The evolution of marsupial and monotreme chromosomes.

Marsupial and monotreme mammals fill an important gap in vertebrate phylogeny between reptile-mammal divergence 310 million years ago (mya) and the eutherian (placental) mammal radiation 105 mya. They possess many unique features including their distinctive chromosomes, which in marsupials are typically very large and well conserved between species. In contrast, monotreme genomes are divided into several large chromosomes and many smaller chromosomes, with a complicated sex chromosome system that forms a translocation chain in male meiosis. The application of molecular cytogenetic techniques has greatly advanced our understanding of the evolution of marsupial chromosomes and allowed the reconstruction of the ancestral marsupial karyotype. Chromosome painting and gene mapping have played a vital role in piecing together the puzzle of monotreme karyotypes, particularly their complicated sex chromosome system. Here, we discuss the significant insight into karyotype evolution afforded by the combination of recently sequenced marsupial and monotreme genomes with cytogenetic analysis, which has provided a greater understanding of the events that have shaped not only marsupial and monotreme genomes, but the genomes of all mammals.

The origin and evolution of vertebrate sex chromosomes and dosage compensation.

In mammals, birds, snakes and many lizards and fish, sex is determined genetically (either male XY heterogamy or female ZW heterogamy), whereas in alligators, and in many reptiles and turtles, the temperature at which eggs are incubated determines sex. Evidently, different sex-determining systems (and sex chromosome pairs) have evolved independently in different vertebrate lineages. Homology shared by Xs and Ys (and Zs and Ws) within species demonstrates that differentiated sex chromosomes were once homologous, and that the sex-specific non-recombining Y (or W) was progressively degraded. Consequently, genes are left in single copy in the heterogametic sex, which results in an imbalance of the dosage of genes on the sex chromosomes between the sexes, and also relative to the autosomes. Dosage compensation has evolved in diverse species to compensate for these dose differences, with the stringency of compensation apparently differing greatly between lineages, perhaps reflecting the concentration of genes on the original autosome pair that required dosage compensation. We discuss the organization and evolution of amniote sex chromosomes, and hypothesize that dosage insensitivity might predispose an autosome to evolving function as a sex chromosome.

A new look at the evolution of avian sex chromosomes.

Birds have a ubiquitous, female heterogametic, ZW sex chromosome system. The current model suggests that the Z chromosome and its degraded partner, the W chromosome, evolved from an ancestral pair of autosomes independently from the mammalian XY male heteromorphic sex chromosomes--which are similar in size, but not gene content (Graves, 1995; Fridolfsson et al., 1998). Furthermore the degradation of the W has been proposed to be progressive, with the basal clade of birds (the ratites) possessing virtually homomorphic sex chromosomes and the more recently derived birds (the carinates) possessing highly heteromorphic sex chromosomes (Ohno, 1967; Solari, 1993). Recent findings have suggested an alternative to independent evolution of bird and mammal chromosomes, in which an XY system took over directly from an ancestral ZW system. Here we examine recent research into avian sex chromosomes and offer alternative suggestions as to their evolution.

Reassignment of chicken W chromosome sequences to the Z chromosome by fluorescence in situ hybridization (FISH).

There is much interest in the gene content of the small heterochromatic W chromosome of the chicken, on the supposition that it may contain sex-determining genes. A considerable region in the chicken genome has been assigned to the W chromosome on the basis of its repetitive sequences. Using fluorescent in situ hybridization (FISH) we localized five Bacterial Artificial Chromosomes (BACs) onto female chicken metaphase spreads. We physically mapped these BACs to the Z chromosome. The chicken genome database, however, assigned all five BACs to the W chromosome. Our results demonstrate that the 17 genes on these BACs are Z-specific, and points to the inadequacy of assigning regions of the genome based exclusively on repetitive sequences.

Class I genes have split from the MHC in the tammar wallaby.

Genes within the Major Histocompatibility Complex (MHC) are critical to the immune response and immunoregulation. Comparative studies have revealed that the MHC has undergone many changes throughout evolution yet in tetrapods the three different classes of MHC genes have maintained linkage, suggesting that there may be some functional advantage obtained by maintaining this clustering of MHC genes. Here we present data showing that class II and III genes, the antigen processing gene TAP2, and MHC framework genes are found together in the tammar wallaby on chromosome 2. Surprisingly class I loci were not found on chromosome 2 but were mapped to ten different locations spread across six chromosomes. This distribution of class I loci in the wallaby on nearly all autosomes is not a characteristic of all marsupials and may be a relatively recent phenomenon. It highlights the need for the inclusion of more than one marsupial species in comparative studies and raises questions regarding the functional significance of the clustering of MHC genes.

Mapping platypus SOX genes; autosomal location of SOX9 excludes it from sex determining role.

