In this review, we describe recent results concerning the genetics of sex determination in mammals. Particularly, we developed the study of the FOXL2 gene and its implication in genetic anomalies in goats (PIS mutation) and humans (BPES). We present the expression of FOXL2 in the ovaries of different species

Baron, Daniel

Batista, Frank

Chaffaux, Stéphane

Cocquet, Julie

Cotinot, Corinne

Cribiu, Edmond

De Baere, Elfride

Fellous, Marc

Guiguen, Yann

Jaubert, Francis

Pailhoux, Eric

Pannetier, Maëlle

Vaiman, Daniel

Veitia, Reiner

Vigier, Bernard

Ghent University Academic Bibliography

377Reprod. Nutr. Dev. 45 (2005) 377–382© INRA, EDP Sciences, 2005DOI: 10.1051/rnd:2005028ReviewFoxl2 gene and the development of the ovary: a story about goat, mouse, fish and woman**Daniel BARONa, Frank BATISTAb, Stéphane CHAFFAUXc, Julie COCQUETb, Corinne COTINOTd, Edmond CRIBIUc, Elfride DE BAEREEe, Yann GUIGUENa, Francis JAUBERTb, Eric PAILHOUXd, Maëlle PANNETIERd, Daniel VAIMANb, Bernard VIGIERd, Reiner VEITIAb, Marc FELLOUSb*a Équipe Sexualité et Reproduction, INRA-SCRIBE, Département PHASE, Campus de Beaulieu, 35042 Rennes Cedex, France b INSERM U709, Pavillon Baudelocque, 123, Boulevard de Port Royal, 75014 Paris, Francec Laboratoire de Génétique Biochimique et Cytogénétique, Domaine du Vilvert Centre INRA, 78350 Jouy-en-Josas, Franced UMR Biologie du Développement et Reproduction, Domaine du Vilvert Centre INRA, 78350 Jouy-en-Josas, Francee University of Gand, BelgiumAbstract – In this review, we describe recent results concerning the genetics of sex determinationin mammals. Particularly, we developed the study of the FOXL2 gene and its implication in geneticanomalies in goats (PIS mutation) and humans (BPES). We present the expression of FOXL2 in theovaries of different species.ovary / comparative genomics / FOXL2 / PIS / BPES1. SEX DETERMINATION IN MAMMALSThe Y chromosome was first revealed in1959 in human male (46 XY) and not infemale (46XX) caryotypes. Since then, itseems clear that the testis determininggene(s) is located on this Y chromosome(see review in [1]). Then, it took thirty yearsto identify this gene by studying sex-reversed46 XX males or 46 XY women. In fact sev-eral of these sex-reversed females carry adeletion of the shorter arm of the Y chro-mosome, a region that is present in sex-reversed 46 XX males. The SRY gene (Sex-determining Region of the Y chromosome)was isolated in this region. After the discov-ery of SRY in 1990 [2], some other genesinvolved in testis differentiation have beenisolated through the observation of clinical* Corresponding author: fellous@cochin.inserm.fr** This presentation reviews a research project carried out for over ten years, starting in 1992, financed by INRA and INSERM, and encouraged by Professor Charles Thibault.Article published by EDP Sciences and available at http://www.edpsciences.org/rnd or http://dx.doi.org/10.1051/rnd:2005028378 D. Baron et al.cases presenting sex-reversal (discordancebetween the cytogenetic and gonadal sex).Nevertheless, up to date many genes remainto be discovered, as well as the molecularmechanisms and gene interactions involvedin the differentiation cascade of the gonad.This is even more relevant for the ovary dif-ferentiation pathway.2. SRYThis single-exon gene is specificallyexpressed in male gonads. It encodes a 204amino acid protein comprising three regions,one of which is the DNA binding domainnamed “HMG Box” (for High MobilityGroup). Sequence homology studies haveshown that the HMG Box is the only regionof SRY conserved among mammal species.Different observations have confirmed thatSRY is the testis determining factor: (i) thepresence of point mutations in this gene in46, XY women with gonadal dysgenesis,(ii) the additive transgenesis of Sry in XXmice which induces a female-to-male sex-reversal [3].Since its discovery, it has been shownthat SRY induces cell proliferation leadingto the following: (i) a size increase of the XYgonads, (ii) differentiation of Sertoli cellsand their organization in seminiferous cords,(iii) differentiation of a testis-specific