## 4- Selected publications

Following are four selected publications that do not necessarily represent the “keystones of my scientific career”, but are rather representative of “my” science the way I like it.

1- Herpin, A., Nakamura, S., Wagner, T., Tanaka, M. and Schartl, M. (2009) A highly conserved mRNA sequence motif directs differential gonadal regulation of mRNA during gonad development, *Nucleic Acids Research.* 37(5):1510-1520.

This is a pure “hard-core” mechanistic paper, where we isolated a conserved cis-regulatory motif involved in the regulation of the Dmrt1 RNA stability in medaka. It is mainly molecular biology experiments (EMSA, cell transfections, DNA constructions and transgenic animals) that show how we can explain the physiology. I am particularly proud of this work because it later allowed clinicians to explain gonadal dysgenesis in a family of patients that were carrying mutation in this evolutionary conserved (from drosophila to mammals) cis regulatory motif we discovered and characterized in medaka (“Novel DMRT1 3’UTR+11 insT mutation associated to XY partial gonadal dysgenesis”, Arq Bras Endocrinol Metab, 2010).

2- Herpin, A., Adolfi, M., Nicol, B., Hinzmann, M., Schmidt, C., Klughammer, J., Engel, M., Tanaka, M., Guiguen, Y. and Schartl, M. (2013) Divergent expression regulation of gonad development genes in medaka shows incomplete conservation of the downstream regulatory network of vertebrate sex determination, *Mol Biol Evol.* 30(10): 2328-46.

With this paper we challenge the paradigm that says that downstream effectors of sex determination are well conserved (“Masters change, slaves remain”, (2)). By saying “we challenge the paradigm”, I mean that for the first time we provide *functional proof* that the paradigm is not that universal. This is what I like, providing functional evidences to test evolutionary hypotheses.

3- Herpin, A., Schartl, M. (2015) Plasticity of the gene regulatory networks controlling sex determination: of masters, slaves, usual suspects, newcomers and usurpators, *EMBO reports.* 16(10):1260-74.

This is a review/perspective paper that sums up all we are working for. Here we challenge and explain most of the theories for sex determination and sex chromosome evolution in the light of functional studies, and give perspectives about it. We eventually conceptualize the notion of interacting gene regulatory networks for sex determination.

4- (Herpin, A., Rafati, N., Chen, J.), Petterson, M., Han, F., Feng, C., Wallerman, O., Rubin, CJ., Péron, S., Cocco, A., Larsson, M., Trötschel, C., Poetsch, A., Korsching, K., Bönigk, W., Körschen, HG., Berg, F., Folkvord, A., Kaupp, UB., Schartl, M. and Andersson, L. (2020) Reconstruction of the birth of an unexpected male sex chromosome in Atlantic herring, *PNAS*.

I like this paper because, using genomics together with functional experiments (from genome to genes and function) we could reconstruct the birth of a sex chromosome...which was, according to some theories, predicted to be absent/unexpected.
# A highly conserved cis-regulatory motif directs differential gonadal synexpression of Dmrt1 transcripts during gonad development

Amaury Herpin<fnref n="1" />*, Shuhei Nakamura<fnref n="2" />, Toni Wagner<fnref n="1" />, Minoru Tanaka<fnref n="2" />* and Manfred Schartl<fnref n="1" />,³

Received November 17, 2008; Revised December 16, 2008; Accepted December 18, 2008

## ABSTRACT

Differential gene expression largely accounts for the coordinated manifestation of the genetic programme underlying embryonic development and cell differentiation. The 3' untranslated region (3'-UTR) of eukaryotic genes can contain motifs involved in regulation of gene expression at the post-transcriptional level. In the 3'-UTR of *dmrt1*, a key gene that functions in gonad development and differentiation, an 11-bp protein-binding motif was identified that mediates gonad-specific mRNA localization during embryonic and larval development of fish. Mutations that disrupt the 11-bp motif leading to *in vitro* protein-binding loss and selective transcript stabilization failure indicate a role for this motif in RNA stabilization through protein binding. The sequence motif was found to be conserved in most of the *dmrt1* homologous genes from flies to humans suggesting a widespread conservation of this specific mechanism.

## INTRODUCTION

Assembly and formation of the gonad primordium is the first step towards gonad differentiation and subsequent sex differentiation (1). Primordial germ cells (PGCs) give rise to the gametes that are responsible for the development of a new organism in the next generation. In many organisms, following their specification the germ cells migrate towards the location of the prospective gonadal primordium (2–5). Similar to other vertebrates, the

structure of fish gonads is composed of germ cells and associated supporting somatic cells (6). The precursors of the somatic cells originate from cells of the lateral plate mesoderm where the gonadal primordium develops, while germ cells are derived from the germline lineage (7,8). To carry out their highly specialized biological functions, together somatic gonadal primordium and germline cells must establish specialized programs of gene expression. However, the early transcriptional and post-transcriptional regulatory events underlying the differentiation of gonad precursor cells through crucial interactions of somatic and germline cells are barely understood.

The *dmrt1* gene is an important regulator of male development in vertebrates (9). It is a highly conserved gene involved in the determination and early differentiation phase of the primordial gonad in vertebrates. In the fish medaka *dmrt1bY*, a functional duplicate of the autosomal *dmrt1a* gene on the Y-chromosome, has been shown to be the master regulator of male gonadal development (10,11), comparable to *Sry* in mammals (12). In males mRNA and protein expression occur before morphological sex differentiation in the somatic cells surrounding PGCs of the gonadal anlage and later on exclusively in Sertoli cells (13). Here it is synexpressed with the autosomal *dmrt1a* (14,15). However, nothing is known about the mechanism(s) that bring about this highly restricted expression pattern.

The expression of most genes is dynamically regulated temporally and spatially. Spatial organization of cells and subcellular compartments arises in part from the sorting and subsequent localization of proteins and RNA. Evidence has been obtained that regulation occurs at multiple steps on the level of gene expression including

<footnote n="1">¹Physiological Chemistry I, University of Würzburg, Biozentrum, Am Hubland, D-97074 Würzburg, Germany,</footnote>

<footnote n="2">²Laboratory of Molecular Genetics for Reproduction, National Institute for Basic Biology 5-1, Higashiyama, Okazaki, Aichi 444-8787, Japan and ³Rudolf-Virchow-Center for Experimental Biomedicine (DFG Research Center), University of Würzburg, D-97078 Würzburg, Germany</footnote>

<footnote>*To whom correspondence should be addressed. Email: amaury.herpin@biozentrum.uni-wuerzburg.de
Correspondence may also be addressed to Minoru Tanaka. Tel/Fax: +81 564 59 5851; Email: mtanaka@nibb.ac.jp</footnote>
transcription, splicing, mRNA transport, mRNA stability, translation, protein stability and post-translational modifications (16,17). Selective advantages could have favored the evolution of regulatory mechanisms at the post-transcriptional level, such as speed of response, reversibility, fine-tuning of protein amounts, coordinated regulation of protein families, potential for spatial control, and efficacy in systems lacking transcriptional control mechanisms. Efficient cell-specific mRNA processing depends on a temporally and spatially orchestrated sequence of protein-protein, protein-RNA and RNA-RNA interactions (16).

Striking examples of localized messengers, transcriptional, post-transcriptional and translational regulations can be found among the maternal mRNAs of fly, fish and frog implicated in the establishment of axial polarity. For example, in the posterior part of the *Drosophila* embryo, Nanos (Nos) protein represses translation of maternal *hb* mRNA (18). Conversely, synthesis of both Bcd and Hb proteins in the anterior of the embryo requires that Nos is limited to the posterior (19). The restricted distribution of Nos is generated by selective translation of a subset of *nos* mRNA that is localized to the germ plasm at the posterior of the embryo coupled with translational repression of *nos* mRNA distributed throughout the whole embryonic cytoplasm (20). Both posterior localization and translational repression of *nos* RNA are mediated by the *nos 3'-untranslated region (3'-UTR)* (21). A nucleotide translational control element (TCE) within the *nos 3'-UTR* confers repression through formation of two stem-loop structures, whose functions are temporally distinct (22).

The zebrafish *nanos1* homologue which is required during germline development (23) has also been shown to be remarkably post-transcriptionally regulated. Here, microRNA miR-430 targets the 3'-UTR of *nanos1* during zebrafish embryogenesis in order to confer restriction of mRNA to PGCs (24). This miR-430 target site was shown to reduce poly(A) tail length, mRNA stability and translation, suggesting that differential susceptibility to microRNAs contributes to tissue-specific gene expression (24). Implicit in these mechanisms are the existence of *cis*-acting signals and *trans*-acting factors forming mRNA-protein complexes (mRNPs) that account for specificity and selectivity.

While the importance of complex post-transcriptional regulation—like in the case of *nanos*— has been widely demonstrated for the development of the germline, such mechanisms have not been uncovered so far for the development of the somatic part of the gonad, which determines the development towards testis or ovary. Studying mechanisms regulating localization and translation of gonad-specific genes during early gonad induction, we demonstrate that an 11-nt protein-binding motif located in the 3'-UTR of *dmrt1bY* mediates gonad-specific mRNA stability during embryonic and larval development. Interestingly, the sequence motif was found to be highly conserved in the homologous genes from flies to humans.

# MATERIALS AND METHODS

## Fish maintenance and breeding

Medaka were taken from closed breeding stocks of the Carbio strain and kept under standard conditions. Medaka embryos were staged according to Iwamatsu (25).

## Whole-mount in situ hybridization

RNA whole-mount *in situ* hybridization using GFP digoxigenin (DIG)-labeled probe was performed as described for Medaka (26). Briefly, after capped mRNA injection, embryos of the desired stage were fixed with 4% paraformaldehyde and dehydrated with methanol. Anti-sense-DIG-labeled RNA probes were synthesized according to the manufacturers' instructions (Roche, Meylan). Hybridization and detection with alkaline phosphatase (AP)-coupled anti-DIG antibody (Roche, Meylan) were performed according to Thisse *et al.* (27).

## Plasmid constructs and RNA injections

To obtain RNA transcripts of *eGFP* the *GFP* open reading frame (ORF) from *pEGFP-N1* (Clontech Laboratories) was inserted (*BamH1/Not1*) into *pCS2+* plasmid (*pCS2:GFP*). To produce *pCS2:GFP:dmrt1bY 3'-UTR*, a PCR product containing the entire 3'-UTR of *dmrt1bY* flanked by *Not1* sites, was amplified from medaka testes and inserted into the *Not1* sites of *pCS2:GFP*. Similarly, *pCS2:GFP:dmrt1a 3'-UTR*, *pCS2:GFP:d. rerio dmrt1bY 3'-UTR*, *pCS2:GFP:h. sapiens 3'-UTR*, *pCS2:GFP:O. curvinotus dmrt1bY 3'-UTR* and *pCS2:GFP:fugu 3'-UTR* and deletion constructs (Supplementary Figure 2) were constructed the same way. Corresponding RFP plasmids were constructed by replacing the *GFP* ORF by *RFP*. *Xenopus* *β-globin* constructs were produced by inserting the *xenopus* *β-globin 3'-UTR (Not1/Kpn1)* from plasmid pRN3 (28) into *pCS2:GFP* (*pCS2:GFP:xlβ-globin 3'-UTR*). For constructing the *pCS2:GFP:BOXxlβ-globin 3'-UTR* plasmid, the *dmrt1bY* box was inserted between the *Not1* sites. The *GFP/RFP 3'-UTR* constructs include the mmGFP5/RFP ORF cloned upstream of the 3'-UTR of the zebrafish *nanos1* gene (23,29). All constructs were checked by restriction digests, diagnostic PCRs and sequencing. Sertoli cell-specific Ds-Red expressing sox9prom:DsRed transgenic medaka fish (30) was provided by Prof. Tanaka.

Capped RNAs for injections were transcribed from linearized vectors using the SP6/T3/T7 m MESSAGE mMACHINE Kit (Ambion). One nanoliter was injected into the cytoplasm of one-cell stage Medaka embryos as described (31).

## Cell culture and transfection

Medaka spermatogonial (Sg3), embryonic stem (MES-1) or fibroblast (OL-17) cells were cultured as described (32–34). Cells were grown to 70–80% confluency in six-well plates and then transfected with 5 µg expression vector using GeneJuice reagent (Novagen) as described by the manufacturer. Luciferase activity was then quantified using the Luciferase Reporter Assay System from
Promega and luciferase activity was normalized against
mRNA luciferase copy number. Transcriptional differ-
ences between luciferase constructs were evaluated statis-
tically by paired Student's test.

Electromobility shift assay

Nearly confluent cells [Medaka spermatogonial (Sg3),
mouse Sertoli (TM4) and Medaka embryonic stem
(MES-1)] cell lines grown on plates were washed with
ice-cold phosphate-buffered saline (PBS) and removed
from the plates with 1 mM EDTA in PBS. The cells
were centrifuged at low speed and then resuspended in
Passive Lysis Buffer (Promega) supplemented with leupep-
tin (0.2 µg/ml) and aprotinin (10 µg/ml). Binding assays
were carried out using the Gel Shift Assay System
(Promega) using radiolabeled RNA oligonucleotides: (Y)
UGGUUCACGUCUGCUGCAGGUCUCUGACUCU
for the native box target sequence and Mut(2)-box UGG
UUCACGUUUGGUCGGGATCUCUGACUCU;
Mut(3)-box UGGUUCACGUUCUUCACAUGUCUCU
GACUCU; Mut(4)-box UGGUUCACGUCUGCUGAG
ACGCUCUGACUCU; Mut(5)-box UGGUUCACGUC
UGCCAUAGGUCUCUGACUCU as competitors.

RESULTS AND DISCUSSION

*Dmrt1a/dmrt1bY 3'-UTRs regulate spatial and temporal expression during early Medaka development*

While searching for potentially conserved regulatory
sequences in genes involved in gonad induction and for-
mation we analyzed the tightly regulated Medaka *dmrt1a*
and *dmrt1bY* duplicated gene pair. Postulating that
important *cis*-regulatory motifs required for mRNA reg-
ulation in the context of gonad formation might have been
retained between the duplicates despite of the processes of
co-ortholog gene specialization and subfunctionalization,
we noticed that the *dmrt1a* and *dmrt1bY* 3'-UTRs
appeared more conserved than expected for independently
diverging genes. To test whether this conservation of
*dmrt1a/dmrt1bY* UTRs implied common regulatory
mechanisms, we examined GFP expression of reporter
constructs that contained either the *dmrt1a* or *dmrt1bY*
3'-UTR or the *Xenopus* β-globin 3'-UTR as a control
(Figure 1). After injection of the different constructs into
one cell stage embryos we initially observed a high and
uniformly distributed GFP expression in the whole
embryo (Figure 1). After 2 days of development (stages
22-24; 12-16 somites), GFP fluorescence slowly vanished
elsewhere except in the primordial gonad area of the fish
injected with either *dmrt1a* or *dmrt1bY* UTR (Figures 1A,
B, C, D compared to 1E, F, G, H).

GFP fluorescence was clearly detectable in the primor-
dial gonad area until more than 7 days after hatching
 stage 40; first fry stage, 2.5 weeks after fertilization)
 (Supplementary Figure 1). In controls with the β-globin
 3'-UTR GFP remained ubiquitously expressed through-
 out the whole embryonic development. Obviously, the
 *dmrt1a/dmrt1bY* 3'-UTRs are responsible for specific
 expression of the GFP protein in the primordial gonad
 area.

Surprisingly, fusing either human (AJ276801)
(Figure 1I, J and K) or takifugu (CAC42778)
(Figure 1L, M and N) *dmrt1* 3'-UTRs to GFP mRNA
also drove primordial gonad area-specific fluorescence in
Medaka (Figure 1I–N compared to 1A–D). In addition,
injection of GFP mRNA fused to Medaka *dmrt1bY*
3'-UTR in zebrafish resulted in a similar gonadal persis-
tence of GFP fluorescence (Figure 1O, P and Q) indicating
a functional cross-species conserved mechanism mediated
by *cis*-regulatory element(s) in these *dmrt1* 3'-UTRs.

A short, highly conserved *cis*-regulatory motif located
*in dmrt1a/dmrt1bY 3'-UTR is responsible for gonadal*
*differential regulation*

To delineate the precise RNA sequence and/or secondary
structures involved in gonadal-specific fluorescence, GFP
expression of a series of reporters (Supplementary
Figure 2) that contained deletion mutants of the *dmrt1a/*
*dmrt1bY* 3'-UTRs was investigated (Supplementary
Figure 2B, C and D). As a result a core 11-nt box located
in the 5' region of the Medaka *dmrt1a/dmrt1bY* UTRs was
isolated and shown to be responsible for gonad-specific
fluorescence. Consequently, when the box was inserted
into the *Xenopus* β-globin 3'-UTR, GFP expression was
identical to *GFP::dmrt1a* and *dmrt1bY* 3'-UTR con-
structs, namely gonad-specific expression (Figure 1R, S,
T and U). Conversely, deletion of the box sequence
from the *dmrt1bY* 3'-UTR drastically extinguished
gonad-specific expression (Supplementary Figure 2D com-
pared to 2B and C).

Considering the functional conservation of gonad-spe-
cific expression seen with other fish and human *dmrt1*
UTRs we then searched for similar motifs in the 3'-UTR
of *dmrt1* genes of other organisms (Table 1). The motif
was found to be highly conserved in the fish lineage
(*Oryzias latipes*, *Oryzias curvinotus*, *Takifugu rubripes*,
*Tetraodon nigroviridis*, *Epinephelus coioides* and *Danio*
*rerio*), but as well in the *dmrt1* 3'-UTR of other vertebrates
including man (*Mus musculus*, *Pan troglodytes*, *Macaca*
*mulatta* and *Homo sapiens*) and most surprisingly even
in the ecdysozoan clade. For the *doublesex* (*dsx*, the
*dmrt1* orthologue) of *Anopheles gambiae* (Table 1), inter-
estingly, the sex-specific differentially spliced anopheles
*dsx* transcript results in a male *dsx* form where
the box is largely conserved while this is not the case for
the female splice form (Table 1). This situation is similar
for *dsx* of the olive fruit fly (*Bactocera oleae*) for which a
male-specific splicing leads to the preservation of a highly
conserved box in the ORF while this fragment is spliced
out in the female form (Table 1).

Taken together, a short highly conserved *cis*-regulatory
motif located in *dmrt1a* and *dmrt1bY* 3'-UTRs (CUGCU
GCAGGU) appears to be mostly responsible for differen-
tial expression of the transcripts.

The *dmrt1* box drives specific stability in the somatic
mesoderm anlage of the gonadal primordium as well as in
a sub-population of PGCs

*Dmrt1* is expressed in most species specifically in
Sertoli cells and PGCs. To find out the contribution of
<figure><img src="image_47.png" /><figcaption>**Figure 1.** A short highly conserved cis-regulatory motif located in *dmrt1bY/dmrt1a* 3'-UTRs regulates spatial and temporal expression during early development. (A–D) GFP expression of a reporter construct that contains Medaka *dmrt1bY* 3'-UTR during somitogenesis (A and B) and at hatching stage (C and D). (E–H) GFP expression of a control reporter construct that contains *Xenopus* β-globin 3'-UTR during somitogenesis (E and F) and at hatching stage (G and H). (I–Q) GFP expression of reporter constructs that contain either human *dmrt1* 3'-UTR (I–K) or takifugu *dmrt1* 3'-UTR (L–N) in Medaka embryos during somitogenesis (I, J and L, M) and at hatching stage (K and N). (O–Q) GFP expression of a reporter construct that contains Medaka *dmrt1bY* 3'-UTR in zebrafish embryos during somitogenesis (O and P) and at hatching stage (Q). (R–U) GFP expression in Medaka embryos of a reporter construct that contains *Xenopus* β-globin 3'-UTR in which the Box was inserted. Specific GFP expression in PGCs is indicated (arrow heads).</figcaption></figure>
<table><caption>**Table 1.** The *dmrt1* 3' UTR cis-regulatory motif is well conserved from ecdysozoans to mammals</caption><tr><td>Oryzias latipes</td><td>Dmrt1a3'-UTR(AF319994):</td><td>CUGCUGCAGGU</td></tr><tr><td>Oryzias latipes</td><td>Dmrt1bY 3'-UTR (AB071534):</td><td>CUGCUGCAGGU</td></tr><tr><td>Oryzias curvinotus</td><td>Dmrt1bY 3'-UTR (AB091695):</td><td>CUGCUGCAGGU</td></tr><tr><td>Oryzias curvinotus</td><td>Dmrt1ba 3'-TR (AB091696):</td><td>CUGCUGCAGGU</td></tr><tr><td>Takifugu rubripes</td><td>Dmrt1 3'-UR (CAC42778):</td><td>CUGCUGCAGGU</td></tr><tr><td>Tetraodon nigrovihdis</td><td>Dmrt1 3'-UTR (CAC42783):</td><td>CUGCUGCAGGU</td></tr><tr><td>Epinephelus coioides</td><td>Dmrt1 3'-UTR(EF017802):</td><td>TUGCUGCAGGU</td></tr><tr><td>Danio rerio</td><td>Dmrt1 3'-UTR(AAU11586):</td><td>CUGCUGCAGAU</td></tr><tr><td>Homo sapiens</td><td>Dmrt1 3'-UTR(AJ276801):</td><td>CUGCUGCCGAU</td></tr><tr><td>Pan troglodytes</td><td>Dmrt1 3'-UTR (XM528528):</td><td>CUGCUGCCGAU</td></tr><tr><td>Macaca mulatta</td><td>Dmrt1 3'-UTR (XM001090081):</td><td>CUGCUGCCGAU</td></tr><tr><td>Mus musculus</td><td>Dmrt1 3'-UTR (NM175647):</td><td>CUGGUGCUGAU</td></tr><tr><td>Anophele gambia</td><td>Dsx 3'-UR (Male-spe, AY903307):</td><td>CUGCUGUAAGU</td></tr><tr><td>Anophele gambia</td><td>Dsx 3'-UTR (Female-spe, AY903308):</td><td>CUG_UGCAGAA</td></tr><tr><td>Bactocera oleae (olive fruit fly) Doublesex ORF (AJ547622):</td><td></td><td>CUGCUGCAGCU</td></tr><tr><td colspan="3">'Box' present in Male form (dsxm) but absent in Female form (dsxf)</td></tr><tr><td>Musca domestica Doublesex ORF (AY461854/AY461853):</td><td></td><td>CUGCUGCAGGA</td></tr><tr><td colspan="3">'Box' present for both dsxm anf dsxf</td></tr></table>

<figure><img src="image_48.png" /></figure>

post-transcriptional regulation by this cis-regulatory motif to the restricted expression pattern, Medaka *dmrt1bY* 3'-UTR was fused to the monomeric RFP mCherry and injected either together with *GFP::Nos* 3'-UTR into one-cell stage embryos of wild-type Medaka or of the Olvas transgenic strain. The *GFP::Nos* 3'-UTR construct was previously shown to drive PGC-specific fluorescence in medaka (35-37). Similarly, in Olvas fish the PGCs are marked by GFP expression from stage 25 [18-19 somite stage (25)] onwards (38). RFP expression was compared to germ cell-specific GFP expression due to the *nanos* 3'-UTR or the *vasa* promoter (Figure 2A-F). Starting at stages 14-18 fluorescence was exclusively observed from GFP in PGCs (data not shown; Figure 3A). Only by stages 22-24 (nine somite stage) red fluorescence could also be observed with specific gonadal localization (Figure 2D-F). Noteworthy, although at this stage both fluorescences (green and red) were confined to the same embryonic structures, they are clearly expressed from different cell populations (Figure 2A-C). Additionally, around stage 24-26 (16-22 somite stage) cells expressing both fluorochromes could be observed (Figure 2A-C). Subsequently, at hatching stage, gonadal *dmrt1bY* 3'-UTR-driven GFP expression was then investigated in Sertoli cell-specific RFP expressing *sox9prom:DsRed* transgenic fish (30) either at stage 34 (Figures 2G, I, H and J) or just after hatching (Figure 2K, L and M) in

males and females, respectively. It revealed that although no more supporting cell expression was apparent for these later stages, two different populations of germ cells could be discriminated according to their high or just above background GFP fluorescence expression (arrowheads in Figure 2G, H, K and L). The fact that the RFP reporter gene product was only observed at later stages indicated that mRNAs with the *dmrt1bY* 3'-UTR are translationally repressed in migrating PGCs and in the somatic gonad precursor cells.

