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RESEARCH ARTICLE

Open Access

<figure><img src="image_76.png" /></figure>

CrossMark

# Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements

Manfred Schartl<sup>1,2,3</sup>, Susanne Schories<sup>1</sup>, Yuko Wakamatsu<sup>1</sup>, Yusuke Nagao<sup>4</sup>, Hisashi Hashimoto<sup>4</sup>, Chloé Bertin<sup>5</sup>, Brigitte Mourot<sup>5</sup>, Cornelia Schmidt<sup>1</sup>, Dagmar Wilhelm<sup>6</sup>, Lazaro Centanin<sup>7</sup>, Yann Guiguen<sup>5</sup> and Amaury Herpin<sup>1,5* ID</sup>

**Abstract**

**Background:** Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene *dmrt1bY* underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called *Izanagi*, which is co-opted to act as a silencer to turn off the *dmrt1bY* gene after it performed its function in sex determination.

**Results:** We now show that a second TE, *Rex1*, has been incorporated into *Izanagi*. The insertion of *Rex1* brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of *dmrt1bY*. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a *dmrt1bY*-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal.

**Conclusions:** Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of *dmrt1* transcriptional activity during primary sex determination due to exaptation of the *Rex1* transposable element.

**Keywords:** Exaptation, Master sex-determining gene, Transcriptional rewiring, Medaka, *Dmrt1bY*, *Sox5*

**Background**

Sex determination (SD) is one of the most plastic processes in evolution. The trigger for the bipotential undifferentiated embryonic gonad anlage to develop into either testis or ovary can be provided by various signals from the environment, the genetic constitution of the individual, or a mixture of both [1, 2]. Studies of the modes of genetic SD revealed that the genes at the top of the regulatory network and the genes of the network itself are subject to rapid changes in evolution. New master SD genes evolved repeatedly and independently

[3]. This situation is particularly obvious in fish, since closely related sister species can have different genetic SD systems or master SD genes [3-7].

Clearly, such a high turnover of genetic determinants can work only if the evolutionary innovations are accompanied by the ability of the respective genes to neo-functionalize or sub-functionalize quickly and efficiently [8, 9]. In addition to changes in protein structure, differences in gene regulation have an important role in evolution and are considered a quick and effective way to adapt gene functions to novelty [10-12]. Hence, the necessity for the transcriptional rewiring of the architecture of the SD regulatory network and connecting novel master SD genes to it requires high-capacity and fast mechanisms. Such a mechanism was proposed by Britten and Davidson

<footnote>* Correspondence: amaury.herpin@inra.fr</footnote>

<footnote n="1">¹Physiological Chemistry, Biocenter, University of Würzburg, 97074 Würzburg, Germany</footnote>

<footnote n="5">⁵INRA, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France
Full list of author information is available at the end of the article</footnote>
almost 50 years ago [13, 14]. They hypothesized that transposable elements (TEs) carry preformed transcription factor binding sites, which, after mobilization and insertion into novel locations of the genome, would contribute novel regulatory features to nearby genes through these motifs. However, examples of authentic co-option, or exaptation [15], of TEs, where most or all gene transcriptions initiate within a TE, remain sparse (see [16] for a review).

Intriguingly, of the handful of examples of this process, one of the best documented comes from a novel SD gene. The master male-determining gene *dmrt1bY* of the teleost fish *Oryzias latipes* (medaka) arose approximately 5 to 10 million years ago from an autosome encompassing the *dmrt1* gene. *Dmrt1* is a highly conserved transcription factor that usually functions at a downstream position of the sexual regulatory cascade. In medaka, the *dmrt1* gene was locally duplicated and inserted into another chromosome that became the Y-chromosome [5]. To exert its novel function at its new upstream position, *dmrt1bY* acquired a divergent expression pattern and effector gene profile compared to its autosomal ancestor, *dmrt1a* [5, 17]. We previously showed that this evolutionary innovation, which required a complete rewiring of the regulatory network, was partly brought about by exaptation of a ready-to-use pre-existing *cis*-regulatory element contributed by a TE, called *Izanagi* [17]. This element acts as a silencer. It recruits proteins *Dmrt1bY* and *Dmrt1a* to turn off the *dmrt1bY* gene after it has fulfilled its function as the primary male SD gene.

We report here that TE-mediated transcriptional rewiring can reach an unexpected level of complexity that exceeds this simple feedback regulation. We find that another TE, *Rex1*, has jumped into *Izanagi*. Through the disruption of *Izanagi*, *Rex1* immobilized this TE and fixed the *Dmrt1*-mediated downregulation. Moreover, *Rex1* brought in a preformed regulatory element for the transcription factor *Sox5*. We show that medaka *Sox5* binds to the *sox5*-responsive elements of the *dmrt1bY* promoter and downregulates its transcriptional activity. Interestingly, in vivo analysis of double transgenic fluorescent reporter fish additionally revealed a complementary pattern of expression of both genes. The higher expression of *sox5* correlates with a lower expression of *dmrt1bY* and vice versa. Our results underpin the importance of the *Rex1* TE for the establishment of a new SD mechanism in medaka and likely contribute in establishing the temporal and cell-type specific expression pattern of *dmrt1bY*.

Several transcription factors of the *Sox* family (*Sox3*, *SRY*, *Sox 9*, and *sox8*) play crucial roles in SD, but *Sox5* has not been previously implicated in SD in any metazoan so far (neither *Sox5* in vertebrates, nor its

*Drosophila* homologue *Sox102F*). Interestingly, in medaka, disruption of *sox5* leads to XX female-to-male sex reversal. From an analysis of mutants, we find the critical involvement in gonadal development in medaka by regulating primordial germ cells (PGCs). In overexpression experiments, there is an ectopic induction of germ-cell markers including *dmrt1*. With all necessary notes of caution, our preliminary expression pattern data, also detecting SOX5 expression in the fetal gonad of mice, may indicate an evolutionarily conserved role for SOX5 during early mammalian gonad development.

Our work reveals a dual role for *sox5* during SD: (i) first being an evolutionarily conserved important regulator of germ-cell number in medaka and possibly beyond and (ii) second, de novo regulation of medaka *dmrt1* transcriptional activity during primary SD after it has been recruited for transcriptional rewiring of the *dmrt1* promoter due to exaptation of a TE.

## Results

### Identification of putative *Sox5* transcription factor binding sites in the *dmrt1bY* promoter

In an initial analysis of the promoter of the medaka male-determining gene *dmrt1bY*, we found that after duplication from its autosomal progenitor *dmrt1a*, an insertion of an *Izanagi* DNA transposon brought in a novel transcriptional repressor element [17]. It functions by binding *Dmrt1a* and *Dmrt1bY* transcription factors and is essential for the downregulation of *dmrt1bY* after fulfilling its SD function in the male gonad.

In addition to this repressor element [17], the promoter region contains an unexpectedly high density of putative binding sites for *Sox5* (see the [β] region in Fig. 1a and [17]). It harbors seven *Sox5* binding sites; a random prediction would expect only 0.46 sites over the whole sequence. Interestingly, a unique putative *Sox5* binding site is also found within a *Rex1* TE [α], and two within repeat 3 [γ] of the *dmrt1bY* proximal promoter region (see [α], [β], or [γ] in Fig. 1a and Additional file 1: Figure S1 for *sox5* binding site locations). These regions were all inserted into the promoter after the duplication event and, thus, during the evolution of the novel male-determining function of *dmrt1bY*.

In particular, the *Sox5* binding site nested within the *Rex1* TE of the *dmrt1bY* proximal active promoter showed high prediction probability (weight 9.4, *p* value 5.4 × 10⁻⁵, lnPval -9;831; [α] in Fig. 1a, see also "Methods" for the positional weight matrix employed). To address the question whether this *Sox5* binding site has evolved de novo after insertion or was already an integral part of the *Rex1* element that was inserted into the *dmrt1bY* promoter, we blasted the *Rex1* sequence of the *dmrt1bY* promoter against the medaka and other fish genomes (Fig. 1b,c). *Rex1* elements are present in
<figure><img src="image_77.png" /></figure>

<table><thead><tr><th></th><th># of Hits<br>(partial Rex1)</th><th># encompassing<br>Sox5 binding site</th><th># with intact<br>Sox5 binding site</th></tr></thead><tbody><tr><td>'Xenopus laevis'</td><td>0</td><td>0</td><td>0</td></tr><tr><td>'Gallus gallus'</td><td>0</td><td>0</td><td>0</td></tr><tr><td>'Mus musculus'</td><td>0</td><td>0</td><td>0</td></tr><tr><td>'Homo sapiens'</td><td>0</td><td>0</td><td>0</td></tr><tr><td>'Latimeria chalumnae'</td><td>0</td><td>0</td><td>0</td></tr><tr><td>'Lepisosteus oculatus'</td><td>10</td><td>0</td><td>0</td></tr><tr><td>'Astyanax mexicanus'</td><td>20</td><td>5</td><td>0</td></tr><tr><td>'Danio rerio'</td><td>59</td><td>10</td><td>0</td></tr><tr><td>'Gadus morhua'</td><td>15</td><td>2</td><td>0</td></tr><tr><td>'Gasterosteus aculeatus'</td><td>3</td><td>0</td><td>0</td></tr><tr><td>Tetraodon</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Takifugu</td><td>0</td><td>0</td><td>0</td></tr><tr><td>'Oreochromis niloticus'</td><td>100</td><td>47</td><td>1</td></tr><tr><td>'Oryzias latipes'</td><td>100</td><td>20</td><td>20</td></tr><tr><td>'Poecilia formosa'</td><td>2</td><td>1</td><td>0</td></tr><tr><td>'Xiphophorus maculatus'</td><td>4</td><td>0</td><td>0</td></tr></tbody></table>

**Fig. 1** Comparative analysis of the *dmrt1a* and *dmrt1bY* co-ortholog promoters and presence of *Rex1* element sequences in the genomes of selected fish species. **a** Comparative analysis of the medaka *dmrt1* co-ortholog promoter regions. Differences in length for the two promoter regions are caused by *Rex1* and *Izanagi* transposable elements as well as repeats 3 and 4 that were inserted into the *dmrt1bY* promoter after the duplication event that gave rise to the *dmrt1bY* gene approximately 10 million years ago [17]. Regions α, β, or γ (brackets [] underlined in red) contain multiple Sox5 binding sites within *Rex1*, *Izanagi*, and repeat 3, respectively, that have been subjected to chromatin immunoprecipitation (ChIP) (see also Additional file 1: Figure S1). The red star (*) identifies the *Dmrt1* binding site described in [17]. **b** Alignment of the Y-chromosomal *Rex1* element together with the 19 remaining *Rex1* copies encompassing the *sox5* binding site in the medaka genome. Dots indicate conserved nucleotides. Black arrows define primers used for chromatin immunoprecipitation. **c** Presence of *Rex1* element (i) partial sequences, (ii) sequences encompassing the *dmrt1bY*-nested *sox5* binding site, and (iii) sequences encompassing the *dmrt1bY*-nested *sox5* binding site with the intact *sox5* binding site in the genomes of medaka (*Oryzias latipes*), tilapia (*Oreochromis niloticus*), zebrafish (*Danio rerio*), cavefish (*Astyanax mexicanus*), cod (*Gadus morhua*), gar (*Lepisosteus oculatus*), stickleback (*Gasterosteus aculeatus*), platy (*Xiphophorus maculatus*), Amazon molly (*Poecilia formosa*), fugu, tetraodon, and coelacanth (*Latimeria chalumnae*)

100 copies in the genome of medaka. Many copies are also found in tilapia (100) and zebrafish (59), but there are fewer in cavefish (20), cod (15) and gar (10). They are scarce in stickleback (3), platyfish (4), and Amazon molly (2), and absent in fugu, tetraodon, and coelacanth

(Fig. 1c). Interestingly, among *Rex1* elements, the region encompassing the predicted *sox5*-binding site is very poorly conserved despite being part of the reverse transcriptase-coding region (region 6) of the *Rex1* element (Additional file 2: Figure S2). It can be detected with
some divergence to the consensus sequence in only 47, 20, 10, 5, 2, and 1 copies in tilapia, medaka, zebrafish, cavefish, cod, and Amazon molly, respectively (Fig. 1c). An intact sox5 binding site is predicted in only two species. In tilapia, a single copy has a high-fidelity site, whereas 20 copies in medaka, including the one in the *dmrt1bY* promoter, have putatively intact sox5 binding sites (Fig. 1b,c and Additional file 3: Table S1). Hence, *Rex1*-nested sox5 binding sites appear to be a medaka-specific feature.

Sox5 has been correlated with *dmrt1* expression in zebrafish [18] and the wrasse, *Halichoeres tenuispinis* [19], in in vitro promoter studies. Thus, we hypothesized that the identified sox5 binding sites could be involved in the transcriptional rewiring of *dmrt1bY*.

## Sox5 binds to the putative Sox5-responsive elements of the dmrt1bY promoter with different affinities

To assess the relevance of the predicted Sox5 binding sites [α], [β], and [γ] (see Fig. 1a,b) in the *dmrt1bY* promoter, two different medaka cell lines, *Oryzias latipes* spermatogonial (Sg3) and fibroblast (OLF) cells, were transfected with a FLAG-tagged version of Sox5 and then subjected to chromatin immunoprecipitation (ChIP) using an anti-FLAG antibody. DNA amplification with specific sets of primers from the Sox5 immunoprecipitates revealed that the Sox5 protein binds to the predicted sites ([α], [β], and [γ]) of the *dmrt1bY* promoter. However, much stronger binding is apparent for the proximal site [α] (up to tenfold higher enrichment, Fig. 2a) located within the *Rex1* element. Of note, although two DNA-binding sequences for Sox5 were predicted within the *dmrt1a* proximal promoter (at positions [-286/-300] and [-1100/-1116] upstream of the ATG), they do not appear to be functional, as Sox5 does not target them in the ChIP experiments (data not shown).

Of note, the predicted sox5 binding region [β] has previously been shown to overlap with a high affinity binding site for Dmrt1 [17]. Hence, competition between Sox5 and Dmrt1 for access to this sequence motif cannot be excluded. If Dmrt1 already occupies the motif, this might explain the low amount of recovery in the Sox5 ChIP experiment.

## Sox5 downregulates the activity of the dmrt1bY promoter

Next, we examined (i) the respective contributions of each part of the promoter for *dmrt1bY* transcriptional regulation and (ii) the direction of that regulation (up- or downregulation) using transcriptional reporter assays. Thus, 5' deletions of the *dmrt1bY* promoter ([0/-1593], [0/-2995], [0/-6207], and [0/-8996]) were produced and analyzed after transient transfection for their ability to drive luciferase expression (Fig. 2b).

In the medaka spermatogonial cell line, the highest promoter activity was detectable for the [0/-2995] proximal promoter region encompassing sites [α] and [β] (Fig. 2b2). Promoter activity was significantly lower (Fig. 2b3,4), when more distal sequences ([2996/-6207]) containing site [γ] were present in the construct. The shortest proximal promoter region (encompassing site [α] only) had intermediate transcriptional activity (Fig. 2b1). Interestingly, the transcriptional activity of the most proximal parts of the *dmrt1bY* promoter—encompassing the sites [α] in *Rex1* (Fig. 2b1) and [β] in *Izanagi* (Fig. 2b2)—was reduced by between 43% and 68% when Sox5 was overexpressed (Fig. 2b1,2). This effect of Sox5 overexpression was not apparent for the longer constructs including further distal sequences displaying strong constitutive repression (Fig. 2b3,4). Of note, the highest repression of *dmrt1bY* promoter transcriptional activity was observed for the proximal promoter region encompassing the [α] site in *Rex1* (68% in Fig. 2b1). Next, modulation of transcriptional activity was tested for the [α], [α]-MUT, and [β] regions alone (Fig. 2c). Interestingly, only the [α] region was able to downregulate promoter activity (by about 40%) when fused to a minimal thymidine kinase promoter. Neither the [β] or a mutated version of the [α] region ([α]-MUT) were able to modulate the activity of the minimal thymidine kinase promoter (Fig. 2c).

To obtain a more precise readout for the regulation of medaka *dmrt1bY* expression by Sox5, spermatogonial and fibroblast medaka cell lines were transiently transfected with a Sox5-expressing construct and endogenous *dmrt1bY* expression was quantified by the real-time polymerase chain reaction (RT-PCR) at different time points post-transfection (24, 48, and 72 h). Our findings reveal that the transcription of *dmrt1bY* is highly repressed in the presence of Sox5 (up to 90% 48 h after transfection, Fig. 3a) in both the OLF and Sg3 medaka cell lines.

Next, to validate our in vitro results, Sox5 coding mRNAs were microinjected into one-cell-stage embryos, and the expression of *dmrt1bY* and several germ-cell markers (*nanos2*, *nanos3*, *dead-end*, *vasa*, *tra2a*, and *piwi*) was monitored (Fig. 3b). The results confirm the transient cell transfection experiments and demonstrated that in vivo Sox5 can act as a negative modulator of *dmrt1bY* expression (Fig. 3b). Overexpression of Sox5 also resulted in the repression of all analyzed germ-cell genes regardless of their intrinsic endogenous expression levels (Fig. 3b). Our in vivo results identify Sox5 as a strong negative regulator of germ-cell gene expression, including *dmrt1bY*. Interestingly, although most of the germ-cell marker genes (*nanos3*, *dead-end*, *vasa*, and *piwi*) are maternally deposited, their lower relative abundances compared to controls after Sox5 overexpression
<figure><img src="image_78.png" /><figcaption>**Fig. 2** Analysis of Sox5 binding to the *dmrt1bY* promoter and regulation of *dmrt1bY* promoter activity upon modulation of Sox5 expression. **a** Chromatin immunoprecipitation (ChIP) of Sox5 binding to regions α, β, or γ of the *dmrt1bY* proximal promoter. Transient transfection of a flagged and tagged version of Sox5 into either medaka spermatogonial or fibroblast cell lines and subsequent immunoprecipitation (FLAG antibody) followed by the quantitative real-time polymerase chain reaction. Results are presented as enrichment compared to the input and correspond to three independent immunoprecipitations for each region (α, β, or γ). Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 3). **b1–b4** Analysis of *dmrt1bY* proximal promoter activity after Sox5 transient transfection into the medaka spermatogonial cell line (Sg3). Deletions of the 5' *dmrt1bY* promoter were generated: **b1** α region, **b2** α and β regions, **b3**, **b4** α, β, and γ regions. Transcriptional activity was quantified in the absence (control, -Sox5) or presence (+Sox5) of Sox5. Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 4). **c** Detailed analysis of the transcriptional activity of the alpha (α), alpha-mutant (α-MUT), and beta (β) fragments. Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 4). \* *p* value ≤ 0.05, \*\* *p* value ≤ 0.01. ns non-significant, OLF *Oryzias latipes* fibroblast, Sg3 *Oryzias latipes* spermatogonial cell</figcaption></figure>

are likely attributable to a total arrest of background
transcription after zygotic transcription started, or pos-
sibly accelerated mRNA decay.