vascu-lature. However, for the moment, the molec-ular target(s) of SRY remain unknown.Furthermore, mutations of this gene areinvolved in only 20% of 46, XY sex-reversedcases and 80% of 46, XX sex-reversedmales. Thus, there must be other genesinvolved in the testis differentiation cas-cade. By positional cloning, some of themhave been isolated in 46, XY females withgonadal dysgenesis (WT1, SOX9, DAX1,etc.). On the contrary, no gene responsiblefor XX male sex-reversal has been identi-fied, but we propose a model in order toexplain the development of an XX malephenotype in the absence of SRY (Fig. 1, Zmodel). An animal model, fitting with thecharacteristics of this theoretical model hasbeen recently studied in mutated goats.3. PIS MUTATION IN GOATSThe PIS syndrome (Polled Intersex Syn-drome) was first observed in the early 20thcentury. It associates the absence of horns(dominant) to sex-reversal in XX animals(recessive). When heterozygous hornlessgoats are crossed among them, a Mendeliansegregation for the hornless phenotype isobserved. On the contrary, XX animals(homozygous for the hornless mutation)have a male sex-reversal phenotype similarto 46, XX SRY-negative masculin pheno-type in men. The histology and immunohis-tochemistry of the gonads of these animalshave confirmed the presence of seminifer-ous cords and the expression of AMH(Anti-Müllerian Hormone) produced bySertoli cells; however, no germ cells arefound at puberty [4].After several years of goat genome car-tography and genetic linkage analysis, thePIS locus has been mapped on the caprinechromosome 1. In humans, the Blepharo-phimosis Ptosis Epicantus inversus Syn-drome (BPES) has been mapped on thechromosome 3 equivalent region. This syn-drome associates eyelid malformations toovarian dysgenesis in 46, XX patients. Ithas been shown that loss-of-function muta-tions of FOXL2 are responsible for this syn-drome [5].After a long work of positional cloning,the PIS mutation has been characterized asa deletion of a 12kb region responsible forthe cis-regulation of at least three genes:FOXL2, PISRT1 and PFOXic. This corre-sponds to a metagenomic complex: the PISregion acts as a regulatory element control-ling the transcriptional expression of sev-eral genes [6]. The abnormal expression ofthese three genes is in accordance with themode of inheritance observed for the differ-ent phenotypes: dominant by a gain-of-function through over expression of theseFoxl2 gene and the development of the ovary 379three genes in horns, since the heterozygousstate; recessive for the sex-reversal pheno-type due to an absence of expression ofthese three genes in the developing ovaryleading to testis differentiation.In wild-type animals, the regulatory ele-ment PIS inhibits the expression of thesethree genes in horns (Fig. 2). By contrast,the PIS region is necessary for their expres-sion in the ovaries beginning with the firststages of their differentiation. In XY males,SRY would block the regulatory elementPIS and then prevent the gonad expressionof these three genes. The deletion of this    Figure 1. Regulatory cascade hypothesis for mammalian sex determination and an explanation forsex reversal in XX  male SRY– and XY SRY+  individuals. (A) In wild-type males, the SRY proteinis present and inhibits Z by acting as a repressor of Z synthesis (as drawn) or by inhibiting theactivity of Z (not drawn). Male sex determination is therefore uninhibited by Z and can proceed.(B) In wild-type femalas, the SRY protein is lacking. Therefore, the Z protein is able to inhibit malesex determination, and female sex determination occurs. (C) XX individuals lacking SRY whoexhibit male phenotypes (male with sexual ambiguities or true hermaphroditism) are proposed tobe defective in the Z gene since they are homozygous for a Z– mutation (indicated by –). Theseindividuals are therefore able to express male sex determination. (D) XY individuals who carry anintact SRY gene can exhibit female phenotypes if they carry a mutation at the Z locus (Zi) thatrenders Z insensitive to repression by the SRY protein. The Z protein is