Enkhjargal Tsend-Ayush

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.

Platypus Pou5f1 reveals the first steps in the evolution of trophectoderm differentiation and pluripotency in mammals

SUMMARY Uterine nourishment of embryos by the placenta is a key feature of mammals. Although a variety of placenta types exist, they are all derived from the trophectoderm (TE) cell layer of the developing embryo. Egg‐laying mammals (platypus and echidnas) are distinguished by a very short intrauterine embryo development, in which a simple placenta forms from TE‐like cells. The Pou5f1 gene encodes a class V POU family transcription factor Oct3/4. In mice, Oct3/4 together with the highly conserved caudal‐related homeobox transcription factor Cdx2, determines TE fate in pre‐implantation development. In contrast to Cdx2, Pou5f1 has only been identified in eutherian mammals and marsupials, whereas, in other vertebrates, pou2 is considered to be the Pou5f1 ortholog. Here, we show that platypus and opossum genomes contain a Pou5f1 and pou2 homolog, pou2‐related, indicating that these two genes are paralogues and arose by gene duplication in early mammalian evolution. In a complementation assay, we found that platypus or human Pou5f1, but not opossum or zebrafish pou2, restores self‐renewal in Pou5f1‐null mouse ES cells, showing that platypus possess a fully functional Pou5f1 gene. Interestingly, we discovered that parts of one of the conserved regions (CR4) is missing from the platypus Pou5f1 promoter, suggesting that the autoregulation and reciprocal inhibition between Pou5f1 and Cdx2 evolved after the divergence of monotremes and may be linked to the development of more elaborate placental types in marsupial and eutherian mammals.

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes.

BackgroundThe monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.ResultsWe characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.ConclusionMolecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.

RBMX Gene Is Essential for Brain Development in Zebrafish

The human RBMX gene was discovered recently through its homology to the spermatogenesis candidate gene RBMY. Its position on the human X chromosome suggests that it may be involved in X‐linked mental retardation syndromes. However, to date there is scant information on the in vivo role of RBMX. To address this issue, we have isolated a zebrafish rbmx orthologue and characterized its embryonic expression pattern. Zebrafish rbmx is maternally expressed and then widely expressed in the embryo up to 24 hr postfertilization. In later stages of embryonic development, rbmx transcripts are localized predominantly in the brain, branchial arches, and liver primordium. The function of rbmx during embryonic development was examined by the use of an antisense morpholino targeting rbmx. The rbmx‐morphants displayed an underdeveloped head and eyes, reduced body size, defective somite patterning, and absence of jaws. Furthermore, in the absence of functional rbmx, expression of specific markers for the fore‐ and hindbrain (otx2, krox20) was severely reduced. These studies demonstrate for the first time that rbmx is required for normal embryonic development, in particular of the brain, consistent with a role in X‐linked mental retardation. Developmental Dynamics 234:682–688, 2005.

Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution

In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently.

Conservation and Expression of PIWI-Interacting RNA Pathway Genes in Male and Female Adult Gonad of Amniotes

ABSTRACT The PIWI-interacting RNA (piRNA) pathway is essential for germline development and transposable element repression. Key elements of this pathway are members of the piRNA-binding PIWI/Argonaute protein family and associated factors (e.g., VASA, MAELSTROM, and TUDOR domain proteins). PIWI-interacting RNAs have been identified in mouse testis and oocytes, but information about the expression of the different piRNA pathway genes, in particular in the mammalian ovary, remains incomplete. We investigated the evolution and expression of piRNA pathway genes in gonads of amniote species (chicken, platypus, and mouse). Database searches confirm a high level of conservation and revealed lineage-specific gain and loss of Piwi genes in vertebrates. Expression analysis in mammals shows that orthologs of Piwi-like (Piwil) genes, Mael (Maelstrom), Mvh (mouse vasa homolog), and Tdrd1 (Tudor domain-containing protein 1) are expressed in platypus adult testis. In contrast to mouse, Piwil4 is expressed in platypus and human adult testis. We found evidence for Mael and Piwil2 expression in mouse Sertoli cells. Importantly, we show mRNA expression of Piwil2, Piwil4, and Mael in oocytes and supporting cells of human, mouse, and platypus ovary. We found no Piwil1 expression in mouse and chicken ovary. The conservation of gene expression in somatic parts of the gonad and germ cells of species that diverged over 800 million yr ago indicates an important role in adult male and female gonad.

