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. 2005 Oct 18;102(42):15134-9.
doi: 10.1073/pnas.0503640102. Epub 2005 Oct 7.

Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis

Yasunori Sasakura  1 , Keisuke Nakashima, Satoko Awazu, Terumi Matsuoka, Akie Nakayama, Jun-ichi Azuma, Nori Satoh
Affiliations

Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis

Yasunori Sasakura et al. Proc Natl Acad Sci U S A. .

Abstract

Tunicates are the only animals that perform cellulose biosynthesis. The tunicate gene for cellulose synthase, Ci-CesA, was likely acquired by horizontal transfer from bacteria and was a key innovation in the evolution of tunicates. Transposon-based mutagenesis in an ascidian, Ciona intestinalis, has generated a mutant, swimming juvenile (sj). Ci-CesA is the gene responsible for the sj mutant, in which a drastic reduction in cellulose was observed in the tunic. Furthermore, during metamorphosis, which in ascidians convert the vertebrate-like larva into a sessile filter feeder, sj showed abnormalities in the order of metamorphic events. In normal larvae, the metamorphic events in the trunk region are initiated after tail resorption. In contrast, sj mutant larvae initiated the metamorphic events in the trunk without tail resorption. Thus, sj larvae show a "swimming juvenile" phenotype, the juvenile-like trunk structure with a complete tail and the ability to swim. It is likely that ascidian cellulose synthase is required for the coordination of the metamorphic events in the trunk and tail in addition to cellulose biosynthesis.

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Figures

Fig. 1.
Fig. 1.
Metamorphosis of C. intestinalis and phenotypes of the sj mutant. (A) A swimming larva. PL, preoral lobe. (B) A larva during tail resorption. (C) After the completion of tail resorption, the preoral lobe becomes long and transparent and is called the stalk (St in D). (D) During the formation of the stalk, body axis rotation is initiated, and the trunk rotates by 90°. The oral siphon is shown by an arrow. (E) A juvenile that has completed body axis rotation. Adult organs such as gill slits (Gs), endostyle (Es), and heart (H) are formed. The position of the oral siphon (arrow) is completely different from the initial position (an arrow in D). (F) At 1 dpf, the larval tunic (Tu) of a sj mutant larva (sj) was narrower at the tail (arrows), whereas it swelled in the trunk region (arrowhead), compared with a normal larva (normal). The adult tunic (aTu) of a sj larva was normally formed inside the larval tunic. (Bar, 100 μm.) (G) At 2 dpf, a sj mutant started body axis rotation without tail resorption. (Bar, 100 μm.) (Insets) At 6 dpf, the preoral lobe of a sj mutant larva became long and transparent to form the stalk (St). The endostyle (Es) became longer in the sj larva than a normal larva. (H) A metamorphosed sj mutant (Lower) showed normal body organization compared with normal juveniles (Upper). (Bar, 100 μm.)
Fig. 2.
Fig. 2.
Mapping of the insertion site of Minos transposon in the sj mutant genome. (A) The design of a Minos transposon used for the insertional mutagenesis. E and H indicate the restriction sites of EcoRI and HindIII, respectively. (B) Location of the Minos insertion site in the sj mutant genome. Open and filled boxes represent exons of Ci-CesA corresponding to the UTR and ORF, respectively. A gray box indicates the position of an EST (rciad083d01) in the public genome browser of C. intestinalis (http://genome.jgi-psf.org/ciona4/ciona4.home.html). H, the restriction sites of HindIII. One restriction site showed polymorphism in the Japanese population of C. intestinalis and is shown as (H). pCesA(-2080)-GFP and pCesA(-327)-GFP indicate constructs used for the promoter analyses in Fig. 3C. (C) Genomic Southern blot analyses of sj mutants. (Left) Genomic DNA from a sj heterozygous animal (sj/+) digested with EcoRI was detected with the probe shown in A (dotted line). Multiple bands indicate the formation of a tandem repeat array. (Right) Genomic DNA from wild-type animals (+/+) and sj heterozygous animals (sj/+) digested with HindIII were detected by the probe shown in B (dotted line). M indicates the size marker. sj heterozygous animals showed specific bands (arrow) that were expected from the fusion of a part of pMiTFr3dTPO-gfp and genomic DNA. (D) An example of PCR analysis showing the homozygosity of the Minos insertion in sj mutants. DNA from larvae with normal appearance without GFP signal (N), normal appearance with GFP expression (P), and sj mutants (sj) were subjected to PCR by primers shown in B (small arrows). The absence of a band indicates the homozygosity of insertion. (Lower) The results of PCR that detects gfp (positive control). (E) A dramatic reduction of Ci-CesA expression in sj mutants (sj) revealed by RT-PCR. Ci-Epi1, which is expressed in epidermis (36), was used as a positive control. The expression of ciad083d01 was not affected in sj mutants. Negative controls without reverse transcription are shown by RT – lanes.
Fig. 3.
Fig. 3.
Ci-CesA is the gene responsible for the sj mutation. (A) Expression of Ci-CesA was not detected in a sj mutant (sj), whereas the expression is evident at the trunk of a normal larva at the same age (normal). (Bar, 100 μm.) (B) Microinjection of MO specific to Ci-CesA caused the same phenotype as the sj mutant. A larva (Left) that developed from an egg into which Ci-CesA MO was injected showed the abnormal tunic, retraction of papillae, rotation of body axis, and stalk formation. A larva injected with control MO did not show such phenotypes (Right). (C) GFP expression from pCesA(-2080)-GFP and pCesA(-327)-GFP. (D) The reduction in cellulose in the sj mutant larva (sj), compared with the presence of cellulose in a normal larva at the same age (normal), as shown by dark blue signals.
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