Avian sex determination: what, when and where?

Abstract

Sex is determined genetically in all birds, but the underlying mechanism remains unknown. All species have a ZZ/ZW sex chromosome system characterised by female (ZW) heterogamety, but the chromosomes themselves can be heteromorphic (in most birds) or homomorphic (in the flightless ratites). Sex in birds might be determined by the dosage of a Z-linked gene (two in males, one in females) or by a dominant ovary-determining gene carried on the W sex chromosome, or both. Sex chromosome aneuploidy has not been conclusively documented in birds to differentiate between these possibilities. By definition, the sex chromosomes of birds must carry one or more sex-determining genes. In this review of avian sex determination, we ask what, when and where? What is the nature of the avian sex determinant? When should it be expressed in the developing embryo, and where is it expressed? The last two questions arise due to evidence suggesting that sex-determining genes in birds might be operating prior to overt sexual differentiation of the gonads into testes or ovaries, and in tissues other than the urogenital system.

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Gene Expression
Cytogenet Genome Res 117:165–173 (2007)
DOI: 10.1159/000103177
Avian sex determination: what, when and
where?
C.A. Smith a K.N. Roeszler a Q.J. Hudson b A.H. Sinclair a
a
Royal Childrens Hospital, Parkville (Australia); b
Murdoch Childrens Research Institute and University of Melbourne, Department of Paediatrics,
The Jackson Laboratory, Bar Harbor , ME (USA)
though SRY is absent in birds, it is generally thought that the
principle is the same. That is, a sex determinant is first ex-
pressed in the embryonic gonads, triggering gonadal sex
differentiation, followed by hormone production to induce
secondary sexual differentiation (ducts, external genitalia
and brain sex). In this review, we consider the evidence for
this idea in birds. Firstly, what is the nature of the avian sex
determinant? Does is depend on the presence/absence of a
single gene, as in mammals? Secondly, when is it expressed?
The two most promising sex-determining candidates in
birds, the Z-linked DMRT1 and W-linked HINTW genes,
are expressed in the gonads well prior to sexual differentia-
tion. Lastly, where is the sex determinant(s) expressed?
There is evidence that candidate sex-determining genes
may be expressed independently in other tissues in addition
to the gonads, as occurs in marsupials (O et al., 1988) and
probably also in eutherian mammals (Dewing et al., 2003).
Chicken sex chromosomes and sex determination
The sex chromosomes in most birds are heteromorphic,
characterised by a large Z chromosome and smaller hetero-
chromatic W chromosome (Takagi and Sasaki, 1974; Solari,
Abstract. Sex is determined genetically in all birds, but
the underlying mechanism remains unknown. All species
have a ZZ/ZW sex chromosome system characterised by fe-
male (ZW) heterogamety, but the chromosomes themselves
can be heteromorphic (in most birds) or homomorphic (in
the flightless ratites). Sex in birds might be determined by
the dosage of a Z-linked gene (two in males, one in females)
or by a dominant ovary-determining gene carried on the W
sex chromosome, or both. Sex chromosome aneuploidy has
not been conclusively documented in birds to differentiate
between these possibilities. By definition, the sex chromo-
Request reprints from Craig A. Smith
Murdoch Childrens Research Institute and University of Melbourne
Department of Paediatrics, Royal Childrens Hospital
Parkville, Vic. 3052 (Australia)
telephone: +61 3 8341 6353; fax: + 61 3 8341 6429
e-mail: craig.smith@mcri.edu.au
© 2007 S. Karger AG, Basel
1424–8581/07/1174–0165$23.50/0
Accessible online at:
www.karger.com/cgr
somes of birds must carry one or more sex-determining
genes. In this review of avian sex determination, we ask
what, when and where? What is the nature of the avian sex
determinant? When should it be expressed in the develop-
ing embryo, and where is it expressed? The last two ques-
tions arise due to evidence suggesting that sex-determining
genes in birds might be operating prior to overt sexual dif-
ferentiation of the gonads into testes or ovaries, and in tis-
sues other than the urogenital system.
