Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioicasuggests the presence of three chromosomes.

Andrew W Liu,Charles Plessy,Nicholas M Luscombe,Yongkai Tan,Aki Masunaga

doi:10.12688/f1000research.25019.1

Abstract

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals' chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies' staining properties for use in epifluorescent and confocal based fluorescent microscopy.

Highlights

Karyotyping is a long-established histochemical method to visualize chromosomes of eukaryotes (Hsu & Benirschke, 1967; Tjio & Levan, 1950)
Considering the discrepancy of past findings, and the fact that our laboratory strain originates from a geographically distinct ocean, we applied H3S28P staining on intact embryos and oocytes to confirm the chromosome count and validate our genome sequencing assemblies of Okinawan O. dioica marine populations among the Ryukyu Islands of southern Japan
In order to eliminate possible miscounts and other Giemsa staining artifacts, immunostaining was used to count individual chromosomes using a centromere-specific primary antibody directed against H3S28P and a secondary antibody conjugated to Alexa488 directed against the primary antibody

Summary

Introduction

Karyotyping is a long-established histochemical method to visualize chromosomes of eukaryotes (Hsu & Benirschke, 1967; Tjio & Levan, 1950). A multi-dye reagent developed at the turn of the 20th century for the diagnosis of infections in human histological preparations (Giemsa, 1902; Giemsa, 1904) was later used to stain chromosomes themselves in order to study their numbers, translocations, and other aberrations This rapid technique, involving the use of stains including methylene blue, eosin, and azure B allows for observation of chromosomes with a simple light microscope, naturally lending itself to a first attempt for karyotyping analysis. A structure in which genetic material is sequestered in a ∏-shaped conformation has been observed during meiotic cell divisions between the final phases of oogenesis and mature oocytes (Ganot et al, 2008) These results were all obtained from the same laboratory strain originating from the Atlantic Ocean. Considering the discrepancy of past findings, and the fact that our laboratory strain originates from a geographically distinct ocean, we applied H3S28P staining on intact embryos and oocytes to confirm the chromosome count and validate our genome sequencing assemblies of Okinawan O. dioica marine populations among the Ryukyu Islands of southern Japan

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Conclusion