C 2014 Wiley Periodicals, Inc.

V genesis 53:160–169 (2015)

REVIEW

Mutation Studies in Ascidians: A Review

Fabio Crocetta,1 Rita Marino,1 Paola Cirino,2 Alberto Macina,2 Leopoldo Staiano,3
Rosaria Esposito,1 Maria Rosa Pezzotti,1 Claudia Racioppi,1 Francesco Toscano,1 Elena De Felice,1
Annamaria Locascio,1 Filomena Ristoratore,1 Antonietta Spagnuolo,1 Laura Zanetti,1
Margherita Branno,1 and Paolo Sordino1,4*
1
Laboratory of Cellular and Developmental Biology, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy
2
Marine Resources for Research, Stazione Zoologica Anton Dohrn, Villa Comunale, Naples, Italy
3
TIGEM, Telethon Institute of Genetics and Medicine, Via P. Castellino 111, Naples, Italy
4
CNR ISAFOM, Institute for Agricultural and Forest Systems in the Mediterranean, Via Empedocle 58, Catania, Italy

Received 6 June 2014; Revised 6 November 2014; Accepted 11 November 2014

Summary: Historically, mutations have had a significant
impact on the study of developmental processes and
phenotypic evolution. Lesions in DNA are created by
artificial methods or detected by natural genetic varia-
tion. Random mutations are then ascribed to genetic
change by direct sequencing or positional cloning.
Tunicate species of the ascidian genus Ciona represent
nearly fully realized model systems in which gene func-
tion can be investigated in depth. Additionally, tuni-
cates are valuable organisms for the study of naturally
occurring mutations due to the capability to exploit
genetic variation down to the molecular level. Here, we
summarize the available information about how muta-
tions are studied in ascidians with examples of insights
that have resulted from these applications. We also
describe notions and methodologies that might be use-
ful for the implementation of easy and tight procedures
for mutations studies in Ciona. genesis 53:160–169,
2015. V C 2014 Wiley Periodicals, Inc.
Key words: mutations; genetics; ascidians; Ciona
INTRODUCTION
broad-scale screening of animals that carry heritable
random mutations for particular phenotypes. In geneti-
cally tractable organisms, high-density linkage maps are
used for rapid and precise correlation between pheno-
types and the causative mutation. With the develop-
ment of reverse genetic manipulations to select
mutations in known genes, the rapid creation of inter-
esting null alleles has propelled the study of animal
development. Similar methods for the study of gene
function and regulation are becoming available in sim-
ple animal models at distant branches of the tree of life
(Abzhanov et al., 2008; Zantke et al., 2014). Marine
invertebrates that are amenable to genetic approaches
include species belonging to the Ascidiacea class of
tunicates, namely Ciona intestinalis sp. A, C. intestina-
lis sp. B and C. savignyi. The phylogenetic position of
the closest relatives of vertebrates renders their study
of relevance in development and evolution (Delsuc
et al., 2006). The most favorable characteristics that
have contributed to the popularity of tunicates are sim-
ple, stereotyped and rapid development (Corbo et al.,
2001; Kumano and Nishida, 2007; Lemaire, 2009; Satoh,

Forward genetics seeks to identify genes and gene
functions with no prior assumption of biological path-
ways or processes. Historically, mutational phenotypes
defined on the basis of existing variation have repre-
sented major experimental models for understanding
genetic factors and processes. The application of
standard mutagenizing techniques has allowed for the
Additional Supporting Information may be found in the online version
of this article.
* Correspondence to: Paolo Sordino, Stazione Zoologica Anton Dohrn,
Naples, Italy. E-mail: paolo.sordino@szn.it
Contract grant sponsor: Assemble; Contract grant number: EU-FP7;
Progetto Premiale 2011 (MIUR); SZN PhD Fellowship
Published online 14 November 2014 in
Wiley Online Library (wileyonlinelibrary.com).
DOI: 10.1002/dvg.22837
 ASCIDIAN MUTATIONS 161

FIG. 1. Natural populations of Ciona intestinalis in the Fusaro Lake at different locations and seasons. a. Typical crowded assemblage of
individuals growing horizontally on a wooden column in spring months. b. A less dense population with distributed individuals in the hori-
zontal position. c. Dying Ciona during the warm season. d. Young recruitants. Photos by F. Toscano.

