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Developmental Table (4 pages version)

Developmental Table (2 pages version)

 C. intestinalis type A (C. robusta) adults for time-lapse imaging and for confocal scanning laser microscopy (CLSM) were provided by NBRP from the Maizuru bay and Tokyo bay areas in Japan. For histology, adults were obtained from the Lagoon of Venice, Italy. Species determination was performed checking the discriminating factor “trunk shape” of late larvae, described in Pennati et al., 2015. Specimens collected in different sites possessed the same anatomical and developmental features.

Eggs and sperm were obtained surgically from gonoducts. After insemination, eggs were maintained in agarose-coated dishes with Millipore-filtered seawater (MFSW) containing 50 µg/ml streptomycin sulfate, and the early cleavages were uniformly synchronized (data not shown). To keep the temperature stable, we used a Peltier-type incubator (CN-25B, Mitsubishi, Japan) without any vibration to prevent embryo fusion. Embryos developed in hatched larvae approximately 18 h after insemination.
The naturally hatched larvae derived from egg with chorion were maintained in plastic dishes on the thermo-plate at 20°C to acquire images. Using a digital camera (Olympus SP-350) mounted on a microscope, images were acquired every 3 to 10 min for 7 days (Supplementary Video S4). After 3 days post-fertilization, food was given (vegetal plankton, sun culture).

Fixed specimens were prepared at different timings of development, from hatching larva up to 7dpf juvenile. Samples incubated at 18 ℃ were fixed for 30 min – 1 day at room temperature in 4% EM grade paraformaldehyde (nacalai tesque code 00380) in MOPS buffer (0.1 M 3-(N-Morpholino) propanesulfonic acid), adjusted to pH 7.5. Specimens were then washed three times with phosphate-buffered saline (PBS) and incubated in Alexa-546 phalloidin (Molecular Probes, Eugene, OR) in PBS containing 0.01% Triton X-100 (PBST) either overnight at 4°C or at room temperature for 1-2 h. Specimens were then rinsed for 3 min in PBS, attached to glass slide dishes, dehydrated through an isopropanol series, and finally cleared using Murray clear, a 2:1 mixture of benzyl benzoate and benzyl alcohol. Alexa 546 phalloidin was used to visualize cell membranes because it stains mainly cortical actin filaments.
Images were collected with a CLSM on a Zeiss LSM510 META with 40X oil objective or on an OLYMPUS fv1000. To reconstruct the 3D images, 100 cross-section images from top to bottom per sample were acquired (LSM image browser, Zeiss, Germany). The focus interval depended on the sample (from 0.5 to 1.2 µm). The resulting stacks were then exported to raw image series or to 3D image data for database integration. Although the timing of metamorphosis showed a huge deviation depending on the timing of adhesion, we considered an average timing, looking at animals exhibiting a representative morphology. Lastly, these stacks were integrated into the database TunicAnatO.

Histology

Authors thank Yutaka Satou Lab at Kyoto University, Manabu Yoshida Lab at the University of Tokyo and Onagawa Marine Station for providing individuals of Ciona samples with support by the National Bio-Resource Project of AMED, Japan;

Akitsu Fukuzawa for collection of CLSM image stacks;

Takumi T. Shito for the construction of the flash-independent version (version 3.0) of TunicAnatO;

Paolo Burighel for the excellent comments and suggestions regarding the ontology.