# Evolution and plasticity of the gene regulatory networks controlling sex determination: of masters, slaves, usual suspects and usurpators.

Amaury Herpin

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Amaury Herpin. Evolution and plasticity of the gene regulatory networks controlling sex determination: of masters, slaves, usual suspects and usurpators.. Life Sciences [q-bio]. University of Rennes, 2022. tel-03833228

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# HABILITATION à DIRIGER les RECHERCHES (HDR)

Ecole Doctorale EGAAL

Amaury HERPIN

INRAE, Fish Physiology and Genomics Institute

Evolution and plasticity of the gene regulatory networks controlling sex determination:
of masters, slaves, usual suspects and usurpators.

-Emphasis on fish sex determination, differentiation and maintenance-

## Referees:

-Dr Marie-Christine CHABOISSIER, Institut de Biologie Valrose (iBV), Nice, France.

-Dr Nathalie Di CLEMENTE, Hôpital St-Antoine, Site Bâtiment Kourilsky, Paris, France.

-Pr Lukas KRATOCHVIL Lukas, Department of Ecology, Faculty of Science Charles University Vinicna, Praha, Czech Republic.

## Examiners:

-Dr Eric Pailhoux, UMR BDR, INRAE, ENVA, Université Paris Saclay, France.

-Pr Gilles Salbert, Institut Génétique et Développement de Rennes, UMR 6290 CNRS, France.
Table of Contents

<table><tr><td>Presentation of the candidate</td><td>02</td></tr><tr><td>I- Experience and Current Research Work</td><td>12</td></tr><tr><td>1- Past and current scientific research career</td><td>12</td></tr><tr><td>2- Sex determination in vertebrates: current concerns</td><td>13</td></tr><tr><td>3- Research strategy: animal model and scientific value</td><td>13</td></tr><tr><td>3.1- Sex determination evolution and the central role of the dmrt1 gene in fish</td><td>16</td></tr><tr><td>3.2- Characterization of the molecular network regulating gonad differentiation in medaka</td><td>21</td></tr><tr><td>II- Current Research Work, Objectives and Perspectives</td><td>26</td></tr><tr><td>1- Context and positioning of our current research work at INRAE</td><td>26</td></tr><tr><td>(i) Functions and evolution of master sex determining genes and systems: cases studies</td><td>27</td></tr><tr><td>(ia) Dmrt1bY the medaka master sex determining gene: function and evolution</td><td>30</td></tr><tr><td>(ib) sdY: an unusual and conserved SD gene in Salmonids</td><td>34</td></tr><tr><td>(ic) Esocidae species challenge the fitness advantage and sexually antagonistic models</td><td>34</td></tr><tr><td>(id) The “improbable” sex determination in the Atlantic herring</td><td>35</td></tr><tr><td>(ii) Functional analysis of the gene regulatory networks underlying sex determination and differentiation</td><td>37</td></tr><tr><td>(iia) The dmrt1bY regulatory network of medaka</td><td>38</td></tr><tr><td>(iib) Allelic diversification after transposable element exaptation into its proximal regulatory region promoted Gsdf as the master sex determining gene of sablefish</td><td>42</td></tr><tr><td>(iii) Germline/soma interactions, gonadal morphogenesis and plasticity</td><td>46</td></tr><tr><td>(iia) The autosomal gsdf gene acts as a male sex initiator in medaka fish</td><td>46</td></tr><tr><td>(iib) Of the pronephric contribution to the differentiating gonads in fish</td><td>47</td></tr><tr><td>(iv) Autophagy in fish</td><td>54</td></tr><tr><td>2- Discussion (evolution of master sex determining genes)</td><td>56</td></tr><tr><td>3- Perspectives</td><td>58</td></tr><tr><td>(i) TGF-β molecules challenge the paradigms</td><td>58</td></tr><tr><td>(ia) Emergence of the TGF-β signalling molecules ahead of the sex determining networks in fish</td><td>58</td></tr><tr><td>(ib) A central role for gonadal TGF-β signalling?</td><td>58</td></tr><tr><td>(ic) TGF-β signalling specificity</td><td>60</td></tr><tr><td>(ii) Physiological readout of the neo-functionalization of the medaka Dmrt1 paralogs (Dmrt1a vs Dmrt1bY)</td><td>65</td></tr><tr><td>(iii) Of the pronephric contribution to the differentiating gonads in fish... at a cellular resolution</td><td>68</td></tr><tr><td>4- References</td><td>69</td></tr><tr><td>5- Selected publications</td><td>77</td></tr></table>
# Presentation of the candidate

## Amaury Herpin

**Chargé de Recherches**, INRA Rennes (France) Unit 1037, Fish Physiology and Genomics, team "Sexe, Ovogenèse et ComportementS".

