Comparison of the Z and W sex chromosomal architectures in elegant crested tinamou (Eudromia elegans) and ostrich (Struthio camelus) and the process of sex chromosome differentiation in palaeognathous birds
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To clarify the process of avian sex chromosome differentiation in palaeognathous birds, we performed molecular and cytogenetic characterization of W chromosome-specific repetitive DNA sequences for elegant crested tinamou (Eudromia elegans, Tinamiformes) and constructed comparative cytogenetic maps of the Z and W chromosomes with nine chicken Z-linked gene homologues for E. elegans and ostrich (Struthio camelus, Struthioniformes). A novel family of W-specific repetitive sequences isolated from E. elegans was found to be composed of guanine- and cytosine-rich 293-bp elements that were tandemly arrayed in the genome as satellite DNA. No nucleotide sequence homologies were found for the Struthioniformes and neognathous birds. The comparative cytogenetic maps of the Z and W chromosomes of E. elegans and S. camelus revealed that there are partial deletions in the proximal regions of the W chromosomes in the two species, and the W chromosome is more differentiated in E. elegans than in S. camelus. These results suggest that a deletion firstly occurred in the proximal region close to the centromere of the acrocentric proto-W chromosome and advanced toward the distal region. In E. elegans, the W-specific repeated sequence elements were amplified site-specifically after deletion of a large part of the W chromosome occurred.
Avian species are categorized into two large groups: the Palaeognathae [ratites and palaeognathous carinates (tinamous)] and the Neognathae (all other carinates). They are classified morphologically based on the palatal form, and this classification has been confirmed at the molecular level by DNA–DNA hybridization and nucleotide sequencing of the nuclear and mitochondrial ribosomal RNA genes (Sibley and Ahlquist 1990; van Tuinen et al. 1998, 2000). There are remarkable differences in the constitution of sex chromosomes between the two groups. Neognathous birds have highly differentiated W chromosomes that are comparatively smaller than the Z chromosome, highly heterochromatized and late replicating (Takagi 1972; Takagi and Sasaki 1974; Schmid et al. 1989). In contrast, the palaeognathous ratites (the Struthioniformes) retain the most primitive forms of avian sex chromosomes, which are largely homomorphic between the Z and W chromosomes (Takagi et al. 1972; de Boer 1980; Ansari et al. 1988) since palaeognathous birds and neognathous birds diverged about 120 million years ago (MYA) (van Tuinen and Hedges 2001). Comparative chromosome painting with the chicken Z chromosome-specific DNA revealed that the extensive homology between the Z and W chromosomes is also preserved on a molecular basis in emu (Dromaius novaehollandiae) (Shetty et al. 1999). The homology was also confirmed by comparative mapping of the sex chromosomes of emu, ostrich and double-wattled cassowary, which demonstrated that the Z and W chromosomes are homomorphic except for some marginally differentiated regions (Ogawa et al. 1998; Nishida-Umehara et al. 1999; Shetty et al. 2002). These data suggest that the W chromosomes of the Struthioniformes are hardly differentiated molecularly and still retain much Z homology.
Tinamous are classified as palaeognathous carinates and are phylogenetically positioned as a sister group to the ratites (van Tuinen et al. 1998, 2000; Cracraft 2001). One half to two thirds of the W chromosomes consist of heterochromatin in elegant crested tinamou (Eudromia elegans) (Sasaki et al. 1980), red-winged tinamou (Rhynchotus rufescens) and spotted tinamou (Nothura maculosa) (Pigozzi and Solari 1999, 2005). Therefore, the W chromosomes of the Tinamiformes are considered to be at an intermediate stage in heterochromatization between the largely euchromatic W chromosomes of the palaeognathous ratites and the highly heterochromatic W chromosomes of neognathous birds. This has been confirmed by cytogenetic studies of meiotic chromosome pairing. In R. rufescens and N. maculosa, the recombination nodules on the Z and W chromosomal pair are distributed in much longer regions than in neognathous birds but are restricted to shorter segments than those of the two rhea species, Pterocnemia pennata and Rhea americana (Pigozzi and Solari 1997, 1999, 2005). However, W-heterochromatin has not been molecularly cloned from any Tinamiformes species, and there is little information about comparative chromosome mapping in these taxa either.