Interestingly, such variation and diversity in germ cell-specific *Dmrt1* expression is also reported for mouse gonads for which two populations of germ cells could be observed according to *Dmrt1* protein presence or absence (39). Consequently, akin to what was observed during medaka gonad formation (Figure 2G-N), it might be possible that mouse *Dmrt1<sup>+</sup>* and *Dmrt1<sup>-</sup>* expressing germ cells are the result of a similar mechanism since the 'box' motif is also found to be highly conserved in mouse *dmrt1* 3'-UTR (Table 1).

Embryonic expression of *dmrt1* mRNA has been examined in various vertebrates, including mammals (40-42), birds (41), reptiles (41,43) and fish (44). In most cases, *dmrt1* is expressed very early in the genital ridge-the structure from which the gonad derives-and later on in germ and Sertoli cells of the male gonadal primordium [see Zarkower *et al.* (45) for review]. It is then evident that
<figure><img src="image_49.png" /><figcaption>**Figure 2.** The *dmrt1* box drives specific stability in the somatic mesoderm of the gonadal primordium as well as in a sub-population of PGCs. (A–F) RFP expression from *dmrt1bY* 3'-UTR containing capped RNA compared to germ cell-specific GFP expression due to the *nanos* 3'-UTR at stages 24–26 (A–C). (D–F) RFP expression compared to germ cell specific GFP expression achieved with *vasa* promoter around hatching stage. (G–N) Gonadal *dmrt1bY* 3'-UTR-driven GFP expression investigated in Sertoli cell specific DsRed expressing *sox9prom*:DsRed transgenic fish either at stage 34 (G, I and H, J) or just after hatching (K, M and L, N) in males (XY) and females (XX). Arrow heads indicate either putative somatic gonadal precursor cells (C) or different sub-populations of germ cells (A, B and D–N). Blue: DAPI or Olvas; Red: Sertoli cell specific expression (*sox9prom*:DsRed transgenic fish) and Green: gonadal *dmrt1bY* 3'-UTR-driven GFP expression.</figcaption></figure>
<figure><img src="image_50.png" /><figcaption>**Figure 3.** Tissue-specific and temporal-restricted expression by a combination of *dmrt1* 3'-UTR induced differential mRNA stability and translational regulation. (A) GFP:*dmrt1bY* 3'-UTR was injected. As controls, mRNA constructs such as GFP:*nos* 3'-UTR, *olvas*:GFP, GFP:*zfvasa* 3'-UTR and GFP:*xlβ-globin* 3'-UTR were also injected to be able to compare the expression with the pattern of known post-transcriptional mechanisms such as micro-RNA mRNA induced decay, specific PGC translational regulation, and ubiquitous stability, respectively. (B) Subsequently *in situ* hybridization using an antisense GFP probe was performed at different stages of development to reveal the spatial distribution of the injected GFP:*dmrt1bY* 3'-UTR RNAs. GFP fluorescence and the distribution of RNA were followed at different stages of development. Arrowheads and circles indicate where the PGCs are or should be located respectively. (C) Luciferase expression in different cell lines transfected with a *dmrt1bY* 3'-UTR containing construct reveals that translation of the reporter gene was significantly enhanced by the presence of *dmrt1bY* 3'-UTR in Medaka spermatogonial cells in contrast to either Medaka embryonic stem or fibroblast cells.</figcaption></figure>
GFP expressing cell are PGCs; likewise are the cells
expressing jointly both GFP and RFP. The question
remains what the identity of the RFP only expressing
cells at early stages (stages 24–26) is. Considering the tem-
poral appearance of these cells and a report identifying
and tracing different populations of gonadal precursor
cells in medaka (7), it is most likely that these cells are
representatives of a subset of gonadal precursors.

Interestingly, like observed for mouse Dmrt1, these
results point to the existence of at least two populations
of PGCs within the primordial gonad of Medaka with
possibly different regulations of *dmrt1* in relation to the
above described *cis*-acting element located in its 3'-UTR.

Tissue-specific and temporal-restricted expression is caused by a combination of *dmrt1* 3'-UTR-induced differential mRNA stability and translational regulation

Independently of promoter-induced transcription striking
examples of specifically localized messengers can be found
among the mRNAs of fly, fish and frog, most of them
being post-transcriptionally (dynamic relocalization and
stabilization) and translationally regulated. To test
whether *dmrt1bY* 3'-UTR was more involved in regulating
mRNA stability or controlling its translational regulation,
embryos were injected with *GFP:dmrt1bY* 3'-UTR and
subsequently analyzed for the spatial distribution of the
injected RNA by RNA *in situ* hybridization at different
stages of development (Figure 3). As controls, *GFP:nos*
3'-UTR, *olvas:GFP*, *GFP:zfvasa* 3'-UTR and *GFP:xlβ-
globin* 3'-UTR mRNA constructs were also injected in
order to compare the expression with the pattern of
known post-transcriptional mechanisms such as micro-
RNA mRNA-induced decay, specific PGC translational
regulation and ubiquitous stability, respectively
(Figure 3). GFP fluorescence and the distribution of
RNA were followed at different stages of development.
Remarkably, GFP expressed from the construct contain-
ing the *dmrt1bY* 3'-UTR, was shown to be quite stable in
the whole body, including the primordial gonad area like
with the *GFP:zfvasa* 3'-UTR and *GFP:xlβ-globin* 3'-UTR
mRNAs (Figure 3). This is in striking contrast to *GFP:nos*
3'-UTR construct for which mRNA underwent rapid
somatic degradation [Figure 3; (22,24,36,46)]. This pattern
of decay is typical for a microRNA-mediated process (24).
Although an analogous germ cell-specific fluorescence was
observed in germ cells of *olvas:GFP* injected embryos,
*in situ* hybridizations showed homogenous olvas
RNA distribution. This pattern implies germ cell-specific
translational regulation (35). Accordingly GFP *in situ*
hybridization after *GFP:dmrt1bY* 3'-UTR injection
revealed that the fused mRNA is first homogenously dis-
tributed in the whole embryonic body until stage 24 and
only then becomes progressively restricted to the gonad
area to be exclusively present in the gonadal primordium
around hatching (stage 39) (Figure 3). Hence, it can be
inferred that the apparent specific *GFP:dmrt1*-UTR
driven expression in primordial gonad is the result of dif-
ferential RNA stabilization.

Interestingly these results reflect also the probability
of another mechanism affecting mRNAs containing the

*dmrt1* UTR. The fact that strong fluorescence is observed
in the whole body (including PGCs) at early stages sug-
gests that the *dmrt1bY* box here contributes to enhanced
translation. To test whether the *dmrt1bY* 3'-UTR is able to
enhance translation, a plasmid construct containing a thy-
midine kinase promoter driven luciferase fused either to
SV40 3'-UTR or *dmrt1bY* 3'-UTR was transfected in dif-
ferent cell lines and translation efficiency measured.
Translation of the reporter gene was significantly
(*P* < 0.01) enhanced in the presence of *dmrt1bY* 3'-UTR
in Medaka spermatogonial cells in contrast to either
Medaka embryonic stem cells or fibroblast cells where
protein production was even reduced (Figure 3). The sup-
pression of translation in non-gonad cell types is interest-
ing to note in relation to the non-gonad-specific expression
of *dmrt1bY* transcripts during early Medaka embryogen-
esis and in adult male spleen (10,14).

In summary, taking into account the endogenous
expression of *dmrt1a* exclusively in the developing gonad
and the here observed late gonad-specific translation of
injected RFP:*dmrt1bY* 3'-UTR mRNA, it appears that
the UTR mediates a translational regulation process spe-
cifically in a subset of PGCs and certain somatic cells of
the gonad. From RNA localization studies it is apparent
that the later gonad-specific expression is then primarily
due to the 11-bp *cis*-acting *dmrt1* motif conferring differ-
ential RNA stabilization in primordial gonad cells.

The *dmrt1* 3'-UTR box specifically binds a protein possibly involved in gonad-restricted expression

Obviously the 11-mer is involved in a cell-type-specific
stability of *dmrt1* RNAs. One explanation could be that
it is the target for a protein or small ribonucleoproteins
particles. To test this hypothesis, electrophoretic mobil-
ity shift assays (EMSAs) were performed using the
11-mer box as a probe and different mutated boxes as
competitors (Figure 4). In a first step cell extracts from
three different cell lines were tested: (i) a medaka sperma-
togonia cell line, (ii) a medaka embryonic stem cell line
and (iii) a mouse sertoli cell line (TM4) (Figure 4A). While
no mobility shift was observed for the medaka embryonic
stem cell-like line in all conditions tested, a weak shift was
apparent for the TM4 cells and a quite robust one for
the Medaka spermatogonial cell line (Sg3) (Figure 4A).
As controls, using the medaka spermatogonial cell line,
a mutated version of the box used as competitor
(Mut(3)-Box) did not interfere significantly with the
*Dmrt1bY*-Box binding, indicating the specificity of the
interaction (Figure 4B). Furthermore, competition of
radioactively with non-radioactively labeled box probes
(*Dmrt1bY*-Box) resulted in progressive loss of the appar-
ent shift (Figure 4C). Next, to better characterize the inter-
action domain(s) of the box, different point mutations
were then introduced to the native *dmrt1bY*-box
(Figure 4D).

The first mutated RNA oligonucleotide [Mut(2)-Box]
regrouped the three main mutations observed for the dif-
ferent dmrt1 boxes within fish and mammals (see also
Table 1). These introduced mutations did not significantly
interfere for binding (Figure 4D). Interestingly, this result
<figure><img src="image_51.png" /><figcaption>**Figure 4.** A RNA-binding factor that recognizes the Box could be responsible for primordial gonad restricted stability. (A) Electrophoretic Mobility Shift Assay (EMSA) using cell extracts from three different cell lines (i) a medaka spermatogonial cell line (Sg3), (ii) a medaka embryonic stem cell line (MES-1) and (iii) a mouse sertoli cell line (TM4) and the 11-mer box as a probe shows shift for Sg3 and TM4 cells. (B) A mutated version of the box [Mut(3)-Box] used as competitor (1–1, 1–5 and 1–10 ratios) did not interfere significantly with the binding, indicating the specificity of the binding. (C) As control, using the spermatogonial cell line, competition of radioactively and non-radioactively labeled box probes resulted in progressive loss of the apparent shift (1–1, 1–5 and 1–10 ratios). (D) Different mutated versions of the box [Mut(2–5)-Box] were then tested for binding, and resulted in apparent different binding affinities. (E–H) Relative robustness of the shift was then tested by mean of competition assays among the different mutated versions of the box (E: 1–1, 1–5 and 1–10; F: 1–1, 1–2 and 1–5; G and H: 1–1 and 1–5 ratios).</figcaption></figure>

corroborates *in vivo* observations showing GFP stabilization after injection of either human or zebrafish *dmrt1* 3'-UTR containing constructs in medaka (Figure 4D). Nevertheless, this affinity seems to be significantly weaker when competing against the native *Dmrt1bY-Box* (Figure 4E). Noticeably, *Mut(2)-Box* competition against another box mutated in its 3' region [*Mut(4)-Box*] could not appreciably interfere with the binding (Figure 4F). Similarly, using this box [*Mut(4)-Box*] and a box mutated in its core region [*Mut(5)-Box*] alternatively against each other, revealed that although the entire native box is required for efficient binding, its 3' region is important while the 5' and core regions are more likely involved in modulating the affinity (Figures 4G and 4H).

In summary these experiments suggest that the 11-mer box is a preferential target for RNA-binding proteins that may be involved in specifically regulating transcript stability.

## CONCLUSIONS

The great majority of sequences that direct the subcellular localization, translation and degradation have been found within the 3'-UTRs [see (17,47) for review]. UTRs are highly diverse in sequence, but often contain regulatory motifs that are common to members of the same metabolic family (48). Nevertheless, the analysis of such *cis*-acting elements has been rather unsatisfying with respect to the identification of common sequences that direct localization of different RNAs (49).

We have identified an 11-nt motif in the *Dmrt1* 3'-UTR, *CUGCUGCAGGU*, common to the great majority of *Dmrt1* orthologs genes from fly to mammals that is responsible for specific stabilization and translational control of *dmrt1* mRNA in the forming primordial gonad of fish and probably of other species. Interestingly, for the first time such stabilizing motif involving *cis*-regulatory actor(s) has been shown to be not only present in a
single organism [see Kloc *et al.* (49) for review], co-reg-
lating a very specific pool of few synexpressed transcripts
(50), but rather to be highly conserved across species for a
given transcript family, namely the Doublesex/Mab-3/
Dmrt1 genes. The occurrence of such an 11-mer in the
3'-UTR of orthologs genes over a wide range of organisms
already indicates that different systems may employ
common RNA regulatory mechanisms. Hence, other
than a motif involved in specific localization of mRNAs
to a well defined subtype of cells, this box would be
responsible for mRNA-specific preservation of different
subsets of cells all together involved in primordial gonad
assembly and formation, namely putative gonadal pre-
cursor cells and a specific subclass of PGCs.
Consequently, like synexpressed groups reflect the func-
tional compartmentalization of the eukaryote genome
(51) and have a striking parallel to the prokaryote
operon, our finding might be of particular interest since
it might reveal an otherwise hidden logic of cellular regu-
lation where cis-regulatory motifs couple spatial and tem-
poral gene expression in different subset of cells during
organogenesis.

Finally, our data indicate that transcript stabilization is achieved by interaction of a specific protein with a cis-acting stability element located in the *dmrt1* 3'-UTR. Although the identity and the dynamic of action of this stabilizing factor has still to be resolved, our findings point to an obvious level of integrated regulation, namely the presence and the accessibility of this *cis*-regulatory element. The occurrence of multiple *dmrt1*-related spliced variants in corals (52), insects (53–55), lizards (56), fish (57,58), chicken (59), mice (60) and human (61) selecting or splicing out parts of the UTRs also argue for such way of regulation.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

This work was supported by a grant of the Rudolf-
Virchow-Zentrum for Experimental Medicine (DFG
Forschungszentrum) and DFG-Graduiertenkolleg 1048
(Molecular Basis of Organ Development in Vertebrates)
through a PhD fellowship to T.W. Funding for open
access charge: XXX.

*Conflict of interest statement.* None declared.

REFERENCES

1. Wilhelm,D., Palmer,S. and Koopman,P. (2007) Sex determination and gonadal development in mammals. *Physiol. Rev.*, **87**, 1-28.

2. Santos,A.C. and Lehmann,R. (2004) Germ cell specification and migration in Drosophila and beyond. *Curr. Biol.*, **14**, R578–R589.

3. McLaren,A. (2001) Mammalian germ cells: birth, sex, and immor-
tality. *Cell Struct. Funct.*, **26**, 119–122.

4. Raz,E. (2004) Guidance of primordial germ cell migration. *Curr. Opin. Cell Biol.*, **16**, 169–173.

5. Saito,D., Morinaga,C., Aoki,Y., Nakamura,S., Mitani,H., Furutani-Seiki,M., Kondoh,H. and Tanaka,M. (2007) Proliferation

of germ cells during gonadal sex differentiation in medaka: Insights
from germ cell-depleted mutant zenzai. *Dev. Biol.*, **310**, 280-290.

6. Delvin,R.H. and Nagahama,Y. (2002) Sex determination and sex differentiation in fish. *Aquaculture*, **208**, 191–364.

7. Nakamura,S., Kobayashi,D., Aoki,Y., Yokoi,H., Ebe,Y.,
Wittbrodt,J. and Tanaka,M. (2006) Identification and lineage tra-
cing of two populations of somatic gonadal precursors in medaka
embryos. *Dev. Biol.*, **295**, 678–688.

8. Kurokawa,H., Saito,D., Nakamura,S., Katoh-Fukui,Y., Ohta,K., Baba,T., Morohashi.K. and Tanaka,M. (2007) Germ cells are essential for sexual dimorphism in the medaka gonad. *Proc. Natl Acad. Sci. USA*, **104**, 16958–16963.

9. Morrish,B.C. and Sinclair,A.H. (2002) Vertebrate sex determina-
tion: many means to an end. *Reproduction*, **124**, 447–457.

10. Nanda,I., Kondo,M., Hornung,U., Asakawa,S., Winkler,C., Shimizu,A., Shan,Z., Haaf,T., Shimizu,N., Shima,A. et al. (2002) A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. *Proc. Natl Acad. Sci. USA*, **99**, 11778–11783.

11. Matsuda,M., Nagahama,Y., Shinomiya,A., Sato,T., Matsuda,C., Kobayashi,T., Morrey,C.E., Shibata,N., Asakawa,S., Shimizu,N. et al. (2002) DMY is a Y-specific DM-domain gene required for male development in the medaka fish. *Nature*, **417**, 559–563.

12. Brennan,J. and Capel,B. (2004) One tissue, two fates: molecular genetic events that underlie testis versus ovary development. *Nat. Rev. Genet.*, **5**, 509–521.

13. Kobayashi,T., Matsuda,M., Kajiura-Kobayashi,H., Suzuki,A., Saito,N., Nakamoto,M., Shibata,N. and Nagahama,Y. (2004) Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes. *Dev. Dyn.*, **231**, 518–526.

14. Hornung,U., Herpin,A. and Schartl,M. (2007) Expression of the male determining gene dmrt1bY and its autosomal coorthologue dmrt1a in medaka. *Sex Dev.*, **1**, 197–206.

15. Winkler,C., Hornung,U., Kondo,M., Neuner,C., Duschl,J., Shima,A. and Schartl,M. (2004) Developmentally regulated and non-sex-specific expression of autosomal dmrt genes in embryos of the Medaka fish (Oryzias latipes). *Mech. Dev.*, **121**, 997–1005.

16. Moore,M.J. (2005) From birth to death: the complex lives of
eukaryotic mRNAs. *Science*, **309**, 1514–1518.

17. Keene,J.D. (2007) RNA regulons: coordination of post-transcrip-
tional events. *Nat. Rev. Genet.*, **8**, 533–543.

18. Irish,V., Lehmann,R and Akam,M. (1989) The Drosophila posterior-group gene nanos functions by repressing hunchback activity. *Nature*, **338**, 646–648.

19. Struhl,G., Johnston,P. and Lawrence,P.A. (1992) Control of Drosophila body pattern by the hunchback morphogen gradient. *Cell*, **69**, 237–249.

20. Deshpande,G., Calhoun,G. and Schedl,P. (2004) Overlapping
mechanisms function to establish transcriptional quiescence in the
embryonic Drosophila germline. *Development*, **131**, 1247–1257.

21. Dahanukar,A. and Wharton,R.P. (1996) The Nanos gradient in Drosophila embryos is generated by translational regulation. *Genes Dev.*, **10**, 2610–2620.

22. Duchow,H.K., Brechbiel,J.L., Chatterjee,S. and Gavis,E.R. (2005) The nanos translational control element represses translation in somatic cells by a Bearded box-like motif. *Dev. Biol.*, **282**, 207–217.

23. Koprunner,M., Thisse,C., Thisse,B. and Raz,E. (2001) A zebrafish nanos-related gene is essential for the development of primordial germ cells. *Genes Dev.*, **15**, 2877–2885.

24. Mishima,Y., Giraldez,A.J., Takeda,Y., Fujiwara,T., Sakamoto,H., Schier,A.F. and Inoue,K. (2006) Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. *Curr. Biol.*, **16**, 2135–2142.

25. Iwamatsu,T. (2004) Stages of normal development in the medaka
Oryzias latipes. *Mech. Dev.*, **121**, 605–618.

26. Kluver,N., Kondo,M., Herpin,A., Mitani,H. and Schartl,M. (2005) Divergent expression patterns of Sox9 duplicates in teleosts indicate a lineage specific subfunctionalization. *Dev. Genes Evol.*, **215**, 297–305.

27. Thisse,C., Thisse,B., Schilling,T.F. and Postlethwait,J.H. (1993) Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. *Development*, **119**, 1203–1215.
28. Lemaire,P., Garrett,N. and Gurdon,J.B. (1995) Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. *Cell*, **81**, 85-94.

29. Herpin,A., Fischer,P., Liedtke,D., Kluever,N., Neuner,C., Raz,E. and Schartl,M. (2008) Sequential SDF1a and b-induced mobility guides Medaka PGC migration. *Dev. Biol.*, **320**, 319-327.

30. Nakamura,S., Aoki,Y., Saito,D., Kuroki,Y., Fujiyama,A., Naruse,K. and Tanaka,M. (2008) Sox9b/sox9a2-EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells. *Mol. Reprod. Dev.*, **75**, 472-476.

31. Koster,R., Stick,R., Loosli,F. and Wittbrodt,J. (1997) Medaka spalt acts as a target gene of hedgehog signaling. *Development*, **124**, 3147-3156.

32. Hong,Y., Liu,T., Zhao,H., Xu,H., Wang,W., Liu,R., Chen,T., Deng,J. and Gui,J. (2004) Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. *Proc. Natl Acad. Sci. USA*, **101**, 8011–8016.

33. Komura,J., Mitani,H. and Shima,A. (1988) Fish cell culture: Establishment of two fibroblast-like cell lines (OL-17 and OL-32) from fins of the medaka, Oryzias latipes. *In Vitro Cell Dev. Biol.*, **24**, 294-298.

34. Hong,Y., Winkler,C. and Schartl,M. (1996) Pluripotency and differentiation of embryonic stem cell lines from the medakafish (Oryzias latipes). *Mech. Dev.*, **60**, 33-44.

35. Herpin,A., Rohr,S., Riedel,D., Kluever,N., Raz,E. and Schartl,M. (2007) Specification of primordial germ cells in medaka (Oryzias latipes). *BMC Dev. Biol.*, **7**, 3.

