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Somatic sex identity is cell-autonomous in the chicken

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In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in birds, sexual differentiation is substantively cell autonomous.
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ARTICLES
Somatic sex identity is cell autonomous in
the chicken
D. Zhao
1
*, D. McBride
1
*, S. Nandi
1
, H. A. McQueen
3
, M. J. McGrew
1
, P. M. Hocking
2
, P. D. Lewis
4
, H. M. Sang
1
& M. Clinton
1
In the mammalian model of sex determination, embryos are considered to be sexually indifferent until the transient action of
a sex-determining gene initiates gonadal differentiation. Although this model is thought to apply to all vertebrates, this has
yet to be established. Here we have examined three lateral gynandromorph chickens (a rare, naturally occurring
phenomenon in which one side of the animal appears male and the other female) to investigate the sex-determining
mechanism in birds. These studies demonstrated that gynandromorph birds are genuine male:female chimaeras, and
indicated that male and female avian somatic cells may have an inherent sex identity. To test this hypothesis, we
transplanted presumptive mesoderm between embryos of reciprocal sexes to generate embryos containing male:female
chimaeric gonads. In contrast to the outcome for mammalian mixed-sex chimaeras, in chicken mixed-sex chimaeras the
donor cells were excluded from the functional structures of the host gonad. In an example where female tissue was
transplanted into a male host, donor cells contributing to the developing testis retained a female identity and expressed a
marker of female function. Our study demonstrates that avian somatic cells possess an inherent sex identity and that, in
birds, sexual differentiation is substantively cell autonomous.
Sexual development in vertebrates is thought to be governed by
general principles defined in the early to mid-twentieth century
1,2
.
These principles state that the sexual phenotype of individuals is
dependent on the gonad: male and female somatic cells and tissues
are initially sexually indifferent and sexual dimorphism is imposed by
the type of gonad that develops. Although these principles have been
challenged, most notably by work on songbird neural development
3–6
and marsupial development
7,8
, these observations are generally con-
sidered as exceptions. In the currently acceptedmodel, gonadal differ-
entiation is triggered in sexually indifferent embryos by the transient
action of a sex-determining gene. In mammals, the sex-determining
gene is known to be the testis-determining Sry gene carried by the
male-specific Y chromosome
9
. Although all vertebrates are thought
to conform to this general model, with the exception of Sry in mam-
mals and Dmy in medaka
10,11
, no other vertebrate sex-determining
genes have been confirmed.
In terms of morphology, birds seem to conform to the mammalian
pattern: male and female embryos are sexually indistinguishable until
around days 5–6 of incubation (Hamburger and Hamilton
12
(H&H)
stage 28/29) when the action of a sex-determining gene is thought to
initiate testis or ovary development
13
. However, in birds, not only is
the identity of the putative sex-determining gene unknown, the
nature of the sex-determining mechanism has not been established.
Current theories of sex determination in birds include the presence of
an ovary-determining gene on the female-specific W chromosome,
and a dosage mechanism based on the number of Z chromosomes
(females have one Z and one W sex chromosome whereas males have
two Z sex chromosomes)
14
. Currently, the best candidate for a testis-
determining gene in birds is DMRT1 (doublesex and mab-3-related
transcription factor 1). Expression of DMRT1 is restricted to the
gonads and it has recently been shown that repressing levels of
DMRT1 in male embryos has a ‘feminizing’ effect on the developing
testis
15
.
In an attempt to clarify the nature of the sex-determining mech-
anism in birds, we have investigated the composition of a number of
gynandromorph chickens. These birds are rare, naturally occurring
phenomena in which one side of the animal appears male and the
other female
16
. We investigated these birds with the expectation that
this condition resulted from a sex-chromosome aneuploidy on one
side of the bird, and that our analysis would provide evidence regard-
ing the nature of the avian sex-determining mechanism. Contrary to
expectations, our analysis established that the gynandromorphs were
in fact male:female chimaeras, and that the gynandromorphic pheno-
type was due to ZZ (male) and ZW (female) somaticcells responding
in different ways to the same profile of gonadal hormones. These
observations led to a series of transcriptome screens and embryonic
transplantation studies showing that male and female avian cells
possessed an inherent sex identity. Our studies demonstrate that in
chickens, gonadal development and the sexual phenotype are largely
cell autonomous and not principally dependent on sex hormones.
Gynandromorph birds are mixed-sex chimaeras
We obtained three adult lateral gynandromorph birds (designated
G1, G2 and G3) which we maintained and observed over a period of
24 months. These birds occur naturally and it has been suggested that
this condition results from the loss of a single sex chromosome at the
two-cell stage
17
. All three birds were ISA brown commercial hybrids
with sex-linked plumage colour. ISA brown males are heterozygous
for the dominant silver and recessive gold genes (Ss) and so have
white plumage; females possess only the gold gene (s-) and have
brown plumage. The birds displayed a marked bilateral asymmetry,
where one side of the animal appeared phenotypically female and the
*These authors contributed equally to this work.
1
Division of Developmental Biology and
2
Division of Genetics and Genomics, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin,
Midlothian EH25 9PS, UK.
3
Institute of Cell Biology, University of Edinburgh, West Mains Road, Edinburgh EH9 3JR, UK.
4
Animal and Poultry Science Department, University of
KwaZulu-Natal, Pietermaritzburg, South Africa.
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other side phenotypically male (Supplementary Fig. 1). Figure 1
shows a picture of G1 where the right side of the bird is female in
coloration (brown) and has a small wattle and small leg spur. In
contrast, the left side is male coloured (predominantly white), has
a large wattle and a large leg spur, a heavier leg structure and an
obviously greater mass of breast muscle, typical of a cockerel. Post
mortem, whole tissues from both sides were weighed and measured
and samples of all tissues were taken for later analysis. The measure-
ments performed on individual tissues from both sides of all three
animals supported the observation that these animals were, at least
phenotypically, half male and half female. On the side that appeared
male, tissues were larger and heavier and bones were longer and
denser than corresponding tissues and bones from the side with a
female appearance (Supplementary Table 1). Fluorescent in situ
hybridization (FISH) analysis using Z and W chromosome probes
was performed on preparations of blood cells from all three animals
and on multiple preparations of cultured skin cells from both sides
of birds G2 and G3. Whereas an autosomal probe demonstrated a
diploid chromosome constitution for G1 blood cells, the sex chro-
mosome probes demonstrated that approximately half of gynand-
romorph G1 cells were female (ZW) and half were male (ZZ)
(Fig. 2a). Similar FISH analyses of blood and primary fibroblast
cultures from birds G2 and G3 demonstrated that all three animals
were composed of a mixture of normal diploid male and female cells
(Supplementary Fig. 2 and Supplementary Table 2). Although a
recent analysis of a gynandromorph zebra finch
3
demonstrated that
both Z-chromosome and W-chromosome containing cells were pre-
sent, the possibility remained that such animals were composed of a
mixture of ZW and Z0 cells. Here we show that gynandromorph
birds are genuine male:female chimaeras and provide an explanation
for a phenomenon that has been debated for centuries
18
.
We next investigated whether the apparent bilateral asymmetry
reflected the distribution of ZZ and ZW cells by examining the cellular
composition of a variety of tissues from both sides of the individual
birds. Southern analysis using sex chromosome probes on genomic
DNA extracted from multiple tissues revealed that none of the tissues
from either side was composed exclusively of either ZZ- or ZW-
containing cells; that is, all tissues examined comprised a mixture of
both female and male cells(examples shown in Supplementary Fig. 3).
Multiple Southern analyses were performed on separate DNA samples
extracted from different regions of skin, from wattle and from breast
muscle from both sides of all three birds, to quantify the relative
proportions of male and female cells. Phosphorimager analyses com-
paring the hybridization signal obtained from DNA from gynandro-
morphic tissues with that obtainedfrom known amounts of male and
female DNA produced a measure of the relative proportion of male
and female cells in each tissue. Figure 2b shows the mean proportion
of female and male cells in skin, wattle and breast muscle from the
‘male’ side and ‘female’ side of all three birds. It is clear that tissues
from the side that appeared female contained more ZW (female) than
ZZ (male) cells, whereas tissues from the side that appeared male were
composed predominantly of ZZ cells (Supplementary Table 2). Our
data establishing the presence of both ZZ- and ZW-containing cells
indicate that it is highly unlikely that these birds arise as a consequence
of mutation at the two-cell stage of development, and would support
the hypothesis that gynandromorphs arise as a result of failure of
extrusion of a polar body during meiosis and subsequent fertilization
of both a Z- and W-bearing female pronucleus
19
.
The development of gonads in the gynandromorph birds was of
obvious interest (Supplementary Fig. 4). The type of gonad present
did not correspond to the external appearance but rather reflected the
cellular composition of the individual organs. The gonads differed
for each gynandromorph: G1 contained a testis-like gonad on the left
side, G2 contained an ovary-like gonad on the left side, and G3
contained a swollen testis-like structure on the left side (in contrast
to G1 and G2, G3 appeared female on the left side and male on the
right). The G1 testis-like gonad was composed primarily of sperm-
containing seminiferous tubules, whereas the G2 ovary-like gonad
was composed predominantly of large and small follicles. The gonad
from G3 comprised a mixture of empty tubules and small follicular-
like structures (ovo-testis). Southern analyses demonstrated that the
morphological appearance of the gonads conformed to the cellular
composition in that the structures that appeared to be testis and
ovary were composed principally of ZZ- and ZW-containing cells,
Right Left
Figure 1
|
Image of gynandromorph bird (G1). ISA brown bird where the
right side has female characteristics and left side has male characteristics
(white colour and larger wattle, breast musculature and spur).
