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.

Asymmetric larval head and mandibles of Hydrophilus acuminatus (Insecta: Coleoptera, Hydrophilidae): Fine structure and embryonic development.

The larvae of a water scavenger beetle, Hydrophilus acuminatus, have strongly asymmetric mandibles; the right one is long and slender, whereas the left one is short and stout. The fine structure and embryonic development of the head capsule and mandibles of this species were examined using light and scanning electron microscopy, and asymmetries in shape were detected in these structures applying an elliptic Fourier analysis. The larval mandibles are asymmetric in the following aspects: whole length, the number, structure and arrangement of retinacula (inner teeth), and size and shape of both the molar and incisor regions. The larval head is also asymmetric; the left half of the head capsule is larger than the right, and the left adductor muscle of the mandible is much thicker than the right. The origin and developmental process of asymmetric mandibles were traced in developing embryos whose developmental period is about 270 h and divided into 10 stages. Mandibular asymmetries are produced by the cumulative effects of six stepwise modifications that occur from about 36% of the total developmental time onward. The significance of these modifications was discussed with respect to the functional advantages of asymmetries and the phylogeny of members of the Hydrophilidae.

Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.

Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for genome editing. In this study, using a microinjection-based CRISPR/Cas9 system, we efficiently generated mouse lines carrying mutations at the Irx3 and Irx5 loci, which are located in close proximity on a chromosome and are functionally redundant. During the generation of Irx3/Irx5 double mutant mice, a deletion of ~0.5 Mb between the Irx3 and Irx5 loci was unintentionally identified in 6 out of 27 living pups by PCR based genotyping analysis. This deletion was confirmed by DNA fluorescence in situ hybridization analysis of fibroblasts. These results indicate that the mutant mice with a deletion of at least 0.5 Mb in their genome can be generated by the CRISPR/Cas9 system through microinjection into fertilized eggs. Our findings expand the utility of the CRISPR/Cas9 system in production of disease model animals with large deletions.

The inheritance of intrasexual dimorphism in female diving beetles (Coleoptera: Dytiscidae).

Many species of Dytiscus diving beetles exhibit intrasexual dimorphism, e.g., the elytra is smooth in some females and grooved in others. However, the expression of the grooves and whether they are a product of heredity or the environment remain unknown. One Japanese species, Dytiscus sharpi sharpi Wehncke, 1875 , also shows female dimorphism, with grooved and smooth morphs, while D. sharpi validus Régimbart, 1899, only has a single morph (the grooved type). A hybrid of the two species should therefore provide a means of sorting out how the grooves are inherited. We found two independent wetlands of D. sharpi sharpi in Chiba Prefecture, Japan. One was a place where a high proportion of grooved females lived, and the others had high proportions of smooth females. After five to eight generations of beetles from two populations with different proportions of grooved females were reared under aquarium conditions constituting a common garden design, i.e., water temperature, water depth, and presence of a plant for oviposition, the differences remained. We mated smooth virgin females of D. sharpi sharpi with males of D. sharpi validus to obtain hybrid offspring. The elytral traits of the hybrid females produced only grooved forms. These results suggested that the female dimorphism is determined by genetics, and that the grooved morph was dominant over the smooth one, independent of environmental factors. In addition, the hybrid insects did not differ from the two subspecies insects in larval survivorship, pupation success, or sex ratio. They also showed neither morphological abnormality nor reduced survival.

Whole chromosome elimination and chromosome terminus elimination both contribute to somatic differentiation in Taiwanese hagfish Paramyxine sheni.

Chromosome elimination is a process in which some chromatins are discarded from the presumptive somatic cells during early embryogenesis. Eliminated chromatins in hagfish generally consist of repetitive sequences, and they are highly heterochromatinized in germ cells. In this study, we characterized four novel eliminated DNA families, EEPs1-4, from the Taiwanese hagfish Paramyxine sheni. Sequences of these four elements occupied 20-27% of eliminated DNA in total, and each family was arranged mainly in tandem in the germline genome with high copy numbers. Although most of these elements were eliminated, a minor fraction remained in somatic cells. Some eliminated DNA families are shared as eliminated sequences between Eptatretidae and Myxinidae. Fluorescence in situ hybridization (FISH) of these elements showed that not only heterochromatic chromosomes but also both ends of euchromatic chromosomes in germ cells are absent in somatic cells of P. sheni. It strongly suggests that chromosome terminus elimination, in addition to whole chromosome elimination, contributes to somatic chromosome differentiation. Telomere-FISH further showed that chromosome fragmentation and the subsequent de novo addition of telomeric repeats are the likely mechanisms underlying chromosome terminus elimination. These characteristics make it indispensable to study the evolution and mechanisms underlying chromosome elimination in hagfish.

Molecular organization of 5S rDNA in bitterlings (Cyprinidae).

Molecular organization and nucleotide sequences of the 5S rRNA gene and NTS were investigated in freshwater fish, bitterlings (Acheilognathinae), including 10 species/subspecies of four genera, Acheilognathus, Pseudoperilampus, Rhodeus, and Tanakia, to understand the evolutionary trait of 5S rDNA arrays. Southern hybridization analysis revealed a general trend with tandem repeats of 5S rDNA in all the examined bitterlings. Sequence analysis demonstrated a conserved 120 bp sequence of the 5S rRNA gene and a short NTS of 56-67 bp with two distinct portions, a conserved (5'-flanking portion; at positions -1 to -38) and a variable part (3'-flanking portion), in 6 of 10 species/subspecies examined. The conserved NTS region was most likely an external promoter so far observed in various vertebrates, whereas the variable NTS region could be divided into two types due to its nucleotide polymorphisms. Molecular phylogeny using the 5S rRNA gene and NTS sequences suggested the occurrence of 5S rDNA duplication before speciation and a concerted evolution for the gene and conserved NTS regions, but a birth-and-death process to maintain the variable NTS region. Thus, the 5S rDNA in the examined bitterlings might have evolved under a mixed process of evolution.