Sex chromosome evolution in Vertebrates Frédéric VEYRUNES Institut des Sciences de l’Evolution Department Genome Team Sex & Speciation CNRS / University Montpellier 2 email: frederic.veyrunes@univ-montp2.fr Comparative Genomics Lyon 2011

I. Origin and Evolution of sex chromosomes in Vertebrates. II. The “weird” sex chromosome system of the platypus. III. New data from the complete sequencing genome of the platypus IV. Sex chromosomes and sex determination in African pygmy mice.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y X Y

X and Y chromosomes evolved from an ancestral pair of autosomes > 1000 genes Y >45 genes (SRY) SRY Bien que très différents, ils sont issus et ont évolués à partir d’une même paire d’autosome. En quelques mots pour faire simple: un autosome a acquis un gène déterminant la fonction mâle, Sry, et devint de ce fait un proto-Y, entraînant sous l’action de la sélection l’accumulation d’autres gènes spécifiques et avantageux chez les mâles. Pour préserver cette association de gènes liés à la fonction mâle, la recombinaison a été sélectivement interrompue. L’arrêt de la recombinaison a notamment empêché la purge des allèles délétères et a entraîné l’accumulation d’accidents génétiques qui progressivement dégradent le chromosome Y ayant ainsi perdu 955 gènes. Le X quant à lui continue de recombiner avec son homologue chez les femelles. proto-X -Y X Y

Y = by-product of perverse evolution leading to its extinction ? The Human Y chromosome rapidly degenerates. Y = by-product of perverse evolution leading to its extinction ? Graves Cell 2006 Well… Human Y may disappear, but probably not males Ellobius lutescens, a species without Y, nor SRY (Just et al Nature Genet 1995) The ancestral Y of D. pseudoobcura has been lost, and a novel Y has arisen de novo (Carvalho & Clarck Science 2005)

Gradual degeneration of snake W chromosome No genes shared between Z and W (W has lost most genes) 11/11 genes shared between Z and W (small sex-determining region) 3/11 genes shared between Z and W Gradual degeneration of snake W chromosome (Matsubara et al PNAS 2006) Pythonidae Colubridae Viperidae Z W Non-recombining region has remained small Large non-recombining region Ratite bird sex chromosomes are not evolving at the same rate than other birds (Nanda et al Cytogenet Genome Res 2008) FOUR active genes in human and gorilla became pseudogenes in chimpanzee, suggesting a faster Y degrading process in the chimp than in the other lineages (Goto et al J Mol Evol 2009)

Most of the gametologues present on the human Y are found in the youngest strata (3 and 4) of the X This indicates that the older strata are genetically degenerated and have lost most of the genes that were once on the Y Lahn & Page Science 1999

The Y chromosome determines the male sex, regardless of which other chromosomes are present Female development: « default pathway » The Y chromosome is a by-product of perverse evolution leading to its degeneration BUT… AT CONTRARY: The X chromosome is gene-rich, and notably with an over-representation of genes involved in sex, reproduction, and cognitive (“intelligence genes”) functions. IN FACT, Rapid Selection for: - male-advantage recessive mutations on the X chromosome (hemizygous males) - female-advantage dominant mutations on the X chromosome (ratio 2:1)

Genetic Sex Determination In Vertebrates, sex is determined by many different strategies Environmental Sex Determination Genetic Sex Determination Z W X Y Male heterogametic: Female heterogametic :

sp. with Environmental SD sp. hermaphrodite Silverside Platyfish sp. with Genetic SD sp. with Environmental SD sp. hermaphrodite Conover et al Evolution 1992 Volff & Schartl Genetica 2001 Polyfactorial Genetic Sex Determination Females: XX, XW or ZW Males: XY or YY Nile Tilapia XX/XY species But temperature induces sex reversal Stickleback Ross et al PLoS Genet 2009 sp. with XX/XY sp. with ZZ/ZW Baroiller & D’Cotta Comp Biochem Phys C 2001

In the Japanese frog, some populations are XY and others ZW The XY pair switched into a ZW pair Rana rugosa Uno et al Chrom Res 2008 Pogona vitticeps Quinn et al Science 2007 Temperature induces sex reversal in a ZZ/ZW lizard