ZZ/ZW
ba
Mean percentage
of cell type
0
20
40
60
80
100
Sk. Wa. BM Sk. Wa. BM
Female side’ Male side’
ZZ cells
ZW cells
Figure 2
|
Male and female cells in gynandromorph birds. a, FISH analysis
of sex chromosomes in gynandromorph blood cells. Shown are interphase
nuclei prepared from cultured blood cells from gynandromorph G1
hybridized according to standard FISH procedures with probes specific to
both the W and Z chromosome (XhoI repeat on W chromosome, and Z
chromosome bacterial artificial chromosome (BAC) containing the VLDL
receptor gene identified by screening the HGMP chicken BAC library).
Erythrocytes were hybridized with probes for Z chromosome (green) and W
chromosome (red). Cells contain either two Z chromosomes or one Z and
one W chromosome. b, Mean relative proportions of ZZ and ZW cells in
tissues from male and female sides of gynandromorph birds. The average
percentage of ZW and ZZ cells (Supplementary Table 2) in three tissues from
the phenotypically female side and from the phenotypically male side of
three gynandromorph birds is shown. Tissues from the sides that appear
female contain more ZW (female) than ZZ (male) cells, whereas tissues from
the sides that appear male are composed predominantly of ZZ cells. BM,
breast muscle; Sk., skin; Wa., wattle.
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respectively, whereas the ‘ovo-testis’ comprised a mixture of ZZ- and
ZW-containing cells.
Although the findings from our gynandromorph analyses are
uninformative in terms of elucidating the avian sex-determining
mechanism, they do lead to the conclusion that the classical dogma
of sex differentiation, where the phenotype is mainly determined by
gonadal hormonal secretions, does not apply to birds. These results
strongly indicate that the avian phenotype is dependent on the nature
of the cells comprising the individual tissue rather than being
imposed by the type of gonad formed: both sides of these animals
are exposed to an identical profile of gonadal products yet each side
responds differently to these stimuli. For example, although it is well
established that growth of the wattle is sensitive to testosterone
20
,itis
clear from Fig. 1 that a major determinant in wattle size is the cellular
composition of the tissue, and therefore cellular identity and gonadal
hormones both have a significant role in establishing the sexual
phenotype of this tissue. Our analyses led us to conclude that male
and female chicken somatic cells may have a cell-autonomous sex
identity.
Sex differences precede gonadal hormone influences
To investigate whether differences exist between male and female
cells independently of any possible gonadal influences we compared
the transcriptomes of male and female embryos at developmental
stages before the formation of the gonads (data not shown). These
analyses identified both messenger RNAs and microRNAs (miRNAs)
that were expressed in a sexually dimorphic fashion throughout the
embryos at stages before the formation of the gonadal precursor (the
genital ridge; H&H stage 21) and well before the generally accepted
point of sex determination in the chicken (that is, around day 5/6 of
incubation). Screening for mRNAs expressed exclusively in male or
in female embryos led to the identification of an mRNA encoded by a
W chromosome gene that was expressed ubiquitously in females.
This gene was designated FAF for female-associated factor and
sequences were deposited in the EMBL/GenBank databases (acces-
sion numbers AJ606294–AJ606297). Whole-mount in situ hybridiza-
tion analysis of embryos at stages before genital ridge formation
showed that FAF mRNA is expressed throughout the female embryo
as early as 18 h of incubation (H&H stage 4) (Fig. 3a). We also
identified a ubiquitously expressed miRNA that is present at levels
approximately tenfold higher in males than in females throughout
development, including at stages before the expected point of sex
determination (Fig. 3b and Supplementary Fig. 5). This miRNA is
encoded on the Z chromosome and the sequence has not previously
been reported in any other species (Gallus gallus mir-2954, accession
number AM691163). These observations are in agreement with other
studies that have identified sexually dimorphic gene expression in the
brain preceding morphological differentiation of the gonads, in both
chicken and mouse
21,22
. Although the functions of these particular
transcripts are unknown, these findings not only supported the
concept that the tissue phenotype was not dependent on gonadal
products, but also reinforced the suggestion that the phenotype
was defined by inherent differences between the male and female cells.
Chimaeras confirm cell-autonomous sexual differentiation
To test the hypothesis that the male and female cellular composition
defines phenotype, we generated embryos containing chimaeric
gonads comprised of a mixture of male and female cells. Gonadal
chimaeras were generated by transplantation of sections of presump-
tive mesoderm from green fluorescent protein (GFP)-expressing
embryos
23
at developmental stage 12 (day 2) to replace the equivalent
tissue of non-GFP embryos at the samestage of development (Fig. 4a).
Donor tissue was transplanted only to the left side of recipient
embryos as only the left ovary develops fully in the chick. Trans-
planted embryos were returned to the incubator and allowed to
develop until stage 35 (day 9). By stage 35, donor cells were incor-
porated into tissues on the left side of the embryo in the region
between the fore and hind limbs (Supplementary Fig. 6). In addition
to the gonads, donor cells were observed in a variety of tissues includ-
ing skin, muscle, mesonephros, Wolffian duct and Mu
¨llerian duct. A
minimum of four donor:host chimaeric gonads were generated for
each of the four possible combinations: male:male, female:female,
male:female and female:male. For each of these donor:host com-
binations, chimaeras were generated with different levels of donor
contribution—ranging from examples where the contribution of
donor cells was limited to isolated individual cells dispersed through-
out the host gonad, to instances where areas of the host gonad were
almost exclusively composed of donor cells. Gonad:mesonephros
pairs were collected at stage 35 and longitudinal frozen sections
were prepared for confocal microscopy. GFP expression was used to
estimate the extent of donor contribution to the individual chimaeric
gonads and immunohistochemistry (IHC) analysis was performed
with antibodies for both anti-Mu
¨llerian hormone (AMH) and aro-
matase. AMH is a marker of functionally ‘male’ cells
24
(expressed by
precursor Sertoli cells of the sex cords) whereas aromatase is a marker
of functionally ‘female’ cells
25
(expressed by cells in the female medul-
lary region). At stage 35 of development, the male gonad is composed
of a thin layer of cortex tissue surrounding a medullary region which
contains the developing sex cords (expressing AMH) separated by
interstitial connective tissue. In contrast, the female left ovary com-
prises a greatly thickened cortex surrounding a smaller less-structured
medullary region (expressing aromatase). Figure 4b shows the normal
expression of AMH and aromatase in stage 35 male and female
gonads, respectively. It is clear that the testis is composed almost
exclusively of medullary tissue and that AMH is expressed in distinct
cord-like structures within this tissue. In contrast, the developing
ovary comprises a definitive cortex enclosing a reduced medulla and
aromatase is expressed in cells throughout the medulla. Examples of
a
Stage 14 Stage 20
mmff
miRNA
U6
H&H stage 4 H&H stage 14 H&H stage 20
m
m
f
m
f
f
b
Figure 3
|
Sexually dimorphic expression in early chick embryos.
a, Expression of FAF in male and female embryos before development of
genital ridge/gonads. Whole-mount ISH showing expression of FAF
(purple) in embryos at 18 h, 48 h and 72 h of development (H&H stages 4
(original magnification, 340), 14 (320) and 20 (310), respectively). FAF is
clearly expressed throughout the female embryos at all developmental stages
and is not expressed in male embryos. FAF is not expressed in extra-
embryonic tissues of the female. The FAF transcript is encoded by the
genomic DNA complementary to the intergenic regions between copies of
the W-chromosome repeat gene Wpkci (also called HINTW)
34,35
and
transcribed in the opposite orientation. f, female; m, male. b, Expression of
novel chicken miRNA (Gallus gallus mir-2954). Expression in whole
embryos at 48 h (H&H 14) and 72 h (H&H 20) of development is shown.
This miRNA is clearly expressed in a sexually dimorphic fashion at stages
before the sexual differentiation of the gonads. This miRNA matches
sequence present in chicken Z-chromosome BAC clones AC192757 and
AC187119. U6 RNA was used as a loading control.
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all four donor:host combinations of chimaeric gonads are shown in
Fig. 4c and Supplementary Fig. 7. It is clear that GFP expression did
not affect the ability of donor cells to contribute to host tissues and to
function normally: in each case of same-sex chimaeras, either male or
female donor cells were integrated into all somatic compartments of
the respective host testis and ovary (cortex, sex cords and interstitial
tissue). Moreover, when integrated into the appropriate ‘functional’
compartment, donor male cells expressed AMH and donor female
cells expressed aromatase. In contrast, in mixed-sex chimaeras the
donor cells did not integrate into the ‘functional’ structures of the
host gonad: female donor cells in host testis medulla were not
recruited into the AMH-expressing sex cords and were restricted to
the interstitial tissue, whereas male donor cells in host ovary were
excluded from the aromatase-expressing structures. In mixed-sex chi-
maeras the inability of donor cells to form functional host structures
was evident regardless of the relative contribution of male and female
cells (Supplementary Fig. 7). The fact that female chicken cells in an
environment and location that induces testiculardevelopment cannot
be recruited into the functionally ‘male’ Sertoli cell compartment, and
male cells in an ovary-inducing environment are excluded from a
functionally ‘female’ compartment, strongly supports the suggestion
that chicken somatic cells possess a cell-autonomous sexual identity.
