Molecular Analysis of the Sex-Determining Region of the Platyfish Xiphophorus maculatus

ZEBRAFISHVolume 3, Number 3, 2006© Mary Ann Liebert, Inc.

Molecular Analysis of the Sex-Determining Region of the Platyfish Xiphophorus maculatus

CHRISTINA SCHULTHEIS,1 QINGCHUN ZHOU,1,6 ALEXANDER FROSCHAUER,1,7

INDRAJIT NANDA,2 YVONNE SELZ,1 CORNELIA SCHMIDT,1 SABINE MATSCHL,1MARINA WENNING,1 ANNE-MARIE VEITH,1 MARIAM NACIRI,1,8

REINHOLD HANEL,1,9 INGO BRAASCH,1 AGNÈS DETTAI,1,3 ASTRID BÖHNE,1CATHERINE OZOUF-COSTAZ,3 STEFAN CHILMONCZYK,4 BÉATRICE SÉGURENS,5

ARNAUD COULOUX,5 SYLVIE BERNARD-SAMAIN,5 MICHAEL SCHMID,2MANFRED SCHARTL,1 and JEAN-NICOLAS VOLFF1

ABSTRACT

Due to the presence of genetically well-defined sex chromosomes, with a relatively restricted sex-determinationregion containing markers identified at the molecular level, the platyfish Xiphophorus maculatus is one of the bestmodels for the positional cloning of a master sex-determining gene in fish. Both male and female heterogametesand three different types of sex chromosomes have been described in the platyfish, with several loci involved inpigmentation, melanoma formation, and sexual maturity closely linked to the master sex-determining locus. Us-ing the melanoma-inducing oncogene Xmrk, its protooncogenic counterpart egfrb, as well as other X- and Y-linkedmolecular markers, bacterial artificial chromosome (BAC) contigs have been assembled for the sex-determiningregion of X. maculatus, which was mapped by fluorescent in situ hybridization to the subtelomeric region of thesex chromosomes. Initial sequence analysis of these contigs revealed several gene candidates and uncovered syn-tenies with different mammalian and chicken autosomes, supporting an independent origin of sex chromosomesin platyfish and tetrapods. Strikingly, the sex determination region of the platyfish is very instable and frequentlyundergoes duplications, deletions, and transpositions. This instability might be linked to the high genetic vari-ability affecting sex determination and other sex-linked traits in Xiphophorus.

299

1Physiologische Chemie I, Biozentrum and 2Department of Human Genetics, University of Würzburg, Würzburg,Germany.

3Département Systématique et Evolution, Muséum National d’Histoire Naturelle, Paris, France.4Laboratoire de Virologie et Immunologie Molécularies, INRA, Jouy en Josas, France.5Genoscope/Centre National de Séquençage, CNRS-UMR, Evry, France.6Present address: Department of Zoology and Stephenson Research and Technology Center, University of Okla-

homa, Norman, Oklahoma.7Present address: Institut für Zoologie, Technische Universität Dresden, Dresden, Germany.8Present address: Université Mohamed V, Faculté des Sciences, Rabat, Morocco.9Present address: Leibniz Institut für Meereswissenschaften, IFM-GEOMAR, Kiel, Germany.This work is funded by grants from the German Federal Ministry of Education and Research (BMBF, Biofuture

programme, to JNV) and from the German Research Society (DFG, to MS and JNV). AD and MN are recipients ofpostdoctoral research fellowships from the Alexander von Humboldt Foundation and the German Academic Ex-change Service (DAAD), respectively.

SEX DETERMINATION AND SEXCHROMOSOMES IN VERTEBRATES

SEXUAL DIMORPHISM is driven by the activationof regulator genes located at the top of the

sex-determining cascade. The diversity of mech-anisms inducing these regulators in vertebratescontrasts with the higher conservation observeddownstream in the cascade.1–5 In environmen-tal sex determination, regulator genes are dif-ferentially activated by external factors in malesand females. For example, the temperature ofegg incubation determines the sex of individu-als in some reptiles: higher temperature leads tothe male phenotype in crocodiles, but inducesthe formation of females in turtles (temperature-dependent sex determination).5

In species with genetic sex determination,different copy numbers (or different alleles) ofthe master regulators are present in the genomeof males and females. Frequently, master sex-determining genes are carried by sex chromo-somes (gonosomes) having evolved from a pairof autosomes.6,7 Major genetic sex determina-tion systems well conserved in distant speciesare present in mammalian and bird lineages. Inplacental mammals and marsupials, maleshave one X and one Y chromosome, and fe-males two X chromosomes (XY system, maleheterogamety). The master sex-determininggene inducing the male pathway is the Y-linkedSry gene, a member of the Sox transcription fac-tor gene family.8,9 Sry is at least 180 millionyears old and has been occasionally lost dur-ing evolution, as observed in some mole volespecies of the genus Ellobius (rodents). The hu-man Y chromosome might also disappear in5–10 millions of years.10 Birds have a sex-de-termining system with female heterogamety(ZZ males and WZ females). An excellent can-didate for a Z-linked male-inducing mastergene is dmrt1, encoding a putative transcriptionfactor involved in sex determination/differen-tiation in tetrapods and fish.11 However, twoputative female-inducing genes have been alsoidentified on the W chromosome.12,13 Bird andplacental mammalian/marsupial sex chromo-somes have apparently evolved independentlyfrom different autosomes and are therefore nothomologous.3,11 The platypus Ornithorhynchusanatinus, which belongs to a basal mammalian

sublineage called monotremes, has five Y andfive X chromosomes. Some of these sex chro-mosomes are related either to the “classical”mammalian X chromosome or to the bird Zchromosome, suggesting an evolutionary linkbetween mammalian and bird sex determina-tion systems.14 All snakes and most lizards, aswell as amphibians, also have a genetic sex de-termination system. Genetic sex determinationis very variable in amphibians, and can be in-fluenced by the temperature.5