Sex determining systems mapped onto a molecular phylogeny of Squamates - Numerous transitions from TSD to GSD (but GSD > TSD ??) - A few XY  ZW Pokorna & Kratochvil Zool J Linn Soc 2009

All Therian mammals have the same sex chromosomes

But some exceptions exist… Why? Heteromorphic sex chromosomes, with a highly degraded and specialized Y chromosome, are a strong barrier to transitions between sex-determining systems. Due to formation of lethal genotypes (YY) Vs. YY The potential adverse effects of Y-specific genes on the feminization process of neo-XY females And conversely, the lack of necessary Y-specific genes in neo-XX males But some exceptions exist…

The X and Y are extremely conserved Any modifications generally lead to infertility/sterility Human patients with Klinefelter syndrom (XXY) or Turner (XO) The X and Y are very ancient Murphy et al Genome Res 1999

Origin of sex chromosomes in Mammals. Large-scale gene-mapping comparisons 5 Graves 1995 BioEssays X X Recent autosomal addition on the Eutherian X chromosome

Independent origin of sex chromosomes in mammals, birds, and snakes 1 12 4 Fridolfsson et al 1998 PNAS Z 4 5 8 9 18 Nanda et al 1999 Nature Genet 2 Matsubara et al 2006 PNAS Independent origin of sex chromosomes in mammals, birds, and snakes

Origin of sex chromosomes in Mammals. Differentiation of mammalian X-Y started 310 Mya, just after mammal / reptile split (Lahn & Page Science 1999)

? Amniote sex chromosomes systems Mammals ? Monotremes: pivotal position for genomic comparisons/origin of mammal XY …But their sex chromosome system remained a long mystery until recently Lizards Snakes Birds Crocodilians Turtles Monotremes Marsupials Eutherians XY XY ZW ZW Z W Z W X Y X Y 245mya 150mya 220mya 165mya 270mya 285mya 310mya

II. The “weird” sex chromosome system of the platypus. I. Origin and Evolution of sex chromosomes in Vertebrates. II. The “weird” sex chromosome system of the platypus. III. New data from the complete sequencing genome of the platypus IV. Sex chromosomes and sex determination in African pygmy mice.

10 sex chromosomes !! X1Y1X2Y2X3Y3X4Y4X5Y5 Karyotype X1 X4 X2 Y2 Y3 X3 Y1 Y5 X5 Y4 10 sex chromosomes !! X1Y1X2Y2X3Y3X4Y4X5Y5 5 Ys: male-specific 5 Xs: in pairs in female Unprecedented sex chromosome system Rens et al PNAS 2004

An exceptional system (1) Extraordinary meiotic chain: 5Xs 5Ys Grutzner et al Nature 2004

Déterminisme du sexe inconnu An exceptional system (2) Déterminisme du sexe inconnu orthologues Mammalian X Watson et al PNAS 1990 Grutzner et al Nature 2004 El Morghabel et al Genomics 2007 DMRT1 gene Avian Z X5 DMRT1 X1 Y1 X2 Y2 X3 Y3 X4 Y4 X5 Y5 absence SRY

Search for the sex determining switch in Monotremes SRY absent DMRT1 X5, but in opposite dosage than in birds (2:1 in : ) DAX1 15 Mapped on autosomes SOX9, DAX1, SF1, WT1, etc… GATA4 X2 Y1/X2 BUT… On the pseudo-autosomal region (same dose between sexes) Sex determining gene remains unknown Graphodatskaya et al Chrom Res 2007 unpublished data

And the Echidna ? Need a comparative sex chromosome map between Echidna and Platypus Chromosome Painting allows to establish the chromosomal homologies according to the DNA content, by fluorescence in situ hybridization

Genomic comparison Human/Pangolin How does it work? Amplification by DOP-PCR Labelling with fluorochrome Chromosome probe Flow-sorted chromosomes are separated Pics are isolated Genomic comparison Human/Pangolin Human chromosome 1 Human chromosome 19 Yang et al Chrom Res 2006

Platypus X5 and Y5 are not the last chromosomes in echidna chain Cross-species Chromosome Painting Echidna Y2 Echidna X5 Platypus X5 and Y5 are not the last chromosomes in echidna chain Sex chromosome system still evolved after platypus-echidna divergence Echidna = “only” 9 sex chromosomes Rens et al. Genome Biol 2007