In the absence of an SRY orthologue the platypus sex determining gene is unknown, so genes in the human testis determining pathway are of particular interest as candidates. SOX9 is an attractive choice because SOX9 deletions cause male-to-female sex reversal in humans and mice, and SOX9 duplications cause female-to-male sex reversal. We have localized platypus SOX9, as well as the related SOX10, to platypus chromosomes 15 and 10, respectively, the first assignments to these platypus chromosomes, and the first comparative mapping markers from human chromosomes 17 and 22. The autosomal localization of platypus SOX9 in this study contradicts the hypothesis that SOX9 acts as the sex determining switch in platypus.

Characterization of two whey protein genes in the Australian dasyurid marsupial, the stripe-faced dunnart (Sminthopsis macroura).

We report the first isolation and sequencing of genomic BAC clones containing the marsupial milk protein genes Whey Acidic Protein (WAP) and Early Lactation Protein (ELP). The stripe-faced dunnart WAPgene sequence contained five exons, the middle three of which code for the WAPmotifs and four disulphide core domains which characterize WAP. The dunnart ELPgene sequence contained three exons encoding a protein with a Kunitz motif common to serine protease inhibitors. Fluorescence in situ hybridization located the WAPgene to chromosome 1p in the stripe-faced dunnart, and the ELPgene to 2q. Northern blot analysis of lactating mammary tissue of the closely related fat-tailed dunnart has shown asynchronous expression of these milk protein genes. ELPwas expressed at only the earlier phase of lactation and WAPonly at the later phase of lactation, in contrast to beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) genes, which were expressed in both phases of lactation. This asynchronous expression during the lactation cycle in the fat-tailed dunnart is similar to other marsupials and it probably represents a pattern that is ancestral to Australian marsupials.

Linkage mapping and physical localization of the major histocompatibility complex region of the marsupial Monodelphis domestica.

We used genetic linkage mapping and fluorescence in situ hybridization (FISH) to conduct the first analysis of genic organization and chromosome localization of the major histocompatibility complex (MHC) of a marsupial, the gray, short-tailed opossum Monodelphis domestica. Family based linkage analyses of two M. domestica MHC Class I genes (UA1, UG) and three MHC Class II genes (DAB, DMA, and DMB) revealed that these genes were tightly linked and positioned in the central region of linkage group 3 (LG3). This cluster of MHC genes was physically mapped to the centromeric region of chromosome 2q by FISH using a BAC clone containing the UA1 gene. An interesting finding from the linkage analyses is that sex-specific recombination rates were virtually identical within the MHC region. This stands in stark contrast to the genome-wide situation, wherein males exhibit approximately twice as much recombination as females, and could have evolutionary implications for maintaining equality between males and females in the ability to generate haplotype diversity in this region. These analyses also showed that three non-MHC genes that flank the MHC region on human chromosome 6, myelin oligodendrocyte glycoprotein (MOG), bone morphogenetic protein 6 (BMP6), and prolactin (PRL), are split among two separate linkage groups (chromosomes) in M. domestica. Comparative analysis with eight other vertebrate species suggests strong conservation of the BMP6-PRL synteny among birds and mammals, although the BMP6-PRL-MHC-ME1 synteny is not conserved.

Sequencing and mapping hemoglobin gene clusters in the Australian model dasyurid marsupial Sminthopsis macroura.

Comparing globin genes and their flanking sequences across many species has allowed globin gene evolution to be reconstructed in great detail. Marsupial globin sequences have proved to be of exceptional significance. A previous finding of a beta(beta)-like omega(omega) gene in the alpha(alpha) cluster in the tammar wallaby suggested that the alpha and beta cluster evolved via genome duplication and loss rather than tandem duplication. To confirm and extend this important finding we isolated and sequenced BACs containing the alpha and beta loci from the distantly related Australian marsupial Sminthopsis macroura. We report that the alpha gene lies in the same BAC as the beta-like omega gene, implying that the alpha-omega juxtaposition is likely to be conserved in all marsupials. The LUC7L gene was found 3' of the S. macroura alpha locus, a gene order shared with humans but not mouse, chicken or fugu. Sequencing a BAC contig that contained the S. macroura beta globin and epsilon globin loci showed that the globin cluster is flanked by olfactory genes, demonstrating a gene arrangement conserved for over 180 MY. Analysis of the region 5' to the S. macroura epsilon (epsilon) globin gene revealed a region similar to the eutherian LCR, containing sequences and potential transcription factor binding sites with homology to eutherian hypersensitive sites 1 to 5. FISH mapping of BACs containing S. macroura alpha and beta globin genes located the beta globin cluster on chromosome 3q and the alpha locus close to the centromere on 1q, resolving contradictory map locations obtained by previous radioactive in situ hybridization.

Sex and death in birds: a model of dosage compensation that predicts lethality of sex chromosome aneuploids.

