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\DOI{10.5802/crbiol.177}
\datereceived{2024-09-28}
\daterevised{2025-04-21}
\dateaccepted{2025-05-03}
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\dateposted{2025-06-04}
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\title{Uncovering the mechanisms of sexual differentiation: insights
from\break \textit{Drosophila} research}

\alttitle{La drosophile, un mod\`{e}le pour comprendre les bases
biologiques de la diff\'{e}renciation sexuelle}

\author{\firstname{Bruno} \lastname{Hudry}\CDRorcid{0000-0003-1299-843X}}
\address{Universit\'{e} C\^{o}te d'Azur, CNRS, Inserm, Institut de Biologie Valrose, Nice 06108, France}
\email{Bruno.HUDRY@univ-cotedazur.fr}

\keywords{\kwd{Sex determination}
\kwd{Sex chromosomes}
\kwd{Drosophila}
\kwd{Development}
\kwd{Physiology}}

\altkeywords{\kwd{D\'{e}termination du sexe}
\kwd{Chromosomes sexuels}
\kwd{Drosophile}
\kwd{D\'{e}veloppement}
\kwd{Physiologie}}

\thanks{Universit\'{e} C\^{o}te d'Azur, CNRS (ATIP-Avenir program),
INSERM, European Research Council  (ERC starting grant CellSex, 
ERC-2019-STG\#850934), French Government (National Research Agency,
ANR)  through the ``Investments for the Future'' programs  LABEX
SIGNALIFE ANR-11-LABX-0028-01 and IDEX UCAJedi ANR-15-IDEX-01.}

\thanks{\textbf{Note.} Bruno Hudry is the recipient of the 2022 Dr. and
Mme Henri Labb\'e prize.}

\begin{abstract}
The differences between males and females represent the largest
phenotypic dimorphism observed in most species. In humans, this
variation contributes to disparities in the risk, incidence, and
treatment responses for numerous diseases, with many of these
significant differences remaining unexplained. While hormones derived
from sex organs play critical roles in shaping and maintaining certain
sex differences, recent research using the \textit{Drosophila} model
underscores the significance of cell-intrinsic mechanisms linked to the
sex chromosomes.
\end{abstract}

\begin{altabstract}
Les diff\'{e}rences entre m\^{a}les et femelles repr\'{e}sentent le
plus grand dimorphisme ph\'{e}notypique observ\'{e} chez la plupart des
esp\`{e}ces. Chez l'homme, cette variation contribue \`{a} des
disparit\'{e}s dans le risque, l'incidence et les r\'{e}ponses au
traitement de nombreuses maladies, et beaucoup de ces diff\'{e}rences
significatives restent inexpliqu\'{e}es. Alors que les hormones
d\'{e}riv\'{e}es des organes sexuels jouent un r\^{o}le essentiel dans
la formation et le maintien de certaines diff\'{e}rences entre les
sexes, des recherches r\'{e}centes utilisant le mod\`{e}le de la
drosophile soulignent l'importance des m\'{e}canismes
intrins\`{e}ques aux cellules li\'{e}s aux chromosomes sexuels.
\end{altabstract}

\maketitle

\vspace*{.6pc}

\twocolumngrid

\end{noXML}

\section{Introduction}

Living organisms use two main biological mechanisms to generate sex
differences~\cite{1,2,3}. The first mechanism involves the
development of male- and female-specific sex organs that produce
systemic signals. These gonadal hormones can shape and maintain somatic
sex differences over time, regardless of the sex of the receiving
cells. The second mechanism relies on cell-intrinsic mechanisms, where
sex-chromosomal genes create distinctions in equivalent differentiated
cells in both males and females. Sex chromosome functions have
traditionally been explored using invertebrate model systems. This
review will outline how research on the \textit{Drosophila} model has
uncovered fundamental principles of sexual differentiation that are
broadly applicable across various species.

\begin{figure*}
\includegraphics{fig01}
\vspace*{2pt}
\caption{The sex determination process in \textit{Drosophila melanogaster}.\label{fig1}}
\vspace*{3pt}
\end{figure*}


\section{\textit{Drosophila} as a model for the study of\newline sexual
differentiation}

\subsection{X-Chromosome dose determines sexual\newline fate}

In many species, the development of testes or ovaries is determined by
specific genetic elements known as sex chromosomes, which carry the
loci responsible for sex determination~\cite{4}. In
\textit{Drosophila melanogaster}, females possess two X chromosomes,
while males have one X and one Y chromosome~\cite{5}. The number of X
chromosomes acts as the primary sex-determination signal in this
species (Figure~\ref{fig1}). Individuals with two X chromosomes develop as
females, whereas those with a single X chromosome develop as males.
This mechanism is referred to as dose-dependent X sex determination.
Notably, the fly's Y chromosome is irrelevant and does not play a
role in sex determination. The immediate target of the X chromosome
dosage is the X-linked switch gene, \textit{Sex-lethal} (\textit{Sxl}),
which directs female differentiation~\cite{6}. Sxl functions as a
female determinant, produced exclusively in female cells; its absence
leads to male developmental commitment. Cells determine whether to
express \textit{Sxl} by counting their X chromosomes through a small
set of specific X-linked numerator genes that convey the X-chromosome
dose to an \textit{Sxl} promoter~\cite{7}. A diplo-X dose, involving
four genes---\textit{scute}, \textit{sisterless A},
\textit{unpaired}, and \textit{runt}---activates \textit{Sxl}
transcription, initiating female development. Conversely, a haplo-X
dose is insufficient to activate \textit{Sxl}, resulting in male
development. This X chromosome-counting mechanism occurs during a brief
30--40~min period just before the onset of
cellularisation. Afterward, Sxl engages in a positive autoregulatory
loop that sustains its protein production in female cells. Ultimately,
a transient twofold concentration difference in these four X-linked
proteins determines sex, leading to distinct sexual fates.
\looseness=-1