Copyright © 2007 S. Karger AG, Basel
Sex is determined in birds at the time of fertilisation
through the inheritance of the sex chromosomes (ZZ male
versus ZW female). While the mechanism of avian sex de-
termination is still unknown, it must involve a sex-linked
gene carried on either or both of the sex chromosomes. It
has long been thought that, as in eutherian mammals, a sex-
determining gene/s carried on the sex chromosome acts
solely in the embryonic gonads, initiating testicular or ovar-
ian differentiation. The developing gonads then synthesise
and release sex steroid hormones to masculinise or feminise
the gonadal ducts, external genitalia, brain and other tissues
(Romanoff, 1960). In mammals, the key sex determinant is
SRY , carried on the Y chromosome and transiently ex-
pressed in the embryonic male gonad to trigger a genetic
cascade leading to testis formation. Testosterone released
from the male gonad then masculinizes the tissues, which
otherwise follow a female developmental program. Al-
Manuscript received 21 July 2006; accepted in revised form for publication by I. Nanda, 13 September 2006.

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Cytogenet Genome Res 117:165–173 (2007) 166
1993; Schmid et al., 2005). The exception is seen in the
flightless ratites (emus, ostriches, etc.), in which the Z and
W are essentially identical in size (homomorphic) (Takagi
et al., 1972). The differentiated sex chromosomes of ad-
vanced, modern birds (carinates) have derived from a ho-
momorphic autosomal chromosome pair that pre-dates
their divergence from ratites (Fridolfsson et al., 1998; Oga-
wa et al., 1998). In the ratites, these chromosomes have dif-
ferentiated little, but they are homologous to those of the
chicken (Shetty et al., 1999) and they must carry sex deter-
minants, as in all other birds. Our current understanding of
avian sex determination largely derives from research on
the chicken (Gallus gallus domesticus) . This species is wide-
ly used in developmental biology, it is of agricultural impor-
tance and its genome has now been sequenced (Hillier et al.,
2004). As in other gallinaceous birds, the chicken has het-
eromorphic sex chromosomes. The large chicken Z sex
chromosome has over 350 genes, while the smaller, hetero-
chromatic W chromosome has very few, probably less than
twenty (Mizuno et al., 2002; Stiglec et al., 2007). Recent
studies indicate that the Z sex chromosome is strongly con-
served across all birds, while the W is far less conserved and
has undergone varying degrees of degradation (Shetty et al.,
1999; Nanda and Schmid, 2002; Raudsepp et al., 2002; Ber-
lin and Ellegren, 2006).
Sex could be determined by Z chromosome dosage (two
for males, one for females), by a dominant W gene carried
only in females, or by both mechanisms. The Z dosage hy-
pothesis requires that the relevant gene is not subject to dos-
age compensation between the sexes. Indeed, the chicken Z
chromosome does not appear to undergo widespread inac-
tivation typical of the mammalian X chromosome (Kuroda
et al., 2001; Ellegren, 2002). Although RT-PCR studies sug-
gest dosage equalization between the sexes for several Z-
linked genes (McQueen et al., 2001), recent microarray data
indicate that dosage compensation in birds is weak in com-
parison to that seen in mammals (Itoh et al., 2007). Defini-
tive sex chromosome aneuploidy (2A:ZZW or 2A:ZO) has
not been reported in birds, and it may be lethal in embryo,
at least in the case of ZO individuals (Graves, 2003). How-
ever, a line of triploid chickens with a 3A:ZZW genotype,
reported by Thorne and Sheldon (1993), developed as inter-
sexes. At hatching, these birds had a right testis and tran-
sient left ovotestis, and a female external phenotype. The
ovarian component degenerated with age. This suggests
that the W chromosome carries a female determinant, be-
cause some ovarian tissue could form despite the presence
of two Z chromosomes (Lin et al., 1995). However, regres-
sion of this ovarian tissue in the ZZW triploid chickens im-
plies that the putative W-linked female determinant is not
dominant and that it can be ‘overridden’ by two Z chromo-
somes. A complication here, however, is the fact that these
birds were complete triploids, so three copies of the entire
genome may have influenced gonadal development. Re-
cently, Arit et al. (2004) reported a female great reed warbler
with an inferred 2A:ZZW genotype, based on the heterozy-
gous inheritance of two Z-linked microsatellite markers.