Analysis of SINE and LINE repeat content of Y chromosomes in the platypus, Ornithorhynchus anatinus

Monotremes feature an extraordinary sex-chromosome system that consists of five X and five Y chromosomes in males. These sex chromosomes share homology with bird sex chromosomes but no homology with the therian X. The genome of a female platypus was recently completed, providing unique insights into sequence and gene content of autosomes and X chromosomes, but no Y-specific sequence has so far been analysed. Here we report the isolation, sequencing and analysis of approximately 700 kb of sequence of the non-recombining regions of Y2, Y3 and Y5, which revealed differences in base composition and repeat content between autosomes and sex chromosomes, and within the sex chromosomes themselves. This provides the first insights into repeat content of Y chromosomes in platypus, which overall show similar patterns of repeat composition to Y chromosomes in other species. Interestingly, we also observed differences between the various Y chromosomes, and in combination with timing and activity patterns we provide an approach that can be used to examine the evolutionary history of the platypus sex-chromosome chain.

Genomic structure and paralogous regions of the inversion breakpoint occurring between human chromosome 3p12.3 and orangutan chromosome 2

Intrachromosomal duplications play a significant role in human genome pathology and evolution. To better understand the molecular basis of evolutionary chromosome rearrangements, we performed molecular cytogenetic and sequence analyses of the breakpoint region that distinguishes human chromosome 3p12.3 and orangutan chromosome 2. FISH with region-specific BAC clones demonstrated that the breakpoint-flanking sequences are duplicated intrachromosomally on orangutan 2 and human 3q21 as well as at many pericentromeric and subtelomeric sites throughout the genomes. Breakage and rearrangement of the human 3p12.3-homologous region in the orangutan lineage were associated with a partial loss of duplicated sequences in the breakpoint region. Consistent with our FISH mapping results, computational analysis of the human chromosome 3 genomic sequence revealed three 3p12.3-paralogous sequence blocks on human chromosome 3q21 and smaller blocks on the short arm end 3p26→p25. This is consistent with the view that sequences from an ancestral site at 3q21 were duplicated at 3p12.3 in a common ancestor of orangutan and humans. Our results show that evolutionary chromosome rearrangements are associated with microduplications and microdeletions, contributing to the DNA differences between closely related species.

Characterisation of ATRX, DMRT1, DMRT7 and WT1 in the platypus (Ornithorhynchus anatinus)

One of the most puzzling aspects of monotreme reproductive biology is how they determine sex in the absence of the SRY gene that triggers testis development in most other mammals. Although monotremes share a XX female/XY male sex chromosome system with other mammals, their sex chromosomes show homology to the chicken Z chromosome, including the DMRT1 gene, which is a dosage-dependent sex determination gene in birds. In addition, monotremes feature an extraordinary multiple sex chromosome system. However, no sex determination gene has been identified as yet on any of the five X or five Y chromosomes and there is very little knowledge about the conservation and function of other known genes in the monotreme sex determination and differentiation pathway. We have analysed the expression pattern of four evolutionarily conserved genes that are important at different stages of sexual development in therian mammals. DMRT1 is a conserved sex-determination gene that is upregulated in the male developing gonad in vertebrates, while DMRT7 is a mammal-specific spermatogenesis gene. ATRX, a chromatin remodelling protein, lies on the therian X but there is a testis-expressed Y-copy in marsupials. However, in monotremes, the ATRX orthologue is autosomal. WT1 is an evolutionarily conserved gene essential for early gonadal formation in both sexes and later in testis development. We show that these four genes in the adult platypus have the same expression pattern as in other mammals, suggesting that they have a conserved role in sexual development independent of genomic location.