This is further supported by a striking example where the degree of
the female donor contribution was sufficient to effectively generate
an ‘ovo-testis’ in the host embryo (Fig. 4d). This mixed-sex chimaera
contained a gonad with an anterior portion composed almost exclu-
sively of female cells. Whereas the posterior portion contained testis-
like medulla with AMH-expressing sex cords, the region composed of
female cells did not form sex cords and did not express AMH. Most
surprisingly, the female cells in this region expressed aromatase. This
demonstratesthat although female cells in a male embryo can correctly
interpret gonadal location and differentiation signals, they respond in
a cell-autonomous manner characteristic of a female genotype (and
express aromatase). Our findings are in contrast with those from
mammalian mixed-sex chimaeras, where XX cells can become func-
tional Sertoli cells and XY cells can become functional granulosa
cells
26,27
.
These studies demonstrate that avian somatic cells possess a cell-
autonomous sex identity. Our results support and extend previous
findings
3
that showed that differences between male and female zebra
finch brains were a result of endogenous genetic differences in the
brain cells themselves. Our analysis of lateral gynandromorph birds,
showing that they are male:female chimaeras, and our experimental
generation of embryos with mixed-sex chimaeric gonads, together
gg
mm
md
c
t
m
o
m
o
m
m
t
t
b
a
d
Transplant
HostDonor
GFP/AMH
GFP/AROM
GFP AMH
GFP AROM GFP/AMHGFP AMH
GFP/AROMGFP AROM
GFP/AROMGFP AROM
GFP/AMHGFP AMH
Figure 4
|
Expression of male and female markers in chimaeric gonads.
a, Generation of chimaeras. Left: schematic illustrating transplantation of
presumptive mesoderm from GFP-expressing embryo to non-GFP embryo
at day 2. Right: image of mesonephros and gonads from chimaeric embryo at
day 9 showing donor contribution to left gonad (g), mesonephros (m) and
Mu
¨llerian duct (md). Original magnification, 320. b, Expression of female
and male markers in embryonic gonads. Expression of aromatase (AROM)
in ovary and anti-Mu
¨llerian hormone (AMH) in testis at day 9 is shown by
IHC. Original magnification, 3400. c, Integration of GFP-expressing donor
cells into host gonads. Panels in the first column show a low-magnification
view of sections through host gonads and illustrate the extent of donor cell
contribution. Panels to the right show higher-magnification views of
highlighted areas. Using IHC, donor cells are marked by GFP (green)
whereas expression of AMH and aromatase are shown in red. The fourth
column is a merged image of the images from the second and third columns.
In same-sex chimaeras, GFP-expressing donor cells co-localize with AMH-
expressing and aromatase-expressing cells in host testis and ovary,
respectively (yellow/orange in the fourth column). In mixed-sex chimaeras,
GFP-expressing donor cells do not co-localize with AMH or aromatase. m,
mesonephros; o, ovary; t, testis. d, Retention of female donor phenotype in
mixed-sex chimaeras. IHC showing expression of AMH (red in top row) and
aromatase (red in bottom row) in neighbouring sections from the gonad of a
female:male (donor:host) chimaera. Donor contribution is illustrated by
GFP (green) expression. The right column shows a merged image of the
images in left and middle columns. Regions containing a significant host
contribution (defined by the bottom bracket) formed sex-cord-like
structures and expressed AMH. Female donor cells were not incorporated
into AMH-expressing sex cords, as shown by the lack of GFP and AMH co-
localization. Regions composed primarily of female donor cells (defined by
top bracket) behaved as ovarian-like tissue and expressed aromatase, as
shown by co-localization of GFP and aromatase (yellow/orange). Scale bars
in cand dindicate 100 mm. IHC was performed following standard
procedures. Primary antibodies were (1:100) goat anti-human AMH (Santa
Cruz Biotechnology), (1:200) mouse anti-human cytochrome P450
aromatase (AbD Serotec) and (1:250) rabbit anti-GFP conjugated to Alexa
Fluor 488 (Invitrogen). Secondary antibodies were conjugated to Alexa
Fluor 594 (Invitrogen).
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indicate that male and female somatic cells possess a sex identity.
These observations indicate that there is a molecular mechanism
functioning in every cell that confers a sex-specific identity that
influences how individual cells respond to developmental and hor-
monal signals. We propose that cell-autonomous sex identity is
dependent on sexually dimorphic gene expression resulting from
the ‘dosage compensation’ system that operates to equalize the
phenotypic effects of characteristics determined by genes on the Z
chromosome. Recent evidence has shown that this system in birds is
not chromosome-wide and results in a large number of gene expres-
sion differences between male and female cells
28–31
. We have esti-
mated that this system of dosage compensation would result in at
least 300 non-compensated Z-chromosome genes
31
. Our identifica-
tion of sexually dimorphic transcripts that are expressed ubiquitously
from very early in development adds to these observations. On the
basis of our findings, and from evidence of the dosage compensation
system in birds, we propose that the overall mechanism of sex deter-
mination in birds differs significantly from the mammalian model
(Fig. 5). Although sexually dimorphic differentiation of the gonads
may be regulated independently from other somatic tissues, we pro-
pose that a male or female sex identity is imposed on the chicken
soma early in development by sex chromosome transcription and it is
this inherent molecular identity that triggers the appropriate testis or
ovary gene cascade in the developing genital ridge (for example, via
DMRT1 (ref. 15)). Although the gonads clearly have a significant
influence on the adult phenotype they do not dictate somatic differ-
ences to the same extent as in mammals. It is also possible that
elements of such a system are retained in certain mammals: previous
studies have shown that, in a marsupial mammal, the wallaby, forma-
tion of the mammary gland and scrotum is independent of gonadal
hormones
32
, and rather than exhibiting transient localized expres-
sion, Sry shows widespread expression in multiple tissues well before
the point of gonadal differentiation
33
. As Sry-type sex-determining
mechanisms have not yet been established for all vertebrate species, it
is possible that the model we propose where the phenotype of indi-
vidual tissues is largely defined by an inherent sex identity of the
somatic cells is not restricted to birds.
METHODS SUMMARY
Generation of chimaeric embryos:GFP embryos
23
and ISA brown embryos at
H&H stage 11/12 (13–15 somites) were used as donor and host, respectively. The
blunt end of donor eggs was pierced to create an air hole and a ‘window’ cut on
the midline. The embryos were removed and pinned on a 3% agarose surface
containing 0.5% India ink. Embryos were kept moist by the addition of PBS
containing 100 units ml
21
penicillin and 100 mgml
21
streptomycin (PBS-Pen/
Strep). A strip of presumptive mesoderm flanking presumptive somites 21–23
was removed and stored in CO
2
-independent medium (Invitrogen) containing
10% FBS and Pen/Strep. Host eggs were windowed as above and kept moist by
the addition of PBS-Pen/Strep. To help visualize somites, sterile India ink (20%
in PBS-Pen/Strep) was injected under the host embryos. Using a microneedle,
the vitelline membrane and a flap of ectoderm were folded back from the under-
lying mesoderm. A strip of presumptive mesoderm was then removed from the
host embryo taking care to leave the endoderm intact. The GFP-donor tissue was
then inserted into the host site and the ectodermal flap replaced. Two millilitres
of albumen was then withdrawn from the host eggs using a hypodermic syringe.
Transplanted eggs were tightly sealed with tape and incubated at 37 uCina
humidified incubator. All other methods are standard.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 10 August 2009; accepted 18 January 2010.
1. Lillie, F. R. Sex-determination and sex-differentiation in mammals. Proc. Natl Acad.
Sci. USA 3, 464
470 (1917).
2. Jost, A. Hormonal factors in the sex differentiation of the mammalian foetus. Phil.
Trans. R. Soc. Lond. B 259, 119
131 (1970).
3. Agate, R. J. et al. Neural, not gonadal, origin of brain sex differences in a
gynandromorph finch. Proc. Natl Acad. Sci. USA 100, 4873
4878 (2003).
4. Wade, J. & Arnold, A. P. Functional testicular tissue does not masculinize
development of the zebra finch song system. Proc. Natl Acad. Sci. USA 93,
5264
5268 (1996).
5. Arnold, A. P. Sexual differentiation of the zebra finch song system: positive
evidence, negative evidence, null hypothesis, and a paradigm shift. J. Neurobiol. 33,
572
584 (1997).
6. Wade, J. & Arnold, A. P. Sexual differentiation of the zebra finch song system. Ann.
NY Acad. Sci. 1016, 540
559 (2004).
7. Renfree, M. B. & Short, R. V. Sex determination in marsupials: evidence for a
marsupial-eutherian dichotomy. Phil. Trans. R. Soc. Lond. B 322, 41
53 (1988).
8. Glickman, S. E., Short, R. V. & Renfree, M. B. Sexual differentiation in three
unconventional mammals: spotted hyenas, elephants and tammar wallabies.
Horm. Behav. 48, 403
417 (2005).
9. Sekido, R. & Lovell-Badge, R. Sex determination and SRY: down to a wink and a
nudge? Trends Genet. 25, 19
29 (2009).
10. Matsuda, M. et al. DMY gene induces male development in genetically female
(XX) fish. Proc. Natl Acad. Sci. USA 104, 3865
3870 (2007).