This also supports the notion that the W carries a female
determinant, regardless of the number of Z chromosomes.
However, the two Z alleles were apparently not passed on to
the male offspring of this bird and its ZZW status was not
confirmed by karyotyping. Both the Z dosage and domi-
nant W hypotheses therefore remain viable.
Gonadal sex differentiation and conserved
sex-determining genes
As in mammals, studies on chicken sex determination
have focussed on the embryonic gonads, where sex-deter-
mining genes are logically thought to operate. The gonadal
rudiments form on the medial surface of the embryonic
(mesonephric) kidneys at approximately day 3.5 of develop-
ment in the chicken embryo (developmental stage 20; Ham-
burger and Hamilton, 1951) ( Fig. 1 ). At this stage, gonads
are morphologically identical between the sexes (or ‘bipo-
tential’) ( Fig. 1 A–C). Recent studies have shown that fibro-
blast growth factor signalling is important for the formation
of the gonad during the indifferent stage (Yoshioka et al.,
2005). Sexual differentiation is first detectable at the histo-
logical level at day 6.5 (developmental stage 30), when sem-
iniferous cords begin to coalesce in the interior (medulla) of
the male gonad, while the outer cortical layer starts to pro-
liferate in females ( Fig. 1 B, C) (van Limbough, 1973; Ebens-
perger et al., 1988). Thus, morphological differentiation be-
gins at the same time in the two sexes (in contrast to mam-
mals, in which overt signs of sexual differentiation are first
seen in males). Some researchers have reported sex differ-
ences in the size and distribution of primordial germ cells
prior to day 6.5 (Zaccanti et al., 1990) but, since germ cells
are not required for somatic differentiation of the gonads
(McCarrey and Abbott, 1978, 1982), they will not be consid-
ered further here. Ovarian differentiation in the chicken
embryo is characterised by asymmetry; although both go-
nads initially undergo cortical proliferation, this process is
not maintained in the right gonad ( Fig. 1 A). As embryogen-
esis proceeds beyond day 6.5, cortical proliferation stops in
the right gonad and, by day 8.5, the right cortex is reduced
to a flattened epithelial monolayer. In contrast, the left go-
nad has a highly thickened cortex, populated by proliferat-
ing germ cells. Both female gonads have an underlying me-
dulla, which is steroidogenic and characterised morpholog-
ically by extensive lacunae (fluid-filled cavities). In males,
bilateral testes are characterised by well-developed seminif-
erous cords, containing supportive Sertoli cells and mitoti-
cally arrested germ cells.
Based on this morphology, sex-determining genes in the
chicken embryo are expected to operate within the gonads
just prior to the onset of sexual differentiation, which begins
at day 6.5 (stage 30). Two genes expressed just prior to mor-
phological differentiation are SOX9 (SRY-like BOX, no. 9)
and CYP19A1 (aromatase ) ( Fig. 2 ). SOX9 is expressed from
stage 30 (day 6) only in male gonads, while CYP19A1 is ex-
pressed from the same stage, but only in female gonads
( Fig. 2 ) (Smith et al., 1997, 2005; Nakabayashi et al., 1998).
SOX9 is required for testis development in mammals, where

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Cytogenet Genome Res 117:165–173 (2007) 167
it regulates Sertoli cell differentiation and seminiferous cord
formation. It is also likely to perform the same role in birds
(Kent et al., 1996). CYP19A1 encodes the rate-limiting en-
zyme responsible for estrogen synthesis, which is required
for ovarian differentiation in birds (reviewed in Smith and
Sinclair, 2004). The embryonic female gonad in birds there-
fore synthesises estrogen from an early age (reviewed in
Scheib, 1983). Estrogen receptor alpha (ER ? ) is expressed in
both sexes, but expression declines in males as development
proceeds (Andrews et al., 1997; Smith et al., 1997; Naka-
bayashi et al., 1998). ER ? is primarily expressed in the go-
nadal cortex and may underlie the cortical hypertrophy typ-
ical of ovarian differentiation (Nakabayashi et al., 1998).
