered," before he was nineteen. Milton was
an unremitting student at ten. Southey began to write verses before
he was eleven, Chaucer and Cowley at twelve, and Leigh Hunt at
about the same age. Pope,[36] like so many others, began to write
poetry as a child, thus proving that "poets are born and not made."
Chatterton, the remarkable literary prodigy, died at eighteen, but not
until he had established a lasting reputation. Bulwer-Lytton was a
successful author at about the same age, and so were Keats and
Bayard Taylor. Dickens produced the "Pickwick Papers" before he
was twenty-five, and it may safely be said that in wit, humor, and
originality he never surpassed that delicious book. These seem
interesting facts to remember, though they do not establish any
actual criterion, since the thoughtful student of the past can adduce
many notable examples of mature development in art and literature.
Among these is that of Edmund Burke, on the whole the greatest of
English philosophical statesmen. He is the most remarkable instance
of a number of men of genius who seem to have grown younger as
they grew older,—that is, mentally and morally. Macaulay has
noticed that Bacon's writings towards the close of his career
exceeded those of his youth and manhood "in eloquence, in
sweetness and variety of expression, and in richness of illustration."
[37] He adds: "In this respect the history of his mind bears some
resemblance to the history of the mind of Burke. The treatises on
the 'Sublime and Beautiful,'[38] though written on a subject which
the coldest metaphysician could hardly treat without being
occasionally betrayed into florid writing, is the most unadorned of
Burke's works. It appeared when he was twenty-five or twenty-six.
When, at forty, he wrote the 'Thoughts on the Causes of the Present
Discontents,' his reason and judgment had reached their full
maturity, but his eloquence was in its splendid dawn. At fifty his
rhetoric was as rich as good taste would admit; and when he died,
at almost seventy, it had become ungracefully gorgeous. In his youth
he wrote on the emotions produced by mountains and cascades, by
the masterpieces of painting and sculpture, by the faces and necks
of beautiful women, in the style of a Parliamentary report. In his old
age he discussed treaties and tariffs in the most fervid and brilliant
language of romance."
Socrates learned to play on musical instruments in his old age. Cato
at eighty first studied the Greek language, and Plutarch did not apply
himself to learn the Latin language until about the same age.
Theophrastus[39] began his "Character of Man" on his ninetieth
birthday. Peter Rusard, one of the fathers of French poetry, did not
develop his poetic faculty until nearly fifty. Arnauld, the learned
French theologian and philosopher, translated Josephus in his
eightieth year. Lope de Vega, one of the most learned men of the
sixteenth century, wrote his best at seventy years of age. Dr.
Johnson applied himself to learn the Dutch language at seventy. At
seventy-three, when quite feeble, he composed a Latin prayer to test
to his own satisfaction the loss or retention of his mental faculties.
Chaucer's "Canterbury Tales" were the work of the author's last
years. Franklin's philosophical pursuits were but fairly begun at fifty.
La Mothe le Vayer's best treatises were written after he was eighty
years of age, and Izaak Walton's when he was nearly ninety.
Thomas Hobbes, the remarkable English philosopher and author,
published his version of the Odyssey in his eighty-seventh year, and
his Iliad in his eighty-eighth. Winckelmann,[40] author of the "History
of Ancient Art," lived in ignorance and obscurity until the prime of his
life, when he became famous. Landor was busy with authorship until
after he was eighty. The Earl of Chatham made his most remarkable
oratorical effort at seventy, and our own American orator and
statesman, Robert C. Winthrop, at a still later period of his life.
Fontenelle continued his literary pursuits until he was ninety-nine,
"blossoming in the winter of his days," as Lord Orrery wrote of him.
Ménage, the celebrated French critic and scholar, wrote sonnets and
epigrams at ninety. Julius Scaliger, the renowned Italian scholar and
poet, dictated to his son, at the age of seventy, two hundred verses
of his own composition from memory. Mr. Gladstone and John
Bright, the English statesmen, are more recent examples of
oratorical, mental, and physical powers in advanced years. George
Bancroft the American historian, in his eighty-sixth year is still
engaged in authorship, and Whittier and Holmes are writing with
unabated vigor at nearly eighty years of age. Miss Elizabeth Peabody
at eighty-four is still a vigorous writer and active philanthropist, and
the same may be said of Mrs. Julia Ward Howe at the age of sixty-
six. Mrs. Howe, indeed, is one of the foremost of American women,
whether we regard the ripeness of her scholarship, the breadth of
her understanding, the richness of her imagination, or the quiet
intrepidity with which she champions great reforms.

CHAPTER II.