11. Volff, J.-N., Kondo, M. & Schartl, M. Medaka dmY/dmrt1Y is not the universal
primary sex-determining gene in fish. Trends Genet. 19, 196
199 (2003).
12. Hamburger, V. & Hamilton, H. L. A series of normal stages in the development of
the chick embryo. J. Morphol. 88, 49
92 (1951).
13. Smith, C. A. & Sinclair, A. H. Sex determination in the chicken embryo. J. Exp. Zool.
290, 691
699 (2001).
14. Clinton, M. Sex determination and gonadal development: a bird’s eye view. J. Exp.
Zool. 281, 457
465 (1998).
15. Smith, C. A. et al. The avian Z-linked gene DMRT1 is required for male sex
determination in the chicken. Nature 461, 267
271 (2009).
16. Hutt, F. B. Genetics of the Fowl (McGraw-Hill, 1949).
17. Cock, A. G. Half-and-half mosaics in the fowl. J. Genet. 53, 49
80 (1955).
18. Birkhead, T. The Wisdom of Birds.An Illustrated History of Ornithology (Bloomsbury,
2008).
19. Hollander, W. F. Sectorial mosaics in the domestic pigeon: 25 more years. J. Hered.
66, 197
202 (1975).
20. Owens, I. P. F. & Short, R. V. Hormonal basis of sexual dimorphism in birds:
implications for new theories of sexual selection. Trends Ecol. Evol. 10, 44
47
(1995).
21. Scholz, B. et al. Sex-dependent gene expression in early brain development of
chicken embryos. BMC Neurosci. 7, 12 (2006).
22. Dewing, P., Shi, T., Horvath, S. & Vilain, E. Sexually dimorphic gene expression in
mouse brain precedes gonadal differentiation. Brain Res. Mol. Brain Res. 118,
82
90 (2003).
23. McGrew, M. et al. Localised axial progenitor cell populations in the avian tail bud
are not committed to a posterior Hox identity. Development 135, 2289
2299
(2008).
24. Nishikimi, H. et al. Sex differentiation and mRNA expression of P450c17,
P450arom and AMH in gonads of the chicken. Mol. Reprod. Dev. 55, 20
30
(2000).
25. Nomura, O., Nakabayashi, O., Nishimori, K., Yasue, H. & Mizuno, S. Expression of
five steroidogenic genes including aromatase gene at early developmental stages
of chicken male and female embryos. J. Steroid Biochem. Mol. Biol. 71, 103
109
(1999).
26. Patek, C. E. et al. Sex chimaerism, fertility and sex determination in the mouse.
Development 113, 311
325 (1991).
27. Burgoyne, P. S., Buehr, M. & McLaren, A. XY follicle cells in ovaries of XX
XY
female mouse chimaeras. Development 104, 683
688 (1988).
Testis
Ovary
Mammalian model
Indifferent
gonad
Genital
ridge
Male
soma
Female
soma
Chicken model
Sry
Genital
ridge DMRT1(?)
Testis
Ovary
Male
phenotype
Male
phenotype
Female
phenotype
Female
phenotype
Hormones
Hormones
Hormones
Hormones
Figure 5
|
A novel mechanism of sex determination in the chicken. A sexual
identity is genetically imposed on the male and female chicken soma at
fertilization and is the major factor in determining the adult sexual
phenotype. At the appropriate stage in development, the sexually-dimorphic
transcripts underlying the male/female identity trigger expression in the
genital ridge of the gene cascade responsible for testis/ovary development.
The gonads have limited effects on the sexual phenotype. In contrast, in
mammals, gonadal fate is dependent on transient expression of the testis-
determining Sry gene in the indifferent early gonad. The mammalian gonads
have a major influence on the sexual phenotype.
NATURE
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|
11 March 2010 ARTICLES
241
Macmillan Publishers Limited. All rights reserved
©2010
28. McQueen, H. A. et al. Dosage compensation in birds. Curr. Biol. 11, 253
257
(2001).
29. Melamed, E. & Arnold, A. P. Regional differences in dosage compensation on the
chicken Z-chromosome. Genome Biol. 8, R202 (2007).
30. Ellegren, H. et al. Faced with inequality: chicken do not have a general dosage
compensation of sex-linked genes. BMC Biol. 5, 40 (2007).
31. McQueen, H. A. & Clinton, M. Avian sex chromosomes: dosage compensation
matters. Chrom. Res. 17, 687
697 (2009).
32. O, W.-S., Short, R. V., Renfree, M. B. & Shaw, G. Primary genetic control of somatic
sexual differentiation in a mammal. Nature 331, 716
717 (1988).
33. Harry, J. L., Koopman, P., Brennan , F. E., Graves, J. A. & Renfree, M. B. Widespread
expression of the testis-determining gene SRY in a marsupial. Nature Genet. 11,
347
349 (1995).
34. Hori, T., Asakawa, S., Itoh, Y., Shimizu, N. & Mizuno, S. Wpkci, encoding an altered
form of PKCI, is conserved widely on the avian W chromosome and expressed in
early female embryos: implications of its role in female sex determination. Mol.
Biol. Cell 11, 3645
3660 (2000).
35. Smith, C. A., Roeszler, K. N. & Sinclair, A. H. Genetic evidence against a role for
W-linked histidine triad nucleotide binding protein (HINTW) in avian sex
determination. Int. J. Dev. Biol. 53, 59
67 (2009).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements This work was supported by DEFRA and BBSRC (BB/
E015425/1). We thank G. Miele, G. Robertson, S. Wilson, A. Sherman,
M. Hutchison, F. Thomson and R. Mitchell for technical support and for provision of
fertilized eggs and embryos. We also thank T. Cannon for donation of
gynandromorph bird G1, and R. Field and N. Russell for photography.
Author Contributions D.Z. and D.M. performed transplantation studies,
transcriptome screens, Southern analyses and general molecular biology. S.N.
performed immunostaining, H.A.M. performed FISH analyses and P.M.H.
performed dissections and post-mortem measurements. M.J.M. performed ISH
and suggested transplantation strategy and P.D.L. obtained gynandromorph birds.
Overall project was conceived by M.C. and H.M.S. M.C. carried out day-to-day
supervision and wrote the manuscript. All authors edited the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to M.C.
(Michael.clinton@roslin.ed.ac.uk).
ARTICLES NATURE
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METHODS
Embryo transplantation. Generation of chimaeric embryos:GFP embryos
23
and
ISA brown embryos at H&H stage 11/12 (13–15 somites) were used as donor and
host, respectively. The blunt end of donor eggs was pierced to create an air hole
and a ‘window’ cut on the midline. The embryos were removed and pinned on a
3% agarose surface containing 0.5% India ink. Embryos were kept moist by the
addition of phosphate buffered saline (PBS) containing 100 units ml
21
penicillin
and 100 mgml
21
streptomycin (PBS-Pen/Strep). A strip of lateral plate meso-
derm flanking presumptive somites 21–23 was removed and stored in CO
2
-
independent medium (Invitrogen) containing 10% FBS and Pen/Strep. Host
eggs were windowed as above and kept moist by the addition of PBS-Pen/
Strep. To help visualize somites, sterile India ink (20% in PBS-Pen/Strep) was
injected under the host embryos. Using a microneedle, the vitelline membrane
and a flap of ectoderm were folded back from the underlying mesoderm. A strip
of lateral plate mesoderm was then removed from the host embryo, taking care to
leave the endoderm intact. The GFP-donor tissue was then inserted into the host
site and the ectodermal flap replaced. Two millilitres of albumen was then
withdrawn from the host eggs using a hypodermic syringe. Transplanted eggs
were tightly sealed with tape and incubated at 37 uC in a humidified incubator.
Immunostaining. Immunohistochemistry was carried out as described previ-
ously
36
. Briefly, tissues were fixed in 4% paraformaldehyde for 2 h at 4 uC,
equilibrated in 15% sucrose then embedded in 15% sucrose plus 7.5% gelatin
in PBS, pH 7.2. Sections, 15 mm thick, mounted on Superfrost slides (Menzel)
were washed for 30 min in PBS at 37 uC and blocked in PBS containing 10%
donkey serum, 1% BSA, 0.3% Triton X-100 and 0.05% Tween 20 for 2 h at 22–24
uC. Incubation with primary antibodies was carried out overnight at 4 uC, fol-
lowed by washing in PBS containing 0.3% Triton X-100 and 0.05% Tween 20,
and then incubation with secondary antibodies for 2 h at room temperature.
After washing, the sections were treated with Hoechst solution (10 mgml
21
) for
5 min to stain nuclei.
Fluorescent in situ hybridization (FISH). FISH analysis of metaphase or inter-
phase preparations of chicken cells was performed by standard procedures
37
.
BAC clones containing the VLDL receptor, aldolase B, CHRN or SCII genes were
identified by screening the HGMP chicken BAC library and used to identify Z
chromosomes. A probe for the W chromosome was prepared by polymerase
chain reaction (PCR) amplification of a portion of the XhoI repeat region from
the W chromosome
38
. After gel purification, the probe was labelled by incor-
poration of either biotin-16-dUTP (Roche) or digoxigenin-11-dUTP (Roche)
during a further round of PCR. BAC DNA was prepared using Qiagen plasmid
columns following recommendations for low-copy plasmid purification. Biotin-
16-dUTP and digoxigenin-11-dUTP were incorporated into BAC DNA by nick
translation and labelled probes were concentrated by precipitation in the pres-
ence of 5 mg of salmon sperm DNA as a carrier and 2 mg of sonicated chicken
genomic DNA as competitor. The pellet was resuspended in 15 ml of hybridiza-
tion mix, denatured and pre-annealed for 15 min at 37 uC to block repetitive
sequences.