A number of other genes shown to play a role in mam-
malian gonadal sex differentiation have homologues in
birds. These include SF1 ( S teroidogenic Factor-1, also called
Ad4BP ) and DAX1 (Dosage sensitive sex reversal, A drenal
hypoplasia congenita, X linked, no. 1). These genes encode
E5.5
(st. 27-28)
E6.5
(st.30)
ZZ
male
ZW
female
Bipotential
gonad
ovarian
cortex
md
md
c
C
100µm
ZZ
male
ZW
female
Testis
Ovary
Bipotential
gonad
B
c
md
pgc
testis
cord
proliferated
cortex
lacunae
(cavities)
500µm
L
R
L
R
A
ZZ
male
ZW
female
Bipotential
gonad
Testis
Ovary
E3.5
(st. 19-20)
E8.5
(st. 35)
cord
interstitium
cord
interstitium
germ cells
E6.5
(st. 30)
L
R
L
R
Fig. 1. Gonadal development and sexual
differentiation in the chicken embryo. ( A )
Schematic of gonadal anatomy. At embryonic
day 3.5 (stage 19–20), the gonads are undif-
ferentiated or bipotential (shown in blue), on
the medial surface of the mesonephric kid-
neys (pale brown). In ZZ males, bilateral tes-
tes develop, while, in ZW females, the left go-
nad becomes an ovary and the right regresses.
( B ) Schematic of gonadal histology. The bipo-
tential gonad comprises an outer cortical layer
(c), and underlying medulla (md). Primordial
germ cells (pgc) are concentrated mainly in
the cortex. Testis formation involves the con-
densation of medullary cords into seminifer-
ous (testis) cords. The (left) ovary is charac-
terised by cortical proliferation, while the me-
dulla becomes reticulated, with numerous
cavities (lacunae). ( C ) Gonadal histology in
the chicken embryo. At embryonic day 5.5
(E5.5; stage 27–28) the gonads are histologi-
cally undifferentiated. The cortex (c) is dis-
tinct from the underlying medulla (md). In
ZZ males, condensing cords are apparent by
E6.5 (stage 30). The interstitium (site of Ley-
dig cell development and testosterone synthe-
sis) is present between the cords. In contrast,
cortex proliferation, including germ cell pro-
liferation, is apparent in ZW females.

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Cytogenet Genome Res 117:165–173 (2007) 168

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Cytogenet Genome Res 117:165–173 (2007) 169
orphan nuclear receptors. Other factors include signalling
molecules such as WNT4 (Wingless, Int-related, no. 4), and
the AMH (Anti-Müllerian Hormone) gene (Smith et al.,
1999a, b, 2000; Oreal et al., 2002). In the chicken embryo,
these genes have expression profiles consistent with con-
served roles in gonadal sex differentiation. However, none
of the above genes are sex-linked, including SOX9 and
CYP19A1 . While SRY represents the sex-determining trig-
ger in mammals, this gene is absent in birds and an alterna-
tive genetic trigger(s) must exist.
What is the nature of the sex determinant(s)?
In mammals, the SRY gene encodes a transcription factor
regulating gonadal sex differentiation. There is no reason to
expect a priori that the avian sex determinant also encodes a
transcription factor, although the only other master sex de-
terminant identified in vertebrates, the DMY gene in some
teleost fish species, also encodes a transcription factor (Mat-
suda et al., 2002; Nanda et al., 2002). The two most promising
candidate avian sex determining genes are currently DMRT1
(related to DMY ), located on the Z sex chromosome, and
HINTW , located on the W chromosome. DMRT1 (Doublesex
and Mab-3 Related Transcription factor, no. 1) encodes a
novel nuclear transcription factor with a DNA-binding motif
(the DM domain). DMRT1 is unrelated to SRY . Deletions of
human DMRT1 are associated with XY male-to-female sex
reversal (Raymond et al., 1999a) while mouse Dmrt1 null
mutants have (postnatal) testicular dysfunction, pointing to
a role in male development (Raymond et al., 2000). DMRT1
is conserved across vertebrates and even has homologues in-
volved in sex determination in Drosophila and Caenorhabdi-
tis elegans (Raymond et al., 1998; Nanda et al., 1999). DMRT1
is expressed specifically in the embryonic urogenital system
of all vertebrates that have been examined, from fishes to
birds and mammals, and it is more highly expressed in male
gonads (Raymond et al., 1999b; Smith et al., 1999c; Shan et
al., 2000; reviewed in Zarkower, 2001). Chicken DMRT1 is
Z-linked and is absent from the W sex chromosome. It is
therefore present in two doses in males and in one dose in
females. Furthermore, it is not dosage compensated (Nanda
et al., 2000). In chicken embryos, it is expressed only in the
gonads and Müllerian ducts, where expression is higher in
males compared to females, from as early as day 3.5–4.5
(stage 20–25) ( Fig. 2 ). Significantly, while many Z-linked
genes in chicken map to both Z and W chromosomes of rat-
ites, excluding them as universal avian sex determinants,
DMRT1 maps only to the Z chromosome of at least one ratite,
the emu (Dromaius novaehollandiae) (Shetty et al., 2002).