Expression of *sox5* during early gonad primordium
formation

Medaka *sox5* mRNA, which is expressed in embryonic
and early larval development, has a distinct spatially and
temporarily restricted expression pattern (Fig. 4). Be-
tween stages 18 and 22, *sox5* transcripts localize mainly

in the head and tail bud regions of the embryos
(Fig. 4a–d). At stage 22, expression is additionally de-
tected in the lateral plate mesoderm (arrowheads in
Fig. 4c,d), from which the somatic gonadal primordium
will develop shortly thereafter [20]. Later, *sox5* expres-
sion is observed over the entire dorsal neural tube
(Fig. 4e,e',f,f') and pre-migratory neural crest cells
(NCCs, Fig. 4f'). At stage 29, *sox5* transcripts are present
in migrating NCCs ventrally [21] (Fig. 4g and arrow-
heads in g'). A higher resolution of the dynamic
<figure><img src="image_79.png" /><figcaption>**Fig. 3** Effects of Sox5 modulation on *dmrt1bY* gene expression. Analysis of *dmrtbY* a transcriptional regulation after *sox5* transient transfection in either spermatogonial or fibroblast medaka cell lines at 24 or 48 h post-transfection. Dataset results of four independent transfections. Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 4; \* *p* value ≤ 0.05, \*\* *p* value ≤ 0.01). b Overexpression of *sox5* was stimulated in fish eggs. The expression of *dmrt1bY* and germ-cell markers (*nanos2*, *nanos3*, *dead-end*, *vasa*, *tra2a*, and *piwi*) were monitored at stage 18 of development and compared to wild-type fish embryos. Dataset results of three independent transfections. Statistical significance was assessed with the t-test (N = 3 and each replicate is a pool of 25 eggs; \* *p* value ≤ 0.05, \*\* *p* value ≤ 0.01). ns non-significant, OLF *Onyzias latipes* fibroblast, Sg3 *Onyzias latipes* spermatogonial cell</figcaption></figure>

expression pattern of *sox5* in vivo was obtained with a transgenic line, which has the 3288-bp upstream promoter region of *sox5* fused to a fluorescent (*mCherry*) reporter. The reliability of *sox5* gene expression was confirmed by comparison of the observed fluorescence with the in situ hybridization data (Fig. 4c and f compared to d, h, and i, respectively). *Sox5* promoter-driven fluorescence was detected as early as stage 22 in the lateral plate mesoderm (Fig. 4d). This region has been shown to have already the properties of a gonadal field because somatic gonadal precursors arise from the most posterior part of the lateral plate mesoderm [20]. Consistently, at stages 26/28, when the gonadal primordium just has formed, faint *sox5* expression is still observable in the somatic tissues surrounding the germ cells that express *Dmrt1bY* (Fig. 5a–c).

At stages 33 to 34, the gonadal expression of *sox5* is restricted to the germ cells (Fig. 5d–i). Of note, variations in the levels of *sox5* expression are clearly visible between different germ cells (Fig. 5d,e). Interestingly,

*dmrt1bY* (*Dmrt1bY:GFP*) is also expressed in germ cells at that specific stage of development [22] and displays variations in expression between individual germ cells [23, 24] (Fig. 5d,f). Analysis of the [Sox5:mCherry and Dmrt1bY:GFP] double transgenic line revealed a complementary pattern of expression of both genes: those germ cells that have a higher expression of *sox5* have a lower expression of *dmrt1bY* and vice versa (Fig. 5d–i).

During the following developmental stages (stages 38/39), the expression of *sox5:mCherry* increases in all germ cells, whereas *dmrt1bY:GFP* concurrently switches from germ cells to the somatic, germ-cell-surrounding cells (Fig. 5j,k). Taken together, these results show that expression of *sox5* and *dmrt1bY* is highly dynamic during gonadal primordium formation, switching from germ cells to somatic cells (Fig. 5l).

To determine whether the expression of *sox5* during early gonadal development is a medaka-specific feature or is more widely conserved, we used immunofluorescence on 13.5 and 14.5 days post coitum (dpc) mouse embryos.
<figure><img src="image_80.png" /><figcaption>**Fig. 4** Expression of medaka *sox5* during embryogenesis. **a** to **c** and **e** to **g** Medaka *sox5* expression investigated by whole-mount in situ hybridization or **d**, **h**, and **i** fluorescence using a transgenic reporter line for which a 3288-bp *sox5* promoter fragment drives the expression of mCherry. **a–c** Between stages 18 and 22, *sox5* mRNA localizes predominantly in the head and tail bud regions of the embryos. **c,d** At stage 22, additional expression is detected in the lateral plate mesoderm of the embryos (arrows). **e–g'** From stage 24 onward, *sox5* expression spans over the dorsal neural tube and pre-migratory neural crest cells (arrowheads). **g,g'** At stage 29, *sox5* expression is also seen in ventral migrating neural crest cells (arrowheads). **h** and **i** Fluorescent *sox5* expression is monitored in the neural tube and neural crest cells of hatching embryos (stages 38/39). **h** For comparison, *patched2* highlights the notochord at stage 39 [11]</figcaption></figure>

This revealed that SOX5 was expressed in peritubular myoid cells surrounding cords in the fetal testis (Additional file 4: Figure S3, left and middle panels) and in a subset of somatic and germ cells in the fetal ovary (Additional file 4: Figure S3, right panels). Of note, a substantial fraction of SOX5 expression also displayed cytoplasmic localization. Although reported for other SOX factors (see SOX2 [25], SOX9 [26, 27], and [28] for reviews), the functional significance of the SOX5 non-exclusive nuclear localization in mice gonads remains to be investigated.

## Expression of *sox5* in adult gonads

Given reduced *dmrt1bY* expression in the fully developed testis [29], we also monitored the expression of *sox5* in fully mature gonads of both sexes (Fig. 6). In adult testes, *sox5* fluorescence was mainly restricted to the interlobular cells (see Fig. 6a–d). In contrast, *dmrt1bY* expression is clearly localized within the Sertoli cells [17] of the testicular lobules (Fig. 6a). No co-localization of *dmrt1bY* and *sox5* transcripts whatsoever was observed (Fig. 6a). To define the nature of the interlobular *sox5*-positive cells better,

immunofluorescence of 11-β-hydroxylase protein, a specific marker of Leydig cells, was performed (Fig. 6b). Interstitial cells of Leydig are found adjacent to the seminiferous lobules in the testes and produce androgens [30]. A perfect co-localization of *sox5* transcripts and 11-β-hydroxylase protein confirmed that the interlobular *sox5*-positive cells are indeed Leydig cells (Fig. 6c,d). Interestingly, another discrete population of *sox5*-positive cells is discernable between but close to the lobules (Fig. 6e–g). These cells are very small compared to their neighboring germ cells, and their nuclei appear compact (Fig. 6h–j). The expression of the germ-cell marker *vasa* (Fig. 6e–j) assigns these cells to the germ-cell lineage, which are probably at a very early stage of differentiation. These germ cells do not express *dmrt1bY* (Fig. 6k–n). The adult ovary displays only a very few *sox5*-positive cells with small and condensed nuclei (Fig. 6o–q). At present, the identity of these cells is difficult to ascertain, but oocytes and somatic supporting cells (granulosa or theca cells) can be excluded from their morphology and location.
<figure><img src="image_81.png" /><figcaption>**Fig. 5** Comparative analysis of *sox5* and *dmrt1bY* expression dynamics during gonadal primordium formation. Expression of *sox5* compared to *dmrt1bY* in a double transgenic fluorescent reporter line. **a–c** During early gonadal formation, *sox5* is first detected in the somatic tissues surrounding the germ cells at stages 26 to 28. At the same time, *dmrt1bY* is expressed in germ cells. **d–i** By stages 33 to 34, *sox5* expression becomes restricted to the germ cells. *dmrt1bY* is also expressed in the germ cells at those specific stages of development. Variations within the respective levels of *sox5* and *dmrt1bY* expression are clearly observable (d compared to e and f and g compared to h and i). **j** *dk* Around hatching (stages 38/39), the expression of *sox5* strengthens in all germ cells while parallel *dmrt1bY* expression quickly switches from germ cells only to somatic germ-cell-surrounding cells only. **l** In vivo visualization of the dynamics of expression localization of *sox5* and *dmrt1bY* during male gonadal primordium development. The expression of *sox5* and *dmrt1bY* is highly dynamic during primordium gonadal formation, switching from somatic to germ cells and vice versa, respectively, from stage 26 until hatching. Being mutual repressors of each other, a seesaw of expression is observed, finally finely restricting *dmrt1bY* expression in the somatic part of the primordium gonad. Blue and red represent cellular expression localizations only and should not been interpreted as expression levels</figcaption></figure>

## PGC number is decreased in *sox5* mutants

To obtain functional data on the role of *sox5* in gonadal development, we next analyzed early gonadal development in mutants. The *ml-3* mutant (N541S) is a naturally occurring mutation, for which a

premature stop codon results in the production of a truncated *Sox5* protein lacking the HMG box domain and causing *Sox5* loss of function [21, 31, 32]. Considering that *sox5* is first expressed in the lateral plate mesoderm and then in germ cells during
<figure><img src="image_82.png" /><figcaption>**Fig. 6** Expression of *sox5* in adult gonads. **a–d** In adult testes, sox5 fluorescence is restricted to the cells located between the lobules where the germ and Sertoli cells lie. **a,b** In double transgenic reporter fish, fluorescence of sox5 and *dmrt1bY* (marking the Sertoli cells) is distinct. **c, d** The interlobular expression of sox5 co-localizes with 11-β-hydroxylase, a marker of Leydig cells. **e–j** Expression of sox5 is also detected in another discrete population of cells between but always close to the germinal lobules. **h–j** Small in size, these sox5-positive cells also express *vasa*, a specific marker of germ cells. **k–n** The sox5- and *vasa*-positive cells do not express *dmrt1bY*. **o–q** In adult ovaries, only extremely few sox5 positive cells are detected.</figcaption></figure>

primordial gonad formation, we investigated whether Sox5 plays a role in regulating PGC number. Whole-mount in situ hybridizations utilizing the PGC-specific marker *vasa* were performed in wild-type and *sox5*⁻/⁻ mutant fish embryos (Fig. 7a,b). At stage 22, during the formation of the primordial gonad when *sox5* has a first expression peak in wild-type embryos, a drastic reduction in the PGC number is evident in mutants (Fig. 7a compared to b). This emphasizes a possible role for *Sox5* as being a regulator of PGC proliferation although such a reduction in PGC numbers might also be ascribed to reduced proliferation, reduced survival, or defects in fate specification.

### PGC number is rescued in sox5 mutants by in vivo conditional knock-in of Sox5

Given that absence of functional *Sox5* expression in the *sox5*⁻/⁻ mutant resulted in a reduced number of PGCs, we next attempted to rescue the gonadal phenotype by wild-type *sox5* expression to show that this gene is crucial in regulating PGC numbers. Thus, we established a transgenic line that expresses *sox5* after controlled homologous recombination in the *sox5* mutant genetic background (Fig. 7c). In this line, the *sox5* promoter drives the expression of a mCherry-stop cassette flanked by *LoxP* sites. This cassette is followed by the wild-type *sox5* open reading frame (ORF) fused to a 2A self-cleaving system [33] with nuclear-addressed green
<figure><img src="image_83.png" /><figcaption>**Fig. 7** Regulation of PGC numbers by Sox5. **a,b** As early as stage 22, a drastic reduction of the germ-cell number (vasa in situ hybridization) is observed in *sox5*⁻/⁻ mutants compared to wild-type embryos. **c** For conditional knock-in and rescue of the *sox5*⁻/⁻ mutant fish, a transgenic line expressing *sox5*⁻ was produced. See "Methods" for details. **d,e** In vivo visualization of the effective recombination and expression of *sox5*⁻ is apparent after a switch from red cytoplasmic to green nuclear-localized fluorescence. After Cre recombinase injection at the one-cell stage, an almost total recombination is observed, leading to the expression of the *sox5*⁻ transcript as monitored by green fluorescence. Stars indicate auto-fluorescent pigment cells and arrows indicate recombined germ cells. **f** Germ-cell numbers in *sox5*⁻ mutant medaka after in vivo recombination and expression of *sox5*. Of note, for that specific experiment, embryos were additionally injected with a GFP-Nanos 3' untranslated region (UTR) mRNA construct allowing effective PGC monitoring. Statistical significance was assessed with the Wilcoxon–Mann–Whitney test (N = 30, 28, and 42 for *Sox5*⁻/⁻, *Sox5*⁻/⁻ recombined, and wild-type embryos, respectively; \* *p* value ≤ 0.05, \*\* *p* value ≤ 0.01). GFP green fluorescent protein, ns non-significant, PGC primordial germ cell</figcaption></figure>

fluorescent protein (GFP) (H2B-GFP) (Fig. 7c). After injection of mRNAs encoding for the Cre-NLS protein and effective recombination, the mCherry-stop cassette is excised, and the *sox5* ORF is expressed together with nuclear-localized GFP (see recombination in Fig. 7d for the trunk and 7e for germ cells). We employed this system as being preferable to conventional overexpression because it allows us to bypass the deleterious effect of overexpressing the pleiotropic *sox5* gene during early development. Using this system, we show that in terms of germ-cell numbers, a partial rescue of the *Sox5*⁻/⁻ phenotype at stage 20 occurred in the *Sox5*⁻/⁻ mutant genetic background (Fig. 7f). This provides evidence that *Sox5* is indeed required for controlling PGC numbers during the

formation of the early gonadal primordium as early as stage 20.

## Female-to-male sex reversal of *Sox5* mutant fish

Since germ-cell number was reduced in the *sox5*⁻/⁻ mutant fish, we further investigated whether sexual development was affected. Phenotypic and genotypic sex was determined in a *sox5*⁻/⁻ mutant line [31, 34]. A complete XX female-to-male sex reversal (up to 95%) was recorded (Fig. 8a). The female-to-male sex-reversed phenotypic males were fully fertile, so this *sox5*⁻/⁻ mutant line could be maintained exclusively on a XX genotypic background (see Fig. 8b for phenotypes). Genotypic XY *sox5*⁻/⁻ mutant fish were never detected in the progeny (Fig. 8b) nor in outcrosses.
<figure><img src="image_84.png" /></figure>

The absence of *XY* *sox5*<sup>-/-</sup> male-to-female sex-reversed fish, predicted to overexpress *dmrt1bY* according to our results, is in line with the scarcity of surviving *YY* zygotes—carrying two copies of the *dmrt1bY* gene—reported in the literature [35].

Monitoring gene expression in the XX embryos of that line, we find the upregulation of PGC marker genes, including *nanos2*, *nanos3*, *dead-end*, and *piwi*, whereas *tra2a* and *vasa* did not show significant changes compared to wild-type embryos (Fig. 8c). Of note is the
upregulation of the autosomal *dmrt1a* in the Sox5 mu-
tant background. The precocious expression of this gene
at early stages of development (stages 28 and 32 in
Fig. 8c) is intriguing because *dmrt1a* expression is not
expected before 10 days after hatching. This untimely
expression may be related to the XX female-to-male sex
reversal, because a similar untimely expression of
dmrt1a has been seen in high-temperature-induced XX
male-to-female sex reversals [36] (and our own unpub-
lished data).

**Discussion**

SD relies on the proper control of a hierarchically struc-
tured, multilayered network of genes. The genes at the
top orchestrate complex transcriptional and post-
transcriptional regulations (see [3, 4, 6, 37] for reviews).
Despite such a critical function, they appear to be dis-
pensable in evolutionary terms and can be quickly re-
placed with the emergence of new lineages. Our present
analysis provides evidence that concomitantly to the ac-
quisition of a dominant position within the SD network,
the medaka master male determiner, *dmrt1bY*, was sub-
jected to a profound rearrangement of its regulatory
landscape. We found that sequential insertions of both
*Izanagi* and the *Rex1* transposon were instrumental for
rewiring the *dmrt1bY* promoter in the process of diversi-
fication from its autosomal progenitor *dmrt1a*.

First, the integration site itself appears to be highly
relevant. We have previously reported that a *P*-element-
like DNA transposon, *Izanagi*, brought in a regulatory
sequence that mediates specific transcriptional regula-
tion of *dmrt1bY*, which was important for the Y-
chromosomal duplicate to evolve its new function [17].
A common feature of class II transposons is that they
can excise. The insertion of the *Rex1* element in the
*dmrt1bY* promoter occurred in the DNA-binding
domain of the transposase and thereby fixed the *Izanagi*
element and the contained *Dmrt1* transcription factor
binding motif to the promoter of the new SD gene.

Second, the *Rex1* transposon contributed a functional
high-affinity binding site for the transcription factor
Sox5 as a novel regulatory element for *dmrt1bY* expres-
sion. Thus far, neither Sox5 in vertebrates nor its *Dros-
ophila* homolog Sox102F [38] have been shown to be
implicated in SD. In medaka, the expression pattern of
*sox5* already indicated a function in gonad formation. In-
deed, independently of *dmrt1bY*, *sox5* is expressed in the
lateral plate mesoderm that later gives rise to the gonad.
Thereafter, during gonadogenesis stages, *sox5* expression
switches toward the germ-cell lineage. There the *sox5*
expression pattern in PGCs is mutually exclusive with
the expression of the master SD gene *dmrt1bY*, which,
during the formation of the gonad primordium, concur-
rently changes from germ cell to somatic cell expression

during this period. This is consistent with the in vitro
findings of a suppressive action of Sox5 on the *dmrt1bY*
promoter, although the early expression of *sox5* in the
lateral plate mesoderm as well as in the adult gonads
suggests other additional gonadal functions de-
correlated from *dmrt1bY* activity.

It has been shown that *Dmrt1bY* has a suppressive
effect on cell proliferation by mediating a G2 arrest
[39]. Thus, continued expression of *dmrt1bY* in PGCs
during early embryonic stages, which precede the
actual SD stage at hatching, could have a negative ef-
fect on the number of PGCs that is presumed to be
decisive at the SD stage [40, 41]. Clearly the observed
suppressive action of *sox5* toward *dmrt1bY* expression
in vitro and in vivo at stages 33/34, down-modulates
this negative effect.

Later, the persistence of *sox5* expression—indepen-
dently of *dmrt1bY* expression—in germ cells within the
gonadal primordium and in the early-differentiated germ
cells of mature gonads indicates another independent
major role in germ-cell physiology from gonad induction
to adult development and maintenance. Hence, it is
likely that following the transcriptional rewiring of
*dmrt1bY* first by *Izanagi* and then by *Rex1* TEs, Sox5
has been hijacked in the primary SD cascade for control-
ling and fine-tuning *dmrt1bY* expression during the
male-determining period. Independent of the regulatory
function of *dmrt1bY*, Sox5 appears to have a more gen-
eral involvement during gonadal formation (visible by
expression in the lateral plate mesoderm) and germ-cell
physiology (apparent from persistence of *sox5* expression
in germ cells).

Although not directly related to *dmrt1bY* regulation
during the formation of the male gonadal primordium,
the most convincing evidence for a sexual development
function of Sox5 comes from medaka strains that carry
knockout alleles for this gene. We find that lack of
Sox5 leads to a decrease in PGC numbers, which is res-
cued by re-introducing the wild-type version of the
gene in mutant embryos. Strikingly, at the molecular
level, we found an upregulation of several germ-cell
markers in the mutants, even though the germ-cell
number is sensibly reduced. It can be assumed that
such overexpression in mutants is an indication of an
insufficient compensatory mechanism needed to rescue
germ-cell numbers properly. These findings demon-
strate that germ-cell marker expression levels upon
Sox5 modulation are primarily the result of gene ex-
pression regulation and are not due to the number of
cells that express these genes.

The XX female-to-male sex reversal in the Sox5 mu-
tant is in line with an inferred important function of
maintaining the appropriate number of PGCs. The num-
ber of PGCs at the SD stage is critical for determining
male or female sex in medaka [39, 42]. It is higher in fe-
males at the SD stage. Lowering the number of germ
cells in medaka or zebrafish results in female-to-male
sex reversal [43, 44]. Hence, when the number of PGCs
falls below the threshold in sox5<sup>-/-</sup> mutant XX fish, it
will permit male development. Interestingly, we find an
ectopic and earlier than normal expression of *dmrt1a* in
the primordial gonad of the mutants. This has also been
observed in environmentally induced XX female-to-male
sex reversal in medaka and has been interpreted as a
compensatory mechanism to supply the necessary
trigger for testis development in the absence of
*dmrt1bY* [36, 45].

Sox5 regulation of *dmrt1bY* and the importance of this
gene for sexual development in medaka raise the ques-
tion of whether this co-option of Sox5 regulation
through *Rex1* insertion brought a novel member into
the SD regulatory network as a medaka-specific evolu-
tionary innovation or whether this event provided a
necessary connection to an indispensable gonad-
development downstream pathway. This is difficult to
answer at present but should motivate further studies on
the role of Sox5 in the formation of ovaries and testes in
other species.

