Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

Uno, Yoshinobu; Nozu, Ryo; Kiyatake, Itsuki; Higashiguchi, Nobuyuki; Sodeyama, Shuji; Murakumo, Kiyomi; Sato, Keiichi; Kuraku, Shigehiro

doi:10.1038/s42003-020-01373-7

Cited by 24 publications

(24 citation statements)

References 53 publications

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“…On the other hand, our time-lapse recordings rarely showed cell division, which is usually observed in primary cultured cells from embryos (Additional file 1). Although further quantitative examination using BrdU is necessary to confirm this, the frequency of cell division might be lower in our experiments than it has been in reports using other vertebrates including sharks [31] [32]. One possible reason for this low frequency of cell division is the low incubation temperature.…”

Section: Discussionmentioning

confidence: 55%

In vitro and in vivo gene introduction in the cloudy catshark (Scyliorhinus torazame), a cartilaginous fish

Fujimori

Umatani

Chimura

et al. 2022

Preprint

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Cartilaginous fishes have various unique physiological features such as cartilaginous skeletons and a urea-based osmoregulation strategy for adaptation to their marine environment. Also, because they are considered a sister group of bony vertebrates, understanding their unique features is important from an evolutionary perspective. However, experimental approaches are limited in cartilaginous fishes. Particularly, genetic engineering, which can analyze gene functions as well as cellular behavior, has not been effectively utilized in cartilaginous fishes. This is partly because their reproductive strategy involves internal fertilization, which results in difficulty in microinjection into fertilized eggs at the early developmental stage. Trials of gene transfer have also been limited both in in vitro cultured cells and in vivo. Here, to identify efficient gene transfer methods in cartilaginous fishes, we examined the effects of various methods both in vitro and in vivo using the cloudy catshark, a candidate model cartilaginous fish species. In all methods, green fluorescent protein (GFP) expression was used to evaluate exogenous gene introduction. First, we established a primary cell culture containing fibroblast-like and epithelial-like cells from cloudy catshark embryos. Using these primary cultured cells, we attempted gene transfection by lipofection, polyethylenimine (PEI), adenovirus, baculovirus and electroporation. Among the methods tested, lipofection, electroporation and baculovirus infection enabled the successful introduction of exogenous genes into primary cultured cells, allowing us to study physiological mechanisms at a single-cell level in culture conditions close to those in a living cartilaginous fish. We also attempted in vivo transfection into cloudy catshark embryos by electroporation and baculovirus infection. Although baculovirus-injected groups did not show GFP fluorescence, electroporation successfully introduced GFP into various tissues including muscle cells. Furthermore, we succeeded in GFP introduction into adult testis by electroporation. The in vitro and in vivo gene introduction methods that worked in this study may identify paths for future genetic manipulation including knockout experiments and cellular linage analysis in cartilaginous fishes.

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Section: Discussionmentioning

confidence: 55%

In vitro and in vivo gene introduction in the cloudy catshark (Scyliorhinus torazame), a cartilaginous fish

Fujimori

Umatani

Chimura

et al. 2022

Preprint

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“…Our results suggest that their karyotypes are organized by chromosomes of gradual sizes marked with size-dependent sequence properties (Figure 2). The pattern in the shark chromosomal organization is unique in its diversity among vertebrates (Uno et al 2020), which is characterized by abundant chromosomes (up to 106 for diploids) and variable chromosome sizes.…”

Section: Discussionmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted October 21, 2022. ; https://doi.org/10.1101/2022.10.17.512540 doi: bioRxiv preprint 4 (blood or embryos) (e.g., at aquariums) enabled karyotyping using culture cells for four orectolobiform shark species in Elasmobranchii (Uno et al 2020). This has paved the way for rigid evaluation of whole genome sequences.…”

Section: Introductionmentioning

confidence: 99%

Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization

Yamaguchi

Uno

Kadota

et al. 2022

Preprint

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Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra sharkStegostoma tigrinum, an endangered species that has the smallest genome sequenced to date among sharks (3.71 Gb), as well as for the whale sharkRhincodon typus. Our analysis employing a male–female comparison identified an X chromosome, the first genomically characterized shark sex chromosome. The X chromosome harbors a Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes exhibit a gradualism of chromosome length with remarkable length-dependent characteristics—shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length, which resemble the edges of longer chromosomes. This pattern of intragenomic heterogeneity, previously recognized as peculiar to species with so-called microchromosomes, occurs in more vertebrates including elasmobranchs. We challenge the traditional binary classification of karyotypes as with and without microchromosomes, as even without microchromosomes, shorter chromosomes tend to have higher contents of GC and simple tandem repeats and harbor shorter and more rapid-evolving genes. Such characteristics also appear on the edges of longer chromosomes. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout.

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“…Access to fresh tissues from local aquaria facilitates embryological analysis, genome size quantification with flow cytometry, and karyotyping from cell cultures ( Figure 3 ). Remarkably, cell culture in cartilaginous fishes, which was long thought difficult because of their high body fluid osmolarity, was enabled by modifying the culture medium with balancing osmolytes ( Uno et al , 2020 ). Our cytological expertise also allowed various epigenomic analyses that benefit from whole genome sequencing, on transcription factor binding with ChIP-seq ( Hara et al , 2018 ) and chromatin openness with ATAC-seq, in addition to long-range DNA interactions with Hi-C ( Kadota et al , 2020 ; Onimaru et al , 2021 ).…”

Section: Versatile Sample Collection Featuring the Local Faunamentioning

confidence: 99%

Squalomix: shark and ray genome analysis consortium and its data sharing platform

et al. 2022

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The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.

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Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis

Cited by 24 publications

References 53 publications

In vitro and in vivo gene introduction in the cloudy catshark (Scyliorhinus torazame), a cartilaginous fish

In vitro and in vivo gene introduction in the cloudy catshark (Scyliorhinus torazame), a cartilaginous fish

Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization

Squalomix: shark and ray genome analysis consortium and its data sharing platform

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