Who does not enjoy recalling these silent friends, favorite authors
grown dear to us by age and long association? Some one has said
that authors, like coins, grow dearer as they grow old. Indeed,
Samuel Rogers, the banker and poet, declared that when friends at
his famous "breakfasts" were praising a new book, he forthwith
began to re-read an old one. All these writers were double-sided, so
to speak; they had their book natures and their human natures, and
it is when we prefer to contemplate them in the latter aspect that we
like them best. Carlyle calls them "the vanguard in the march of
mind, the intellectual backwoodsmen reclaiming from the idle
wilderness new territory for the thought and activity of their happier
brethren." It is true that we can form but a partial judgment of
authors by their books, their motives being not always as pure as we
are inclined to believe.[41] A traitor like Bolingbroke is quite capable
of writing a captivating book on patriotism; and it has been said if
Satan were to write one, it would be upon the advantages of virtue.
It is certain he has ever shown such a hearty appreciation of virtue
that he holds in highest estimation his success in corrupting it.
Examples flash across the memory. There was Sir Thomas More
advocating toleration, while he was himself a fierce persecutor;
Sallust declaring against the licentiousness of his age, yet addicted
to habitual debaucheries; Byron assuming a misanthropy which he
never felt; and Cowley boasting of his mistresses, though he had not
the courage even to address one. Smollett's descriptions and scenes
were often indelicate, though he was himself in that respect a
faultless man. "As a rule, the author who is not in genius far above
his productions must be a second-rate one at best," says Bulwer-
Lytton. Sometimes we detect striking likenesses between the author
and his works. Goldsmith, for instance, was the same hero to low-
bred women, and the same coward to ladies, that he depicts in his
charming comedy. It is difficult, however, in the light of Handel's
inspired music, to realize what an animal nature possessed him in
his every-day mood,—what a glutton he was at table; or to reconcile
the sublime strains of Mozart with his trivial personality.[42] Still,
Buffon persistently declares, "Le style c'est l'homme."
Addison, recognized as the purest and most perspicuous writer of
the English language, though exercising such mastership of the pen,
had no oral ability, and rarely attempted to talk in social circles. He
said of himself that though he had a hundred pounds in the bank, he
had no small coin in his pocket.[43]
Dr. Johnson and Coleridge were famous for their colloquial facility,
but both of these were rather lecturers than talkers, however
delightful in this respect the latter may have been. Johnson during
his life was undoubtedly more of a power as a talker than as a
writer. It has been said that Scott talked more poetry and Edmund
Burke more eloquence than they ever wrote. Emerson thought that
"better things are said, more incisive, more wit and insight are
dropped in talk and forgotten, than gets into books." E. H. Chapin
and H. W. Beecher have talked sounder and more brilliant theology
than they ever preached from the pulpit. Spontaneous thoughts
come from our inner consciousness; sermons and essays, from the
cooler action of the brain. Coleridge, on first meeting Byron,
entertained the poet with one of his monologues, wherein he
ascended into the seventh heaven upon wings of theology and
metaphysics. Leigh Hunt described the scene to Charles Lamb, and
expressed his wonder that Coleridge should have chosen so
unsympathetic an auditor. "Oh, it was only his fun," explained Lamb;
"there's an immense deal of quiet humor about Coleridge!"
Wordsworth speaks of him as the "rapt one, with the godlike
forehead," the "heaven-eyed creature." Hazlitt says that "no idea
ever entered the mind of man, but at some period or other it had
passed over his head with rustling pinions." Talfourd writes of seeing
"the palm-trees wave, and the pyramids tower, in the long
perspective of his style." When Coleridge once asked Lamb,
"Charles, did you ever hear me preach?" he received the quiet reply,
"I never heard you do anything else." Rogers tells us: "Coleridge was
a marvellous talker. One morning, when Hookham Frere also
breakfasted with me, Coleridge talked for three hours without
intermission about poetry, and so admirably that I wish every word
he uttered had been written down." Madame de Staël said of him
that he was great in monologue, but that he had no idea of
dialogue.
Macaulay was also remarkable for his conversational powers, which
were greatly aided by an excellent memory. He has been accused of
talking too much; and Sydney Smith once said of him: "He is
certainly more agreeable since his return from India. His enemies
might perhaps have said before—though I never did so—that he
talked rather too much; but now he has occasional flashes of silence
that make his conversation perfectly delightful!" In a party in which
eminent men are present, the rule is said to be that, for good
conversation, the number of talkers should never be fewer than the
Graces or more than the Muses. Goldsmith, who wrote so
charmingly and exhibited such a remarkable versatility with the pen,
could make no figure in conversation. Fox, Bentley, Burke, Curran,
and Swift were all brilliant talkers; Tasso, Dante, Gray, and
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