Shigeki Mizuno

Conserved synteny between the chicken Z sex chromosome and human chromosome 9 includes the male regulatory gene DMRT1: a comparative (re)view on avian sex determination

Sex-determination mechanisms in birds and mammals evolved independently for more than 300 million years. Unlike mammals, sex determination in birds operates through a ZZ/ZW sex chromosome system, in which the female is the heterogametic sex. However, the molecular mechanism remains to be elucidated. Comparative gene mapping revealed that several genes on human chromosome 9 (HSA 9) have homologs on the chicken Z chromosome (GGA Z), indicating the common ancestry of large parts of GGA Z and HSA 9. Based on chromosome homology maps, we isolated a Z-linked chicken ortholog of DMRT1, which has been implicated in XY sex reversal in humans. Its location on the avian Z and within the sex-reversal region on HSA 9p suggests that DMRT1 represents an ancestral dosage-sensitive gene for vertebrate sex-determination. Z dosage may be crucial for male sexual differentiation/determination in birds.

Absence of Z-chromosome inactivation for five genes in male chickens

In order to examine if Z-chromosome inactivation, which is analogous to X-chromosome inactivation in mammals, takes place in male birds having ZZ sex chromosomes, five Z-linked genes of chickens which are expressed in both sexes in certain tissues were selected: i.e. genes for growth hormone receptor, nicotinic acetylcholine receptor β3, aldolase B, β1,4-galactosyltransferase I, and iron-responsive element-binding protein (also known as cytosolic aconitase). Antisense or sense riboprobe was prepared from an intronic sequence of each gene and subjected to fluorescence in situ hybridization to nascent transcripts of each gene in a nucleus. Each antisense riboprobe hyridized to two spots of nascent RNA which corresponded to its gene loci on the two Z chromosomes in a majority of nuclei in a tissue of the male. The efficiency of detection of two spots per nucleus was comparable to that for the glyceraldehyde-3-phosphate dehydrogenase gene, an autosomal housekeeping gene. These results suggest strongly that Z-chromosome inactivation, i.e. virtual silence of transcription at one of the alleles, does not take place for these five Z-linked genes in male chickens.

Chicken spindlin genes on W and Z chromosomes: Transcriptional expression of both genes and dynamic behavior of spindlin in interphase and mitotic cells

Contigs of genomic clones covering about 480 kb on the terminal region of the short arm of chicken W chromosome were obtained. By applying the exon trapping procedure on this whole region, a chicken homolog of spindling gene, chSpin-W, was identified and subcloned. A counterpart gene, chSpin-Z, was found near the centromere on the long arm of Z chromosome. Although protein-coding regions of both genes are nearly identical, a part of the 3′-untranslated region is sufficiently different to distinguish the transcript of chSpin-W. Both chSpin-W and chSpin-Z are transcribed in early embryos. chSpin-Z is transcribed in various tissues of adult chickens, while chSpin-W is transcribed most prominently in ovarian granulosa and thecal cells. When female chicken embryonic fibroblasts were transfected with a cDNA construct for red fluorescent protein or green fluorescent protein-fused spindlin or FLAG-tagged spindlin, the expressed spindlin was co-localized with SUMO-1 in nuclear dots, ND10, in interphase cells, while the expressed spindlin was localized on entire chromosomes during mitosis. The localization of spindlin in ND10 reappeared after mitosis in daughter cell nuclei. A C-terminal region of spindlin was suggested to be required for the localization of spindlin to ND10.

Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation

Since the discovery of SRY/Sry as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/Sry and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/Sry, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.

Molecular and cytological characterization of SspI-family repetitive sequence on the chicken W chromosome

A genomic clone, pWS44, isolated from the chicken W chromosome-specific genomic library contained a partial (226-bp) sequence of a novel SspI-family repetitive sequence. A genomic clone, pWPRS09, containing a 508-bp SspI fragment (a repeating unit of the family) was subsequently obtained and sequenced. This 0.5-kb unit is tandemly repeated about 11 300 times. FISH to mitotic and lampbrush W chromosomes indicates that the SspI-family is located on the chromomere 6 between heterochromatic and distal non-heterochromatic regions on the short arm. The SspI-family sequence was proved to be a good positional marker in FISH mapping of active genes in the non-heterochromatic region on the lampbrush W chromosome. The presence of SspI-family repetitive sequence is limited to the genus Gallus (chickens and jungle fowls). The 0.5-kb repeating unit contains a 120-bp stretch of polypurine/polypyrimidine sequence (GGAGA repeats), shows no DNA curvature, and rapid electrophoretic mobility in 4% polyacrylamide gel at 4°C. The SspI-family forms a relatively diffused chromatin structure in nuclei. These features are distinctly different from those of XhoI- and EcoRI-family sequences on the W chromosome. The total amount of non-repetitive DNA in the chicken W chromosome is estimated to be about 10 Mb.

