Takao Namikawa

Thyrotrophin in the pars tuberalis triggers photoperiodic response

Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin (TSH) β-subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV) administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor–cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long-day photoinduced seasonal breeding.

Molecular analysis of avian circadian clock genes.

Unlike mammals, avian circadian rhythms are regulated by a multiple oscillatory system consisting of the retina, the pineal and the suprachiasmatic nucleus in the hypothalamus. To understand avian circadian system, we have cloned Clock and Period homologs (qClock, qPer2 and qPer3) and characterized these genes in Japanese quail. Overall, qCLOCK, qPER2 and qPER3 showed approximately 79%, approximately 46% and approximately 33% amino acid identity to mCLOCK, mPER2, mPER3, respectively. Clock was mapped to quail chromosome 4 and chicken chromosome 4q1.6-q2.1. Per2 and Per3 genes were both localized to microchromosomes. qClock mRNA was expressed throughout the day, while qPer2 and qPer3 showed robust circadian oscillation in the eye and the pineal gland. All three genes were expressed in various tissues. In addition, qPer2 mRNA was induced by light, but neither qClock nor qPer3 was induced. These results can explain the molecular basis for circadian entrainment in Japanese quail and also provide new avenues for molecular understanding of avian circadian clock and photoperiodism.

A comparative cytogenetic study of chromosome homology between chicken and Japanese quail

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1–8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.

Phylogenetic relationship among all living species of the genusBubalus based on DNA sequences of the cytochromeb gene

The cytochromeb genes of all living species ofBubalus, including the river type and the swamp type of domestic buffaloes (Bubalus bubalis), were sequenced to clarify their phylogenetic relationships. These sequences were compared together with the African buffalo (Syncerus caffer) and banteng (Bos javanicus) sequences as an outgroup. Phylogenetic trees ofBubalus species based on the DNA sequences of the cytochromeb gene demonstrated that the tamaraw (Bubalus mindorensis), endemic to the Philippines, could be classified into the subgenusBubalus, not the subgenusAnoa. The divergence time between the lowland anoa (B. depressicornis) and the mountain anoa (B. quarlesi) was estimated at approximately 2.0 million years (Myr), which is almost the same as the coalescence time for theBubalus sequences. This large genetic distance supports the idea that the lowland anoa and the mountain anoa are different species. An unexpectedly large genetic distance between the river and the swamp type of domestic buffaloes suggests a divergence time of about 1.7 Myr, while the swamp type was noticed to have the closest relationship with the tamaraw (1.5 Myr). This result implies that the two types of domestic buffaloes have differentiated at the full species level.

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).

Detection of quantitative trait loci for body weight at 10 weeks from Philippine wild mice

Abstract. A genome-wide scan for quantitative trait loci (QTLs) controlling body weight at 10 weeks after birth was carried out in a population of 387 intersubspecific backcross mice derived from a cross between C57BL/6J inbred mice (Mus musculus domesticus) and wild mice (M. m. castaneus) captured in the Philippines, in order to discover novel QTLs from the wild mice that have about 60% lower body weight than C57BL/6J. By interval mapping, we detected four QTLs: a highly significant QTL on Chromosome (Chr) 2, which was common in both sexes; two significant QTLs on Chr 13, one male-specific and the other female-specific; and a suggestive male-specific QTL on X Chr. By composite interval mapping, we confirmed the presence of the three QTLs on Chrs 2 and 13, but not of the male-specific X-linked QTL. The composite interval mapping analysis newly identified three QTLs: a significant male-specific QTL on Chr 11 and two highly significant female-specific QTLs on Chrs 9 and X. Individual QTLs explained 3.8–11.6% of the phenotypic variance, and all the QTL alleles derived from the wild mice decreased body weight. A two-way analysis of variance revealed a significant epistatic interaction between the Chr 2 QTL and the background marker locus D12Mit4 on Chr 12 only in males. The interaction effect unexpectedly increased body weight. The chromosomal region containing the Chr 2 QTL did not coincide with those of growth or fatness QTLs mapped in previous studies. These results suggest that a population of wild mice may play an important role as new sources of valuable QTLs.

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.