SEX DETERMINATION AND SEX CHROMOSOMES IN FISH:

VARIATIONS ON A THEME

In sharp contrast to the situation observed inmammals and birds, but more similar to whathas been reported in amphibians, all possibleforms of genetic sex determination have beendescribed in fish, with variable influence of en-vironmental factors such as the temperature orthe pH of the water.15–18 In addition, numer-ous fish are hermaphrodites. Different types ofsex determination can coexist within the samespecies (e.g., genetic sex determination and in-fluence of temperature in the Nile tilapia Ore-ochromis niloticus), and different systems of ge-netic sex determination can be found within thesame fish genus (e.g., in Oreochromis spp.) andeven within the same species (e.g., in the platy-fish Xiphophorus maculatus). The variability ofsex determination might play a role in the as-tonishing organismal diversity and speciesrichness observed in teleost fish.19

The molecular and evolutionary mechanismsdriving the variability of sex determination infish have not been elucidated so far. A better un-derstanding of this phenomenon requires thecomparative analysis of sex determination andsex chromosomes from different species. Unfor-tunately, most fish with important genomic re-sources are not the best models to study sex de-termination. The zebrafish Danio rerio and thepufferfish Takifugu rubripes and Tetraodon ni-groviridis, all subjects of very advanced or com-pleted genome sequencing projects, do not haveany recognizable sex chromosomes, and no sex-linked genetic loci or molecular markers havebeen identified so far in these species.18

SCHULTHEIS ET AL.300

In contrast, analysis of the sex chromosomesof the medaka Oryzias latipes, another specieswith an almost completely sequenced genome,revealed important insights into the formationand evolution of sex chromosomes infish.17,18,20 The male-inducing master sex-de-termining gene of the medaka is dmrt1bY (a.k.a.DMY), a Y-specific duplicate of an autosomalgene called dmrt1 involved in sex determina-tion/differentiation in different vertebrate sub-lineages.21,22 Dmrt1bY was the first master sex-determining gene to be identified in a non-mammalian vertebrate species. Natural muta-tions affecting this gene result in XY sex-re-versed females.21 The gene duplication eventthat led to the formation of dmrt1bY is evolu-tionarily rather recent and took place approxi-mately 10 million years ago.23 Consequently,dmrt1bY is present only in certain Oryziasspecies and does not function as the generalmaster sex-determining gene in fish.24–27

Therefore, other models are required for theidentification of alternative master sex-deter-mining genes. Beside the three-spined stickle-back Gasterosteus aculeatus,28 the Nile tilapiaOreochromis niloticus,29,30 and several species ofsalmonids,31 the platyfish Xiphophorus macula-tus is one of the most promising models to an-alyze sex determination in fish.

SEX DETERMINATION AND SEXCHROMOSOMES IN THE PLATYFISH

XIPHOPHORUS MACULATUS

The platyfish Xiphophorus maculatus belongsto the family Poeciliidae, which perfectly ex-emplifies the diversity of genetic sex determi-nation systems observed in teleost fish.32,33 Theplatyfish has three different sex chromosomeswell defined at the genetic level: males are ei-ther XY or YY; females are XX, XW, or YW.Hence, strains with male heterogamety (XYmales and XX females) or female heterogamety(YY males and YW females) can be obtained inthe platyfish. Xiphophorus maculatus is thereforean excellent model to study the relationship be-tween both types of genetic sex determinationin the absence of environmental influence. WWfemales have not been observed under naturalconditions but can be obtained in the labora-

tory. The W chromosome is apparently absentfrom certain populations.

Several models have been proposed to ex-plain the existence of three types of sex chro-mosomes in the platyfish. Male-determininggenes might be present on all types of gono-somes. Only the Y-linked allele might be active,alleles on the X and W being suppressed by au-tosomal repressors. The W chromosome itselfmight carry a suppressor for the Y allele, ex-plaining why YW individuals are females.32 Inanother model, different copy numbers of amale-inducing sex-determining gene are pre-sent on the Y (two copies), X (one copy), andW (no copy) chromosomes. A total of threecopies or more of this gene is predicted to in-duce the male phenotype in XY (three copies)and YY (four copies) individuals, whereas in-dividuals with two copies or less would be fe-males.33 A similar mechanism can be imaginedwith a female-inducing gene, with two, one,and null copies on the W, X, and Y chromo-somes, respectively.

In the XY system, the sex-determining regionof the platyfish is located in the subtelomericregion of the long arm of a pair of medium-sized telocentric chromosomes34 and belongsto linkage group 24.35 As observed in most poe-ciliids, gonosomes are homomorphic, with nomorphological differences between differenttypes of sex chromosomes in the karyotype ofthe heterogametic sex. No differentiation be-tween X and Y chromosomes has been detectedthrough synaptonemal complex analysis andcomparative genomic hybridization.36 Recom-bination has been observed over almost the en-tire length of the X and Y chromosomes, withpossible suppression around the sex-determin-ing locus.37–40 Hence, platyfish sex chromo-somes are at an early step of differentiation, asobserved in a number of other fish species.16

However, accumulation of a repeat called XIR41

was observed on the Y but not on the X chro-mosome in the population Rio Jamapa, but notin other populations.34 This recent amplifica-tion might correspond to an initial event to-ward the suppression of recombination be-tween the X and Y chromosomes in thesex-determining region. Taken together, theseobservations indicate that the region specifyingthe respective identity of the X and Y chromo-

SEX-DETERMINING REGION OF XIPHOPHORUS 301

somes is relatively small, thus restricting thepart of the chromosomes to be analyzed for theidentification of the sex-determining gene.