The in vitro data of *sox5* effects on *dmrt1* transcriptional regulation in zebrafish and wrasse [18, 19] point to an evolutionarily conserved function of *Sox5* that have been unnoticed so far. Members of the SOX family of transcription factors play essential roles during SD in mammals. Both the founding member of the SRY family and the closely related factor SOX9 have been shown to be necessary and sufficient for mammalian male SD [46, 47]. In addition, other SOX family members, such as SOX3 and SOX10, can take over this role if they are expressed ectopically in the developing testis at the time of SD, as demonstrated in transgenic mice and in human patients with duplications in these genes [48–53]. In contrast, *Sox5* has been implicated in spermatogenesis in the adult [54, 55] but not in embryonic gonad development and/or SD in mouse. With all necessary notes of caution, our preliminary data detecting SOX5 expression in the fetal gonad of mice may indicate an additional role for SOX5 during embryonic gonad development in mice after SD.

The finding that a preformed transcription factor
binding site contributed by the *Rex1* transposon modu-
lates the regulation of *dmrt1bY* promoter highlights the
important role that mobile elements play in the genome
for shaping the evolution of new functions. Intriguingly,
although bona fide examples of this process are still rare
[16], *Rex1* is the second such event found in the same
promoter. It will be interesting to analyze whether the
other repeats present in the *dmrt1bY*, but not in the pro-
moter region of *dmrt1a*, provide further instances of TE

exaptation. Genes that arose by gene duplication such as *dmrt1bY* are primarily dispensable and can only escape degeneration through sub- or neo-functionalization. As *dmrt1bY* and *dmrt1a* both have exclusive functions in male sexual development in line with the highly conserved role of *dmrt1* in invertebrates and vertebrates [56], a change in transcriptional control via the insertion of two different TEs might initially have led to sub-functionalization; *dmrt1bY* acquired its transient early expression, whereas the transcription of *dmrt1a* was pushed back to the later testis differentiation phase. In other fish species, and in mouse and chicken, *dmrt1*, which represents the evolutionary precursor of the two genes in medaka, is expressed starting in very early male SD stages and continues to be expressed during testis differentiation and specialization and in the post-pubertal reproductively active organ [6, 37, 57–59].

Conclusion

In summary, the evolutionary history of the promoter of
a newly arising SD gene in medaka not only provides a
new example for TE-mediated rewiring that created evo-
lutionary novelty but also shows the unexpected com-
plexity and richness of such elements. It will be
interesting to have a closer look at the SD genes of other
fish that have been subject to fast evolutionary change
and thus, might also be targets for TE exaptation.

In addition to showing that *sox5* was recruited—or more
exactly promoted thanks to neo-functionalization—to the
very top of the primary SD cascade after insertion of *Rex1*
and that it controls the fine-tuning of *dmrt1bY* expression,
our results provide evidence for a more general and ances-
tral SD function of *Sox5* in regulating germ-cell number
and, in consequence, gonadal identity.

**Methods**

Immunohistochemistry

Testes from adult fish were fixed with 4% paraformalde-
hyde in balanced salt solution (111 mM NaCl, 5.37 mM
KCl, 1 mM CaCl₂H₂O, 0.6 mM MgSO₄·7H₂O, and 5 mM
Hepes, pH 7.3) for 30 minutes on ice. After fixation, sam-
ples were washed three times for 10 minutes with MABT
buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5, and
0.1% Triton X-100) and subsequently twice for 30 minutes
with MABDT buffer (MABT buffer complemented with
1% bovine serum albumin and 1% dimethyl sulfoxide).
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
anti-11-β-hydroxylase primary antibody (1:150 dilution)
overnight at 4 °C. Samples were then washed three times
for 5 minutes in MABDT buffer and washed again four
times for 30 minutes 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 phosphate-buffered saline, stained with Hoechst solution for 3 hours at 4 °C, mounted and imaged with a confocal microscope (Nikon C1 confocal microscope). For Additional file 4: Figure S3, immunohistochemistry was performed according to [60] using the mouse anti-SOX5 antibody from Abcam (ab26041).

## Chromatin immunoprecipitation

For ChIP, the EpiQuik ChIP kit (Epigentek) was used according to the manufacturer's instructions, using 1 million cells (Sg3 spermatogonial or OLF fibroblast cell lines) of transiently transfected cells with FLAG-tagged Sox5 and 3XFLAG antibodies for immunoprecipitation. After fixation and cell re-suspension, DNA was sheared by sonication (nine pulses of 10 seconds with an amplitude of 10%). After immunoprecipitation, specific primer sets were used for enrichment quantification by real-time PCR. For controls, primer sets encompassing regions without any sox binding sites were used. The results are presented as enrichment compared to input. All primer sets were checked for the specificity of the amplifications.

## Bioinformatic analyses

Binding sites for Dmrt1bY were identified using the matrix provided by [61] together with the Regulatory Sequence Analysis Tools portal (RSat) [62]. Sox5 transcription factor binding sites were determined using MatInspector from the Genomatix portal [63] using the following positional weight matrix: [A, C, G, T: (4, 6, 3, 9), (7, 4, 3, 8), (21, 0, 1, 1), (22, 0, 1, 0), (0, 22, 0, 1), (23, 0, 0, 0), (22, 1, 0, 0), (0, 0, 0, 23), (10, 3, 6, 4), (5, 7, 6, 5)].

## In vitro expression regulation analyses and real-time PCR

Medaka spermatogonial (Sg3) and fibroblast-like (OLF) cell lines were cultured as previously described [64–66]. For transfection, cells were grown to 80% confluency in six-well plates and transfected with 5 µg of 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 performed with 2 µg of total RNA using a 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 real-time reactions. Error bars represent the standard deviation of the mean. Relative expression levels were calculated (according to $2^{-\Delta CT}$ where CT is

the number of cycles) after correction of the expression of elongation factor 1 alpha (ef1alpha).

## Luciferase assay

For promoter analysis, a 9107-bp fragment upstream of the Dmrt1bY ORF was isolated by restriction enzyme digestion (*XhoI/EcoRI*) from BAC clone Mn0113N21 [17] and cloned into pBSII-IScelI plasmid (pBSII-IScelI::[0/-8927] Kb Dmrt1bY. Subsequently, the Gaussia luciferase gene from the pGLuc-basic plasmid (New England Biolabs) was inserted between *EcoRI* and *NotI* sites of pBSII-IScelI:: [0/-8927] Kb Dmrt1bY prom (pBSIIIScelI:: [0/-8927] Kb Dmrt1bY prom::GLuc plasmid, Fig. 2). pBSII-IScelI:: [0/-1593] Kb Dmrt1bY prom::GLuc, pBSII-IScelI:: [0/-2963] Kb Dmrt1bY prom::GLuc and pBSII-IScelI:: [0/-6162] Kb Dmrt1bY prom::-GLuc plasmids were constructed in the same way, removing 5' fragments of the 9107-bp Dmrt1bY promoter region using *Kpn1*, *Eco47III*, and *HindIII* restriction enzyme digestion, respectively, and re-ligation.

Gaussia luciferase activity was quantified using the Luciferase Reporter Assay System from Promega and normalized against co-transfected firefly luciferase expressing plasmid (ptkLUC+). For Fig. 2c, [α], [α]-MUT, and [β] fragments (same as used for the ChIP assay shown in Fig. 2a) were PCR amplified and cloned into ptkLUC+ plasmid (accession number AF027128) between *HindIII* and *BamHI* restriction sites.

## Establishment of transgenic reporter lines and in vivo recombination and imaging

A transgenic line was created for the in vivo visualization of endogenous sox5 expression as well as in vivo functional knock-in of sox5. The Sox5 upstream promoter region of the Sox5 gene was cloned in front of an [mCherry-stop] cassette flanked with LoxP sites (Fig. 7c). In detail, in a first line, the sox5 promoter region drives the expression of an mCherry-Stop cassette. This cassette is followed by a Sox5 ORF fused (2A self-cleaving system [33]) with a nuclear-addressed GFP (H2B-GFP). For recombination, direct microinjection of one-cell-stage embryos with mRNA encoding for the Cre-NLS protein was performed (Fig. 7d-f). Sox5 -/ homozygosity of the fish was determined according to the pigmentation pattern of the embryos (see [21]).

To generate stable transgenic lines, the meganuclease protocol was used [67]. Briefly, approximately 10 to 15 pg of total vector DNA in a volume of 500 pl injection solution containing *I-SceI* meganuclease was injected into the cytoplasm of one-cell-stage medaka embryos (Carbio strain). Adult F0 fish were mated to each other and the offspring were tested for the presence of the transgene by checking for fluorescence. Siblings
from positive F1 fish were raised to adulthood and tested
again for fluorescence.

For PGC visualization and counting, the GFP-nos1 3' UTR construct that includes the mmGFP5 ORF cloned upstream of the 3' UTR of the zebrafish nanos1 gene [68, 69] was injected at the one-cell stage (Fig. 7f).

For imaging, embryos, hatchlings, or tissues were
mounted with 1.2% low melting temperature agarose.
Confocal pictures and image stacks were acquired using
Nikon C1 (eclipse Ti) confocal laser scanning and the
NIS element AR software.

Whole-mount in situ hybridization

RNA whole-mount in situ hybridizations were performed as previously described [70]. Hybridization signals were detected using alkaline phosphatase conjugated anti-DIG antibody (Roche) and BM-purple (Roche) as chromogen.

Bioresources and animals

The Sox5 medaka mutant strain (N541S) has been depos-
ited with the National Bioresource Center [71].

Additional files

**Additional file 1: Figure S1.** Annotation of the *dmrt1bY* promoter. Bold underlined: *Rex1* element. Bold red: location of *sox5* binding sites.
*Dmrt1bY* exon 0 is in blue letters. Primer sets used for ChIP are provided. (DOCX 169 kb)

**Additional file 2: Figure S2.** Comparison of medaka Rex1 reverse-transcriptase (RT) sequence with other non-LTR retrotransposons. RT conserved domains are given according to Malik, Burke, and Eickbush [72]. RT sequences are CR1 from *Gallus gallus* (U88211); Maui from *Fugu rubripes* (AF086712); Jockey, R1, and I from *Drosophila melanogaster* (P21328, X51968, and M14954, respectively); and Tad1 from *Neurospora crassa* (L25662). The degree of amino acid conservation between sequences is shown at the foot of the alignment. (JPG 811 kb)

**Additional file 3: Table S1.** Location and adjacent genes of *Rex1* elements containing Sox5 binding sites in the medaka genome. (PDF 70 kb)

**Additional file 4: Figure S3.** SOX5 protein expression in fetal mouse gonads. Double immunofluorescence of SOX5 (green) and MVH (red) on sagittal sections of 13.5 dpc (left panel) and 14.5 dpc (middle panel) mouse testes, as well as 14.5 dpc mouse ovaries (right panel). Gonads are demarcated with dotted lines. The lower panels are a higher magnification image of the area marked by a square in the upper panels. Scale bars 100 µm (upper panels) and 30 µm (lower panels). (JPG 1710 kb)

**Additional file 5:** Supporting data. Raw supporting data for
Figs. 2A,B1–B4,C, 3A,B, 7 F, and 8C. (XLS 215 kb)

Acknowledgements

The authors thank the fish facilities at the University of Würzburg and at Institut national de la recherche agronomique (INRA) Rennes (C. Duret and F. Borel).

Funding

This work was supported by the Deutsche Forschungsgemeinschaft by a
grant (SCHA 408/12-1; HE 7135/2-1) to AH and MS as well as Crédits Incitatifs
Phase 2015/Emergence to AH. AH was additionally funded by the project
AquaCRISPR (ANR-16-COFA-0004-01). This publication was funded by the
German Research Foundation (DFG) and the University of Würzburg in the
funding program Open Access Publishing.

Availability of data and materials

Datasets generated and analyzed during the current study are included in
this published article and its Additional files 1, 2, 3, 4, and 5. The Sox5 medaka
mutant strain (N541S) has been deposited with the National Bioresource Center
(http://www.nbrp.jp/localeAction.do?lang=en). All materials generated during
the current study are available from the corresponding author on reasonable
request.

Authors' contributions

AH, MS, HH, and YG undertook the conceptualization. SS, YW, YN, DW, LC, CB, BM, and CS developed the methodology. AH, MS, DW, and YG did the formal analysis. AH, SS, YW, CB, BM, and CS performed the investigation. AH, MS, DW, and YG wrote the manuscript. AH and MS were responsible for the supervision, project administration, and funding acquisition. All authors read and approved the final manuscript.

Ethics approval

All animals used in this research were treated following the guidelines of the Comité d'Ethique en Expérimentation Animale (CEEA).

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.

Author details

¹Physiological Chemistry, Biocenter, University of Würzburg, 97074 Würzburg, Germany. ²Comprehensive Cancer Center Mainfranken, University Hospital, 97080 Würzburg, Germany. ³Texas Institute for Advanced Study and Department of Biology, Texas A&M University, College Station, TX 77843, USA. ⁴Bioscience and Biotechnology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, Japan. ⁵INRA, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France. ⁶Department of Anatomy & Neuroscience, University of Melbourne, Parkville, Victoria 3010, Australia. ⁷Centre for Organismal Studies (COS), University of Heidelberg, Heidelberg, Germany.

Received: 9 October 2017 Accepted: 11 January 2018

Published online: 29 January 2018

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<figure><img src="image_85.png" /></figure>

# CMA restricted to mammals and birds: myth or reality?

Laury Lescat, Amaury Herpin, Brigitte Mourot, Vincent Véron, Yann Guiguen, Julien Bobe & Iban Seiliez

To cite this article: Laury Lescat, Amaury Herpin, Brigitte Mourot, Vincent Véron, Yann Guiguen, Julien Bobe & Iban Seiliez (2018): CMA restricted to mammals and birds: myth or reality?, Autophagy, DOI: 10.1080/15548627.2018.1460021

To link to this article: https://doi.org/10.1080/15548627.2018.1460021

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Accepted author version posted online: 21 Jun 2018.

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**Publisher:** Taylor & Francis & Taylor and Francis Group, LLC

**Journal:** *Autophagy*

**DOI:** 10.1080/15548627.2018.1460021

**CMA restricted to mammals and birds: myth or reality?**

Laury Lescat¹,†, Amaury Herpin²,†, Brigitte Mourot², Vincent Véron¹, Yann Guiguen², Julien Bobe², Iban Seiliez¹,*

¹ INRA-UPPA, UMR1419 Nutrition Metabolisme Aquaculture, F-64310 St-Pée-sur-Nivelle, France.

LPGP, INRA, 35000 Rennes, France.

**\*Corresponding author:**

Iban Seiliez

INRA-UPPA, UMR 1419 Nutrition, Métabolisme, Aquaculture
F-64310 Saint Pée sur Nivelle, France.

Tel: (33) 5 59 51 59 99; Fax: (33) 5 59 54 51 52

e-mail : iban.seiliez@inra.fr

**COMPETING FINANCIAL INTERESTS**

The authors declare no competing financial interests.

<footnote>†These authors contributed equally to this work</footnote>
Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis essential for the control of intermediary metabolism. So far, the absence of any identifiable LAMP2A—a necessary and limiting protein for CMA—outside of the tetrapod clade, led to the paradigm that this cellular function was (presumably) restricted to mammals and birds. However, after we identified expressed sequences displaying high sequence homology with the mammalian *LAMP2A* in several fish species, our findings challenge that view and suggest that CMA likely appeared much earlier during evolution than initially thought. Hence, our results do not only shed an entirely new light on the evolution of CMA, but also bring new perspectives on the possible use of complementary genetic models, such as zebrafish or medaka for studying CMA function from a comparative angle/view.

**Keywords:** chaperone-mediated autophagy; evolution; fish; LAMP2A; RNA-seq
Being one of the main pathways involved in lysosomal proteolysis, chaperone- mediated autophagy (CMA) has been described as a selective mechanism for the degradation of specific soluble proteins within lysosomes [1]. Besides a well described role in protein quality control (resulting from its ability to selectively target damaged or non-functional proteins for degradation), the diversity of the sub-proteome degraded by CMA also associates this function with the regulation of transcriptional programs [2], cell death and cell survival mechanisms [3-5], DNA repair and cell cycle progression [6], as well as a variety of intracellular processes related to the control of cellular energetics [7-10]. Over the last few years, CMA has thus emerged as a major core component in the control of cellular homeostasis [11].

In detail, cytosolic proteins bearing a KFERQ-like motif are first recognized by the chaperone HSPA8/HSC70 [12]. The substrate/chaperone complex then docks at the lysosomal membrane through specific binding to the cytosolic tail of LAMP2A (lysosome-associated membrane protein 2A). Multimerization of LAMP2A will then result in the formation of a translocation complex, and promote translocation of substrate proteins [13]. Following unfolding and internalization, substrate proteins are then rapidly degraded by lysosomal proteases. Next, LAMP2A disassembles from the translocation complex, allowing a new cycle of substrate binding and translocation [14].

LAMP2A originates from the alternative splicing of the *LAMP2* gene, giving rise to 3 different splice variants. These splice variants all share a common lumenal domain but display different cytosolic and transmembrane regions. CMA activity is tightly correlated with (i) the level of LAMP2A (and not those of the 2 other splice variants) at the lysosomal membrane [15] and (ii) the efficiency of assembly/disassembly of LAMP2A in this compartment [14]. As such, LAMP2A is considered to be the necessary and limiting component for CMA activity [15].
In this context, because LAMP2A has, so far, been characterized only in birds and mammals, and not in other clades, functional CMA is thought to be restricted to tetrapods [16]. Interestingly, in *Drosophila*, selective endosomal microautophagy (eMI), a recently identified form of microautophagy which shares together with CMA the dependence on KFERQ-like motifs and HSPA8/HSC70 for substrate targeting, has been suggested to constitute an alternative to CMA [16,17]. Although it is tempting to speculate that in non-tetrapod species eMI might be an ancestral form of selective autophagy for the degradation of substrates that in tetrapods are shared between eMI and CMA, our data reveal that the picture is probably much more complex.

Indeed, homology-based searches on in house-developed RNA-seq databases (PhyloFish), providing consistent and exhaustive gene expression data from 23 different ray-finned fish species [18], resulted in the identification of several contigs displaying high sequence homology with mammalian *LAMP2A*. In detail, the inferred amino acid sequences of the fish Lamp2A contigs not only displayed high homology when compared to the transmembrane and cytoplasmic domains of mammalian LAMP2A, but also high conservation of key motifs shown to be essential for proper function of the protein (Figure 1A). Hence, fish Lamp2A sequences display the typical GY dipeptide conserved in all forms of LAMP2 and required for targeting to lysosomes [19]. Fish Lamp2A present also the hydrophobic phenylalanine (F) residue (not conserved in the other forms of Lamp2) important for lysosomal targeting [20]. Additionally, 3 of the 4 positively charged amino acids necessary for binding substrate proteins [15] are also present in fish sequences, as are the 2 glycine residues (G) involved in the multimeric pattern of Lamp2A [13], at least in some fish species (Figure 1A).

Furthermore, sequence analysis of the complete genomes of several fish species also revealed high conservation of the genomic organization of the *LAMP2* gene across vertebrates
with notably the presence of the 3 alternative exons (B, A and C) encoding the
transmembrane domain and cytoplasmic tail specific of each isoform (LAMP2B, LAMP2A
and LAMP2C, respectively) (Figure 1B). However, the number and size of exons encoding
the lumenal region are only moderately conserved among species (Figure 1B), leading to
lower homology of the corresponding region (from 15 to 20% between analyzed species)
compared to the cytoplasmic tail (from 21 to 85%) (Table S1), thus accounting for different
structure (Figure 1A) and possibly functional variations of Lamp2A among species.

