Willem Rens

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.

In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes

Two centuries after the duck-billed platypus was discovered, monotreme chromosome systems remain deeply puzzling. Karyotypes of males, or of both sexes, were claimed to contain several unpaired chromosomes (including the X chromosome) that form a multi-chromosomal chain at meiosis. Such meiotic chains exist in plants and insects but are rare in vertebrates. How the platypus chromosome system works to determine sex and produce balanced gametes has been controversial for decades. Here we demonstrate that platypus have five male-specific chromosomes (Y chromosomes) and five chromosomes present in one copy in males and two copies in females (X chromosomes). These ten chromosomes form a multivalent chain at male meiosis, adopting an alternating pattern to segregate into XXXXX-bearing and YYYYY-bearing sperm. Which, if any, of these sex chromosomes bears one or more sex-determining genes remains unknown. The largest X chromosome, with homology to the human X chromosome, lies at one end of the chain, and a chromosome with homology to the bird Z chromosome lies near the other end. This suggests an evolutionary link between mammal and bird sex chromosome systems, which were previously thought to have evolved independently.

The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z

BackgroundSex-determining systems have evolved independently in vertebrates. Placental mammals and marsupials have an XY system, birds have a ZW system. Reptiles and amphibians have different systems, including temperature-dependent sex determination, and XY and ZW systems that differ in origin from birds and placental mammals. Monotremes diverged early in mammalian evolution, just after the mammalian clade diverged from the sauropsid clade. Our previous studies showed that male platypus has five X and five Y chromosomes, no SRY, and DMRT1 on an X chromosome. In order to investigate monotreme sex chromosome evolution, we performed a comparative study of platypus and echidna by chromosome painting and comparative gene mapping.ResultsChromosome painting reveals a meiotic chain of nine sex chromosomes in the male echidna and establishes their order in the chain. Two of those differ from those in the platypus, three of the platypus sex chromosomes differ from those of the echidna and the order of several chromosomes is rearranged. Comparative gene mapping shows that, in addition to bird autosome regions, regions of bird Z chromosomes are homologous to regions in four platypus X chromosomes, that is, X1, X2, X3, X5, and in chromosome Y1.ConclusionMonotreme sex chromosomes are easiest to explain on the hypothesis that autosomes were added sequentially to the translocation chain, with the final additions after platypus and echidna divergence. Genome sequencing and contig anchoring show no homology yet between platypus and therian Xs; thus, monotremes have a unique XY sex chromosome system that shares some homology with the avian Z.

Complete homology maps of the rabbit (Oryctolagus cuniculus) and human by reciprocal chromosome painting

Fluorescence in situ hybridization (FISH) was used to construct a homology map to analyse the extent of evolutionary conservation of chromosome segments between human and rabbit (Oryctolagus cuniculus, 2n = 44). Chromosome-specific probes were established by bivariate fluorescence activated flow sorting followed by degenerate oligonucleotide-primed PCR (DOP-PCR). Painting of rabbit probes to human chromosomes and vice versa allowed a detailed analysis of the homology between these species. All rabbit chromosome paints, except for the Y paint, hybridized to human chromosomes. All human chromosome paints, except for the Y paint, hybridized to rabbit chromosomes. The results obtained revealed extensive genome conservation between the two species. Rabbit chromosomes 12, 19 and X were found to be completely homologous to human chromosomes 6, 17 and X, respectively. All other human chromosomes were homologous to two or sometimes three rabbit chromosomes. Many conserved chromosome segments found previously in other mammals (e.g. cat, pig, cattle, Indian muntjac) were also found to be conserved in rabbit chromosomes.

Genomic Instability within Centromeres of Interspecific Marsupial Hybrids

Several lines of evidence suggest that, within a lineage, particular genomic regions are subject to instability that can lead to specific types of chromosome rearrangements important in species incompatibility. Within family Macropodidae (kangaroos, wallabies, bettongs, and potoroos), which exhibit recent and extensive karyotypic evolution, rearrangements involve chiefly the centromere. We propose that centromeres are the primary target for destabilization in cases of genomic instability, such as interspecific hybridization, and participate in the formation of novel chromosome rearrangements. Here we use standard cytological staining, cross-species chromosome painting, DNA probe analyses, and scanning electron microscopy to examine four interspecific macropodid hybrids (Macropus rufogriseus × Macropus agilis). The parental complements share the same centric fusions relative to the presumed macropodid ancestral karyotype, but can be differentiated on the basis of heterochromatic content, M. rufogriseus having larger centromeres with large C-banding positive regions. All hybrids exhibited the same pattern of chromosomal instability and remodeling specifically within the centromeres derived from the maternal (M. rufogriseus) complement. This instability included amplification of a satellite repeat and a transposable element, changes in chromatin structure, and de novo whole-arm rearrangements. We discuss possible reasons and mechanisms for the centromeric instability and remodeling observed in all four macropodid hybrids.

