Table Of ContentAdvances in Experimental Medicine and Biology 1029
Yasunori Sasakura E ditor
Transgenic
Ascidians
Advances in Experimental Medicine
and Biology
Volume 1029
Editorial Board
IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel
ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research,
Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
NIMA REZAEI, Children’s Medical Center, Tehran University of Medical
Sciences, Tehran, Iran
More information about this series at http://www.springer.com/series/5584
Yasunori Sasakura
Editor
Transgenic Ascidians
Editor
Yasunori Sasakura
Shimoda Marine Research Center
University of Tsukuba
Shimoda, Shizuoka, Japan
Additional material to this book can be downloaded from http://extras.springer.com.
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Advances in Experimental Medicine and Biology
ISBN 978-981-10-7544-5 ISBN 978-981-10-7545-2 (eBook)
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Foreword
The ascidian Ciona provides one of the best experimental systems in devel-
opmental biology, developmental genomics, and evolutionary developmental
biology. We ascidian developmental biologists are justifiably proud of the
fact that we established the Ciona system.
In traditional embryology, ascidians were recognized as organisms exhib-
iting a “mosaic-mode of embryogenesis”, in contrast to the regulatory mode
of embryogenesis seen, for example, in sea urchins. A detailed description of
embryonic cell lineage by Edwin G. Conklin revealed that the mosaic-mode
of embryogenesis is determined by determinate cleavage of the embryo and a
robust pattern of differentiation and morphogenesis. Maternal factors and cel-
lular communications that play essential roles in gastrulation, neurulation,
tailbud-embryo formation, and tadpole-type larval development work
together under the harmonious, but rigid control of a gene regulatory net-
work. In addition, newly hatched larvae comprise only about 2600 cells,
including 40 notochord cells and 38 muscle cells. Thus, ascidians are consid-
ered embryonically simple compared to vertebrates and other deuterostomes,
such as sea urchins. In other words, ascidians represent a system to challenge
the most basic question of developmental biology: How does a complex, mul-
ticellular, metazoan body arise from a single cell, the fertilized egg?
Decoding of the draft genome of Ciona intestinalis in 2002 identified
almost all of the developmentally relevant genes in its genome, which repre-
sent the basic set of gene components in chordates prior to the two rounds of
genome duplication that occurred in the vertebrate lineage. The absence of
redundancy in ascidian regulatory gene functions makes it easier to ascertain
developmental roles of individual genes. In association with the genome
sequencing project, a cDNA sequencing project was also carried out, provid-
ing a great quantity of information on expression profiles of regulatory genes.
Many whole-mount in situ hybridization studies reveal distinct gene expres-
sion profiles in embryos at the single-cell level. Furthermore, results obtained
from embryological research provide straightforward insights into chordate
evolution as well as the origin of vertebrates, because ascidians are the closest
relatives to vertebrates.
In this research environment, it is desirable to introduce new, state-of-the-
art techniques into the Ciona system, with the most valuable at this time
being “transgenic” techniques. These include microinjection and/or electro-
poration of exogenous DNA to reveal mechanisms involved in gene regula-
tory networks, germ-line transgenesis to create various marker lines for
v
vi Foreword
studies of gene regulation, and TALEN-based and CRISPR/Cas9-based
knockout lines to facilitate studies of gene function.
This book, edited by Prof. Yasunori Sasakura at the University of Tsukuba,
constitutes a very timely discussion of recent advances in this field. In addi-
tion, most authors of this book are new or mid-career researchers. Indeed, we
owe them further development of the ascidian systems. Lastly, I wish to
express once again my conviction that Ciona is the most promising system to
explore molecular and genetic mechanisms involved in animal embryogene-
sis at the single-cell level, the tissue and organ level, and the individual level.
Marine Genomics Unit Noriyuki Satoh
Okinawa Institute of Science and Technology
Graduate University,
Okinawa, Japan
Preface
Undoubtedly, transgenic technologies are inevitable for studying molecular
functions during development. In several model ascidian species, creation of
transgenic animals has been routinely performed, and the methods are easily
retrieved from many articles and from some book chapters. However, the
details of the methods have not always been provided in the literature. On the
one hand, microinjection is basically a simple method for introducing exog-
enous DNAs without the requirement of an expensive machine. At the same
time, because microinjection can be a relatively difficult process, many labo-
ratories have their own practical tips that are suitable for their study of spe-
cies. These tips are usually not seen in original articles whose main purpose
is describing development mechanisms. To solve the issue, this book has been
purposed to gather and describe the devices that have arisen from our own
great interest and enthusiasm, and that are not usually seen or known as major
methods for performing transgenesis in ascidians.
I hope this book will be useful for all researchers, including beginners in
tunicate research who wish to introduce a tunicate in their laboratories and
also specialists of tunicates who wish to find a clue to improve their
methods.
Shimoda, Japan Yasunori Sasakura
vii
Contents
1 Introduction .................................................................................... 1
Yasunori Sasakura
2 Microinjection of Exogenous Nucleic Acids
into Eggs: Ciona Species ................................................................ 5
Kenji Kobayashi and Yutaka Satou
3 Practical Guide for Ascidian Microinjection:
Phallusia mammillata ..................................................................... 15
Hitoyoshi Yasuo and Alex McDougall
4 Microinjection of Exogenous DNA into Eggs
of Halocynthia roretzi ..................................................................... 25
Gaku Kumano
5 Electroporation in Ascidians: History, Theory
and Protocols .................................................................................. 37
Robert W. Zeller
6 The Use of cis-Regulatory DNAs as Molecular Tools ................. 49
Kotaro Shimai and Takehiro G. Kusakabe
7 Reporter Analyses Reveal Redundant Enhancers
that Confer Robustness on Cis-Regulatory Mechanisms ........... 69
Shigeki Fujiwara and Cristian Cañestro
8 Investigating Evolutionarily Conserved Molecular
Mechanisms Controlling Gene Expression
in the Notochord ............................................................................. 81
Julie E. Maguire, Aakarsha Pandey, Yushi Wu, and Anna
Di Gregorio
9 Purification of Fluorescent Labeled Cells
from Dissociated Ciona Embryos ................................................. 101
Wei Wang, Claudia Racioppi, Basile Gravez,
and Lionel Christiaen
10 Germline Transgenesis in Ciona ................................................... 109
Yasunori Sasakura
ix
x Contents
11 T he Enhancer Trap in Ciona ......................................................... 121
Yasunori Sasakura
12 TALEN-Based Knockout System.................................................. 131
Keita Yoshida and Nicholas Treen
13 C RISPR Knockouts in Ciona Embryos ....................................... 141
Shashank Gandhi, Florian Razy-Krajka, Lionel Christiaen,
and Alberto Stolfi
14 T ransgenic Techniques for Investigating Cell Biology
During Development ...................................................................... 153
Christina D. Cota
15 C ellular Processes of Notochord Formation ................................ 165
William C. Smith
16 M orphology and Physiology of the Ascidian
Nervous Systems and the Effectors .............................................. 179
Atsuo Nishino
Index ........................................................................................................ 197