Whole-mount in situ hybridization. Chicken embryos and isolatedgonads were
fixed in 4% paraformaldehyde for 1 h and whole-mount in situ hybridization was
carried out as described previously
39
. Digoxigenin-labelled probes were prepared
from linearized plasmid clones using a Roche DIG RNA labelling kit to incor-
porate digoxigenin-11-UTP by in vitro transcription with SP6 and T7 RNA
polymerases.
RNA preparation. Total RNA was extracted from pools of male and female chick
embryos and tissues using RNA-Bee (AMS Biotechnology) according to the
manufacturer’s instructions.
Differential display. RNA expression profiles in male and female embryos were
compared by differential display reverse transcription PCR (DDRT–PCR).
Embryoswere sexed
38
and poolsof RNA from male and female embryos generated.
DDRT–PCR was performed as described previously
40
.
miRNA library construction. Low-molecular-mass RNAs (,40 nucleotides
long) were isolated from total RNA by the use of a flashPAGE fractionator
(Ambion). MicroRNA libraries were constructed essentially as described previ-
ously
41,42
.
MicroRNA northern analysis. Five micrograms of total RNA was separated by
electrophoresis through a 15% TBE/urea polyacrylamide gel (Bio-Rad) before
transfer to Hybond-N
1
membrane (GE Healthcare). Locked nucleic acid (LNA)
oligonucleotides antisense to the mature miRNA were end-labelled (mirVana
Probe and Marker kit, Ambion) with
32
P-dATP (Perkin-Elmer) and hybridized
to membranes containing miRNAs. Hybridization was carried out overnight in
ULTRAhyb-oligo (Invitrogen) at 42 uC and membranes washed at 63 uC in 0.13
SSC/0.1% SDS
43
(22 uC below the estimated melting temperature of the LNA,
85 uC).
Southern analysis. High-molecular-mass genomic DNA was extracted from
tissues of embryonic and adult male and female chickens by standard phenol-
chloroform procedures
43
. DNA was digested with restriction endonucleases,
subjected to electrophoresis on a 1% TBE gel and transferred to Hybond-N
membrane. Probes labelled with
32
P-dCTP were hybridized by standard proce-
dures and signal was recorded on high-sensitivity film (Kodak) and by phos-
phorimager analysis.
36. Stern, C. D. In Essential Developmental Biology: A Practical Approach (eds Stern, C.
D. & Holland, P. W. H.) 193
212 (IRL, 1993).
37. McQueen, H. A. et al. CpG islands of chicken are concentrated on
microchromosomes. Nature Genet. 12, 321
324 (1996).
38. Clinton, M., Haines, L., Belloir, B. & Mcbride, D. Sexing chick embryos: a rapid and
simple protocol. Br. Poult. Sci. 42, 134
138 (2001).
39. Henrique, D. et al. Expression of a Delta homologue in prospective neurons in the
chick. Nature 375, 787
790 (1995).
40. Clinton, M., Miele, G., Nandi, S. & McBride, D. In Differential Display Methods and
Protocols 2nd edn (eds Liang, P., Meade, J. D. & Pardee, A. B.) 157
178 (Humana,
2006).
41. Lau, N. C. et al. An abundant class of tiny RNAs with probable regulatory roles in
Caenorhabditis elegans. Science 294, 858
862 (2001).
42. Lee, R. C. & Ambros, V. An extensive class of small RNAs in Caenorhabditis elegans.
Science 294, 862
864 (2001).
43. Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, 2001).
doi:10.1038/nature08852
Macmillan Publishers Limited. All rights reserved
©2010
SUPPLEMENTARY INFORMATION
1
www.nature.com/nature
doi: 10.1038/nature08852
G1
G2
G3 Right Left
ab
Supplementary Figure 1.
Photographs showing a) right and left sides of
gynandromorph birds G1, G2 and G3, and b) typical
female and male ISA Brown birds. Although the males
occasionally show brown patches on the breast the
females are usually uniformly brown. The white
patches seen on the brown ‘female’ side (and vice
versa) most likely represent ‘clones’ of male cells.
Individual gynandromorphs were housed with two egg
laying females and eggs were collected and incubated.
None of these eggs proved to be fertile.
2
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doi: 10.1038/nature08852 SUPPLEMENTARY INFORMATION
Supplementary Figure 2.FISH analysis of sex chromosomes
in blood cells from gynandromorph birds
Interphase nuclei prepared from cultured blood cells from
gynandromorph birds G2 (a) and G3 (b) hybridised according to
standard FISH procedures with probes specific to both the W- and
Z- chromosome (Xho repeat on W- chromosome, and Z-
chromosome BAC containing the VLDL receptor gene identified by
screening the HGMP chicken BAC library). a) G2 erythrocytes
hybridised with probes for Z-chromosome (RED) and
W-chromosome (GREEN)( l) and low abundance ZZ G2 leukocyte
hybridised with probes for Z-chromosome (GREEN) and W-
chromosome (RED) (ll). Images from (l) and (ll) are presented at
similar magnifications.b) G3 blood cells hybridised with probes for
W-chromosome (GREEN) and Z-chromosome (RED).
ZW/ZZ
ZWZZ
lll
b
a
3
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doi: 10.1038/nature08852
Supplementary Figure 3. Examples of Southern analyses of DNA from tissues from right
and left sides of gynandromorph birds.
Equal quantities (2μg) of DNA from individual tissues was hybridised with probe for female
specific sequence (W-chromosome gene Faf). Signal intensity reflects relative proportion of W-
chromosome containing cells (female) on right and left sides of gynandromorph birds. a)
Southern analysis of DNAs from different tissues from gynandromorph bird G1. b) Separate
analyses of DNAs from three tissues from right and left sides of gynandromorph birds G1, G2
and G3. L=left,R=right, Lu-lung, WM-wing muscle, BM-breast muscle, Sk-skin, Go-gonad, Wa-
wattle, H-heart, AD-accessory duct, Li-liver, Sp-spleen.
Gynandromorph 1 Gynandromorph 2 Gynandromorph 3
L
BM R
BM L
Sk R
Sk L
Wa R
Wa L
BM R
BM L
Sk R
Sk L
Wa R
Wa L
BM R
BM L
Sk R
Sk L
Wa R
Wa
L
Lu R
Lu L
WM R
WM L
BM R
BM L
Sk R
Sk L
Go R
Go L
Wa R
Wa H AD Li Sp
a
b
4
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doi: 10.1038/nature08852 SUPPLEMENTARY INFORMATION
a
cSupplementary Figure 4. Gynandromorph
gonads.
a). Photograph of immature right (R) gonad and
testis-like left (L) gonad of gynandromorph G1.
b). Photomicrograph of histological section of
testis-like gonad from gynandromorph G1.
Arrowheads indicate mature sperm.
c). Photograph of ovary-like gonad from left side
of gynandromorph G2. Scale bars represent
10mm (a & c), and 0.1mm (b).
b
R L
5
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SUPPLEMENTARY INFORMATION
doi: 10.1038/nature08852
Lung
Lung
Heart
Heart
Liver
Liver
Brain
Brain
Testis
Ovary
Br.Muscle
Br.Muscle
Kidney
Kidney
Spleen
Spleen
Male Female
miRNA
U6
Supplementary Figure 5.Northern analysis showing expression of novel chicken miRNA
in different tissues from male and female adult birds.
Membrane was hybridised with a LNA probe complementary in sequence to the novel chicken
miRNA (gga-mir-2954) and then stripped and re-probed with a sequence complementary to
chicken U6-RNA.
Upper panel shows expression of miRNA in male and female tissues and demonstrates that
miRNA is expressed at levels 5-10 fold higher in males than in females. Lower panel shows
expression of U6 RNA demonstrating similar loading of RNA quantities from equivalent male
and female tissues. Quantity (µg) of RNA loaded for each tissue : lung-2, heart-4, liver-7, brain-
10, gonads -10, breast muscle-5, kidney-5 and spleen-8
6
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7
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SUPPLEMENTARY INFORMATION
doi: 10.1038/nature08852
female donor
+
female host
male donor
+
male host
AMH GFP AMH +GFP
high
low
AROM GFP AROM +GFP
high
low
a
b
8
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doi: 10.1038/nature08852 SUPPLEMENTARY INFORMATION
female donor
+
male host
AMH GFP AMH +GFP
high
low
male donor
+
female host
high
AROM GFP AROM +GFP
d
low
c
Supplementary Figure 7: Contribution of donor cells in same-sex and
mixed sex chimeras.
Panels show IHC analysis of sections through chimeric gonads. Donor cells
are marked by GFP (green) while AMH and aromatase-expressing cells are
shown in red. For each donor:host combination, examples containing a high
contribution of donor cells (high) and a low contribution of donor cells (low),
are shown. In same sex chimeras (a, b), even examples with a low
contribution of donor cells show co-localisation (yellow/orange) of donor
cells with functional compartments (AMH/ AROM) of the host gonad, i.e.