Finally, fine expression analysis of the newly characterized *lamp2a* splice variants in different fish species shows that they are expressed in different tissues of a large variety of ray-finned fish species, including the medaka (*Oryzias latipes*), a model species widely used in biomedical research (Figure 1C). Although no *lamp2a* transcripts could be identified in zebrafish (*Danio rerio*), RT-PCR analysis using specific primers targeting a conserved region of the *lamp2a* exon from fish nevertheless revealed significant zebrafish *lamp2a* expression in different tissues, including intestine, kidney and liver (data not shown).

Overall, our data show for the first time the existence as well as the expression of *lamp2a* transcripts in different tissues of a large variety of ray-finned fish species, and therefore imply that CMA function might have appeared much earlier during evolution than initially thought. A number of issues remain now to be addressed about the functionality of the Lamp2A sequences found in fish. This includes (i) whether or not in fish *lamp2a* mRNA can be successfully translated to protein, (ii) whether or not these Lamp2A proteins properly localize to lysosomes, and (iii) whether or not the polymerized fish Lamp2A regulates the relocation of the substrates. Further understanding of the structure-function relationship between fish Lamp2A displaying differences in key motifs will also help to complete this picture. The presence of Lamp2A outside of the tetrapod clades opens up new perspectives in autophagy research. Comparative approaches across phylogenetic distant species (fish versus
tetrapods for instance) will certainly provide new insights on selective autophagy by
exploring the extent to which CMA, but also the crosstalk between CMA and other
components of the cellular proteostasis networks (in particular eMI), diverged during
vertebrate evolution.

<figure><img src="image_94.png" /></figure>
ACKNOWLEDGEMENTS

This study was supported by the INRA "Animal Physiology and Livestock Systems" Division, and the French National Research Agency (ANR-17-CE20-0033 "Fish-and-Chap").

We thank AM Cuervo (Albert Einstein College of Medicine, NY) for helpful advice.
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FIGURE LEGEND

<caption>**Figure 1. Protein Structure, gene organization and mRNA expression of Lamp2A in fish**</caption>

(A) Schematic drawing of the structure of vertebrate LAMP2A. The human LAMP2A is used as reference. It shows a lumenal region comprising 2 N-glycosylated LAMP domains of approximately 160 residues (each with 2 disulfide bonds, S-S) separated by a proline-rich, O-glycosylated 'hinge' region of approximately 30 amino acid residues [21]. O- and N-linked glycosylation are indicated in green and red, respectively. The transmembrane (TM) domain, harboring 2 glycine residues (red G) involved in the multimeric pattern of LAMP2A, is followed by a short, C-terminal cytosolic tail that is comprised of 4 positively-charged amino acids (KHHH in blue) required for the binding of substrate proteins as well as motifs for lysosomal targeting (in green). The schematic drawing of fish Lamp2A has been done on the basis of sequence complementarity with the human LAMP2A. Potential O- and N-linked glycosylation are indicated in light green and pink, respectively. Sequence alignment of the boxed region of the 3 LAMP2/Lamp2 variants is shown below. The positively-charged amino acids required for the binding of substrate proteins are colored in blue. The GY dipeptide as well as the hydrophobic F required for targeting of LAMP2A to lysosomes are in green. The glycine residues (G) involved in the multimeric pattern of LAMP2A are in red. (B) The genomic structure of *LAMP2/lamp2* is conserved in vertebrates and contains the 3 alternative exons (B, A and C) encoding the transmembrane domain and cytoplasmic tail specific for each isoform. The size of exons (in base pairs) is shown in italics below or above each exon.

(C) Data from RNAseq show that *lamp2a* is expressed in different tissues of a large number of ray-finned fish. Relative expression of *lamp2a* was expressed in number of reads per kilobase per million reads per species, after normalization of data by the total number of sequences obtained for each tissue and species. The obtained values were then log
transformed and centered to the median (set at 0.00). Ac, *Amia calva* (bowfin); Lo, *Lepisosteus oculatus* (spotted gar); Aa, *Anguilla anguilla* (European eel); Gp, *Gnathonemus petersi* (elephantnose fish); Aal, *Alosa alosa* (allis shad); Ph, *Pangasianodon hypophthalmus* (striped Catfish); Am, *Astyanax mexicanus* (cave Mexican tetra); El, *Esox lucius* (northern pike); Up, *Umbra pygmae* (eastern mudminnow); Tt, *Thymallus thymallus* (grayling); Cl, *Coregonus lavaretus* (European whitefish); Cc, *Coregonus clupeaformis* (American whitefish); St, *Salmo trutta* (brown trout); Om, *Oncorhynchus mykiss* (rainbow trout); Sf, *Salvelinus fontinalis* (brook trout); Gm, *Gadus morhua* (Atlantic cod); *Ol*, *Oryzias latipes* (medaka).

<figure><img src="image_95.png" /></figure>
<figure><img src="image_96.png" /><figcaption>Figure 1</figcaption></figure>
<table><caption>table1</caption><thead><tr><th></th><th>H. sapiens</th><th>L. oculatus</th><th>O. latipes</th><th>D. rerio</th></tr></thead><tbody><tr><td><strong>Lumenal region</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>H. sapiens</td><td></td><td></td><td></td><td></td></tr><tr><td>L. oculatus</td><td>17.89</td><td></td><td></td><td></td></tr><tr><td>O. latipes</td><td>15.24</td><td>19.76</td><td></td><td></td></tr><tr><td>D. rerio</td><td>17.03</td><td>19.21</td><td>15.24</td><td></td></tr><tr><td><strong>Cytoplasmic tail</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>H. sapiens</td><td></td><td></td><td></td><td></td></tr><tr><td>L. oculatus</td><td>46.15</td><td></td><td></td><td></td></tr><tr><td>O. latipes</td><td>38.46</td><td>84.62</td><td></td><td></td></tr><tr><td>D. rerio</td><td>21.43</td><td>64.29</td><td>71.43</td><td></td></tr></tbody></table>
# The unusual rainbow trout sex determination gene hijacked the canonical vertebrate gonadal differentiation pathway

Sylvain Bertho<sup>a,b</sup>, Amaury Herpin<sup>b</sup>, Adèle Branthonne<sup>b</sup>, Elodie Jouanno<sup>b</sup>, Ayaka Yano<sup>b</sup>, Barbara Nicol<sup>b,1</sup>, Thomas Muller<sup>c</sup>, Maëlle Pannetier<sup>d</sup>, Eric Pailhoux<sup>d</sup>, Misako Miwa<sup>e</sup>, Goro Yoshizaki<sup>e</sup>, Manfred Schartl<sup>a,f,g,h,2,3</sup>, and Yann Guiguen<sup>b,2,3</sup>

<sup>a</sup>Physiological Chemistry, Biocenter, University of Wuerzburg, D-97074 Wuerzburg, Germany; <sup>b</sup>Institut National de la Recherche Agronomique, UR1037 Fish Physiology and Genomics, F-35000 Rennes, France; <sup>c</sup>Julius-von-Sachs-Institute, Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, D-97082 Wuerzburg, Germany; <sup>d</sup>UMR Biologie du Développement et Reproduction, Institut National de la Recherche Agronomique, L'École Nationale Vétérinaire d'Alfort, Université Paris Saclay, F-78350 Jouy en Josas, France; <sup>e</sup>Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 108-8477 Tokyo, Japan; <sup>f</sup>Comprehensive Cancer Center Mainfranken, University Hospital, D-97080 Wuerzburg, Germany; <sup>g</sup>Hagler Institute for Advanced Study, Texas A&M University, College Station, TX 77843; and <sup>h</sup>Department of Biology, Texas A&M University, College Station, TX 77843

Edited by Catherine L. Peichel, University of Bern, Bern, Switzerland, and accepted by Editorial Board Member Hopi E. Hoekstra October 23, 2018 (received for review March 5, 2018)

Evolutionary novelties require rewiring of transcriptional networks and/or the evolution of new gene functions. Sex determination (SD), one of the most plastic evolutionary processes, requires such novelties. Studies on the evolution of vertebrate SD revealed that new master SD genes are generally recruited from genes involved in the downstream SD regulatory genetic network. Only a single exception to this rule is currently known in vertebrates: the intriguing case of the salmonid master SD gene (sdY), which arose from duplication of an immune-related gene. This exception immediately posed the question of how a gene outside from the classical sex differentiation cascade could acquire its function as a male SD gene. Here we show that SdY became integrated in the classical vertebrate sex differentiation cascade by interacting with the Forkhead box domain of the female-determining transcription factor, Foxl2. In the presence of Foxl2, SdY is translocated to the nucleus where the SdY:Foxl2 complex prevents activation of the aromatase (cyp19a1a) promoter in cooperation with Nr5a1 (Sf1). Hence, by blocking a positive loop of regulation needed for the synthesis of estrogens in the early differentiating gonad, SdY disrupts a preset female differentiation pathway, consequently allowing testicular differentiation to proceed. These results also suggest that the evolution of unusual vertebrate master sex determination genes recruited from outside the classical pathway like sdY is strongly constrained by their ability to interact with the canonical gonadal differentiation pathway.

sex determination | Forkhead box proteins | sex differentiation |
fish | evolution

Sexual development is a fundamental process that shapes animal morphology, physiology, and behavior. The development of the undifferentiated embryonic gonad toward a testis or an ovary is regulated by a complex network of genes, where the initial triggers for male or female sex differentiation can come from the environment or the genome (1). A great number of studies revealed that the chromosomal, molecular, and cellular mechanisms of genetic sex determination (SD) are highly variable (1–4). It is now particularly clear that SD mechanisms evolved frequently and independently, leading to a high turnover of the genes governing sexual development (1, 5), even between closely related organisms. For instance, the therian sex-determining gene SRY is not found in other vertebrates (6), and recent studies have identified many different master SD genes in birds, amphibians, and fish (7–14). In these species, known members of the downstream regulatory sex differentiation network usurped the position at the top of the sex determination cascade to become the master SD gene. However, not all downstream sex differentiation genes are equally able to take the lead as sexual master switches, and currently, only the

genes encoding transcription factors Sox3 and Dmrt1 and several components of TGF-β signaling have been identified as master SD genes in vertebrates. This frequent reuse of the same SD genes led to the hypothesis that there are limited options in becoming a master sex-determining gene, which can be met by only a very limited number of genes from the sex differentiation network. However, this “limited option” hypothesis (15) was challenged by the discovery of the unusual salmonid sex-determining gene (16, 17). This gene, called sdY for “sexually dimorphic on the Y,” turned out to be a duplicated and truncated version of a gene encoding IFN regulatory factor 9 (irf9), which functions in the immune response of vertebrates. Upon IFN binding to its receptor and activation of STAT signaling, IRF9 complexes with both STAT1 and STAT2 in the cytoplasm and then translocates to the nucleus to activate effector genes of the antiviral response through its DNA-binding domain (18). IRF9 nonimmunity

## Significance

Sex determination is one of the most fundamental but also extraordinary plastic processes in nature. Many different master sex-determining genes have been characterized in vertebrates, and most of them are known to fulfill essential functions during sexual development and thus are already tightly linked to the process that they now govern. Only one exception is currently known: the salmonid master sex-determining gene (sdY), which arose from the duplication of an immune-related gene. Here we show that SdY prevents female differentiation by interacting and blocking the action of a key ovarian differentiation factor. These results suggest that the evolution of unusual vertebrate master sex determination genes is strongly constrained by their ability to interact with the canonical gonadal differentiation pathway.

Author contributions: M.S. and Y.G. designed research; S.B., A.H., A.B., E.J., A.Y., B.N., T.M., M.P., E.P., M.M., G.Y., M.S., and Y.G. performed research; M.P. and E.P. contributed new reagents/analytic tools; S.B., A.H., A.B., E.J., A.Y., B.N., T.M., E.P., M.M., G.Y., M.S., and Y.G. analyzed data; and S.B., A.H., M.S., and Y.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. C.L.P. is a guest editor invited by the Editorial Board.

Published under the PNAS license.

¹Present address: Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC 27709.

²M.S. and Y.G. contributed equally to this work.

³To whom correspondence may be addressed. Email: phch1@biozentrum.uni-wuerzburg.de or yann.guiguen@inra.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1803826115/-/DC Supplemental.
roles have been described in neurons and liver and heart patho-
physiology (19), but so far no evidence implicates it in sex de-
termination or sex differentiation processes. The birth of such a
master SD gene recruited from outside of the classical sex differ-
entiation cascade raises the intriguing question of its functional
evolution and how this unusual SD gene determines sex. Did SdY
evolve a new function to be able to interact directly with the classical
gonadal sex differentiation cascade, or does it use part of its ancestral
pathway, that is, the IFN immune-related response, for its action?

Results and Discussion

During evolution of SdY, the DNA-binding domain and the nu-
clear localization signals of Irf9 were lost, while the protein–protein
interaction domain (IRF association domain, or IAD) was pre-
served and underwent some sequence diversification (16). To test
the hypothesis that the IAD domain of SdY still functions in pro-
tein binding, we first performed molecular modeling and found that
the 3D structure of SdY strongly overlaps the IAD domain of IRF
proteins (Fig. 1A). As the IAD domain is the only domain of known
function predicted from the primary sequence of the *sdY* gene (16,
17), we hypothesized that SdY could still exert its function based on
protein–protein interactions. We thus searched for SdY inter-
acting proteins using a yeast two-hybrid (Y2H) screen with SdY
used as bait and with a rainbow trout prey cDNA library prepared
from late differentiating testes sampled when *sdY* expression is still
high (16). Among the 46 different putative interacting proteins there
were none of the known Irf9 partners like Stat1 or Stat2. Instead, we
found a very strong enrichment of many members of the Forkhead
box (FOX) family (11 FOX proteins, *SI Appendix*, Tables S1–S3).
The Forkhead box, a highly conserved DNA-binding domain (20)
common to all FOX proteins, was identified as the minimum do-
main needed for an effective interaction with SdY (Fig. 1B and *SI
Appendix*, Table S3). Interestingly, among all of the FOX proteins
interacting with SdY in yeast, we found the well-known female sex
differentiation protein Foxl2 (21, 22). Taking into account the
importance of Foxl2 in vertebrate sex differentiation, we reasoned
that this would be an interesting and biologically relevant SdY
partner. We then explored the interaction of SdY with trout Foxl2
in a direct yeast interaction assay and confirmed that SdY and
Foxl2 can interact together (*SI Appendix*, Fig. S1).

To better characterize this interaction, an in vitro approach
was developed using cell transfection assays. In HEK 293T cells

transfected only with *sdY* plasmid, SdY protein was localized
predominantly in the cytoplasm (Fig. 2 *A–A''* and *SI Appendix*,
Fig. S2). However, when cotransfected along with Foxl2, SdY
was completely translocated into the nucleus (Fig. 2 *B–B''* and *SI
Appendix*, Fig. S2). Such a complete SdY nuclear translocation
was observed only with fish Foxl2 proteins (Fig. 2H), including
the two rainbow trout paralogous gene products (Foxl2b1 and
Foxl2b2) resulting from the salmonid whole-genome duplication
(23) (Fig. 2 *I–O*) and the medaka, *Oryzias latipes*, Foxl2 (*SI Ap-
pendix*, Figs. S3 and S4). No complete nuclear translocation was
observed with some other rainbow trout Fox proteins (Fig. 2 *C–H*
and *SI Appendix*, Fig. S2), with some mammalian Foxl2, that is,
mouse and goat (*SI Appendix*, Fig. S3), and with the rainbow trout
Foxl2b2 containing a modified mouselike Forkhead box domain
(*SI Appendix*, Fig. S4). This complete nuclear relocalization of
SdY with trout and medaka Foxl2s indicated some specific protein–
protein interaction and that this interaction required the con-
formation of a fish Forkhead domain. This interaction was also
confirmed in vitro by co-immunoprecipitation experiments (Fig.
2 *P* and *Q* and *SI Appendix*, Fig. S5A) and in vivo by showing that
SdY was also translocated into the nucleus following coinjection
with Foxl2 in medaka embryos (*SI Appendix*, Fig. S5 *B* and *C*).

To obtain further insights into the physiological relevance of
the SdY and Foxl2 interaction in vivo, a gene expression time
course of *foxl2*, *nr5a1*, and *sdY* genes in differentiating trout
gonads was performed. In agreement with its male-determining
role, *sdY* expression was detected only in male gonads, with a
peak of expression around 45 d postfertilization (dpf) (Fig. 3A).
In contrast, *foxl2b1*, *foxl2b2*, and *nr5a1* were not expressed in a
sexually dimorphic fashion before the time point at which *sdY*
peaks in males; after this time point *foxl2b1* and *foxl2b2* are
markedly up-regulated in females and down-regulated in males
(Fig. 3 *A* and *B* and *SI Appendix*, Fig. S6). We also explored
expression of gonadal aromatase (*cyp19a1a*), as this gene is a
well-known direct target of Foxl2 (24). We found that *cyp19a1a*
is expressed only in female gonads and its expression parallels
the expression of the trout *foxl2* genes (Fig. 3A). These *cyp19a1a*,
*nr5a1*, and *foxl2* expression patterns are consistent with the
critical role of Foxl2 in the up-regulation of *Cyp19a1a/cyp19a1a*
(24, 25), in cooperation with Nr5a1 (steroidogenic factor 1, Sf1)
for driving ovarian differentiation (26). In addition, *sdY* and *foxl2*
are colocalized in some somatic cells of the early differentiating
gonad in the male rainbow trout (Fig. 3C), suggesting that they
could interact to modulate *cyp19a1a* expression. Interestingly,
trout *foxl2* genes are also strongly and positively regulated by
estrogens (Fig. 3B), the steroid end products of the aromatase
enzyme (21). This points to a positive regulatory loop with Foxl2
inducing *cyp19a1a* expression and thus increasing estrogen syn-
thesis that will, in return, stimulate the expression of *foxl2* (21).
Taking into account the pivotal role of Cyp19a1a and estrogens
in fish ovarian differentiation (27) and our results on a specific
interaction of SdY with Foxl2, we proposed that SdY exerts its
sex-determining function by suppressing this positive regulatory
loop through its interaction with Foxl2. To evaluate this, we first
confirmed, using a luciferase reporter assay, that activation of
the medaka *cyp19a1a* promoter requires the presence of both
Foxl2 and Nr5a1, which work in synergy to activate *cyp19a1a*
(25) (Fig. 4A). We then tested the effect of SdY on the tran-
scriptional activity of the *cyp19a1a* promoter and demonstrated
that SdY strongly represses the synergistic Foxl2- and Nr5a1-
induced *cyp19a1a* expression (Fig. 4B) but not the Foxl2-alone-
or Nr5a1-alone-induced *cyp19a1a* expression (Fig. 4C).