DMRT1 is therefore a candidate avian sex determinant under
the Z dosage hypothesis, with higher expression correlating
with male (testicular) differentiation.
The dominant W hypothesis predicts an ovary determi-
nant carried on the W sex chromosome that directs female
development. In the absence of the W, male development
would occur. One such candidate female gene is HINTW
(HIstidine triad NucleoTide binding protein, W-linked).
This gene was initially identified independently by two
groups and was called WPKCI, W-linked Protein Kinase C
inhibitor (Hori et al., 2000) and ASW, Avian Sex related, W-
linked (O’Neill et al., 2000). Because it is now clear that this
gene encodes a derived version of a histidine triad nucleo-
tide binding protein (HINT), rather than a protein kinase
inhibitor (PKCI), HINTW is the more appropriate and ac-
cepted nomenclature (see Ceplitis and Ellegren, 2004).
HINT proteins form a branch of the HIT family of nucleo-
tide hydrolase enzymes, they are conserved across animals
and they specifically hydrolyse adenosine from lysine resi-
dues. However, HINTW is very unusual because it lacks the
key catalytic motif (the histidine triad) that confers enzyme
activity to all other bona fide HINT proteins, including a
HINT homologue, HINTZ , which resides on the chicken Z
sex chromosome. HINTW is reiterated over 40 times on the
chicken W sex chromosome, it is conserved on the W in
carinate birds, and phylogenetic studies indicate that this
gene has undergone positive selection during evolution
(Ceplitis and Ellegren, 2004; Backström et al., 2005).
HINTW is widely expressed in female chicken embryos, in-
cluding strong expression in the gonads ( Fig. 2 ). The HINTZ
gene, encoding a bona fide HINT enzyme, is expressed in
the gonads of both sexes (Hori et al., 2000). Given its lack of
a catalytic domain, it has been hypothesised that HINTW
acts in a dominant negative fashion to block HINTZ func-
tion in the gonads, leading to female sexual differentiation
(Pace and Brenner, 2003). This idea is supported by recent
in vitro studies showing a direct interaction between re-
combinant HINTW and HINTZ, which inhibits the bio-
chemical function of the latter (Moriyama et al., 2006). Tak-
en together, these data point to a role for HINTW in avian
sex determination. In the ratites, Southern blot analysis de-
tects one band in both sexes; this indicates that the gene is
not reiterated on the ratite W chromosome (Hori et al.,
2000), and it is unclear at this stage whether a distinct
HINTW gene exists in this group. If HINTW is absent in
ratites, it negates this gene as a universal avian sex determi-
nant. Alternatively, sex could be determined differently in
ratites. This seems unlikely, given the common ancestry of
ratite and carinate sex chromosomes.
Fig. 2. Timing of gene expression in embryonic chicken gonads, as
assessed by whole mount and tissue section in situ hybridisation. The
onset of morphological differentiation into testes or ovaries is shown
(from day 6.5; stage 30). In ZZ males, DMRT1 mRNA expression is de-
tectable from day 3.5–4.5 (stages 20–25). In comparison, SOX9 in males
is first detectable at day 6.0 (stage 29). In ZW females, HINTW mRNA
is expressed from days 3.5–4.5 (stages 20–25). FET1 mRNA is also ex-
pressed from days 3.5–4.5, but asymmetrically expressed, with stron-
ger expression in the left gonad. FET1 expression is down-regulated in
the gonads by day 6.5 (stage 30). In comparison, CYP19A1 is first de-
tectable at day 6.0 (stage 29). AMH is first detectable at stage 25 in both
sexes, but appears higher in males, according to tissue section in situ
hybridisation. By stage 28, this dimorphism in AMH is clear (left go-
nads only are shown). The onset of AMH expression precedes SOX9
expression in males, and CYP19A1 expression in females. The AMH
expression is taken from Oreal et al. (1998) with permission.