1994), as well as a growing number of sequenced and
annotated genomes (Dehal et al., 2002; Denoud et al.,
2010; Hill et al., 2008; Satou et al., 2008; Small et al.,
2007). In addition, ascidians have the practical advan-
tages of being hermaphrodites and broadcast spawners
(Lambert, 2005), so that deleterious alleles that individ-
ually produce visible phenotypes are screened in the F1
generation (Hendrickson et al., 2004; Veeman et al.,
2011). In addition, techniques for systematic mainte-
nance and breeding of Ciona remain understudied
(Cirino et al., 2002; Joly et al., 2007; Mita et al., 2012).
Standard operating procedures are not available and
any advances in husbandry are often employed in isola-
tion, so that newly established ascidian facilities likely
operate at sub-optimal levels. In culturing, species with
high genetic variability and weak genetic structure are
confronted with a progressive loss of genetic diversity
(Stohler et al., 2004). Heterozygosity decline in isolated
strains may be further increased by inconsistent
breeding schemes and methodologies, and may lead to
decreases in production and long-term viability. The
implication is that ascidian research is based upon a
continuous supply of wild-caught individuals.
Ciona have lecithotrophic larvae and sessile adults.
Dispersal processes may last hours to days, affecting the
larval phase on a variety of spatial and temporal scales
(Tremblay and Sinclair, 1992). Larval settlement and
subsequent metamorphosis depend on available space
in suitable habitats, and may be limited by population
size (Tursi, 1980). The ecology of these ascidians dis-
plays high colonization capabilities in coastal biotopes,
where the species may act as sentinels of eutrophic
conditions following anthropization (Therriault and
Herborg, 2008). Population dynamics is primarily influ-
enced by environmental conditions, e.g., salinity and
temperature, and is characterized by dense and eventu-
ally transient aggregates (Fig. 1). Interestingly, popula-
tions of Ciona intestinalis from the northern (sp. B)
162 CROCETTA ET AL.
and southern (sp. A) European coasts are subject to plu-
riannual cycles, with 1–2 years of die-off (Caputi et al.,
in press; Lundalv and Christie, 1986).
Ascidians present different types of evolutionary
processes acting at the population level, such as sea-
sonal forms, morphotypes, cryptic species, hybrids,
etc. (Caputi et al., 2007, 2008; Kano et al., 2001;
Nydam and Harrison, 2007, 2010, 2011a,b; Roux et al.,
2013; Sato et al., 2014; Tsagkogeorga et al., 2012; Zhan
et al., 2010, 2012) Records of naturally occurring, herit-
able abnormalities affecting the ascidian body plan may
considerably enhance our understanding of how
genetic variation influences adaptation and divergence
in marine habitats (Chang and Shaw, 2003; Drake et al.,
1998; Mitchell-Olds et al., 2007; Rudall and Bateman,
2003). In C. savignyi and C. intestinalis sp. A, phylomi-
micking mutations that affect larval settlement and dis-
persal capabilities may be used to address questions
related to the genetic basis of morphological evolution.
Several methods and resources have been developed
that enable relatively straightforward genetic manipula-
tion in the Ciona species. Except for targeted mutations
and transposon insertions, for which the identification
of the gene of interest is made possible by locating
sequences near the tag, methods for positional cloning
of genetic mutations are rapidly progressing (Abdul-
Wajid et al., 2014). While reverse and forward genetics
approaches in Ciona have been extensively summar-
ized in reviews (Hendrickson et al., 2004; Stolfi and
Christiaen, 2012; Veeman et al., 2011), details for the
design of mutation studies remain fragmented. Muta-
tion studies in ascidians require notions and methods
from different scientific fields. In this review, we
attempt to integrate the available knowledge related to
ascidian biology, ecology, genetics and husbandry that
can be used to improve the efficiency with which these
studies are executed in these promising model species.
In detail, we focus on the current status of mutation
research in ascidians and on the developmental insights
that have come from studies of tunicate mutant pheno-
types. Finally, a small-scale screening for naturally
occurring mutations in C. intestinalis sp. A allowed for
new interesting phenotypes and operational solutions.
MUTATION STUDIES IN ASCIDIANS: METHODS
AND FINDINGS
Insertional Mutagenesis
Transposable DNA constructs are powerful tools for
genetic analysis. The Tc1/mariner superfamily Minos is
efficiently employed for transposon-based transgenesis