Karyotype relationships between four distantly related marsupials revealed by reciprocal chromosome painting.

Marsupial karyotypes have shown extensive conservation even between distantly related groups with a high diversity of life forms and reproductive biology. Banding analysis has been the main test for assessing their homologies and chromosome rearrangements. More recently, cross-species reciprocal chromosome painting has been developed and applied to several mammalian species and has shown homologies and rearrangements not revealed by banding analysis. Karyotype relationships between four marsupial species, Sminthopsis crassicaudata, Potorous tridactylus, Trichosurus vulpecula and Macropus eugenii, which are from different families in two orders, were investigated and presented in the form of comparative chromosome maps. These show that only a limited number of chromosomal rearrangements have occurred during their evolution. A karyotype phylogeny of the four marsupials was derived from these maps. A comparison between published gene location and the comparative chromosome maps for these species is presented and inconsistencies with previous gene mapping data indicated.

Reversal and convergence in marsupial chromosome evolution

The karyotypes of marsupial species are characterized by their relatively low number of chromosomes, and their conservation. Most species have diploid numbers lying between the two modes, 2n = 14 and 2n = 22, but the karyotype of Aepyprymnus rufescens is exceptional in containing 2n = 32 chromosomes. Many differences in diploid number between marsupial species can be accounted for by particular fissions and fusions, which are easy to detect because of the low numbers of chromosomes in each karyotype. This should be a system in which it is possible to detect reversals and repeated chromosome rearrangements. We have used chromosome-specific paints derived from A. rufescens to compare the karyotypes of eight marsupial species, representing closely and distantly related taxa, to trace chromosome change during evolution, and especially to detect reversals and convergence. From these and other painting comparisons, we conclude that there have been at least three reversals of fusions by fissions, and at least three fusions or fissions that have occurred independently in different lineages.

Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of a Transmissible Tumour

Devil facial tumour disease (DFTD) is a fatal, transmissible malignancy that threatens the worlds largest marsupial carnivore, the Tasmanian devil, with extinction. First recognised in 1996, DFTD has had a catastrophic effect on wild devil numbers, and intense research efforts to understand and contain the disease have since demonstrated that the tumour is a clonal cell line transmitted by allograft. We used chromosome painting and gene mapping to deconstruct the DFTD karyotype and determine the chromosome and gene rearrangements involved in carcinogenesis. Chromosome painting on three different DFTD tumour strains determined the origins of marker chromosomes and provided a general overview of the rearrangement in DFTD karyotypes. Mapping of 105 BAC clones by fluorescence in situ hybridisation provided a finer level of resolution of genome rearrangements in DFTD strains. Our findings demonstrate that only limited regions of the genome, mainly chromosomes 1 and X, are rearranged in DFTD. Regions rearranged in DFTD are also highly rearranged between different marsupials. Differences between strains are limited, reflecting the unusually stable nature of DFTD. Finally, our detailed maps of both the devil and tumour karyotypes provide a physical framework for future genomic investigations into DFTD.

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence

The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.

Epigenetic modifications on X chromosomes in marsupial and monotreme mammals and implications for evolution of dosage compensation

X chromosome dosage compensation in female eutherian mammals is regulated by the noncoding Xist RNA and is associated with the differential acquisition of active and repressive histone modifications, resulting in repression of most genes on one of the two X chromosome homologs. Marsupial mammals exhibit dosage compensation; however, they lack Xist, and the mechanisms conferring epigenetic control of X chromosome dosage compensation remain elusive. Oviparous mammals, the monotremes, have multiple X chromosomes, and it is not clear whether they undergo dosage compensation and whether there is epigenetic dimorphism between homologous pairs in female monotremes. Here, using antibodies against DNA methylation, eight different histone modifications, and HP1, we conduct immunofluorescence on somatic cells of the female Australian marsupial possum Trichosurus vulpecula, the female platypus Ornithorhynchus anatinus, and control mouse cells. The two marsupial Xs were different for all epigenetic features tested. In particular, unlike in the mouse, both repressive modifications, H3K9me3 and H4K20Me3, are enriched on one of the X chromosomes, and this is associated with the presence of HP1 and hypomethylation of DNA. Using sequential labeling, we determine that this DNA hypomethylated X correlates with histone marks of inactivity. These results suggest that female marsupials use a repressive histone-mediated inactivation mechanism and that this may represent an ancestral dosage compensation process that differs from eutherians that require Xist transcription and DNA methylation. In comparison to the marsupial, the monotreme exhibited no epigenetic differences between homologous X chromosomes, suggesting the absence of a dosage compensation process comparable to that in therians.