therefore produced and caninhibit male sex determination, allowing female sex determination to occur.380 D. Baron et al.regulatory element in the XX gonads isresponsible for a female-to-male sex reversal.Currently, we are trying to confirm thehypothetical roles of the three genes bytransgenesis experiments in goats.4. GENETICS OF BPES IN HUMANS BPES is a genetic autosomal dominantdisease which associates an eyelid malfor-mation (in both sexes) and an ovarian dys-genesis responsible for a primary amenor-rhea in 46, XX women (Fig. 3). As describedabove, the BPES mutation is located onhuman chromosome 3 (band q23), homol-ogous to the PIS region in goats. RecentlyFOXL2 has been shown to be responsiblefor BPES [5].FOXL2 is a single-exon gene encodinga transcription factor presenting a “fork-head” DNA binding domain. FOXL2 pro-tein also has a polyalanine tract (n = 14)with a currently unknown function. Note-worthy, 30% of mutations consist in theexpansion of this poly-ALA tract. TheFOXL2 gene is highly conserved among allstudied vertebrates; however its conserva-tion is more pronounced in the C-terminaldomain rather than in the N-terminal part ofthe protein. This high conservation is alsofound in fish like Fugu, Tilapia and Trout.The expression of FOXL2 has been ana-lyzed with a polyclonal antibody. In humans,we detected the FOLX2 protein in thenucleus of fetal eyelid muscular cells and inthe nucleus of fetal granulosa cells. On thecontrary, FOXL2 could not be detected nei-ther in the oocyte, nor in the fetal or adulttestes. Identical results have been observedin the mouse, goat, chicken and fish [9, 10].5. FOXL2: AN OVARIAN DIFFERENTIATING GENE The expression of FOXL2 takes place atan early stage in the development of mam-mal ovaries. Indeed, it has been detected inXX genital ridges. This observation sug-gests that FOXL2 could be an ovarian deter-mining gene. Nevertheless, Foxl2 invalida-tion was reported in 2004: the heterozygousFoxl2+/– mice have a normal phenotype [11,12]. However, the homozygous Foxl2–/– hasa phenotype similar to the one observed inBPES patients. These mice present eyelidmalformation and the females develop ste-rility while males remain fertile. Even dis-playing a normal ovarian differentiation atbirth, XX Foxl2–/– mice later develop pre-mature ovarian failure. This failure is due toaltered differentiation of granulosa cellswhich are not able to form follicles. Conse-quently, Foxl2 KO-mice are an excellentanimal model of human BPES. This modelwill allow us to study the mechanism of pri-mary amenorrhea in BPES patients.6. FOXL2 IN FISHES The rainbow trout is an interesting modelfor the study of Foxl2 for the following rea-sons: (i) the trout genome went through atetraploid phase, then a diploid phase inevolution; (ii) sexual differentiation, evengenetically controlled by an XY/XX mech-anism, can be modified by environmentalfactors such as hormones, leading to thedevelopment of fertile neo-XX males orneo-XY females.Figure 2. Schematic representation of the PISregion. The PIS mutation is represented on theright. It contains transcriptional regulatoryelements of three genes (arrowheads), PISRT1,PFOXic and FOXL2, located respectively at 20and 300 kb from PIS. The lines represent thedifferent BACs encompassing the region.BACs’ addresses are given in italic.Foxl2 gene and the development of the ovary 381The study of the FOXL2 gene in trout ledus to two main conclusions:1. There are two FOXL2 genetically inde-pendent paralogue genes: FOXL2a andFOXL2b. FOXL2a expression is similar tothat observed for the mammalian FOXL2gene. FOXL2b, being specific of somaticcells of the ovary, is expressed later thanFOXL2a.2. The expression of FOXL2 in the ovariansomatic compartment is also found in theovaries of estrogen-treated neo-XY fema-les. Conversely, FOXL2 expression is notfound in androgen-treated neo-XX males.Accordingly, the expression of FOXL2 shar-ply decreases in XX females treated witharomatase inhibitors [10].Very recently identical results wereobtained in Tilapia.In conclusion, the FOXL2 gene appearshighly conserved in vertebrates regardingits sequence and its expression in the ova-ries. We still have to identify the targets ofthis transcription factor, which must play animportant role in the differentiation of ovar-ian follicular cells.REFERENCES[1] Cotinot C, Pailhoux E, Ottolenghi C, Veitia R,Fellous M. Le déterminisme du sexe: son con-trôle génétique. In: Thibault C, Levasseur MC(Eds), La reproduction chez les mammifèreset l’homme, INRA, Paris, 2001, p 215–234.