GFP-expressing cells co-localise with AMH-expressing and aromatase-
expressing cells in host testis and ovary respectively. In contrast, in mixed-
sex chimeras (c, d), donor cells are not integrated into the functional
compartments of the host gonad i.e. no co-localisation of GFP and AMH or
aromatase even in examples showing a high contribution of donor cells.
Scale bar represents 100 μm.
9
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SUPPLEMENTARY INFORMATION
doi: 10.1038/nature08852
G1
(left /right ) G2
(left /right ) G3
(left /right )
Side Value Ratio* Value Ratio Value Ratio
right dark** dark light Feather
colour left light** - light - dark -
right 1.2 0.13 3.3
Wattle (g) left 2.9 2.4 0.54 4.1 1.4 0.4
right 180 138 172 Breast muscle
(g) left 199 1.1 150 1.1 135 0.8
right 400 203 319
Leg muscle (g) left 480 1.2 247 1.2 259 0.8
right immature
testis none immature
testis
Gonad
left testis-
like
-
ovary-
like
- ovo-testis -
right 91 97 105 Femur length
(mm) left 99 1.1 107 1.1 95 0.9
right 73 73 78 Femur
circumference
(mm) left 81 1.1 80 1.1 70 0.9
right 3.26 3.09 7.26 Femur density
(mmAl) left 3.47 1.1 4.28 1.4 3.50 0.5
Supplementary Table 1. Physical properties of gynandromorph birds
*: Ratio of left side measurements to right side measurements.
**: feathers on the “dark” side were predominantly brown while feathers on the
“light” side were predominantly pale.
mmAl = mm of Aluminium.
10
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doi: 10.1038/nature08852 SUPPLEMENTARY INFORMATION
Sample
(days in
culture)
Probes Female (%) Male (%)
G2-R1 (17) Z-bio: W-dig 76 24
Z-dig: W-bio 79 21
G2-R2 (17) Z-bio: W-dig 48 52
Z-dig: W-bio 47 53
G2-L (7) Z-dig: W-bio 75 25
Z-bio: W-dig 60 40
G3- R (13) Z-dig: W-bio 48 52
(44) Z-dig: W-bio 46 54
G3- L (17) Z-bio: W-dig 94 6
Z-dig: W-bio 89 11
Table 2a
Bird ZZ (%) ZW (%)
G1 53 47
G2 <1* >99
G3 892
Table 2b.
Supplementary Table 2. Proportion of ZW (female) and ZZ (male) cells in
cultures derived from skin and blood samples from gynandromorph
birds.
Cultured skin cells or circulating blood cells were fixed and prepared for FISH
analysis by standard procedures. >100 informative interphase nuclei were
hybridised with probes detecting the W and Z- chromosomes (XhoI repeat on
W and Z chromosome BACs containing either aldolase B, CHRN, VLDL
receptor or SCII genes identified by screening the HGMP chicken BAC library)
and were scored in each case.
a. Proportion of ZW (female) and ZZ (male) cells in cultures derived from
gynandromorph skin samples.
Skin samples were collected from different sites from the right (R) and left (L)
sides of gynandromorph birds (G2 & G3) and were cultured for 7-44 days (as
indicated in parenthesis) before fixing and preparing for FISH by standard
procedures. Many hybridisations were performed in duplicate with Z and W
probes reciprocally labelled with either Biotin-16-dUTP (bio) or digoxigenin 11-
dUTP (dig). G2 R1 was prepared from a patch of skin located approximately 2
cm from G2R2.
b. Proportion of ZW (female) and ZZ (male) cells in cultures derived from
blood samples of gynandromorph birds (G1, G2 & G3).
The majority of nuclei scored were small erythrocytes but a minority of larger
leukocytes were also scored and showed the sex chromosome identity
associated with the phenotypic sex identity of the left side of the bird. *Only
one such leukocyte was seen from many hundreds of cells analysed for G2.
11
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SUPPLEMENTARY INFORMATION
doi: 10.1038/nature08852
Contribution of female cells (%)
Breast Skin Wattle
Southern
analysis left right left right left right
I 22 46 18 60 18 57
II 34 59 18 41 13 32
III 30 69 15 66 10 68
IV 20 60 20 76
V 27 71 14 42 23 38
VI 27 60 16 51 27 57
G1
(left/right )
Mean
percentage 27 61 17 56 18 50
I 24 42 15 72 37 52
II 17 28 11 54 22 52
III 21 66 33 54
IV 21 53 17 56 25 51
V 28 50 16 57 23 52
G2
(left/right )
Mean
percentage 22 43 16 61 28 52
I 45 26 85 28 74 51
II 43 13 60 26 80 60
III 53 15 58 24 69 45
IV 37 16 67 25 74 52
V 49 15 64 20 63 23
VI 41 23 73 26 80 63
G3
(left/right )
Mean
percentage 45 18 68 25 73 49
Supplementary Table 3: Contribution of female cells to individual tissues from
right and left sides from three gynandromorph birds.
Tables represent a minimum of four separate Southern analyses on DNA from
each gynandromorph bird, e.g. see Figure S3. Membranes were hybridised with
probe for W-chromosome sequence.
Phosphorimager values obtained from known quantities of DNA from individual
tissues from right and left sides were compared to signals obtained from equivalent
quantities of purified female genomic DNA. Results are presented as proportion of
individual tissues composed of female cells. Tissues on right side of G1 and G2
contain a higher proportion of female cells than tissues on the left side, whereas in
G3, the left side contains the higher proportion of female cells.

Supplementary resource (1)

... All three of the main mechanisms of action identified for mammalian Y chromosomes above seem plausible for avian W chromosomes as well. Because all functional W-linked genes seem to be broadly expressed, variation in amino acid sequence or expression levels of protein-coding genes (category 1) in somatic tissues could have widespread effects [194]. W-linked modulation of expression of other genes (category 2) is also possible. ...
... Finally, the W could also potentially influence non-sexual traits via hormonal effects (category 3), although this mechanism may be of lesser importance in birds than in mammals. Evidence of cell-autonomous sex determination in chickens has emerged from the study of lateral gynandromorphs [194], along with sexually dimorphic gene expression that precedes gonadal differentiation [191,194], suggesting that many sex differences are established independently of the action of sex hormones in birds. In addition, it is currently unclear whether W-linked genes have important effects on sex steroid levels in birds. ...
... Finally, the W could also potentially influence non-sexual traits via hormonal effects (category 3), although this mechanism may be of lesser importance in birds than in mammals. Evidence of cell-autonomous sex determination in chickens has emerged from the study of lateral gynandromorphs [194], along with sexually dimorphic gene expression that precedes gonadal differentiation [191,194], suggesting that many sex differences are established independently of the action of sex hormones in birds. In addition, it is currently unclear whether W-linked genes have important effects on sex steroid levels in birds. ...
Article
Full-text available
Sex chromosomes are typically viewed as having originated from a pair of autosomes, and differentiated as the sex-limited chromosome (e.g. Y) has degenerated by losing most genes through cessation of recombination. While often thought that degenerated sex-limited chromosomes primarily affect traits involved in sex determination and sex cell production, accumulating evidence suggests they also influence traits not sex-limited or directly involved in reproduction. Here, we provide an overview of the effects of sex-limited chromosomes on non-reproductive traits in XY, ZW or UV sex determination systems, and discuss evolutionary processes maintaining variation at sex-limited chromosomes and molecular mechanisms affecting non-reproductive traits.
... Gynandromorphs are bilateral sex chimeras, male on one side of the body and female on the other. Such birds are rare, but they have been reported in chickens and some other birds [15,16]. In the few gynandromorphic chickens that have been described, the "male" side of the body is predominantly composed of ZZ cells, while the female side is predominantly ZW. ...
... The male side has large comb and wattle, legs spurs, male feathering and thicker breast muscle. The female side has smaller wattle, small or absent spurs, female feathering and lighter breast muscle [15]. The gonads of these birds reflect the relative proportions of ZZ or ZW cells; testes when ZZ cells are present, and ovaries when ZW cells are predominant [15,17]. ...
... The female side has smaller wattle, small or absent spurs, female feathering and lighter breast muscle [15]. The gonads of these birds reflect the relative proportions of ZZ or ZW cells; testes when ZZ cells are present, and ovaries when ZW cells are predominant [15,17]. Gynandromorphs are unlikely to be generated by aberrant endocrine signalling, because hormones would be expected to flow equally to both sides of the body. ...
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As in other vertebrates, avian testes are the site of spermatogenesis and androgen production. The paired testes of birds differentiate during embryogenesis, first marked by the development of pre-Sertoli cells in the gonadal primordium and their condensation into seminiferous cords. Germ cells become enclosed in these cords and enter mitotic arrest, while steroidogenic Leydig cells subsequently differentiate around the cords. This review describes our current understanding of avian testis development at the cell biology and genetic levels. Most of this knowledge has come from studies on the chicken embryo, though other species are increasingly being examined. In chicken, testis development is governed by the Z-chromosome-linked DMRT1 gene, which directly or indirectly activates the male factors, HEMGN, SOX9 and AMH. Recent single cell RNA-seq has defined cell lineage specification during chicken testis development, while comparative studies point to deep conservation of avian testis formation. Lastly, we identify areas of future research on the genetics of avian testis development.
... In more recent publications, the cause of gynandromorphism has been considered as the presence of a mosaic of cells with male and female karyotype during the development of the embryo (e.g. Zalokar, Erk & Santamaria 1980;Zusman & Wieschaus 1987;Zhao et al. 2010). Intersex individuals are genetically uniform (i.e. ...