36. Kurokawa,H., Aoki,Y., Nakamura,S., Ebe,Y., Kobayashi,D. and Tanaka,M. (2006) Time-lapse analysis reveals different modes of primordial germ cell migration in the medaka Oryzias latipes. *Dev. Growth Differ.*, **48**, 209-221.

37. Saito,T., Fujimoto,T., Maegawa,S., Inoue,K., Tanaka,M., Arai,K. and Yamaha,E. (2006) Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA. *Int. J. Dev. Biol.*, **50**, 691-699.

38. Tanaka,M., Kinoshita,M., Kobayashi,D. and Nagahama,Y. (2001) Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: a useful model to monitor germ cells in a live vertebrate. *Proc. Natl Acad. Sci. USA*, **98**, 2544-2549.

39. Lei,N., Hornbaker,K.I., Rice,D.A., Karpova,T., Agbor,V.A. and Heckert,L.L. (2007) Sex-specific differences in mouse DMRT1 expression are both cell type- and stage-dependent during gonad development. *Biol. Reprod.*, **77**, 466-475.

40. De Grandi,A., Calvari,V., Bertini,V., Bulfone,A., Peverali,G., Camerino,G., Borsani,G. and Guioli,S. (2000) The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation. *Mech. Dev.*, **90**, 323-326.

41. Smith,C.A., McClive,P.J., Western,P.S., Reed,K.J. and Sinclair,A.H. (1999) Conservation of a sex-determining gene. *Nature*, **402**, 601-602.

42. Moniot,B., Berta,P., Scherer,G., Sudbeck,P. and Poulat,F. (2000) Male specific expression suggests role of DMRT1 in human sex determination. *Mech. Dev.*, **91**, 323-325.

43. Kettlewell,J.R., Raymond,C.S. and Zarkower,D. (2000) Temperature-dependent expression of turtle *Dmrt1* prior to sexual differentiation. *Genesis*, **26**, 174-178.

44. Marchand,O., Govoroun,M., D'Cotta,H., McMeel,O., Lareyre,J., Bernot,A., Laudet,V. and Guiguen,Y. (2000) DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss. *Biochem. Biophys. Acta*, **1493**, 180-187.

45. Zarkower,D. (2001) Establishing sexual dimorphism: conservation amidst diversity? *Nat. Rev. Genet.*, **2**, 175-185.

46. Aoki,Y., Nagao,I., Saito,D., Ebe,Y., Kinjo,M. and Tanaka,M. (2008) Temporal and spatial localization of three germline-specific proteins in medaka. *Dev. Dyn.*, **237**, 800-807.

47. Schier,A.F. (2007) The maternal-zygotic transition: death and birth of RNAs. *Science*, **316**, 406-407.

48. Mazumder,B., Seshadri,V. and Fox,P.L. (2003) Translational control by the 3'-UTR: the ends specify the means. *Trends Biochem. Sci.*, **28**, 91-98.

49. Kloc,M., Zearfoss,N.R. and Etkin,L.D. (2002) Mechanisms of subcellular mRNA localization. *Cell*, **108**, 533-544.

50. Gao,F.B., Carson,C.C., Levine,T. and Keene,J.D. (1994) Selection of a subset of mRNAs from combinatorial 3' untranslated region libraries using neuronal RNA-binding protein Hel-N1. *Proc. Natl Acad. Sci. USA*, **91**, 11207-11211.

51. Niehrs,C. and Pollet,N. (1999) Synexpression groups in eukaryotes. *Nature*, **402**, 483-487.

52. Miller,S.W., Hayward,D.C., Bunch,T.A., Miller,D.J., Ball,E.E., Bardwell,V.J., Zarkower,D. and Brower,D.L. (2003) A DM domain protein from a coral, Acropora millepora, homologous to proteins important for sex determination. *Evol. Dev.*, **5**, 251-258.

53. Scali,C., Catteruccia,F., Li,Q. and Crisanti,A. (2005) Identification of sex-specific transcripts of the Anopheles gambiae doublesex gene. *J. Exp. Biol.*, **208**, 3701-3709.

54. Nagoshi,R.N. and Baker,B.S. (1990) Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns. *Genes Dev.*, **4**, 89-97.

55. Burtis,K.C. and Baker,B.S. (1989) Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. *Cell*, **56**, 997-1010.

56. Sreenivasulu,K., Ganesh,S. and Raman,R. (2002) Evolutionarily conserved, DMRT1, encodes alternatively spliced transcripts and shows dimorphic expression during gonadal differentiation in the lizard, Calotes versicolor. *Mech. Dev.*, **119**(Suppl. 1), S55-S64.

57. Guo,Y., Cheng,H., Huang,X., Gao,S., Yu,H. and Zhou,R. (2005) Gene structure, multiple alternative splicing, and expression in gonads of zebrafish *Dmrt1*. *Biochem. Biophys. Res. Commun.*, **330**, 950-957.

58. Huang,X., Guo,Y., Shui,Y., Gao,S., Yu,H., Cheng,H. and Zhou,R. (2005) Multiple alternative splicing and differential expression of *dmrt1* during gonad transformation of the rice field eel. *Biol. Reprod.*, **73**, 1017-1024.

59. Zhao,Y., Lu,H., Yu,H., Cheng,H. and Zhou,R. (2007) Multiple alternative splicing in gonads of chicken DMRT1. *Dev. Genes Evol.*, **217**, 119-126.

60. Lu,H., Huang,X., Zhang,L., Guo,Y., Cheng,H. and Zhou,R. (2007) Multiple alternative splicing of mouse *Dmrt1* during gonadal differentiation. *Biochem. Biophys. Res. Commun.*, **352**, 630-634.

61. Cheng,H.H., Ying,M., Tian,Y.H., Guo,Y., McElreavey,K. and Zhou,R.J. (2006) Transcriptional diversity of DMRT1 (dsx- and mab3-related transcription factor 1) in human testis. *Cell Res.*, **16**, 389-393.
# Divergent Expression Regulation of Gonad Development Genes in Medaka Shows Incomplete Conservation of the Downstream Regulatory Network of Vertebrate Sex Determination

Amaury Herpin,*,<fnref n="1" /> Mateus C. Adolfí,<fnref n="1" /> Barbara Nicol,<fnref n="2" /> Maria Hinzmann,<fnref n="1" /> Cornelia Schmidt,<fnref n="1" />
Johanna Klughammer,<fnref n="1" /> Mareen Engel,<fnref n="1" /> Minoru Tanaka,<fnref n="3" /> Yann Guiguen,<fnref n="2" /> and Manfred Schartl<fnref n="1" />

## Abstract

Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of “master sex-determining genes” at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is *dmrt1bY*, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of *Dmrt1bY*. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type *aromatase* transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

**Key words:** gene regulatory network evolution, divergent expression regulation, gonadal development, adaptive evolution.

## Introduction

Sex determination, the decision whether the bipotential gonad anlage will become a testis or an ovary, is a complex and tightly controlled developmental process. The fate determination and cell differentiation programs are regulated and tuned by cascades or networks of genes. Comparative studies on sex determination cascades of different organisms revealed a remarkable diversity of “master sex-determining genes” at the top of the genetic hierarchies, whereas downstream components surprisingly appeared to be evolutionarily more conserved and tend to converge upon the regulation of common effectors. Hence, a comparative view on genetic sex determination mechanisms led to the paradigm that “masters change, slaves remain” (Graham et al. 2003). A well-known example illustrating this paradigm is the *SRY* gene, the master sex-determining gene of mammals, which has not been detected outside of the therian mammals. However, its subordinated genes (*SOX9*, *WT1*, *DMRT1*, *AMH*, *SF1*, *FOXL2*) or signaling pathways (TGF-beta, WNT4/ beta-catenin, Hedgehog) have homologs in a much broader spectrum of species, including nonvertebrates, where they apparently are also involved in sex determination. These

observations led to the emergence of the subversive stereotype that master sex-determining genes individually spark and orchestrate the irreversible action of uniform and integrated gender-specific pathways. Conservation at the bottom and diversity at the top could be convincingly explained by an evolutionary scenario in which these hierarchies evolve from common core downstream components that acquire new upstream regulators (Wilkins 2007). Although the global rule of sex determination evolution is intuitively appealing and well accepted, only the variety at the top is well supported by comparative experimental data (Graham et al. 2003; Haag and Doty 2005; Herpin and Schartl 2008). The downstream conservation is less studied and relies only on a few gene expression studies. We have used the medaka as a versatile model system to study gene regulatory interactions and their evolutionary conservation (Wittbrodt et al. 2002; Herpin and Schartl 2009).

In the medaka fish, which has XY-XX sex determination, *dmrt1bY*, the duplicated copy on the Y-chromosome of *dmrt1a*, was shown to be the dominant master regulator of male development (Matsuda et al. 2002; Nanda et al. 2002), similar to *Sry* in mammals. Interestingly, *dmrt1*, the ancestor of *dmrt1bY*, is one of the downstream effectors of *SRY* in the

<footnote n="1">¹University of Wuerzburg, Physiological Chemistry, Biocenter, Am Hubland, Wuerzburg, Germany</footnote>

<footnote n="2">²INRA, UR1037, LPGP, Fish Physiology and Genomics, Rennes, France</footnote>

<footnote n="3">³Laboratory of Molecular Genetics for Reproduction, National Institute for Basic Biology, Okazaki, Japan</footnote>

<footnote>*Corresponding author: E-mail: amaury.herpin@biozentrum.uni-wuerzburg.de.</footnote>

<footnote>Associate editor: Takashi Gojobori</footnote>
mammalian male pathway. The duplicated copy of *dmrt1* on the Y-chromosome has acquired an upstream position in the sex-determining cascade. Remarkably this evolutionary novelty, requiring a rewiring of the regulatory network, was brought about by co-optation of "ready-to-use" pre-existing *cis*-regulatory elements contributed by transposable elements (Herpin et al. 2010). With respect to their biochemical functions, both medaka *dmrt1* paralogs act as transcriptional regulators (Herpin et al. 2010). *Dmrt1bY* was shown to be responsible for male-specific primordial germ cell mitotic arrest in the developing gonad at the sex-determination stage (Herpin et al. 2007). In contrast, the autosomal *dmrt1a* medaka gene is essential for testis maintenance (Masuyama et al. 2012).

The medaka gonad is formed by the coordinated development of two different cell lineages: the germ cells and the somatic gonadal mesoderm surrounding the germ cells. Shortly before hatching, at the time of expression of *dmrt1bY* in the male gonad primordium, the germ cells in the female gonad actively proliferate and undergo meiosis, whereas this is not observed in male gonads (Kobayashi et al. 2004; Herpin et al. 2007). It is only 10 days later that the first somatic gonadal dimorphisms are apparent with the formation of the acinus (the seminiferous tubule precursor) and the follicles in gonads of male and female, respectively. Interestingly, ovarian cords within the germinal epithelia of medaka ovaries have been recently characterized (Nakamura et al. 2010). These cords composed of somatic *sox9b*-expressing cells and mitotic *nos2*-expressing oogonia continually give rise to germ cells and form a stem cell niche referred to as the germinal cradle (Nakamura et al. 2010). These cradles containing germline stem cells contribute to the production of fertile eggs during the life cycle of the adult ovary.

Like in other vertebrates studied so far, many components of the classical repertoire of mammalian sex-determining genes could be inventoried in medaka as well (Matsuda 2005; Siegfried 2010). To elucidate the gene regulatory network that controls specification and patterning of the gonads in the medaka fish, in this study we report the in vivo expression dynamics of classical mammalian markers within the forming and the adult gonads in medaka.

Because of their important roles in initiating male or female gonadal development in mammals, the respective implications of either *Dmrt1* or *Foxl2* transcription factors were examined. Further on, two of the major signaling pathways central for early gonadal induction and maintenance in mammals, namely the canonical Hedgehog and Wnt4/β-catenin signaling pathways (Wilhelm et al. 2007; Liu et al. 2010; Franco and Yao 2012), were investigated.

The Hedgehog (HH) signaling pathway plays an essential role in a wide variety of developmental processes (Ingham and McMahon 2001). Three HH proteins have been identified in mammals; SONIC (SHH), INDIAN (IHH), and DESERT (DHH) (Hooper and Scott 2005). *Dhh* and *Ihh* are co-expressed in the adult ovary where they stimulate proliferation and steroidogenesis of theca cells (Spicer et al. 2009). *Dhh* is also required for maintenance of the male germ line and spermatogenesis in mice (Bitgood et al. 1996; Clark et al.

2000). In mammals all three HH ligands signal by binding to one of two homologous transmembrane receptors, PATCHED homolog 1 and 2 (PTCH1 and 2). HH signaling is modulated by HH-induced transcription of the HH antagonistic interacting protein (HHIP) that binds to HH ligands and prevents their interaction with PTCH receptors (Chuang and McMahon 1999).

*R-spondin1* (*Rspo1*), a member of a small family of secreted growth factors, is a key female-determining factor. RSPO protein operates through the canonical WNT signaling pathway (Tomizuka et al. 2008) to activate the β-catenin pathway as well as via upregulation of *Follistatin* (*Fst*) through WNT4 (Yao et al. 2004). It is well established that mammalian R-SPO-1, WNT4, β-catenin, and FST are components of a single pathway that promotes ovarian development and suppresses the formation of testis cord (Chassot et al. 2008).

The winged helix/forkhead transcription factor FOXL2 is mainly expressed in the somatic cells of the female gonad (Crisponi et al. 2001). The major role of *Foxl2* during gonadal differentiation and maintenance has recently been shown via the mutual antagonistic relationship of *Foxl2* and *Dmrt1*. FOXL2 suppresses expression of *Dmrt1* and vice versa for maintaining female or male gonadal fate, respectively (Uhlenhaut et al. 2009; Matson et al. 2011). Additionally it has been reported that FOXL2 and WNT4 (Yao 2005; Ottolenghi et al. 2007; Garcia-Ortiz et al. 2009) cooperate in regulating FST expression during ovarian development. Interestingly in the ovary the expression profiles of *Foxl2* highly correlate with that of *Aromatase* (*Cyp19*), suggesting that *Foxl2* is involved in the regulation of estrogen synthesis via direct transcriptional upregulation of *Aromatase* (Pannetier et al. 2006). On the other hand, several other factors (e.g., testosterone, TGF-β1, TNF-α, and glucocorticoids) have been shown to direct the expression of the aromatase gene in Sertoli, Leydig, and germ cells of rat testis (see Bourguiba et al. 2003 for review).

Analyzing several fluorescent reporter lines established from a bacterial artificial chromosome recombination (BAC recombination) method resulting in optimal spatial resolution and high reliability of gene expression (Giraldo and Montoliu 2001; Nakamura et al. 2008b; Suster et al. 2011), we find major discrepancies between mammals and medaka, notably amongst spatial and temporal expression patterns of the canonical signaling pathways. Using in vivo whole tissues chromatin immunoprecipitation and in vitro gene regulation assays, we can reveal possible interactions between these pathways that emphasize the importance of the cellular context on modulating these regulations and call into question a strict conservation of regulatory and functional interactions of sexual development genes in vertebrates.

## Results

### Patched-2 Expression and Hedgehog Pathway Regulation

To determine the role of the hedgehog signaling pathway in gonadal development of medaka, we recorded the temporal and spatial expression patterns of the key receptor, Ptch2.
Although expressed quite early on during somatogenesis (data not shown), *patched-2* was surprisingly neither detected throughout the early phase of gonadogenesis when the undifferentiated gonad anlage grows up to hatching stage (fig. 1A) nor further on up to the stage when the dimorphic gonad develops 10 days after hatching (fig. 1B and C). The first specific gonadal *ptch-2* expression was detected in the young ovary (fig. 1D–H). Here expression is restricted to the somatic cells that express *sox9b* and surround the germline stem cells of the germinal cradle within the ovarian cord (Nakamura et al. 2010) (fig. 1G and H). Interestingly, *r-spo-1* is also co-expressed with *sox9b* in these cells (figs. 1I–K and 2A–D compared with fig. 8A–F). In ovaries of fish of later reproductive phase, when the cradle number has declined, *ptch-2* expression could be only noticed in the interstitium (fig. 2E–G). Unexpectedly and in contrast to *patched* expression in mammals, *ptch-2* expression was only detected at background levels throughout stages of testis development (data not shown). This inconspicuous role for the Hedgehog signaling in gonad development and maintenance was also apparent in real-time polymerase chain reaction (PCR) analysis. Although similarly expressed in different tissues, both *patched* receptor transcripts (1 and 2) are only expressed at background levels in adult gonads of both sexes (fig. 3A).

## *In vivo Dmrt1bY Binding to hhip Promoter Region*

Given the pivotal role of Dmrt1 transcription factors during medaka sex determination, *patched-2* and the antagonistic regulatory HH interacting protein (*hipp*) promoter regions were scanned for putative Dmrt1bY and Dmrt1a target sites. Although no Dmrt1-binding sites could be identified in the *patched-2* promoter region (10 kb upstream scanned), two sites were predicted with high fidelity in the *hipp* 5' region (fig. 4B). To assess the in vivo relevance of the predicted Dmrt1 interaction, two stable transgenic lines expressing either the full-length Dmrt1bY protein (Dmrt1::GFP) or a truncated form lacking the DNA-binding domain (ΔDmrt1::GFP), both fused to GFP, were utilized (Herpin et al. 2010). These two lines were used for in vivo tissue chromatin immunoprecipitation (in vivo tissue ChIP) on testis tissue using GFP antibody for immunoprecipitation (fig. 4A). For the predicted two proximal Dmrt1-binding sites in the *hipp* promoter, a more than 2.3-fold enrichment after immunoprecipitation validated Dmrt1 binding (fig. 4B). Under the same conditions, no binding was detected in ovary (data not shown). This indicates that, in vivo, Dmrt1bY and/or Dmrt1a are potentially regulating the gonadal HH signaling through direct transcriptional regulation of the antagonist hedgehog interacting protein, *hipp*.

## *Dmrt1-Induced Hedgehog Pathway Regulation*

Further functional characterization of Dmrt1-induced *hipp* transcriptional regulation was performed by overexpression of Dmrt1a or Dmrt1bY in spermatogonial (SG3) or fibroblast (OLF) cell lines of medaka. In both cell lines, *hipp* transcription was clearly induced (fig. 5A–D). Consistently, examination of the *hipp* expression pattern disclosed high and specific expression in testes (fig. 3A). Interestingly, at the receptor level (*patched* 1 and 2), although no direct interaction with

Dmrt1bY could be demonstrated, transcriptional down regulation of *patched-2* was observed after Dmrt1bY overexpression in the spermatogonial cell line (fig. 5E–H). In line with the in vitro regulation data, *patched-1/2* expression in gonads of both sexes was not above background (fig. 3A). Due to the absence of the *dmrt1bY* gene in females, and since a background level of *dmrt1a* expression is detected in ovary (Hornung et al. 2007), the high expression of *hipp* and the suppression of *ptch-2* must be exclusively regulated by the autosomal *dmrt1a* ortholog in the ovary of medaka.

## *R-spondin 1 and Follistatin Expression and Wnt Pathway Regulation*

Although ubiquitously expressed at early stages of development, neither medaka *r-spo-1* nor *fst* expressions could be detected in the presumptive gonadal mesoderm before the PGCs reach the undifferentiated gonadal primordium at stage 30 (fig. 7A–C; supplementary fig. S1, Supplementary Material online). Subsequently, in the dorsal region of the hindgut, a very furtive and time restricted pulse of *r-spo-1* and *fst* expression appears in the male gonadal primordium between stages 33 and 35 (fig. 7D–F). Interestingly, this very brief pulse of *r-spo-1* and *fst* expression occurs shortly before the rise of *dmrt1bY* expression at that stage (Kobayashi et al. 2004; Hornung et al. 2007). At hatching stage when sex determination occurs, no *r-spo-1* or *fst* expression could be detected in the gonadal primordia of males and females (fig. 7G–I). This lack of later gonadal expression is in line with similar findings in zebrafish (Zhang et al. 2011), turtle (Smith et al. 2008), chicken (Smith et al. 2008), and mice (Yao et al. 2004; Parma et al. 2006). Further on, while the ovary develops in juvenile females, *r-spo-1* expression is restricted to few somatic cells surrounding germline stem cells of the germinal cradle within the ovarian cord (fig. 8A–F). These somatic cells of the germinal cradle are the same that also co-express *sox9b* and *patched-2* (fig. 2A–D compared with fig. 8A–F). Similar to zebrafish (Zhang et al. 2011) and mouse (Smith et al. 2008), expression of medaka *r-spo-1* was also detected in granulosa cells around young oocytes (fig. 8G–L and table 1). Granulosa expression is then progressively lost while the oocytes are growing. In male gonads, only a low expression of *r-spo-1* is detected (fig. 3B–D). Medaka *fst* could not be detected in the ovary at this stage. In contrast to germ cell expression of *r-spo-1* in chicken (Smith et al. 2008), zebrafish (Zhang et al. 2011) and mouse (Smith et al. 2008) ovaries or zebrafish testis (Zhang et al. 2011) or *fst* in rat testis (Meinhardt et al. 1998), neither *r-spo-1* nor *fst* could be detected in gonadal germ cells or spermatogonia in medaka (fig. 8 and table 1). In older ovaries, *r-spo-1* and *fst* are also expressed in the interstitium as well as in the ovarian epithelium (fig. 8M, N, and Q–S). Of note, sparse clusters of *fst* expressing interstitial somatic cells were detected in testes (fig. 8O and P).

## *In vivo Dmrt1bY Binding to the r-spo-1 and dkk1 Promoter Regions and Regulation of the r-spo-1 Pathway*

We next analyzed the capacity of Dmrt1 to transcriptionally regulate positive (*r-spo-1* and *fst*) and negative (*dkk1*)
<figure><img src="image_52.png" /><figcaption>FIG. 1. Spatial and temporal expression patterns of *patched-2* during medaka early gonad formation and in adult gonads. A *patched-2* BAC reporter transgenic medaka line expressing GFP was established to follow *patched-2* expression dynamics in vivo during gonad formation. Although expressed early on during somatogenesis (A), *patched-2* expression was never detected during the early phase of gonadogenesis at hatching stage (B and C). In the young ovary, *patched-2* expression is restricted to somatic cells of the ovarian cord (D–H) where it is co-expressed together with *sox9b* (I–K).</figcaption></figure>
<figure><img src="image_53.png" /><figcaption>FIG. 2. Specific *patched-2* expression in the germinal cradle of the ovary. *Patched-2* expression is specifically restricted to the somatic cells that express *sox9b* and surround the germ line stem cells of the germinal cradle within the ovarian cord but is absent in the germ line stem cells (A–D). In ovaries of fish of later reproductive phase, *patched-2* expression is only apparent in the interstitium (E–G).</figcaption></figure>
<figure><img src="image_54.png" /><figcaption>**FIG. 3.** Real-time PCR expression patterns of Hedgehog and Wnt pathway components and in vivo reporter expression of *R-spondin1* in medaka adult testis. Expression patterns of different components of the Hedgehog (A) and Wnt (B) pathways in organs of adult male and female medaka determined from pooled (3–4 animals) total RNA extracts. In adult testes, background levels of *R-spondin1* expression are detected either by real-time PCR (B) or BAC reporter fluorescence (C and D) methods.</figcaption></figure>
<figure><img src="image_55.png" /><figcaption>In vivo tissue ChIP of Dmrt1bY binding to R-spondin 1 promoter</figcaption></figure>

<figure><img src="image_56.png" /></figure>

FIG. 4. In vivo tissue chromatin immunoprecipitation (in vivo tissue-ChIP) analysis of Dmrt1bY targets. Chromatin immunoprecipitation using both Dmrt1bY::GFP and deltaDmrt1bY transgenic lines respectively expressing either Dmrt1bY or as control a truncated Dmrt1bY (delta DM form lacking the DNA-binding domain) fused to GFP revealed in vivo specific Dmrt1bY protein affinities to target sites nested within hedgehog interacting protein 1 (hip1), r-spondin-1, and dkk-1 respective promoter regions. (A) Transgenic lines established for in vivo tissue-ChIP. (B-D) Specific enrichment of hhip-1 (B), r-spondin-1 (C), and dkk-1 (D) promoter-nested Dmrt1 binding sites subsequent to Dmrt1bY immunoprecipitation.