The W chromosome has been poorly charac-terized so far and is apparently more divergent.Numerous gene loci identified on the X and Yhave not been detected on the W chromosome.However, crossovers between the W and the Ychromosomes have been described, demon-strating the relationship between both types ofgonosomes.32

GENE LOCI AND MOLECULARMARKERS IN THE SEX-DETERMINING

REGION OF THE PLATYFISH

Several gene loci are linked to the sex-deter-mining locus SD in the sex-determining regionof X. maculatus (Fig. 1).33,37,40 Of particular in-terest are two closely linked but distinct sexchromosomal loci, Tu and Mdl, which are bothinvolved in the formation of melanoma in hy-brids between X. maculatus and other Xiphopho-rus species.42–49 The tumor locus Tu encodesthe oncogenic activity derepressed in hybridsleading to the formation of melanoma. Themacromelanophore-determining locus Mdl isnecessary for the formation of large melanin-producing pigment cells called macrome-lanophores, which are the progenitors of

melanoma cells. Mdl determines not only thephenotype of macromelanophore pigmenta-tion patterns but also, presumably in combina-tion with Tu, the pathophysiological propertiesof the derived melanoma. Therefore, Mdl canbe considered as a tumor modifier locus.

Other gene loci linked to SD include thered–yellow locus (RY, a.k.a. Xant/Ptr), whichis responsible for red, brown, orange, and yel-low pigmentation patterns in the iris, on thebody, and in the fins.38 This pattern is producedby pigment cells from the xanthophore/ery-throphore lineage.50 Also located in this regionbut not precisely mapped relative to other lociis the P locus (for “puberty” or “pituitary”,a.k.a. PIT), which determines the onset of sex-ual maturity in fish.51,52 Interestingly, this traitis highly polymorphic in Xiphophorus, withmultiple phenotypes ranging from very earlyto very late maturating animals. Macrome-lanophore pigment patterns and melanomaphenotypes, which are also encoded by locilinked to the sex-determining gene, are alsohighly polymorphic.

The only sex chromosomal gene locus iden-tified so far at the molecular level in this regionis the Tu locus, which corresponds to theXiphophorus melanoma receptor tyrosine kinaseoncogene Xmrk.53 Xmrk encodes a Xiphophorus-specific oncogenic member of the epidermalgrowth factor receptor family, which was


FIG. 1. (A) Gene loci linked to thesex-determining region of the platy-fish Xiphophorus maculatus and (B, C)fluorescent in situ co-hybridization ofBAC clones linked to Xmrk (B6, digox-igenin-labeled, red) and egfrb (G1, bi-otin-labeled, green) on chromosomesof XY males (B) and XX females (C).Chromosome preparation, probe la-beling, and hybridization conditionswere performed according to Ref. 34.BAC, bacterial artificial chromosome;Cen, centromere; egfrb, epidermalgrowth factor receptor b; Mdl,macromelanophore-determining lo-cus; P, puberty locus; RY, red–yellowlocus; SD, sex-determining locus; Tel,telomere; Xmrk, Xiphophorus mela-noma receptor tyrosine kinase onco-gene.

formed through duplication of the proto-onco-gene egfrb.54,55 Both Xmrk and egfrb are locatedon the sex chromosomes on different sides ofthe master sex-determining gene (Fig. 1). Thissituation is obviously favorable for the posi-tional cloning of the SD locus.

MOLECULAR ANALYSIS OF THE SEX-DETERMINING REGION:

CANDIDATE GENES AND SYNTENIES

As a first step toward the identification ofgenes linked to the sex determination region ofthe platyfish, a bacterial artificial chromosome(BAC) genomic library was constructed usingXY males from the Rio Jamapa population.BAC clones containing either Xmrk or egfrbwere isolated, and assigned though allele-spe-cific PCR and restriction fragment length poly-morphism analysis to the X or the Y chromo-some.56 BAC contigs were assembled throughre-screening of the genomic library with endprobes of already isolated BAC clones. The con-struction of the sex chromosomal BAC contigsis still in progress, and the junction betweenXmrk and egfr contigs has not been achieved sofar.

In order to estimate the physical distance be-tween Xmrk and egfrb, BAC clones linked tothese genes were cohybridized by fluorescentin situ hybridization on chromosomes of XYmales and XX females (Fig. 1). The hybridiza-tion pattern obtained confirmed that Xmrk andegfrb are located in the same subtelomeric re-gion of the X and Y chromosomes. The signalsfor each probe were very close but not super-posed. According to the limit of resolution ofthe method, this suggests that the physical dis-tance between Xmrk and egfrb is at least onemegabase. No obvious difference was observedbetween the X and the Y chromosomes, con-firming that the gonosomes of the platyfish areat an early stage of molecular differentiation.