**Senior Scientist**, Hunan Normal University (China), State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences.

Tel: +33 02 23 48 50 18

Email: amaury.herpin@inra.fr

Profile: http://www.researcherid.com/rid/B-8950-2011

Citizenship: French

Date and place of Birth: 06/23/1975, Blois (41) Loir et Cher, France

Marital Status: married, two children (Nils, 2003 and Louison, 2008)

Languages: fluent in French and English (written and spoken), skills in Norwegian and German.

## Education Background - Employments

### Baccalaureat:

June 1993 – Section C (mathematics and physics) – Lycée Dessaignes – BLOIS (41), France.

### University:

1994 – 1996 **DEUG** Sciences Cursus II; University of Orléans (45).

1996 – 1997 **Licence** of Biology, Geology, and Biotechnologies; University of Orléans.

1997 – 1998 **Master** in Biology and Cellular Physiology; University of Orléans.

1998 – 1999 **DEA** in Cellular Biology (Rouen-Caen).

*Diploma thesis: « Identification and characterisation of a cDNA coding for a TGF-β receptor in the oyster Crassostrea gigas ».*

1999-2003 **Joint Ph.D.:** Universities of Caen (France) and University of Bergen (Norway)
-Laboratoire de Biologie et Biotechnologies Marines (France, M. Mathieu)
-Sars International Centre for Marine Molecular Biology / EMBL (Norway, D. Chourrout)

*Thesis: "Structural and functional characterisation of TGF-β family members in the oyster Crassostrea gigas."*

2003 - 2005 **Post-Doc.** University of Wuerzburg (Germany), Group of Manfred Schartl.

2005 - 2006 **Scientific Assistant.** University of Wuerzburg (Germany), Physiological Chemistry I.

2006 - 2014 **Civil Servant.** University of Wuerzburg (Germany), Physiological Chemistry I.

2014 – present **Chargé de Recherches I**, INRA Rennes (France), Fish Physiology and Genomics Institute, SOCS team.

2020 – present **Senior Scientist**, Hunan Normal University (China), State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences.
-**Reviewer** (peer review) for: Development, Gene, PLoS One, PLoS Genetics, Marine Biotechnology, Protein and Cell, Journal of Genetics (...)

-**Reviewer** for the National Science Foundation (NSF), USA; Agence Nationale de la Recherche (ANR), France; and Czech Science Foundation, Czech Republic.

-**Guest Editor** (2007, 2011) for a FEBS journal mini review series dealing with BMPSignalling and Vertebrate Sex Determination.

-**Editorial Board Member** at "Reproduction and Breeding" (Elsevier).

-**Member of the exam committee** for bachelor and master students at the University of Wuerzburg, Germany (2003-2014).

-**PhD referee** for the University of Singapore (2014).

-**PhD committees** INRA (2014-present).

-**Co-coordination** of the "CRISPR/Cas9 club", INRA, CNRS, INSERM, Rennes (2015-2016).

-**Coordinator** of the scientific animation at Fish Physiology and Genomics Laboratory, Rennes, France.

-**Member** of a special-topic-network (Connecting population genetics and developmental biology to elucidate vertebrate sex evolution, CONGEN-DEVSEX), ESEB, European Society for Evolutionary Biology, 2017-2020).

-**Active Member** of a working group on Genome Editing, INRA (2019-2020).

**Meetings (speaker/invited speaker):**

* 2002 Norwegian Biochemical Society Meeting, Roros, Norway. Oral Communication.

* 2003 International Society of Developmental and Comparative Immunology meeting, Scotland. Oral Communication.

* 2003 Marine Biotechnology Conference, Chiba, Japan. Oral Communication: "TGF-β superfamily receptors in the bivalve mollusc Crassostrea gigas"

* 2006 4th International Symposium on Vertebrate Sex Determination, Hawaii, USA. Oral Communication: "Inhibition of male germ cell proliferation by Dmrt1bY".