<figure><img src="image_57.png" /><figcaption>B In vivo tissue ChIP of Dmrt1bY binding to Hhip1 promoter</figcaption></figure>

effectors of the Wnt/Rspo1/Fst pathway. Hence, the ability of Dmrt1 to directly bind in vivo to the *fst*, *r-spo-1*, and *dkk1* promoters was investigated. Out of several putative Dmrt1-binding sites within the *r-spo-1* and *dkk1* promoter regions, in vivo ChIP revealed direct Dmrt1bY interaction with both promoters (fig. 4C and D). Of note, robust binding of Dmrt1bY to *dkk1* promoter region was seen in testes, but similar interactions did not occur in ovaries (fig. 4D). Although not highly significant, an analogous trend is observed for Dmrt1bY binding to *r-spo-1* promoter (fig. 4C). Furthermore cell transfection experiments overexpressing either Dmrt1a or Dmrt1bY showed a Dmrt1bY-specific slight transcriptional upregulation of *dkk1* (fig. 6A-D). Interestingly, although *fst* does not seem to be under Dmrt1bY regulation (fig. 6I and L), Dmrt1a does upregulate *fst* transcription (fig. 6I and K). Astonishingly and in contrast to our in vivo expression data, Dmrt1-induced transcriptional upregulation was observed for *r-spondin-1* (fig. 6E-H). Real-time PCR quantification nevertheless revealed *r-spo-1* and *fst* to be only expressed at background levels in

gonads (fig. 3B), surprisingly also including adult testis (fig. 3C and D).

## Foxl2 Expression in the Adult Gonad

To investigate the role of Foxl2 in ovarian differentiation, we analyzed Foxl2 protein distribution in the medaka ovary (figs. 9 and 10). During the transition process of germ line stem cells to oocytes within the germinal cradle, medaka Foxl2 expression starts within the germ line stem cells and continues during meiosis until early oogenesis (fig. 9A-L). On the contrary, no Foxl2 protein could be detected in the interwoven threadlike ovarian cords of *sox9b*-expressing cells where the supporting follicular cells reside (fig. 9A-L). During the following steps of oogenesis, the accompanying cells of the supporting layer progressively loose *sox9b* expression while Foxl2 expression rises (fig. 9A-L). Consistent with mRNA localization (Nakamoto et al. 2006), Foxl2 protein in the medaka ovary was localized within the follicular cells of the previtellogenic and vitellogenic follicles and then gradually lost while maturation proceeds (fig. 10A).
<caption>A</caption>

HHIP regulation
(Dmrt1a transfection; Sg3 cells)

<figure><img src="image_58.png" /><figcaption>B</figcaption></figure>

HHIP regulation
(Dmrt1bY transfection; Sg3 cells)

<figure><img src="image_59.png" /><figcaption>C</figcaption></figure>

HHIP regulation
(Dmrt1a transfection; OLF cells)

<figure><img src="image_60.png" /><figcaption>D</figcaption></figure>

HHIP regulation
(Dmrt1bY transfection; OLF cells)

<figure><img src="image_61.png" /><figcaption>E</figcaption></figure>

Ptch1 regulation
(Dmrt1a transfection; Sg3 cells)

<figure><img src="image_62.png" /><figcaption>F</figcaption></figure>

Ptch1 regulation
(Dmrt1bY transfection; Sg3 cells)

<figure><img src="image_63.png" /><figcaption>G</figcaption></figure>

Ptch2 regulation
(Dmrt1a transfection; Sg3 cells)

<figure><img src="image_64.png" /><figcaption>H</figcaption></figure>

Ptch2 regulation
(Dmrt1bY transfection; Sg3 cells)

<figure><img src="image_65.png" /><figcaption>FIG. 5. Hedgehog pathway transcriptional regulation after Dmrt1a/Dmrt1bY overexpression in different cell lines. *hip (A–D)*, *patched-1 (E and F)*, and *patched-2 (G and H)* expression were monitored in different cell lines (SG3 and OLF) after Dmrt1a or Dmrt1bY overexpression. *hip transcription is mainly upregulated by both Dmrt1s (A–D).* *patched-2 transcription is only clearly downregulated by Dmrt1bY (G and H)* and *patched-1 expression levels remain unaffected (E and F).*</figcaption></figure>

Particularly, Foxl2 is present in the nuclei of all granulosa cells (fig. 10B–D). Interestingly, while Foxl2 has been reported to be a strong inducer of the steroidogenic activity of granulosa cells via upregulation of the aromatase gene (the ovarian-type Cyp19a1) (Hudson et al. 2005; Wang et al. 2007; Guiguen et al. 2010), unexpectedly, and in contrast to mammals, a minority of theca cells do also express Foxl2 in medaka (fig. 10E–G). Our results reveal two subpopulations of *cyp19a1*-positive theca cells, which are either Foxl2 positive or do not express the transcription factor (fig. 10H–K).

Discussion

Comparative studies on sex determination cascades of different organisms revealed that the genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly were found more widespread and are evolutionarily more conserved. Without much further studies, these observations led to the reasoning that conservation of sequence equalizes to conservation of function. While vertebrates have at least a common set of transcriptional regulators, including DMRT1 and FOXL2, as well as some signaling molecules and pathways such as the Hedgehog and R-spo-1/Wnt4 pathways, their molecular interplay and epistatic

relationships are nevertheless far from being understood. The purpose of our work was to examine this molecular interplay in fish.

Absence of *R-spo-1*, *fst*, and *ptch-2* in Medaka Germ Cells

In the mouse it was proposed that activation of the R-spo-1/Wnt/Fst signaling pathway in both somatic and germ cells, besides triggering meiosis in fetal germ cells, is required for ovarian differentiation and maintenance of ovarian cell identity (Chassot et al. 2008). Indeed inherent to their inductive epigenetic mode of specification, the germline sex is likely to be determined early on by *Sry* acting in the somatic cells (McLaren and Southee 1997; Sekido and Lovell-Badge 2008; Bowles et al. 2010) in a noncell-autonomous manner (see De Felici 2009 for review). *Sry* controls whether bipotential precursor cells differentiate into testicular Sertoli cells or ovarian granulosa cells (Koopman et al. 1991). This pivotal decision in a single gonadal cell type ultimately controls sexual differentiation throughout the body. Sex determination can be viewed as a battle for primacy in the fetal gonad between a male regulatory gene network in which *Sry* activates *Sox9* and a female network involving WNT/β-catenin signaling (Uhlenhaut et al. 2009; Herpin and Schartl 2011; Matson et al. 2011).
<figure><img src="image_66.png" /><figcaption>FIG. 6. Canonical Wnt pathway (dkk-1, r-spondin-1, and fst) transcriptional regulation after Dmrt1a/Dmrt1bY overexpression in different cell lines. dkk-1 (A–D), r-spondin-1 (E–H), and fst (I–L) expression was monitored in different cell lines (SG3 and OLF) following Dmrt1a or Dmrt1bY overexpression. Transcription of dkk-1 and r-spondin-1 is upregulated by both Dmrt1s (A–H). Differential regulation of fst is observed. While no regulation was observed after Dmrt1bY overexpression, fst transcription is clearly upregulated in both cell lines overexpressing Dmrt1a (I–L).</figcaption></figure>

In contrast, the sexual plasticity of medaka germ cells seems to be retained much longer than in mammals as illustrated by XX/XY transplantation chimeras. Although XY somatic cells differentiate into male cells according to their sex chromosome composition, in this environment XX germ cells differentiate into male cells regardless of their sex chromosome composition (Shinomiya et al. 2002). Hence, while unlike their mammalian counterparts none of the different medaka marker genes analyzed (*r-spo-1*, *fst* or *ptch-2*) were detected in germ cells at any time of gonadal development, this major inconsistency between mammals and fish certainly reflects intrinsically divergent modes of germ cell commitment and interaction between germ and somatic

cells possibly accounting for a higher sexual plasticity of germ cells in fish.

## Expression of the Rspo1/Fst and Hedgehog Pathways
### Role of the Rspo1/Wnt/Fst Pathway during Gonad Development and Maintenance

In line with observations made in zebrafish (Zhang et al. 2011), turtle (Smith et al. 2008), chicken (Smith et al. 2008), and mice (Yao et al. 2004; Parma et al. 2006), the absence in medaka of *r-spo-1* and *fst* dimorphic expression during sex determination stages does not support a role during gonad induction. Of particular interest and unlike in mammals, the lack of medaka *ptch-2* expression during the same
<figure><img src="image_67.png" /><figcaption>FIG. 7. *R-spondin-1* and *follistatin* spatial and temporal expression patterns during medaka gonad primordium formation. Two BAC reporter fish transgenic lines expressing either GFP or mCherry were established to follow *r-spo-1* and *fst* expression dynamics during gonad formation in vivo. Neither *r-spo-1* nor *fst* expression was detected in the presumptive gonadal mesoderm at early stages of development (A–C). Between stages 33 and 35 in the dorsal region of the hindgut *r-spo-1* and *fst* are co-expressed (D–F). At hatching stage, although ubiquitously expressed, neither *r-spo-1* nor *fst* were detected in the gonadal primordium (G–I).</figcaption></figure>

period additionally rules out any involvement of the Hedgehog pathway for gonad induction. Further on, while the ovary develops in the juvenile female, the strict co-expression of *r-spo-1* and *ptch-2*, together with *sox9b* in the somatic cells of the germinal cradles, likely indicates a role in differentiating and specifying the somatic supporting lineage of the ovary. Of note the early decoupling of *r-spo-1* and *fst* expression patterns, although expected to be involved in the same signaling pathway in mammals (Yao et al. 2004), de facto invalidates such an interplay in the medaka ovarian cradle.

Our results support the view that *r-spo-1* has a globally conserved female-specific expression profile in vertebrate gonads despite some slight but intriguing divergences. In all analyzed vertebrate species it is apparent that *r-spo-1* expression goes along with the process of somatic cell organization within the young ovary (table 1). In mouse, chicken, and medaka, *r-spo-1* expression at that time, predominantly or

exclusively in the somatic cells, suggests its implication in a conserved pathway leading to folliculogenesis. On the other hand, the absence of medaka *r-spo-1* expression in gonad embedded germ cells, unlike in zebrafish, mice, chicken, and turtle, reveals that a role for germ cell development might not be accordingly conserved in the adult gonad of medaka.

In mammals, *R-spo-1* engages the effector pathway of Wnt signaling and β-catenin and thereby activates *Fst* expression (Carmon et al. 2011). Hence, also medaka *fst* spatial and temporal expression pattern was expected to overlap with *r-spo-1*. While medaka *r-spo-1* and *fst* indeed display similar expression in adult tissues (fig. 3B), medaka *fst* was, however, absent during the early and late stages of ovarian induction and development. *Fst* expression was perceptible only in the interstitium and epithelium of old ovaries together with *r-spo-1* (fig. 8M, N, and Q-S). Unexpectedly, *fst* was also
<figure><img src="image_68.png" /><figcaption>FIG. 8. *R-spondin-1* and *follistatin* expression in the adults gonads. In the ovary of juvenile females, *r-spondin* expression is restricted to somatic cells surrounding the germ line stem cells of the germinal cradle within the ovarian cord (A–F). Co-expression with *sox9b* (A to F) and *patched-2* (see fig. 2A–D) is observed. *R-spondin-1* expression in granulosa cells is also detected around young oocytes (G–L). In the ovary, *follistatin* expression was only detected in the interstitium (M and N) and in the ovarian epithelium together with *r-spondin-1* (Q–S). Sparse clusters of *follistatin* expressing interstitial somatic cells are also detected in adult testes (O and P).</figcaption></figure>
<table><caption>**Table 1.** Comparative Analysis of Gonadal Expression of R-spondin1, Follistatin, Patched2, and Foxl2 in Vertebrates.</caption><thead><tr><th rowspan="2">Genes</th><th colspan="3">Gonadal Expression Patterns (Adult Ovaries/Testes)</th></tr><tr><th>Medaka</th><th>Mouse</th><th>Other Vertebrates</th></tr></thead><tbody><tr><td><i>R-spondin 1</i></td><td>O (A) Somatic cells surrounding the germ cells and germline stem cells of the ovarian cord. Granulosa cells of the young oocytes. (E) Not expressed during early gonadal development.<br>T N.D.</td><td>(E) Predominantly in the somatic cells of the developing ovary (Smith et al. 2008). (E) Germ cells (during meiosis at low levels) (Smith et al. 2008).<br>N.D. (Smith et al. 2008).</td><td>Chicken: (E) outer cortical zone of the developing ovary and germ cells during meiosis (Smith et al. 2008). <i>Danio:</i> (A) granulosa and theca cells. Premature germ cells, oogonia, primary oocytes (Zhang et al. 2011).<br>Chicken: (E) not detected (Smith et al. 2008). <i>Danio:</i> (A) Leydig cells, spermatogonia, and spermatocytes (Zhang et al. 2011).</td></tr><tr><td><i>Follistatin</i></td><td>O (A) Ovarian epithelium and interstitium of old ovaries.<br>T N.D.</td><td>(E) Somatic cells of the embryonic ovary (Menke and Page 2002; Yao et al. 2004), (A) co-localization with Foxl2 (Kashimada et al. 2011).<br>(E) Not detected in embryonic testes (Menke and Page 2002).</td><td>Sheep: (A) granulosa cells of the growing follicles II and III (Tisdall et al. 1994).<br>Rat: (A) Sertoli and endothelial cells, germ cells, spermatogonia, spermatocytes, and round spermatids (Meinhardt et al. 1998).</td></tr><tr><td><i>Patched 2</i></td><td>O (E) Not expressed during early gonadal development. (A) Somatic cells surrounding the germ cells and germline stem cells of the ovarian cord (co-expression with r-spondin1).<br>T N.D.</td><td>(A) Highly expressed (testis-specific splice variants) (Szczepny et al. 2006).<br>(A) Lowly expressed (Spicer et al. 2009).</td><td>Tammar wallaby: (E) expressed throughout the development of the embryonic ovary. (A) Abundant in granulosa, cumulus, and theca cells of the adult ovary. Very weak in germ cells (O'Hara et al. 2011).<br>Tammar: (E) Leydig cells in the interstitium of the developing testes. (A) Restricted to Sertoli cells of the adult testes (O'Hara et al. 2011).</td></tr><tr><td><i>FoxL 2</i></td><td>O (A) Ovarian germline stem cells, initial stage of post meiotic oocytes. Sub-population of theca and granulosa cells together with aromatase (Cyp19a1) expression.<br>T N.D.</td><td>(E) From 12.5 dpc in mesenchymal pre-granulosa cells and (A) later in granulosa cells (Schmidt et al. 2004). (A) Small and medium size follicles (Pisarska et al. 2004).<br>N.D.</td><td>Chicken: (A) medullar part of the ovary, maturing and ovulated oocytes. Granulosa cells, weak in theca cells layer (Govoroun et al. 2004).</td></tr></tbody></table>

NOTE.—(E) embryonic expression; (A) adult expression.

detected in testes (fig. 8O and P). This expression pattern indicates that in medaka and unlike in mammals, Fst is probably neither acting throughout early female gonad patterning nor during maintenance of cell identity in the adult ovary. Instead medaka *fst* expression appears to be more a marker of the aging ovary, probably acting during follicular atresia. Of interest, while exclusive ovarian expression is generally described for *R-spo-1* and *Fst* in vertebrates, such strict female dimorphism was not observed for *r-spo-1* and *fst* in zebrafish, rat, and medaka (table 1 and fig. 3).

## Role of the Hedgehog Pathway during Gonad Development and Maintenance

The gonadal expression pattern of two components of the Hedgehog pathway in medaka is peculiar and different from what has so far been reported for other vertebrates including mammals (table 1). Unlike in mammals it appears that medaka gonadal HH signaling through the *patched-2* receptor

is not involved in inducing and specifying the gonad primordia. It would rather act late exclusively in the process of somatic cell differentiation in the ovarian cradle. In strict contrast to its mammalian counterparts, the quasi-absence of medaka *patched-2* expression during testis formation in larvae and for testis cell identity maintenance in the adult rules out any functions during these processes (table 1). Certainly, the low testicular expression of *patched-2* together with *Dmrt1a/1bY*-induced transcriptional upregulation of the HH antagonist *hipp* indicates a general function of *Dmrt1* in actively downregulating the Hedgehog pathway in medaka testes. Taken together, we can conclude that although apparently downregulated at the transcriptional level, a background expression of *patched-2* remains. This phenomenon is known as illegitimate transcription (Chelly et al. 1989; McLeod and Cooke 1989). We speculate that the high expression of the *hipp* hedgehog pathway antagonist in gonads (about 5 to 10 times higher than *patched1/2*
<figure><img src="image_69.png" /><figcaption>FIG. 9. *Foxl2* protein localization in the ovarian cradle. During development from germ line stem cells to oocytes within the germinal cradle, *foxl2* localization is first detected in the germ line stem cells and remains during meiosis until early oogenesis (A–L). Concomitantly, the accompanying somatic cells of the supporting layer progressively lose *sox9b* expression while *foxl2* expression rises (A–L).</figcaption></figure>

expression [fig. 3]) likely prevents any hedgehog activation
resulting from leaky background expression of patched recep-
tors. Interestingly, several putative Sox9 binding sites are pre-
sent in the patched-2 promoter region and might explain the
strict *sox9b/patched-2* co-expression observed in the support-
ing cells of the ovarian cradle.

A Newly Identified Subpopulation of Theca Cells
Expressing *Aromatase* but Not *Foxl2*

Examination of Foxl2 protein distribution in the medaka
ovary allowed us to define a new subpopulation of theca
cells expressing Foxl2. Also expressing *cyp19a1* (aromatase)
these cells are then suspected of having a steroidogenic
<figure><img src="image_70.png" /><figcaption>FIG. 10. Protein expression and localization of *foxl2* in the ovary. *foxl2* immunostaining is present in the nuclei of the follicular cells of the previtellogenic and vitellogenic follicles (A). In vitellogenic follicles, *foxl2* protein is detected in all granulosa cells (B–D). Nuclear localization of *foxl2* in few theca cells (E–G). *foxl2* expression occurs only in a sub-population of theca cells (arrow heads vs. asterisk in E–G) as shown by comparison with the thecal layer marker aromatase *cyp19a1* (H–K).</figcaption></figure>

activity. In contrast to mammals where ovarian-type *aromatase* is only produced by granulosa cells, the biological significance of such cells expressing *cyp19a1* and *Foxl2* within the thecal layer remains unclear. Of note, in ovaries of *cyp19a1/p450c17l* double transgenic medaka reporter lines, two subpopulations of theca cells were previously identified, being either *cyp19a1* or *p450c17l* positive in a mutually exclusive manner (Nakamura et al. 2009). The *cyp19a1* expressing subpopulation of theca cells was already considered as the precursors of the theca lineage (Nakamura et al. 2009).

The strict co-expression of *Foxl2* and *Aromatase* (*Cyp19*) in the mammalian ovary led to the further demonstration that *Foxl2* is involved in the regulation of estrogen synthesis via direct transcriptional upregulation of ovarian-type *Aromatase* (see Pannetier et al. 2006 for review). Surprisingly in medaka we found, within the thecal layer, aromatase-only positive theca cells that remained Foxl2-negative. In that perspective it is interesting to note that birds also have multiple

populations of theca cells some of which are also steroidogenic (Nitta et al. 1991). In contrast to the main consensus, the discordance of spatial expression patterns of *Foxl2* and ovarian-type *aromatase* (*cyp19a1*) calls into question an exclusive transcriptional regulation of *cyp19a1* by *Foxl2* in the ovary of medaka (fig. 11). Although we cannot exclude that aromatase-only positive cells have not been previously also positives for *Foxl2*, implying the requirement of *Foxl2* for the induction of the aromatase expression, our results indicate that *foxl2* is nevertheless not required for the maintenance of the aromatase expression.

## Variable Molecular Interplay among the Repertoire of Gonadal Markers during Medaka Gonad Formation and Maintenance

We could show a direct regulation of the Hedgehog and R-spo-1 pathways by *Dmrt1bY* (fig. 12). It is thus becoming apparent that despite its tangible requirement for mammalian testis formation and later on in regulating
<figure><img src="image_71.png" /><figcaption>FIG. 11. Schematic representation of granulosa and theca cell populations in mouse and medaka. In mammals, granulosa cells are the only cell type with steroidogenic activity, expressing both *foxl2* and *aromatase*. *Aromatase* expression is directly induced by *foxl2*. In medaka, like in mammals, granulosa cells express both *Foxl2* and *aromatase*. Examination of Foxl2 protein distribution in the medaka ovary revealed a new subpopulation of theca cells expressing Foxl2.</figcaption></figure>

Leydig and myoid cell function (Clark et al. 2000; Pierucci-Alves et al. 2001; Canto et al. 2004), the Hedgehog pathway might not only be dispensable during medaka male gonado-genesis and maintenance but even needs to be suppressed as it is actively repressed by *Dmrt1* genes. This would also explain the specific lack of *ptch-2* expression in medaka testes shown to be indirectly downregulated by *Dmrt1bY* (see fig. 12 for summary). In contrast the female-specific, *Dmrt1a*-triggerd upregulation of *follistatin* transcription might nevertheless point out the importance of the R-spo-1 pathway during female gonad differentiation although the upstream compo-nents of this same pathway are tightly regulated by the *Dmrt1* co-orthologs (fig. 12).

Importantly, showing that under certain conditions *Dmrt1* paralogs are able to strongly upregulate the female-specific *r-spondin-1* gene expression, we could also demonstrate that *Dmrt1bY/Dmrt1a*-triggered regulations are highly dependent of the cellular context and might suggest requirement of co-factors (fig. 12). These findings are reminiscent of obser-vations showing *Stra8* transcription to be directly repressed by *DMRT1* in mouse testes while activated in the fetal ovary (Krentz et al. 2011).

