Yuichiro Itoh

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken—the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

Dosage compensation is less effective in birds than in mammals

Background In animals with heteromorphic sex chromosomes, dosage compensation of sex-chromosome genes is thought to be critical for species survival. Diverse molecular mechanisms have evolved to effectively balance the expressed dose of X-linked genes between XX and XY animals, and to balance expression of X and autosomal genes. Dosage compensation is not understood in birds, in which females (ZW) and males (ZZ) differ in the number of Z chromosomes. Results Using microarray analysis, we compared the male:female ratio of expression of sets of Z-linked and autosomal genes in two bird species, zebra finch and chicken, and in two mammalian species, mouse and human. Male:female ratios of expression were significantly higher for Z genes than for autosomal genes in several finch and chicken tissues. In contrast, in mouse and human the male:female ratio of expression of X-linked genes is quite similar to that of autosomal genes, indicating effective dosage compensation even in humans, in which a significant percentage of genes escape X-inactivation. Conclusion Birds represent an unprecedented case in which genes on one sex chromosome are expressed on average at constitutively higher levels in one sex compared with the other. Sex-chromosome dosage compensation is surprisingly ineffective in birds, suggesting that some genomes can do without effective sex-specific sex-chromosome dosage compensation mechanisms.

Chromosomal polymorphism and comparative painting analysis in the zebra finch.

The zebra finch (Taeniopygia guttata) is often studied because of its interesting behaviour and neurobiology. Genetic information on this species has been lacking, making analysis of informative mutants difficult. Here we report on an improved cytological method for preparation of metaphase chromosomes suitable for fluorescent in situ hybridization of adult birds. We found that individual chicken chromosome paints usually hybridized to single zebra finch chromosomes, indicating only minor chromosomal rearrangements since the evolutionary divergence of these two species, and suggesting that the genomic location of chicken genes will predict the location of zebra finch orthologues. Chicken chromosome 1 appears to have split into two macrochromosomes in zebra finches, and chicken chromosome 4 paint hybridizes to a zebra finch macrochromosome and a microchromosome. This pattern was confirmed by mapping the androgen receptor (AR), which is located on chicken chromosome 4 but on a zebra finch microchromosome. We detected a telocentric/submetacentric polymorphism of chromosome 6 in our colony of zebra finches, and found that the polymorphism was inherited in a Mendelian pattern

Sex bias and dosage compensation in the zebra finch versus chicken genomes: General and specialized patterns among birds

We compared global patterns of gene expression between two bird species, the chicken and zebra finch, with regard to sex bias of autosomal versus Z chromosome genes, dosage compensation, and evolution of sex bias. Both species appear to lack a Z chromosome-wide mechanism of dosage compensation, because both have a similar pattern of significantly higher expression of Z genes in males relative to females. Unlike the chicken Z chromosome, which has female-specific expression of the noncoding RNA MHM (male hypermethylated) and acetylation of histone 4 lysine 16 (H4K16) near MHM, the zebra finch Z chromosome appears to lack the MHM sequence and acetylation of H4K16. The zebra finch also does not show the reduced male-to-female (M:F) ratio of gene expression near MHM similar to that found in the chicken. Although the M:F ratios of Z chromosome gene expression are similar across tissues and ages within each species, they differ between the two species. Z genes showing the greatest species difference in M:F ratio were concentrated near the MHM region of the chicken Z chromosome. This study shows that the zebra finch differs from the chicken because it lacks a specialized region of greater dosage compensation along the Z chromosome, and shows other differences in sex bias. These patterns suggest that different avian taxa may have evolved specific compensatory mechanisms.

A Bird's-Eye View of Sex Chromosome Dosage Compensation

Intensive study of a few genetically tractable species with XX/XY sex chromosomes has produced generalizations about the process of sex chromosome dosage compensation that do not fare well when applied to ZZ/ZW sex chromosome systems, such as those in birds. The inherent sexual imbalance in dose of sex chromosome genes has led to the evolution of sex-chromosome-wide mechanisms for balancing gene dosage between the sexes and relative to autosomal genes. Recent advances in our knowledge of avian genomes have led to a reexamination of sex-specific dosage compensation (SSDC) in birds, which is less effective than in known XX/XY systems. Insights about the mechanisms of SSDC in birds also suggest similarities to and differences from those in XX/XY species. Birds are thus offering new opportunities for studying dosage compensation in a ZZ/ZW system, which should shed light on the evolution of SSDC more broadly.