\subsection{Sex-specific splicing mediates sexual determination} 

Sxl is an RNA-binding protein that regulates pre-mRNA splicing to drive
female development~\cite{6}. It controls the alternative splicing of
its own gene, as well as those of the SR splicing factor
\textit{transformer} (\textit{tra}) and \textit{male-specific-lethal~2}
(\textit{msl-2})~\cite{5}. These three female-specific splicing
events are crucial for governing all aspects of sexual differentiation.
Sxl sustains its own expression by preventing the inclusion of a poison
cassette exon that contains a premature stop codon. For its targets
\textit{tra} and \textit{msl-2}, Sxl interferes with U2 snRNP auxiliary
factor (U2AF) binding, redirecting splicing to an alternative $3'$
splice site or promoting intron retention. As a result, the female
\textit{tra} isoform contains an extended open reading frame that
bypasses the introduction of a premature stop codon. In the case of
\textit{msl-2}, intron retention leads to translational repression in
the cytoplasm. Sexual fate in \textit{Drosophila} is mainly controlled
by alternative splicing, but two additional biological processes---RNA
methylation and splicing factor phosphorylation---also play critical
roles in sex determination (Figure~\ref{fig1}). Specifically,
N6-methyladenosine (m6A) RNA methylation near the poison cassette exon
is important for the female-specific alternative splicing of
\textit{Sxl}, as demonstrated by the knockout of several subunits of
the m6A methylosome
complex~\cite{8,9,10,11,12,13,14}. It is
hypothesised that m6A readers bind to these methylated sites, and
modulate the splicing machinery to promote skipping of the poison exon
in females. Although m6A writers and readers are not absolutely
required for sex determination---since null mutants are viable and
fertile---multiple components of the m6A pathway genetically interact
with \textit{Sxl} in sensitised backgrounds. These findings suggest
that RNA methylation contributes to the robustness of \textit{Sxl}
alternative splicing, highlighting its role in fine-tuning the sex
determination process. Phosphorylation of the female-specific splicing
factor Tra is essential for its activity and for sex
determination~\cite{15}. The fly CDC-like kinase (CLK), known as
Darkener of apricot (Doa), mediates this phosphorylation~\cite{16}.
Mutations in \textit{Doa} lead to somatic female-to-male sex
transformations, underscoring its critical role in establishing female
fate~\cite{15,16}.


\subsection{Doublesex is the terminal effector of sexual differentiation} 

Sxl regulates the sex-specific splicing of its target \textit{tra},
which in turn influences the splicing of two critical genes:
\textit{doublesex} (\textit{dsx})~\cite{17,18,19} and
\textit{fruitless}
(\textit{fru})~\cite{20,21,22,23,24,25,26,27,28}.
These two key factors are the terminal effectors of sexual
differentiation and shape most of the sex differences in
flies~\cite{5}. The male-specific isoform of \textit{fru}
(\textit{fruM}) is essential for producing transcription factors that
govern specific neuronal circuits involved in sexual orientation and
aggression~\cite{20,21,22,23,24,25,26,27,28}. 
Both male (\textit{dsxM}) and female (\textit{dsxF}) isoforms of
\textit{dsx} encode transcription factors, though only a few direct
targets of Dsx are well characterised~\cite{29}. DsxM plays a
crucial role in regulating sexual behaviours by controlling the
development of male-specific neurons~\cite{30,31}. DsxF supports
egg growth by activating \textit{yolk protein} (\textit{Yp}) genes in
fat body cells~\cite{32} and promotes pheromone production by
regulating the desaturase DesatF~\cite{33}. Sex-specific gonadal
development is the last and critical aspect of sexual\unskip\break differentiation
regulated by \textit{dsx} (Figure~\ref{fig1})~\cite{5,34}. Although
\textit{dsx} is expressed in only a subset of cells and required at
specific developmental stages to form dimorphic
structures~\cite{35,36,37}, its continued expression can be
essential for maintaining sex differences throughout adulthood. For
instance, thermosensitive alleles have highlighted the importance of
the sex determination cascade in sustaining sex-specific \textit{Yp}
synthesis~\cite{38,39} and \textit{desatF}
expression~\cite{29}. In the adult gonads, sexual plasticity is also
evident; continuous expression of DsxM in testis cyst cells is
necessary to preserve their male identity, while ectopic expression of
DsxF can feminise these cells into a follicle-like
phenotype~\cite{34,40}. Additionally, \textit{dsx} plays a role
in adulthood by inhibiting male-male courtship
behaviours~\cite{41,42,43}.