Interestingly, the canonical Wnt/ β-catenin (R-spo-1 and Fst) pathway strongly antagonizes FGF9, a robust component of the male sex-determining cascade in mammals (Kim et al. 2006; Matson et al. 2011). However, FGF9 is absent in the fish lineage and no indication of a redundant action of related FGF has been obtained, questioning the importance of FGF signaling in fish sex determination (Forconi et al. 2013). Into that direction the importance of the FGF9 signaling in vertebrates is actually also challenged by the finding that in chicken embryos *Fgf9* does not show any sexually dimorphic

expression pattern during gonadal differentiation (Cutting et al. 2013). Similarly, although phylogenetically preserved, the Sox9 gene, a direct target of Sry in mammals, has been shown to be functionally dispensable for medaka testis de-termination (Nakamura et al. 2008a, 2012). In this context our data might reflect a profound reorganization of that part of the fish gonadal regulatory network compared with mammals. While some components such as DMRT1, SOX9, FOXL2 and pathways such as Hedgehog or R-spo1/Wnt/Fst of the gonadal gene regulatory network are conserved on the DNA sequence level across phyla, their functions, regulation, and interplays might be considerably different.

## Materials and Methods

### BAC Recombination

Bacterial artificial chromosome clones encompassing medaka *patched-2* (ola1-199K19), *follistatin* (ola1-124N21), or *r-spondin-1* (ola1-158A23) genomic regions were obtained from NRPB Medaka (http://www.shigen.nig.ac.jp/medaka/, last accessed August 12, 2013). A BAC transgenic method using homologous recombination was employed to generate the reporter constructs as previously described (Nakamura et al. 2008b; Herpin et al. 2010). The following primers were used to amplify *eGFP/mCherry* fragments for homologous recombina-nation into the different BAC clones: BAC-Ptchd2-GFP-Fw: GCTGAACTCGCACCGATTCTGCGTCGCCTCCTGTTACCCGTCTTTGGACTATGGATATCATTTCTGTCGCCTTAAAG, BAC-Ptchd2-GFP-Rv: CGCAAGCGGCTGGGAGCGCGTATAACTCGGGGGTAAATCTCCAAAGACGCCAGAACAAACGACCCAA CACCGTGCG; BAC-Fst-Cherry-Fw: CTTTTGCGCTGCTTGTG TCAAATACGTGGCTCACTTTGCCTCTCCATCATGCTTGGG CCACCGGTCGCCACCATGGT, BAC-Fst-Cherry-Rv: CTTACC
<figure><img src="image_72.png" /><figcaption>FIG. 12. The *Dmrt1bY/Dmrt1a* gene regulatory networks during gonadal formation in medaka. Interaction scheme of the possible Dmrt1bY/Dmrt1a-triggered regulations of the Hedgehog and Wnt4 pathways during gonadal formation in medaka. Solid red arrows indicate for both Dmrt1bY and Dmrt1a positive regulation while dashed lines indicate sex-specific regulations. The green dashed line indicates a Dmrt1a, female-specific, indirect positive regulation favoring the expression of *follistatin*, while the blue dashed line reports a Dmrt1bY, male-specific, indirect repression of *patched-2* transcription. For the Dmrt1bY/Dmrt1a-triggered transcriptional regulation of *R-spondin1*, depending on the cellular contexts, the involvement of a sex-specific co-factor is proposed.</figcaption></figure>

TTGAACCTTCTGATGTTCCATGAGGTGACAAAGCCACATG AAGAAGAGAGTCGACCAGTTGGTGATTTTG; BAC-Rspo1- GFP-Fw: GATCCATCTGGTTGCAGGGGGGACCTTCACA GCCTGGAAGGCAGCAGGGACTCCACCGTCCGCCACCATG GTG, BAC-Rspo1-GFP-Rv: CTTCTCGCCTTGGAGAGTTTGAC AACATCGCTGTGACCCATGGAGCTGAGAATGAGTCGACC AGTTGGTATTTTG. After homologous recombination, the generated fragments were inserted into the BAC clones in frame downstream of the translation initiation site of the targeted genes.

## Generation of BAC Transgenic Medaka Lines and Imaging Analyses

The Carbio (WLC# 2674) strain of medaka (*Oryzias latipes*) was used for establishment of the transgenic lines. Microinjection of DNA was performed as described previously (Herpin et al. 2009) using BAC clone DNA at a concentration of 50–100 ng/mL. Adult G0 fish were then screened for fluorescence, and positive individuals were raised to adulthood. Siblings from positive G0 fish were mated to each other and the offspring were again sorted for fluorescence. *Sox9b*

and *cyp19a1* (*aromatase*) transgenic lines were described earlier (Nakamura et al. 2008a, 2009). For imaging embryos, hatchlings or tissues were mounted with 1.2% low melting temperature agarose. Confocal pictures and image stacks were acquired using a Nikon C1 (eclipse Ti) confocal laser scanning microscope and the NIS elements AR software.

## Immunochemistry

Ovaries or testes from juvenile and adult fish were fixed with 4% paraformaldehyde/balanced salt solution (111 mM NaCl, 5.37 mM KCl, 1 mM CaCl₂·H₂O, 0.6 mM MgSO₄·7H₂O, 5 mM Hepes, pH 7.3) for 30 min on ice. After fixation samples were washed three times for 10 min with MABT buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5, 0.1% Triton X-100) and subsequently twice for 30 min with MABDT buffer (MABT buffer complemented with 1% BSA and 1% DMSO). After blocking in MABDT-blocking buffer (MABDT buffer supplemented with 2% lamb or sheep serum), the tissues were incubated in MABDT-blocking buffer together with the primary antibody (1:150 dilution) overnight at 4 °C. Samples were then washed three times 5 min in MABDT buffer and
washed again four times for 30 min in MABDT-blocking buffer on ice. Thereafter samples were incubated overnight at 4°C with the secondary antibody diluted at 1:600 in MABDT-blocking buffer. Finally the tissues were washed in PBS, stained with Hoechst solution for 3 h at 4°C, mounted, and imaged with a confocal microscope (Nikon C1 confocal microscope).

## In vivo Chromatin Immunoprecipitation

For in vivo chromatin immunoprecipitation, the EpiQuik Tissue Chromatin Immunoprecipitation kit (Epigentek) was used according to the manufacturer's instructions, using 20 mg of testis tissue samples either from *dmrt1bY::GFP* or *deltadmrt1bY::GFP* transgenic fish (Hornung et al. 2007; Herpin et al. 2010) (20 testes for each) and GFP antibody (3 µg, Upstate) for immunoprecipitation. After tissue disaggregation and cell re-suspension, DNA was sheared by sonication (9 pulses of 10 s with an amplitude of 10%). After immunoprecipitation ([DKK1-(1/2) Fw01]: 5'-GATAACTCC GGCTGGGACGTTGAC-3'/[DKK-(1/2) Rv01]: 5'-ACAACAC TGAAGTGCTACAGAAGTC-3'; [DKK1-(3) Fw02]: 5'-AGTAT CAAGTGCTCAAGACGATCC-3'/[DKK1-(3) Rv02]: 5'-TACGA GCTGACATGTTCACATCTGCC-3'; [DKK1-(4/5) Fw03]: 5'-GC TGCAAGACAGGAAGAC-3'/[DKK1-(4/5) Rv03]: 5'-GTTAAT AGTCATGCTCAGTCTG-3'; [R-spo1-(1) Fw01]: 5'-CATCGGA TTTAACAGTTATGATTGC-3'/[R-spo1-(1) Rv01]: 5'-CGATAG TGATTGGTCAGTTA-3'; [R-spo1-(2/3) Fw02]: 5'-CATCGTGC CAACTTACAGCCAATC-3'/[R-spo1-(2/3) Rv02]: 5'-CTACCA AGACACGCTAGAAGCTCC; [R-spo1-(4) Fw03]: 5'-AAGTTG CTCAACACTTGTACAC-3'/[R-spo1-(4) Rv03]: 5'-AAGCAGA GACAATAGAATGCATC-3'; [R-spo1-(5) Fw04]: 5'-ATAAAC ATGTACAACAGTCATCTG-3'/[R-spo1-(5) Rv04]: 5'-TTCCA CTCTCGGCAAGAAATCAG-3'; [HHIP-Fw01]: 5'-TAGAGTAC GTCCGTCTACTG-3'/[HHIP-Rv01]: 5'-TGACAACAAAGTCG CAA-3') primer sets were used for enrichment quantification by real-time PCR.

## Bioinformatic Analyses

Binding sites for *Dmrt1bY* were identified using the matrix provided by (Murphy et al. 2007) together with the Regulatory Sequence Analysis Tools portal; RSat (http://rsat.ulb.ac.be/rsat/, last accessed August 12, 2013).

## In Vitro Expression Regulation Analyses and Real-Time PCR

Medaka spermatogonial (SG3) and fibroblast-like (OLF) cell lines were cultured as described (Etoh 1988; Hong et al. 2004). For transfection cells were grown to 80% confluency in 6-well plates and transfected with 5 µg expression vector using FuGene (Roche) reagent as described by the manufacturer.

Total RNA was extracted from fish tissues or transfected cells using the TRIZOL reagent (Invitrogen) according to the supplier's recommendation. After DNase treatment, reverse transcription was done with 2 µg total RNA using RevertAid First Strand Synthesis kit (Fermentas) and random primers. Real-time quantitative PCR was carried out with SYBR Green

reagents and amplifications were detected with an i-Cycler (Biorad). All results are averages of at least two independent reverse transcription reactions. Error bars represent the standard deviation of the mean. Relative expression levels (according to the equation 2-DeltaCT) were calculated after correction of expression of elongation factor 1 alpha (ef1alpha) and brain expression was set to 1 as a reference.

## Supplementary Material

Supplementary figure S1 is available at *Molecular Biology and Evolution* online (http://www.mbe.oxfordjournals.org/).

## Acknowledgments

This work was supported by a grant of the Graduate Training Program of the DFG (GK 1048, Molecular Basis of Organ Development in Vertebrates). Part of this study was also supported by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 222719-LIFECYCLE (to Y.G and B.N). This study was also partly funded by a grant dedicated to the "Mechanisms of sex determination in zebrafish" (UO Subaward Number 212791A).

## References

* Bitgood MJ, Shen L, McMahon AP. 1996. Sertoli cell signaling by Desert hedgehog regulates the male germline. *Curr Biol.* 6:298-304.

* Bourguiba S, Genissel C, Lambard S, Bouraima H, Carreau S. 2003. Regulation of aromatase gene expression in Leydig cells and germ cells. *J Steroid Biochem Mol Biol.* 86:335-343.

* Bowles J, Feng CW, Spiller C, Davidson TL, Jackson A, Koopman P. 2010. FGF9 suppresses meiosis and promotes male germ cell fate in mice. *Dev Cell.* 19:440-449.

* Canto P, Soderlund D, Reyes E, Mendez JP. 2004. Mutations in the desert hedgehog (DHH) gene in patients with 46,XY complete pure gonadal dysgenesis. *J Clin Endocrinol Metab.* 89:4480-4483.

* Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. 2011. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. *Proc Natl Acad Sci U S A.* 108:11452-11457.

* Chassot AA, Gregoire EP, Magliano M, Lavery R, Chaboissier MC. 2008. Genetics of ovarian differentiation: Rspo1, a major player. *Sex Dev.* 2: 219-227.

* Chelly J, Concordet JP, Kaplan JC, Kahn A. 1989. Illegitimate transcription: transcription of any gene in any cell type. *Proc Natl Acad Sci U S A.* 86:2617-2621.

* Chuang PT, McMahon AP. 1999. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. *Nature* 397: 617-621.

* Clark AM, Garland KK, Russell LD. 2000. Desert hedgehog (Dhh) gene is required in the mouse testis for formation of adult-type Leydig cells and normal development of peritubular cells and seminiferous tubules. *Biol Reprod.* 63:1825-1838.

* Crisponi L, Deiana M, Loi A, et al. (22 co-authors). 2001. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. *Nat Genet.* 27:159-166.

* Cutting A, Chue J, Smith CA. 2013. Just how conserved is vertebrate sex determination? *Dev Dyn.* 242:380-387.

* De Felici M. 2009. Primordial germ cell biology at the beginning of the XXI century. *Int J Dev Biol.* 53:891-894.

* Etoh. 1988. Establishment and characterization of various cell lines from Medaka (Teleostei) Invertebrate and fish tissue culture, p 266-269.

* Forconi M, Canapa A, Barucca M, et al. (14 co-authors). 2013. Characterization of sex determination and sex differentiation genes in latimeria. *PLoS One* 8:e56006.
Franco HL, Yao HH. 2012. Sex and hedgehog: roles of genes in the hedgehog signaling pathway in mammalian sexual differentiation. *Chromosome Res.* 20:247–258.

Garcia-Ortiz JE, Pelosi E, Omari S, et al. (12 co-authors). 2009. Foxl2 functions in sex determination and histogenesis throughout mouse ovary development. *BMC Dev Biol.* 9:36.

Giraldo P, Montoliu L. 2001. Size matters: use of YACs, BACs and PACs in transgenic animals. *Transgenic Res.* 10:83–103.

Govoroun MS, Pannetier M, Pailhoux E, Cocquet J, Brillard JP, Couty I, Batellier F, Cotinot C. 2004. Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development. *Dev Dyn.* 231:859–870.

Graham P, Penn JK, Schedl P. 2003. Masters change, slaves remain. *Bioessays* 25:1–4.

Guiguen Y, Fostier A, Piferrer F, Chang CF. 2010. Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. *Gen Comp Endocrinol.* 165:352–366.

Haag ES, Doty AV. 2005. Sex determination across evolution: connecting the dots. *PLoS Biol.* 3:e21.

Herpin A, Braasch I, Kraeussling M, Schmidt C, Thoma EC, Nakamura S, Tanaka M, Schartl M. 2010. Transcriptional rewiring of the sex determining dmrt1 gene duplicate by transposable elements. *PLoS Genet.* 6:e1000844.

Herpin A, Nakamura S, Wagner TU, Tanaka M, Schartl M. 2009. A highly conserved cis-regulatory motif directs differential gonadal synexpression of Dmrt1 transcripts during gonad development. *Nucleic Acids Res.* 37:1510–1520.

Herpin A, Schartl M. 2009. Molecular mechanisms of sex determination and evolution of the Y-chromosome: insights from the medakafish (Oryzias latipes). *Mol Cell Endocrinol.* 306:51–58.

Herpin A, Schartl M. 2008. Regulatory putsches create new ways of determining sexual development. *EMBO Rep.* 9:966–968.

Herpin A, Schartl M. 2011. Sex determination: switch and suppress. *Curr Biol.* 21:R656–R659.

Herpin A, Schindler D, Kraiss A, Hornung U, Winkler C, Schartl M. 2007. Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt I bY. *BMC Dev Biol.* 7:99.

Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, Chen T, Deng J, Gui J. 2004. Establishment of a normal medakafish spermatogonial cell line capable of sperm production in vitro. *Proc Natl Acad Sci U S A.* 101:8011–8016.

Hooper JE, Scott MP. 2005. Communicating with Hedgehogs. *Nat Rev Mol Cell Biol.* 6: 306–317.

Hornung U, Herpin A, Schartl M. 2007. Expression of the male determining gene dmrt1bY and its autosomal coorthologue dmrt1a in medaka. *Sex Dev.* 1:197–206.

Hudson QJ, Smith CA, Sinclair AH. 2005. Aromatase inhibition reduces expression of FOXL2 in the embryonic chicken ovary. *Dev Dyn.* 233: 1052–1055.

Ingham PW, McMahon AP. 2001. Hedgehog signaling in animal development: paradigms and principles. *Genes Dev.* 15:3059–3087.

Kashimada K, Pelosi E, Chen H, Schlessinger D, Wilhelm D, Koopman P. 2011. FOXL2 and BMP2 act cooperatively to regulate follistatin gene expression during ovarian development. *Endocrinology* 152:272–280.

Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, Chaboissier MC, Poulat F, Behringer RR, Lovell-Badge R, Capel B. 2006. Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. *PLoS Biol.* 4:e187.

Kobayashi T, Matsuda M, Kajiura-Kobayashi H, Suzuki A, Saito N, Nakamoto M, Shibata N, Nagahama Y. 2004. Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes. *Dev Dyn.* 231:518–526.

Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R. 1991. Male development of chromosomally female mice transgenic for Sry. *Nature* 351:117–121.

Krentz AD, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D. 2011. DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary. *Dev Biol.* 356: 63–70.

Liu CF, Liu C, Yao HH. 2010. Building pathways for ovary organogenesis in the mouse embryo. *Curr Top Dev Biol.* 90:263–290.

Masuyama H, Yamada M, Kamei Y, Fujiwara-Ishikawa T, Todo T, Nagahama Y, Matsuda M. 2012. Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka. *Chromosome Res.* 20:163–176.

Matson CK, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D. 2011. DMRT1 prevents female reprogramming in the postnatal mammalian testis. *Nature* 476:101–104.

Matsuda M. 2005. Sex determination in the teleost medaka, Oryzias latipes. *Annu Rev Genet.* 39:293–307.

Matsuda M, Nagahama Y, Shinomiya A, et al. (13 co-authors). 2002. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. *Nature* 417:559–563.

McLaren A, Southee D. 1997. Entry of mouse embryonic germ cells into meiosis. *Dev Biol.* 187:107–113.

McLeod JF, Cooke NE. 1989. The vitamin D-binding protein, alpha-fetoprotein, albumin multigene family: detection of transcripts in multiple tissues. *J Biol Chem.* 264:21760–21769.

Meinhardt A, O'Bryan MK, McFarlane JR, Loveland KL, Mallidis C, Foulds LM, Phillips DJ, de Kretser DM. 1998. Localization of follistatin in the rat testis. *J Reprod Fertil.* 112:233–241.

Menke DB, Page DC. 2002. Sexually dimorphic gene expression in the developing mouse gonad. *Gene Expr Patterns.* 2:359–367.

Murphy MW, Zarkower D, Bardwell VJ. 2007. Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA. *BMC Mol Biol.* 8:58.

Nakamoto M, Matsuda M, Wang DS, Nagahama Y, Shibata N. 2006. Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes. *Biochem Biophys Res Commun.* 344: 353–361.

Nakamura S, Aoki Y, Saito D, Kuroki Y, Fujiyama A, Naruse K, Tanaka M. 2008a. Sox9b/sox9a2-EGFP transgenic medaka reveals the morphological reorganization of the gonads and a common precursor of both the female and male supporting cells. *Mol Reprod Dev.* 75: 472–476.

Nakamura S, Kobayashi K, Nishimura T, Higashijima S, Tanaka M. 2010. Identification of germline stem cells in the ovary of the teleost medaka. *Science* 328:1561–1563.

Nakamura S, Kurokawa H, Asakawa S, Shimizu N, Tanaka M. 2009. Two distinct types of theca cells in the medaka gonad: germ cell-dependent maintenance of cyp19a1-expressing theca cells. *Dev Dyn.* 238:2652–2657.

Nakamura S, Saito D, Tanaka M. 2008b. Generation of transgenic medaka using modified bacterial artificial chromosome. *Dev Growth Differ.* 50:415–419.

Nakamura S, Watakabe I, Nishimura T, Toyoda A, Taniguchi Y, Tanaka M. 2012. Analysis of medaka sox9 orthologue reveals a conserved role in germ cell maintenance. *PLoS One* 7:e29982.

Nanda I, Kondo M, Hornung U, et al. (12 co-authors). 2002. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. *Proc Natl Acad Sci U S A.* 99: 11778–11783.

Nitta H, Osawa Y, Bahr JM. 1991. Multiple steroidogenic cell populations in the thecal layer of preovulatory follicles of the chicken ovary. *Endocrinology* 129:2033–2040.

O'Hara WA, Azar WJ, Behringer RR, Renfree MB, Pask AJ. 2011. Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial. *BMC Dev Biol.* 11:72.

Ottolenghi C, Pelosi E, Tran J, Colombino M, Douglass E, Nedorezov T, Cao A, Forabosco A, Schlessinger D. 2007. Loss of Wnt4 and Foxl2 leads to female-to-male sex reversal extending to germ cells. *Hum Mol Genet.* 16:2795–2804.

Pannetier M, Fabre S, Batista F, Kocer A, Renault L, Jolivet G, Mandon-Pepin B, Cotinot C, Veitia R, Pailhoux E. 2006. FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development. *J Mol Endocrinol.* 36:399–413.
Parma P, Radi O, Vidal V, Chaboissier MC, Dellambra E, Valentini S, Guerra L, Schedl A, Camerino G. 2006. R-spondin1 is essential in sex determination, skin differentiation and malignancy. *Nat Genet.* 38: 1304–1309.

Pierucci-Alves F, Clark AM, Russell LD. 2001. A developmental study of the Desert hedgehog-null mouse testis. *Biol Reprod.* 65:1392–1402.

Pisarska MD, Bae J, Klein C, Hsueh AJ. 2004. Forkhead l2 is expressed in the ovary and represses the promoter activity of the steroidogenic acute regulatory gene. *Endocrinology* 145:3424–3433.

Schmidt D, Ovitt CE, Anlag K, Fehsenfeld S, Gredsted L, Treier AC, Treier M. 2004. The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance. *Development* 131:933–942.

Sekido R, Lovell-Badge R. 2008. Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. *Nature* 453: 930–934.

Shinomiya A, Shibata N, Sakaizumi M, Hamaguchi S. 2002. Sex reversal of genetic females (XX) induced by the transplantation of XY somatic cells in the medaka, Oryzias latipes. *Int J Dev Biol.* 46: 711–717.

Siegfried KR. 2010. In search of determinants: gene expression during gonadal sex differentiation. *J Fish Biol.* 76:1879–1902.

Smith CA, Shoemaker CM, Roeszler KN, Queen J, Crews D, Sinclair AH. 2008. Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development. *BMC Dev Biol.* 8:72.

Spicer LJ, Sudo S, Aad PY, Wang LS, Chun SY, Ben-Shlomo I, Klein C, Hsueh AJ. 2009. The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells. *Reproduction* 138:329–339.

Suster ML, Abe G, Schouw A, Kawakami K. 2011. Transposon-mediated BAC transgenesis in zebrafish. *Nat Protoc.* 6:1998–2021.

Szczepny A, Hime GR, Loveland KL. 2006. Expression of hedgehog signalling components in adult mouse testis. *Dev Dyn.* 235: 3063–3070.

Tisdall DJ, Hudson N, Smith P, McNatty KP. 1994. Localization of ovine follistatin and alpha and beta A inhibin mRNA in the sheep ovary during the oestrous cycle. *J Mol Endocrinol.* 12:181–193.

Tomizuka K, Horikoshi K, Kitada R, et al. (12 co-authors). 2008. R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. *Hum Mol Genet.* 17:1278–1291.

Uhlenhaut NH, Jakob S, Anlag K, et al. (15 co-authors). 2009. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. *Cell* 139:1130–1142.

Wang DS, Kobayashi T, Zhou LY, Paul-Prasanth B, Ijiri S, Sakai F, Okubo K, Morohashi K, Nagahama Y. 2007. Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with ad4 binding protein/steroidogenic factor 1. *Mol Endocrinol.* 21:712–725.

