1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, Centre National de la Recherche Scientifique, Institut National de
la Santé et de la Recherche Médicale, Université Louis
Pasteur, B.P. 163, 67404 Illkirch, Strasbourg, France
2 Università San Raffaele, Via Olgettina 58, 20132 Milano, Italy
* Authors for correspondence (e-mail: ninino{at}igbmc.u-strasbg.fr; paolosc{at}igbmc.u-strasbg.fr)
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Summary |
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Key words: Sex determination, Splicing factors, Developmental pathways, Sox factor
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Introduction |
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A cornucopia of sexual strategies |
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Alternative splicing, sex and death in Drosophila melanogaster |
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In D. melanogaster a ratio of X chromosomes to autosomes equal to
1 produces females; a ratio of 0.5 produces males. X-linked genes are dosage
compensated in males by transcriptional upregulation. The X-to-autosome ratio
signal impinges upon the expression of the sex-lethal (Sxl)
gene, which encodes an RNA-binding protein endowed with two RNP-type
RNA-binding domains that have affinity to uridinerich sequences. In females a
high level of SXL prevents assembly of spliceosomes on the male-specific exon
3 within its own transcript. SXL also represses translation of MSL-2, a
component of the dosage compensation complex, by binding to untranslated
regions of its mRNA. In the absence of Sxl, overexpression of
X-linked genes in female flies is lethal. SXL facilitates the use of a
downstream alternative 3' splice site in the transformer
(tra) pre-mRNA, binding to a sequence next to a stronger upstream
3' splice site and blocking access of U2AF. Alternative splicing allows
the production of a full-length TRA protein in females but not in males. TRA,
which contains an RS domain, heterodimerizes with the product of the
transformer2 (tra2) gene, which encodes an RNA-binding
protein endowed with one RNP-type RNA-binding domain and two RS domains.
TRA/TRA2 heterodimers bind to a splicing enhancer situated within the
female-specific exon 4 of the doublesex (dsx) gene and
stimulate the use of the 3' splice site of this exon, through formation
of a complex containing SR proteins and RS-domain-mediated interactions with
the U2AF small subunit. This triggers expression of a short isoform of the
dsx gene in females (DSXF), whereas males express a longer
isoform (DSXM). Both isoforms are transcription factors, binding
sequences present in the promoters of genes involved in sexual differentiation
through an N-terminal DNA-binding domain, the DM domain
(Raymond et al., 1998).
Specificity in the effects of DSXF compared with DSXM is
obtained through the sex-specific protein C-terminus. Sexual differentiation
in the nervous system is also under the control of the TRA-TRA2 complex, which
activates a female-specific 5' splice site in the fruitless
gene and regulates production of sex-specific isoforms (FRUF and
FRUM). Sxl is also required for oogenesis in the germline,
whereas tra2 plays a role in spermatogenesis.
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Patterns of mammalian sex differentiation |
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In mammals, an undifferentiated gonad forms from the intermediate mesoderm. During this bipotential stage, two ductal structures are present: the Wolffian duct, which will differentiate into the epidydimis and vas deferens in the male; and the Müllerian duct, which is the progenitor of the oviducts, uterus and upper vagina in the female. A few genes are known to play a master role in gonadogenesis, namely Wilms' tumor (Wt1), the orphan nuclear receptor Sf1, the LIM homeobox Lhx1 and Lhx9, the homeobox Emx2 and the Polycomb group M33. Once the bipotential gonad is formed, the critical event in sex determination is the formation of Sertoli cells rather than ovarian follicle cells. Sertoli cells release the Müllerian-inhibiting substance (MIS), which triggers the regression of Müllerian ducts in males. In the absence of MIS production, the default female differentiation program is activated. This critical stage is regulated by the testis-determining factor SRY, whose gene is present on the Y chromosome. SRY is the founder member of the SOX family, whose members share similarity in their HMG domains (see below). Another SOX factor, SOX9, is also required for testis differentiation. In the absence of SRY, ovaries are developed. Another important determinant of testis differentiation is the FGF9 growth factor, which appears to function downstream of SRY to stimulate mesenchymal proliferation, cell migration and Sertoli cell differentiation. Finally, once gonadal sex has been determined, gonad differentiation ensues. In males, genes involved in testis differentiation include Wt1, Sf1, Gata4, Dhh, Sox9 and Dmrt1. Conversely, the Wnt4 signalling molecule suppresses Leydig cell differentiation in the ovary.