Initial partial sequencing of BAC clones re-vealed sequences with significant similarity toknown genes, particularly from other verte-brates (Table 1). None of them correspond orare related to genes involved in the sex deter-mination cascade of other organisms. Syntenieswere observed with chromosomes 2 and 12 of

zebrafish, as well as with several nonmappedgenomic scaffolds from Fugu and Tetraodon,opening the possibility that these regions mightbe involved in sex determination in these fish.Syntenic relationships were detected neitherwith the sex-determining region of sticklebackand medaka,22,28 nor with the sex chromo-somes of human, mouse, or chicken. Even ifmore sequence data are clearly required for theplatyfish, these data support an independentorigin of the sex chromosomes in these differ-ent vertebrate species.

Since the DM domain protein-encoding genedmrt1 is involved in sex determination and/ordifferentiation in mammals and probably alsoin fish, and a Y-specific duplicate of this geneis the male-determining gene of the medakaOryzias latipes,21,22 the dmrt1 gene of the platy-fish was characterized as well as other mem-bers of the same gene family.26,57,58 None ofthese genes could be identified in the BAC con-tigs linked to SD, strongly suggesting that themaster sex-determining gene of the platyfish isnot a dmrt gene.

TURNOVER OF CODING SEQUENCES INTHE SEX-DETERMINING REGION OF

THE PLATYFISH

Southern blot hybridization analysis of platy-fish genomic DNA using probes specific of dif-ferent gene candidates revealed that most ofthem are multicopy in the platyfish, while theyare generally single-copy in other fish and ver-tebrates. This phenomenon was observed fordrosha and msh2 (Fig. 2), as well as for othergenes (data not shown). Numerous restrictionfragments observed in XY males were absentfrom XX females, suggesting the presence of sev-eral duplicates on the Y chromosome. This wasconfirmed by hybridization on X- and Y-linkedBAC clones, revealing several copies not only onthe Y but also on the X chromosome (notshown). Added to the fact that both Xmrk andits molecular progenitor egfrb are also linked toSD, these observations suggest that the regioncontaining the sex-determining gene of theplatyfish is a hot spot for gene duplications.

Complete sequencing of some SD-linkedcopies of cript, mc4r, msh2, and drosha showed


TA

BL

E1.

GE

NE

CA

ND

IDA

TE

SO

NT

HE

X A

ND

Y C

HR

OM

OSO

ME

SO

FT

HE

PLA

TY

FISH

XIP

HO

PH

OR

US

MA

CU

LAT

US

AN

DSY

NT

EN

IES

WIT

HO

TH

ER

VE

RT

EB

RA

TE

GE

NO

ME

S

Gen

esP

rodu

ctZ

ebra

fish

Med

aka

Fugu

Tet

raod

onH

uman

Mou

seC

hick

en

crip

tPo

stsy

napt

ic p

rote

inD

re12

Scaf

1652

M00

3100

SCA

F797

1H

sa2p

1M

mu1

7G

ga3

msh

2M

ism

atch

rep

air

prot

ein

Dre

12Sc

af18

5M

0041

24SC

AF7

971

Hsa

2p22

-21

Mm

u17

Gga

3fb

xo11

F-bo

x pr

otei

nD

re2

Scaf

185

M00

4124

SCA

F797

1H

sa2p

16M

mu1

7G

ga3

chrn

dA

cety

lcho

line

rece

ptor

Dre

21Sc

af26

2M

0001

96SC

AF1

2416

Hsa

2q33

-34

Mm

u1G

ga9

bgd

DN

A b

ind

ing

prot

ein

Dre

5Sc

af10

29M

0039

06SC

AF1

0808

Hsa

7q11

Mm

u5nd

dros

haR

ibon

ucle

ase

III

Dre

12Sc

af27

_4M

0006

22SC

AF1

4565

Hsa

5p13

Mm

u15

Gga

2cd

h6C

adhe

rin

Dre

2Sc

af27

_4M

0006

22SC

AF1

4565

Hsa

5p15

-14

Mm

u15

Gga

2m

c4r

Mel

anoc

orti

n re

cept

orD

re2

Scaf

27_4

M00

0622

SCA

F145

65H

sa18

q22

Mm

u18

Gga

2cd

h20

Cad

heri

nD

re2

Scaf

27_2

M00

1690

SCA

F145

65H

sa18

q22-

23M

mu1

Gga

2eg

frb/

Xm

rkE

GF

rece

ptor

�Sc

af76

5M

0003

99SC

AF1

4764

Hsa

7p13

-12

Mm

u11

Gga

2

Dre

, ch

rom

osom

e of

Dan

io r

erio

(zeb

rafi

sh);

EG

F, e

pid

erm

al g

row

th f

acto

r; G

ga,

chro

mos

ome

of G

allu

s ga

llus

(chi

cken

); H

sa,

chro

mos

ome

of H

omo

sapi

ens

(hum

an);

Mm

u, c

hrom

osom

e of

Mus

mus

culu

s(m

ouse

); nd

, not

det

ecte

d; S

caf,

scaf

fold

; �, i

den

tifi

ed b

ut n

ot m

appe

d. S

eque

nces

and

gen

e lo

caliz

atio

ns w

ere

obta

ined

dir

ectl

y fr

omZ

FIN

(ht

tp:/

/zf

in.o

rg)

or N

CB

I (h

ttp:

//w

ww

.ncb

i.nlm

.nih

.gov

). Se

quen

ces

not

char

acte

rize

d s

o fa

r w

ere

iden

tifi

ed t

hrou

gh B

LA

ST a

naly

sis

of t

he g

enom

e of

the

zeb

ra-

fish

(ht

tp:/

/w

ww

.ens

embl

.org

/D

anio

_rer

io),

med

aka

(htt

p://

dol

phin

.lab.

nig.

ac.jp

/m

edak

a),

Fugu

(ht

tp:/

/fu

gu.b

iolo

gy.q

mul

.ac.

uk/

blas

t/),

Tet

raod

onan

d t

etra

pod

s(h

ttp:

//w

ww

.ncb

i.nlm

.nih

.gov

/B

LA

ST).

that they correspond to pseudogenes inactivatedby partial deletions and other types of re-arrangements.55 The Xmrk oncogene itself canalso be affected by this phenomenon and inacti-vated by deletion or insertional mutagenesis.47,59

TRANSPOSABLE ELEMENTS AND OTHER REPEATS IN THE

SEX-DETERMINING REGION OF THE PLATYFISH

Initial analysis of genomic clones linked tothe master sex-determining gene SD uncoverednumerous transposable elements and other re-peated sequences, some of them identified forthe first time in fish and even in vertebrates.Examples include non-long terminal repeat(non-LTR) retrotransposons, LTR retrotrans-posons, and endogenous retroviruses, as wellas several types of DNA transposons (Table 2).More than 80% of the 33 kb region located 3�of Xmrk is constituted by retroelements.55

Hence, the region linked to the sex-deter-mining gene is frequently subject to transposi-tion events, confirming its high level of insta-bility. Some of these transposable elements arestill active, as demonstrated by the insertionalinactivation of the Xmrk oncogene by thenonautonomous LTR retrotransposon Tx1.59

There is also substantial evidence that homol-ogous recombination between nonallelic copies

of a same element is involved in some re-arrangements observed on the sex chromo-somes (data not shown). XIR repeats have beenpossibly involved in the duplication event hav-ing generated the Xmrk oncogene.55 Generally,insertions of transposable elements are locatedat the same position on the X and the Y, ex-cluding a direct role in the suppression ofrecombination between both types of chromo-somes. However, the Helitron DNA transpo-son has more copies on the Y than on the Xchromosome in the sex-determining region,60

and other examples of insertions found on theY but not on the X chromosome have been re-ported.55 In addition, the XIR repeat, whichmight correspond to the LTR of a retrotrans-poson,41 has been recently amplified on the Ybut not on the X chromosome in the Rio Jamapapopulation.34 Southern blot hybridization ofprobes from transposable elements on genomicDNA of males and females revealed in somecases Y-specific restriction fragments, whichcan be subsequently used as markers for the ex-tension of the sex chromosomal BAC contigs(Fig. 2, endogenous retrovirus T-Rex).

CONCLUSIONS AND PERSPECTIVES

The initial sequence analysis of genomicBAC clones linked to the sex-determining lo-cus of the platyfish suggests that the evolution


FIG. 2. Multiple copies of the T-Rex endogenous retrovirus and of the msh2 and drosha genes in the genome of theplatyfish Xiphophorus maculatus. Y-specific fragments are present in males (M) but absent in females (F). Southern blothybridization was performed as described.55

of sex chromosomes in X. maculatus followssome general patterns observed in other fishspecies and more distant organisms.5,61,62 Asobserved in the medaka Oryzias latipes and thethree-spined stickleback Gasterosteus aculea-tus,22,28 the sex determination region of theplatyfish is rich in transposable elements andother kinds of repeats and frequently under-goes genomic rearrangements, particularly duplications and deletions. Some of these re-arrangements might correspond to initialevents of molecular differentiation leading tothe suppression of recombination between theX and Y chromosomes around the sex-deter-mining locus. The observed genetic instabilitymight be involved in the high genetic poly-morphism reported in Xiphophorus for traitslike sex determination, onset of puberty,macromelanophore pigmentation, and mela-noma phenotype, which are controlled by sexchromosomal gene loci.

No synteny could be identified so far withthe sex chromosomes of medaka and stickle-back. Accordingly, the gonosomes of bothspecies are not older than 10 millions ofyears.23,28 These observations suggest an inde-pendent origin of the gonosomes of medaka,stickleback, and platyfish. Teleost fish genomes

have apparently the astonishing ability to cre-ate new sex chromosomes. This phenomenonmight be linked to the high diversity of sex de-termination systems observed in fish.

The master sex-determining gene of theplatyfish has not been characterized so far, andno obvious candidate has been identified dur-ing the initial analysis of SD-linked BACclones. This is principally due to the lowamount of sequence data obtained until nowand to the presence of gaps in the BAC con-tigs. These contigs are currently extended,particularly through the use of sex chromoso-mal duplicates and transposable elementsinsertions as markers (Fig. 2). The sex chro-mosomal contigs will be completely sequencedin a collaborative effort between the Univer-sity of Würzburg (Germany), the Genoscopesequencing center (Evry, France) and theMuséum National d’Histoire Naturelle (Paris,France). This should lead to the identificationof candidates for the sex-determining gene andother sex chromosomal loci and shed a newlight on the molecular mechanisms driving theevolution of the sex-determining region of theplatyfish. Of particular interest is the functionof the master sex-determining gene in otherfish species and other vertebrates, particularly


