Japanese Hagfish Research Articles

Bioinformatic and fine-scale chromosomal mapping reveal the nature and evolution of eliminated chromosomes in the Japanese hagfish, Eptatretus burgeri, through analysis of repetitive DNA families.

In the Japanese hagfish, Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n = 52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n = 36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome of E. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1-6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes of E. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7-10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1-10) by fluorescence in situ hybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2-10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

Novel selectively amplified DNA sequences in the germline genome of the Japanese hagfish, Eptatretus burgeri

In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n = 36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n = 52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E.burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3-6) through PCR amplification and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE elements. EEEb4 was GC-rich repeats and has one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats with one pair and two pairs of sub-repeats, respectively. Interestingly, all repeat classes except EEEb3 were transcribed in the testes, although no open reading frames (ORF) were identified. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3-6 and EEEb2, which was previously isolated from the germline genome of E. burgeri. All sequences were only found on all EEEb1-positive E-chromosomes. Copy number estimation of the repeated elements by slot-blot hybridization revealed that (i) the EEEb1-6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting that there is incomplete elimination of the repeated elements. These results provide new insights into the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.

Internal deletion of highly repetitive DNA families from the non-eliminated chromosome in a Japanese hagfish, Eptatretus burgeri: First finding in this species

Internal deletion of highly repetitive DNA families from the non-eliminated chromosome in a Japanese hagfish, <i>Eptatretus burgeri</i>: First finding in this species

Highly repetitive DNA families restricted to germ cells in a Japanese hagfish (Eptatretus burgeri): a hierarchical and mosaic structure in eliminated chromosomes.

It is known that in eight hagfishes chromosome elimination occurs during early embryogenesis. The eliminated chromosomes are mostly C-band positive, so that none of the somatic cells have any C-band-positive chromatin. Recently, some highly repetitive DNA sequences have been reported as eliminated elements in these hagfishes based on molecular biological methods. However, no germline-restricted repetitive DNA have been directly isolated from the Japanese hagfish Eptatretus burgeri, from which approximately 21% of the total DNA is eliminated from presumptive somatic cells. Through electrophoretic investigation after digestion with restriction endonucleases, two DNA families that are restricted to germline DNA were isolated. Molecular cloning and sequence analysis revealed that these families are composed of closely related sequences of 64 and 57bp in length, respectively. Southern blot hybridization revealed that the two DNA families are restricted to germline DNA and were thus named EEEb1 and EEEb2, respectively. Moreover, these eliminated elements were highly and tandemly repeated, and it is predicted that they might amplify by saltatory replication and have evolved in a concerted manner. By densitometric scanning, EEEb1 and EEEb2 were found to amount to make up approximately 18.5 and 0.024% of the total germline genomic DNA, accounting for 88.6% of the total eliminated DNA. A fluorescence in situ hybridization experiment demonstrated that EEEb1 is located on all C-band-positive chromosomes that are limited to germ cells, suggesting that EEEb1 is the primary component of eliminated DNA of E. burgeri.

A germline restricted, highly repetitive DNA sequence in Paramyxine atami: an interspecifically conserved, but somatically eliminated, element.

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83 bp long, has been designated "EEPa1", for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms. The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution.

Analysis of DNA Levels During Gonomery in Early Cleavage Divisions of the Freshwater Copepod Mesocyclops Edax

Gonomery, the separate grouping of maternal and paternal chromosomes during the first few mitoses following fertilization (Fig. 1) and chromatin diminution, the fragmentation and elimination of whole chromosome regions during specific stages of early cleavage in all primordial cells of the soma, occur in several species of copepods, ascarid nematodes, ciliated protozoa, Japanese hagfish 9 and a few other invertebrates. Because of the marked loss of DNA from all presumptive somatic cells by selective chromatin diminution during the 4th cleavage division in M. edax, it was of interest to determine DNA levels in chromosomes during gonomery in anaphases preceding diminution (Fig. 1). It was also important to estimate the DNA content of sperm as an index of the relative contribution by the male genome at fertilization in this species.Mature males and egg-carrying females were fixed in 3:1 methanol/acetic acid for 3-5 mins, swollen in 45% acetic acid for 2-3 mins, squashed, frozen in liquid N2 for coverslip removal, thawed in 2 changes of absolute ETOH and air dried.

Chromosome elimination in three Baltic, south Pacific and north-east Pacific hagfish species.

We have confirmed that chromosome elimination occurs in the cells of Myxine glutinosa, collected from the Baltic Sea off Sweden, Eptatretus cirrhatus from the south Pacific Ocean off the east coast of New Zealand, and E. stoutii from the north-east Pacific Ocean off Canada, similar to cells of four Japanese hagfish species. In M. Glutinosa, E. cirrhatus type A, E. cirrhatus type B and E. stoutii, the differences in chromosome number between spermatogonia (44, 72, 80 and 48) and somatic cells (28, 34, 34 and 34) were 16, 38, 46 and 14 respectively. The amount of DNA eliminated from presumptive somatic cells averaged 43.5%, 48.7%, 54.6% and 52.8% respectively. Euchromatic chromosomes and/or parts of chromosomes in addition to heterochromatic chromosomes were clearly eliminated in E. cirrhatus and E. stoutii. Adding our previous observations of four Japanese hagfish species, chromosome elimination occurs in all seven of the hagfish species. These results suggest that this phenomenon, chromosome elimination, generally occurs in the order Myxinida. In addition, B-chromosomes were observed in the germ cells of E. cirrhatus and E. stoutii, similar to the cells of E. okinoseanus, E. burgeri and Paramyxine atami (E. atami). This fact suggests that B-chromosomes might exist generally in the family Eptatretidae.