Several female-specific repetitive DNA sequences have been cloned for some neognathous birds: the XhoI family, EcoRI family and SspI family of chicken (Gallus gallus) (Tone et al. 1982, 1984; Kodama et al. 1987; Saitoh et al. 1991; Saitoh and Mizuno 1992; Itoh and Mizuno 2002), the PstI family of turkey (Meleagris gallopavo), the TaqI family of Japanese common pheasant (Phasianus versicolor) (Saitoh et al. 1989) and LfW-1 of lesser black-backed gull (Larus fuscus) (Griffiths and Holland 1990). Some of them are major components of the W-heterochromatin, and their nucleotide sequences are highly diverged between different species as rapidly evolved molecules. We recently cloned a novel family of repetitive sequences from Galliformes species, which is an interspersed-type repetitive sequence amplified site-specifically on the W chromosome (Yamada et al. 2006). This family of repetitive sequences is highly conserved in neognathous birds but not in palaeognathous birds. All these results collectively suggest that the W-heterochromatin of E. elegans is composed of other types of repetitive sequences whose origins are different from those of the known W-specific repetitive sequences.
To define the sex chromosomal architecture of palaeognathous birds and to elucidate the process of avian sex chromosome differentiation, we first molecularly cloned a novel family of W-specific repetitive sequences from E. elegans and characterized them by nucleotide sequencing and chromosomal and filter hybridization. Secondly, we cloned eight and nine homologues of chicken Z-linked genes from S. camelus and E. elegans, respectively, and localized them to the Z and W chromosomes of these two species. Finally, we discuss the process of sex chromosome differentiation that occurred in palaeognathous birds.
Materials and methods
Specimens, cell cultures and chromosome preparation
The lymphocyte cells prepared from the blood of one male and female each of E. elegans were cultured in RPMI 1640 Medium supplemented with 18% fetal bovine serum, 3 μg/ml concanavalin A (Sigma), 10 μg/ml lipopolysaccharide (Sigma), 90 μg/ml phytohaemagglutinin (HA15, Murex) and 5 × 10−5 M mercaptoethanol. Cell cultures were incubated at 39°C in a humidified atmosphere of 5% CO2 in air. Bromodeoxyuridine (BrdU; 25 μg/ml) was added to the culture 48 h later, and cell culturing was continued for an additional 5 h including 1 h of colcemid treatment (0.025 μg/ml) before harvesting. The cells were collected, suspended in 0.075 M KCl and fixed with 3:1 methanol/glacial acetic acid following a standard protocol. The cell suspension was dropped on glass slides and air-dried. After staining of the chromosome slides with Hoechst 33258 (1 μg/ml) for 5 min, R-bands were obtained by heating the slides for 5 min at 65°C and exposing them to UV light at 65°C for an additional 3 min (Matsuda and Chapman 1995). The fibroblast cells prepared from skin tissues of one male and female each of E. elegans and one female of Struthio camelus were cultured in 199 Medium supplemented with 18% fetal bovine serum under the same conditions used for the lymphocyte cell cultures. BrdU (25 μg/ml) was added at log phase, and the cell culturing was continued for an additional 5 h. The cells were harvested after 30 min of colcemid treatment (0.025 μg/ml) and fixed in the same way as in the case of lymphocyte cell cultures, and chromosome preparations were made. For C-banding analysis, the chromosome slides were prepared from fibroblast cells cultured without BrdU treatment.
To examine the chromosomal distribution of constitutive heterochromatin in male and female E. elegans, chromosome C-banding was made with the BSG (barium hydroxide/saline/Giemsa) method (Summer 1972) with slight modification.
