Masaoki Tsudzuki

Karyotypic evolution in the Galliformes: an examination of the process of karyotypic evolution by comparison of the molecular cytogenetic findings with the molecular phylogeny

To define the process of karyotypic evolution in the Galliformes on a molecular basis, we conducted genome-wide comparative chromosome painting for eight species, i.e. silver pheasant (Lophura nycthemera), Lady Amherst’s pheasant (Chrysolophus amherstiae), ring-necked pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), Western capercaillie (Tetrao urogallus), Chinese bamboo-partridge (Bambusicola thoracica) and common peafowl (Pavo cristatus) of the Phasianidae, and plain chachalaca (Ortalis vetula) of the Cracidae, with chicken DNA probes of chromosomes 1–9 and Z. Including our previous data from five other species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and blue-breasted quail (Coturnix chinensis) of the Phasianidae, guinea fowl (Numida meleagris) of the Numididae and California quail (Callipepla californica) of the Odontophoridae, we represented the evolutionary changes of karyotypes in the 13 species of the Galliformes. In addition, we compared the cytogenetic data with the molecular phylogeny of the 13 species constructed with the nucleotide sequences of the mitochondrial cytochrome b gene, and discussed the process of karyotypic evolution in the Galliformes. Comparative chromosome painting confirmed the previous data on chromosome rearrangements obtained by G-banding analysis, and identified several novel chromosome rearrangements. The process of the evolutionary changes of macrochromosomes in the 13 species was in good accordance with the molecular phylogeny, and the ancestral karyotype of the Galliformes is represented.

Evolution of the vertebrate DNMT3 gene family : a possible link between existence of DNMT3L and genomic imprinting

DNA methylation plays an essential role in genomic imprinting observed in eutherian mammals and marsupials. In mouse, one of the two de novo DNA methyltransferases, Dnmt3a, and a related protein, Dnmt3L have been shown to be essential for imprint establishment in the parental germline. To gain insights into the evolution of imprinting mechanisms, we have identified and characterized the DNMT3 family genes in other vertebrate species. We cloned cDNAs for chicken DNMT3A and DNMT3B, whose putative protein products shared 81.5% and 48.6% amino acid sequence identity with their mouse orthologues. Using computer-assisted database searches, we also identified DNMT3A and DNMT3B orthologues in fish (fugu and zebrafish) and marsupials (opossum). We found that, while opossums had an orthologue for DNMT3L, chickens and fish did not have this gene. Thus, unlike the other DNMT3 members, DNMT3L was restricted to the species in which imprinting occurs. The acquisition of DNMT3L by a common ancestor of eutherians and marsupials might have been closely related to the evolution of imprinting.

Chymase Inhibitor Improves Dermatitis in NC/Nga Mice

Background: Mast cell chymase is thought to participate in allergic inflammation, but its precise role remains undetermined. Inbred NC/Nga mice develop skin lesions similar to atopic dermatitis (AD) when they grow up in a conventional environment. To elucidate the possible role of chymase in AD, we examined the effect of a chymase inhibitor on skin lesions of NC/Nga mice. Methods: NC/Nga mice were given the chymase inhibitor SUN-C8257 daily at 150 mg/kg/day with drinking water, and the severity of the dermatitis was evaluated on day 35 of the experiment. The role of chymase in dermatitis was further investigated in vitro and in vivo using recombinant mouse mast cell protease-4 (mMCP-4). Results: Administration of SUN-C8257 significantly reduced the clinical skin and histological score in NC/Nga mice. SUN-C8257 also inhibited the accumulation of inflammatory cells, such as eosinophils and mast cells, in the affected lesions in this model. mMCP-4 stimulated eosinophil migration in vitro, and intradermal injection of the enzyme resulted in a significant accumulation of inflammatory cells, including eosinophils, at the injection site. Thus amelioration of the skin lesions in NC/Nga mice by SUN-C8257 might be, at least in part, due to the suppression of cell infiltration in the lesions. Conclusions: Mast cell chymase may contribute to the pathogenesis of AD, and SUN-C8257 will be beneficial to the treatment of the skin disorder.

Sequence polymorphisms, allelic expression status and chromosome locations of the chicken IGF2 and MPR1 genes

By screening 26 chicken breeds and lines, DNA polymorphisms were identified in the IGF2 and MPR1 genes, of which mammalian homologues are parentally imprinted, and the GAPD gene, a housekeeping control. Using the polymorphisms as genetic markers, we found that all three genes are expressed biallelically in embryonic tissues. IGF2 and MPR1 were mapped on chicken chromosomes 5 and 3, respectively, by fluorescence in situ hybridization, demonstrating conserved linkage homology between mammals and birds.