\section{Sex-determining mechanisms are diverse and can evolve rapidly}

Dose-dependent X or Z chromosome sex determination has been observed in
two other model organisms following its discovery in
\textit{Drosophila}: the invertebrate \textit{Caenorhabditis
elegans}~\cite{44} and the chicken~\cite{45}. In nematodes, males
have an X0 genotype, while hermaphrodites are XX. The X-chromosome
counting mechanism in \textit{C.~elegans} regulates a male-specific
switch gene called \textit{xol-1}. Interestingly, the effect of
X-chromosome dosage on \textit{xol-1} is the opposite of that on
\textit{Sxl} in \textit{Drosophila}: a single X chromosome (haplo dose)
leads to high levels of \textit{xol-1} transcripts, while two X
chromosomes (diplo dose) result in low levels. X-linked numerator genes
convey the X-chromosome dose by repressing \textit{xol-1} in a
cumulative, dose-dependent manner at both transcriptional and
post-transcriptional levels in XX embryos. Xol-1 encodes a GHMP kinase
that promotes male development by repressing the hermaphrodite fate. 

\begin{figure*}
\includegraphics{fig02}
\vspace*{2pt}
\caption{Sex-determining mechanisms are diverse and can evolve rapidly.
(A) Beyond dose-dependent X chromosome systems, sex
determination can also involve dominant Y/W-linked factors,
haplodiploid systems, and allele heterozygosity at a single locus.
(B) In many non-Drosophilid Diptera, the primary signal for sex
determination is a dominant male-determining factor located on the Y
chromosome. While many primary sex determinants encode proteins, in
\textit{Bombyx mori}, sex is determined by a single female-specific
piRNA, \textit{Fem}, and in the Argentine ant (\textit{Linepithema
humile}), the primary sex determinant is a long noncoding RNA
(\textit{lncRNA}).\label{fig2}}
\vspace*{2pt}
\end{figure*}

In chickens, as in most birds, males are the homogametic sex (ZZ), and
females are heterogametic (ZW). Avian gonadal sex determination
primarily depends on the dosage of a single Z-linked gene,
\textit{Dmrt1}, which is homologous to \textit{dsx} in
flies~\cite{45}. During the critical period of gonadal development,
males (ZZ) express Dmrt1 at significantly higher levels than females
(ZW), with Dmrt1 functioning as the primary testis-determining factor.
Deleting one copy of \textit{Dmrt1} in chromosomally male (ZZ) chickens
leads to ovarian development~\cite{46,47}, while overexpression
of \textit{Dmrt1} in genetically female (ZW) gonads results in their
masculinisation~\cite{48}. Unlike the ubiquitous expression of
sex-determining genes, Sxl and xol-1, Dmrt1 expression is specifically
restricted to the sex organs. Thus, \textit{Dmrt1} dosage is the
central switch in avian gonadal sex determination. 

\begin{figure*}
\includegraphics{fig03}
{\vspace*{2pt}}
\caption{The TGF-$\upbeta$ signalling pathway as a hotspot for the
evolution of new master sex-determining genes in teleosts.
Teleost fish represent a prime example of a taxonomic group
characterised by frequent turnovers of Y-linked factors that govern
maleness. Within this group, various components of the gonadal
TGF-${\upbeta}$ signalling pathway---including ligands (\textit{Amhy,
Gsdfy, Gdf6}), receptors (\textit{Amhr, Bmpr}), and regulators
(\textit{Id2bby})---have been independently recruited to the top of the
sex determination cascade.\label{fig3}}
\end{figure*}



Accurate differentiation into fertile males and females is a
fundamental developmental process in animals, yet the primary signals
for sex determination are not conserved across species. Diverse
mechanisms are employed to determine sex
(Figure~\ref{fig2}A)~\cite{4}. Another common mechanism for sex
determination, besides dose-dependent X chromosome systems, involves
dominant Y-linked factors that confer maleness (Figure~\ref{fig2}A). In
many non-Drosophilid Diptera, the primary signal for sex determination
is a dominant male-determining factor (often called the M factor)
located either on the Y chromosome or at a male-determining locus on a
homomorphic sex chromosome. Examples include the male determiner Mdmd
in \textit{Musca domestica}~\cite{49,50}, Maleness-on-the-Y (MoY)
in \textit{Ceratitis capitata}~\cite{51}, Yob in \textit{Anopheles
gambiae}~\cite{52,53}, Nix in \textit{Aedes
aegypti}~\cite{54,55,56,57,58,59,60}, AsuMf in
\textit{Armigeres subalbatus}~\cite{61} and Guy1 in
\textit{Anopheles stephensi}~\cite{62,63} (Figure~\ref{fig2}B).
These Y-linked dominant M factors are both necessary and sufficient to
initiate male development during embryogenesis, primarily by inhibiting
the activity of Tra homologs, thereby suppressing female fate.
Similarly, in therian mammals (placental mammals and marsupials), male
sex determination is controlled by a Y-linked testis-determining
factor, SRY~\cite{64}. Teleost fish represent another prime example
of a taxonomic group characterised by frequent turnovers of Y-linked
factors that govern maleness~\cite{65,66} (Figure~\ref{fig3}).
Within this group, various components of the gonadal TGF-${\upbeta}$
signalling pathway---including ligands
(Amhy~\cite{67,68,69,70,71}, Gsdfy~\cite{72},
Gdf6~\cite{73,74}), receptors (Amhr~\cite{75,76,77},
Bmpr~\cite{78}), and regulators (Id2bby~\cite{79})---have been
independently recruited to the top of the sex determination cascade
(Figure~\ref{fig3}). This underscores the TGF-${\upbeta}$ signalling
pathway as a hotspot for the evolution of new master sex-determining
genes in teleosts~\cite{65}.