Molecular cloning of female-specific repetitive DNA sequences
High molecular weight genomic DNA was extracted from the blood of one male and female each of E. elegans. The genomic DNA was digested with 23 restriction endonucleases: AluI, ApaI, BglII, BamHI, BstXI, DdeI, EcoRI, EcoRV, HaeIII, HapII, HindIII, HinfI, MboI, NotI, PstI, PvuII, SacI, SalI, SmaI, SphI, TaqI, XbaI and XhoI and fractionated by electrophoresis with 1 and 3% agarose gels and stained with ethidium bromide. Female-specific DNA bands were isolated from the gel, and the DNA fragments were eluted using a QIAquick Gel Extraction Kit (Qiagen) and ligated into pBluescript II SK (+) and transformed into competent Escherichia coli JM109 cells (Takara Bio). The sizes of the DNA fragments inserted in the vector were confirmed by electrophoresis of the polymerase chain reaction (PCR) products that were amplified with T3 and T7 primers, and the clones were used for fluorescence in situ hybridization (FISH).
Nucleotide sequences were determined using an ABI PRISM3100 DNA Analyzer (Applied Biosystems) after the sequencing reaction with a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems).
Southern blot hybridization
The genomic DNA digested with restriction endonucleases was fractionated on a 1% agarose gel by electrophoresis and transferred onto a Hybond N+ nylon membrane (Amersham Biosciences). The DNA fragment cloned from the female-specific DNA band was labeled with digoxigenin-11-dUTP (dUTP, deoxyuridine 5-triphosphate) using a PCR DIG Labeling Mix (Roche Diagnostics) and was hybridized to the membrane as a probe overnight at 42°C using DIG Easy Hyb (Roche Diagnostics). The membrane was washed sequentially at 42°C in 2 × saline sodium citrate (SSC), 1 × SSC, 0.5 × SSC and 0.1 × SSC for 15 min each. The chemiluminescent signals were detected with anti-digoxigenin-alkaline phosphatase (AP) Fab fragments and CDP-Star (Roche Diagnostics) and exposed to BioMax MS Autoradiography Film (Kodak).
To examine the nucleotide sequence divergence of the repetitive DNA sequences, slot-blot hybridization probed with a repeated sequence element was performed. Genomic DNA was extracted from blood samples collected from one male and female each of the following 11 species of five orders and used for slot-blot hybridization: elegant crested tinamou (E. elegans) of the Tinamiformes, emu (D. novaehollandiae), double-wattled cassowary (Casuarius casuarius), greater rhea (R. americana), lesser rhea (P. pennata) and ostrich (S. camelus) of the Struthioniformes, chicken (G. gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris) of the Galliformes, Siberian crane (Grus leucogeranus) of the Gruiformes and Blakiston’s fish owl (Ketupa blakistoni) of the Strigiformes. The DNA was denatured with NaOH and blotted onto a Hybond N+ nylon membrane using BIO-DOT SF blotting equipment (Bio-Rad Laboratories). The probe DNA was labeled with digoxigenin-11-dUTP using a PCR DIG Labeling Mix (Roche Diagnostics) and hybridized to the membrane overnight at 42°C using DIG Easy Hyb (Roche Diagnostics). Then the membrane was washed, and the chemiluminescent signals were detected using the same procedure as for Southern blot hybridization.
To estimate the amount of the repetitive DNA sequences in the genome, eight different concentrations of female genomic DNA and the repetitive sequences were prepared. Slot-blot analysis was performed using a DNA fragment of the repetitive sequence labeled with digoxigenin-11-dUTP as probe. The luminescent hybridization signals were measured using Bio-Profile Image Analysis Software (Vilber Lourmat), and the intensity of the signals was compared between the genomic DNA and the repetitive sequence.