In our attempt to understand how *irf9*, a transcriptional regulator
of the immune system, has evolved into a master SD gene whose
expression is necessary and sufficient to drive testicular differenti-
ation (16), we obtained multiple sets of evidence supporting the
hypothesis that SdY exerts its sex-determining function by inter-
acting with the female-determining factor Foxl2. An interaction of
SdY with Foxl2 was not anticipated, but some Forkhead box
proteins such as the FOXO proteins have been shown to have direct
protein-protein interactions with a variety of unrelated transcription

<figure><img src="image_97.png" /><figcaption>**Fig. 1.** SdY conserves the structure of the IRF protein–protein interaction domain and interacts with the Forkhead box domain of Fox proteins. (A) SdY shares structural homologies with IAD, a protein–protein interaction domain. The structure of SdY (in gray) was modeled using the crystal structure of IRF5 as a template (in green). This SdY structure reveals eight β-sheets forming a β-sandwich and three α-helices that are highly conserved with IRF5. (B) SdY interacts in yeast with Fox proteins through their highly conserved DNA-binding domain. The alignments of the SdY-Fox interacting clone sequences (gray lines) delineate the minimum domain or selected interacting domain needed for an effective interaction with SdY in yeast, which is the Forkhead box domain (110 aa, black lines). The 11 Fox proteins characterized in the Y2H screen are represented by open cylinders with numbers of interacting clones indicated on the right side.</figcaption></figure>
**Fig. 2.** SdY interacts with Foxl2, resulting in its nuclear translocation. (A–H) GFP:SdY alone (A–A") and GFP:SdY in combination with different trout Fox proteins, Foxl2 (Foxl2b1) (B–B"), Foxd2 (C–C"), Foxd3 (D–D"), Foxn2 (E–E"), Foxn3 (F–F"), Foxo3 (G–G"), were cotransfected in HEK 293T cells (delimited by white dotted lines). GFP:SdY is translocated in the nucleus (delimited by yellow dotted lines and stained in blue with Hoechst staining) only in the presence of Foxl2 (B–B"). (Scale bar, 5 µm.) (H) Percentage of transfected cells (mean ± standard deviation on 200 cells) in which SdY is completely translocated in the nucleus after three independent cotransfection experiments with different trout Fox proteins. Significant differences compared with SdY alone were calculated using an unpaired two-tailed Student's t test. ***P < 0.001; ns, nonsignificant. (I–O) Foxl2b1 and Foxl2b2 are both able to drive SdY complete nuclear translocation (delimited by yellow dotted lines and stained in blue with Hoechst staining). Confocal images of HEK 293T cells (delimited by white dotted lines) transiently transfected with SdY (I–I"), mCherry:Foxl2b1 alone (J–J"), SdY and mCherry:Foxl2b1 (K–K"), mCherry:Foxl2b2 alone (L–L"), SdY and mCherry:Foxl2b2 (M–M"). (Scale bar, 10 µm.) (N) Quantitative analysis in the presence or absence of Foxl2b1 and Foxl2b2. Percentage of complete SdY nuclear translocation (mean ± standard deviation on 100 cells) after three independent cotransfection experiments. Statistical significances compared with SdY alone were calculated using an unpaired two-tailed Student's t test. (O) SdY colocalizes with Foxl2 in the nucleus. SdY, SdY-Foxl2b1, and SdY-Foxl2b2 colocalizations were measured in the nucleus for SdY (n = 5), SdY and Foxl2b1 (n = 5), and SdY and Foxl2b2 (n = 5) with Pearson's correlation. Statistical significance was calculated using an unpaired two-tailed Student's t test, ***P < 0.001. (P and Q) SdY binds with Foxl2 in co-immunoprecipitation (IP) experiments. HEK 293T cells were transiently transfected with expression plasmids for SdY fused to a hemagglutinin tag (3xHA:SdY) and for Foxl2 fused to a 3xFlag tag (3xFlag:Foxl2b1 or 3xFlag:Foxl2b2). Whole-cell lysates were used for immunoprecipitation with anti-Flag or anti-Foxl2 (P) and with anti-HA or anti-SdY (Q) followed by immunoblotting with the appropriate antibodies. Input represents 10% whole-cell lysate. IgG mouse antibody was used as the control. In P, 3xFlag:Foxl2b1 or 3xFlag:Foxl2b2 was immunoprecipitated with either Flag (Top) or Foxl2 (Bottom) antibodies followed by immunoblotting with an antibody against the HA tag to reveal the interaction with 3xHA:SdY (SdY). In Q, 3xHA:SdY was immunoprecipitated with an HA or SdY antibody, followed by immunoblotting with an antibody against the Flag tag to reveal 3xFlag:Foxl2b1 (Foxl2b1) (Top) or 3xFlag:Foxl2b2 (Foxl2b2) (Bottom).

<figure><img src="image_98.png" /></figure>

<figure><img src="image_99.png" /></figure>

factors (28). In zebrafish the Foxo3b protein is even able to repress the transcriptional activity of *irf* genes by a direct IRF/FOX protein-protein interaction (29). This may suggest that SdY acquired its ability to specifically bind to fish Foxl2 proteins based on a preexisting possibility of interaction of Irf9 with the FOX protein.

The specific interaction of SdY with Foxl2 carries SdY into the nucleus, where it can prevent, by blocking the synergistic action of Nr5a1 (Sf1) and Foxl2, the implementation of a positive regulatory loop controlling the expression of *cyp19a1a*. By doing so very early in the differentiation process of the gonads, i.e., long before implementation of the *cyp19a1a* loop of regulation in females (Fig. 3A), SdY would completely prevent estrogen production in the differentiating male gonads. The absence of estrogen production subsequently triggers masculinization as it has been demonstrated in many fish species, including rainbow trout, using treatments with an aromatase inhibitor (30) or, more recently, by direct inactivation of the *cyp19a1a* gene (26, 31, 32). Such masculinization following the blockade of estrogen production is even effective in adult females (27), showing that estrogens in fish are needed not only for ovarian differentiation but also for ovarian maintenance. However, the fact that *foxl2b1* and *foxl2b2* gene expression is not down-regulated in the male gonad before 45–50 dpf suggests that the inhibition of the positive regulatory loop between *cyp19a1a* expression, estrogen production, and *foxl2* gene expression is not active at these early testicular differentiation stages. This absence of inhibition of *foxl2* expression in the early male differentiating gonad could suggest that there is additional regulation of this positive loop or that expression of *foxl2* is not sensitive to estrogen at these early developmental

stages. Such a mechanism of action through the blockage of *cyp19a1a* and estrogen production assigns to SdY an activity as an antiovarian determining factor directly preventing the ovarian differentiation pathway instead of activating the male pathway. However, it cannot be totally excluded that SdY, besides suppressing the female pathway, may also affect directly the activation of the male pathway. Nevertheless, known important male developmental actors such as Dmrt1, Amh, and Sox9 (1) were not identified in the Y2H screen, and this along with their late expression during rainbow trout male gonad development (33) compared with *sdY* expression suggests that they are not direct interacting partners of SdY.

In summary, we provide strong evidence that SdY determines sex in rainbow trout not by using part of its ancestral Irf9 pathway but by directly interacting with Foxl2, an important member of the classical gonadal sex differentiation cascade. This suggests that innovation at the top of the vertebrate sex determination cascade may be constrained because novel master SD genes have to cope with the regulation of the conserved vertebrate sex differentiation cascade. The “limited option” hypothesis is mainly based on the idea that only a small subset of genes and chromosomes, because they are better at doing the job, would be independently and repeatedly selected as new vertebrate master SD genes (15). We now propose that the limited option is actually more constrained by the conservation of the sex differentiation pathway and that evolution of SD genes may include some innovations like SdY if these unusual SD genes evolved a means to build an interface with the sex differentiation cascade.
<figure><img src="image_100.png" /><figcaption>Fig. 3. The gonadal expression patterns of *sdY*, *foxl2*, *nr5a1*, and *cyp19a1a* (A and B) are in agreement with a repressive effect of SdY on the Foxl2 positive regulation of the aromatase promoter (C). (A) Gene expression profiles of *sdY*, *nr5a1*, *foxl2b2*, and *cyp19a1a* in male and female gonads from 33 to 125 dpf. All values represent the mean ± standard deviation of three biological replicates (in percentage of the highest measured value). The gray area highlights the period before the sexually dimorphic expression of trout *foxl2* genes during gonadal differentiation. (B) Gonadal localization of *foxl2b2* transcripts (NBT/BCIP signal in blue) in male and female gonads. *foxl2b2* is expressed in somatic cells of both female and male gonads at 50 dpf and only in female gonads at later stages (60, 70, and 85 dpf). In males fed with estrogens (male E2) *foxl2b2* is strongly up-regulated compared with control males quickly after (60 dpf) the application of the treatment (55 dpf). (Scale bar, 20 µm.) (C) Colocalization by double in situ hybridization of *sdY* (NBT/BCIP signal in blue) and *foxl2b2* (HNPP/Fast Red signal in red fluorescence) in somatic cells of a rainbow trout male differentiating gonad at 50 dpf. Cell nuclei are shown in the dark-field panels stained with DAPI either with or without the HNPP fluorescent detection of *foxl2b2*. Germ cells are shown by an asterisk. (Scale bar, 20 µm.)</figcaption></figure>

## Materials and Methods

**Protein Structure Prediction.** Three-dimensional homology modeling of SdY was predicted with the software SWISS-MODEL (https://swissmodel.expasy.org/) using the structure of the dimeric IRF5 (PDB ID 3DSH) transactivation domain (34) as a template. The resulting model was obtained by the superposition of the template and SdY. The 3D views of SdY were made with PyMOL (molecular graphics system, version 1.7.4; Schrödinger).

**Yeast Two-Hybrid Screen.** Yeast two-hybrid screening was performed by Hybrigenics Services (https://www.hybrigenics-services.com). The coding sequence for SdY (amino acids 1–215) (GenBank accession number GI:392583258) was PCR amplified and cloned into pB27_A as a C-terminal fusion to LexA (N-LexA-SdY-C) and into pB66_A as a C-terminal fusion to the Gal4 DNA-binding domain (N-Gal4-SdY-C). These constructs were checked by sequencing and used as baits to screen a random-primed *Oncorhynchus mykiss* immature male gonad (gonads sampled around 75 dpf) cDNA library; 112 million interactions were tested with pB27_SdY, and 71.3 million interactions were tested with p66_SdY, leading to the detection of 24 and 178 processed clones, respectively. The prey fragments of these 202 positive clones were amplified by PCR and sequenced at their 5' and 3' junctions. Each fragment corresponding to an interacting protein was identified using GenBank release 192 (National Center for Biotechnology Information). The common sequence shared by all prey fragments of the same protein defines the selected interacting domain containing all of the structural determinants required for a given interaction to occur. A confidence score [predicted biological score (PBS)] that outlines the reliability of the interaction is given to each interaction as previously described (35). PBS scores were divided into four categories, from A (highest confidence) to D (lowest confidence).

**Genomic DNA Extraction.** To clone rainbow trout *foxl2b1* and *foxl2b2*, genomic DNA was extracted from liver tissue with lysis buffer (10 mM Tris-HCl, 100 mM EDTA, 0.5% SDS, and 20 µg/mL RNase A). Proteinase K was added to 150 mg/mL, and the sample was incubated at 56 °C overnight. A double-extraction phenol-chloroform (1:1) followed by chloroform-isoamyl alcohol

(24:1) extraction was done. DNA precipitation was performed with an equal volume of isopropanol (1:1). The precipitate was pelleted by centrifugation at 16,000 × g and washed twice in 70% ethanol, dried at room temperature, and dissolved in 2 mL of distilled water.

**Cloning.** Plasmids and primers used are listed in the SI Appendix, Tables S4 and S5. The coding sequence of SdY was amplified from the psdy:sdy-prcy: cfp plasmid (16) and inserted into pCS2⁺. From this plasmid, the PCR-amplified fragment was inserted into pCS2⁺:HA:mCherry (gift from Manfred Gessler, University of Wuerzburg, Wuerzburg, Germany), pCS2⁺-emGFP, and pCS2⁺-3xHA expression plasmids. Rainbow trout *foxl2b1* and *foxl2b2* and medaka *foxl2* (*Ol-foxl2*) were cloned by PCR amplification on genomic DNA and inserted in pCS2⁺ plasmid, pCS2⁺:HA:mCherry, and pCS2⁺:3xFlag between the EcoRI-Xhol restriction sites. The constructs 3xHA-pCS2⁺ and 3xFlag-pCS2⁺ were obtained by concatemerization of three single HA sequences (3xHA) or three single FLAG sequences (3xFLAG) flanked by HindIII restrictions sites. pCS2⁺-emGFP:SdY was obtained by inserting a PCR-amplified fragment corresponding to emGFP in frame into the EcoRI site. To explore the hypothesis that SdY could be able to interact with all Fox proteins through an interaction with their highly conserved Forkhead domain, we selected different rainbow trout Fox proteins from an EST resource collection in which ESTs were cloned into a CMV expression (pCMV-Sport6) plasmid (36). Five trout cDNA clones encoding for Foxd2, Foxd3, Foxl3, Foxn3, and Foxn2 were found to have a complete open coding frame, including Foxn3 and Foxn2, which were identified as SdY partners in the Y2H screen. The rainbow trout Foxl2b2 sequence with threonine 64 and threonine 79 replaced by adenines (T64A; T79A) was synthesized (Genscript) by replacing in the *foxl2b2* cDNA sequence the threonine codon sequence ACC or ACT with an adenine codon sequence GCC at amino acid positions 64 and 79, respectively. The corresponding sequence was inserted in a pcDNA3.1 plasmid between the EcoRI and Xhol restriction enzyme sites and checked by sequencing.

**Cell Culture.** HEK 293T cells were cultured and maintained in DMEM (PAN Biotech), supplemented with 10% FCS (PAN Biotech) and 1% penicillin-streptomycin (PAN Biotech) at 37 °C with 5% CO₂. Transfections for HEK 293T cells were performed by incubating cells with polyethylenimine (PEI) (100 mg/mL PEI diluted 1:100 in 150 mM NaCl) and respective plasmids (10 µg for 10-cm dishes, 2 µg for six-well plates) for 6–8 h in fresh medium. Then, the medium was discarded, and fresh medium was added.

Rainbow trout gonadal (RTG2) cells were cultured and maintained in L15 medium, 20 mM glutamine (PAN Biotech), supplemented with 10% FCS (PAN Biotech) and 1% penicillin-streptomycin (PAN Biotech), at 20 °C in an atmosphere of air. For transfection, RTG2 cells were detached by Trypsin-EDTA (P0781; Sigma-Aldrich) and pelleted by centrifugation at 1,000 × g for 5 min, washed once with medium and once with PBS. The pellet was drained and resuspended in solution V (Amaxa Kit) at a density of 10⁶ cells/mL; 2 µg of plasmid were added to the suspension. After mixing, the suspension was transferred to a cuvette (Kit V; Amaxa). Program D-23 was used to electroporate the cells. After transfection, cells were immediately transferred to six-well plates filled with medium. All experiments were performed 72 h after transfection.

**Immunofluorescence.** HEK 293T cells were seeded in a 6-well plate containing coverslips. After pCS2⁺-meGFP-SdY and pCS2⁺-mCherry-Foxl2b1 (or pCS2⁺-3xFLAG:Foxl2b2 or pCS2⁺-3xFLAG-Ol-Foxl2) cotransfection for 48 h, cells were fixed in 4% fresh paraformaldehyde for 15 min, extensively washed, and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Then cells were blocked with 1% BSA for 20 min. The primary antibody (SI Appendix, Table S6) was incubated overnight at 4 °C. After extensive washing with PBS, cells were incubated with Alexa 488 or Alexa 594 conjugated secondary antibodies in 1% BSA for 1 h, followed by Hoechst 33342 (Invitrogen) staining for 5 min (1 µg/mL final concentration). Cells were mounted using Mowiol 4-88 (Roth). Confocal images were acquired using a Nikon Eclipse C1 laser-scanning microscope (Nikon), fitted with a 60× Nikon objective (PL APO, 1.4 N.A.), and Nikon image software. Images were collected at 1,024 × 1,024 pixel resolution. The stained cells were optically sectioned in the z axis. The step size in the z axis varied from 0.2 to 0.25 mm to obtain 50 slices per imaged file. All experiments were independently repeated at least three times.

**Colocalization Analyses.** The Nikon NIS-Elements imaging analysis software was used for the colocalization analyses. Confocal images of double-stained sections were first subjected to background correction. SdY nuclear translocation was counted as complete translocation when the majority of the GFP:SdY signal was found in the nucleus. Pearson's correlation was calculated and used to obtain the colocalization values as percentages of SdY overlapping with Foxl2b1 or Foxl2b2 for a minimum of five cells (n = 5). The Pearson's coefficient values were defined as very strong colocalization
<figure><img src="image_101.png" /><figcaption>**Fig. 4.** SdY prevents the Foxl2/Nr5a1 positive regulation of the *cyp19a1a* promoter. Medaka *cyp19a1a* promoter activity (pGL3-cyp19a1a) promoter coupled to a firefly luciferase) was measured using a luciferase reporter assay after HEK 293T cell cotransfection with either medaka *nr5a1* and/or *foxl2* expression plasmids and variable concentrations of the rainbow trout *sdY* expression plasmid. All results were calculated as the mean ± SEM of three biological replicates in two independent experiments. (A) Foxl2 and Nr5a1 act in synergy to induce *cyp19a1a* expression. Medaka *cyp19a1a* luciferase assay with variable concentrations of *foxl2* (50–400 ng) and *nr5a1* (50–200 ng) or 100 ng of *nr5a1* with variable concentrations of *foxl2* (50–400 ng). Statistical significances of luciferase activity within treatments were tested using an unpaired two-tailed Student's t test. Effects of *foxl2* alone and *nr5a1* alone compared with their synergistic effect (shown by asterisks on the dotted lines joining the different groups) were tested by a one-way ANOVA with a post hoc Dunnett test. (B) SdY prevents Foxl2/Nr5a1 positive regulation of the *cyp19a1a* promoter. Medaka *cyp19a1a* luciferase assay with variable concentrations of *foxl2* (50–400 ng) combined with a fixed concentration of *nr5a1* (100 ng) and a fixed concentration of *foxl2* (200 ng) and *nr5a1* (100 ng) combined with variable concentrations of *sdY* (25–300 ng). Empty plasmid control (pGL3) and Foxl2 alone (200 ng) are depicted by a + sign. Statistical significances of luciferase activity were tested using a Mann-Whitney *U* test. (C) SdY does not repress *cyp19a1a* promoter expression induced by medaka Foxl2 alone or Nr5a1 alone. Medaka *cyp19a1a* luciferase assay with fixed *foxl2* (200 ng) or *nr5a1* (100 ng) concentrations combined with variable concentrations of *sdY* (25–300 ng). Statistical significances of luciferase activity within treatments were tested using an unpaired two-tailed Student's t test. The effect of *foxl2* or *nr5a1* alone compared with *foxl2* or *nr5a1* and variable concentrations of *sdY* was tested by a one-way ANOVA with post hoc Tukey tests.
***P < 0.001; **P < 0.01; *P* < 0.1; ns, *P* > 0.05 (nonsignificant).</figcaption></figure>

between 0.85 and 1, strong colocalization between 0.5 and 0.85, and weak or no colocalization between -1 and 0.5.

**Co-Immunoprecipitation.** HEK-293T cells were transfected with pCS2\*-3xHA: SdY and pCS2\*-3xFlag-Foxl2b1(or pCS2\*-3xFlag:Foxl2b2 or pCS2\*-3xFlag-Ol-

Foxl2) constructs to be assessed for their ability to co-immunoprecipitate. After 48 h cells were scraped and resuspended in 50 µL lysis buffer [20 mM Hepes (pH 7.8), 500 mM NaCl, 5 mM MgCl₂, 5 mM KCl, 0.1% deoxycholate, 0.5% Nonidet-P40, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 200 mM Na₃VO₄, 1 mM phenylmethanesulfonyl fluoride, and 100 mM NaF]. Cells were incubated in lysis buffer for 30 min at 4 °C and then cleared by high-speed centrifugation for 20 min. After Bradford protein concentration measurement, HNTG buffer (20 mM Hepes pH 7.5; 150 mM NaCl; 10% glycerol; 0.1% Triton X-100) was added (1:1) to 250 µg of the whole-cell lysate. After preclearing with IgG antibodies for 1 h at 4 °C, whole-cell lysates were used for immunoprecipitation with the corresponding antibodies. One microgram of anti-Flag, anti-HA, or IgG antibody was added to 500 µL of cell lysate or 5 µg of anti-SdY or anti-Foxl2 (SI Appendix, Table S6) and then incubated at 4 °C overnight. After the addition of washed protein G agarose beads (Pierce, 20398), incubation in HNTG buffer was continued for another 2 h. Immunoprecipitates were washed (five times with centrifugation at 1,000 × g, supernatant discarded, HNTG lysis buffer added) and eluted with SDS/PAGE loading buffer by boiling for 10 min. Co-immunoprecipitation was detected by standard Western blot analysis procedure.