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New cases of abnormalities in the development of the copulatory organs in several species of wolf spiders are described: three specimens with side by side duplicated epigynes, one gynandromorph, four intersexes, and one with pathologically asymmetric epigyne. The potential causes of such disorders are discussed.
... In addition, chicken embryonic gonadal differentiation is also an excellent model for studying key factors of vertebrate sex determination [6] and human sexual development disorders [7,8]. Although many investigations have focused on chicken sex determination and differentiation [9][10][11][12][13][14][15][16][17][18][19][20], the mechanisms underlying gonadal differentiation are still elusive. ...
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Background As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. Results The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2 , SMAD2 , SOX9 and CYP19A1 , which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9 , in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. Conclusions This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.
... Coincidentally, most of the DEPs were related to sex-related diseases (Lee et al., 2017) and metabolic pathways (Jones et al., 2019), which were regulated by sex hormones directly or indirectly (Keyvanshokooh et al., 2009;Lee et al., 2017;Jones et al., 2019). Moreover, sex hormones have been reported as gender markers for hatched eggs and played an important role in gender differentiation for chicks (embryos) (Weissmann et al., 2013;Zhao et al., 2010). Not accidentally, it could be speculated that there may be a strong association between sex-related proteins and hormones. ...
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There is a huge demand to identify the sex of unhatched fertilized eggs for laying industry and to understand the differences between male and female eggs as early as possible. Then the molecular mechanisms of sex determination and sex allocation in chicken were revealed. Therefore, TMT proteomic was applied to characterize the variation of molecular matrix between unhatched male and female egg yolks. A total of 411 proteins were identified and 35 differentially expressed proteins (DEPs), including 375332005, 015809562, 763550308 (up-regulated, UPs) and 1337178851, 89000557, 89000581 (down-regulated, DPs), etc. were confirmed between them. Gene ontology analyses showed that DEPs were mainly involved in response to stimulus, distributed in the extracellular region and participated in binding; KEGG analyses showed that few DPs were participated in cell growth and death, transport and catabolism, signaling molecules, interaction and were enriched in ubiquitin mediated proteolysis, endocytosis, ferroptosis, etc. metabolic pathways. Moreover, most of the DEPs and related metabolic pathways were associated with sex hormones. More importantly, this study supports maternal sex-allocation theory and extends our understanding of the molecular mechanism of sex determination and differentiation in avian. Which also provides a powerful evidence for ovo sexing of unhatched fertilized domestic chicken eggs by non-destructive approach and will be of great significance to eggs processing and production.
... Most of the egg-laying vertebrates are sensitive to oestrogen (Fig 1). Besides, the ZZ/ZW chimaeras can develop as gynandromorphs: the side with a higher percentage of ZZ cell develops into male with male sexual characteristics, while the other side with more ZW cells develops into female (Arnold, 2004;Arnold et al., 2013;Zhao et al., 2010). ...
Thesis
Sex determination is a highly sophisticated and ordered process where both male and female gonads develop from a common bipotential gonad depending on different activated signaling pathways. XY gonads develop into a testis promoted by the SRY/SOX9 pathway, whereas XX gonads develop into an ovary through the action of the RSPO1/WNT4/ß-Catenin pathway. R-spondin (Rspo) genes encode one kind of secreted proteins that activate the canonical WNT/β-Catenin pathway by inhibiting the degradation of WNT receptors. After binding to its receptors LGR4/5, RSPO1-LGR4/5 recruit E3 Ubiquitin-Protein Ligases ZNRF3 and RNF43 to release WNT receptors from being degraded by ubiquitination process, therefore WNT/β-Catenin signaling can be activated continuously. Rspo1 is a major regulator of ovary development across species. In the developing mouse gonad, Rspo1 is mainly expressed in the female supporting cells, and Rspo1 XX mutant gonads undergo female-to-male sex reversal by developing into ovotestis, gonads with both male and female characteristics. The molecular and cellular mechanisms behind this partial sex reversal remain unclear. In this work, we have developed a new mouse model allowing a conditional mutation of Rspo1. We have established that the critical window for Rspo1 requirement in the developing XX mouse gonad is around E11.5, and that Rspo1 function is dispensable for ovarian differentiation after this time point. We have shown that ectopic steroidogenesis is an early event in the phenotypic changes of XX Rspo1 mutant gonads. Through pharmacological inhibition of the androgen receptor we have identified androgen signaling pathway as a major player in the female-to-male sex reversal of XX Rspo1 mutant gonads. In the second part of this work we have studied the phenotype of Sox9cKOWnt4KO double mutants where both the male and female pathway are impaired. We have found that XX Sox9cKOWnt4KO gonads develop as ovotestis indicating that the additional deletion of Sox9 did not rescue the female-to-male sex reversal caused by the Wnt4 mutation. We have also shown that XY Sox9cKOWnt4KO double knockout mutants undergo a transient female-like developmental phase before the gonads develop into ovotestis. This result demonstrates that Wnt4 deletion cannot rescue the initial steps of the male-to-female sex reversal caused by the Sox9 mutation. Together, these results reveal the timing of requirement of Rspo1 in ovarian development and highlight the pro-male role of androgen signaling in the XX female-to-male sex reversal process. It also rises new thoughts on the interactions between male and female pathway in mouse sex determination.
... In bird species, males are the homogametic sex and contain two Z sex chromosomes while females are heterogametic and contain a single Z and one W chromosome. Avian PGCs form precociously in the pre-laid egg before the formation of the embryonic germ cells. In chicken, somatic cells were shown to have a cell autonomous sex identity that was independent of gonadally produced sex hormones (Zhao et al., 2010). PGCs at migratory stages also show a sex-specific proteome (M. ...
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In birds, males are the homogametic sex (ZZ) and females are the heterogametic sex (ZW). Here, we investigate the role of chromosomal sex and germ cell competition on avian germ cell differentiation. We recently developed genetically sterile layer cockerels and hens for use as surrogate hosts for primordial germ cell (PGC) transplantation. Using in vitro propagated and cryopreserved PGCs from a pedigree Silkie broiler breed, we now demonstrate that sterile surrogate layer hosts injected with same sex PGCs have normal fertility and produced pure breed Silkie broiler offspring when directly mated to each other in Sire Dam Surrogate mating. We found that female sterile hosts carrying chromosomally male (ZZ) PGCs formed functional oocytes and eggs, which gave rise to 100% male offspring after fertilization. Unexpectedly, we also observed that chromosomally female (ZW) PGCs carried by male sterile hosts formed functional spermatozoa and produced viable offspring. These findings demonstrate that avian PGCs are not sexually restricted for functional gamete formation and provide new insights for the cryopreservation of poultry and other bird species using diploid stage germ cells.
... In agreement with this observation, a previous study that compared gene expression and protein abundance in male and female adult chicken tissues described that 30% of all proteins encoded from Z-linked genes showed a significant change in the male/female ratio compared with the corresponding ratio at the RNA level 31,32 . The differential expression of both autosomal and Z-linked genes in male PGCs in the absence of surrounding gonadal somatic cells or sexual hormones suggests that their molecular sex differentiation is cell autonomous, as previously suggested in studies performed in murine PGCs and chicken embryonic tissues [32][33][34] . The substantial sexual differences in the protein abundances in pregonadal PGCs observed in this study may be the consequence of the cumulative effect of uncompensated Z-linked gene products. ...
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In poultry, in vitro propagated primordial germ cells (PGCs) represent an important tool for the cryopreservation of avian genetic resources. However, several studies have highlighted sexual differences exhibited by PGCs during in vitro propagation, which may compromise their reproductive capacities. To understand this phenomenon, we compared the proteome of pregonadal migratory male (ZZ) and female (ZW) chicken PGCs propagated in vitro by quantitative proteomic analysis using a GeLC-MS/MS strategy. Many proteins were found to be differentially abundant in chicken male and female PGCs indicating their early sexual identity. Many of the proteins more highly expressed in male PGCs were encoded by genes localised to the Z sex chromosome. This suggests that the known lack of dosage compensation of the transcription of Z-linked genes between sexes persists at the protein level in PGCs, and that this may be a key factor of their autonomous sex differentiation. We also found that globally, protein differences do not closely correlate with transcript differences indicating a selective translational mechanism in PGCs. Male and female PGC expressed protein sets were associated with differential biological processes and contained proteins known to be biologically relevant for male and female germ cell development, respectively. We also discovered that female PGCs have a higher capacity to uptake proteins from the cell culture medium than male PGCs. This study presents the first evidence of an early predetermined sex specific cell fate of chicken PGCs and their sexual molecular specificities which will enable the development of more precise sex-specific in vitro culture conditions for the preservation of avian genetic resources.
Chapter
In mammals, sex is determined genetically and is static. Under natural conditions it does not change throughout life. In contrast, sex determination mechanisms are much more diverse and plastic among non‐mammalian vertebrates. In some teleost fish and reptiles, sex is determined primarily by environmental factors. Although most teleost fish and amphibian species do possess sex chromosomes, their effect can be overridden by environmental effects. Even sex reversal in adulthood is reported in teleost species. The molecular mechanisms underlying such plasticity have been the focus of research for a long time, and recent advances in this field have revealed that epigenetic regulation is prominent in sex determination. In this chapter, sex‐determining mechanisms and the involvement of epigenetic regulation in non‐mammalian vertebrates are described. In addition, the effect of epigenetic toxicants on sex determination and the possible application of non‐mammalian vertebrates for biomarkers as such pollutants are discussed.