Molecular cloning of chicken Z-linked gene homologues
Degenerate oligonucleotide primers used for cloning cDNA fragments of the chicken Z-linked gene homologues from S. camelus and E. elegans and temperatures at annealing step in PCR reaction
Forward primer (5′–3′)
Reverse primer (5′–3′)
Temperature at annealing step (°C)
Accession number of cDNA fragments
Fluorescence in situ hybridization
FISH analysis was performed for chromosomal localization of the repetitive DNA sequences and the functional genes as described previously by Matsuda and Chapman (1995). The DNA fragments of the repetitive DNA sequences were labeled with biotin-16-dUTP using a nick translation kit (Roche Diagnostics) and ethanol precipitated with salmon sperm DNA and E. coli transfer RNA. After hybridization, the slides were incubated with avidin-fluorescein (Roche Diagnostics) and stained with 0.75 μg/ml propidium iodide (PI).
For chromosome mapping of the chicken Z-linked gene homologues, multiple cDNA fragments isolated for each gene were mixed and used as probes (Table 1). The cDNA fragments of GHR and ATP5A1 of S. camelus and ATP5A1 of E. elegans were labeled with CyDye 3-dUTP (Amersham Biosciences) using a nick translation kit, and other cDNA fragments were labeled with biotin-16-dUTP. After hybridization with the cDNA fragments labeled with biotin-16-dUTP, the probes were reacted with goat anti-biotin antibody (Vector Laboratories) and then stained with Alexa Fluor 488 rabbit anti-goat IgG (H + L) conjugate (Molecular Probes). The chromosome slides were counterstained with 0.75 μg/ml PI. The flourescein isothiocyanate (FITC) signals of the repetitive sequences and Cy3 fluorescence signals of cDNA fragments were captured using a cooled CCD camera (MicroMAX 782Y, Princeton Instruments) mounted on a Leica DMRA microscope and were analysed with the 550CW-QFISH application program of Leica Microsystems Imaging Solution (Cambridge, UK). The Alexa signals were observed under a Nikon fluorescence microscope using Nikon filter sets B-2A and UV-2A. DYNA HG ASA 100 films (Kodak) were used for microphotography.
C-banded karyotype of E. elegans
Molecular cloning of female-specific repetitive DNA sequences
Chromosomal location of female-specific repetitive DNA sequences
Nucleotide sequences of W-specific repeated sequence elements
Genomic organization of W-specific repetitive sequences
Nucleotide sequence conservation of W-specific repetitive sequences
Chromosomal locations of chicken Z-linked gene homologues in S. camelus and E. elegans
The length of cDNA fragments of the chicken Z-linked gene homologues cloned from S. camelus and E. elegans
The length of cDNA fragments (bp) and accession number
ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle
Chromodomain helicase DNA binding protein 1
1,771a (AB254867, 254868)
1,727a (AB254883, 254884)
Growth hormone receptor
Neurotrophic tyrosine kinase receptor, type 2
806a (AB254872, 254873)
905a (AB254888, 254889)
Protein kinase C inhibitor
498a (AB254874, 254875)
492a (AB254890, 254891)
Ribosomal protein S6
786a (AB254876, 254877)
747a (AB254892, 254893)
903a (AB254895, 254896)
Soluble aconitase 1/iron-responsive element binding protein
1,145a (AB254897, 254898)
Nucleotide sequence similarities of Z-linked genes among chicken, S. camelus and E. elegans
Nucleotide sequence identities of cDNA fragments of nine Z-linked genes among G. gallus (GGA), S. camelus (SCA) and E. elegans (EEL)
Comparison of nucleotide sequences between the Z and W forms of the CHD1 gene in E. elegans
In this paper, a novel BamHI family of female-specific repetitive DNA sequences was molecularly cloned from E. elegans. The hybridization signals of the sequences were localized to the proximal region of the long arm of the W chromosome, which accounts for about two thirds of the C-positive heterochromatin region, suggesting that the repeated sequence family is a major component of the W chromosome heterochromatin of E. elegans. The W-specific repetitive sequences are composed of GC-rich 291- to 293-bp elements and organized in tandem arrays as satellite DNA (stDNA). Internal restriction sites of BglII and BstXI are present in almost all of the BamHI repeated sequence fragments. Southern blot hybridization probed with the BamHI repeated sequence element revealed that the sizes and the polymeric ladder patterns of hybridized bands in the BamHI digest were completely the same as those in the BglII and BstXI digests. These results clearly indicate that the BamHI repeated sequence is the same as the BglII and BstXI sequence families. The repeated sequence family was not found in the ZZ male of E. elegans, other palaeognathous ratites or neognathous birds. The novel stDNA sequences might have been amplified on the W chromosome independently in the Tinamiformes lineage after the Struthioniformes and the Tinamiformes diverged from the common ancestor of palaeognathous birds. The hypermethylation status of the W-specific stDNA suggests that the repetitive sequence has some roles in chromatin organization of the W chromosome in interphase nuclei, and replication timing at S phase and chromosome condensation at metaphase.