Wilhelm D, Palmer S, Koopman P. 2007. Sex determination and gonadal development in mammals. *Physiol Rev.* 87:1–28.

Wilkins AS. 2007. Between "design" and "bricolage": genetic networks, levels of selection, and adaptive evolution. *Proc Natl Acad Sci U S A.* 104(Suppl. 1), 8590–8596.

Wittbrodt J, Shima A, Schartl M. 2002. Medaka—a model organism from the far East. *Nat Rev Genet.* 3:53–64.

Yao HH. 2005. The pathway to femaleness: current knowledge on embryonic development of the ovary. *Mol Cell Endocrinol.* 230: 87–93.

Yao HH, Matzuk MM, Jorgez CJ, Menke DB, Page DC, Swain A, Capel B. 2004. Follistatin operates downstream of Wnt4 in mammalian ovary organogenesis. *Dev Dyn.* 230:210–215.

Zhang Y, Li F, Sun D, Liu J, Liu N, Yu Q. 2011. Molecular analysis shows differential expression of R-spondin1 in zebrafish (*Danio rerio*) gonads. *Mol Biol Rep.* 38:275–282.
# Plasticity of gene-regulatory networks controlling sex determination: of masters, slaves, usual suspects, newcomers, and usurpators

Amaury Herpin<fnref n="1" />,<fnref n="2" /> & Manfred Schartl<fnref n="1" />,<fnref n="3" />,*

## Abstract

Sexual dimorphism is one of the most pervasive and diverse features of animal morphology, physiology, and behavior. Despite the generality of the phenomenon itself, the mechanisms controlling how sex is determined differ considerably among various organismic groups, have evolved repeatedly and independently, and the underlying molecular pathways can change quickly during evolution. Even within closely related groups of organisms for which the development of gonads on the morphological, histological, and cell biological level is undistinguishable, the molecular control and the regulation of the factors involved in sex determination and gonad differentiation can be substantially different. The biological meaning of the high molecular plasticity of an otherwise common developmental program is unknown. While comparative studies suggest that the downstream effectors of sex-determining pathways tend to be more stable than the triggering mechanisms at the top, it is still unclear how conserved the downstream networks are and how all components work together. After many years of stasis, when the molecular basis of sex determination was amenable only in the few classical model organisms (fly, worm, mouse), recently, sex-determining genes from several animal species have been identified and new studies have elucidated some novel regulatory interactions and biological functions of the downstream network, particularly in vertebrates. These data have considerably changed our classical perception of a simple linear developmental cascade that makes the decision for the embryo to develop as male or female, and how it evolves.

**Keywords** Dmrt1; ovary; SRY; testis; transcription factor

**DOI** 10.15252/embr.201540667 | Received 13 May 2015 | Revised 28 July 2015 | Accepted 31 July 2015

See the Glossary for abbreviations used in this article.

## Introduction

Developmental cascades are generally headed by evolutionary conserved master regulators that determine the developmental fate of a cell lineage toward distinct tissues or organs during embryogenesis. In contrast, determination of the development of the reproductive organs does not follow this rule. Studies over the last decades have revealed that the gene-regulatory cascades triggering sexual differentiation from worms and flies to mammals are composed of substantially different factors. In particular, a remarkable diversity of master sex-determining genes that govern the genetic hierarchies has become apparent. On the other hand, the downstream components seemed to be evolutionarily more conserved and appear to converge on the regulation of a few central common effectors. A well-known example illustrating this paradigm is the master sex-determining gene of mammals, the *SRY* gene. A corresponding homolog has not been detected outside of therian mammals (Marsupials and Placentalia). Conversely, those genes that act downstream of *SRY* as transcription factors (*SOX9*, *DMRT1*) or signaling pathways (TGF-β/Amh, Wnt4/β-catenin, Hedgehog), and genes involved in *SRY* regulation (*SF1*, *WT1*) have homologs with a known or presumed role in gonadogenesis or gonadal differentiation in many vertebrate species, and some even in non-vertebrate deuterostomes and protostomes. These findings suggested that a central paradigm of sex determination is that “masters change, slaves remain”.

This appealing global rule was quickly commonly accepted, in particular as the diversity at the top was confirmed experimentally [1–3]. Remarkably, some master sex-determining genes were recurrently identified and became the “usual suspects” for future studies in the search for master regulators (Table 1). All of these are genes, or duplicates and paralogs of genes, which were previously known to act in the regulatory network of gonad development. Much progress has also been made in understanding some of the regulatory interactions of the networks or cascades governed by the long known master sex-determining genes as well as, although to a lower extent, for the newly detected ones. We review here the current knowledge about the different molecules that have been demonstrated

<footnote n="1">1 Department Physiological Chemistry, Biocenter, University of Würzburg, Würzburg, Germany</footnote>

<footnote n="2">2 INRA, UR1037 Fish Physiology and Genomics, Sex Differentiation and Oogenesis Group (SDOG), Rennes, France</footnote>

<footnote n="3">3 Comprehensive Cancer Center Mainfranken, University Clinic Würzburg, Würzburg, Germany
*Corresponding author. Tel: +49 931 318 4148; E-mail: phch1@biozentrum.uni-wuerzburg.de</footnote>
## Glossary

**Amh**

Anti-Müllerian hormone

**Autosome**

On contrary to a sex chromosome, autosomal chromosomes are chromosomes that are not involved in primary sex determination

**Csd**

Complementary sex determiner

**CTD**

C-terminal domain

**DKK1**

Dickkopf-related protein 1

**Dmd3**

Doublesex and Mab-3 domain family member 3

**DMRT1 or 3**

Doublesex and Mab-3 related transcription factor 1 or 3

**Dosage sensitive gene**

Gene where the amount of gene product that determines the phenotype is dependent on the number of copies. Two copies are usually sufficient to establish the phenotype, while one is not (haploinsufficiency). For example, in birds two copies of the *Dmrt1* gene trigger male gonadal development, while one copy is not sufficient to make a male and then leads to female development

**Dsx**

Doublesex

**Environmental sex determination (ESD)**

When the sex of an individual is driven by different external factors including temperature, pH, social interactions (dominance, stress...)

**Esr1**

Estrogen receptor 1 is the human estrogen receptor alpha

**Fem**

Feminizer

**FGF9**

Fibroblast growth factor 9

**Foxl2**

Forkhead box transcription factor L2

**Fru**

Fruitless

**Fst**

Follistatin

**Gene regulatory network**

Set of interactions between different regulators (DNA, RNA, proteins) leading to their interdependent modulation of expression and regulation

**Genotypic sex determination (GSD)**

When the sex of an individual is triggered by its genotype only (can be mono or polygenic)

**Gonadal maintenance**

Establishment of a genetic program in order to maintain the fate and differentiation state of the different cellular types composing the gonad, keeping either the male or female identity

**Gsdf**

Gonadal soma derived factor

**Her-1**

Hermaphroditization of XO-1

**Hetero-/homo- gamety**

When individuals produce gametes with either different sex chromosomes (hetero-) or similar sex chromosomes (homo-). It is referred to male heterogamety when males produce X and Y chromosome-containing gametes or female homogamety for females producing only X chromosome-containing gametes (XX-XY sex determination system, like in most mammals). For instance in birds, snakes and butterflies males are (ZZ) homogametic and females (ZW) heterogametic (ZZ-ZW sex determination system)

**Heteromorphic sex chromosomes**

When sexual chromosomes are morphologically distinguishable (different degrees of heteromorphism exist, depending on the age of the sex chromosomes)

**Hhip**

Hedgehog-interacting protein

**HMG**

High mobility group

**irf9**

Interferon regulatory factor 9

**Mab-3**

Male abnormal 3

**masc**

Masculinizer

**Master sex-determining gene**

A gene (not necessarily coding for a protein) responsible for the initial trigger leading to sex determination

**Neofunctionalization**

The process by which a gene changes its function or adds a new one by mutations that change the structure of its gene product and/or its expression pattern

**Nix**

Male-determining factor in the mosquito *Aedes aegypti*

**NTD**

N-terminal domain

**piRNA**

PIWI-interacting RNA

**Primordial germ cells**

In the embryo the precursors of the stem cells that will give rise to the germ cell lineage. During sex determination and gonad differentiation they become committed to either produce male or female germ cells as spermatogonia or oogonia, which after meiosis will become the gametes. Primordial germ cells continuously express a certain set of genes in order to maintain their unique undifferentiated/pluripotent state

**Ptch**

Patched

**Rspo1**

R-spondin 1

**Sdc**

Sex determination and dosage compensation defective

**SdY**

Sexual dimorphic on the Y chromosome

**Sex chromosome**

Chromosome involved in the primary sex determination. They usually harbour a master sex determining gene/trigger

**Sex determination**

Primary mechanism leading to the expression of the phenotypic sex. Sex determination is mostly triggered either by the genome (genotypic sex determination) or by the environment (environmental sex determination)

**Sexual differentiation**

Developmental consequence of the sex determination process. Regroups the events dealing with internal and external genitalia and secondary sex characters

**SF1**

steroidogenic factor-1

**Somatic gonad**

The non-germ line component of the gonad. The somatic gonad consists of mainly two characteristic cell types in female: the granulosa and theca cells of the ovary and three specific cell types in the testis: Sertoli, Leydig and peritubular myoid cells

**SOX9**

Sry-related HMG box 9
**SRY**

Sex determining region Y

**STRA8**

Stimulated by retinoic acid gene 8

**Sxl**

Sex lethal

**TAD**

Transactivation domain

**TESCO**

Testis-specific enhancer core

**TGF-β**

Transforming growth factor beta

**Therian mammals**

Non-egg-laying = marsupials and placental mammals

**TRA**

Transformer

**Wnt**

Wingless-related MMTV integration site

**WT1**

Wilm's tumor gene 1

**Xol**

XO lethal

<table><caption>Table 1. Master sex-determining genes in vertebrates.</caption><thead><tr><td>Master SD gene</td><td>Organism</td><td>SD system</td><td>SD gene ancestor</td><td>SD gene generated from ancestor by</td><td>Ancestor gene function</td></tr></thead><tbody><tr><td>SRY</td><td>Therian mammals</td><td>XY</td><td>Sox3</td><td>Allelic diversification</td><td>Transcription factor, required in formation of the hypothalamo-pituitary axis, functions in neuronal differentiation, expressed in developing gonads</td></tr><tr><td>Dmrt1</td><td>Birds</td><td>WZ</td><td>Dmrt1</td><td>Allelic diversification</td><td>Transcription factor, key role in male sex determination and differentiation</td></tr><tr><td>DM-Y</td><td>Xenopus laevis</td><td>WZ</td><td>Dmrt1</td><td>Gene duplication</td><td>Transcription factor, key role in male sex determination and differentiation</td></tr><tr><td>Dmrt1bY</td><td>Medaka (Oryzias latipes, O. curvinotus)</td><td>XY</td><td>Dmrt1</td><td>Gene duplication</td><td>Transcription factor, key role in male sex determination and differentiation</td></tr><tr><td>SdY</td><td>Rainbow trout (Oncorhynchus mykiss)</td><td>XY</td><td>Irf9</td><td>Gene duplication</td><td>Interferon response factor, no gonadal function known</td></tr><tr><td>GsdfY</td><td>Luzon ricefish (Oryzias luzonensis)</td><td>XY</td><td>Gsdf</td><td>Allelic diversification</td><td>TGF-β factor, important role in fish gonad development</td></tr><tr><td>Sox3Y</td><td>Indian ricefish (Oryzias dancena)</td><td>XY</td><td>Sox3</td><td>Allelic diversification</td><td>Transcription factor, required in formation of the hypothalamo-pituitary axis, functions in neuronal differentiation, expressed in developing gonads</td></tr><tr><td>amhY</td><td>Perjerrey (Odontesthes hatcheri)</td><td>XY</td><td>Amh</td><td>Gene duplication</td><td>Anti-Muellerian hormone, growth factor</td></tr><tr><td>amhr2Y</td><td>Fugu (Takifugu rubripes)</td><td>XY</td><td>Amh receptor 2</td><td>Allelic diversification</td><td>Type II receptor for Amh, important function in gonad development, medaka mutant shows sex reversal</td></tr><tr><td>Dmrt1</td><td>Chinese tongue sole (Cynoglossus semilaevis)</td><td>WZ</td><td>Dmrt1</td><td>Allelic diversification</td><td>Transcription factor, key role in male sex determination and differentiation</td></tr><tr><td>GsdfY</td><td>Sablefish (Anoplopoma fimbria)</td><td>XY</td><td>Gsdf</td><td>Allelic diversification</td><td>TGF-β factor, important role in fish gonad development</td></tr></tbody></table>

to determine sex in a variety of animals and what has been learned about the maintenance of the sexual identity of ovary and testis.

## Master sex-determining genes: case studies from Sox and DM domain factors to emerging “unusual” suspects

### From Sry down to Sox3 across vertebrates

SRY belongs to a family of transcription factors, which are characterized by an evolutionary conserved high-mobility group (HMG box) DNA-binding domain flanked by weakly conserved N- and C-terminal sequences. In mice, both, gain- and loss-of-function studies have shown that SRY is not only sufficient but also necessary for triggering testis development [4,5]. With the exception of only two species (the mole vole *Ellobius* [6] and the spiny rat [7]) which have

probably lost the gene), SRY is the universal master male sex regulator of all therian mammals [8]. Cytogenetic and comparative molecular studies of mammalian sex chromosomes provided evidence that SRY most probably arose after two major events: (i) a dominant mutation of the *SOX3* allele (giving rise to the proto-Y) as well as (ii) fusion of the gene with regulatory sequences from another gene already located on the X chromosome [9] (Fig 1). Necessarily occurring before the divergence of the therian lineage, these events could be estimated to have happened ~146–166 million years ago [10,11]. Sharing an overall identity of 67% at the amino acid level and up to 90% identity when specifically considering the HMG DNA-binding domain, the X-chromosomal *SOX3*-encoded protein is most similar to SRY [12]. Consistent with this hypothesis, the expression of *SOX3* has been documented in the developing gonads of mice, chicken [13], fish [14], and frog [15]. Only the absence of *SOX3* expression
<figure><img src="image_73.png" /><figcaption>**Figure 1.** Independent evolution of *SOX3* genes toward a master sex-determining function in mice and Indian rice fish.</figcaption></figure>

While SRY appears to be restricted to the therian mammals, evidence accumulates that SOX3 has independently been recruited as a "precursor" of master sex-determining genes also outside mammals. Hence, although not a priori destined to have a direct function during sex determination, common mechanisms of evolution seem to be repeatedly employed. Given that SOX3 is not generally expressed during gonadal induction or during gonadal development, the first step toward a sex-determining function is a transcriptional rewiring in order to acquire a timed pattern of expression compatible with sex determination. Such transcriptional rewiring, although not unique to SOX3 (see Dmrt1bY in medaka fish for example [56]), generally involves either fusion of the gene to new promoters or insertions of transposable elements into their pre-existing promoter, bringing in cis-regulatory elements compatible with the timing of gonadal induction. Interestingly and surprisingly, it seems that at least in mice and rice fish, this step alone was sufficient to endow SOX3 with a sex-determining function. Usually, the transcriptional rewiring steps seem to be accompanied by neo-functionalization or functional specialization processes. These include specialization of the protein activity itself in therian mammals (adapted from reference [20]) or more surprisingly adaptation of the downstream gene-regulatory network (target genes) in the Indian rice fish.

in the developing marsupial gonad is not consistent with a conserved role in mammalian sex determination [16,17]. Although SOX3 has no obvious primary function in sex determination, as the *Sox3* knockout mice have no gonadal phenotype [18], the clear evolutionary relationship between SOX3 and SRY raised the question whether gain-of-function point mutations may account for SOX3-induced XX male sex reversal in mice or humans. This has been shown only recently using a transgenic mouse model in which ectopic expression of SOX3 in the developing XX gonads resulted in complete XX female to male sex reversal [19]. Interestingly, the XX gonads of the transgenic hemizygous mice (Tg+) did not only display an up-regulation of *Sox9* but also started to differentiate Sertoli cells, forming testis cords together with the appearance of a male-specific vasculature. Interestingly, using co-transfection assays it was shown that, similar to SRY, SOX3 only modestly trans-activated the SOX9 testis-specific enhancer "TESCO" element [20] and synergistically interacted with steroidogenic factor-1 (SF1).

Interestingly, the development of SOX3-triggered testes in XX animals was not possible in the absence of *Sox9*. In the same direction, patients displaying XX female to male sex reversal due to rearrangements of the genomic regions encompassing the regulatory sequences of SOX3 have been reported [19]. Together, these data suggest that gain of function of SOX3 during gonadal development can in principle substitute for SRY to trigger testis development. These findings provide functional evidence supporting the long-standing hypothesis that SOX3 is the evolutionary precursor of SRY (Fig 1). It is also reasonable to postulate that rearrangements of the SOX3 gene might be an underappreciated cause of XX female to male sex reversal in human patients [19].

While SRY appears to be specific to the therian mammals, there is accumulating evidence that SOX3 has spawned independently other sex chromosomes outside mammals. Though being expressed in the ovary of frogs [21] without any sex-determining function determined so far, *sox3* might be involved in the switch responsible
for sex determination in the Japanese wrinkled frog (Rana rugosa). Members of this species are either ZW or XY depending on which side of the island they are located [22]. Curiously, the Z and X chromosomes are not only homologous but share many genes with the X chromosome of humans including the *sox3* gene. Further molecular characterization and genetic mapping could disclose the presence of a Y-specific allele for *sox3* [23,24]. So far, this is an intriguing finding, but further studies are needed to ascertain a function for *sox3* in the sex developmental decision process of the embryonic gonad. If *sox3* has such a function, then the next question would be how the different genetic systems (ZW or XY) impact on *sox3* function.

Stronger evidence comes from the Indian ricefish (*Oryzias dancena*) (Fig 1), in which the XY sex chromosome pair also shares homology with the human X, including the presence of the *sox3* gene [14]. Using positional cloning to identify the sex-determining locus, it was found that the male-specific region on the Y chromosome harbors a *cis*-regulatory DNA segment that up-regulates expression of the Y-chromosomal copy of *sox3* during gonadal development (Fig 1). Sex reversal of XX fish transgenic for the regulatory segment linked to *sox3* to become males, and fish with targeted deletion of the Y-chromosomal *sox3* gene developing as females confirmed its major role during sex determination. Furthermore, it was demonstrated that Sox3 initiated testicular differentiation by up-regulating expression of *gsdf*, a gene highly conserved in fish male sex differentiation pathways [14]. Interestingly, a BAC clone carrying the *sox3* gene of *O. dancena* was not able to induce male gonadal development in the closely related species *O. latipes*, which has a different male sex determination gene. This supports the hypothesis that the acquisition of Sox3 function as a master sex-determining gene has occurred with a concomitant change in the downstream gonadal gene-regulatory network (Fig 1). Taken together, the results provided strong evidence for the recruitment—even in distantly related species—of Sox3 into the pathway leading to male gonadal development.

**SRY reveals plasticity of sex-determining mechanisms among mammals** Despite substantial variations in expression profiles, structure, and amino acid sequences within mammals, the function of SRY to activate a conserved target gene—SOX9—during testis development appears to be conserved [20]. SRY directly binds to the TESCO sequence of the *SOX9* gene [20]. Once activated, the SOX9 protein initiates the differentiation of somatic precursors into Sertoli cells that will then coordinate the gonadal development toward testes [25]. In the absence of *SOX9* activation, the fetal gonad will develop toward ovaries. While the function of SRY as a regulator of *SOX9* appears to be conserved, the molecular details underlying transcriptional regulation of *SOX9* by SRY [26] are not fully known and their conservation among mammals has not been deeply investigated. Such information would be important to evaluate whether under a conserved master determiner, the subordinate network is strictly conserved as well or shows variation in its regulatory interactions.

In contrast to most known transcriptional activators, most SRY proteins that have been studied in different mammalian species do not exhibit a well-defined transactivation domain (TAD). For instance, the N- and C-terminal domains (NTD and CTD) flanking the evolutionary conserved DNA-binding domain of human SRY are

poorly preserved and do not seem to display any intrinsic transactivation activity [27]. Hence, it is assumed that the transcriptional activation of the human *SOX9* gene by SRY is possible only after the recruitment of a transactivating protein partner through its NTD and/or CTD sequences [28]. However, mouse SRY does not only lack the NTD but also displays an unusual CTD made of a bridge domain together with a poly-glutamine (polyQ) tract encoded by a CAG-repeat microsatellite [27]. It has recently been shown that this poly-glutamine domain does not only prevent mouse SRY from proteasomal degradation, but additionally functions as a bona fide TAD. Due to the fact that it allows the direct transcriptional induction of Sox9, this poly-Q domain plays a central role for the male-determining function of SRY *in vivo* [27]. Such data suggest that during evolution, mouse SRY has gained a functional unit, which is absent in other mammals [27]. Given such important transactivating properties for that poly-Q CTD in mice, it is puzzling that SRY proteins from either human or goat lacking a TAD are able to induce testicular development in transgenic XX mice embryos [29,30]. It appears reasonable to consider that both human and goat SRY proteins are able to bind to the highly conserved mouse TESCO target sequence using their respective DNA-binding HMG boxes. For the activation of *SOX9* transcription, it is assumed that transactivation is then mediated after the recruitment of a third TAD-containing protein partner. It can be further hypothesized that acquisition of a poly-glutamine stretch after insertion of a CAG microsatellite in a rodent ancestor made the recruitment of a transactivating partner unnecessary. Consequently, it is assumed that mouse SRY's ability to employ such a transactivating partner was lost during evolution. This assumption is supported by the observation that the acquisition of the poly-glutamine stretch is concomitant with an increase of variation in different parts of the SRY protein. These include the loss of the NTD as well as accumulation of deleterious amino acid substitutions in the HMG box [31]. Though no longer required, the third partner protein—probably a pleiotropic effector—may still be expressed at the sex determination stage. It would then potentially enable human and goat SRYs to trigger male gonadal development when expressed in transgenic mice. This reveals an unanticipated level of plasticity of the molecular mechanisms in the implementation of the primary sex-determining signal even among mammals. Identification of such putative partners of SRY may help in understanding human primary sex reversal pathologies, which are not explained by alterations in the known players of male sexual development [32].