A third strategy for sex determination is the haplodiploid mechanism
observed in many hymenopteran insects~\cite{80}. In this system,
males are haploid and develop from unfertilised eggs, while females are
diploid and develop from fertilised eggs. In other words, a paternal
genome is essential for initiating female development; in its absence,
uniparental haploid males arise. At the molecular level, the master
switch gene for sex determination displays a parent-of-origin effect,
being maternally silenced and transcribed only from the paternal allele
in fertilised eggs to induce female development. This mechanism is
exemplified by the \textit{wasp overruler of masculinisation}
(\textit{wom}) gene in \textit{Nasonia vitripennis}~\cite{80}.

Lastly, a recently discovered mechanism of sex determination in the
butterfly \textit{Bicyclus anynana} involves allele heterozygosity at a
single gene called \textit{Masculinizer} (\textit{Masc}), which acts as
the primary sex-determining switch~\cite{81}. Embryos with either
one or two identical alleles (monoallelic) develop into females, while
those with two distinct alleles (polyallelic) develop into males.
Heterozygosity activates the Masc factor, triggering male development,
whereas hemizygosity or homozygosity result in an inactive Masc state,
leading to the default female developmental pathway.

The remarkable diversity of sex determination mechanisms is accompanied
by the rapid evolution of the primary sex determinants, which often
vary even among closely related species. This phenomenon is exemplified
in Diptera, where none of the M factors share a common origin. For
instance, Nix is an SR splicing factor distantly related to the
\textit{transformer2} gene in flies~\cite{60}, \textit{Mdmd}
originated from a duplication of the spliceosomal factor gene
\textit{CWC22}~\cite{49}, \textit{AsuMf} from a duplication of an
autosomal \textit{Drosophila} behaviour/human splicing (DBHS)
gene~\cite{61} and MoY bears no resemblance to any known
proteins~\cite{51}. This evolutionary plasticity is further
highlighted by the variety in the molecular nature of master switch
genes. While many primary sex determinants encode proteins, in
\textit{Bombyx mori}, sex is determined by a single female-specific
piRNA, \textit{Fem}~\cite{82}. The silkworm utilises a WZ sex
determination system similar to birds, where males have two Z
chromosomes and females have one Z and one W chromosome. The W-linked
\textit{Fem} piRNA dominantly silences \textit{Masc}, which is crucial
for male development. In the Argentine ant (\textit{Linepithema
humile}), which follows a haplodiploid system, the primary sex
determinant is a long noncoding RNA (lncRNA)~\cite{83} (Figure~\ref{fig2}B).


\section{Sex-specific splicing is a widespread mechanism for sex determination}

While master sex determinants often evolve rapidly, the phenomenon of
sex-specific alternative splicing may serve as a more conserved
transduction mechanism of sex determination. The sex-specific splicing
of \textit{dsx} is observed across most insects and predates the last
common ancestor of hexapods
(Figure~\ref{fig4})~\cite{84,85,86,87,88,89,90,91,92}.
In many insect species, male and female sexual development is governed
by this splicing process. Notably, in early-branching insect orders,
\textit{dsx} primarily functions as a male-determining gene, crucial
for male-specific sexual differentiation but not for female
development~\cite{86,93}. Over time, the female-specific
\textit{dsx} isoforms evolved additional functions essential for female
differentiation. 

\begin{figure*}
\includegraphics{fig04}
\caption{The sex-specific splicing of \textit{dsx}
governs sexual differentiation across most insects and predates
the last common ancestor of hexapods.\label{fig4}}
\end{figure*}