and insertional mutagenesis in C. savignyi and C. intes-
tinalis (Matsuoka et al., 2004, 2005; Sasakura et al.,
2003a,b). Germline integration of transgenes is
achieved by co-injection or co-electroporation of Minos
transposase mRNA and plasmid carrying the transgene
flanked by Minos terminal repeats. Furthermore,
transposon-based insertional mutagenesis has the
potential to disrupt the endogenous gene at the site of
integration. For instance, swimming juvenile contains a
Minos insertion in the cellulose synthase gene, illustrat-
ing a new function of this gene in the coordination of
morphogenetic events in the trunk and tail during meta-
morphosis that leads to premature body plan reorgan-
ization prior to settlement of larvae (Sasakura et al.,
2005). Minos activity in generating mutant and trans-
genic animals differs between C. savignyi and C. intesti-
nalis, being slightly stronger in the second species
(Matsuoka et al., 2004). It has been shown that the
unique sequence of the Minos transgene can be used to
easily locate the insertion site. The high-throughput
remobilization of single Minos integration events from
DNA to DNA in enhancer trap screenings contains great
promise for its potential for insertional mutagenesis
(Awazu et al., 2007; Matsuoka et al., 2004; Sasakura
et al., 2012).
Chemical Mutations
Early attempts to apply forward genetics in ascidians
consisted of chemical mutagenesis in C. savignyi and C.
intestinalis (Moody et al., 1999; Nakatani et al., 1999;
Sordino et al., 2000, 2001). This method was based on
the use of the powerful alkylating agent N-ethyl-N-nitro-
sourea (ENU), a point mutagen originally applied to
Drosophila and Caenorhabditis and, later, to Xenopus,
zebrafish and mouse (Ashburner and Roote, 2007; De
Stasio and Dorman, 2001; Goda et al., 2006; Justice
et al., 1999). Chongmague is a mutation in the C.
savignyi ortholog of the vertebrate alpha 3/4/5 family
of laminins that may be downstream targets of the pla-
nar cell polarity (PCP) pathway (Jiang et al., 2005a).
Notochord progenitor cells abnormally adopt a
mesenchyme-like fate, thus suggesting that PCP signal-
ing plays distinct functions in notochord morphogene-
sis (Nakatani et al., 1999; Veeman et al., 2008). The
phenotype of an ENU mutation lacking a functional bra-
chyury gene shows stochastic fate transformation and a
major defect in tail resorption (Chiba et al., 2009). Fur-
thermore, a point mutation in the C. savignyi tyrosin-
ase gene, named spotless, specifically disrupts sensory
organ pigmentation, so that mutant larvae are unable to
respond properly to gravity and illumination cues while
settling (Jiang et al., 2005b). Genetic disturbances
affecting different stages in sensory organ formation,
from specification to terminal differentiation, are com-
monly encountered in C. intestinalis sp. A (Sordino
et al., 2001). A null mutation in the doublesex/mab3
related-1 gene causes severe abnormalities in the sen-
sory vesicle (brain), palps and oral siphon primordium
(Tresser et al., 2010). In addition to these findings, the
 ASCIDIAN MUTATIONS
use of ENU for mutagenesis screenings has been aban-
doned because seawater salinity appears to reduce
mutagenic activity (Hendrickson et al., 2004).
Naturally Occurring Mutations
The study of changes induced in the laboratory has
been complemented by the discovery of spontaneous
mutations in natural populations of Ciona species (Hen-
drickson et al., 2004; Sordino et al., 2008). The
frequency of heterozygote carriers with obvious pre-
metamorphic phenotypes varies among ascidian
aggregates, reaching one fifth of the analyzed adults.
The research group of Bill Smith (University of Califor-
nia Santa Barbara, USA) was the first to conduct a
detailed examination of naturally occurring mutation
phenotypes, providing new insights into the anterior
neural fate (frimousse, C. intestinalis sp. A) (Deschet
and Smith, 2004) and anterior-posterior polarity of the
notochord (aimless, C. savignyi) (Jiang et al., 2005a).
In particular, the analysis of aimless, a mutant of the
ortholog of the PCP pathway component prickle,
revealed a role for the PCP signaling pathway in the
establishment of antero-posterior polarity in notochord
cells (Jiang et al., 2005a).
Sequence-Specific Mutations
Genome editing methodologies that promote tar-
geted genetic mutations by means of DNA endonucle-
ases (e.g., zinc fingers: ZFNs; transcriptional activator-
like effector nucleases: TALENs) and the CRISPR/Cas
system have the potential to facilitate the field of
reverse genetics in less conventional model organisms.
Recently, the laboratory of Yasunori Sasakura (Shimoda