Development of the skeleton in Japanese quail embryos.

In the research fields of experimental embryology, teratological testing, and developmental engineering in avian species, a knowledge of normal embryonic development is necessary so that research may be performed efficiently and precisely. A series of normal stages based on external appearance has been established in both chicken and quail embryos. Those based on skeletal features, however, have not been elucidated. The present study newly established a series of normal stages for the development of the Japanese quail embryo skeleton. This series is composed of 15 stages determined by observing the timing of chondrification and calcification of the skeleton every 24 h, from 3 to 17 days of incubation. Cartilage and ossified bones were stained blue and red with Alcian blue 8GX and alizarin red S, respectively. These skeletogenous stages of the Japanese quail embryo will be useful as a normal control not only in studies of experimental embryology, teratological testing, and developmental engineering, but also in the analysis of mutant embryos with skeletal abnormalities.

Genetic analyses for dermatitis and IgE hyperproduction in the NC/Nga mouse

Atopic dermatitis (AD) is a severe problem in the human species, and many approaches have been taken to reveal the causes of the disease and to develop therapeutical methods to combat it. However, the fundamental causes of AD are still unknown, and no radical therapy exists (Leung 1997; van Bever 1992), mainly due to lack of a suitable animal model for studying the mechanisms through which AD arises. We recently determined that the NC/Nga (NC) mice would be an excellent animal model for human AD, because they show severe hereditary dermatitis with hyperproduction of immunoglobulin E (IgE), similar to the situation in humans (Matsuda et al. 1997). The NC strain was established as an inbred strain by K. Kondo in 1957 based on Japanese fancy mice (Festing 1996; Kondo 1984), and is now a highly inbred strain (more than 100 generations). The dermatitis and IgE hyperproduction occur spontaneously in all NC mice reared under non-sterile (conventional) conditions, but no genetic analyses for these characters have been done so far. When trying to use the NC mice as an efficient animal model for human AD, it is necessary to reveal the mode of inheritance of the expression of dermatitis and IgE hyperproduction, and we describe it in this paper. All animals used in this study were handled according to the rules described in Guide for the Care and Use of Laboratory Animals (NIH publication no. 86-23, 1985); Standards Relating to the Care and Management of Experimental Animals, Prime Ministers Office, Japan, 1980; and Guide for the Use of Experimental Animals in Universities, The Ministry of Education, Science, and Culture, Japan, 1987. NC and BALB/cA (BALB) strains and their crossbreds were used, with the BALB mice serving as a control. All mice were raised in a clean, conventional animal room with temperature and relative humidity adjusted to 23+2 °C and 50+10%, respectively. The room was illuminated by fluorescent light from 5 am until 9 pm. Mice were kept in a polycarbonate cage (Clea Japan, Inc., Osaka, Japan) that was bedded with clean wood chips (Clea Japan, Inc.), and tap water and a commercial diet for small rodents (CE-2, Clea Japan, Inc.) were provided for ad libitum consumption. We reciprocally paired NC mice with BALB mice to produce F1 progeny. Subsequently, F2 and backcross generations were also produced. In these mice, we scored the incidence of abnormal mice showing dermatitis or high levels of plasmic total IgE at 12 weeks of age. The resulting segregation ratios were analyzed by the chi-square test. To obtain segregation data for the mice with dermatitis, we classified the animals on the basis of the absence or presence of scratching, erythema, hemorrhage, edema, superficial erosion, deep excoriation, and/or scaling and dryness of the skin, because all conventional NC mice are characterized by these symptoms. The BALB mice, however, showed no such symptoms even though they were reared together with the severely lesioned NC mice in the same cage for several months (Matsuda et al. 1997). To obtain segregation data for the mice showing IgE hyperproduction, we classified the animals with an IgE level less than 1500 ng/ml as a low-level group and those with more than 9000 ng/ml as a high-level group, because the F2 and M. Tsudzuki ( ) Laboratory of Animal Breeding and Genetics, Faculty of Applied Biological Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739, Japan

Biallelic expression of Z-linked genes in male chickens

In birds, females are heterogametic (ZW), while males are homogametic (ZZ). It has been proposed that there is no dosage compensation for the expression of Z-linked genes in birds. In order to examine if the genes are inactivated on one of the two Z chromosomes, we analyzed the allelic expression of the B4GALT1 and CHD-Z genes on Z chromosomes in male chickens. One base substitution was detected among 15 chicken breeds and lines examined for each gene, and cross mating was made between the breeds or lines with polymorphism. cDNAs were synthesized from cultured cell colonies each derived from a single cell of an F1 male embryo. The allelic expression of the B4GALT1 gene was examined by restriction fragment length polymorphism analysis of the PCR products digested with RsaI, and that of the CHD-Z gene by the single nucleotide primer extension (SNuPE) method. Both of the genes displayed biallelic expression, suggesting that these Z-linked genes were not subject to inactivation in male chickens. Comparison between expression levels in males and females by real-time quantitative PCR suggested that expression was compensated for the CHD-Z gene but not for the B4GALT1 gene.