Assembly of the silk fibroin elementary unit in endoplasmic reticulum and a role of L‐chain for protection of α1,2‐mannose residues in N‐linked oligosaccharide chains of fibrohexamerin/P25

Silk fibroin of Bombyx mori is secreted from the posterior silk gland (PSG) as a 2.3‐MDa elementary unit, consisting of six sets of a disulfide‐linked heavy chain (H‐chain)–light chain (L‐chain) heterodimer and one molecule of fibrohexamerin (fhx)/P25. Fhx/P25, a glycoprotein, associates noncovalently with the H–L heterodimers. The elementary unit was found and purified from the endoplasmic reticulum (ER) extract of PSG cells. A substantial amount of fhx/P25 unassembled into the elementary unit was also present in ER. In normal‐level fibroin‐producing breeds (J‐139 and C108), the elementary unit contained fhx/P25 of either 30 kDa (major) or 27 kDa (minor). The 27‐kDa fhx/P25 was produced from the 30‐kDa form by digestion with the bacterial α1,2‐mannosidase in vitro. The elementary unit in the ER extract contained only the 30‐kDa fhx/P25, whereas both 30‐ and 27‐kDa forms of fhx/P25 were present in the ER plus Golgi mixed extracts. In naked‐pupa mutants [Nd(2), Nd‐s and Nd‐s D], extremely small amounts of fibroin were produced and they consisted of one molecule of 27‐kDa fhx/P25 and six molecules of H‐chain but no L‐chain. When the Nd‐s D mutant was subjected to transgenesis with the normal L‐chain gene, the (H‐L)6fhx1‐type elementary unit containing the 30‐kDa fhx/P25, was produced. These results suggest that fhx/P25 in the elementary unit is largely protected from digestion with Golgi α1,2‐mannosidases when L‐chains are present in the unit. Models suggesting a role of L‐chain for the protection of α1,2‐mannose residues of fhx/P25 are presented.

Comprehensive search for chicken W chromosome-linked genes expressed in early female embryos from the female-minus-male subtracted cDNA macroarray.

In order to seek chicken W chromosome-linked genes expressed significantly earlier than the time of gonadal differentiation, female-minus-male-subtracted cDNA macroarrays were prepared from day 2 (Hamburger-Hamilton stages 12-13), day 3 (stages 19-20) and day 4 (stages 24-25) embryos. From a total of 15-744 macroarrayed cDNA clones, 610 clones exhibiting significantly female-specific expression were selected. When each one of the 610 cDNA clones was used as a probe in Southern blot hybridization with male or female chicken genomic DNA, 62 clones, grouped into eight (A-H) types according to their patterns of hybridization, were considered to be derived from W chromosome-linked genes. When representative cDNA clones in each type were sequenced, clones derived from two known W-linked genes; SPIN-W and ATP5A1W, and from two hitherto unknown W-linked genes, represented by 2d-2D9 and 2d-2F9 clones, were identified and their localizations on the W chromosome were confirmed by fluorescence in-situ hybridization. The 2d-2D9 sequence has no significant homology with other genes in databases but 2d-2F9 has a region which shows partial homology to the consensus sequence of the AAA ATPase superfamily. Both 2d-2D9 and 2d-2F9 sequences are found in contigs of undetermined chromosome-linkage in the Draft Chicken Genome Sequence.

Sequence Analysis of Full-Length cDNA of Sex Chromosome-Linked Novel Gene 2d-2F9 in Gallus gallus

We obtained two novel W chromosome-linked chick genes by the use of female-male subtraction macroarrays, one of which, 2d-2F9, (recorded as AB188527 in DDBJ) did not have sufficient length (776 bp) to reveal its real form or characteristics. Hence, we obtained full-length Z-linked and W-linked 2d-2F9 genes of 2596 bp and 2589 bp respectively by the oligo-capping and RACE methods. Sequence analysis of these genes not only revealed that there is a counterpart of the W-linked 2d-2F9 gene on the Z chromosome, but also that there is a low homologous area at 5′-UTR between the W- and Z-kinked genes. Using this information, we designed a set of primers to identify sex and to select clones having the Z and W-linked gene (named 2d-2F9-Z and 2d-2F9-W), and also prepared two sets of primers for RT-PCR. These genes were found to be expressed constitutively and ubiquitously from the early embryo to the hatched chick, and they were assigned to the AAA ATP-superfamily.

The Male Hypermethylation (MHM) Region on the Chicken Z Chromosome: Female-Specific Transcription and its Biological Implication

The human DM-related transcript 1 (DMRT1) and DMRT2 genes are located at the distal region of chromosome 9p24.3 and suggested to be involved in the differentiation of testis in a dosage dependent manner, because XY individuals hemizygous for these genes exhibit a high frequency of XY feminization (Raymond et al., 1999a). In mouse embryos, the expression of DMRT1 gene is first detected by RT-PCR at E9.5, the earliest stage of genital ridge formation, and its expression in genital ridges and in early gonads, as detected by RT-PCR and in situ hybridization, continues until E14.5 in both male and female embryos. At E15.5, its expression in the female declines significantly and becomes testis-specific in adults (Raymond et al., 1999b). In chickens, a single DMRT1 gene is located on the short arm of the Z chromosome, expressed in genital ridges of both male and female embryos at as early as stage 19 (~3-day) (Raymond et al., 1999b) but later its expression becomes male (testis)-specific as in mammals (this study; Figure 2A,B). In early chicken embryos at stages 25–31 (4.5- to 7-day), the level of DMRT1 mRNA is about 2-fold higher in males than in females (Raymond et al., 1999b and this study), which might have been attained by the absence of a mechanism to shut off the transcription on one of the Z chromosomes in males (Kuroda et al., 2001). It has been speculated that the apparently dosage-dependent expression of the DMRT1 gene in early chicken embryos may have a role in the sex determination (Raymond et al., 1999b). The exclusive expression of the DMRT1 gene in adult testis both in mammals and birds may also suggest that its gene expression is required for some essential functions in the differentiated testis.