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Conservation of downstream effectors |
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How does SRY work? |
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SRY is thought to work as an architectural factor modulating local
chromatin structure in the vicinity of target genes to favour the assembly of
the transcriptional machinery (Wegner,
1999; Kamachi et al.,
2000
). However, target genes for SRY remain unknown. One idea is
that SRY directly activates the MIS gene promoter
(Haqq et al., 1994
), but the
time lag between SRY and MIS expression in the male gonad
and the absence of transactivation properties in human SRY suggest that other
genes directly regulated by SRY mediate its effects. One of these is probably
SOX9. Conversely, the existence of phenotypically male XX individuals lacking
SRY has led to the hypothesis that SRY works as a negative regulator of a gene
(Z) that in turn negatively regulates the male sex determination pathway
(McElreavey et al., 1993
).
According to this hypothesis, XX males lacking SRY harbor mutations that
inactivate the Z gene. Recessive mutations causing female-to-male sex reversal
are known in a variety of species (Vaiman
and Pailhoux, 2000
). In the goat, the polled intersex syndrome
(PIS) associates polledness and female-to-male sex reversal. A chromosomal
deletion responsible for PIS in goats alters the expression of two
transcripts: PISRT1, a non-coding RNA; and FOXL2, a putative forkhead
transcription factor (Pailhoux et al.,
2001
). These two are potential candidates for Z, together with
WNT4, which represses Leydig cell differentiation in the ovary
(Vainio et al., 1999
) and the
products of genes present on human distal chromosome 9p
(Ottolenghi and McElreavey,
2000
).
Recent studies showing that the HMG domains of two different Sox proteins,
Sox3 and Sox9, can substitute for the Sry HMG domain and trigger male sex
determination in transgenic mice further complicate matters
(Bergstrom et al., 2000). Since
multiple Sox genes, including Sox3, are expressed in the developing
XX and XY gonads during the period critical for sex determination
(Collignon et al., 1996
), it is
not clear what makes SRY act in such a specific way to trigger the male
differentiation pathway.
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Splicing regulators in mammalian sex determination |
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Another gene that plays a crucial role in mammalian sex determination and
encodes splicing-regulating proteins is Wt1. The WT1 isoforms that
include a lysine-threonine-serine (KTS) sequence at the end of exon 9 localize
to nuclear speckles, interact with the U2AF65 splicing factor and associate
with spliceosomes (Larsson et al.,
1995; Davies et al.,
1998
). WT1 also interacts with WTAP, the mammalian homologue of
D. melanogaster female-lethal(2)d, which is required for the
female-specific splicing of the sxl and tra pre-mRNAs
(Little et al., 2000
).
Interestingly, WTAP has been isolated as a component of active human splice
complexes (Zhou et al.,
2002
).
Dominant donor splice site mutations impairing WT1(+KTS) expression produce
Frasier syndrome, which is characterized by progressive glomerulopathy and
male-to-female sex reversal (Barbaux et
al., 1997). Hammes et al. have recently described a mouse model
for Frasier syndrome (Hammes et al.,
2001
). Homozygous XY Frasier mice show, in addition to
glomerulosclerosis, significantly reduced Sry expression and complete
sex reversal. Conversely, XY mice in which the expression of WT1(KTS)
isoforms has been selectively ablated still express male-specific genes in
severely hypoplasic gonads. These findings strongly argue for a specific role
of WT1 (+KTS) isoforms in the sex determination process.
On the basis of these findings, we suggest that WT1 (+KTS) isoforms and SRY regulate the expression of critical testis differentiation genes at the post-transcriptional level, with WT1 (+KTS) lying upstream in the cascade and also controlling genes required for kidney development. SRY, in turn, would repress the expression of one or more genes acting negatively upon male sex determination. The pathways of sex determination in D. melanogaster and mammals, showing the positive and negative effects of gene products upon subsequent steps in the cascade, are shown in parallel in Fig. 2.
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Conclusions |
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Acknowledgments |
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