Marine Reseach Center, Japan) developed functional
tools that introduce custom-made DNA lesions into
Ciona genomes using ZFN, TALEN and CRISPR/Cas9
(Kawai et al., 2012; Sasaki et al., 2014; Yoshida et al.,
2014). Promising results that resulted from the first
knockout approaches in tunicates revealed a role for
fgf3 in metamorphosis induction (Treen et al., 2014).
TOWARD A FRAMEWORK FOR MUTATION
STUDIES IN ASCIDIANS
The full potential of mutations in genetics studies of the
Ciona species has yet to be elucidated. High-
throughput approaches require concepts and methodo-
logical solutions that may influence experimental
design, including culturing, phenotyping, mapping, and
storage. Detailed protocols for large-scale studies of nat-
urally occurring mutations are available for C. intestina-
lis (Veeman et al., 2011) and C. savignyi (Hendrickson
et al., 2004; Jiang et al., 2005b). Ciona cryptic species
differ in several biological, ecological and genomic
aspects, highlighting the requirement of species-
specific solutions in research design (Hoshino and Nish-
163
ikawa, 1985; Roux et al., 2013; Tsagkogeorga et al.,
2012; Vercaemer et al., 2011). Here, we present a
step-by-step summary of the current information rela-
tive to mutation studies in Ciona.
Classification
The two more widespread C. intestinalis species are
globally encountered in temperate areas of both hemi-
spheres, where they occupy confined coastal environ-
ments, such as harbors and lagoons, (e.g., Lambert and
Lambert, 1998; Procaccini et al., 2011; Vercaemer
et al., 2011). Geographical distribution of these two
types is largely disjointed (Caputi et al., 2007). How-
ever, evidence of phenotypic consistency, lack of exter-
nal diagnostic characters and continuous human-
mediated introduction of Ciona species encourage
PCR-based identification of the population of interest
before approaching natural genetic variation by forward
genetics (Caputi et al., 2007; Sato et al., 2012, 2014;
Suzuki et al., 2005).
Self-Fertilization
F1 screenings to identify induced and naturally occur-
ring mutations in Ciona individuals take advantage of
self-fertilization compatibility. Self and non-self fertiliza-
tion rates in C. intestinalis change seasonally and differ
from one population to another, but remain consis-
tently high (usually >40%) (Caputi et al., in press; Mor-
gan, 1938; Murabe and Hoshi, 2002). A recent report
on the high degree of transgenerational phenotypic
plasticity in Ciona intestinalis sp. B populations sug-
gests that local forces, such as adaptation and habitat,
may control gamete compatibility and mating strategies
(Renborg et al., 2013). Together, these findings high-
light the importance of assaying self-fertilization over
time and among sites to choose the best population and
season for mutagenesis (Jiang and Smith, 2005). Self-
fertilization may be achieved by the light/dark-induced
spawning method to avoid the sacrifice of animals, but
this approach is not compatible with systematic sperm
freezing because of uncertainty surrounding the even-
tual production of new semen. Indeed, repetitive pho-
toperiod alterations and induced spawning can stress
the mutation carrier, causing a lack of sperm produc-
tion and therefore the possibility of cryopreserving all
mutations. We found that the alternative approach of
dissecting gametes for in vivo self-fertilization from
anesthetized C. intestinalis sp. A, which implies a pri-
ori sperm cryopreservation, ensures safer maintenance
of genetic lines.
Culturing
Worldwide research on the Ciona species is based on
wild-collected specimens. With no available isogenic or
semi-isogenic lines, culturing of transgenic and mutant
164 CROCETTA ET AL.
lines is essential for genetic studies of these species
(Kano, 2007). In general, Ciona are crossed and grown
to sexual maturity with a 1–3 month generation time in
both natural (Cirino et al., 2002; Hendrickson et al.,
2004; Veeman et al., 2011; this work) and artificial sea-
water (Joly et al., 2007). Ciona larvae are allowed to
settle on plastic supports. Then, juveniles are allowed
to grow erect, horizontally but also downwards, a
condition not frequently encountered in nature (Fig. 1).
Undisturbed ascidians filter water at constant rates,
with a continuous supply ensuring suspended food in
the ascidian environment. Implementation of ascidian
culturing protocols enables a reduction in mortality and
supports healthy growth. Using live microalgae poses
less operational problems on a daily routine basis (e.g.,
a diet based on equal amount of Isochrysis galbana
and Tetraselmis suecica; Supporting Information).