Splicing has also been linked to sex determination in vertebrates, a
process that can be influenced by a number of different factors in this
taxonomic group, ranging from genes on sex chromosomes, as seen in
flies, to environmental factors such as temperature. In the red-eared
slider turtle (\textit{Trachemys scripta}), which exhibits
temperature-dependent sex \mbox{determination} (TSD), functional studies have
shown that the histone demethylase Kdm6b, an epigenetic modifier, plays
a crucial role~\cite{94,95}. Kdm6b binds to and demethylates
the promoter of the dominant male determinant gene, \textit{Dmrt1} (a
\textit{dsx} homologue), thereby activating male development.
Remarkably, loss of \textit{Kdm6b} function at the male-producing
temperature leads to male-to-female sex reversal~\cite{94}.
Interestingly, in this species, \textit{Kdm6b} undergoes
temperature-dependent alternative splicing (Figure~\ref{fig5}A)~\cite{96}, a
phenomenon also observed in other reptilian species with TSD, such as
the American alligator (\textit{Alligator
mississippiensis})~\cite{97} and the central bearded dragon
(\textit{Pogona vitticeps})~\cite{97}. While \textit{P.~vitticeps}
has a ZZ/ZW chromosomal sex determination system, high temperatures can
induce sex reversal from the ZZ genotype to a female phenotype. In
sex-reversed individuals, high temperature incubation eliminates an
intron retention event in \textit{Kdm6b} gene, and products a specific
\textit{Kdm6b} isoform~\cite{97}. The functional significance of
these alternative splicing events in triggering the male sex
determination cascade remains to be fully understood. 

\begin{figure*}
\includegraphics{fig05}
\vspace*{4pt}
\caption{Splicing and its role in vertebrate sex determination.
(A) In the red-eared slider turtle (\textit{Trachemys
scripta}), which exhibits temperature-dependent sex determination
(TSD), \textit{kdm6b} undergoes temperature-sensitive alternative
splicing. Kdm6b binds to and demethylates the promoter of the dominant
male determinant gene~\textit{dmrt1} (a homologue of
\textit{doublesex}), thereby promoting male development. In this
species, temperature-dependent CLK kinase activity regulates
TSD-associated alternative splicing~of \textit{kdm6b}. (B) In
mammals, the ovarian-determining factor has recently been identified as
an alternatively spliced isoform of the Wilms' tumour suppressor gene
\textit{Wt1}, specifically the Wt1-KTS\unskip\break isoform.\label{fig5}}
\vspace*{4pt}
\end{figure*}

What factors regulate the temperature-dependent splicing of
\textit{Kdm6b}? Notably, homologs of the fly kinase Doa, such as CLK1,
have been implicated in linking temperature changes to alternative
splicing through the phosphorylation of SR splicing
factors~\cite{98}. The temperature-activity profile of
alligator~CLK4 and turtle~CLK1 correspond closely with the critical
temperature ranges for temperature-dependent sex determination (TSD) in
these species. For instance, CLK1 is active below 26~\textdegree C, the
male-producing temperature, but its activity significantly decreases at
the female-producing temperature above 31~\textdegree C. Moreover,
inhibition of Clk1-4 prevents intron retention at lower temperatures,
providing evidence that temperature-dependent CLK activity controls
TSD-associated alternative splicing events. CLK kinases may act as
molecular thermometers, functioning as an on-off switch for sex
determination by regulating SR factor phosphorylation in response to
temperature changes (Figure~\ref{fig5}A). However, the specific splicing
factors involved in this process have yet to be identified.

Although functional studies in reptiles are still lacking, the role of
Kdm6b alternative splicing has been validated in a fish species with
temperature-induced sex reversal, the \textit{Nile
tilapia}~\cite{99}. In many fish species, sex determination is
notably plastic and temperature-sensitive. \textit{Nile tilapia} have a
genetic sex-determination system (XX/XY); however, exposure to high
temperatures during a critical thermosensitive period can induce XX
females to\unskip\break develop as pseudo-males. In this species, alternative
splicing of \textit{Kdm6bb}, a paralogue of \textit{Kdm6b} in
\textit{P.~vitticeps}, produces two isoforms: \textit{Kdm6bb\_tv1},
lacking intron~5, and \textit{Kdm6bb\_tv2}, which includes intron~5.
Notably, overexpression of \textit{Kdm6bb\_tv1}, but not
\textit{Kdm6bb\_tv2}, induces sex reversal in XX fish. Furthermore,
high temperatures specifically upregulate \textit{Kdm6bb\_tv1},
directly linking elevated temperatures to the activation of
\textit{Dmrt1} expression by demethylating H3K27me3, ultimately leading
to male sex determination.

In mammals, while the Y-linked testis-determining gene \textit{Sry} has
been known for some time, the ovarian-determining factor was only
recently identified as an alternatively spliced isoform of the Wilms'
tumour suppressor gene, \textit{Wt1} (specifically the \textit{Wt1-KTS}
isoform) (Figure~\ref{fig5}B)~\cite{100}. Elevated expression of
this variant, as observed in Frasier syndrome, can lead to
male-to-female sex reversal by promoting ovarian development
independently of chromosomal sex. Interestingly, in the turtle
\textit{Chelydra serpentina}, which exhibits temperature-dependent sex
determination, a rapid increase in \textit{Wt1-KTS} isoform expression
is triggered when eggs are shifted from a male- to a female-promoting
temperature~\cite{101}. Although the exact mechanism by which
Wt1-KTS induces ovarian development remains unclear, it is known that
Wt1 interacts with Wilms' tumor~1-associated protein (WTAP) to form a
splicing complex. The \textit{Drosophila} homolog of WTAP, Fl(2)d, is a
key component of the m6A writer complex, which is implicated in the
female-specific splicing of \textit{Sxl} and
\textit{tra}~\cite{12,13}. In mammals, WTAP acts also as a
regulatory subunit of the m6A methylome complex, guiding the complex to
nuclear speckles and target~mRNAs~\cite{102}. Therefore, Wt1-KTS may
drive ovarian differentiation by influencing sex-specific splicing or
by modulating m6A modifications, either directly or indirectly, through
its interaction\break with WTAP.

