3D reconstruction of structures of hatched larva and young juvenile of the larvacean Oikopleura dioica using SBF-SEM

Abstract

The larvacean Oikopleura dioica is a planktonic chordate and an emerging model organism with a short life cycle of 5 days that belongs toTunicata (Urochordata), the sister clade of vertebrates. It is characterized by the rapid development of a tadpole-shaped body. Organ formation in the trunk proceeds within 7 h after the hatching of the tailbud larvae at 3 h after fertilization (hpf) and is completed at 10 hpf, giving rise to fully functional juveniles as miniature adult form. Serial block face scanning electron microscopy was used to acquire ~ 2000 serial transverse section images of a 3 hpf larva and a 10 hpf juvenile to characterize the structures and cellular composition of the trunk and organs using 3D images and movies. Germ cells were found to fuse and establish a central syncytial cell in the gonad as early as 10 hpf. Larval development gave rise to functional organs after several rounds of cell division through trunk morphogenesis. The feature would make O. dioica ideal for analyzing cellular behaviors during morphogenetic processes using live imaging. The detailed descriptions of the larvae and juveniles provided in this study can be utilized as the start and end points of organ morphogenesis in this rapidly developing organism.

The pericardium of Oikopleura dioica (Tunicata, Appendicularia) contains two distinct cell types and is rotated by 90 degrees to the left

“…Based on ultrastructural observations, we could unequivocally demonstrate that two cell types are present in the epithelial lining of the pericardium: six myocardial cells on the left side of the pericardium and ten peritoneal cells on its right. Although these had not been characterized in detail for O. dioica before, it was not an unexpected finding, because Onuma et al (2017) and Nishida et al (2021) had already noticed a difference in thickness between the left and right sides of the pericardial epithelium. In addition, Savelieva and Temereva (2020) described the ultrastructure of myocardial and peritoneal cells for the closely related species O. gracilis.…”

“…These data were compiled using serial block face scanning electron microscopy. For further details see Nishida et al (2021). The resulting image stacks were used for segmentation and 3D-reconstruction using the Amira software package (version 6.4.0).…”

“…Oikopleura dioica is by far the best studied species of the approximately 69 appendicularian species and many morphological details such as the fine anatomy of the nervous system and the sensory organs (e.g., Holmberg 1984Holmberg , 1986Olsson et al 1990;Canestro et al 2005), the ultrastructure of the epidermis (Burighel et al 1989;Nakashima et al 2011), the digestive tract (Burighel et al 2001), the morphogenetic patterning of the oikoplastic epithelium (Kishi et al 2017), or the detailed cell lineage (Stach et al 2008) are only known in this appendicularian model species. Recently, Nishida et al (2021) have complimented the earlier studies with a detailed morphological description of a hatchling and a juvenile of O. dioica based on serial block face scanning electron microscopy (SBF-SEM), a technique that allows for the three-dimensional visualization of anatomical features at near ultrastructural level.…”

“…Stach (2009) investigated the general anatomy of the mesoderm in tunicates and concerning the pericardium of O. dioica only briefly mentions that "the internal lumen … is completely lined by flat mesodermal cells" (p. 101) and that "the major part of their cytoplasm is occupied by myofilaments" (p. 102). Nishida et al (2021) reconstruct the three-dimensional anatomy of the pericardium of a juvenile. These authors were able to determine the number of nuclei, and thus the number of cells that make up the pericardium.…”

“…They also confirmed a difference between the thickness of the right and left sides of the pericardium that had been noticed by Onuma et al (2017) before. Nishida et al (2021) concluded that the left side consisted of six long muscle cells (here termed myocardial cells), whereas the right side was built by ten pericardial epithelium cells (here termed peritoneal pericardial cells). The authors state that with the SBF-SEM method they used "it was difficult to recognize muscle fibers in the SBF-SEM images."…”