*Roles of DM domain factors in sex determination, differentiation, and gonadal maintenance*

**DMRT1, wherever you look** Among the evolutionary conserved downstream effector genes of genetic sex-determining cascades, the DMRT gene family holds an outstanding position. This family is involved in sexual development of organisms as phylogenetically diverse as mammals, birds, fish, frogs, flies, worms, and corals [33–38] (Figs 2 and 3). Characterized by a highly conserved DNA-binding core motif—known as the DM (Doublesex and Mab-3) domain—, DMRT proteins act as transcription factors. Initially described to be involved in sex determination in worms and flies, they have been shown to regulate diverse aspects of somatic sexual dimorphism in these organisms. The ability to functionally
<figure><img src="image_74.png" /><figcaption>**Figure 2.** Gene-regulatory network of gonadal sex induction and maintenance in vertebrates.</figcaption></figure>

Schematic representation of main interactions within the regulatory network. In gonadal fate determination of mammals, Sry initiates activation of the male pathway (blue) through up-regulation of Sox9. Dmrt1 is not only important for keeping the male pathway on but also in suppressing the two female networks (red). These two female networks involve Foxl2 as well as the Wnt/β-catenin signaling pathways. Maintenance of gonadal identity in the differentiated gonads is a result of the cross-inhibition activities of Dmrt1 and Foxl2. A critical equilibrium between these conflicting pathways underlies the bipotentiality of the gonadal somatic cells. Tipping the balance into one direction or the other will regulate the gonadal fate as a consequence of the activation of the male or female pathways. Solid lines define negative regulations. Dashed lines designate positive regulations. Beside the Sry ancestor *Sox3* and *Dmrt1*, other genes (pink) can become the master sex-determining genes by similarly impacting on the seesaw between the male and female programme.

substitute for each other across species led to the picture that sex determination cascades might—at least partially—rely on preserved molecules and pathways [37] (Figs 2 and 3; Table 1). Consistent with this, many of the DMRT homologs so far characterized among metazoans have been shown to be predominantly expressed during the development of the primordial gonad [35]. Interestingly, DM domain genes have also recently been described to be primarily involved in gonadal differentiation of the male flatworm (*Schmidtea mediterranea*) [39]. Similarly, in the water flea *Daphnia magna*, a crustacean with environmental sex determination, DMRT homologs have been found to trigger the switch in male versus female development of many dimorphic structures [40]. Thus, this widespread gene family appears to be directly involved in sexual development in all major animal groups. Nevertheless, DM domain factors were long considered as one of the underdogs of sexual determination

because of their recurrent subordinate role in the cascade. A deeper interest in the field of sex determination for this group of genes only came with the discovery of a *dmrt1* homolog located on the Y chromosome of the medakafish (*Oryzias latipes*). Resulting from a gene duplication of the autosomal *dmrt1a* gene, it was designated *dmrt1bY* [41] or *dmy* [42]. It is the only functional gene in the Y-specific region of the sex chromosome, and it was shown to be not only necessary but also sufficient for triggering male development (see also Fig 2).

In humans, haploinsufficiency of the genomic region that includes *DMRT1* and its paralogs *DMRT2* and *DMRT3* leads to XY male to female sex reversal [43]. This suggested that the *DMRT1* gene is an important dosage-sensitive regulator of male development in vertebrates. In chicken and other avian species and in a fish, the smooth tongue sole (*Cynoglossus semilaevis*) [44]), *DMRT1* is located on the Z chromosome, but absent from W, and shows the expected expression pattern for a dosage-dependent male sex-determining gene of birds [45] and flatfish. In chicken, it was demonstrated through RNA interference experiments that *DMRT1* is indeed required for male gonad development [45]. While in these organisms *DMRT1* acts as a dosage-dependent male determiner, in *Xenopus laevis*, a duplicated copy of *dmrt1* on the W, which lacks the dimerization domain, appears to fulfill its function as a dominant-negative version. It is proposed to interfere with the transcriptional activation of the target genes of Dmrt1 and thus acts as a suppressor of male development [46].

Remarkably, all these *DMRT1* genes have acquired their new roles as master sex determination genes through different mechanisms: via gene duplication and translocation in medaka, duplication, translocation and truncation in *Xenopus*, or loss of function of the W allele in birds or tongue sole (Table 1).

In mice, it is apparent that *Dmrt1* is not required for male primary sex determination since newborn Dmrt1 mutants are males with testes [36]. However, *Dmrt1* is required for male gonadal differentiation of somatic cells and germ cells [47–49]. This is a parallel situation to mammalian *Foxl2* [50], which plays a conserved role in ovarian development but in mouse (opposed to some other mammals, including human and goat [51]) is not required for initiation of female development (see [52] for review). Targeted deletion of mouse *Dmrt1* and also of the autosomal *dmrt1a* of medaka, which is not involved in primary male sex determination, have revealed a major role in male gonad maintenance: when *Dmrt1* is lost, even in adults, this triggers sexual cell-fate reprogramming, in which male Sertoli cells trans-differentiate into their female counterparts, the granulosa cells [49]. This is accompanied by testicular reorganization toward a more ovarian morphology [49]. Ectopic DMRT1 expression in the ovary silenced the female sex-maintenance gene *Foxl2* and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures, which resemble male seminiferous tubules [53]. In the same direction, deletion of the *dmrt1* gene in medaka resulted in transition of the developing testis to ovary [54]. Hence, DMRT1's range of action is not limited to function in initiating the male gonadal phenotype during early development but also accounts for the livelong active repression of the two "anti-testis" pathways of FOXL2 and WNT4/β-catenin [49], and can do so even in the absence of the testis-determining genes *SOX8* and *SOX9* (Fig 2). Additionally, mRNA profiling revealed that DMRT1 activates many testicular genes and
down-regulates ovarian genes [53]. Interestingly, transient expression of DMRT1 has also been reported in the fetal gonad of both sexes. The involvement in the regulation of germ cell development in testes and ovaries indicates that DMRT1 has different functions in males and females [55].

DMRT1 is required in female germ cells for entry into meiotic prophase, and in male germ cells for the control of mitotic arrest until birth [55]. Control of the decision to enter meiosis versus mitotic arrest is mediated by the ability of DMRT1 to selectively modulate retinoic acid signaling through context-dependent regulation of STRA8. DMRT1, for example, directly represses STRA8 transcription during testicular differentiation [55]. Thus, a picture emerges where DMRT1 controls a regulatory network that on the one hand can drive sexual fate and on the other hand can maintain the program of sexually differentiated cells, depending on the cellular context.

**DMRT1, a jack-of-all-trade** From studies in mouse and medaka [49,53,54,56,57], it is emerging that *DMRT1* holds a key position as the master switch or gatekeeper controlling the cell fate of the somatic cells of the gonads in female and male [33,34,53,58,59]. If this is so, then one could ask, why such a complex regulatory network upstream of *DMRT1* would be necessary to flip the switch, because numerous examples indicate that DMRT1 can do it on its own as for instance in birds, *Xenopus* and medaka [41,42,45,46]. *DMRT1* orthologs in these species appear to have undergone mutational events causing either loss or gain of function. Such altered DMRT1 activity may have favored evolutionary transitions leading to new genetic sex determination systems (see [59] for review). The ability of DMRT1 to toggle Sertoli/granulosa cell fate supports the hypothesis that loss- or gain-of-function mutations in *DMRT1* can elevate it into a master sex-determining role. Such mutations would help to promote changes between genetic sex determination mechanisms that are commonly observed among vertebrates.

*DMRT1* is one of the sex determination network genes that appears more often also as master regulator (Table 1). It can be hypothesized that its strategic position at the interface of sex determination and the process of sex-specific gonadal differentiation, integrating a developmental fate decision with activation of organ differentiation programmes (Fig 2), made *DMRT1* suitable to be selected either as new controller at the top or at least for being one of the few key genes to be regulated.

*Emerging suspects from gonadal TGF-β signaling*

The anti-Müllerian hormone (Amh) is a growth factor from the TGF-β family and plays a major role in mammals for the degradation of the Müllerian duct-forming part of the female reproductive tract in male embryos. It is not required for mouse testis development. However, in non-mammalian vertebrates, it appears to play a central role in testis formation. For instance, in chicken embryonic gonads, AMH is expressed much higher in males and is predicted to be responsible for organizing the early testis in birds [60]. In the medaka *hotei* mutant, Amh signaling is disrupted by a mutation in the type II receptor for Amh. As a consequence, a male to female sex reversal with an over-proliferation of germline stem cells occurs [61].

Although being clearly a subordinate member of the sex regulatory network in mammals and at least in those species that make use of *DMRT1* as master regulator of male development, the Amh/

Amh-receptor system has, like *DMRT1*, sometimes made it to the top (Table 1). In the pejerrey, a freshwater fish species from Patagonia, a duplicated version of the *amh* gene became the male sex-determining gene on the Y chromosome [62], reminiscent of the situation for *dmrt1* in medaka fish. In the pufferfish, *Fugu rubripes*, the receptor for Amh exists in two versions that differ by one amino acid (H384D) in the kinase domain [63]. The 384<sup>His</sup> allele is a Fugu-specific (conserved in several other pufferfishes) mutation that confers lower activity to the receptor and is encoded on the X chromosome [63]. Thus, a quantitative difference in Amh signal transduction in females, which are homozygous for the mutant, versus males, which have kept one allele of the wild-type receptor on their Y, is responsible for male development [63]. Like in the medaka *hotei* mutant [61], low signaling from the receptor is connected to feminization of the gonad.

Gonadal soma-derived factor (Gsdf) is another growth factor from the TGF-β family that is closely related to Amh. It is only found in fish, and its biochemical function is not well studied. It is assumed to have a role in male gonad development due to its exclusive expression in the early differentiating testis of all fish looked at so far [64–68]. Despite its proposed role in the downstream regulatory network, *gsdf* has made it up to the top in *Oryzias luzonensis* [69] a sister species to medaka, and most likely also in the sablefish [70].

Taken together, it appears that certain genes, which are members of the regulatory network, namely *sox3*, *dmrt1*, and TGF-β signaling components, can become the master sex-determining gene independently again and again, while other important components of the sex-determining pathways have not appeared as masters so far (Fig 2 and Table 1). Whether we just have to wait for the analyses of primary genes for sexual development in more species, in order to put genes like *foxl2*, *sox9*, *sox8*, *wnt4*, etc., on the list of usual suspects, or whether there is a biological reason that makes some genes more prone to become the top regulator, is currently unsolved. We could imagine that some genes remain “too difficult to recruit” as master regulators, for instance if they have also non-reproductive but vital functions in other organs. In such case, interferences between a duplicated new master gene and its homolog may not be tolerated, except for the case that the neo-gene would have an appropriate gonad-specific regulation as soon as the founder event occurs. Many of those genes that did not appear as master sex determiners so far indeed have important functions in other tissues and organs.

*Recurrent actors in invertebrate sex determination*

**The invertebrate ancestors of DMRT1** DM domain-containing genes have been shown to be primarily involved in gonad differentiation in a flatworm [39] and to direct male versus female development of dimorphic structures in water flea [40]. Interestingly, this functional convergence is common among insects (see [3,71–73] for reviews). In *Drosophila*, the initial trigger of sex is dependent on the ratio of the number of X chromosomes versus the haploid autosome complement (X:A). In the female situation, an X:A ratio of one will enable the transcription of the *Sex lethal* gene (*Sxl*), a splicing regulator. The SXL protein will then promote the female-specific splicing of *Transformer* (Tra), a direct downstream target, and lead to the production of functional TRA proteins. Similarly, a complex made of TRA and TRA-2 proteins will then favor the female-specific
splicing of the *Doublesex* (*Dsx*, the *Dmrt1* homolog) gene tran-
scripts. This results in the production of the female-type DSX protein
DSX<sup>F</sup>, which initiates up-regulation of the downstream gene-
regulatory network for female development. In males, an X:A ratio
of 0.5 will prevent the production of the SXL protein and, by default,
results in the production of the male-specific splice form of the *Tra*
gene. This splice variant translates into a non-functional protein due
to a premature stop codon. In the absence of TRA, by default the
male-specific splice form from the *Dsx* gene will be produced. The
male-type DSX protein DSX<sup>M</sup> will then orchestrate the downstream
gene-regulatory network for male development [71,74] (Fig 3).
Orthologs of *Drosophila dsx* have been identified and studied in a
large number of insects [75–77]. Mediation of alternative sex-
specific splicing of *dsx* by TRA and TRA2 is also widely conserved
in insects although variations of the sex determination systems

occur [3], suggesting that different molecular mechanisms involving
splicing activators or repressors are employed to preferentially
generate sex-specific variants of dsx mRNA [78].

Despite considerable efforts, similar sex-specific alternative splic-
ing events in the molecular regulation of sex determination of verte-
brates have not been shown. Conceptually similar is the fact that
DSX translates the sexual determination process of a cascade of
alternative splicing events into the transcriptional control of a large
number of sex-specific effector genes. Similarly, DMRT1 in verte-
brates appears to hold such a “translational” function at the inter-
face where a fate-determining signal is put into effect at the level of
sex-specific somatic cell differentiation (Figs 2 and 3).

In invertebrates, the homologs of vertebrate *Dmrt1* (e.g. *Dsx* in *Drosophila* and *Mab3* in *C. elegans*) are typical downstream factors of sex determination and so far, it is not reported that a DM domain

<figure><img src="image_75.png" /><figcaption>Figure 3. Sex-determining cascades in *C. elegans* and some insects.</figcaption></figure>

Molecular and genetic pathways leading to the formation of the gonad in the worm *C. elegans*, the mosquito *Aedes aegypti*, the fly *D. melanogaster*, the honey bee *A. mellifera*, and the silkworm *B. mori*. Conservation of the *Dsx*, *Mab-3* and *Dmrt1*, *Tra*-like, (*Tra-2*), or *Fru* homologs is designated with either pale brown, pale blue, pale green, or pale orange boxes, respectively. *Tra*-*(1, 2 or 3)* of *C. elegans* are not phylogenetically related to *Tra* of *Drosophila*. *Fem*-*(1, 2 or 3)* of *C. elegans* are not phylogenetically related to *fem* of *Bombyx mori*. In *C. elegans* and *D. melanogaster*, a ratio between X chromosomes and autosomes determines the sex. This leads to the on/off state of *Xol* or *Sxl*, respectively. Heterozygosity turns on *Csd* in the honeybee *Apis mellifera*, leading to female development, and hemizygosity or homozygosity leaves *Csd* unexpressed and produces a drone. In the mosquito (*Aedes aegypti*), sex determination is triggered by a dominant male determiner (*Nix*). *Nix* is a distant homolog of the splicing factor *Tra*-2 of *Drosophila* and likely regulates the sex-specific splicing of *Fru* and *Dsx*. Sex in the silkworm *Bombyx mori* is controlled via a ZW sex chromosome system. Produced only from the sex-determining locus on the W, the piRNAs suppress the male sex-determining factor MASC.
gene has made it up to the top in any invertebrate species [3]. But like in vertebrates, genes that are known as downstream members in one species can also usurpate a position as an initial genetic trigger in another species [3]. In insects, paralogs of the gene *tra* that is a well-studied component of the sex determination cascade in *Drosophila*, evolved as the master sex-determining switch gene in the housefly (*Musca domestica*), a wasp (*Nasonia vitripennis*), and the honeybee (*Apis mellifera*) [72,79,80]. In this regard, studies about complementary sex determination in the honeybee give exciting insights into how molecular diversity of regulatory pathways can evolve [81,82], as discussed in more detail below.

**Complementary sex determination in honeybees uses a conserved module from chromosomal sex determination** Genetic sex determination in the honeybee does not depend on the presence of hetero- or homomorphic sex chromosomes with different genetic compositions but rather follows a haplodiploid mode. Males develop from haploid unfertilized eggs, while diploid fertilized eggs develop into females. Hence, male or female sexual development occurs as the result of a signal originating from either a single or two different alleles from one gene, called *complementary sex determiner* (*Csd*) (Fig 3). Consequently, maleness or femaleness is determined by either homo-, hemi-, or heterozygosity of the *Csd* locus. The *Csd* gene products belong to an arginine-/serine-rich protein family. Interestingly, the C-terminal end of *Csd* also displays high similarity with the TRA protein, an essential downstream genetic factor of the sex-determining pathway in *Drosophila* ([81] and Fig 3).

Intriguingly and in contrast to the situation in *Drosophila* with *Tra* and other downstream genes (see Fig 3), neither transcriptional nor splicing variations of the *Csd* gene could be detected as sex-specific triggers. It is currently presumed that the regulation of the downstream regulatory network is mediated by the tendency of the CSD proteins to form heterodimers. Interestingly, the sex determination locus of the honeybee harbors a second gene also required for sex determination: *feminizer* (*Fem*) [82]. Further, phylogenetic studies revealed that *Fem*—as *Csd*—is also a close homolog of the *Tra* gene from *Drosophila*. It has been shown that *Csd* arose after duplication of the *Fem* gene 10–70 million years ago while the honeybee lineage was specifying. Knockdown experiments using RNA interference (RNAi) of either *Csd* or *Fem* resulted in female to male phenotypic sex reversions, implying that both factors are required for sex determination in the honeybee downstream of sex-specific splicing of the *Fem* gene by the CSD protein ([81,83] and [3] for review).

The situation in the honeybee resembles the roles of *dmrt1* in medaka and *Xenopus* and of *amh* in the pejerrey: A highly conserved downstream component of the network underwent a gene duplication, and then, one of the duplicates evolved a new function at the top of the cascade (Figs 2 and 3).

**Another usurpator in mosquito?** In the yellow fever mosquito, *Aedes aegypti*, like *Drosophila* a member of the order Diptera, sex is dependent on the presence or absence of a Y chromosome. Recent work has uncovered the molecular nature of the male-determining gene [84]. Intriguingly, this gene, called *Nix*, shows some sequence similarity to the *Tra-2* gene. This gene in *Drosophila melanogaster* is a downstream member of the sex determination cascade. Further downstream in the fruitfly cascade are the *Fru* and *Dsx* genes, and also in *Aedes aegypti*, both genes are regulated by the *Tra-2*

homolog *Nix* (Fig 3). It is tempting to propose that in the mosquito, we have another example of a subordinate sex determination gene that has made it to the top.

*The “unusual” suspects*

All the above discussed cases of turnovers and novel master sex determiners include genes that have been previously known as components of downstream sex determination networks, for example, from mouse, human, *Drosophila*, and *C. elegans*. Unexpectedly, there are two recent reports on sex-determining genes which were neither known nor suspected to be involved in the molecular regulation of this process.

**An immune-related gene evolved into the master sex-determining gene in rainbow trout** In the rainbow trout *Oncorhynchus mykiss*, a gene expressed only in the testis, predominantly during testicular differentiation, was recently characterized [85]. Localized at the sex-determining locus, this gene was named *sdY* for sexual dimorphic on the Y chromosome. Astonishingly and unlike other master sex-determining genes characterized so far, *sdY* has no homology with any known gene in sex determination pathways but with an immunity-related gene, the interferon regulatory factor *irf9* [85]. *SdY* arose by duplication and truncation of the autosomal *irf9* gene (Table 1). It lost the DNA-binding domain but preserved its protein-protein interaction domain. So far, the molecular mechanism through which *SdY* triggers male gonad development is unknown.

**A single female-specific piRNA is the primary determiner of sex in the silk worm** Sex in the silkworm *Bombyx mori* and all butterflies is determined by a ZW sex chromosome system. The W chromosome lacks any protein-coding genes but consists predominantly of transposons and non-coding RNAs. The only transcripts produced from the sex-determining region on the W are PIWI-interacting RNAs (piRNAs). After deep sequencing and isolation of dimorphically expressed RNAs, the *Fem* piRNA (*Fem* standing for “feminizing factor”) was shown to be specifically expressed in females at all stages of development [86]. Furthermore, *Fem* piRNA targets and cleaves the *Masculinizer* (*Masc*) RNA molecule transcribed from a gene located on the Z chromosome. Interestingly, MASC, a CCCH-type zinc finger protein, favors male-specific splicing of *Bm-dsx*, leading to male development [86]. Hence, in ZW embryos, *Masc* RNA level is down-regulated by *fem* piRNAs, inhibiting male development. By default, female-specific splicing of *Bm-dsx* then occurs, triggering female development [86] (Fig 3). Interestingly, genetic inhibition of *Masc* resulted in the premature death of ZZ embryos before they hatched. In light of this observation, it was shown that the MASC protein is necessary for dosage compensation in order to lower Z gene transcription in ZZ embryos to the same level as in ZW embryos [86]. Whether or not this sex determination pathway is conserved across all lepidopterans remains to be explored, but coupling two important mechanisms namely sex determination and dosage compensation within the same genetic pathway and additionally distributing their genes onto the sex chromosomes should strongly promote evolutionary conservation.

*SdY* from rainbow trout and *Fem* piRNA are paradigms showing that unrelated genes are able to acquire *de novo* sex-determining functions. It can, however, not be excluded that they are representing
factors of the sex determination regulatory network that have been
overlooked so far.

Plasticity of the downstream sex determination
regulatory network

What happens when “masters change”? The slogan “slaves remain”
could imply that not much happens downstream of the changing
master sex determiner. However, the findings on the diversity of
SRY structure and its way to act as a transcriptional activator (see
above) indicate that even under the same master gene, the regula-
tory interactions of the network undergo changes and that biology is
not that simple.

In *Drosophila*, it has been shown that at the very downstream end of the sex determination, cascade pathways diverge by cooption of new effector genes [73] explaining the divergence of secondary sex characters between species. In vertebrates, some transcription factors like *DMRT1*, *FOXL2*, *SOX9*, and components of pathways such as *Rspo1/Wnt/Fst* or Hedgehog of the gonadal gene-regulatory network are well conserved on the DNA sequence level; however, their specific functions, regulations, and interplays can be substantially different. In medaka, down-regulation of the Hedgehog pathway by *Dmrt1bY* was shown [87]: Transcription of the Hedgehog receptor *Ptch-2* in medaka testis is down-regulated by *Dmrt1bY/* *Dmrt1a*, while the antagonist *Hhip* is up-regulated [87]. The Hedgehog pathway is usually up-regulated by *DMRT1* in mammals. It appears that despite its necessity for mammalian testis induction and development and later on in regulating Leydig and myoid cell function [88–90], the Hedgehog pathway might not only be dispensable during medaka male gonadogenesis and maintenance, but needs to be suppressed by *DMRT1* genes.

For R-spondin 1 (Rspo1), preferential ovarian expression is
generally described. However, such strict female dimorphism was
not observed in zebrafish [91], where the gene is also expressed in
adult testes. Here, Rspo1 has a crucial role in testis cell proliferation
[92] and it has further been shown to be involved in skin and
mammary gland differentiation in mammals [93]. Follistatin (Fst)
expression in the mouse co-localizes with *Foxl2* in the ovary [94],
but in rat, it is expressed very broadly in germ and somatic cells of
the testis [95]. Sparse expression of *fst* was also noted in the intersti-
tial cells of the medaka testis, together with an up-regulation of *fst*
expression *in vitro* after transfection of *dmrt1a* [87].