TABLE 2. TRANSPOSABLE ELEMENTS AND OTHER REPEATED SEQUENCES ON THE

SEX CHROMOSOMES OF THE PLATYFISH XIPHOPHORUS MACULATUS

Element Type References

Rex1 Non-LTR retrotransposon, AP endonuclease [65]Rex2 Non-LTR retrotransposon, AP endonuclease [55]Rex3 Non-LTR retrotransposon, AP endonuclease [66,67]Rex5 Non-LTR retrotransposon, AP endonuclease [55]L1-like Non-LTR retrotransposon, AP endonuclease unpublishedRex6 Non-LTR retrotransposon, REL endonuclease [68]Rex7 Ty3/gypsy LTR retrotransposon [55]Rex8 Ty3/gypsy LTR retrotransposon [55]Jule Ty3/gypsy LTR retrotransposon [69]T-Rex Endogenous retrovirus this workRex4 Endogenous retrovirus [55]Tx1 Non-coding LTR retrotransposon [59]Bbb Non-coding SINE retrotransposon Dettai et al. unpublishedHelitron Rolling circle DNA transposon [60]Tcl1-like DNA transposon unpublishedPiggyBac DNA transposon [55]D-locus DNA transposon [70]MITai Non-coding MITE DNA transposon Schultheis et al. unpublishedXIR DNA repeat, LTR-like [34,55]

AP, apurinic/apyrimidinic-like; LTR, long terminal repeat; MITE, miniature inverted repeat transposable element;REL, restriction enzyme-like; SINE, short interspersed nuclear element.

in humans. On the one hand, the function ofSD in sex determination might be restricted toseveral fish species. On the other hand, thisgene might correspond or be related to a genemember of the sex determination/differentia-tion cascade in more distant taxa. Interestingly,human chromosome 2 and mouse chromo-some 17, with regions syntenic to the sex-determining region of the platyfish, contain an unknown sex-determining gene detectedthrough the mapping of mutations causingmale-to-female sex reversal.63,64 Hence, mole-cular analysis of the sex-determining region ofthe platyfish might lead to the identification ofa new member of the sex determination cas-cade in mammals.

REFERENCES

1. Marin I, Baker BS. The evolutionary dynamics of sexdetermination. Science 1998;281:1990–1998.

2. Raymond CS, Shamu CE, Shen MM, Seifert KJ, HirschB, Hodgkin J, et al. Evidence for evolutionary con-servation of sex-determining genes. Nature 1998;391:691–695.

3. Marshall Graves JA, Shetty S. Sex from W to Z: evo-lution of vertebrate sex chromosomes and sex deter-mining genes. J Exp Zool 2001;290:449–462.

4. Zarkower D. Establishing sexual dimorphism: con-servation amidst diversity? Nat Rev Genet 2001;2:175–185.

5. Schartl M. Sex chromosome evolution in non-mam-malian vertebrates. Curr Opin Genet Dev 2004;14:634–641.

6. Muller HJ. Genetic variability, twin hybrids and con-stant hybrids in a case of balanced lethal factors. Ge-netics 1918;3:422–499.

7. Ohno S. Sex Chromosomes and Sex-Linked Genes.Springer Verlag, Berlin, 1967.

8. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Grif-fiths BL, Smith MJ, et al. A gene from the human sex-determining region encodes a protein with homologyto a conserved DNA-binding motif. Nature 1990;346:240–244.

9. Foster JW, Brennan FE, Hampikian GK, GoodfellowPN, Sinclair AH, Lovell-Badge R, et al. Evolution of sex determination and the Y chromosome: SRY-related sequences in marsupials. Nature 1992;359:531–533.

10. Marshall Graves JA. The rise and fall of SRY. TrendsGenet 2002;18:259–264.

11. Nanda I, Shan Z, Schartl M, Burt DW, Koehler M,Nothwang H, et al. 300 million years of conservedsynteny between chicken Z and human chromosome9. Nat Genet 1999;21:258–259.

12. Reed KJ, Sinclair AH. FET-1: a novel W-linked, femalespecific gene upregulated in the embryonic chickenovary. Mech Dev 2002;119 Suppl 1:S87–S90.

13. Pace HC, Brenner C. Feminizing chicks: a model for avian sex determination based on titration of Hint enzyme activity and the predicted structure of an Asw–Hint heterodimer. Genome Biol 2003;4:R18.

14. Grützner F, Rens W, Tsend-Ayush E, El-MogharbelN, O’Brien PC, Jones RC, et al. In the platypus a mei-otic chain of ten sex chromosomes shares genes withthe bird Z and mammal X chromosomes. Nature 2004;432:913–917.

15. Baroiller JF, D’Cotta H. Environment and sex deter-mination in farmed fish. Comp Biochem Physiol CToxicol Pharmacol 2001;130:399–409.

16. Devlin R, Nagahama Y. Sex determination and sexdifferentiation in fish: an overview of genetic, physi-ological and environmental influences. Aquaculture2002;208:191–364.

17. Volff JN, Schartl M. Sex determination and sex chro-mosome evolution in the medaka, Oryzias latipes, andthe platyfish, Xiphophorus maculatus. CytogenetGenome Res 2002;99:170–177.

18. Schartl M. A comparative view on sex determinationin medaka. Mech Dev 2004;121:639–645.

19. Volff JN. Genome evolution and biodiversity inteleost fish. Heredity 2005;94:280–294.

20. Matsuda M. Sex determination in the teleost medaka,Oryzias latipes. Annu Rev Genet 2005;39:293–307.

21. Matsuda M, Nagahama Y, Shinomiya A, Sato T, Mat-suda C, Kobayashi T, et al. DMY is a Y-specific DM-domain gene required for male development in themedaka fish. Nature 2002;417:559–563.