Amniote sex chromosomes systems Mammals Lizards Snakes Birds Crocodilians Turtles Monotremes Marsupials Eutherians XY XY ZW ZW Z W Z W X Y X Y 245mya 150mya 220mya 165mya 270mya 285mya 310mya proto X -Y A link between the bird ZW and mammal XY systems ? Ezaz et al Current Biol 2006

III. New data from the complete sequencing genome of the platypus I. Origin and Evolution of sex chromosomes in Vertebrates. II. The “weird” sex chromosome system of the platypus. III. New data from the complete sequencing genome of the platypus IV. Sex chromosomes and sex determination in African pygmy mice.

Physical map of the platypus genome correspondence « karyotype – genotype » Y2 X2 X3 Warren et al Nature 2008

Platypus genome: a patchwork of mammalian, reptilian and avian characters miRNAs Some are shared with others mammals but not birds (mammal-specific), and others are bird-specific Egg - Only one vitellogenin gene, whereas chicken has 3, but other mammals 0 - Formation of zona pellucida: 4 genes shared with other mammals + 2 ZPAX genes present only in birds, fishes, amphibians Milk Duplication/Cluster of 5 casein genes Venom Like in reptiles, cocktail of variations of 3 peptide families, but arose from duplications of different set of genes convergence with snake venom.

human X chicken Z Sex Chromosomes Comparative Genomic : Platypus – Human - Chicken chicken Z human X X1 X4 X2 Y2 Y3 X3 Y1 Y5 X5 Y4 1 3 6 18 15 Sex chrom autosomes Veyrunes et al Genome Res 2008

the 10 sex chromosomes of the platypus(5 Xs and 5 Ys) had no gene homology with the “mammalian” XY, while they did have substantial homology with the bird Z chromosome.

Sex chromosome evolution Mammals Lizards Snakes Birds Crocodilians Turtles Monotremes Marsupials Eutherians XY XY ZW ZW Z W Z W X Y X Y Z W Gekko hokouensis Kawai et al Chromosoma 2008 245mya 150mya 220mya 165mya 270mya 285mya proto-X -Y 310mya 145My + jeune proto-Z -W Veyrunes et al Genome Res 2008

Is homology a relic of shared ancestry or just convergence? Do particular portions of genome make better sex chromosomes? Graves & Peichel Genome Biol 2010

Mutations on DMRT1 induce sex reversal in human In Vertebrates, DMRT1 is expressed specifically in male gonads just after sex determination DMRT1 is homolog of the genes doublesex in Drosophila and mab-3 in C. elegans, both involved in the downstream events of sex differentiation DMRT1 is a critical gene near the top of the sex determination cascade in both vertebrates and invertebrates Review in Ferguson-Smith Sex Dev 2007

In addition to DMRT1 (birds) and SRY (mammals) Sinclair et al Nature 1990 Smith et al Nature 2009 Only two other vertebrate sex determining genes are known: Both homologs of DMRT1 ! - DMY a duplicated copy of DMRT1 in the Medaka fish Matsuda et al. Nature 2002 Nanda et al PNAS 2002 - DM-W a truncated copy of DMRT1 in the frog Xenopus laevis Yoshimoto et al PNAS 2008 Repeated use of the same gene for sex determination Some parts of the genome may make better sex chromosomes

Conclusions The 10 platypus’ sex chromosomes do not share gene homology with the “mammal” X, but with the bird Z chromosome Two independent sex chromosome systems among mammals The human XY are 145My younger than previously thought. Consequences on rates of gene evolution and Y degeneration Platypus genome: genomic features of two separate lineages (mammals and sauropsids) coexist in a single organism Platypus sex determining gene remains unknown Warren et al Nature 2008 Veyrunes et al Genome Res 2008

IV. Sex chromosomes and sex determination in African pygmy mice. I. Origin and Evolution of sex chromosomes in Vertebrates. II. The “weird” sex chromosome system of the platypus. III. New data from the complete sequencing genome of the platypus IV. Sex chromosomes and sex determination in African pygmy mice.