Comparison of the chicken and zebra finch Z chromosomes shows evolutionary rearrangements

Using fluorescent in-situ hybridization (FISH) of zebra finch (Taeniopygia guttata) bacterial artificial chromosome (BAC) clones, we determined the chromosomal localizations of 14 zebra finch genes that are Z-linked in chickens: ATP5A1, CHD1, NR2F1, DMRT1, PAM, GHR, HSD17B4, NIPBL, ACO1, HINT1, SMAD2, SPIN, NTRK2 and UBE2R2. All 14 genes also map to the zebra finch Z chromosome, indicating substantial conservation of gene content on the Z chromosome in the two avian lineages. However, the physical order of these genes on the zebra finch Z chromosome differed from that of the chicken, in a pattern that would have required several inversions since the two lineages diverged. Eight of 14 zebra finch BAC DNA showed cross-hybridization to the W chromosome, usually to the entire W chromosome, suggesting that repetitive sequences are shared by the W and Z chromosomes. These repetitive sequences likely evolved in the finch lineage after it diverged from the Galliform lineage.

The importance of having two X chromosomes.

Historically, it was thought that the number of X chromosomes plays little role in causing sex differences in traits. Recently, selected mouse models have been used increasingly to compare mice with the same type of gonad but with one versus two copies of the X chromosome. Study of these models demonstrates that mice with one X chromosome can be strikingly different from those with two X chromosomes, when the differences are not attributable to confounding group differences in gonadal hormones. The number of X chromosomes affects adiposity and metabolic disease, cardiovascular ischaemia/reperfusion injury and behaviour. The effects of X chromosome number are likely the result of inherent differences in expression of X genes that escape inactivation, and are therefore expressed from both X chromosomes in XX mice, resulting in a higher level of expression when two X chromosomes are present. The effects of X chromosome number contribute to sex differences in disease phenotypes, and may explain some features of X chromosome aneuploidies such as in Turner and Klinefelter syndromes.

What a Difference an X or Y Makes: Sex Chromosomes, Gene Dose, and Epigenetics in Sexual Differentiation

A modern general theory of sex determination and sexual differentiation identifies the factors that cause sexual bias in gene networks, leading to sex differences in physiology and disease. The primary sex-biasing factors are those encoded on the sex chromosomes that are inherently different in the male and female zygotes. These factors, and downstream factors such as gonadal hormones, act directly on tissues to produce sex differences and antagonize each other to reduce sex differences. Recent studies of mouse models such as the four core genotypes have begun to distinguish between the direct effects of sex chromosome complement (XX vs. XY) and hormonal effects. Several lines of evidence implicate epigenetic processes in the control of sex differences, although a great deal of information is needed about sex differences in the epigenome.

Cell-autonomous sex determination outside of the gonad

Background: The classic model of sex determination in mammals states that the sex of the individual is determined by the type of gonad that develops, which in turn determines the gonadal hormonal milieu that creates sex differences outside of the gonads. However, XX and XY cells are intrinsically different because of the cell‐autonomous sex‐biasing action of X and Y genes. Results: Recent studies of mice, in which sex chromosome complement is independent of gonadal sex, reveal that sex chromosome complement has strong effects contributing to sex differences in phenotypes such as metabolism. Adult mice with two X chromosomes (relative to mice with one X chromosome) show dramatically greater increases in body weight and adiposity after gonadectomy, irrespective of their gonadal sex. When fed a high‐fat diet, XX mice develop striking hyperinsulinemia and fatty liver, relative to XY mice. The sex chromosome effects are modulated by the presence of gonadal hormones, indicating an interaction of the sex‐biasing effects of gonadal hormones and sex chromosome genes. Conclusions: Other cell‐autonomous sex chromosome effects are detected in mice in many phenotypes. Birds (relative to eutherian mammals) are expected to show more widespread cell‐autonomous sex determination in non‐gonadal tissues, because of ineffective sex chromosome dosage compensation mechanisms. Developmental Dynamics 242:371–379, 2013.

Four core genotypes mouse model: localization of the Sry transgene and bioassay for testicular hormone levels.

BackgroundThe “four core genotypes” (FCG) mouse model has emerged as a major model testing if sex differences in phenotypes are caused by sex chromosome complement (XX vs. XY) or gonadal hormones or both. The model involves deletion of the testis-determining gene Sry from the Y chromosome and insertion of an Sry transgene onto an autosome. It produces XX and XY mice with testes, and XX and XY mice with ovaries, so that XX and XY mice with the same type of gonad can be compared to assess phenotypic effects of sex chromosome complement in cells and tissues.FindingsWe used PCR to amplify the Sry transgene and adjacent genomic sequences, to resolve the location of the Sry transgene to chromosome 3 and confirmed this location by fluorescence in situ hybridization (FISH) of the Sry construct to metaphase chromosomes. Using quantitative PCR, we estimate that 12–14 copies of the transgene were inserted. The anogenital distance (AGD) of FCG pups at 27–29 days after birth was not different in XX vs. XY males, or XX vs. XY females, suggesting that differences between XX and XY mice with the same type of gonad are not caused by difference in prenatal androgen levels.ConclusionThe Sry transgene in FCG mice is present in multiple copies at one locus on chromosome 3, which does not interrupt known genes. XX and XY mice with the same type of gonad do not show evidence of different androgen levels prenatally.