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Cytogenet Genome Res 117:165–173 (2007)170
The potential disruption of HINTZ by HINTW de-
scribed above indicates that the Z and W sex chromosomes
may directly interact to regulate avian sex determination.
Another study also suggests direct interaction between
these chromosomes, but in a different manner. Teranishi et
al. (2001) identified a tandem repeat region on the chicken
Z chromosome that is highly methylated in the two Z chro-
mosomes of male cells (the male hypermethylated region,
MHM), but far less so in females. Due to the lack of exten-
sive methylation, the region is transcribed from the single Z
of female cells, resulting in non-coding mRNA that accu-
mulates at the site of transcription, near the DMRT1 locus
(within about 23 kb). This is reminiscent of Xist , the X-in-
activation transcript in mammals. Interestingly, in triploid
ZZW cells, the two Z chromosomes are hypomethylated
and transcribed. In contrast, ZZZ triploid cells remain hy-
permethylated on the Z. This clearly points to the W chro-
mosome as a potential regulator of methylation on the Z.
The W may therefore encode a factor, such as a demethylat-
ing enzyme, that allows transcription of the non-coding
RNA in females. This RNA then coats the Z and could si-
lence or down-regulate potential male genes, such as DMRT1
(Teranishi et al., 2001). Such potential interplay between the
two sex chromosomes to control sex determination has not
been reported in mammals or other vertebrates. The puta-
tive factor derived from the W chromosome remains to be
identified. Interestingly, it has recently been reported that Z
chromatin is hyperacetylated at histone 4 in female but not
male chicken fibroblasts. This hyperacetylation is adjacent
to the MHM locus and suggests a functional link, because
hyperacetylation predicts chromatin hyperactivity (Bisoni
et al., 2005). However, it is unclear at present whether female
hyperacetylation is a cause or an effect of female transcrip-
tion of the neighbouring MHM. Indeed, both processes
could be related to dosage compensation and unrelated to
sex determination.
When are sex-determining genes operating?
It is generally expected that the expression of sex-deter-
mining genes will begin in embryonic gonads just prior to
overt gonadal sex differentiation (day 6.5, stage 30, in the
chicken). However, current candidate genes do not meet
this expectation. As shown in Fig. 2 , the candidate male and
female genes, DMRT1 and HINTW , are both expressed in
the gonads from at least as early as day 4.5, well prior to the
onset of morphological differentiation and prior to the ex-
pression of the key genes, SOX9 and CYP19A1 (day 6.0–6.5).
Therefore, these genes may be initiating sexual differentia-
tion at the molecular level in a way that is not detectable
morphologically until later. If so, they would be activating
the SOX9 and CYP19A1 genes indirectly. This would make
the avian system dissimilar to the mammalian system, in
which SRY, for example, sets in motion SOX9 expression
and seminiferous cord formation over a short, tightly regu-
lated developmental window. In birds, the sex determinants
may be expressed very early, leading to further downstream
gene expression well before histological differentiation be-
comes apparent. A potential intermediate gene in the chick-
en is the candidate aromatase regulator, FOXL2 , which is
expressed in the gonads female-specifically from day 5
(stage 28) (Loffler et al., 2003; Gorovoun et al., 2004; Hud-
son et al., 2005). FOXL2 is autosomal but is likely to repre-
sent a link between the female determinant and CYP19A1
(aromatase).
Another potential intermediate in the chicken sex-deter-
mining pathway may be Anti-Müllerian hormone (AMH).
AMH is a hormone secreted by Sertoli cells, and it induces
regression of the paired Müllerian ducts, which otherwise
form oviducts (Fallopian tubes) in females. Accordingly, in
the mammalian embryo, AMH is only expressed in males.
