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A mutational approach to the study of development of the protochordate Ciona
intestinalis (Tunicata, Chordata)
Paolo Sordino, Carl-Philipp Heisenberg, Paola Cirino, Alfonso Toscano, Paola Giuliano, Rita Marino,
Maria Rosaria Pinto & Rosaria De Santis
SARSIA
Sordino P, Heisenberg C-P, Cirino P, Toscano A, Giuliano P, Marino R, Pinto MR, De Santis R. 2000.
A mutational approach to the study of development of the protochordate Ciona intestinalis (Tunicata,
Chordata). Sarsia 85:173-176.
We have developed a protocol to perform a genetic screen for zygotic mutations affecting embryogenesis on the protochordate Ciona intestinalis. The choice of this taxon, whose phylogenetic position
places it at the basis of the chordates as one the most primitive vertebrate relatives, could allow to
address several evolutionary questions. The protochordates share many morphological features with
the vertebrates, in primis the presence of a notochord. Ciona intestinalis shows several ideal features for
a mutational analysis, such as external development and larvae made of a limited number of cells and
cell types. Detailed cell lineage studies are available. The haploid genome size is comparable to the size
of the Drosophila haploid genome. We have optimised conditions for chemical mutagenesis studying
the efficiency at which different concentration of N-ethyl-N-nitrosourea (ENU) can induce mutations.
Because the adult Ciona are hermaphrodites, we are performing a one-generation screen. The induced
mutations are identified by visual inspection of developmental stages. We report the preliminary results
from our screen including examples of the different classes of mutant phenotypes found so far.
Paolo Sordino*, Carl-Philipp Heisenberg, Department of Anatomy and Developmental Biology, University College London, Gower Street, WC1E 6BT, London, United Kingdom. – Paola Giuliano, Rita
Marino, Rosaria De Santis, Laboratory of Cell Biology, Stazione Zoologica Anton Dohrn, Villa Comunale,
80121, Napoli, Italy. – Maria Rosaria Pinto, Laboratory of Cell Biology, Stazione Zoologica Anton
Dohrn, Villa Comunale, 80121, Napoli, Italy and Institute of Protein Biochemistry and Enzymology,
CNR, Via Marconi 10, 80125, Napoli, Italy.
* Corresponding author. E-mail: [email protected]
Keywords: Ciona intestinalis; Urochordata; mutagenesis.
INTRODUCTION
In the last two decades, mutagenesis screens have
strongly impacted upon our comprehension of developmental genetics, from early pattern formation to morphogenesis and behaviour. In a classic genetic approach,
random mutagenesis makes it possible to survey the genome for genes that function in particular embryonic
pathways. This approach allows the introduction of random mutations into individual genes causing phenotypic
alterations which may be helpful to unravel the wildtype
function of the mutated gene(s).
Until now, mutagenesis screens applied to animal organisms have been focusing on two invertebrate and one
vertebrate species. While for the nematode Caenorhabditis elegans and the insect Drosophila melanogaster
these screens have led to the sequencing of a large
number of molecules crucial in development (NüssleinVolhard & Wieschaus 1980; Chalfie 1993), the outcome
of the mutagenesis screen in the zebrafish Brachydanio
rerio (Mullins & al. 1994; Solnica-Krezel & al. 1994)
has yet to await the identification of the majority of the
mutated genes. The zebrafish has been chosen mainly
on the basis that it is easy to keep and raise in large
number under laboratory conditions and that zebrafish
embryos are well suited for morphological examination
and experimental manipulation during the whole course
of embryonic development (Westerfield 1993). In the
light of substantial recent progress in the establishment
of genetic maps (Postlethwaite & al. 1998) and insertional mutagenesis methods (reviewed by Weinberg
1998), it is reasonable to expect that the cloning of mutated genes will be facilitated. The limitations of vertebrate screens may however become more evident by
observations that many developmental control genes are
found in multiple copies especially in the zebrafish genome, as a result of gene or subgenomic duplications or
losses, indicating that there may be a high degree of partial redundancy for the function of these genes (see
Wittbrodt & al. 1998). Therefore mutations in these genes
174
Sarsia 85:173-176 – 2000
ENU
WT
sperm
egg
self-fertilisation
Fig. 1. Scheme for F1 genetic screen in C. intestinalis. N-ethylN-nitrosourea (ENU) was used to generate mutations (+*) in
pre-meiotic spermatozoa. Adults belonging to the F1 progeny,
obtained by crossing wild-type eggs with mutagenized spermatozoa, were self-fertilised and the F2 progeny was scored for
morphological or heterochronic defects.
may only lead to relatively mild phenotypes which could
be the reason that a considerable portion of mutants may
not have been identified in the zebrafish screens reported
yet (Driever & al. 1996; Haffter & al. 1996).
Developmental evolutionary biology is providing
strong evidence that embryonic processes are conserved
between basal chordates and vertebrates (e.g. Corbo &
al. 1997; Shimeld 1997; Glardon & al. 1998; Marino &
al. 1998). Consistent with this, an extension of the mutagenesis approach to the protochordate Tunicata, with their
simpler genetics and development (Satoh 1994; Di
Gregorio & Levine 1998), could facilitate dissection of
programmes common for all chordates. While revealing
parallels in developmental mechanisms, it can also offer
the opportunity to uncover novel genes or gene functions and, ultimately, unravel the evolutionary emergence
of vertebrate developmental processes.