22. Nanda I, Kondo M, Hornung U, Asakawa S, WinklerC, Shimizu A, et al. A duplicated copy of DMRT1 inthe sex-determining region of the Y chromosome ofthe medaka, Oryzias latipes. Proc Natl Acad Sci USA2002;99:11778–11783.

23. Kondo M, Nanda I, Hornung U, Schmid M, SchartlM. Evolutionary origin of the medaka Y chromosome.Curr Biol 2004;14:1664–1669.

24. Kondo M, Nanda I, Hornung U, Asakawa S, ShimizuN, Mitani H, et al. Absence of the candidate male sex-determining gene dmrt1b(Y) of medaka from otherfish species. Curr Biol 2003;13:416–420.

25. Matsuda M, Sato T, Toyazaki Y, Nagahama Y, Ham-aguchi S, Sakaizumi M. Oryzias curvinotus has DMY,a gene that is required for male development in themedaka, O. latipes. Zoolog Sci 2003;20:159–161.

26. Veith AM, Froschauer A, Körting C, Nanda I, HanelR, Schmid M, et al. Cloning of the dmrt1 gene ofXiphophorus maculatus: dmY/dmrt1Y is not the mastersex-determining gene in the platyfish. Gene 2003;317:59–66.

27. Volff JN, Kondo M, Schartl M. Medaka dmY/dmrt1Yis not the universal primary sex-determining gene infish. Trends Genet 2003;19:196–199.

28. Peichel CL, Ross JA, Matson CK, Dickson M, Grim-wood J, Schmutz J, et al. The master sex-determina-


tion locus in threespine sticklebacks is on a nascent Ychromosome. Curr Biol 2004;14:1416–1424.

29. Griffin DK, Harvey SC, Campos-Ramos R, Ayling LJ,Bromage NR, Masabanda JS, et al. Early origins of theX and Y chromosomes: lessons from tilapia. Cyto-genet Genome Res 2002;99:157–163.

30. Lee BY, Penman DJ, Kocher TD. Identification of asex-determining region in Nile tilapia (Oreochromisniloticus) using bulked segregant analysis. AnimGenet 2003;34:379–383.

31. Woram RA, Gharbi K, Sakamoto T, Hoyheim B, HolmLE, Naish K, et al. Comparative genome analysis ofthe primary sex-determining locus in salmonid fish.Genome Res 2003;13:272–280.

32. Kallman KD. A new look at sex determination in poe-ciliid fish. In: Evolutionary Genetics in Fish. Turner BJ,(ed), pp. 95–171, Plenum Publishing, New York, 1984.

33. Volff JN, Schartl M. Variability of genetic sex deter-mination in poeciliid fish. Genetica 2001;111:101–110.

34. Nanda I, Volff JN, Weis S, Körting C, Froschauer A,Schmid M, et al. Amplification of a long terminal re-peat-like element on the Y chromosome of the platy-fish, Xiphophorus maculatus. Chromosoma 2000;109:173–180.

35. Kazianis S, Nairn RS, Walter RB, Johnston DA, Ku-mar J, Trono D, et al. The genetic map of Xiphophorusfish represented by 24 multipoint linkage groups. Ze-brafish 2004;1:287–304.

36. Traut W, Winking H. Meiotic chromosomes andstages of sex chromosome evolution in fish: zebra-fish, platyfish and guppy. Chromosome Res 2001;9:659–672.

37. Bellamy AW, Queal ML. Heterosomal inheritance andsex determination in Platypoecilus maculatus. Genetics1951;36:93–107.

38. Kallman KD. The platyfish Xiphophorus maculatus. In:Handbook of Genetics Vol. 4. King RC, (ed), pp. 81–132, Plenum Press, New York, 1975.

39. Morizot DC, Slaugenhaupt SA, Kallman KD,Chakravarti A. Genetic linkage map of fish of thegenus Xiphophorus (Teleostei: Poeciliidae). Genetics1991;127:399–410.

40. Gutbrod H, Schartl M. Intragenic sex-chromosomalcrossovers of Xmrk oncogene alleles affect pigmentpattern formation and the severity of melanoma inXiphophorus. Genetics 1999;151:773–783.

41. Roushdy J, Michel J, Petry H, Anders A, Anders F.Paragenetic suppressors of suppressor genes—a newclass of oncodeterminants. J Cancer Res Clin Oncol1999;125:123–133.

42. Gordon M. The genetics of viviparous top-minnowPlatypoecilus: The inheritance of two kinds of melano-phores. Genetics 1927;12:253–283.

43. Kosswig C. Über Kreuzungen zwischen denTeleostiern Xiphophorus helleri und Platypoecilus mac-ulatus. Z Indukt Abstammungs-Vererbungsl 1928;47:150–158.

44. Anders F. Contributions of the Gordon–Kosswigmelanoma system to the present concept of neopla-sia. Pigment Cell Res 1991;3:7–29.

45. Anders A, Anders F. Etiology of cancer as studied inthe platyfish-swordtail system. Biochim Biophys Acta1978;516:61–95.

46. Schartl M. Platyfish and swordtails: a genetic systemfor the analysis of molecular mechanisms in tumorformation. Trends Genet 1995;11:185–189.

47. Weis S, Schartl M. The macromelanophore locus andthe melanoma oncogene Xmrk are separate genetic en-tities in the genome of Xiphophorus. Genetics 1998;149:1909–1920.