In contrast, AMH is expressed in both male and female
chicken embryos because the right Müllerian duct in fe-
males follows the male pattern and regresses. In mammals,
AMH is expressed downstream in the male pathway. In the
mouse, for example, Amh is expressed after Sry and Sox9 ,
and indeed it has been shown that SOX9 acts together with
WT1, SF1 and GATA4 to regulate the Amh gene (reviewed
in Brennan and Capel, 2004). However, in the chicken em-
bryo, AMH expression begins very early. According to tis-
sue section and whole mount in situ hybridisation, low lev-
els of AMH expression are first detectable in the gonads of
both sexes stage 25–27 (stage day 4.5–5.0) (Oreal et al., 1998;
Nishikimi et al., 2000). This is prior to the onset of histo-
logical differentiation and prior to the onset of SOX9 ex-
pression in males (day 6.0; stage 29; Oreal et al., 1998). Re-
cent evidence suggests that SF1 may be at least partly re-
sponsible to activating AMH gene expression in avian
embryos (Takada et al., 2006). While AMH expression ap-
pears similar in both sexes at the very early stages, it be-
comes stronger in male chicken embryos by stage 28 (day
5.5–6) ( Fig. 2 ). This again points to sexually dimorphic gene
regulation prior to overt histological differentiation of the
gonads. Because it is male-enriched at this early stage, AMH
may respond to the higher levels of DMRT1 or another un-
known male factor in ZZ embryos. Therefore, AMH may
have a more ‘upstream’ position in the sex determining
pathway of birds compared to mammals. However, a linear
cascade linking DMRT1, AMH and genes such as SOX9 has
not been demonstrated in birds.
If sex is determined by gene dosage in birds, as suggested
by the expression of DMRT1 , then cumulative or threshold
expression levels could be important. For example, although
DMRT1 is expressed sexually dimorphically from as early
as day 3.5, it is possible that its function in sex determina-
tion requires a specific threshold level of expression to be
achieved. DMRT1 mRNA expression levels in male gonads
appear to be highest at day 6 (stage 29) (Smith et al., 2003),
which is just prior to morphological differentiation and co-
incides with the onset of SOX9 expression. Similarly,
HINTW , although expressed from day 2–3 in females, is
most highly expressed in the gonads at day 5–6 (stages 27–
29), just prior to aromatase activation (day 6; stage 30) (Hori
et al., 2000). DMRT1 and/or HINTW expression could reg-
ulate their own expression, leading to a threshold level re-

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Cytogenet Genome Res 117:165–173 (2007) 171
quired for triggering gonadal sex differentiation. In the case
of DMRT1 , alternative splicing may also play a role in defin-
ing its function. DMRT1 is alternatively spliced in human
testis (Cheng et al., 2006) and in the gonads of lower verte-
brates (Sreenivasulu et al., 2002; Guo et al., 2005). Most re-
cently this has also been shown for DMRT1 transcripts in
embryonic chicken gonads, with a male specific splice vari-
ant detected at stage 31, the time of gonadal sex differentia-
tion (Zhao et al., 2007). It is noteworthy that differential
splicing of the Drosophila homologue, Doublesex, is crucial
to its sex-specific function. Therefore, different DMRT1 iso-
forms may be produced at different embryonic stages in the
avian embryo, with an isoform specific to days 5 and 6 trig-
gering gonadal sex differentiation. This does imply, how-
ever, that another sex-specific factor controls sexually di-
morphic alternative splicing of DMRT1 , unless it is auto-
regulated.