XO / XY XX, XY / XY XO / XO XX / XX Dicrostonyx torquatus Myopus schistocolor Microtus oregoni XO / XY XX, XY / XY Akodon sp. Mus minutoides Ellobius lutescens Ellobius tancrei XO / XO XX / XX Tokudaia osimensis

Very low morphological differentiation (species-complexes) Biological Model: African pygmy mice  Genus : Mus (40 sp.)  Subgenera Mus sensu stricto Coelomys Pyromys Nannomys Laboratory mouse 19 sp. Sub-Saharan Africa Very low morphological differentiation (species-complexes) Chromosomal radiation involving in particular the sex chromosomes

The sex chromosomes of the African pygmy mice show three extraordinary deviations from the norm: Unusual meiotic behaviour Addition of genetic material on the sex chromosomes (multiple sex-autosome fusions) Modifications of sex determination systems

Unusual meiotic behaviour During male meiosis, sex chromosomes must pair to ensure balanced gamete formation. However, in a very few species, the sex chromosomes do not pair (asynaptic). X Y Mus mattheyi Loss of the Pseudo-Autosomal Region ? How these species manage to ensure a proper segregation is still under discussion Mus minutoides X Y 1

Multiple formations Neo-sex chromosomes Multiple sex-autosome fusions a paradox … Sex-autosome fusions : rare and deleterious among the mammals - Gametogenesis disruptions - Spreading of X-inactivation to autosome (silencing autosomal genes) Y-autosome = azoospermia X-autosome = sterility + other symptoms (congenital malformations …) Human pathologic cases: X chromosome autosome Extremely diverse among the Nannomys but… 1 X Y  7 13 16 15 Genome modif/plasticity allowing a higher rate of appearance/fixation of these fusions Multiple formations Neo-sex chromosomes Veyrunes et al Chrom Res 2004, 2007

Very high proportion of fertile sex-reversed females Modification of sex determining systems (1) Y Y mouse probe 1 X Y deleted X? Y Mus minutoides: XY XX Very high proportion of fertile sex-reversed females Stellenbosch Caledon NR Kuruman Belfast Willem Pretorius NR Mutanda Sterkfontein NR Sandveld NR 14 15 4 24 11 23 Unexplained strong bias in favor of the XY females Veyrunes et al Proc R Soc B 2010

SRY sequencing = male & female have the same haplotype Modification of sex determining systems (2) Sex reversal is probably not due to a mutation on SRY gene SRY sequencing = male & female have the same haplotype Females are XX, XX* or X*Y, and Males only XY Two morphologically different X chromosomes, one always associated to sex-reversed females X X* Y female XX female XX* female X*Y 1 male XY … but rather on a X-linked mutation Male XY Female XX Female XX* Female X*Y asterisk designates the still unknown mutation converting X*Y individuals into females Novel sex determination system in a Therian species Veyrunes et al Proc R Soc B 2010

Modification of sex determining systems (3) Identification of the Mutation TEST ON CANDIDATE GENES + ovulation? Meiotic drive? BREEDING PROGRAM Litter sizes & Offspring sex-ratio Consequences on mating system ? Reproductive Cost Loss YY Evolutionary Processes: Impact on sex-ratio Population Dynamics, etc… FIELD TRIPS Temporal survey in natural pop.

Complex meiotic configurations Modification of sex determining systems (4) Selective pressures favouring such system? Different fitness for each type of female? XX XX* X*Y 1:1 3:1 2:1 50% XX females 50% XY males 25% XX females 25% XX* females 25% X*Y females 25% XY males 25% XX* females 25% X*Y females 25% XY males 25% dead embryos In addition, in one pop., the X* has been translocated to another autosome 1 X* 16 Y 1 X 16 X* Sex chromosomes Complex meiotic configurations bivalent Ring of 4 Chain of 4 Y 1 13 16 X* X How could explain the high prevalence of X*Y females ?

Modification of sex determining systems (5) In addition to the standard XX / XY, and the new sex determining system described here: Mus triton in Burundi has been described with an XO / XO sex chromosome system (Jotterand-Bellomo 1988) Mus indutus, among five : three were XX, and two XO Maybe four different sex chromosome systems recorded within the African pygmy mice !!

Evolutionary cul-de-sac species ? Finally, a few mammal species have evolved to escape convention, and present aberrant sex chromosome systems But all new variants restricted to only one species Evolutionary cul-de-sac species ?