**Western Blotting.** Cells were lysed in a Hepes-based lysis buffer [20 mM Hepes (pH 7.8), 500 mM NaCl, 5 mM MgCl₂, 5 mM KCl, 0.1% deoxycholate, 0.5% Nonidet-P40, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 200 mM Na₃VO₄, 1 mM phenylmethanesulfonyl fluoride, and 100 mM NaF] for 3 h. Cells debris was pelleted by centrifugation for 15 min at 16,000 × g. Cell lysate protein concentration was measured with a Bradford assay (Cary 50 Spectrophotometer; Varian). The protein lysates (30–50 µg) were resolved by SDS/PAGE on 12% Tris-glycine gels followed by transfer to nitrocellulose membranes. Unspecific binding was blocked with 5% BSA in Tris buffered saline with Tween-20 (TBST) [10 mM Tris (pH 7.9); 150 mM NaCl; 0.1% Tween] for 1 h at room temperature. Incubation with primary antibodies was performed overnight at 4 °C. After three washes with TBST, HRP conjugated antibodies were incubated with blocking solution for 1 h. Following the washes, membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) for 1 min. The signal from the membrane was detected using a Photo Image Station 4000MM (Kodak). At least two independent experiments were performed, and representative protein blot images are shown.

**Quantitative PCR.** Expression levels of *sdY*, *foxl2b1*, *foxl2b2*, *nr5a1*, and *cyp19a1a* were measured by qPCR as previously described (37). Gonads (15–20 pairs of gonads per time points) were sampled in triplicate at 33, 35, 37, 40, 44, 50, 61, 85, and 125 dpf in both genetic all-male (XY) and all-female (XX) populations of rainbow trout. Total RNA was extracted using the RNAqueous-Micro Kit (Ambion, Life Technologies) for the 33- to 50-dpf samples and the RNAqueous-Kit (Ambion, Life Technologies) for the 61- to 125-dpf samples. All samples were then treated with the TURBO DNA-free Kit (Ambion, Life Technologies) to remove any leftover genomic DNA. Reverse transcriptions were carried out using 150 ng of total RNA as the starting material with the Ovation RNA Amplification System (NuGEN Technologies), following the manufacturer's recommendations. Quantitative PCR was performed using the StepOnePlus system (Applied Biosystems) using 4 µL of reverse-transcribed cDNA (single tube quantification per sample, with three biological replicates for each sex and time point) diluted to 1:90, the Fast SYBR Green Master Mix (Applied Biosystem), and 600 nmol of each primer listed in SI Appendix, Table S7. The enzyme was activated for 20 s at 95 °C, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing and elongation at 60 °C for 30 s.

**Whole-Mount in Situ Hybridization.** In situ hybridization was performed as previously described (38). RNA probes were produced from PCR products obtained by amplification of *foxl2b2*. Ten nanograms of the PCR product were used as a template for digoxigenin-labeled RNA probe synthesis using digoxigenin 11-UTP (Roche Diagnostics Corp.) and T3 or T7 RNA polymerase (Promega) following standard protocols. Whole-mount in situ hybridization was carried out using an in situ Pro, Intavis AG robotic station. Male and female embryos were fixed overnight in 4% paraformaldehyde at 4 °C, dehydrated in 100% methanol, and stored at -20 °C. Before in situ hybridization they were rehydrated, permeabilized by proteinase K treatment (25 µg/mL, 30 min, at room temperature), and postfixed (4% paraformaldehyde and glutaraldehyde 0.2%, for 20 min). Prehybridization and hybridization media contained 50% formamide, 5XSSC, 0.1% Tween 20, 0.005% heparin, 0.1 mg/mL tRNA. Hybridization was carried out at 65 °C for 16 h. After post-hybridization washes, embryos were incubated in blocking buffer (PBS/Triton 0.1%/Tween 20 0.2%, containing 2% serum) for 2 h before the addition of the alkaline phosphatase coupled anti-digoxigenin antibody (1:2,000; Roche Diagnostics Corp.) for 6 h. After washing, the color reaction was performed in
the presence of nitro-blue tetrazolium/5-Bromo-4-chloro-3'-indolyl phosphate (NBT/BCIP) (Roche). Briefly, dehydration and paraffin infiltration were performed in a Citadel 1000 tissue processor (Shandon). Dehydrated tissues were embedded in plastic molds in paraffin using a HistoEmbedder (TBS88; Medite). Each embedded sample was sectioned 5 µm thick on a MICRO HM355 (Thermo Fisher Scientific).

**Colocalization of sdY and foxl2 by in Situ Hybridization.** Fifty-dpf male rainbow trout were fixed in Bouin's fixative at 4 °C. One hour after fixation and dehydration, the tissues were embedded in paraffin and cut into 4-µm sections. Antisense *sdY* and *foxl2b2* RNA probes were synthesized using in vitro transcription with a fluorescein RNA labeling mix (Roche) and a DIG RNA labeling mix (Roche), respectively. Sections were deparaffinized, rehydrated, and treated with 1% H₂O₂ in TBST buffer at room temperature for 30 min and 1 µg/mL proteinase K (Roche) at 37 °C for 13 min. After the enzymatic treatment, sections were dehydrated with ethanol and chloroform and then hybridized with *sdY* and *foxl2* probes simultaneously at 60 °C for 18 h. Fluorescein was visualized by using an anti-fluorescein-alkaline phosphatase (anti-fluorescein-AP) Fab fragment (Roche) (1:1,000) and the HNPP Fluorescent Detection Set (Roche). Digoxigenin (DIG) was visualized by using an anti-digoxigenin-AP Fab fragment (Roche) (1:500) and NBT/BCIP. Before the DIG visualization, alkaline phosphatase was inactivated in 0.1 M glycine/0.1% Tween 20 at room temperature for 30 min. DAPI staining was performed to visualize nuclei.

**Luciferase Assay.** HEK 293T cells were transfected using PEI with the following plasmids: 0.3 µg of pGL3-OlaCyp19a1a sequence (kindly provided by D. Wang Deshou, Key Laboratory of Aquatic Science of Chongqing, School of Life Sciences, Southwest University, Chongqing, China), 0.05-0.4 µg of pCS2⁺-SdY expression plasmid, 0.05-0.4 µg of pCS2⁺-OlaFoxl2, 0.1 µg of pcDNA3.1-OlaNrs1a1, and 0.1 µg of plasmid thymidine kinase-*Renilla* used for calibration. Each experiment was performed with a 1.0-µg final amount. Adjustments were made with empty plasmid (pCS2+) accordingly. Firefly luciferase and *Renilla* luciferase readings were obtained using the Dual-Luciferase Reporter Assay System (Promega) and LUMAT LB 9501 luminometer (Berthold Technologies).

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**One-by-one Yeast Two-Hybrid Assay.** Diploid cells containing the same bait construct of the yeast two-hybrid assay (p66_sdY) and a prey plasmid construct coding for Foxl2b2 cloned in frame with the activation domain of GAL4 (p14-N-GAL4-Foxl2b1-C) were mated and spotted on selective media. The medium lacking tryptophan and leucine was used as a control for the yeast growth test and to check for the presence of the bait or the prey. The assay is based on the histidine reporter gene. A triple-negative medium (tryptophan, leucine, and histidine) selects yeast growth if interaction occurs. Interaction pairs were tested at decreasing concentrations (10⁻¹, 10⁻², 10⁻³, 10⁻⁴) from two independent clones. An inhibitor of the histidine gene product 3-AT was used to increase stringency at four different concentrations (1, 5, 10, 50 mM).

**RNA Injections.** For injections capped RNA GFP-*sdY*, mCherry-*foxl2b2*, Olafoxl2 from pCS2⁺-meGFP-*sdY*, pCS2⁺-HA:mCherry-*Foxl2b2*, and pCS2⁺-OlaFoxl2, respectively, was transcribed from linearized pCS2+ plasmid using the SP6/T3/T7 m MESSAGE mMACHINE Kit (Ambion). One nanoliter was injected into the cytoplasm of one-cell stage Medaka embryos.

**Statistical Analysis.** Most of the data were analyzed using an unpaired Student's t test. A Mann-Whitney U test was then used to compare the median value when the Kolmogorov-Smirnov test was negative. Statistical analysis of multiple groups was performed using an ANOVA one-way test with post hoc Dunnett or Tukey tests for multiple comparison (a control group compared with an experimental group or a control sample compared to an experimental group, respectively). All statistical analyses were performed with GraphPad Prism (version 5; GraphPad Software). Significant differences are symbolized in figures by asterisks if *P* < 0.001 (***), *P* < 0.05 (**), or *P* < 0.01 (*) or indicated by ns if not significant.

**ACKNOWLEDGMENTS.** We acknowledge Wang Deshou for the medaka *cyp19a1a* promoter and Reiner Veitia for helpful discussions. This work was supported by Agence Nationale de la Recherche (ANR) Grant ANR-11-BSV7-0016 (SDS project) and grants from the Deutsche Forschungsgemeinschaft (Scha408/12-1, 10-1; to M.S.).

22. Boulanger L, et al. (2014) FOXL2 is a female sex-determining gene in the goat. *Curr Biol* 24:404-408.

23. Bertho S, et al. (2016) Foxl2 and its relatives are evolutionary conserved players in gonadal sex differentiation. *Dev Sex* 10:111-129.

24. Pannetier M, et al. (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.

25. Wang D-S, et al. (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.

26. Li M-H, et al. (2013) Antagonistic roles of Dmrt1 and Foxl2 in sex differentiation via estrogen production in tilapia as demonstrated by TALENs. *Endocrinology* 154:4814-4825.

27. Guiguen Y, Fostier A, Piferrer F, Chang C-F (2010) Ovarian aromatase and estrogens: A pivotal role for gonadal sex differentiation and sex change in fish. *Gen Comp Endocrinol* 165:352-366.

28. van der Vos KE, Coffer PJ (2008) FOXO-binding partners: It takes two to tango. *Oncogene* 27:2289-2299.

29. Liu X, Cai X, Zhang D, Xu C, Xiao W (2016) Zebrafish *foxo3b* negatively regulates antiviral response through suppressing the transactivity of *irf3* and *irf7*. *J Immunol* 197:4736-4749.

30. Guiguen Y, et al. (1999) Involvement of estrogens in the process of sex differentiation in two fish species: the rainbow trout (Oncorhynchus mykiss) and a tilapia (Oreochromis niloticus). *Mol Reprod Dev* 54:154-162.

31. Dranow DB, et al. (2016) Bmp15 is an Oocyte-produced signal required for maintenance of the adult female sexual phenotype in zebrafish. *PLoS Genet* 12:e1006323.

32. Lau ES-W, Zhang Z, Qin M, Ge W (2016) Knockout of zebrafish ovarian aromatase gene (*cyp19a1a*) by TALEN and CRISPR/Cas9 leads to all-male offspring due to failed ovarian differentiation. *Sci Rep* 6:37357.

33. Vizziano D, Randuineau G, Baron D, Cauty C, Guiguen Y (2007) Characterization of early molecular sex differentiation in rainbow trout, Oncorhynchus mykiss. *Dev Dyn* 236:2198-2206.

34. Chen W, et al. (2008) Insights into interferon regulatory factor activation from the crystal structure of dimeric IRF5. *Nat Struct Mol Biol* 15:1213-1220.

35. Formstecher E, et al. (2005) Protein interaction mapping: A Drosophila case study. *Genome Res* 15:376-384.

36. Rerox CE, 3rd, et al. (2003) Sequence analysis of a rainbow trout cDNA library and creation of a gene index. *Cytogenet Genome Res* 102:347-354.

37. Marivin E, et al. (2014) Sex hormone-binding globulins characterization and gonadal gene expression during sex differentiation in the rainbow trout, Oncorhynchus mykiss. *Mol Reprod Dev* 81:757-765.

38. Yano A, Nicol B, Guerin A, Guiguen Y (2011) The duplicated rainbow trout (Oncorhynchus mykiss) T-box transcription factors 1, tbx1a and tbx1b, are up-regulated during testicular development. *Mol Reprod Dev* 78:172-180.
RESEARCH ARTICLE

# A novel evolutionary conserved mechanism of RNA stability regulates synexpression of primordial germ cell-specific genes prior to the sex-determination stage in medaka

Amaury Herpin<sup>1,2*</sup>, Cornelia Schmidt<sup>2</sup>, Susanne Kneitz<sup>2</sup>, Clara Gobé<sup>1,3</sup>, Martina Regensburger<sup>2</sup>, Aurélie Le Cam<sup>1</sup>, Jérôme Montfort<sup>1</sup>, Mateus C. Adolfí<sup>2</sup>, Christina Lillesaar<sup>2</sup>, Dagmar Wilhelm<sup>4</sup>, Michael Kraeusling<sup>2¶</sup>, Brigitte Mourot<sup>1,5</sup>, Béatrice Porcon<sup>1</sup>, Maëlle Pannetier<sup>1,3</sup>, Eric Pailhoux<sup>3</sup>, Laurence Ettwiller<sup>1,5</sup>, Dirk Dolle<sup>5</sup>, Yann Guiguen<sup>1,1</sup>, Manfred Schartl<sup>2,6,7</sup>

1 INRA, UR1037 Fish Physiology and Genomics, Rennes, France, 2 University of Wuerzburg, Physiological Chemistry, Biocenter, Wuerzburg, Germany, 3 UMR BDR, INRA, ENVA, Université Paris Saclay, Jouy en Josas, France, 4 University of Melbourne, Department of Anatomy & Neuroscience, Parkville, Victoria, Australia, 5 University of Heidelberg, Centre for Organismal Studies (COS), Department of Developmental Biology, Heidelberg, Germany, 6 Comprehensive Cancer Center Mainfranken, University Hospital, Wuerzburg, Germany, 7 Hagler Institute for Advanced Study and Department of Biology, Texas A&M University, College Station, Texas, United States of America

¶Authorship confirmed by corresponding author.

* amaury.herpin@inra.fr

<figure><img src="image_102.png" /></figure>

<figure><img src="image_103.png" /></figure>

OPEN ACCESS

**Citation:** Herpin A, Schmidt C, Kneitz S, Gobé C, Regensburger M, Le Cam A, et al. (2019) A novel evolutionary conserved mechanism of RNA stability regulates synexpression of primordial germ cell-specific genes prior to the sex-determination stage in medaka. PLoS Biol 17(4): e3000185. https://doi.org/10.1371/journal.pbio.3000185

**Academic Editor:** Yukiko M Yamashita, University of Michigan, UNITED STATES

**Received:** September 28, 2018

**Accepted:** March 4, 2019

**Published:** April 4, 2019

**Copyright:** © 2019 Herpin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** All relevant data are within the paper and its Supporting Information files.

**Funding:** This work was supported by the Deutsche Forschungsgemeinschaft by a grant (SCHA 408/12-1; HE 7135/2-1) to AH and MS as well as Crédits Incitatifs Phase 2015/Emergence to AH. AH was additionally funded by the project AquaCRISPR (ANR-16-COFA-0004-01). The

## Abstract

Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1—acting as master sex-determining gene—has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within *dmrt1* genes of metazoans—together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells—suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.

## Author summary

The development of the gonads in vertebrates is mainly regulated by *dmrt1*, a master sex-determining gene that has a timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif located in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs. However, this regulation is complex, and
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

**Competing interests:** The authors have declared that no competing interests exist.

**Abbreviations:** BAC, bacterial artificial chromosome; BSA, bovine serum albumin; BSF, bicoid stability factor; Carbio, Carolina Biological Supplies; celf2, CUGBP Elav-like family member 2; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9; D3U-box, dmrt1 3' UTR box; dph, days post hatching; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; GO, gene ontology; Lrpprc, leucine rich pentatricopeptide repeat containing; MES-1, medaka embryonic stem cells; miR-224, microRNA 224; OI-BSF, Oryzias latipes Bicoid Stability Factor; OI-CUG-BP, Oryzias latipes CUG-binding protein; ORF, open reading frame; PARN, poly(a)-specific ribonuclease; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PGC, primordial germ cell; WNT4, Wnt family member 4.

the mechanisms are barely understood. Here, we analyse further dmrt1 regulation in the fish model medaka and show that two RNA-binding proteins with antagonizing properties target the D3U-box and promote either RNA stabilization in germ cells or degradation in the soma. The evolutionary conservation of the D3U-box motif within metazoan *dmrt1* genes—together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells—suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.

## Introduction

The gonads of vertebrates are characterized by the intimate association of germ cells and supporting somatic cells [1–4]. The precursor cells of the soma are derived from the embryonic lateral plate mesoderm, whereas germ cells originate from the germline lineage [5–9]. To carry out their highly specialized biological functions, the somatic gonadal primordium and the germline cells together must establish timely regulated programs of gene expression [1,10,11].

The *mab-3/doublesex/dmrt1* gene orthologs are, among metazoans, the most evolutionary conserved key regulators of the earliest phases of gonad development. They control complex gene regulatory networks specifying male gonadal primordium development as well as gonadal maintenance [12–15]. Remarkably, besides being firmly anchored within the regulatory network at critical nodes, *dmrt1* genes were found to act as upstream male sex determiners in organisms as phylogenetically diverse as flatworm [16], water flea [17], frog [18], flatfish [19], birds [20] and medaka [21,22].

During the last decade, much has been learned about how *dmrt1* as the most versatile sex gene triggers and controls gonad development. In human, it is a critical dosage-sensitive sex-determining gene, such that haploinsufficiency leads to XY male-to-female sex reversal and infertility [23,24]. In mice, it is required for male gonadal differentiation of somatic and germ cells [25–27], although *Dmrt1* appears to be dispensable for primary sex determination [15]. *Dmrt1* also plays the decisive role in maintaining the cellular identity of the adult testis, most obvious from the fact that its malfunction in adult mutant mice leads to transdifferentiation of Sertoli to granulosa-like cells and feminization of a fully developed testis [27]. Consequently, the action range of *Dmrt1* is not restricted to initiation of the male gonadal phenotype during early development but also contributes to the active suppression of the female networks via repression of two ‘anti-testis’ pathways, Foxl2 and *Wnt* family member 4 (*WNT4*/β-catenin (see [28] for review).

In sex determination model fish medaka, male sex determination is implemented by a male-specific primordial germ cell (PGC) mitotic arrest due to the activity of a Y-chromosome-specific duplicate version of *dmrt1*, designated *dmrt1bY* [29]. In *dmrt1* knockout mice, germ cells fail to arrest mitosis [30]. Further work on *dmrt1* has shown it to be a transcriptional gatekeeper controlling mitosis versus meiosis decision in male germ cells [26]. Thus, *dmrt1* in mice and *dmrt1bY* in medaka appear to be regulators of germ cell proliferation.

Despite its well-characterized crucial functions for gonad development in many vertebrates, the mechanisms that regulate the complex temporal and spatial expression pattern and guarantee precise levels of *dmrt1* transcripts are only barely understood. Diverse regulatory mechanisms have been occasionally reported. Indirect transcriptional regulation of *dmrt1* upon steroid treatments has been described in several fish species (see [13] for review). Gonadal dimorphic expression of *dmrt1* has been suggested to be possibly under the control of differential CpG methylation of its promoter in two different flatfish species [31,32]. Similarly, in the
red-eared slider turtle (*Trachemys scripta*), DNA methylation dynamics accounting for *dmrt1* sexual dimorphic expression are tightly correlated with temperature [33]. In vitro transcriptional regulation assays revealed that binding sites for *Sp1*, *Egr1* [34], and *Gata4* [35] factors, which are present in the promoters of many genes, are also involved in transcriptional regulation of the rat *dmrt1* gene. And finally, evidence was presented that microRNA 224 (miR-224) promotes differentiation of mouse spermatogonial stem cells via direct targeting of *dmrt1*, decreasing its expression in testes [36]. Certainly in the context of ‘indirect’ regulation, *dmrt1* is one of the most prominent examples.

In medaka, for which a functional duplicate of the autosomal *dmrt1a* gene on the Y chromosome—*dmrt1bY*—became the master regulator of male sex determination [22,37], transcriptional rewiring was brought about by exaptation of two transposable elements, *Izanagi* and *Rex1*, co-opted to act as silencers. These turn off the somatic and the germ cell-specific expressions of the *dmrt1bY* gene [38, 39]. Thus far, two factors, *dmrt1* itself [38] and *sox5* [39], were identified, which turn off *dmrt1bY* expression after it has fulfilled its function in the early developing gonad [38, 39].