In an effort to generate zygotic mutations affecting
embryogenesis in a protochordate species, we have established a methodology for inducing point-mutations
in the ascidian Ciona intestinalis. Within the subphylum
Tunicata, the ascidian class has been thoroughly studied
in developmental biology most likely because of their
sessility in coastal waters that makes sampling easier than
for the pelagic classes Larvacea and Thaliacea (Jeffery
& Swalla 1997). Ascidian larvae exhibit many vertebratelike anatomical characteristics, such as a dorsal neural
tube, a notochord and tail muscles. Extensive lineage tracings, comparable to that in C. elegans, has been permitted by the simple, invariant embryonic cleavage patterns
(Nishida 1987). Also, the small invertebrate-like genome
of C. intestinalis (Lambert & Laird 1971; Simmen &
al.1998) indicates that functional redundancy of genes
may only be a minor problem for the identification of
mutant phenotypes (Ohno 1970).
One particular trait that separates C. intestinalis from
other most popular research chordates is that it is hermaphrodite and self-sterile, but self-sterility can be abolished by removal of the egg coats or by controlled experimental conditions. This feature allows performing a
one-generation scheme. Likewise, C. intestinalis, a rather
cosmopolitan species, breeds throughout the year with a
high rate of fecundity and its generation time is relatively short (2-3 months). These conditions can be reproduced in the laboratory following an uncomplicated
culture method. In addition, the larva is very transparent
and the cell lineage of the embryo has been described in
detail (e.g. Nicol & Meinertzhagen 1988a, 1988b). Altogether, these represent ideal prerequisites for conducting a mutant screen. Moreover, several laboratories are
expressing an interest in studying C. intestinalis, so that
cloning of genes on the basis of homology to known
developmental control genes in other vertebrate and invertebrate species, is progressing.
MATERIAL AND METHODS
To perform a pilot screen, adult Ciona were treated with
ethylnitrosourea (ENU) (Russell & al. 1979; Yoshikawa
& al. 1984; Zimmering & Thompson 1984; Grunwald &
Streisinger 1992) and mutagenized chromosomes were
driven to homozygosity employing a diploid F1 screen
(Hitotsumachi & al. 1985; Riley & Grunwald 1995).
First, pre-meiotically mutagenised sperm were crossed
with wildtype eggs. Then, gametes from F1 individuals
were self-fertilised and the offspring screened for morphological visible phenotypes at larval stages. Visual
inspection of larval morphologies permitted the identification of several recessive lethal mutants (Fig. 1). Specific locus mutations were consistently represented in
mendelian ratios and were transmitted through the germ
lines of F1 offspring, suggesting that they have been
generated by point-mutations. As for other favourite
model systems, the experimental analysis of mutant
tunicate larvae can be approached by several cellular and
Sordino & al. – Genetic screen for zygotic mutations
175
Fig. 2. Examples of embryonic phenotypes generated during the pilot screen. Tubulin labelled 1 day old larvae. Compared to wild-type larva (A), the mutant big ocellus (bio) (B) is
characterised by an expanded neural vesicle (black arrows) with an enlarged ocellus (arrowhead), while blind (bli) (C) shows no sign of pigmentation in the sensory cells (white arrow).
molecular techniques, such as single-cell manipulations
and injection, expression studies, ectopic or tissue-specific over-expression for functional strategies.
RESULTS AND DISCUSSION
Based on developmental defects, the observed phenotypes fell into several major classes, including defects in
specific processes such as metamorphosis, formation of
tail, head or nervous system (Table 1). We observed
heterochronic changes of metamorphosis consisting, for
instance, in an early, precocious reorganisation and rotation of the endoderm at twelve hours after hatching, not
paralleled by tail resorption, or in the permanence of the
larval stage three days after hatching. Another group of
mutants consistently exhibited abnormalities in differentiation of tail structures, as the notochord which appeared shorter and wider when compared with the wild
type. An interesting class comprises mutants with head
defects ranging from the apical part of the head, to lack
of the anterior portion of the endoderm or abnormal expansion of sensory organs and the neural vesicle. Another mutation lacked pigment in the otolith and ocellus, the two sensory cells of the neural vesicle (Fig. 2).
These and other phenotypes will be described in detail
elsewhere.
Taken together, our preliminary results show that, in a
manner similar to C. elegans, D. melanogaster and the
zebrafish, it is possible to efficiently mutagenise ascidians
and subsequently to identify mutants exhibiting a phenotype during larval development. The relatively small
size of the ascidian genome and the possibility to conduct an F1 screen, indicate that it will be possible in the
future to conduct larger scale mutagenesis screens, including maternal screens (Kimmel 1989), aiming to identify genes with a function in early development of the
ascidian C. intestinalis. The genetic analysis of tunicates
is therefore likely to provide substantial progress in understanding evolutionary modifications of developmental processes leading to the emergence of vertebrates.
ACKNOWLEDGEMENTS
This paper is dedicated to the memory of Prof. Nigel Holder.
We thank W. Smith for sharing data prior to publication and S.
Wilson for helpful comments on the manuscript. This research
was funded by EMBO and EC (PS, CPH). Travel grants were
provided by The Royal Society (CPH, PS) and The Company
of Biologists (PS).
Table 1. F2 developmental phenotypic classes identified using
different ENU concentrations. Numbers in parentheses are
percentages of potential carriers.
Mutagen concentrations
(mM ENU)
2.7
Potential carriers
Lethal early phenotypes
Multiple phenotypes
Distinct phenotypes
Head
Metamorphosis
Notochord/Tail
Neural vescicle
Ocellus
Palps
Gastrulation
Wild-Type
24
14 (58)
4 (17)
9 (37)
2
0
1
1
0
1
0
1
1.35
14
9 (65)
1 (7)
4 (29)
2
1
0
0
0
0
0
1
0.27
24
6 (25)
7 (29)
17 (71)
1
2
1
3
1
0
1
2
176
Sarsia 85:173-176 – 2000
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Accepted 11 January 2000 – Printed 9 June 2000
Editorial responsibility: Jarl Giske