Additionally, sex-specific splicing has been proposed as a crucial
mechanism for sex determination in another phylum, the Platyhelminthes.
In schistosomes, which possess ZW sex chromosomes, the splicing
factor~\textit{u2af2} has been identified as a potential W-linked
candidate for primary sex determination~\cite{103}. This conserved
factor may initiate a sex-specific splicing cascade, akin to the
mechanisms that govern sex determination in\unskip\break \textit{Drosophila}.


\section{Sex determination: it all evolves around Doublesex}

In addition to the conservation of the transduction mechanism, the
terminal effectors of sex determination are also highly conserved
across the animal kingdom. \textit{dsx} and its homologs have emerged
as evolutionarily conserved downstream genes in the sex determination
pathway, regulated by a variety of upstream
factors~\cite{104,105}. In many dioecious species, these
transcription factors play crucial roles in sex-specific development
through their DNA-binding motif, known as the DM domain (named after
\textit{doublesex} and \textit{male-abnormal-3}), suggesting that DM
domain genes and sexual reproduction may have coevolved in metazoans.

\textit{dsx} homologs consistently occupy a key position at the base of
the sex-determination cascade in virtually all species studied
(Figure~\ref{fig6}). In the closest insect relative analysed to date, the
branchiopod crustacean \textit{Daphnia magna}, \textit{Dsx1} is
transcribed specifically in males, orchestrating male trait formation
in both somatic and gonadal tissues~\cite{106,107}. Knockdown of
\textit{DapmaDsx1} in male embryos results in the development of female
traits, including ovarian maturation, while ectopic expression of
DapmaDsx1 in female embryos leads to the development of male-like
phenotypes~\cite{106,107}.

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig06}
\vspace*{-2pt}
\caption{\textit{dsx} homologs occupy a key position at the base of the
sex-determination cascade in virtually all species. In some cases,
\textit{dsx} homologs move up the regulatory hierarchy and act as the
upstream sex-determining factor.\label{fig6}}
\vspace*{-3pt}
\end{figure*}

In molluscs, \textit{Dmrt1} is exclusively expressed in the testes.
Functional experiments in the bivalve species \textit{Tridacna crocea}
have demonstrated that Dmrt1 primarily regulates
spermatogenesis~\cite{108}. Similarly, in the planarian
\textit{Schmidtea mediterranea}, the predicted \textit{dsx} homolog
plays a male-specific role in the maintenance and regeneration of the
testes and male accessory reproductive organs~\cite{109}. 

In vertebrates, the loss of \textit{Dmrt1} is linked to XY sex reversal
in humans, and knockout studies in mice have shown that this gene is
essential for testis development~\cite{104}. \textit{Dmrt1} is also
critical for male sexual differentiation in the Chinese soft-shelled
turtle, \textit{Pelodiscus sinensis}~\cite{110}. In this species,
\textit{Dmrt1} exhibits early male-specific embryonic expression, and
knockdown of \textit{Dmrt1} in ZZ embryos results in male-to-female sex
reversal.

In some cases, \textit{dsx} homologs move up the regulatory hierarchy
and act as the upstream sex-determining factor, as observed in
chickens~\cite{45}. In amphibians, the first primary sex determinant
identified is also a DM domain-containing factor: the dominant
female-specific gene \textit{Dm-W} in the African clawed frog,
\textit{Xenopus laevis}~\cite{111,112}. In \textit{Xenopus},
which has a ZZ/ZW sex chromosome system, a duplicated copy of
\textit{Dmrt1} on the W chromosome, known as \textit{Dm-W}, acts as a
dominant-negative regulator. It interferes with the transcriptional
activation of Dmrt1 target genes, functioning as an anti-male factor
that promotes ovary formation. Similarly, in some fish species, such as
\textit{Cynoglossus semilaevis} (tongue sole)~\cite{113,114} and
\textit{Oryzias latipes} (medaka)~\cite{115,116}, a duplicated
copy of \textit{Dmrt1} on the sex chromosomes serves as a
male-determining factor. In these species, the deficiency of
\textit{Dmrt1} results in male-to-female sex reversal phenotypes,
underscoring its essential role in male sex determination. 

\begin{figure*}
\vspace*{-3pt}
\includegraphics{fig07}
\vspace*{-8pt}
\caption{Cell-autonomous sexual differentiation in \textit{Drosophila} intestinal stem cells.\label{fig7}}
\vspace*{-3pt}
\end{figure*}

These examples highlight the central and conserved role of
\textit{dsx/DMRT1} homologs in sex determination and
differentiation across a wide range of animal taxa, emphasising their
fundamental importance in the evolution of sexual development. The
striking contrast between the strong evolutionary conservation of
\textit{dsx/DMRT1} gene functions and the rapid diversification of
upstream sex-determining mechanisms has spurred investigation into the
evolutionary forces that drive this turnover. A central question is why
sex determination remains highly labile in some taxa, yet remarkably
stable in others. Although this remains an open area of investigation,
mounting evidence suggests that sex chromosomes are frequent hotspots
of genetic conflict---driven by processes such as meiotic drive and
sexually antagonistic selection. These conflicts may play a key role in
fuelling the rapid evolution and recurrent turnover of both sex
chromosomes and their associated master sex-determining loci.