We previously identified a 11-bp sequence motif in the 3' UTR of *dmrt1bY* (D3U-box, for *dmrt1* 3' UTR box). This motif confers stability to the mRNA in the developing embryonic gonad, whereas in other tissues, the transcript is rapidly degraded [40], indicating that a post-transcriptional regulation mechanism could play a role in germline expression of *dmrt1* in medaka.

Here, we show that the dmrt1 11-nucleotide *cis*-regulatory D3U-Box motif is a target for two antagonizing RNA binding proteins, *Oryzias latipes* CUG-binding protein (Ol-cug-bp) and *Oryzias latipes* Bicoid Stability Factor (Ol-bsf)—also known as cugbp Elav-like family member (celf) and leucine rich pentatricopeptide repeat containing (lrpprc), respectively, in mammals. In *Drosophila*, the bicoid stability factor (bsf) has initially been shown to be involved in regulating the stability of *bicoid* transcripts during oogenesis through binding structures within the 3' UTR of transcripts that resemble CUG hairpins [41]. Later, bsf was also reported to have a role in regulation of early zygotic genes by binding a short consensus sequence (CAGGUA) in the 5' UTR of genes expressed in the early zygote [42]. Cug-bp is the human homolog of the *Xenopus* eden-bp, which was shown to bind to mRNAs, such as *c-mos*, that exhibit rapid deadenylation following fertilization of oocytes [43]. Previous studies of cug-bp function have focused mainly on the roles of this protein in regulating alternative splicing [44] and also on its ability to modulate translation of several mRNAs [45]. However, as cug-bp is able to functionally substitute for eden-bp to induce deadenylation in *Xenopus* oocyte extracts [46], it seems likely that it also plays a similar role in regulating poly(A) shortening in mammalian cells. Indeed, it was shown that cug-bp can interact with poly(a)-specific ribonuclease (PARN) deadenylase to promote deadenylation of its substrate RNAs [47].

We find that in medaka, the D3U-box is targeted by these two different RNA binding proteins, with Ol-cug-bp1 leading to dmrt1bY degradation unless Ol-bsf is present in germ cells. Moreover, this new mechanism of dmrt1 RNA stability appears to regulate also the abundance of other transcripts specifically expressed in PGCs.

## Results

### Specific enrichment of a conserved *cis*-regulatory motif (D3U-box) in vertebrate genomes

In an initial analysis of dmrt1 post-transcriptional regulation [40], we found that an 11-bp long *cis*-regulatory motif in the D3U-box confers transcript stability in PGCs (Fig 1A). *In vitro* evidence was obtained that the D3U-box possibly mediates its function through protein
A

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* D3U-box present in male form and spliced out in the female form.

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"Fish-specific" D3U-box consensus motif

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"Full-species" D3U-box consensus motif

B

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**Fig 1. Conservation of the D3U-box motif from ecdysozoans to mammals.** (A) Conservation of the D3U-box motif from *Drosophila* up to mammals. From the different D3U-box sequences among vertebrates, weight-position matrices were deduced and used for genome scans. (B) Occurrences of the D3U-box motif compared to control motifs within the 3' UTR sequences of human, mouse, medaka, or the green spotted puffer (*Tetraodon nigroviridis*). For comparison, scrambled control motifs were used. Absolute values of the occurrences depend on the depth of annotation for every genome. Underlying data for (B) can be found in S1 Data. D3U-box, dmrt1 3' UTR box.

https://doi.org/10.1371/journal.pbio.3000185.g001
binding [40]. Further on this sequence motif was found to be evolutionary conserved in *dmrt1* genes from flies to mammals (Fig 1A; [40]).

To investigate whether this motif is specific for the *dmrt1* genes or also present in other genes, we performed genome-wide searches for human, mouse, medaka, and the green spotted puffer (Fig 1B). The D3U-box was found in more than 250 3' UTRs of genes from human and mice and is also abundant in medaka and green spotted puffer (Fig 1B). Notably, also, a 'split' version of the D3U-box (see Fig 1B) displayed specific enrichment in 3' UTRs (Fig 1B), suggesting that the D3U-box motif might be articulated around two independent cis-regulatory sequences, hence putatively targeted by 2 different RNA binding proteins.

Medaka whole transcriptome scans (3' UTR and coding sequences) using the vertebrate D3U-box consensus motif matrix (Fig 1A: 3' UTR sequences; and S1 Fig: 3' UTR and coding sequences) resulted in several hits, including tra2, sox10, misr2, dead end, and vasa (S1 Fig). Like dmrt1 [14,29,48,49], these RNAs are critically involved in germ cell development and maintenance in medaka and many other organisms ([36,50–55] and [56,57] for review).

Furthermore, bioinformatics analyses and literature searches [45,47] revealed that the D3U-box *cis*-regulatory motif is a putative target for 2 evolutionary conserved RNA binding proteins involved in either mRNA degradation or stabilization. These 2 proteins, Ol-cug-bp (also known as CELF in mammals) and Ol-bsf (also known as LRPPRC in mammals), have been shown to specifically recognize CUG repeats and the CAGGU(AG) motif, respectively, which constitute the D3U-box (see S2 Fig for phylogeny and synteny analysis of *Ol-bsf* and *Ol-cug-bps*).

## Ol-BSF and Ol-CUG-BP1 specifically bind to the D3U-box motif

To confirm our bioinformatics prediction, Ol-bsf and Ol-cug-bp1 and 2 proteins were subjected to electrophoretic mobility shift assay (EMSA) using the D3U-box motif as target and different competitors (Fig 2). The in vitro–translated proteins (Fig 2A and 2F) were assayed for binding with radioactively labelled RNA probes. Using the D3U-box motif, mobility shifts were detected for the 2 proteins tested: Ol-bsf (Fig 2B to 2E) and Ol-cug-bp1 (Fig 2G to 2I), indicating that Ol-bsf as well as Ol-cug-bp1 are, in principle, able to bind the D3U-box in vitro. Binding specificities were confirmed by competition of the medaka D3U-box motif for Ol-bsf or Ol-cug-bp1 interactions with either a scrambled D3U-box–derived motif (Fig 2B and 2I) or a minus CUG repeat motif competitor (Fig 2H). The absence of any significant interference with the D3U-box binding indicated the specificity of the observed interactions (Fig 2B, 2I and 2H). Furthermore, competition experiments between radioactively and nonradioactively labelled D3U-boxes resulted in progressive loss of the apparent shifts (Fig 2C and 2G). Notably, a clearly visible shift was also observed when using the *Drosophila* D3U-box sequence together with the medaka Ol-bsf protein (Fig 2D). Altogether, these experiments suggest that the D3U-box is a preferential target for Ol-bsf and Ol-cug-bp1 binding. Of note, performing the very same set of experiments together with the Ol-cug-bp2 protein did not result in any convincing evidence for specific binding to the D3U-box.

## Ol-bsf and Ol-cug-bps antagonistically regulate the expression and stability of reporter constructs harbouring the D3U-box motif and of dmrt1bY transcripts

To monitor a possible effect of Ol-bsf on regulation of the male sex-determination gene in medaka, we generated a dmrt1bY reporter line by introducing the green fluorescent protein (GFP) open reading frame (ORF) fused to the dmrt1bY 3'UTR (including the D3U-box) into exon 1 of a bacterial artificial chromosome (BAC) clone containing the *dmrt1bY* gene and
<figure><img src="image_108.png" /><figcaption>**Fig 2.** EMSA of in vitro-translated Ol-bsf, Ol-cug-bp1, and Ol-cug-bp2 proteins indicating interactions with the medaka D3U-box target sequence. (A and F) Recombinant protein production of FLAG-tagged versions of Ol-bsf and Ol-cug-bp1 and western blotting detection with an anti-FLAG antibody. (B-E and G-I) EMSA using the recombinant medaka Ol-bsf and Ol-cug-bp1 proteins (in vitro translated) and either the medaka D3U-box sequence (B, C, E, G, H, I), or the *Drosophila* D3U-box sequence (D) as radioactively labelled RNA probes. Increasing amounts of unlabelled probes (scrambled [B, I], medaka [C, G, K], *Drosophila* [D, E], minus CUG [H, L] target sequences) were used as competitors. (B-E and G-I) Apparent shifts are observed for the labelled RNA target probes with the Ol-bsf and Ol-cug-bp1 RNA-binding proteins, likely indicating direct interactions. Ratios of RNA probe to RNA competitor: (B, lane 2: 1/5; C, lane 2: 1/1, lane 3: 1/2, lane 4: 1/4; D, lane 2: 1/1, lane 3: 1/2, lane 4: 1/5, lane 5: 1/10; E, lane 2: 1/1, lane 3: 1/2, lane 4: 1/5, lane 5: 1/10; G, lane 2: 1/1, lane 3: 1/2, lane 4: 1/5; H, lane 2: 1/1, lane 3: 1/2, lane 4: 1/5; I, lane 2: 1/1, lane 3: 1/2, lane 4: 1/5, lane 5: 1/10). D3U-box, dmrt1 3' UTR box; EMSA, electrophoretic mobility shift assay; lrpprc, leucine rich pentatricopeptide repeat containing; Ol-bsf, Oryzias latipes Bicoid Stability Factor; Ol-cug-bp, Oryzias latipes CUG-binding protein.</figcaption></figure>

https://doi.org/10.1371/journal.pbio.3000185.g002

flanking regions (Fig 3A and 3B). The recombined BAC was then used for establishing a stable transgenic line in which GFP expression most reliably indicates endogenous *dmrt1bY* expression [48,58,59]. Expression of *dmrt1bY* is highly dynamic during primordial gonad formation, progressively switching from germ cell expression only between stages 26 (1.25 dpf) and 29 (3.1 dpf) to an exclusive somatic expression from stages 33/34 (5 dpf) up to hatching (9 dpf) when the gonad is formed [39,59]. After injection of the Ol-bsf morpholino (see S3 Fig for validation of the morpholino), we found a significant reduction of GFP expression (Fig 3B) and, after Ol-bsf overexpression, a strong increase of the reporter at both mRNA and protein (fluorescence) levels (Fig 3A and 3B).

Next, to obtain a more physiological readout of the role(s) of Ol-bsf and Ol-cug-bps for RNA stability in vivo, the relative abundances of endogenous *dmrt1bY* transcripts were monitored after modulation of Ol-bsf/Ol-cug-bp1/Ol-cug-bp2 expression in medaka embryos (Fig 3C and 3D). First, we checked for changes in *dmrt1bY* transcript levels after overexpression of the 2 medaka Ol-cug-bp ohnologs (Ol-cug-bp1 and Ol-cug-bp2, Fig 3C). This resulted in
<figure><img src="image_109.png" /><figcaption>**Fig 3.** In vivo modulation of expression and mRNA stability of reporters and *dmrt1bY* transcripts via the D3U-box. (A and B) Effect of the modulation of the Ol-bsf expression on a GFP reporter for *dmrt1bY* expression. Injections of either Ol-bsf morpholino or capped mRNA result in the subsequent modulations of expression of a GFP:(*dmrt1bY* 3' UTR) reporter/sensor construct in a transgenic line (BAC *Dmrt1bY:GFP:3'UTR*) as monitored by fluorescence (A) and RNA quantifications (B). Because endogenous GFP is lowly expressed already before experimental down-regulation, the more sensitive qPCR method was employed to monitor the morpholino effect (B). Dataset results of 4 independent reverse transcription reactions, resulting in 4 different batches of eggs obtained from different couples. Statistical significance has been assessed by mean of the Wilcoxon-Mann-Whitney test (N = 4). (C and D) Real-time qPCR determination of *dmrt1Y* abundances after Ol-cug-bp1 and Ol-cug-bp2 (C) or Ol-bsf (D) modulation of expression in embryos at stage 18 after injection of either capped mRNAs with the full Ol-bsf/Ol-cug-bps ORFs or a splice morpholino targeting the exon2-intron2 splice junction of *Ol-bsf* (S3 Fig). Dataset results of the analysis of 4 batches of eggs injected with either Ol-cug-bp1, Ol-cug-bp2, Ol-bsf, or BSF-MO. Statistical significance has been assessed by mean of the Wilcoxon-Mann-Whitney test (N = 4). \*p ≤ 0.05, \*\*p ≤ 0.01. (E and F) Real-time qPCR determination of the kinetics of RNA stability for D3U-box-containing reporters in presence or in absence of either Ol-cug-bp1 or Ol-bsf coding mRNAs. Datasets are results of 3 independent reverse transcription reactions, resulting from three different batches of injected eggs. Underlying data for (B, C, D, and F) can be found in S1 Data. BAC, bacterial artificial chromosome; D3U-box, *dmrt1* 3' UTR box; GFP, green fluorescent protein; MO, Morpholino; ns, nonsignificant; Ol-BSF, *Oryzias latipes* Bicoid Stability Factor; Ol-CUG-BP, *Oryzias latipes* CUG-binding protein; ORF, open reading frame; qPCR, quantitative PCR; W.T., wild type.</figcaption></figure>

https://doi.org/10.1371/journal.pbio.3000185.g003

decreased *dmrt1bY* mRNA (Fig 3C). Second, the relative abundances of *dmrt1bY* transcripts were recorded after either overexpression or morpholino knockdown of *Ol-bsf* in medaka embryos (Fig 3D). It revealed that higher *Ol-bsf* expression correlates with an increased abundance, while lowering *Ol-bsf* expression resulted in a reduction of *dmrt1bY* transcripts
(Fig 3D). In vivo, D3U-box-induced modulation of RNA stability was further investigated in embryos injected with either control RNAs or RNAs harbouring the D3U-box (Fig 3E). Ratios between control and D3U-box-containing mRNAs were then quantified in absence or in presence of either l-cug-bp1 or Ol-bsf mRNAs (Fig 3F). It revealed that, over time, overexpression of Ol-bsf correlates with an increased stabilization of the D3U-box-containing mRNAs, while overexpression of Ol-cug-bp1 correlates with a decreased stabilization of the D3U-box-containing mRNAs (Fig 3F).

## Expression of Ol-cug-bp1, Ol-cug-bp2, and Ol-bsf during embryonic development and in adult tissues

During the embryonic developmental period, both Ol-cug-bp ohnologs display complementary patterns of expression. Ol-cug-bp1 is expressed at early stages and Ol-cug-bp2 only later when dmrt1bY transcripts appear in the germ cells around stage 25 (S4A and S4C Fig). In adult tissues, Ol-cug-bp1 and Ol-cug-bp2 are expressed at high levels in brain and gonads (S4B and S4D Fig), while Ol-cug-bp2 is additionally expressed in eyes, muscles, and skin (S4D Fig). Of note, both ohnologs are always more expressed in testes compared to ovaries (S4B and S4D Fig). Expression profiling of Ol-bsf revealed that in adult medaka, it is ubiquitously expressed in all adult tissues, with particular high levels in gonads of both sexes (S4E and S4F Fig).

## Ol-bsf is specifically expressed in the germ cells during early gonad primordium formation with correlated levels of expression between Ol-bsf and dmrt1bY at hatching stage

For bioimaging analyses of protein localization over time, we used expression reporter lines for vasa [11] and Ol-bsf (see Materials and methods), respectively. During embryonic development, we noted a distinct spatially and temporarily restricted expression pattern (Fig 4). From fertilization up to stages 16/17, Ol-bsf is expressed throughout the embryo (Fig 4A). Of note, cell transfection of a tagged version of OL-bsf and subsequent immunohistochemistry revealed that bsf protein is localized in the cytoplasm (insert in Fig 4A). From stage 25 onwards—when germ cells line up on both sides of the embryo within the lateral plate mesoderm—progressively, Ol-bsf expression becomes restricted to the PGCs (Fig 4B to 4G) where it is co-expressed with Ol-vas, a specific germ cell marker in medaka [60]. During the following developmental stages (stages 33/34), expression heterogeneity for Ol-bsf between germ cells became obvious (Figs 4H to 4J and 5C and 5D). This heterogeneity was particularly apparent between 4 to 10 days post hatching (dph), when Ol-bsf is higher expressed at the tips of the forming gonads (curly brackets in Fig 4K to 4P and square brackets in Fig 5C and 5D). In summary, Ol-bsf has a highly dynamic expression pattern, switching from an early somatic to a progressively restricted germ cell expression. Within the germ cell pool, the levels of expression show a significant heterogeneity.

Furthermore, dmrt1bY expression shows heterogeneity between individual germ cells (Fig 4Q and 4R). Being also higher expressed at the tip of the primordial gonads, the expression levels of Ol-bsf and dmrt1bY show a clear positive correlation (Fig 4Q and 4R).

## Expression of Ol-bsf in the germ-line stem cells of adult gonads

Given the high abundance of Ol-bsf transcripts in adult gonads detected by qPCR (S4E and S4F Fig), we next monitored expression of Ol-bsf in fully mature gonads of both sexes at cellular resolution (Fig 6). In adult testes, Ol-bsf fluorescence is restricted to two distinct
<figure><img src="image_110.png" /><figcaption>Fig 4. Embryonic expression of medaka Ol-bsf and correlated expression dynamics together with dmrt1bY during gonadal primordium formation. (A to R) Medaka Ol-bsf expression investigated by fluorescence using a transgenic reporter line for which a 732-bp *Ol-bsf* promoter fragment (up to the next upstream gene) drives the expression of the mCherry. Medaka Ol-vas and dmrt1bY expressions were investigated by fluorescence using previously described transgenic reporter lines ([11] and [61], respectively). (A) During neurulation (stages 16/17), Ol-bsf is ubiquitously expressed. (A insert) Cytoplasmic localisation of Ol-bsf after transient transfection of a 3-times FLAG version of Ol-bsf in</figcaption></figure>
a medaka embryonic stem cell line and immunohistochemistry. (B to P) From stage 25 on, Ol-bsf expression is restricted to the germ cells and colocalize with the germ cell marker Ol-vas. (K to P) Four-dph expression of Ol-bsf is apparently heterogeneous within the population of PGCs while dmrt1bY is now expressed in the surrounding somatic cells of the primordial gonad (K to M). (Q and R) By stage 29, Ol-bsf and dmrt1bY are both expressed in the germ cells. Variations within the respective levels of Ol-bsf and dmrt1bY are clearly observable amongst different germ cells although always correlated between the 2 fluorescences (curly brackets compared to square brackets). dph, days post hatching; MES-1, medaka embryonic stem cells; Ol-BSF, Oryzias latipes Bicoid Stability Factor; olf1, *Oryzias latipes* fibroblast-1; PGC, primordial germ cell.

https://doi.org/10.1371/journal.pbio.3000185.g004

subpopulations of germ cells, which are also positive for Ol-vas (Fig 6A to 6C). Diagnosed by condensed nuclear morphology and size [62] and localization [39], these first Ol-bsf-positive cells represent the earliest step of germ cell differentiation while another subpopulation of more mature germ cells is also observed (Fig 6C and 6F). In ovaries, Ol-bsf fluorescence is restricted to the germinal cradle [63] located in the interwoven threadlike ovarian cords at the periphery of the ovary (Fig 6G to 6N). These Ol-bsf-positive cells, representing the smaller-size subpopulation of Ol-vas fluorescent cells, are assigned to germline stem cells and early dividing germ cell lineage [63] (Fig 6O to 6Q).

Furthermore, in mice both bsf (lrpprc) and cug-bp1 (celf1) are expressed in the germ cells within the testis cords and germ cells of the ovary (S5A and S5L Fig). Immunohistochemistry revealed that bsf/lrpprc is expressed only in a subpopulation of germ cells in mice (S5M to S5R Fig).

