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4D time-lapse images of brain and trunk development in the larvacean Oikopleura dioica


Nishida, Hiroki (2023), 4D time-lapse images of brain and trunk development in the larvacean Oikopleura dioica, Dryad, Dataset,


The larvacean, Oikopleura dioica is a planktonic chordate, which is an emerging model organism with a short life cycle of 5 days and belongs to tunicates (urochordates). Organ formation in the trunk proceeds in seven hours from hatching of tailbud larvae at three hours after fertilization (hpf) to completion of organ formation in fully functional juveniles that start feeding at 10 hpf and are just miniature of adult form. Development of O. dioica has been described (Nishida, H., 2008 Development of the appendicularian Oikopleura dioica: culture, genome, and cell lineages. Dev. Growth Differ. 50, S239–S256.). The dataset contains 4D (3D+time) time-lapse images that were acquired during larval development using differential interference contrast optics, and wide-field fluorescent microscope, which visualize cell membrane and nuclei of the entire trunk region. In some cases, animal or vegetal hemisphere blastomeres are labelled to trace the descendants. The dataset would be generally utilized as basic morphological data during the larval development and for tracing cell lineages at a single cell level. This data set is related to the manuscript "Formation of the brain by stem cell divisions of large neuroblasts in Oikopleura dioica, a simple chordate".


To immobilize swimming larvae for time-lapse movies, the tail was cut off immediately after hatching with a fine knife made of tungsten wire. In other experiments, notochord development in the tail was suppressed by Brachyury DNA interference (Omotezako et al., 2015). Double-stranded PCR products (50 ng/µL) of Brachyury were injected into the ovarian cytoplasm. Brachyury DNAi did not affect trunk development.

To visualize the cell membrane, 5 ng/µL FM-1-43 FX (Invitrogen, Carlsbad, CA, USA) was used in seawater. Larvae were transferred into FM seawater for at least 15 min before imaging. Live imaging of nuclei and chromosomes was performed using fluorescent proteins. 2 µg/µL Histone 2B (H2B)-EGFP mRNA (Kishi et al., 2014, 2017; Morita et al., 2020) was injected into the ovary syncytium of females on day five (approximately half a day before spawning) (Omotezako et al., 2015). After spawning and fertilization, embryos with the highest fluorescence intensity were selected and imaged after hatching. Larval development was recorded using a wide-field microscope equipped with DeltaVision (GE Healthcare, Buckinghamshire, UK). 10–20 optical z-sections at a resolution of 896 × 896 pixels were recorded through the trunk, in most cases. The time-lapse interval was one or two minutes.

nls-Kaede mRNA (2 µg/µL) was injected into the ovary syncytium. To label the animal or vegetal hemisphere, eight-cell embryos with vitelline membranes were attached to a poly L-lysine-coated glass-bottomed dish so that the animal-vegetal axis was parallel to the bottom. To convert nls-Kaede fluorescence from green to red, the animal or vegetal cells were exposed to ultraviolet light for three seconds under an Olympus BX-61 microscope with a LUMPlanApo 40 x/0.80 water lens (Kishi et al., 2017; Onuma et al., 2020; Morita et al., 2020). nls-Kaede was dispersed into the cytoplasm during the breakdown of the nuclear membrane when the cells approached metaphase.


  • Kishi K, Onuma TA, Nishida H. (2014) Long-distance cell migration during larval development in the appendicularian, Oikopleura dioica. Dev Biol 395:299-306.
  • Kishi K, Hayashi M, Onuma TA, Nishida H. (2017) Patterning and morphogenesis of the intricate but stereotyped oikoplastic epidermis of the appendicularian, Oikopleura dioica. Dev Biol 428:245-257.
  • Morita R, Onuma TA, Manni L, Ohno N, Nishida H. (2020) Mouth opening is mediated by separation of dorsal and ventral daughter cells of the lip precursor cells in the larvacean, Oikopleura dioica. Dev Genes Evol 230:315–327.
  • Omotezako T, Onuma TA, Nishida H. (2015) DNA interference: DNA-induced gene silencing in the appendicularian Oikopleura dioica. Proc R Soc B 22:282.
  • Onuma, T.A., Hayashi, M., Gyoja, F., Kishi, K., Wang, K., Nishida, H. (2020) A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left-right patterning. Proc. Natl. Acad. Sci. USA.  117:4188-4198.

Usage notes

To look at the image sequence, one can open a single TIFF file in ImageJ/Fiji software. One can select color channels (c), z-sections (z), and run the movie (triangle). Time after hatching is indicated in most image sequences.


Japan Society for the Promotion of Science, Award: 17KT0023

Japan Society for the Promotion of Science, Award: 19H03234