Birds show female heterogamety, with ZZ males and ZW females. It is still not clear whether the W is female-determining, or whether two doses of the Z chromosomes are male-determining, or both. This question could easily be settled by the sexual phenotypes of ZZW and ZO birds, in the same way that the sexual phenotypes of XXY and XO showed that the Y is male determining in humans, but that the dosage of an X-borne gene determines sex in Drosophila. However, despite extensive searches, no ZZW or ZO diploid birds have been satisfactorily documented, so we must assume that these genotypes are embryonic lethals. Given that ZW and ZZ are viable and the W contains few genes it is not clear why this should be so. Here I propose that sex chromosome aneuploids are lethal in chicken because, to achieve dosage compensation, a locus on the W chromosome controls the upregulation of genes on the Z in ZW females. ZO birds would therefore have only half the normal dose of Z-linked gene product and ZZW would have twice the amount, both of which would undoubtedly be incompatible with life. Reports of other aneuploids and triploids are also consistent with this hypothesis.

Evolution of the human X--a smart and sexy chromosome that controls speciation and development.

In humans, as in other mammals, sex is determined by an XX female/XY male chromosome system. Most attention has focused on the small, degenerate Y chromosome, which bears the male-dominant gene SRY. The X, in contrast, has been considered a well-behaved and immaculately conserved element that has hardly changed since the pre-mammal days when it was just another autosome pair. However, the X, uniquely in the genome, is present in two copies in females and only one in males. This has had dire consequences genetically on the evolution of its activity--and now it appears, on its gene content and/or the function of its genes. Here we will discuss the origin of the human X, and the evolution of dosage compensation and gene content, in the light of recent demonstrations that particular functions in sex and reproduction and cognition have accumulated on it.

DMRT1 in a ratite bird: evidence for a role in sex determination and discovery of a putative regulatory element.

Unlike mammals, birds have a ZZ male/ZW female sex-determining system. In most birds, the Z is large and gene rich, whereas the W is small and heterochromatic, but the ancient group of ratite birds are characterized by sex chromosomes that are virtually homomorphic. Any gene differentially present on the ratite Z and W is therefore a strong candidate for a sex-determining role. We have cloned part of the candidate bird sex-determining gene DMRT1 from the emu, a ratite bird, and have shown that it is expressed during the stages of development corresponding to gonadal differentiation in the chicken. The gene maps to the distal region of the Z short arm and is absent from the large W chromosome. Because most sequences on the emu W chromosome are shared with the Z, the Z-specific location constitutes strong evidence that differential dosage of DMRT1 is involved in sex determination in all birds. The sequence of emu DMRT1 has 88% homology with chicken DMRT1 and 65% with human DMRT1. Unexpectedly, an unexpressed 270-bp region in intron 3 of emu DMRT1 showed 90% homology with a sequence in the corresponding intron of human DMRT1. This extraordinarily high conservation across 300 million years of evolution suggests an important function, perhaps involved in control of DMRT1 expression and vertebrate sex determination.

Isolation of chromosomal regions controlling intersex development in a marsupial.

A marsupial (Sminthopsis douglasi) with bilateral intersexuality had a hemiscrotum on the right side and a hemi-pouch with nipples on the left. A normal female karyotype (2n = 14, XX) was present in cells from the right (male) side, while cells from the left (female) side initially had a female karyotype plus two dot-like chromosomes (2n = 14, XX + 2B). It is proposed that the dots represented a region deleted from the X chromosome that contains the "pouch-mammary/scrotum" (PMS) switch gene whose dosage determines development of a pouch and teats (two doses) or a scrotum (one dose). Mis-segregation early in embryonic development produced a lineage with one normal X and one deleted X (male side), and a lineage with a normal and deleted X, plus two copies of the deleted region (female side). The origin of the supernumerary elements was therefore investigated in the expectation that they may contain the long-sought pouch-mammary/scrotum switch gene. Several elements were microdissected, and amplified DNA was used for in situ hybridization, producing signals in five different chromosome regions including the X. This could represent a region of the X that contains, as well as PMS, repetitive DNA that is present also at other chromosomal sites.

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.

Anatomy and chromosomes of two intersexual dasyurid marsupials.

The intersexual phenotypes of marsupials with XXY and XO chromosome constitutions imply that not all sexual dimorphisms are under the control of testicular hormones and, ultimately, the SRY gene on the Y chromosome. It has been hypothesised that there is a gene on the X chromosome that determines whether either a scrotum will form (one copy of the gene) or a pouch with teats (two copies of the gene). Here, we describe the anatomy and chromosomes of two intersexual dasyurid marsupials. One, a Dasyuroides byrnei, had a pouch, but the reproductive tract was essentially male. The other, a Sminthopsis douglasi, had a hemipouch and a hemiscrotum and the reproductive tract was essentially female. The S. douglasi was a mosaic for cells with an apparently normal 2n = 14, XX female karyotype and cells with 2n = 14 plus (usually) two dot-like supernumerary elements 2n = 14, XX + 2B. The D. byrnei cells examined also had a 2n = 14, XX + 2B karyotype. In fibroblasts from the male and female sides of the S. douglasi, it was possible to assign the 2n = 14, XX karyotype to the male side and the 2n = 14, XX + 2B to the female side.