DOI: 10.5061/dryad.5tb2rbp8c
title: 4D time-lapse images of brain and trunk development in the larvacean Oikopleura dioica

description: 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".
description: 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.<br><br>References:
 Kishi K, Onuma TA, Nishida H. (2014) Long-distance
 cell migration during larval development in the appendicularian,
 <em>Oikopleura</em> <em>dioica</em>. 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,
 <em>Oikopleura</em> <em>dioica</em>. 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,
 <em>Oikopleura</em> <em>dioica</em>. Dev Genes
 Evol 230:315–327. Omotezako T, Onuma TA, Nishida H.
 (2015) DNA interference: DNA-induced gene silencing in the appendicularian
 <em>Oikopleura</em> <em>dioica</em>. 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.
description: 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.