The Z- and W-chromosomal forms of the CHD1 gene, CHD1Z and CHD1W, are present in neognathous birds, and the presence of the sex-specific forms facilitates the use of this gene as a molecular marker for sexing (Ellegren 1996; Griffiths et al. 1996, 1998; Fridolfsson and Ellegren 1999). The presence of the Z- and W-chromosomal forms has been reported for five other “gametologous” genes (ATP5A1, UBAP2, SPIN and HINT) in neognathous birds: These are relic genes shared between homologous sex chromosomes as a result of the cessation of recombination (García-Moreno and Mindell 2000; de Kloet and de Kloet 2003; Handley et al. 2004). No W-specific forms of these genes have been reported in palaeognathous ratite birds having the extensively homomorphic Z and W chromosomes. We found sexual dimorphism of the CHD1 gene in E. elegans in this study, indicating that recombination is suppressed around the CHD1 locus between the Z and W chromosomes in E. elegans, leading to the nucleotide sequence divergence between the CHD1Z and CHD1W genes. These data strongly suggest that the sex chromosome differentiation occurred around the CHD1 locus independently in the Tinamiformes lineage after the divergence of the Palaeognathae and the Neognathae. The Z- and W-chromosomal forms have also been reported for the SPIN gene in four Tinamiformes species, including E. elegans (de Kloet 2002; de Kloet and de Kloet 2003). In E. elegans, the SPIN locus is contained in the W-specific heterochromatin region, and chromosomal recombination may be suppressed around this locus. However, no sex-specific products of the SPIN gene were obtained in this study.
Handley et al. (2004) proposed that there were at least two strata in the process of avian sex chromosome differentiation: recombination between Z and W chromosomes initially ceased around the small region partially differentiated from the ancestral homomorphic sex chromosome in the oldest stratum 102–170 MYA, before the split of Neoaves and Eoaves. The disruption of chromosome recombination in the second stratum occurred independently in the different lineages 58–85 MYA, at the time of the major radiation of the existing neognathous birds. Comparative FISH mapping of chicken Z-linked genes to the Z and W chromosomes of S. camelus and E. elegans showed two chromosomal hybridization patterns of the genes: (1) hybridization signals were located on both the Z and W chromosomes, and (2) hybridization signals were detected on the Z chromosome but not on the W chromosome. Two possible evolutionary events might be responsible for the absence of hybridization signals on the W chromosome: (1) deletion of the chromosomal segment that contains the W homologues of the Z-linked genes; and (2) a decrease in hybridization efficiency due to divergence in the nucleotide sequence between the Z- and W-linked genes due to the suppression of recombination. Hybridization signals of TMOD and ACO1/IREBP, which are located near the centromere on the long arm of the Z chromosome, were not detected on the W chromosomes in these two species. In S. camelus, six other genes were localized to both the Z and W chromosomes, suggesting that differentiation due to a small deletion occurred in the proximal region of the W chromosome, as reported by Ogawa et al. (1998). In E. elegans, hybridization signals of RPS6, NTRK2 and PKCI were detected on the Z chromosome but not on the W chromosome. This is indicative of a difference in the state of sex chromosome differentiation between E. elegans and S. camelus, whose lineages diverged around 83 MYA (van Tuinen and Hedges 2001). Considering that the proximal half of the W chromosome of E. elegans is composed of heterochromatin, and that the W chromosome is morphologically shorter than that of S. camelus, the absence of hybridization signals of RPS6, NTRK2 and PKCI may be due to the deletion of about the proximal half of the euchromatic long arm of the ancestral W chromosome, and E. elegans is at an advanced stage of sex chromosome differentiation in comparison with S. camelus.