Germ line-restricted supernumerary (B) chromosomes in Eptatretus okinoseanus.

Cytogenetic studies were performed on two types of Japanese hagfish (Eptatretus okinoseanus) that eliminate about 45% (type A) and 55% (type B) of their DNA from presumptive somatic cells during the differentiation of somatic cells. The observations revealed inter- and intraindividual variations in the number of chromosomes in germ cells of both types of hagfishes. Although the modal number of chromosomes in the germ cells was 54 in both types, the percentage of cells with the modal number was rather low (38.6% [51/132] in five specimens of type A and 22.7% [25/110] in eight specimens of type B). In addition, one of seven type B specimens clearly had a modal number of 62 chromosomes. Another specimen of type B had a bimodal distribution of chromosome numbers, with peaks of 54 and 59 chromosomes. The observation of interindividual variations was supported by data on the amount of DNA in germ cells of type B specimens. However, these variations were rarely observed in somatic cells. These results suggest that supernumerary (B) chromosomes are maintained in germ cells and are eliminated together with some other chromosomes and/or chromatin from somatic cells.

Chromatin diminution and chromosome elimination in four Japanese hagfish species

Cytogenetic examination of four Japanese hagfish species belonging to the order Myxinida (Eptatretus okinoseanus, E. burgeri. Paramyxine atami, and Myxine garmani) revealed differences in chromosome number between germ cells (spermatocytes and spermatogonia) and somatic cells (liver, blood, gill, and kidney). The differences in chromosome number between spermatogonia (54, 52, 48, and 16) and somatic cells (34, 36, 34, and 14) were 20, 16, 14, and 2 in E. okinoseanus, E. burgeri, P. atami, and M. garmani, respectively. The amount of DNA in a somatic cell (2C) relative to that in a germ cell (2C) averaged 54.6% (E. okinoseanus type A), 44.9% (E. okinoseanus type B), 79.1% (E. burgeri), 60.0% (P. atami), and 70.2% (M. garmani). These results clearly indicate that chromosome elimination takes place during early cleavage in the four hagfish species of Myxinida living in Japanese waters, except in the ancestral germline cells. C-banding of metaphase chromosome preparations of germline and somatic cells from each hagfish species revealed that the C-band-positive chromatin in the ancestral somatic cells had been almost completely eliminated. Three patterns of elimination of this chromatin are discussed.

Elimination of the largest chromosome pair during differentiation into somatic cells in the Japanese hagfish, &lt;i&gt;Myxine garmani&lt;/i&gt; (Cyclostomata, Agnatha)

The modal number of chromosomes in &lt;i&gt;Myxine garmani &lt;/i&gt;was 14 in somatic cells from three tissues, 16 in spermatogonia, and eight in first spermatocytes. The amount of DNA in a somatic cell averaged 70.2% of the amount in a spermatogonium. The largest pair of chromosomes present in spermatogonia was absent from the somatic cells. These results indicate that, in &lt;i&gt;M&lt;/i&gt;. &lt;i&gt;garmani, &lt;/i&gt;chromosome elimination takes place during the early stages of cleavage, except in the ancestral germ line cells. The chromosome number of &lt;i&gt;M&lt;/i&gt;. &lt;i&gt;garmani &lt;/i&gt;is extremely small as compared with 48–54 in germ lines and 34–36 in somatic cells of the order Myxinida, and is the lowest in the literature on fishes.

Diurnal variations in the behaviour of the hagfish Eptatretus burgeri

The Japanese hagfish Eptatretus burgeri may be found in relatively shallow water, and is thus accessible to field observations by SCUBA diving. A population of hagfish living in a bay at a depth of about 10 m in Koajiro Bay, Kanagawa Prefecture, Japan has been observed periodically throughout a year by day and night diving. During the day, with rare exceptions, the hagfish remains within its burrow in the soft, muddy substrate; its anterior tip is sometimes just visible within the single opening of the burrow. There are no heaps of mud associated with the burrows, which seem to be relatively impermanent structures. During the night, many hagfish emerge from their burrows, either partially or completely. Partially emerged individuals protrude as much as a third of their length beyond the burrow opening, while completely emerged individuals either rest uncovered on the substrate surface or swim actively near the bottom. An emerged hagfish prefers to reenter a preexisting burrow; when one is not available, however, it quickly excavates a new burrow by swimming vigorously down into the mud. No hagfish were found in the investigated locality from the middle of June to the middle of October.