[2] Berta P, Hawkins JR, Sinclair AH, Taylor A,Griffiths BL, Goodfellow PN, Fellous M.Genetic evidence equating SRY and the testis-determining factor. Nature 1990, 348: 448–450.[3] Koopman P, Gubbay J, Vivian N, GoodfellowP, Lovell-Badge R. Male development ofchromosomally female mice transgenic forSry. Nature 1991, 351: 117–121.[4] Pailhoux E, Vigier B, Vaiman D, Servel N,Chaffaux S, Cribiu EP, Cotinot C. Ontogene-sis of female-to-male sex-reversal in XXpolled goats. Dev Dyn 2002, 224: 39–50.Figure 3. Facial dysmorphy of a BPES patient. Facial malformations in BPES syndromeassociating ptosis and epicantus.382 D. Baron et al.To access this journal online: www.edpsciences.org[5] Crisponi L, Deiana M, Loi A, Chiappe F, UdaM, Amati P, Bisceglia L, Zelante L, NagarajaR, Porcu S, Ristaldi MS, Marzella R, RocchiM, Nicolino M, Lienhardt-Roussie A, NivelonA, Verloes A, Schlessinger D, Gasparini P,Bonneau D, Cao A, Pilia G. The putative fork-head transcription factor FOXL2 is mutated inblepharophimosis/ptosis/epicanthus inversussyndrome. Nat Genet 2001, 27: 159–166. [6] Pailhoux E, Vigier B, Chaffaux S, Servel N,Taourit S, Furet JP, Fellous M, Grosclaude F,Cribiu EP, Cotinot C, Vaiman D. A 11.7kbDeletion triggers intersexuality and polled-ness in goats. Nat Genet 2001, 29: 453–458.[7] Coquet J, Pailhoux E, Jaubert F, Servel N, XiaX, Pannetier M, De Baere E, Messiaen I, Coti-not C, Fellous M, Veitia R. Evolution andexpression of foxl2. J Med Genet 2002, 39:916–922.[8] Cocquet J, De Baere E, Gareil M, Pannetier M,Xia X, Fellous M, Veitia R. Structure, evolu-tion and expression of foxl2 transcription unit.Cytogenet Genome Res 2003, 101: 206–211.[9] Govoroun M, Pannetier M, Pailhoux E,Cocquet J, Brillard JP, Couty I, Batellier F,Cotinot C. Isolation of chicken homolog offoxl2 gene and comparison of its expressionpatterns with those of aromatase during ovar-ian development. Dev Dyn 2004, 231: 859–870.[10] Baron D, Cocquet J, Xia X, Fellous M,Guiguen Y, Veitia RA. An evolutionary andfunctional analysis of FoxL2 in rainbow troutgonad differentiation. J Mol Endocrinol.2004, 33: 705–715.[11] Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S,Gredsted L, Treier AC, Treier M. The murinewinged-helix transcription factor Foxl2 isrequired for granulosa cell differentiation andovary maintenance. Development 2004, 131:933–942.[12] Uda M, Ottolenghi C, Crisponi L, Garcia JE,Deiana M, Kimber W, Forabosco A, Cao A,Schlessinger D, Pilia G. Foxl2 disruptioncauses mouse ovarian failure by pervasiveblockage of follicle development. Hum MolGenet 2004, 13: 1171–1181.

Foxl2 gene and the development of the ovary : a story about goat, mouse, fish and woman

In the article published in Reprod. Nutr. Dev. 45 (2005) 377–382, the name of Elfride De Baere was misspelt. The correct list of authors should be: Daniel Barona, Frank Batistab, Stéphane Chaffauxc, Julie Cocquetb, Corinne Cotinotd, Edmond Cribiuc, Elfride De Baeree, Yann Guiguena, Francis Jaubertb, Eric Pailhouxd, Maëlle Pannetierd, Daniel Vaimanb, Bernard Vigierd, Reiner Veitiab, Marc Fellousb*

Daniel Baron

Frank Batista

Stéphane Chaffaux

Julie Cocquet

Corinne Cotinot

Edmond Cribiu

Elfride De Baere

Yann Guiguen

Francis Jaubert

Eric Pailhoux

Maëlle Pannetier

Daniel Vaiman

Bernard Vigier

Reiner Veitia

Marc Fellous

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Foxl2 gene and the development of the ovary: a story about goat, mouse, fish and woman

Elfride De Baeree

https://biblio.ugent.be/publication/2054916/file/2054940

Foxl2 gene and the development of the ovary : a story about goat, mouse, fish and woman

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