Article
The chicken has a Z-W sex chromosome system, in which the males are the homogametic sex (ZZ) and the females the heterogametic sex (ZW). The smaller W chromosome is generally considered to be a highly degraded copy of the Z chromosome that retains around 28–30 homologous protein-coding genes’ These Z-W homologues are thought to have important, but undefined, roles in development, and here we explore the role of one of these genes, VCP (Valosin Containing Protein) in gonadogenesis. We established RNA expression levels of both Z and W VCP homologues, the levels of VCP protein, and the cellular localization of VCP protein in male and female embryonic gonads during development. We also assessed the effects of female-to-male sex-reversal on VCP expression in developing gonads. The results showed that both VCP RNA and protein are expressed at higher levels in female than male gonads, and the expression levels of VCP protein and VCP-Z transcript, but not VCP-W transcript, are decreased in female-to-male sex reversed gonads. In addition, the spatial expression of VCP protein differs between male and female embryonic gonads: in testes, VCP protein is mainly confined to the medullary sex cords, while in ovaries, VCP protein is expressed throughout the medulla and at higher levels in the cortex. The results suggest that sexually dimorphic expression of chicken VCP reflects differences in gonadal morphology between sexes.
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In 2001 it was established that, contrary to our previous understanding, a mechanism exists that equalises the expression levels of Z chromosome genes found in male (ZZ) and female (ZW) birds (McQueen et al. 2001). More recent large scale studies have revealed that avian dosage compensation is not a chromosome-wide phenomenon and that the degree of dosage compensation can vary between genes (Itoh et al. 2007; Ellegren et al. 2007). Although, surprisingly, dosage compensation has recently been described as absent in birds (Mank and Ellegren 2009b), this interpretation is not supported by the accumulated evidence, which indicates that a significant proportion of Z chromosome genes show robust dosage compensation and that a particular cluster of such dosage compensated genes can be found on the short arm of the Z chromosome. The implications of this new picture of avian dosage compensation for avian sex determination are discussed, along with a possible mechanism of avian dosage compensation.
Article
1 Sex differentiates under genetic control during successive periods. Classical morphological and experimental data have shown the sexual bipotentiality of the developing structures. But, as a matter of fact, several observations indicate that both sexes are not equal or equipotential as to their developmental trends and mechanisms. 2 The developmental analysis of the body sex characteristics reveals a hormonal control. In animal experiments made by the author and by others it has been observed that many structures or systems develop along the feminine type in the absence of testes during several critical developmental stages. These structures include the genital tract, the hypothalamic centres controlling the pituitary function, the nervous structures mediating sex behaviour and possibly other tissues. The ovary is unnecessary for the feminine differentiation of these structures; in males, femaleness has to be repressed and maleness imposed by the testes. 3 The problem of gonadal sex differentiation is re-evaluated; developmental aspects occurring during normal development or in the gonads of freemartins in cattle are examined. During early sexual differentiation of the gonads, testes rapidly differentiate whereas ovaries are first characterized mainly by the fact that they do not become testes. These observations can be interpreted by assuming that in males a signal imposes masculinity on the gonadal primordia which otherwise would slowly become ovaries. 4 It is hypothesized that throughout sexual differentiation in mammals, maleness has to be actively imposed on a system which will become feminine if it escapes this control.
Article
The present study was conducted to reveal effects of in ovo injection of nonsteroidal aromatase inhibitor (Fadrozole) or estradiol at day 3 of incubation on mRNA levels of P45017αhydroxylase (P450c17), P450 aromatase (P450arom) and anti-Müllerian hormone (AMH) in the chicken gonads. The mRNA levels in the gonads at days 4–8 of incubation were assessed by in situ hybridization analysis using digoxigenin labeling method. The in situ hybridization data were analyzed by relative expression of specific hybridizable signals of each mRNA corrected by the non-specific background by employing an image analyzer. P450c17 mRNA expression increased rapidly at day 6 of incubation in the male but decreased thereafter. In contrast to the transient expression in the male, the expression was gradually increased in the female. P450arom mRNA was not expressed in the male but was detectable in the female as early as day 6 and increased subsequently with days of incubation. AMH mRNA was expressed as early as day 5 of incubation followed by a sharp increase on day 6, which was maintained in the male thereafter. In contrast, the female showed very little expression. The injection of Fadrozole caused no effect on P450c17 mRNA expression, while it suppressed P450arom mRNA expression but increased AMH mRNA expression in the female. In contrast, the injection of estradiol induced P450arom mRNA expression significantly but suppressed AMH mRNA expression in the male. These results indicate that expression of P450arom and AMH is sexually dimorphic and is reciprocally regulated during early ontogenic life in chicken gonads. Mol. Reprod. Dev. 55:20–30, 2000. © 2000 Wiley-Liss, Inc.
Article
1. A half-and-half mosaic pullet (case N) from the cross Rhode Island Red ♂ x Light Sussex ♀ is described. The right side of the bird shows all the characters to be expected in a pullet from this cross. The left side is abnormal in the following ways: (i) The shank is yellow instead of white. (ii) The plumage is a darker shade of reddish brown. (iii) The feathers, especially the flight feathers of the wing, have a frayed appearance, due to imperfectly formed barbules. (iv) The whole of the left side of the body is very much smaller than the right, and the limb bones, in particular, are abnormally proportioned. 2. Two females are described which are asymmetrical only in respect of the length of the legs. One of these had originally been regarded as a mosaic, but it now appears that in both cases a copper ring accidentally left too long on the right shank of the young chick hss been the cause of hypertrophy of the right tarsometatarsus and tibiotarsus. 3. The twenty-three known half-and-half mosaic fowls are discussed in relation to the theory of Crew & Munro, which postulates that all such cases are due either to elimination, or to non-disjunction, of a single autosome at first cleavage. The following objections to this theory are pointed out: (i) The data available on the proportions of the limb bones in several cases are inconsistent with the requirements of the theory. (ii) At least five cases, and probably six, are mosaic in respect of a sex-linked character. (iii) Three cases are mosaic in respect of more than one autosomally inherited character (apart from size). In no case do linkage data support the idea that the genes concerned are all situated on the same chromosome, and it is unlikely that this could be true in all cases. (iv) The theory requires unsupported assumptions about the dosage relations between alleles at two loci. (v) The theory assumes that an extra autosome above the normal diploid number will tend to swing the sex in a male direction; this is the reverse of what is to be expected on the sex-chromosome: autosome balance theory of sex determination. 4. An attempt is made to find plausible and consistent explanations for as many as possible of the known cases. These are clearly due to a diversity of causes, and fall into two main types: (i) Those in which somatic segregation has occurred at first cleavage in an otherwise normal zygote. In some, the somatic segregation has taken the form of maldistribution (elimination or non-disjunction) of a single pair of autosomes, or possibly of somatic crossing-over. Simple somatic gene-mutation may account for a few cases. In others (gynandromorphs) elimination of an X-chromosome has occurred. In yet others, multiple irregularities in the distribution of the autosomes seem to have occurred; to this cause are assigned only those cases (of which N is one) which cannot be satisfactorily explained in any other way. (ii) Those originating from a compound zygote; more specifically, in which one side of the body is of purely paternal origin (and diploid), the other side being of normal bisexual origin. This may, or may not, lead to gynandromorphism. 5. The explanations suggested for individual mosaics are all open to some degree of doubt. Certain other difficulties remain, notably the absence of any gynandromorphs genetically female on the right side. There is also some difficulty in reconciling the irregular type of mosaic found in some species of bird (pigeon, turkey) with the bilateral half-and-half characteristic of other species (fowl, finches).
Article
In the course of avian embryo development, estrogen has been indicated to play a key role in gonadal differentiation by the inhibition of aromatase (P-450arom) that synthesizes estrogen from androgen. Biosynthesis of estrogen requires not only P-450arom but also other enzymes for a steroidogenic pathway. To elucidate gonadal differentiation, the steroidogenic pathway should be studied comprehensively in the early developmental stages including that of sex differentiation. Therefore, in the present study, the expressions of the steroidogenic genes, P-450scc, 3β-HSD, P-450c17, 17β-HSD and P-450arom, were measured at the developmental stages (days 2–9 of incubation) of chicken embryos by quantitative RT-PCR. Transcripts for all the genes studied, except for P-450arom were detected in all the developmental stages examined, indicating that mRNAs for the steroidogenic enzymes required to convert cholesterol to androgens are present in the avian embryo before gonadal differentiation. In contrast, P-450arom mRNA was detected in female embryos during days 5–9 of incubation but not in male embryos throughout incubation. The onset of P-450arom gene expression at day 5 coincides with the stage of gonadal differentiation, corroborating the role of estrogen in the process of gonadal differentiation in chicken.
Article
It is widely assumed that the development of male secondary sexual traits in birds and mammals is testosterone-dependent. In birds, however, masculinity has dual origins. Male-type behaviour and morphology, such as spurs and wattles, are usually testosterone-dependent. However, showy male-type plumage is, generally, the neutral state of development. For example, castrating a peacock has no effect on his elaborate plumage whereas ovariectomizing a peahen causes her to develop showy male-type plumage. The surprising relationships between dimorphism and gonadal steroids in birds have important consequences for the current debate concerning the evolution of biological signals and, in particular, the immunocompetence-handicap principle.