Chizuko Nishida

The ZW sex chromosomes of Gekko hokouensis (Gekkonidae, Squamata) represent highly conserved homology with those of avian species.

Populations of the gecko lizard Gekko hokouensis (Gekkonidae, Squamata) on Okinawajima Island and a few other islands of the Ryukyu Archipelago, Japan, have the morphologically differentiated sex chromosomes, the acrocentric Z chromosome and the subtelocentric W chromosome, although the continental representative of this species reportedly shows no sex chromosome heteromorphism. To investigate the origin of sex chromosomes and the process of sex chromosomal differentiation in this species, we molecularly cloned the homologues of six chicken Z-linked genes and mapped them to the metaphase chromosomes of the Okinawajima sample. They were all localized to the Z and W chromosomes in the order ACO1/IREBP–RPS6–DMRT1–CHD1–GHR–ATP5A1, indicating that the origin of ZW chromosomes in G. hokouensis is the same as that in the class Aves, but is different from that in the suborder Ophidia. These results suggest that in reptiles the origin of sex chromosomes varies even within such a small clade as the order Squamata, employing a variety of genetic sex determination. ACO1/IREBP, RPS6, and DMRT1 were located on the Z long arm and the W short arm in the same order, suggesting that multiple rearrangements have occurred in this region of the W chromosome, where genetic differentiation between the Z and W chromosomes has been probably caused by the cessation of meiotic recombination.

Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.

Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication.

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Squamata) by molecular cytogenetic approach.

Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L4m + L2sm + M2m + S4m + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.

Karyotypic evolution in squamate reptiles: comparative gene mapping revealed highly conserved linkage homology between the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Lacertilia) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae, Serpentes)

The butterfly lizard (Leiolepis reevesii rubritaeniata) has the diploid chromosome number of 2n = 36, comprising two distinctive components, macrochromosomes and microchromosomes. To clarify the conserved linkage homology between lizard and snake chromosomes and to delineate the process of karyotypic evolution in Squamata, we constructed a cytogenetic map of L. reevesii rubritaeniata with 54 functional genes and compared it with that of the Japanese four-striped rat snake (E. quadrivirgata, 2n = 36). Six pairs of the lizard macrochromosomes were homologous to eight pairs of the snake macrochromosomes. The lizard chromosomes 1, 2, 4, and 6 corresponded to the snake chromosomes 1, 2, 3, and Z, respectively. LRE3p and LRE3q showed the homology with EQU5 and EQU4, respectively, and LRE5p and LRE5q corresponded to EQU7 and EQU6, respectively. These results suggest that the genetic linkages have been highly conserved between the two species and that their karyotypic difference might be caused by the telomere-to-telomere fusion events followed by inactivation of one of two centromeres on the derived dicentric chromosomes in the lineage of L. reevesii rubritaeniata or the centric fission events of the bi-armed macrochromosomes and subsequent centromere repositioning in the lineage of E. quadrivirgata. The homology with L. reevesii rubritaeniata microchromosomes were also identified in the distal regions of EQU1p and 1q, indicating the occurrence of telomere-to-telomere fusions of microchromosomes to the p and q arms of EQU1.

Characterization of chromosome structures of Falconinae (Falconidae, Falconiformes, Aves) by chromosome painting and delineation of chromosome rearrangements during their differentiation

Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7–10 pairs of large- and medium-sized macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with chicken chromosomes 1–9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S–28S rRNA genes and telomeric (TTAGGG)n sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed, and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently in the lineages of Accipitridae and Falconinae.

The ZW micro-sex chromosomes of the Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) have the same origin as chicken chromosome 15.

The Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) has ZZ/ZW-type micro-sex chromosomes where the 18S-28S ribosomal RNA genes (18S-28S rDNA) are located. The W chromosome is morphologically differentiated from the Z chromosome by partial deletion and amplification of 18S-28S rDNA and W-specific repetitive sequences. We recently found a functional gene (TOP3B) mapped on the P. sinensis Z chromosome, which is located on chicken (Gallus gallus, GGA) chromosome 15. Then we cloned turtle homologues of 4 other GGA15-linked genes (GIT2, NF2, SBNO1, SF3A1) and localized them to P. sinensis chromosomes. The 4 genes all mapped on the Z chromosome, and 2 of them (SBNO1, SF3A1) were also localized to the W chromosome. Our mapping data suggest that at least one large inversion occurred between GGA15 and the P. sinensis Z chromosome, and that there are homologous regions in the distal portions of both the short and long arms between the Z and W chromosomes. W chromosomal differentiation in P. sinensis probably proceeded by the deletion of the proximal chromosomal region followed by 18S-28S rDNA amplification, after a paracentric inversion occurred at the breakpoints between the distal region of 18S-28S rDNA and the proximal region of SBNO1 on the Z chromosome.

Diversity in the origins of sex chromosomes in anurans inferred from comparative mapping of sexual differentiation genes for three species of the Raninae and Xenopodinae

Amphibians employ genetic sex determination systems with male and female heterogamety. The ancestral state of sex determination in amphibians has been suggested to be female heterogamety; however, the origins of the sex chromosomes and the sex-determining genes are still unknown. In Xenopus laevis, chromosome 3 with a candidate for the sex- (ovary-) determining gene (DM-W) was recently identified as the W sex chromosome. This study conducted comparative genomic hybridization for X. laevis and Xenopus tropicalis and FISH mapping of eight sexual differentiation genes for X. laevis, X. tropicalis, and Rana rugosa. Three sex-linked genes of R. rugosa—AR,SF-1/Ad4BP, and Sox3—are all localized to chromosome 10 of X. tropicalis, whereas AR and SF-1/Ad4BP are mapped to chromosome 14 and Sox3 to chromosome 11 in X. laevis. These results suggest that the W sex chromosome was independently acquired in the lineage of X. laevis, and the origins of the ZW sex chromosomes are different between X. laevis and R. rugosa. Cyp17, Cyp19, Dmrt1, Sox9, and WT1 were localized to autosomes in X. laevis and R. rugosa, suggesting that these five genes probably are not candidates for the sex-determining genes in the two anuran species.

Abortive meiosis in the oogenesis of parthenogenetic Daphnia pulex

Most daphnid species adopt parthenogenesis and sexual reproduction differentially in response to varied environmental cues, resulting in the production of diploid progenies in both cases. Previous studies have reportedly suggested that daphnids produce their parthenogenetic eggs via apomixis; the nuclear division of mature oocytes should be an equational division similar to somatic mitosis. However, it seems premature to conclude that this has been unequivocally established in any daphnids. Therefore, the objective of our research was to precisely reveal the process and mechanism of parthenogenetic oogenesis and maintenance of diploidy in Daphnia pulex through histology, karyology, and immunohistochemistry. We found that, when a parthenogenetic egg entered the first meiosis, division was arrested in the early first anaphase. Then, two half-bivalents, which were dismembered from each bivalent, moved back to the equatorial plate and assembled to form a diploid equatorial plate. Finally, the sister chromatids were separated and moved to opposite poles in the same manner as the second meiotic division followed by the extrusion of one extremely small daughter cell (resembling a polar body). These results suggest that parthenogenetic D. pulex do not adopt typical apomixis. We hypothesize that D. pulex switches reproductive mode depending on whether the egg is fertilized or not.