Alternatively, DMRT1 and HINTW may not be involved
in avian sex determination. At present, there are few alter-
native sex-linked candidates. FET1 (Female-Expressed
Transcript, no. 1) is one potential W-linked candidate gene
(Reed and Sinclair, 2002). This gene was isolated from a
screen for novel sex-specific genes in chicken and it is ex-
pressed asymmetrically in female gonads from day 4.5 to
day 6.5 ( Fig. 2 ). While FET1 does not appear to have a ho-
mologue on the chicken Z, its presence in other birds has
not been definitely shown. Recent sequence analysis sug-
gests that it encodes an avian retroviral element (C. Smith,
unpublished), and there is no evidence that is it translated,
which may undermine its possible involvement in sex deter-
mination. In addition, an ovary determinant in the chicken
should be expressed in both left and right gonads, since
FOXL2 and CYP19A1 are expressed in both gonads, while
FET1 is only expressed in the left gonad at the time of sex-
ual differentiation (although it could activate genes in the
contralateral gonad through indirect signalling). In a fe-
male-minus-male subtracted whole embryo cDNA macro-
array, Yamada et al. (2004) isolated two novel W-linked se-
quences in chicken, 2d-2D9 and 2d-2F9. These sequences
have no obvious Z-linked homologues. While 2d-2D9 has
no obvious motifs, 2d-2F9 has homology to an ATPase su-
perfamily. Both genes are expressed very early in the chick-
en embryo (from day 2, stages 12–13, before gonad forma-
tion) although their exact sites of expression are yet to be
determined.
Where are sex-determining genes operating?
The widespread expression of the W-linked gene,
HINTW, throughout the female chicken embryo, and early
expression of the novel sequences 2d-2D9 and 2d-2F9 in
whole embryos raise the question of where potential sex-de-
termining genes are operating. In addition to being ex-
pressed in the gonads, sex-determining genes could be
functioning directly in non-gonadal tissues in a cell auton-
omous fashion. This seems to apply to sexual differentiation
of the avian brain. Scholz et al. (2006) reported sexually di-
morphic gene expression in day 4 embryonic chicken brains,
pre-dating gonadal sex differentiation and prior to aroma-
tase-mediated estradiol synthesis in females. A number of
genes were exclusively or more highly expressed in females,
including HINTW , the Z-linked MHM non-coding RNA
described above, and a novel W-linked sequence, ABTW
(avian brain W-linked transcript). This suggests direct ef-
fects of sex-determining genes in tissues outside the gonads.
The existence of gynandromorphic birds also supports this
idea. Such birds are bilateral sex chimeras, with male fea-
tures on one side of the body and female features on the
other. In the case of a gynandromorphic zebra finch, the
phenotype was female on the left side and male on the right.
This included an ovary and female plumage on the left side
of the body, with a testis and male plumage on the right side.
HINTW was only expressed on the left side of the brain,
which was feminised (Agate et al., 2003). Genotypically, the
left side of the body, including the gonads, was ZW, while
the right side was ZZ. It is highly unlikely that such a phe-
notype could arise from hormonal mechanisms alone, sug-
gesting a direct genetic effect in individual cells. Agate et al.
(2003) concluded that, at least in the brain, sexual differen-
tiation is controlled by both the cell’s genetic sex and its
hormonal environment. This is consistent with the observa-
tion that gonadal steroid hormones alone cannot account
for sexual dimorphisms seen in the neural song circuit of
the brain, in plumage or sexual behaviour (Arnold, 1997;
Gahr, 2003). Avian sex-determining genes might therefore
directly influence sexual differentiation in tissues indepen-
dently of the gonads.
Conclusions
Sex determination in birds involves genes acting in the
gonads, but the exact nature of the sex determinant(s) and
its onset of expression remain undefined. The DMRT1 gene
is currently the most promising candidate gene for avian sex
determination under the Z chromosome dosage hypothesis.
Meanwhile, the W-linked gene, HINTW , represents a very
good candidate under the dominant W hypothesis. If either
or both of these genes are involved in avian sex determina-
tion, their expression profiles in the chicken embryo suggest
that they may act well before the onset of gonadal sex dif-
ferentiation. The widespread expression of HINTW outside
the urogenital system raises the possibility that avian sex
determinants may act directly in tissues independently of
the gonads. Under this scenario, sex in birds may be deter-
mined by direct genetic as well as hormonal mechanisms.
Definitive proof of a role for the current candidates will in-
volve gene over-expression or knockdown in ovo, using avi-
an viral vectors (Chapman et al., 2005; Harpavat and Cep-
ko, 2006). Recently, the isolation and genetic modification
of chicken primordial germ cells for the production of trans-
genic poultry has been reported (van de Lavoir et al., 2006).
This paves the way for the production of sex-reversed trans-
genic birds over-expressing candidate genes such as DMRT1
and HINTW .

Page 8

Cytogenet Genome Res 117:165–173 (2007)172
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