Areas in which further studies are needed to implement
breeding procedures in support of experimental repro-
ducibility and animal welfare include population den-
sity, symbiosis, nutrition and environmental
requirements.
Storage
Once confined to species of patrimonial importance,
semen cryopreservation currently represents a funda-
mental tool for the storage and distribution of trans-
genic and mutant lines of model systems, including
tunicates (C. savigny: Moody et al., 1999; Hendrickson
et al., 2004; C. intestinalis: Sorrenti et al., 2014;
Oikopleura dioica: Ouchi et al., 2011). In all three
species, the use of dimethyl sulfoxide (DMSO) is
uniformly considered to afford the best cryoprotective
effect. When raising lines from sperm whose cryopre-
servation curve is not precisely controlled, some sam-
ples of cryopreserved sperm fail to fertilize eggs due to
variable freezing efficiency (Hendrickson et al., 2004).
While reliable post-thaw fertility of cryopreserved
ascidian sperm remains elusive, efficient storage of
genetic resources may benefit from improved protocols
using controlled rate freezing systems.
Map-Based Cloning
The identification of the exact genetic lesion in natu-
rally occurring and chemical mutations was hindered
by the high genetic polymorphism of the Ciona spe-
cies. Currently, the availability of detailed physical and
genetic maps of C. intestinalis and C. savignyi enables
the discovery of genetic markers with segregation pat-
terns similar to those of interesting gene loci, as in the
case of the self-incompatibility system genes s-Themis
and v-Themis in C. intestinalis (Harada et al., 2008;
Kano et al., 2004; Shoguchi et al., 2006). The genetic
toolbox in the Ciona species was thoroughly revisited
in some recent reviews, including the publication of
detailed protocols for map-based positional cloning of
mutant alleles (Kano, 2007; Stolfi and Christiaen, 2012;
Veeman et al., 2011). Indeed, while high-density maps
with different molecular markers are in progress (AFLP:
Hendrickson et al., 2004; Veeman et al., 2008; SNP:
Chiba et al., 2009; Satou et al., 2012; see Kano, 2007),
the use of next-generation sequencing is likely to facili-
tate the segregation and identification of mutated genes
in ascidians (Abdul-Wajih et al., 2014).
Natural Populations
Currently, the advent of physical, chemical and trans-
genic approaches for generating random and targeted
DNA disruptions of different size, type and frequency
has narrowed research into spontaneous mutations in
the field of evolutionary biology. Understanding popula-
tion dynamics is of great importance for the correct
design of large-scale studies of natural variation. Demo-
graphic fluctuations may drastically reduce the number
of months available for performing mutagenesis screens
on natural populations. In benthic invertebrates, espe-
cially annual species, species abundance is mainly a
function of recruitment variability, competition and
mortality (Coe, 1956; Defeo, 1996; Hughes, 1984). In
the case of species living in habitats characterized by
high environmental stress, changes in several abiotic
factors (temperature, salinity, and oxygen) in small
areas introduce an additional source of variation
(Brown and McLachlan, 1990). At present, we cannot
exclude that other processes, such as hydrodynamic
regimes, parasite–host/predator–prey interactions and
human activity, may deeply affect C. intestinalis demog-
raphy over large geographical areas. In addition, wild
Ciona may modulate its demographic and life history/
cycle traits of adaptive value (e.g., density, sexual selec-
tion, reproductive regimes, larval dispersal) to respond
selectively to the occurrence of unfavorable conditions
at a small geographical scale (Caputi et al., in prepara-
tion). In the central Tyrrhenian sea, monthly averages
of Ciona intestinalis population size show a robust
peak in May to June, a severe demographic minimiza-
tion in August and a smaller peak in October/November
followed by a less pronounced die-off (Caputi et al., in
press).
Screening for Naturally Occurring Mutations
Given the simple anatomy of C. intestinalis larvae,
characterized by few cells and tissues (Katz, 1983), nat-
urally occurring mutations are easily scored by the pres-
ence of obvious phenotypes altering the morphology of
particular organs and tissues. Here, three distinct popu-
lations of C. intestinalis sp. A were screened for natu-
rally occurring mutations by in vivo and in vitro self-
fertilization (Supporting Information). As previously
shown, 20% individuals are heterozygote carriers of
 ASCIDIAN MUTATIONS 165