SOX9 has been shown to be expressed in the developing testes
of all vertebrate embryos examined so far (see [60] for review).
However, whereas SOX9 is upstream of AMH in mammals, the
reverse applies in birds, and in medaka, Sox9 even appears to be
not involved in primary sex determination at all [96,97]. In
mammals, the current understanding is that SRY acts together with
SF1 to activate SOX9, while in return, SRY is turned off by SOX9.
SOX9 further maintains its expression in an autoregulatory loop.
SF1 is still required, but SRY becomes dispensable later during
development [20]. In non-mammalian vertebrates, Sox9 activation
must then rely on other factors than Sry. Intuitively, one could
think that DM domain genes might have taken over. However, in
chicken embryos, *DMRT1* expression is occurring at least 2 days
before that of SOX9 [60], implying that other genes mediating the
DMRT1 signal to SOX9 are involved. In medaka *sox9b*, the

homolog of tetrapod *sox9* genes is rather involved in germ cell
function than gonad determination although being expressed in the
somatic part of the primordial gonads [96]. In addition, while in
mammals, SOX9 activates the expression of *FGF9* [98], the gene
does not exhibit any sexually dimorphic expression in chicken [60]
and has even been lost in fish [99]. It is obvious that the gonadal
function of *SOX9* underwent several changes during vertebrate
evolution.

Genetic networks are indeed more complex than a straight top-
down scenario. We have to add now that the differences in gene
expression do not only reflect differences in cell biology and
morphogenesis of the gonads but definitively are also the conse-
quences of changes in the initial trigger for activating the network.
That master sex-determining genes are prone to regulatory putsches
in order to acquire an upstream position might only be possible
because of the flexibility of the downstream gene-regulatory
network. Hence, while Graham proposed a few years back that
“Masters change, slaves remain” [1], it is now time to change this
paradigm: “When masters change, some slaves remain, others are
dismissed or acquire new tasks, and new ones can be hired”.

Conclusions and perspectives

The variability and plasticity of the mechanisms that govern the
development of the gonads is unmet by any other organ systems or
tissues. While for instance the *Pax6* gene that is a master regulator
of mammalian eye development is highly conserved (ectopic expres-
sion of human *PAX6* is able to induce eye development in *Droso-
phila* [100]), the downstream components of this cascade are not
conserved (the induced eye is a typical composite insect eye).
Surprisingly, it appears to be the other way round for sex determina-
tion genes. The evolution of genetic interactions in the sex-deter-
mining pathways and cascades is characterized by a relative
conservation at the bottom and an apparent diversity at the top.
This was explained in a classical hypothesis by A. Wilkins with an
evolutionary scenario in which these hierarchies during evolution
build up from a common downstream component (Sox or DM
domain factors for instance), which acquires new upstream regula-
tors. Those new additions would naturally vary in different evolu-
tionary lineages [101]. Recent studies on the molecular
identification of such upstream regulators and the downstream regu-
latory network, some of which provided the backbone for this
review, brought new insights into how sexual development is regu-
lated in different organisms, and how new sex determiners have
evolved.

The “bottom-up hypothesis” formulated by Wilkins has to be
revisited now taken into account the discoveries of the new master
regulators. It seems that the master regulator/switch is not necessar-
ily elected from the existing cascade usurping the top position but
could be equally recruited from outside to accomplish a new sex-
determining function after neo-functionalization. We also have to
modify the hypothesis as we now know that in vertebrates, unlike
in invertebrates, sex determination is not brought about by a simple
linear cascade, but by a complex network of multiple regulatory
interactions. Such a network might offer multiple opportunities
where a newly added factor can trigger the outcome of the network
signal toward male or female. There is also evidence accumulating
that regulatory cascades can become shorter, rather than being
topped up, when a new sex determiner appears, for example, in
honeybees [72,102].

Gonad development appears to cope well with such changes of
primary triggers as the many examples of different master sex regu-
lators show, which finally all guarantee the developmental switch to
either a testis or ovary. An intriguing situation has been recently
reported for zebrafish, where the laboratory strains used worldwide
have all lost their original sex-determining chromosome, but still
produce normal males or females [103]. New upstream sex deter-
miners appear to evolve quickly in those domesticated strains—
similar to a situation in the other small aquarium fish model, the
medaka [104]—which might take care in the future of the current
sex bias observed at present for many laboratory strains. These are
instances of “evolution in action,” which offer prospects to observe
in the laboratory how new sex determiners evolve and to obtain
insights into the underlying molecular mechanisms. Certainly, we
also need more information from different species about their
master sex-determining gene and how it acts on the downstream
regulatory network to obtain a reasonable understanding of the vari-
ety of sex-determining mechanisms.

Somehow unexpected are the accumulating findings that also
the downstream network is not as strictly conserved as the
"masters change, slaves remain" paradigm was imposing. Whether
these differences in the expression pattern and function are related
to specific adaptations of varying reproductive biology is a chal-
lenging question for the future. On the other hand, such changes
may be due to the impact of the new upstream regulator. Intrigu-
ingly, even in a setting of the same master sex-determining genes,
intricate differences downstream can be found, as seen for SRY in
different mammals. It has also been argued that genetic networks,
including sex determination, in general can change randomly with-
out necessarily impacting on the final phenotype and thus evolve
neutrally (see Sidebar A). Again, we need more details on the
molecular biology of the sex-determining networks from different
organisms; for instance, on a comparative basis from birds, Xeno-
pus and those fish that all use *dmrt1* as their common master sex-
determining gene.

Unexpectedly, it turned out that sex determination is not only
needed as the molecular switch for the undifferentiated gonad
primordium to develop either as testis or ovary, but that the sexual
identity of the gonadal soma needs to be maintained as long as the
organ has to provide its function(s). In vertebrates, two genes that
appear to have a more downstream function in the determination
network of the embryo are the top players here: *DMRT1* and *FOXL2*.
The dichotomous developmental potency of the gonadal soma is
apparently kept throughout the entire life. The reason for this is
unknown. In particular among fishes, hermaphroditic species are
common. Those fish can switch during their reproductive life from
one sex to the other. Whether these organisms have found a way to
make a controlled use of the livelong plasticity of the gonad or
whether the plasticity seen even in the mammalian gonads is a relic
of an evolutionary past are just two questions that emerge from
those new findings.

The recent progresses reviewed here have considerably increased
our understanding of the diverse molecular mechanisms underlying
the amazing variation and plasticity of sexual development, and we
might so far just only see the tip of the iceberg.

### Sidebar A: Evolutionary concepts for the diversity of sex determination mechanisms

Sex determination is a very basal and ubiquitous developmental
process, and the fact that it is so variable even between closely related
organisms poses many fascinating questions. Molecular biologists are
most interested to understand how these different mechanisms work,
what factors are involved, upstream and downstream, and how they are
regulated to bring about the amazing plasticity of the respective genetic
cascades and networks. These are the so-called proximate causes of the
observed variability. Organismic biologists focus more on the "ultimate"
causes that lead to the changes from one to the other sex determina-
tion mechanism within and between certain lineages. A number of
scenarios and hypotheses have been put forward to explain which
evolutionary forces could favor such transitions and turnovers [105].
One explanation is that a mutation, which creates a new sex determi-
nation mechanism, gives a fitness advantage to its carriers. Then, by
natural selection, this mutation will sweep through the population and
take over, while the previous mechanism is lost [106]. Such new muta-
tions could for instance alter the sex ratio, and if the ecological condi-
tions favor such a bias, this mutation will be beneficial. As another
example, a new sex determination mechanism might for instance be
more efficient under certain ecological conditions, for example, works
faster or is less or more susceptible to environmental influences.
If sex is determined through sex chromosomes, a common feature is
the reduction of recombination around the sex-determining gene,
which spreads out from there over almost the entire chromosome
and finally fully arrests. As a consequence, deleterious loss-of-function
mutations will accumulate in genes on the chromosomes carrying the
sex locus [107]. Hence, such a chromosome will become less fit in
evolutionary terms because of its mutational load, and once these
disadvantages accumulate to a critical level, an emerging "younger"
and less degenerated sex chromosome can take over [108].

Another hypothesis is based on linkage of sex-determining genes to
other genes that favor one sex or are antagonistic to the other sex
[109]. Many examples exist for such genes, which for instance are
involved in gonad development or sexual dimorphism. If such a gene
is closely linked to a gene that can influence the developmental deci-
sion toward male or female, the sex-determining gene will be co-
selected as a hitchhiker and enjoy the fitness advantage that the
linked sex beneficial or sexually antagonistic gene has under condi-
tions of natural or sexual selection.

Rather than postulating a fitness advantage for the emerging novel sex
determination mechanism, it is also considered that neutral or non-
adaptive processes of genetic drift, mutation, and recombination can be
instrumental. Such hypotheses are based on an analysis by M. Lynch how
in general genetic networks can evolve [110]. He pointed out that only
the final gene product of a genetic network or cascade produces a pheno-
type, which is exposed to selection. Thus, many changes in the upstream
system can occur without necessarily altering the finally expressed
phenotype. These changes can become fixed in a population by random
genetic drift. As a result, the regulatory network has changed, but the
phenotype will be constant. Such considerations were then applied to the
genetic cascades and networks that govern sex determination [102].
Indeed, the final outcomes of the sex determination process are morpho-
logically and functionally surprisingly similar in related groups of organ-
isms, which have very different master sex regulators [111].

For all of these theoretical explanations, which appear to be to a certain extent opposing or even contradictory, examples to support them can be found. A single one obviously cannot explain all the different cases of sex determination systems and the multitude of turnovers and transitions. Rather than being alternatives, they may be complementary to explain the biodiversity of mechanisms that make the undifferentiated gonad anlage of an embryo to develop toward testis or ovary. To further our understanding of the trajectories that lead to the evolution of diverse mechanisms, we need not only detailed molecular knowledge about the proximate causes of such diversity but also more information about the ecology and population genetics under which they occur.
## Sidebar B: In need of answers

(i) What are the protein partners of SRY in human and goat that directly activate Sox9 expression?

(ii) Are the differences in the expression pattern and function of the genes in the downstream cascades or networks related to specific adaptations of varying reproductive biology? Or are they the result of neutral evolution and genetic drift?

(iii) Have the naturally occurring hermaphroditic species of fish found a way to make a controlled use of the livelong plasticity of the gonad? Or is the plasticity seen in the mammalian gonads a relic of an evolutionary past?

(iv) What are the evolutionary forces driving the outstanding high variability of molecular and genetic mechanisms of sex determination? Is this all due to stochastic variation? Or is there a global (so far unknown?) reason? Or do all evolutionary mechanisms postulated so far cooperate, with differing importance depending on the species or phylogenetic lineage?

(v) Are Sox3 and Irf9 in vertebrates and *Fem* piRNA components of the downstream sex determination cascades or networks that have been overlooked so far?

(vi) Why do some members of the regulatory networks of sexual development frequently become master sex-determining genes while others never appear at the top position?

## Acknowledgements

We thank Yann Guiguen (INRA, LPGP, Rennes), Mateus Adolfi, Sylvain Bertho, and Alvaro Roco (Biocenter Würzburg) for reading the manuscript and helpful discussions and Monika Niklaus-Ruiz for help in preparing the manuscript. Work of the authors was supported by the Deutsche Forschungsgemeinschaft (Scha408/12-1, 10-1; He7135/2-1) and the ANR (ANR-13-ISV7-0005 PHYLOSEX; Crédits Incitatifs Phase 2015/Emergence to A.H.).

## Conflict of interest

The authors declare that they have no conflict of interest.

## References

1. Graham P, Penn JK, Schedl P (2003) Masters change, slaves remain. *BioEssays* 25: 1–4

2. Haag ES, Doty AV (2005) Sex determination across evolution: connecting the dots. *PLoS Biol* 3: e21

3. Herpin A, Schartl M (2008) Regulatory putsches create new ways of determining sexual development. *EMBO Rep* 9: 966–968

4. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R (1991) Male development of chromosomally female mice transgenic for *Sry*. *Nature* 351: 117–121

5. Lovell-Badge R, Robertson E (1990) XY female mice resulting from a heritable mutation in the primary testis-determining gene, *Tdy*. *Development* 109: 635–646

6. Just W, Rau W, Vogel W, Akhverdian M, Fredga K, Graves JA, Lyapunova E (1995) Absence of *Sry* in species of the vole *Ellobius*. *Nat Genet* 11: 117–118

7. Soullier S, Hanni C, Catzeflis F, Berta P, Laudet V (1998) Male sex determination in the spiny rat *Tokudaia osimensis* (Rodentia: Muridae) is not *Sry* dependent. *Mamm Genome* 9: 590–592

8. Waters PD, Wallis MC, Marshall Graves JA (2007) Mammalian sex-Origin and evolution of the Y chromosome and SRY. *Semin Cell Dev Biol* 18: 389–400

9. Sato Y, Shinka T, Sakamoto K, Ewis AA, Nakahori Y (2010) The male-determining gene SRY is a hybrid of DGCR8 and SOX3, and is regulated by the transcription factor CP2. *Mol Cell Biochem* 337: 267–275

10. Foster JW, Graves JA (1994) An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis-determining gene. *Proc Natl Acad Sci USA* 91: 1927–1931

11. Graves JA (2006) Sex chromosome specialization and degeneration in mammals. *Cell* 124: 901–914

12. Stevanovic M, Lovell-Badge R, Collignon J, Goodfellow PN (1993) SOX3 is an X-linked gene related to SRY. *Hum Mol Genet* 2: 2013–2018

13. Caetano LC, Gennaro FG, Coelho K, Araujo FM, Vila RA, Araujo A, de Melo Bernardo A, Marcondes CR, Chuva de Sousa Lopes SM, Ramos ES (2014) Differential expression of the MHM region and of sex-determining-related genes during gonadal development in chicken embryos. *Genet Mol Res* 13: 838–849

14. Takehana Y, Matsuda M, Myosho T, Suster ML, Kawakami K, Shin IT, Kohara Y, Kuroki Y, Toyoda A, Fujiyama A *et al* (2014) Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish *Oryzias dancena*. *Nat Commun* 5: 4157

15. Oshima Y, Naruse K, Nakamura Y, Nakamura M (2009) Sox3: a transcription factor for Cyp19 expression in the frog *Rana rugosa*. *Gene* 445: 38–48

16. Bowles J, Schepers G, Koopman P (2000) Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators. *Deu Biol* 227: 239–255

17. Pask AJ, Harry JL, Renfree MB, Marshall Graves JA (2000) Absence of SOX3 in the developing marsupial gonad is not consistent with a conserved role in mammalian sex determination. *Genesis* 27: 145–152

18. Rizzoti K, Brunelli S, Carmignac D, Thomas PQ, Robinson IC, Lovell-Badge R (2004) SOX3 is required during the formation of the hypothalamo-pituitary axis. *Nat Genet* 36: 247–255

19. Sutton E, Hughes J, White S, Sekido R, Tan J, Arboleda V, Rogers N, Knower K, Rowley L, Eyre H *et al* (2011) Identification of SOX3 as an XX male sex reversal gene in mice and humans. *J Clin Invest* 121: 328–341

20. Sekido R, Lovell-Badge R (2008) Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. *Nature* 453: 930–934

21. Koyano S, Ito M, Takamatsu N, Takiguchi S, Shiba T (1997) The Xenopus Sox3 gene expressed in oocytes of early stages. *Gene* 188: 101–107

22. Miura I, Ohtani H, Nakamura M, Ichikawa Y, Saitoh K (1998) The origin and differentiation of the heteromorphic sex chromosomes Z, W, X, and Y in the frog *Rana rugosa*, inferred from the sequences of a sex-linked gene, ADP/ATP translocase. *Mol Biol Evol* 15: 1612–1619

23. Graves JA (2013) How to evolve new vertebrate sex determining genes. *Deu Dyn* 242: 354–359

24. Miura IET, Ohtani H, Uno Y, Nishida C, Matsuda Y, Graves JAM (2009) The W chromosome evolution and sex-linked gene expression in the frog *Rana rugosa*. In *Sex Chromosomes: Genetics, Abnormalities and Disorders*, Weingarten CN, Jefferson SE (eds), pp 123–140. Scottsdale, AZ: Nova Science Inc

25. Wilhelm D, Palmer S, Koopman P (2007) Sex determination and gonadal development in mammals. *Physiol Rev* 87: 1–28

26. Kashimada K, Koopman P (2010) *Sry*: the master switch in mammalian sex determination. *Development* 137: 3921–3930

27. Zhao L, Ng ET, Davidson TL, Longmuss E, Urschitz J, Elston M, Moisyadi S, Bowles J, Koopman P (2014) Structure-function analysis of mouse *Sry* reveals dual essential roles of the C-terminal polyglutamine tract in sex determination. *Proc Natl Acad Sci USA* 111: 11768–11773
28. Dubin RA, Ostrer H (1994) Sry is a transcriptional activator. *Mol Endo-crinol* 8: 1182-1192

29. Lovell-Badge R, Canning C, Sekido R (2002) Sex-determining genes in mice: building pathways. *Novartis Found Symp* 244: 4-18; discussion 18-22, 35-42, 253-257

30. Pannetier M, Tilly G, Kocer A, Hudrisier M, Renault L, Chesnais N, Costa J, Le Provost F, Vaiman D, Vilotte JL et al (2006) Goat SRY induces testis development in XX transgenic mice. *FEBS Lett* 580: 3715-3720

31. Chen YS, Racca JD, Sequeira PW, Phillips NB, Weiss MA (2013) Microsatellite-encoded domain in rodent Sry functions as a genetic capacitor to enable the rapid evolution of biological novelty. *Proc Natl Acad Sci USA* 110: E3061-E3070

32. Zhao L, Koopman P (2012) SRY protein function in sex determination: thinking outside the box. *Chromosome Res* 20: 153-162

33. Herpin A, Schartl M (2011) Vertebrate sex determination: questioning the hierarchy. *FEBS J* 278: 1001

34. Herpin A, Schartl M (2011) Dmrt1 genes at the crossroads: a wide-spread and central class of sexual development factors in fish. *FEBS J* 278: 1010-1019

35. Thoma EC, Wagner TU, Weber IP, Herpin A, Fischer A, Schartl M (2011) Ectopic expression of single transcription factors directs differentiation of a medaka spermatogonial cell line. *Stem Cells Dev* 20: 1425-1438

36. Raymond CS, Murphy MW, O'Sullivan MG, Bardwell VJ, Zarkower D (2000) Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. *Genes Dev* 14: 2587-2595

37. Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J, Zarkower D (1998) Evidence for evolutionary conservation of sex-determining genes. *Nature* 391: 691-695

38. Miller SW, Hayward DC, Bunch TA, Miller DJ, Ball EE, Bardwell VJ, Zarkower D, Brower DL (2003) A DM domain protein from a coral, *Acro-pora millepora*, homologous to proteins important for sex determination. *Evol Dev* 5: 251-258

39. Chong T, Collins JJ III, Brubacher JL, Zarkower D, Newmark PA (2013) A sex-specific transcription factor controls male identity in a simultaneous hermaphrodite. *Nat Commun* 4: 1814

40. Kato Y, Kobayashi K, Watanabe H, Iguchi T (2011) Environmental sex determination in the branchiopod crustacean *Daphnia magna*: deep conservation of a Doublesex gene in the sex-determining pathway. *PLoS Genet* 7: e1001345

41. Nanda I, Kondo M, Hornung U, Asakawa S, Winkler C, Shimizu A, Shan Z, Haaf T, Shimizu N, Shima A et al (2002) A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, *Oryzias latipes*. *Proc Natl Acad Sci USA* 99: 11778-11783

42. Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N et al (2002) DMY is a Y-specific DM-domain gene required for male development in the medaka fish. *Nature* 417: 559-563

43. Veitia R, Nunes M, Brauner R, Doco-Fenzy M, Joanny-Flinois O, Jaubert F, Lortat-Jacob S, Fellous M, McElreavey K (1997) Deletions of distal 9p associated with 46, XY male to female sex reversal: definition of the breakpoints at 9p23.3-p24.1. *Genomics* 41: 271-274

44. Chen S, Zhang G, Shao C, Huang Q, Liu G, Zhang P, Song W, An N, Chalopin D, Volff JN et al (2014) Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. *Nat Genet* 46: 253-260

45. Smith CA, Roeszler KN, Ohnesorg T, Cummins DM, Farlie PG, Doran TJ, Sinclair AH (2009) The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. *Nature* 461: 267-271

46. Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, Matsuda Y, Takamatsu N, Shiba T, Ito M (2008) A W-linked DM-domain gene, DM-W, participates in primary ovary development in *Xenopus laevis*. *Proc Natl Acad Sci USA* 105: 2469-2474

47. Kim S, Bardwell VJ, Zarkower D (2007) Cell type-autonomous and non-autonomous requirements for Dmrt1 in postnatal testis differentiation. *Deu Biol* 307: 314-327

48. Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, Zarkower D (2010) The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells. *Deu Cell* 19: 612-624

49. Matson CK, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D (2011) DMRT1 prevents female reprogramming in the postnatal mammalian testis. *Nature* 476: 101-104

50. Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, Treier AC, Klugmann C, Klasen C, Holter NI et al (2009) Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. *Cell* 139: 1130-1142

51. Boulanger L, Pannetier M, Gall L, Allais-Bonnet A, Elzaiat M, Le Bourhis D, Daniel N, Richard C, Cotinot C, Ghyselinck NB et al (2014) FOXL2 is a female sex-determining gene in the goat. *Curr Biol* 24: 404-408

52. Georges A, Auguste A, Bessiere L, Vanet A, Todeschini AL, Veitia RA (2014) FOXL2: a central transcription factor of the ovary. *J Mol Endocrinol* 52: R17-R33

53. Lindeman RE, Gearhart MD, Minkina A, Krentz AD, Bardwell VJ, Zarkower D (2015) Sexual cell-fate reprogramming in the ovary by DMRT1. *Curr Biol* 25: 764-771

54. Masuyama H, Yamada M, Kamei Y, Fujiwara-Ishikawa T, Todo T, Nagahama Y, Matsuda M (2012) Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka. *Chromosome Res* 20: 163-176

55. Krentz AD, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D (2011) DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary. *Deu Biol* 356: 63-70

56. Herpin A, Braasch I, Kraeusling M, Schmidt C, Thoma EC, Nakamura S, Tanaka M, Schartl M (2010) Transcriptional rewiring of the sex determining dmrt1 gene duplicate by transposable elements. *PLoS Genet* 6: e1000844

57. Herpin A, Schindler D, Kraiss A, Hornung U, Winkler C, Schartl M (2007) Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt I by. *BMC Dev Biol* 7: 99

58. Herpin A, Schartl M (2011) Sex determination: switch and suppress. *Curr Biol* 21: R656-R659

59. Matson CK, Zarkower D (2012) Sex and the singular DM domain: insights into sexual regulation, evolution and plasticity. *Nat Rev Genet* 13: 163-174

60. Cutting A, Chue J, Smith CA (2013) Just how conserved is vertebrate sex determination? *Deu Dyn* 242: 380-387

61. Nakamura S, Watakabe I, Nishimura T, Picard JY, Toyoda A, Taniguchi Y, di Clemente N, Tanaka M (2012) Hyperproliferation of mitotically active germ cells due to defective anti-Mullerian hormone signaling mediates sex reversal in medaka. *Development* 139: 2283-2287

62. Hattori RS, Murai Y, Oura M, Masuda S, Majhi SK, Sakamoto T, Fernandino JI, Somoza GM, Yokota M, Strussmann CA (2012) A Y-linked anti-Mullerian hormone duplication takes over a critical role in sex determination. *Proc Natl Acad Sci USA* 109: 2955-2959