Multiple copies of the Z-linked PKCI gene are located on the W chromosome of G. gallus, and their nucleotide sequences are divergent from that of the Z homologue. The WPKCI/ASW gene is highly expressed in the undifferentiated gonads of female chicken embryos (O’Neill et al. 2000; Hori et al. 2000), and it is consequently supposed that this gene has an important role in sex determination followed by gonadal differentiation. In palaeognathous ratites, no W-specific forms of the PKCI gene have been found, and this leads us to predict that the female-specific function of this gene was acquired in the lineage of neognathous birds along with the differentiation of sex chromosomes. The PKCI gene was localized to the same location between the Z and W chromosomes of S. camelus with the same hybridization efficiency in this study, suggesting that the genetic divergence might not have occurred between the Z- and W-linked PKCI in S. camelus (O’Neill et al. 2000). The presence of the W homologue of PKCI was not confirmed in E. elegans either.
The hybridization signals of SPIN were detected on both the Z and W chromosomes in S. camelus and E. elegans, but the locations of the genes on the W chromosomes were different between the two species. The W homologue of SPIN was located around the centre of the long arm in S. camelus, whereas it was mapped to the heterochromatin region near the centromere of the W chromosome in E. elegans. The CHD1 gene of E. elegans was located just distal to the SPIN locus on the Z chromosome, whereas it was localized far from the SPIN locus, located near the centromere, on the W chromosome. The difference in the chromosomal location of SPIN and CHD1 between the Z and W chromosomes in E. elegans suggests two possibilities: One is that the SPIN gene on the W chromosome translocated near the centromere via a paracentric inversion. The other is that the SPIN and CHD1 loci on the W chromosome were kept apart by the BamHI family of W-specific repetitive sequences amplified between the two loci.
An early stage of W chromosome differentiation from the proto-sex chromosomes was clearly demonstrated in S. camelus in this study. This species retains a partially differentiated type of W chromosome in which the chromosomal deletion occurred from a region proximal to the centromere to a locus proximal to RPS6–NTRK2–PKCI. In E. elegans, the deletion occurred in a wider chromosomal region than in S. camelus. Chromosomal deletion advanced from a region near the centromere toward a distal region in E. elegans, and the deleted region consequently extended from the centromere to a locus proximal to SPIN. The absence of recombination in the deleted chromosome region accelerated the site-specific amplification of the W-specific EEL–BamHI repeated sequence due to the absence of recombination subsequently occurring between the SPIN locus and the CHD1 locus. However, the euchromatic region has been preserved between the CHD1 locus and the distal end, and therefore the W chromosome differentiation in E. elegans is at a transitional stage between that in S. camelus, which has a partially deleted W chromosome, and neognathous birds, which have highly degenerated and heterochromatic W chromosomes. The number of genes localized to the sex chromosomes in the two species is still small to confirm this scenario of avian sex chromosome differentiation, and there is no information on the divergence of nucleotide sequences between Z- and W-linked genes except for the SPIN and CHD1 genes of E. elegans. Further investigations will be needed to fully define the process of avian sex chromosome differentiation.
We express our appreciation to Yokohama Zoological Gardens, Yokohama, for providing skin and blood samples of elegant crested tinamou, and Kimiyuki Tsuchiya for skin samples of ostrich. This work was supported by Grants-in-Aid for Scientific Research (nos. 15370001 and 16086201) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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