Identification of quantitative trait loci affecting shank length, body weight and carcass weight from the Japanese cockfighting chicken breed, Oh-Shamo (Japanese Large Game)

We performed a quantitative trait locus (QTL) analysis to map QTLs controlling shank length, body weight, and carcass weight in a resource family of 245 F2 birds developed from a cross of the large-sized, native, Japanese cockfighting breed, Oh-Shamo (Japanese Large Game), and the White Leghorn breed of chickens. Interval mapping revealed three significant QTLs for shank length on chromosomes 1, 4 and 24 at the experiment-wise 5% level, and a suggestive shank length QTL on chromosome 27 at the experiment-wise 10% level. For body weight two QTLs, one significant and the other suggestive, were identified on chromosomes 4 and 24, respectively. As expected, QTLs for carcass weight, which was highly correlated with body weight (r = 0.95), were detected at the same chromosomal locations as the detected body weight QTLs. Interestingly, the chromosomal locations containing these body weight and carcass weight QTLs coincided with those of two of the four shank length QTLs detected. No QTL with an epistatic interaction effect was discovered for any trait. The total contribution of all detected QTLs to genetic variance was 98.4%, 27.0% and 25.9% for shank length, body weight and carcass weight, respectively, indicating that most shank length QTLs have been identified but many body weight and carcass weight QTLs have been overlooked by the present analysis because of a low coverage rate of the 88 microsatellite markers used here (approximately 46% of the whole genome).

High genetic divergence in miniature breeds of Japanese native chickens compared to Red Junglefowl, as revealed by microsatellite analysis

A wide diversity of domesticated chicken breeds exist due to artificial selection on the basis of human interests. Miniature variants (bantams) are eminently illustrative of the large changes from ancestral junglefowls. In this report, the genetic characterization of seven Japanese miniature chicken breeds and varieties, together with institute-kept Red Junglefowl, was conducted by means of typing 40 microsatellites located on 21 autosomes. We drew focus to genetic differentiation between the miniature chicken breeds and Red Junglefowl in particular. A total of 305 alleles were identified: 27 of these alleles (8.9%) were unique to the Red Junglefowl with high frequencies (>20%). Significantly high genetic differences (F(ST)) were obtained between Red Junglefowl and all other breeds with a range of 0.3901-0.5128. Individual clustering (constructed from combinations of the proportion of shared alleles and the neighbour-joining method) indicated high genetic divergence among breeds including Red Junglefowl. There were also individual assignments on the basis of the Bayesian and distance-based approaches. The microsatellite differences in the miniature chicken breeds compared to the presumed wild ancestor reflected the phenotypic diversity among them, indicating that each of these miniature chicken breeds is a unique gene pool.

A comparative karyological study of the blue-breasted quail (Coturnix chinensis, Phasianidae) and California quail (Callipepla californica, Odontophoridae)

We conducted comparative chromosome painting and chromosome mapping with chicken DNA probes against the blue-breasted quail (Coturnix chinensis, CCH) and California quail (Callipepla californica, CCA), which are classified into the Old World quail and the New World quail, respectively. Each chicken probe of chromosomes 1–9 and Z painted a pair of chromosomes in the blue-breasted quail. In California quail, chicken chromosome 2 probe painted chromosomes 3 and 6, and chicken chromosome 4 probe painted chromosomes 4 and a pair of microchromosomes. Comparison of the cytogenetic maps of the two quail species with those of chicken and Japanese quail revealed that there are several intrachromosomal rearrangements, pericentric and/or paracentric inversions, in chromosomes 1, 2 and 4 between chicken and the Old World quail. In addition, a pericentric inversion was found in chromosome 8 between chicken and the three quail species. Ordering of the Z-linked DNA clones revealed the presence of multiple rearrangements in the Z chromosomes of the three quail species. Comparing these results with the molecular phylogeny of Galliformes species, it was also cytogenetically supported that the New World quail is classified into a different clade from the lineage containing chicken and the Old World quail.