\section{Cellular sex and its impact on adult tissue physiology: 
insights from \textit{Drosophila}}

As discussed so far, the process of sex organ determination is
increasingly well understood. However, the influence of the intrinsic
presence of sex chromosomes, referred to as cellular sex, on dimorphic
phenotypes in non-sex organs and physiological traits---especially in
adult tissues---remains less explored. Recent
studies~\cite{117,118,119,120,121,122,123,124,125}
using the \textit{Drosophila} model have begun to shed light on how
cellular sex affects organ physiology beyond the gonads.


To investigate this, we used the adult digestive organs of flies as a
model system. The intestine, one of the best-characterised adult
tissues in \textit{Drosophila}, undergoes self-renewal during adulthood
due to its resident population of adult somatic stem cells
(ISCs)~\cite{122}. Focusing on ISCs, our work highlights the
significant role sex chromosomes play in these cells. We discovered
that the sexual identity of ISCs---determined by the presence or
absence of female sex determinants, Sxl and Tra---profoundly affects
both organ size and the predisposition to genetically induced tumours
by modulating the proliferation rate of ISCs (Figure~\ref{fig7}).

By generating mosaic \textit{Drosophila} in which only certain adult
cells underwent sex reversal, we were able to reduce the size of a
female gut to a male-like size in vivo within 20 days of adult-specific
sex \mbox{reversal} in ISCs. Additionally, this manipulation revealed that
only female guts were susceptible to tumour development. These findings
also demonstrated that the sexual identity of adult ISCs is: (i)
specified during adulthood, (ii) acts in a cell-autonomous manner, and
(iii) is partially reversible. Importantly, ISC sexual identity is
maintained by a novel branch of the sex differentiation pathway
downstream of Tra, independent of the well-known Dsx and Fru pathways.
This shows that even \textit{dsx/fru}-negative cells retain the
capacity for sexual differentiation into adulthood.

Using newly developed genetic models, we further demonstrated that
every cell possesses an intrinsic sexual identity, governed by a binary
and static genetic switch~\cite{125}. Through cell-specific sex
reversals, we and others have shown that cellular sex significantly
influences organ size~\cite{117,125}, body
weight~\cite{118,119,125}, and lifespan~\cite{120,124}.

\section{Beyond hormones: cell-autonomous roles of sex chromosomes in
mammalian physiology}

Building on this body of work, the role of cellular sex in shaping
mammalian physiology has become an active area of investigation. Over
the past five years, multiple studies have identified X-linked and
Y-linked genes as key regulators of sexual differentiation, acting in a
cell-autonomous manner. For decades, it was assumed that Y chromosome
genes were largely inactive outside of the testes. However, research
from David Page's group demonstrated that many Y-linked genes are
also expressed in non-reproductive tissues in humans~\cite{126}.
Similarly, a growing body of evidence shows that some X-linked genes
are expressed at higher levels in XX cells than in XY cells, due to the
presence of two copies of the X chromosome~\cite{127}. These
findings reveal that sex chromosomes can drive functional differences
between male and female cells across the body---not just within the
reproductive system.

One such gene is \textit{Kdm5c}, a histone demethylase that removes
methyl groups from H3K27me3 and is consistently expressed at higher
levels in XX than in XY cells in both mice and
humans~\cite{128,129}. Recent studies implicate \textit{Kdm5c} in
the regulation of metabolism in mice. Experimental data show that mice
with two copies of \textit{Kdm5c} exhibit greater body weight, a higher
proportion of body fat, and increased food intake during the daytime
compared to those with a single copy~\cite{130}. These sex
differences appear to be primarily driven by \textit{Kdm5c} expression
in preadipocytes, where it influences both their development and
metabolic activity~\cite{130}. This example illustrates how the
dosage of an X-linked gene can contribute to sex differences in normal
mammalian physiology.

\section{From \textit{Kdm6a} to \textit{SHOX}: Gene dosage and the
cellular foundations of pathological sex differences}