FIG. 2. Examples of mutant phenotypes at larval stage. a. A mutant larva lacking pigmentation of sensory organs. b. In this mutant,
mRNA transcripts of Arr (green) and Tyrp1/2b (red) have the same distribution and staining intensity as wild-type larvae (not shown). c. A
double ocellus mutant phenotype. d. Riboprobes labelling the ocellus photoreceptor cells (Arr, black arrowhead), retinal ganglion cells
(GlyR, arrow) and sensory vesicle (Six3, white arrowhead) in the mutant in c using the InsituPro VSi device. Arr mRNA signal suggests that
the entire ocellus structure, including pigmented cell and photoreceptors, was duplicated in the mutant shown in c. e, f. An abnormal larva
with a round shaped and poorly organized trunk, including loss of palps. g. Titf1 is expressed in the endoderm of wild type larvae. h. Con-
versely, Titf1 is down-regulated in the mutation shown in e, f. i. Expression patterns of Arr, GlyR and Six3 are not altered (processed as in d).

mutations causing interesting phenotypes in discrete
morphological contexts and with different developmen-
tal timing (Fig. 2). The difficulty of finding representa-
tive mutations to cover the range of phenotypic
variation observed, however, appears to be a drawback
for this type of research. A caveat is the low number of
phenotypic classes, with morphological abnormalities
occurring mainly in sensory organs and the notochord.
In this study, we identified two mutations showing ster-
eotyped defects in larval tunic and endoderm (Fig. 2).
To test the hypothesis that mutations with subtle phe-
notypes are not detected with the traditional visual
inspection of gross morphology, one hundred clutches
of larvae with normal morphology were processed for
automated in situ hybridization (ISH) using a mix of
three riboprobes (Fig. 2d). However, no alteration of
the expression patterns of neural and endodermal land-
mark genes was detected in the analyzed samples (e.g.,
Fig. 2d). While additional markers and improved
robotic procedures may be considered in the future,
these findings suggest that almost all mutations disrupt-
ing expression patterns of a marker gene have a rela-
tively clear phenotype that can be recognized during
visual screening, and also that mutants with normal
166 CROCETTA ET AL.
morphology but abnormal gene expression may appear
very rarely.
CONCLUSIONS
This review only cursorily examined various aspects of
mutation studies in ascidians. Several features make
ascidians suitable for studying the role of developmen-
tal genes in evolutionary processes. An enriched tool-
box is in development for genome-wide studies of gene
functions and interactions among and within members
of the Ciona complex. Mutation genetics targets many
aspects of the ascidian life-cycle, thus allowing for pre-
cise questions about the genetics of phenotypic evolu-
tion: are mutations found in cis-regulatory regions or in
protein-coding regions? Have the same genes and muta-
tions been used to give independent origins to the
same adaptive traits? With more sequence and marker
capability for mapping resolution, the combination of
forward and reverse genetic approaches will allow for
Ciona mutations to be fully exploited for systems-level
analyses of developmental and evolutionary processes.
ACKNOWLEDGMENTS
The authors thank the fishery service at SZN for animal
collection.
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