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Supplementary material 1: Materials and methods used to obtain
original data reported in this article
Animals and embryos
Mature adults of C. intestinalis were collected from harbors in Murotsu,
Hyogo, Japan. The adults were maintained in indoor tanks of artificial seawater (Marine
Art BR, Senju Seiyaku, Osaka, Japan) at 18˚C. The embryos were prepared using
gamates obtained from the gonoducts, as described previously (Nakagawa et al., 1999).
Antibody preparation and immunohistochemical staining
Production of the antibodies against C. intestinalis arrestin (Ci-Arr), Ciopsin3,
Ci-CRALBP and Ci-BCO were previously reported (Tsuda et al. 2003; Takimoto et al.
2006). For preparation of the Ci-RPE65 specific antibody, a cDNA fragment that
encodes the carboxyl (C)-terminal amino acids K431-A524 of Ci-RPE65 was amplified
by PCR (using the primer pair 5’-TTAGATCTGGAGTATATCTTGCCGTCG-3’ and
5’-CTAAGCTTAGTCACGCTTGGAGAATAA-3’) and cloned into the pQE40 vector
(Qiagen GmbH, Hilden, Germany). The plasmid was introduced into the Escherichia
coli strain XL1Blue (Stratagene, La Jolla, CA). The C-terminal region of Ci-RPE65 was
produced as a fusion protein with a dihydrofolate reductase and histidines tag, isolated
and used to immunize mice. Antisera were prepared according to a standard method.
Ciona larvae were fixed with 10% formalin in artificial seawater for 3 h at
4˚C. After fixation, the larvae were washed with PBS containing 0.1% Triton X-100
(T-PBS), and treated with 10% goat serum in T-PBS for 3 h. The larvae were then
incubated overnight with the primary antiserum diluted 1000-fold with the blocking
buffer, and washed with T-PBS for 8 h at 4˚C. The specimens were then incubated with
an Alexa 488-conjugated anti-mouse or anti-rabbit IgG goat antibody (Molecular Probes,
Inc., Eugene, OR). For double labeling, an Alexa 594-conjugated secondary antibody
(Molecular Probes, Inc.) was used. After rinsing several times with T-PBS, the larvae
were mounted in 50% glycerol and observed under a confocal microscope (LSM 510,
Carl Zeiss, Oberkochen, Germany) or a conventional Zeiss microscope (Axioplan 2).
In situ hybridization
We used cDNA clones for Ci-opsin1, Ci-opsin3, Ci-CRALBP, Ci-BCO, and
Ci-RPE65 as the template to synthesize a digoxigenin-labeled antisense RNA probe
using a DIG RNA labeling kit (Roche Diagnostics, Indianapolis, IN). Neural complexes
were dissected out and fixed by overnight incubation in 4% paraformaldehyde in PBS at
4˚C. Whole-mount in situ hybridization of neural complexes was carried out by the
same method previously described for larval specimens (Nakashima et al. 2003).
After coloring reaction, tissues were post-fixed with 4% paraformaldehyde in PBS, the
neural complexes were dehydrated through graded alcohol, embedded in paraffin
mediated with butanol, and sectiond at 8 m. Sections were cleared in xylene and
mounted on slide glasses with NEW M-X (Matsunami Glass, Osaka, Japan) for
microscopic observation.
Identification of amphioxus genes and molecular phylogenetic analysis
The amphioxus Branchiostoma floridae genome was searched for
RPE65/BCO/BCO2 family genes with the TBLASTN algorithm using human RPE65,
BCO, and BCO2 as queries. Because the amphioxus genome is highly polymorphic
(Putnam et al., 2008), haplotypes of the same loci often assembled separately. Among
the six gene models (Brafl1 protein ID: 124233, 218241, 73359, 66862, 115035, and
250066) detected by the TBLASTN search, 73359 and 124223 turned out to be alleles
of the same gene, and similarly, 115035 and 250066 represents alleles of a single gene.
As a result, we concluded that the amphioxus genome contains four RPE65/BCO/BCO2
family genes (Brafl1 protein IDs: 73359/124233, 218241, 66862, and 115035/250066;
named BCO-like1, RPE65-like1, BCO-like2, and RPE65-ilke2, respectively).
The amphioxus orthologues of CRALBP were also found in the genome by
TBLASTN searches using CRALBP from Homo sapiens, C. intestinalis, and
Drosophila melanogaster as queries. Among the three gene models detected (Brafl1
protein IDs: 57347, 59146, and 125389), 57347 and 59146 were found to be alleles of
the same gene, and the gene was named CRALBP1. The other gene (125389) was
named CRALBP2.
Full-length amino acid sequences of RPE65/BCO/BCO2 or CRALBP were
aligned and neighbor-joining trees were constructed using the ClustalW program
(Thompson et al., 1994). Sites with gaps were excluded from the analysis. Evolutionary
distances were estimated using Kimura’s empirical method. Sequences used were:
Homo sapiens BCO AF294900, Gallus gallus BCO AJ271386, Danio rerio BCO
AJ290390, C. intestinalis BCO AK116061, Drosophila melanogaster BCO AJ276682,
H. sapiens RPE65 U18991, G. gallus RPE65 AB017594, Ambystoma tigrinum RPE65
AF047465, C. intestinalis RPE65 AB246321, H. sapiens BCO2 CAC27994, Mus
musculus BCO2 NP_573480, Xenopus tropicalis BCO2 NP_001006739, and D. rerio
BCO2 CAC37567, H. sapiens CRALBP L34219, M. musculus CRALBP AF084642, C.
intestinalis CRALBP AK116914, D. melanogaster CRALBP NM_079215
REFERENCES
Nakagawa, M., Miyamoto, T., Ohkuma, M. & Tsuda, M. 1999 Action spectrum for the
photophobic response of Ciona intestinalis (Ascidieacea, Urochordata) larvae
implicates retinal protein. Photochem. Photobiol. 70, 359-362.
Nakashima, Y., Kusakabe, T., Kusakabe, R., Terakita, A., Shichida, Y. & Tsuda, M. 2003
Origin of the vertebrate visual cycle: genes encoding retinal photoisomerase and two
putative visual cycle proteins are expressed in whole brain of a primitive chordate. J.
Comp. Neurol. 460, 180-190.
Putnam, N. H. et al. 2008 The amphioxus genome and the evolution of the chordate
karyotype. Nature 453, 1064-1071.
Takimoto, N., Kusakabe, T., Horie, T., Miyamoto, Y. & Tsuda, M. 2006 Origin of the
vertebrate visual cycle: III. Distinct distribution of RPE65 and -carotene
15,15’-monooxygenase homologues in Ciona intestinalis. Photochem. Photobiol. 83,
242-247.
Thompson, J. D., Higgins, D. G., & Gibson, T. J. 1994 CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence weighting,
position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,
4673-4680.
Tsuda, M., Kusakabe, T., Iwamoto, H., Horie, T., Nakashima, Y., Nakagawa, M. &
Okunou, K. 2003 Origin of the vertebrate visual cycle: II. Visual cycle proteins are
localized in whole brain including photoreceptor cells of a primitive chordate.
Vision Res. 43, 3045-3053.