## Ol-bsf mutant fish display gonadal phenotypes

To delineate the physiological role of Ol-bsf during gonad formation and maintenance, we generated medaka *Ol-bsf* knockout lines after genome editing using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology (S6 Fig). Homozygous *Ol-bsf* knockout larvae display reduced swimming (S7 Fig) and die within the first 2 weeks after hatching. Heterozygote mutant fish develop normally and produce mature gametes. Histological analysis of gonads of heterozygous mutants of both sexes revealed, however, that ovaries had an accumulation of small-sized oocytes compared to wild type (Fig 7A to 7D, S8 and S9 Figs), whereas testes of heterozygous mutants exhibited reduced amounts of spermatogonial stem cells, with germ cells in advanced stages of spermatogenesis located close to the periphery of the organ (Fig 7E and 7F and S10 Fig). In both sexes, *Ol-bsf* heterozygote mutants present an increase of germ cell committing to gametogenesis. While adult mutant males do not display any observable bias of fertility, adult mutant females have reduced egg production together with lower fertilization rates (S11 Fig).

## Ol-bsf and Ol-cug-bps antagonistically orchestrate expression levels of several germ cell transcripts selectively harbouring the D3U-box

Our matrix scan bioinformatic analysis had revealed the presence of the D3U-box either in 5', 3', or coding regions of several germ cell transcripts (S1 Fig). To find out whether Ol-bsf and Ol-cug-bp might regulate stability of these RNAs in a similar way, like for dmrt1bY, during gonadal development the relative abundances of these transcripts harbouring the box motif (tra2, sox10, misr2, dead end, and vasa; see S1 Fig) were first monitored after either overexpression or morpholino knockdown of Ol-bsf in medaka embryos (Fig 5A1 to 5A10). It revealed that for the majority of these transcripts (sox10, misr2, tra2, and vasa), higher Ol-bsf expression correlated with an increased abundance (Fig 5A1 to 5A5 odd numbers and 5A19), while reduced Ol-bsf expression resulted in lower levels of most of these transcripts (Fig 5A2 to 5A6 even numbers). As exceptions, dead end (Fig 5A7 and 5A8) transcript abundance
Modulation of
Ol-BSF / LRPPRC

<figure><img src="image_111.png" /></figure>

<figure><img src="image_112.png" /></figure>

Modulation of
Ol-CUG-BP1 & Ol-CUG-BP2

<figure><img src="image_113.png" /></figure>

<figure><img src="image_114.png" /></figure>

<figure><img src="image_115.png" /><figcaption>Fig 5. In vivo transcriptional regulation of putative gene candidates harbouring the D3U-box motif in their transcripts after modulation of the Ol-bsf or Ol-cug-bp expressions and correlation between Ol-bsf and Ol-vas expression in germ cells. (A1 to A10) Real-time qPCR determination of expression of putative Ol-BSF-regulated transcripts in embryos at stage 18 after injection of either capped mRNAs with the full Ol-bsf ORF (left odd-numbered panels) or a splice morpholino targeting the exon2-intron2 splice junction (S3 Fig) of the *Ol-bsf* gene (right even-numbered panels) at one cell stage. Ol-bsf overexpression results in an apparent increase of most RNA target candidates, while reduction of the Ol-bsf expression correlates with a reduction of expression. Dataset results of the analysis of 4 batches of eggs injected with either Ol-bsf or Ol-bsf-MO. Statistical significance has been assessed by mean of the Wilcoxon-Mann-Whitney test (N = 4). (B1 to B10) Real-</figcaption></figure>
<figure><img src="image_116.png" /><figcaption>**Fig 6.** Expression of Ol-bsf in adult gonads. (A to F) In adult testes, Ol-bsf fluorescence (same transgenic lines as in Fig 3) is restricted to the cells located within the lobules and colocalize with a subpopulation of Ol-vas-positive cells. (G to Q) Within the ovary, Ol-bsf fluorescence is restricted to the germinal cradle located in the interwoven threadlike ovarian cords at the periphery of the ovary. The Ol-bsf-positive cells represent the smaller-size subpopulation of Ol-vas fluorescent cells (G to N) as well as early dividing germ cells (O to Q). Arrowheads indicate the germinal cradles (G to N), and brackets indicate early dividing mitotic or meiotic germ cell (O to Q). Ol-BSF, *Oryzias latipes* Bicoid Stability Factor.</figcaption></figure>

https://doi.org/10.1371/journal.pbio.3000185.g006
<figure><img src="image_117.png" /><figcaption>**Fig 7.** Ovarian and testicular phenotypes of the *Ol-bsf* mutant fish. Heterozygote mutant fish develop normally. (A to D) In-depth morphological inspection of heterozygote mutant ovaries discloses a significant accumulation of small-sized oocytes compared to wild type (dotted lines in [C] and red arrows in [B] and S8 and S9 Figs). (D) Size distribution of the oocytes in 2 wild-type and 2 *Ol-bsf*(+/-) adult ovaries (see also S8 Fig for details). (E and F) Heterozygote mutant testes exhibit a decreased number of spermatogonia with accumulation of type 2 spermatocytes, spermatids, and sperm within the most external layers of the seminiferous epithelium (see also S10 Fig). The different stages of spermatogenesis were determined according to [64]. Each gonad was sectioned through the mid-sagittal plane. Underlying data for (D) can be found in S1 Data. Ol-BSF, *Oryzias latipes* Bicoid Stability Factor; Sc1, type 1 spermatocytes; Sc2, type 2 spermatocytes; Sg, spermatogonia; Sm, sperm; St, spermatids.</figcaption></figure>

https://doi.org/10.1371/journal.pbio.3000185.g007
characters before the formation of the somatic gonadal primordium depending on dynamic
and tightly timely regulated mechanisms of post-transcriptional regulations [59,70].

We have identified in medaka a *cis*-regulatory 11-bp motif in the 3' UTR of *dmrt1bY* called D3U-box. This motif confers stability to the *dmrt1bY* mRNA in germ cells of the developing embryonic gonad, whereas in other tissues, the transcript is rapidly degraded [40]. The D3U-box motif was found to be highly conserved in the dmrt1 3' UTR in the fish lineage (*O. latipes*, *O. curvinotus*, *Takifugu rubripes*, *Tetraodon nigroviridis*, *Epinephelus coioides*, and *Danio rerio*), as well as in other vertebrates, including *Mus musculus*, *Pan troglodytes*, *Macaca mulatta*, and *Homo sapiens*, and even in the ecdysozoan clade (*Anopheles gambiae* and *Bacto-cera oleae*).

Both phylogenetic conservation and presence of the D3U-box in several germ cell tran-
scripts implied the existence of similarly conserved *trans*-acting factor(s) involved in the
synexpression of those transcripts. To identify such factor(s), we undertook an unbiased
approach centred on the D3U-box sequence and based on the evolutionary conservation of
the 'split' motifs of the D3U-box, implying evolutionary conserved *trans*-acting factors. Fur-
ther bioinformatics analyses and literature searches revealed that the D3U-box motif is a puta-
tive target for 2 RNA-binding proteins, namely cug-bp [45,47] and bsf (also known as lrpprc
in mammals [41,42,71]).

EMSAs indicated that Ol-bsf and Ol-cug-bp1, but not Ol-cug-bp2, specifically target and interact with the different parts of the D3U-box, the 3' and the 5' parts, respectively. Additionally, our results suggested that the observed regulation of *dmrt1bY* transcript abundance is likely to be the result of a differential binding of the 2 RNA-binding proteins (Ol-bsf and Ol-cug-bp1) with antagonistic properties, *trans*-regulating RNA stability via the D3U-box.

Being—like its mammalian counterparts—ubiquitously expressed, Ol-cug-bps are highly expressed in the gonads of both sexes in medaka (S4C to S4F Fig) and mice ([72] and S5 Fig). Interestingly, Ol-bsf is specifically expressed in the germ cells during medaka early gonadal primordium formation as well as in the adult ovary and is cytoplasmically localized. This expression pattern resembles the subcellular localization of *Drosophila* bsf, which is present in cytoplasmic particles in oocytes and surrounding nurse cells [41] and in the cytoplasms and nuclei in early embryos [42].

In vivo, using fluorescent reporter medaka fish lines, we could show that, besides obvious
correlated expression levels between Ol-bsf, *dmrt1bY*, and Ol-vas in a subpopulation of germ
cells of the forming gonadal primordium, medaka Ol-bsf is also preferentially expressed in
adult germline stem cells. This restricted and up to now unreported expression pattern might
reveal another so far underappreciated role for bsf/lrpprc in germ cell physiology. Similarly,
immunohistochemistry localization of mouse bsf/lrpprc revealed its presence in a subpopula-
tion of germ cells in mice (S5 Fig), suggesting an evolutionary conserved function that is not
restricted to exclusive regulation of dmrt1.

Reduced expression of bsf/lrpprc in mammalian cell lines [73,74] or flies [75] or because of
a missense mutation in human (French Canadian Leigh Syndrome [76]) resulted in decreased
levels of mitochondrial mRNAs. This led to respiratory chain dysfunction and increased lac-
tate levels in flies and humans [75,76]. Similarly, in medaka, many transcripts with mitochon-
drial function displayed decreased steady-state levels when Ol-bsf expression was reduced
(S12B Fig). Although knockdown of *bsf* in flies affects climbing ability, fecundity, and life
span [75], mutant medaka hatchlings for *Ol-bsf* comparably displayed a significantly reduced
swimming ability (S7 Fig), suggesting—with all the necessary notes of caution, such as in *Dro-
sophila*—possible muscle weakness in relation to mitochondrial dysfunction and energy
metabolism failure [75].
Ol-bsf-heterozygous mutant fish develop normally. Although adult mutant males do not seem to display any observable bias of fertility, adult mutant females have reduced egg production together with significantly lower fertilization rates. In-depth morphological inspection of heterozygote mutant gonads of both sexes revealed that ovaries display an important accumulation of small-sized oocytes compared to wild type. Mutant testes exhibited a discontinuous spermatogenetic flux, likely reflecting uncontrolled spermatogenesis, independent of the seminiferous epithelial cycle. Such a testicular phenotype can be interpreted in the light of dmrt1 loss of expression as a result of low Ol-bsf expression. Similarly, in mice, loss of dmrt1 in germ cells uncouples meiotic initiation from the seminiferous epithelial cycle, resulting in uncontrolled spermatogenesis, too [26]. The mouse celf1 (also known as cugbp1) is predominantly expressed in testis ([72] and S5E and S5F Fig). There, it was demonstrated that celf1 post-transcriptionally represses cyp19a1 (*aromatase*) mRNA translation, by direct binding, to maintain high concentrations of testosterone compatible with spermiogenesis [77]. This situation is reminiscent of the situation we observed in medaka with a strong repression of aromatase expression when both Ol-cug-bps are overexpressed (S12C1 and S12C2 Fig). Additionally, as observed in medaka, mouse celf/cug-bps and bsf/lrpprc are expressed at relevant levels in germ cells, possibly implying a functional conservation across vertebrates.

Whole transcriptome scans using either the medaka or *Drosophila* D3U-box sequences as query resulted in hits enriched for genes specifically expressed in the germ cells, including tra2, sox10, misr2, dead end, and vasa. We provide evidence that Ol- and Ol-cug-bps antagonistically regulate the expression of germ cell transcripts harbouring the D3U-box motif. Certainly most of these regulations occur via an mRNA decay versus stabilization equilibrium after Ol-cug-bps and Ol-bsf targeted the D3U-box motif.

Lastly, to explain the observed differences in the degree of regulation of D3U-box containing transcripts after either Ol-bsf or Ol-cug-bp1/2 modulations, we consider that this follows the degree of conservation of the D3U-box (S1 Fig). It is also intuitive that the location of the D3U-box (5', 3' or coding regions) within the transcripts is of relevance (S1 Fig). For efficient regulation, reasonably high (tra2) or moderate (sox10) conservation and location within the UTRs appears to be more effective than a moderately conserved motif located in the coding sequences (vasa and dead end). However, multiple, highly conserved D3U-boxes nested within the coding region seem to be efficient as well (misr2). Dead end transcripts, for which the D3U-box was identified within the 5' UTR, however, has only strict conservation for the 3' part (CUGCUG) and is only regulated by Ol-cug-bp1 and Ol-cug-bp2 (Fig 5B7 and 5B8) while it expectedly escapes Ol-bsf regulation (Fig 5A7 and 5A8). Altogether, using complementary approaches, our data suggest that the D3U-box motif is—depending on the cellular context—targeted by 2 antagonizing RNA binding proteins, promoting either RNA stabilization in germ cells or degradation in the soma. This new mechanism of dmrt1 RNA stability appears to also regulate the abundance of other transcripts specifically expressed in PGCs, depending of the preservation of the D3U-box motif.

**Materials and methods**

**Bioinformatic analyses**

Gene, transcript, and UTR annotation, coordinates, and sequence for human, mouse, medaka, and *Tetraodon* were retrieved from EnsEMBL using the EnsEMBL API (version 54). UTR regions spread across several exons were stitched together per transcript, and the resulting sequence was scanned for the presence of the D3U-box consensus matrix and the other motifs.
## Fish maintenance and breeding

All medaka fish used in this study were taken from closed breeding stocks of the wild-type *Carolina Biological Supplies (Carbio)* strain or transgenic lines produced on a wild-type *Carbio* background and were kept under standard conditions [5]. Medaka embryos were staged according to Iwamatsu [78]. The fish used in this study from aquaria housed stocks were kept and sampled in accordance with the applicable EU and national German legislation governing animal experimentation. We hold an authorization (568/300-1870/13) of the Veterinary Office of the District Government of Lower Franconia, Germany, in accordance with the German Animal Protection Law (TierSchG).

## Cell culture, transient cell transfection, and immunohistochemistry

Medaka embryonic stem cells (MES-1) were cultured as described [79]. For transfection, cells were grown to 80% confluence in 6-well plates and transfected with 5 µg expression vector using FuGene (Roche, Germany) reagent as described by the manufacturer. After pCS2:: OL-LRPPRC:3XFLAG transfection for 48 hours, cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes, washed with phosphate-buffered saline (PBS) buffer, and then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes. After blocking in 1% Bovine Serum Albumin (BSA) for 20 minutes, cells were incubated overnight at 4°C in blocking buffer (1% BSA) together with the primary antibody (3-times FLAG, monoclonal anti-FLAG M2, category number F1804; Sigma-Aldrich). After extensive washes in PBS, cells were then incubated with Alexa 488 conjugated secondary antibody in 1% BSA for 1 hour. Cell nuclei were stained with Hoechst 33343 (Invitrogen) for 5 minutes (1 µg/mL final concentration) and subsequently mounted using Mowiol 4-88 (Roth). Confocal images were acquired using a Nikon Eclipse C1 laser-scanning microscope (Nikon) and were fitted with a 60× Nikon objective (PL APO, 1.4 NA) and Nikon image software.

## Phylogenetic analysis

The *lrpprc* phylogenetic tree was built using the online phylogeny.fr automatic pipeline [80]. *lrpprc* sequences were retrieved from public database sequences in the following species and *lrpprc* homologs were retrieved by tblastn searches on the PhyloFish [81] database (http://phylofish.sigenae.org/) using medaka protein (O. latipes, XP_011482612.1) as bait. Sequences were aligned with MUSCLE (version 3.8.31) configured with default settings. After alignment, ambiguous regions were removed with Gblocks (version 0.91b) using the following parameters: minimum length of a block after gap cleaning of 10, no gap positions allowed in the final alignment, rejection of all segments with contiguous nonconserved positions bigger than 4, and a minimum number of sequences for a flank position of 85%. The phylogenetic tree was reconstructed using the neighbour joining method implemented in the BioNJ program [82] with *N* = 100 bootstrapping. The resulting phylogenetic tree was exported as a Newick file and edited in Evolview [83]. The public database for *lrpprc* sequences is as follows: *Cyprinodon variegatus* (XP_015233698.1), *Haplochromis burtoni* (XP_005930623.2), *Fundulus heteroclitus* (XP_012720452.1), *Xiphophorus maculatus* (XP_005804231.1), *Poecilia reticulata* (XP_008426945.1), *Lates calcarifer* (XP_018552533.1), *Salmo salar* (XP_014035445.1), *Kryptolebias marmoratus* (XP_017287636.1), *Larimichthys crocea* (KKF31900.1), *Pygocentrus nattereri* (XP_017546072.1), *Ictalurus punctatus* (XP_017319936.1), *Austrofundulus limnaeus* (XP_013886428.1), *Xenopus tropicalis* (NP_001039203.1), *Cynoglossus semilaevis* (XP_008319858.1), *Callorhinchus milii* (XP_007895038.1), *Astyanax mexicanus* (XP_007255829.1), *Chrysemys picta bellii* (XP_005296166.1), *Scleropages formosus* (KPP63655.1), *Bos taurus* (XP_005212770.1), *Cyprinus carpio* (KTG42350.1 and
XP_018937975.1), *Equus caballus* (XP_005600080.1), *H. sapiens* (BAF82705.1), *Rattus norvegicus* (NP_001008519.1), *M. musculus* (AAH59862.1), *Coturnix japonica* (XP_015712995.1), *Serinus canaria* (XP_009094627.1), *Lepisosteus oculatus* (XP_015218688.1), *Amia calva* (AAC LOC100694568.1), *Esox lucius* (XP_012989242.1), *Latimeria chalumnae* (XP_005999623.1), *Sinocyclocheilus anshuiensis* (XP_016313114.1 and XP_016355337.1), *Sinocyclocheilus rhinocerous* (XP_016417572.1 and XP_016400889.1), *Oreochromis niloticus* (XP_003438484.3), *D. rerio* (NP_001136064.1), *Stegastes partitus* (XP_008296301.1), *T. rubripes* (XP_011601434.1), *Nothobranchius furzeri* (XP_015800103.1), and *Gallus* (XP_001234903.3). The PhyloFish species are as follows:

* (DAA_LPPRC.1).2_Anguilla_anguilla;

* Y_Up_12_k31_Locus_13642_Umbra pygmaea;

* W2_Dp_10_k25_Locus_11398_Dallia pectoralis; S2_Pb_10_k31_Locus_2910_Pantodon bulchozi;

* W_Ha_12_k43_Locus_3235_Hiodon alosoides;

* D2_Om_14_k31_Locus_6093_Oncorhynchus mykiss;

* N_St_1_k25_Locus_84_Salmo trutta;

* O_Ot_10_k25_Locus_4385_Oncorhynchus tshawytscha;

* Q_Sf_1_k25_Locus_1875_Salvelinus fontinalis;

* P_Cl_2_k25_Locus_10853_Coregonus lavaretus;

* I_Tt_3_k25_Locus_2938_Thymallus thymallus;

* E_Aa_2_k25_Locus_4938_Alosa alosa;

* Z_Pa_1_k25_Locus_2290_Plecoglossus altivelis;

* U_Cc_1_k25_Locus_265_Coregonus clupeaformis;

* R_Ok_10_k31_Locus_25108_Oncorhynchus keta;

* G_Ph_4_k37_Locus_379_Pangasianodon hypophthalmus;

* K2_Cb_1_k25_Locus_1206_clarias batrachus;

* M_Pf_1_k25_Locus_1736_Perca fluviatilis;

* G2_Ps_1_k65_Locus_195_Polypterus senegalensis;

* K_Gm_1_k43_Locus_6389_Gadus morhua;

* C_Ob_2_k31_Locus_7388_Osteoglossum bicirrhosum;

* B2_Gp_10_k25_Locus_1874_Gnathonemus petersii;

* V_Sc_10_CL11520Contig1_Scyliorhinus caniculata

The celf2 phylogenetic tree was built using the online phylogeny.fr automatic pipeline [80]. Celf2 sequences were retrieved from Ensembl. Sequences were aligned with MUSCLE (version 3.8.31) configured with default settings. After alignment, ambiguous regions were removed with Gblocks (version 0.91b) using the following parameters: minimum length of a block after gap cleaning of 5, gaps were allowed in the final alignment if they were within an appropriate block, all segments with contiguous nonconserved positions bigger than 8 were rejected, and a