Another compelling example is \textit{Kdm6a} (also known as
\textit{Utx}), a gene encoding a histone demethylase that targets
H3K27me3. \textit{Kdm6a} has emerged as a key agent of sexual
differentiation, contributing to sex differences in both autoimmune
disease and neurodegeneration~\cite{131}. Notably, most autoimmune
diseases are more prevalent in women than in men. Multiple sclerosis
(MS), a putative T cell-mediated disease, exhibits a threefold higher
incidence in females. A similar female bias is observed in Experimental
Autoimmune Encephalomyelitis (EAE), a mouse model of MS induced by
immunisation with myelin protein autoantigens and
adjuvant~\cite{132}. \textit{Kdm6a} is expressed at higher
levels in XX than XY CD4$^{+}$ T cells. Conditional deletion of
\textit{Kdm6a} specifically in CD4$^{+}$ T cells confers strong
protection from EAE and reduces neuroinflammation~\cite{133}.
Transcriptomic analysis revealed that \textit{Kdm6a} loss
upregulates T helper cell--related pathways while
downregulating genes involved in neuroinflammation~\cite{133}. The
gene dosage of \textit{Kdm6a}---two copies in XX cells versus one
in XY---has also been implicated in sex-biased vulnerability in
neurodegenerative diseases. In Alzheimer's disease (AD), men tend to
experience earlier mortality, more severe cognitive decline, and
greater neurodegeneration than women~\cite{134}. In vitro,
treatment of neurons with the neurotoxin A${\upbeta}$42 leads to
greater cell death in XY than XX neurons, correlating with higher
\textit{Kdm6a} expression in XX neurons. Reducing \textit{Kdm6a}
levels in XX neurons to match XY levels increases their vulnerability
to A${\upbeta}$42-induced neurotoxicity. Conversely, elevating
\textit{Kdm6a} expression in XY neurons attenuates this toxicity. In
vivo, overexpression of Kdm6a in the hippocampal dentate gyrus of XY
male mice enhances learning and memory performance.

While lifestyle factors and sex hormones have long been recognised as
contributors to sex differences in cancer incidence and outcomes, three
recent independent studies have uncovered additional cell-intrinsic,
non-hormonal mechanisms involving Y chromosome genes as additional
contributors~\cite{135,136,137}. In the first study,
conducted in a mouse model of colorectal cancer, male mice showed
significantly worse survival than females, mirroring human sex
disparities~\cite{135}. The authors found that the intrinsic
presence of the Y chromosome in cancer cells acted as a risk factor,
driven by upregulation of a Y-linked histone demethylase. In contrast,
the other two studies revealed a protective role for the Y chromosome
in different cancer types. These studies showed that loss of the Y
chromosome (LOY) is extremely common across multiple tumour types,
often occurring at frequencies higher than those of canonical driver
mutations~\cite{137}. While LOY may arise in the context of
genomic instability and might be dismissed as a passenger event due to
the chromosome's small size and low gene density, the authors
demonstrated that Y loss can be a driver event---notably in uveal
melanoma~\cite{137} and bladder cancer~\cite{136}. In bladder    
cancer, experimental removal of the Y chromosome led to increased
tumour burden. Further analyses identified \textit{Uty}, a gene
located in the male-specific region of the Y chromosome, as partly
responsible for this tumour-suppressive effect.

Direct effects of sex chromosome genes on phenotype are also evident
in conditions where sex chromosome dosage is altered. For example, a
hallmark feature of Turner syndrome---caused by the complete or partial
absence of one X chromosome (typically resulting in an XO
karyotype)---is short stature. This phenotype is largely driven by the
dosage of an X-linked gene called \textit{short stature homeobox}
(\textit{SHOX}). \textit{SHOX} is expressed in the developing long
bones of the forearms and lower limbs~\cite{139}. Human studies
have shown that individuals with a deletion of one copy of
\textit{SHOX} exhibit short stature~\cite{140,141}, whereas
individuals with an extra sex chromosome---and therefore an additional
copy of \textit{SHOX}---tend to be taller than
average~\cite{142}. This exemplifies the dose-sensitive role of
\textit{SHOX} in skeletal\break growth.

Together, these studies---all conducted within the past five
years---provide compelling evidence in mammals, building on
foundational work in \textit{Drosophila}, that the intrinsic
expression of sex chromosome-linked genes can drive sex differences.
They also highlight the dynamism and rapid growth of this field.
Crucially, the manifestation of sex differences in cells, organs, or
behaviours varies greatly across species. This diversity reflects the
influence of each species' unique ecology and evolutionary pressures.
There are no universal or fixed male or female traits---what is
sexually dimorphic in one species may be monomorphic in another. While
every cell carries the potential to harness sex chromosome genes to
generate sex-specific traits, this potential is not always realised.
The presence of sex chromosomes does not inherently lead to overt
physiological differences. This underscores the importance of studying
cellular sex: to reveal when and where it matters, and to understand
how it shapes organ development, function, and disease within specific
biological contexts.


\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\section*{Acknowledgments}

I thank all the members of the BH laboratory for~fruitful discussions
and comments. This work was supported by the Universit\'{e} C\^{o}te
d'Azur, CNRS (ATIP-Avenir program), INSERM, European Research Council
(ERC starting grant CellSex, Grant number: ERC-2019-STG\#850934), and
the French Government (National Research Agency, ANR) through the
``Investments for the Future'' programs 
LABEX SIGNALIFE ANR-11-LABX-0028-01 and IDEX UCAJedi ANR-15-IDEX-01.

\CDRGrant[ERC]{ERC-2019-STG\#850934}
\CDRGrant[ANR]{ANR-11-LABX-0028-01}
\CDRGrant[ANR]{ANR-15-IDEX-01}

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