48. Kazianis S, Walter RB. Use of platyfish and sword-tails in biological research. Lab Anim (NY) 2002;31:46–52.

49. Meierjohann S, Schartl M, Volff JN. Genetic, bio-chemical and evolutionary facets of Xmrk-inducedmelanoma formation in the fish Xiphophorus. CompBiochem Physiol C Toxicol Pharmacol 2004;138:281–289.

50. Goodrich HB, Hill GA, Arrick MS. The chemical iden-tification of gene-controlled pigments in Platypoecilusand Xiphophorus and comparisons with other tropicalfish. Genetics 1941;26:573–586.

51. Kallman KD, Schreibman MP, Borkoski V. Geneticcontrol of gonadotrop differentiation in the platyfish,Xiphophorus maculatus (Poeciliidae). Science 1973;181:678–680.

52. Kallman KD, Borkoski V. A sex-linked gene control-ling the onset of sexual maturity in female and maleplatyfish (Xiphophorus maculatus), fecundity in fe-males and adult size in males. Genetics 1978;89:79–119.

53. Wittbrodt J, Adam D, Malitschek B, Maueler W, RaulfF, Telling A, et al. Novel putative receptor tyrosinekinase encoded by the melanoma-inducing Tu locusin Xiphophorus. Nature 1989;341:415–421.

54. Adam D, Dimitrijevic N, Schartl M. Tumor suppres-sion in Xiphophorus by an accidentally acquired pro-moter. Science 1993;259:816–819.

55. Volff JN, Körting C, Froschauer A, Zhou Q, Wilde B,Schultheis C, et al. The Xmrk oncogene can escapenonfunctionalization in a highly unstable subtelom-eric region of the genome of the fish Xiphophorus. Ge-nomics 2003;82:470–479.

56. Froschauer A, Körting C, Katagiri T, Aoki T, AsakawaS, Shimizu N, et al. Construction and initial analysisof bacterial artificial chromosome (BAC) contigs fromthe sex-determining region of the platyfish Xiphopho-rus maculatus. Gene 2002;295:247–254.

57. Kondo M, Froschauer A, Kitano A, Nanda I, HornungU, Volff JN, et al. Molecular cloning and characteri-zation of DMRT genes from the medaka Oryzias latipesand the platyfish Xiphophorus maculatus. Gene 2002;295:213–222.

58. Volff JN, Zarkower D, Bardwell VJ, Schartl M. Evolutionary dynamics of the DM domain gene fam-ily in metazoans. J Mol Evol 2003;57 Suppl 1:S241–S249.

59. Schartl M, Hornung U, Gutbrod H, Volff JN, Wit-tbrodt J. Melanoma loss-of-function mutants inXiphophorus caused by Xmrk-oncogene deletion and


gene disruption by a transposable element. Genetics1999;153:1385–1394.

60. Zhou Q, Froschauer A, Schultheis C, Schmidt C, Bi-enert P, Wenning M, et al. Helitron transposons onthe sex chromosomes of the platyfish Xiphophorusmaculatus and their evolution in animal genomes. Ze-brafish (in press).

61. Charlesworth B. Sex determination: primitive Y chro-mosomes in fish. Curr Biol 2004;14:R745–R747.

62. Fraser JA, Heitman J. Chromosomal sex-determiningregions in animals, plants and fungi. Curr Opin GenetDev 2005;15:645–651.

63. Slavotinek A, Schwarz C, Getty JF, Stecko O, Good-man F, Kingston H. Two cases with interstitial dele-tions of chromosome 2 and sex reversal in one. Am JMed Genet 1999;86:75–81.

64. Washburn LL, Eicher EM. Sex reversal in XY micecaused by dominant mutation on chromosome 17.Nature 1983;303:338–340.

65. Volff JN, Körting C, Schartl M. Multiple lineages ofthe non-LTR retrotransposon Rex1 with varying suc-cess in invading fish genomes. Mol Biol Evol 2000;17:1673–1684.

66. Volff JN, Körting C, Sweeney K, Schartl M. The non-LTR retrotransposon Rex3 from the fish Xiphophorusis widespread among teleosts. Mol Biol Evol 1999;16:1427–1438.

67. Volff JN, Körting C, Meyer A, Schartl M. Evolutionand discontinuous distribution of Rex3 retrotrans-posons in fish. Mol Biol Evol 2001;18:427–431.

68. Volff JN, Körting C, Froschauer A, Sweeney K, SchartlM. Non-LTR retrotransposons encoding a restrictionenzyme-like endonuclease in vertebrates. J Mol Evol2001;52:351–360.

69. Volff JN, Körting C, Altschmied J, Duschl J, SweeneyK, Wichert K, et al. Jule from the fish Xiphophorus isthe first complete vertebrate Ty3/Gypsy retrotrans-poson from the Mag family. Mol Biol Evol 2001;18:101–111.

70. Förnzler D, Altschmied J, Nanda I, Kolb R, BaudlerM, Schmid M, et al. The Xmrk oncogene promoter isderived from a novel amplified locus of unusual or-ganization. Genome Res 1996;6:102–113.

Address reprint requests to:Jean-Nicolas Volff

Biofuture Research GroupBiozentrum

University of Würzburgam Hubland

D-97074 Würzburg, Germany

E-mail: [email protected]


Documents

Molecular Analysis of the Sex-Determining Region of the Platyfish Xiphophorus maculatus