Further mapping of quantitative trait loci for postnatal growth in an intersubspecific backcross of wild Mus musculus castaneus and C57BL/6J mice

We performed a quantitative trait locus (QTL) analysis of eight body weights recorded weekly from 3 weeks to 10 weeks after birth and two weight gains recorded between 3 weeks and 6 weeks, and between 6 weeks and 10 weeks in an inter-sub-specific backcross population of wild Mus musculus castaneus mice captured in the Philippines and the common inbred strain C57BL/6J ( M. musculus domesticus ), to elucidate the complex genetic architecture of body weight and growth. Interval mapping identified 17 significant QTLs with main effects on 11 chromosomes. In particular, the main effect of the most potent QTL on proximal chromosome 2 increased linearly with age, whereas other QTLs exerted effects on either the early or late growth period. Surprisingly, although wild mice displayed 60% of the body size of their C57BL/6J counterparts, the wild-derived allele enhanced growth at two QTLs. Interestingly, five of the 17 main-effect QTLs identified had significant epistatic interaction effects. Five new epistatic QTLs with no main effects were identified on different chromosomes or regions. For one pair of epistatic QTLs, mice that were heterozygous for the wild-derived allele at one QTL and homozygous for that allele at another QTL exhibited the most rapid growth in all four possible genotypic combinations. Out of the identified QTLs, several showed significant sex-specific effects.

Fatty liver and hyperlipidemia in iddm (insulin-dependent diabetes mellitus) of streptozotocin-treated shrews

Severe IDDM (insulin-dependent diabetes mellitus) was produced in the musk shrew (Suncus murimus, Insectivora) by a high dose (a single intraperitoneal injection of 100 mg/kg Body Weight) of streptozotocin (STZ) injection. All shrews that were administered a high dose of STZ exhibited hyperglycemia (449 +/- 16 mg/dl vs 73 +/- 4 mg/dl in controls) and hypoinsulinemia(0.25 +/- 0.07 ng/ml vs 10.96 +/- 1.97 ng/ml in controls) with ketosuria 10 days after injection. Their livers were enlarged and exhibited ayellowish-brown color with marked triglyceride (TG) accumulation (63.25 +/- 7.10 mg/g Liver vs 2.11 +/- 0.19 mg/g Liver in controls). It is probable that the increased influx of fatty acids into the liver induced by hypoinsulinemia and the low capacity of excretion of lipoprotein secretion from liver in the musk shrew resulting from a deficiency of apolipoprotein B synthesis play important roles in fatty liver formation. Hyperlipidemia was another feature in shrews with severe IDDM. The blood TG level was especially high in these shrews (899 +/- 178 mg/dl vs 23 +/- 5 mg/dl in controls). These results indicate that the IDDM shrew, induced by high doses of STZ, is a unique model characterized by fatty liver and hyperlipidemia and may be useful for studying lipid metabolism of IDDM.

Quantitative trait locus analysis of abnormal circadian period in CS mice.

Abstract. CS mice show a free-running period (κ) longer than 24 h and rhythm splitting in constant darkness (DD). These features in behavioral circadian rhythms are distinctive as compared with other inbred strains of mice, which exhibit robust free-running rhythms with κ shorter than 24 h. To identify the genes affecting κ, quantitative trait locus (QTL) analysis was initially conducted by using 289 F2 mice derived from a cross between CS and C57BL/6J strain. A suggestive QTL (LOD = 3.71) with CS allele increasing κ was detected on the distal region of Chromosome (Chr) 19. Next, using 192 F2 mice from a cross between CS and MSM strain, the presence of the QTL on Chr 19 was examined, and we confirmed the QTL at the genome-wide significant level (LOD = 4.61 with 10.4% of the total variance explained). This QTL was named long free-running period (Lfp). Three other suggestive QTLs (LOD = 3.24–4.28) were mapped to the midportion of Chr 12 in (CS×C57BL/6J)F2 mice, and to the proximal and middle region of Chr 19 in (CS×MSM)F2 mice, respectively, of which, CS alleles for two QTLs on Chr 19 have the effect of lengthening κ. None of these QTLs were mapped to the chromosomal regions of previously described QTLs for κ and known clock genes (Clock, mPer1, Bmal1, mCry1, mCry2, mTim, and Csnk1e).