* 2008 International Symposium for Gonad and Brain Sex Differentiation, Fukuoka, Japan. Oral Communication: "Expression, Regulation and Sex Determining Function of Dmrt1bY in Medaka".

* 2014 5th EFOR Annual Meeting, Paris, France. Invited Speaker: "Transcriptional rewiring of the sex-determining dmrt1 gene: how Dmrt1bY was born in Medaka fish".

* 2014 2nd Strategic Meeting for Medaka research (PI meeting), Sevilla, Spain. Invited speaker.
*2014 27th Conference of European Comparative Endocrinologists, Rennes, France.
Invited speaker.

*2015 7th International Symposium on Vertebrate Sex Determination, Hawai, USA. Oral Communication: "A novel evolutionary conserved mechanism of RNA stability regulates dmrt1bY expression in PGCs prior to the sex determination stage". 1st *ex aequo* best talk.

*2017 1st European Symposium on sex determination in vertebrates, Dinard, France.
Invited speaker. "Insertion of a transposable element rewires transcriptional regulation of a novel sex-determining gene".

*2018 European Society for Evolutionary Biology (ESEB) Special Topic Network, Prague, Czech Republic. Invited speaker. "Connecting genome-wide approaches and functional genomics for elucidating evolution in motion in fish."

*2018 8th International Symposium on Vertebrate Sex Determination, Hawai, USA. Oral Communication: "Sox5 is involved in germ-cell regulation and sex determination in medaka following co-option of nested transposable elements".

*2019 Workshop: "Paradigm shift in sex chromosome evolution", Berlin, Germany.
Invited speaker.

*2019 Workshop: "CRISPR/Cas9 improvement in bivalve molluscs, IFREMER",
Nantes, France. Invited speaker.

**Teaching Experience:**

* 1999 Lab course (128 hours) Reproduction and development, University of Caen, France.

* 2004-2014 Lab course, seminar and tutorials, Developmental biology using Medaka, University of Wuerzburg. Medical students (2nd year, 22 hours per semester).

* 2015-2018 Master II, Biology of reproduction; University of Tours (4 hours).

**Students:**

* 2008 J. Driessle, Bachelor Biomedicine (supervisor).

* 2010 J. Klughammer, Bachelor Biomedicine (supervisor).

* 2010 M. Engel, Bachelor Biomedicine. (supervisor).

* 2010-2012 M. Zehner, Ph.D. medical (supervisor).

* 2010-2012 M. Hinzmann, Bachelor/Diploma Biomedicine (supervisor).

* 2008-2014 P. Fischer, Ph.D. biology (co-supervisor).

* 2012-2016 M. Adolfi, Ph.D. biology (co-supervision). Sao Paulo University, Brasil.

* 2012-2016 S. Bertho, Ph.D. biology (co-supervision/Ph.D committee).

* 2014-2017 M. Pan, PhD. biology (co-supervision/Ph.D committee).

* 2015-2019 A. Martinez, PhD. biology (supervisor).

* 2017-2020 B. Imarazene, PhD. biology (co-supervisor).

* 2017 C. Pagneux, DUT biology (supervisor).

* 2017 C. Lane, Master II biology (supervisor).

* 2016-2019 L. Lescat, Ph.D biology (co-supervisor).

* 2018-2020 J. Kempf, Ph.D biology (supervisor).

* 2020 S. Petit, DUT biology (supervisor).
## Grants:

* 2007-2010 GRK1048, #7F57C Pluripotency and differentiation of the germ line. (Partner/ PI: M. Schartl)

* 2010-2013 NSF (1R01GM085318) Mechanisms of sex determination in zebrafish. (Partner/ PI: J. Postlethwait)

* 2012-2015 ANR (11-BSV7-0016) Role and evolution of SdY in salmonids. (Partner/ PI: Y. Guiguen)

* 2015-2019 DFG (HE7135/2-1) Post-transcriptional regulation of Dmrt1 during sexual development. (Coordinator)

* 2015-2017 ACI /dept. PHASE INRA (Coordinator)

* 2017-2021 COFASP ERA-net (ANR-16-COFA-0004-01) AquaCRISPR (Co-coordinator)

* 2018-2021 ANR Fish'n Chap (Partner)

* 2019-2021 AquaExcel 3.0, European Project (Task leader)

* 2020-2023 TUNESAL Robust Atlantic Salmon Through Fine-Tuned Genome Editing (Research Project- HAVBRUK2, PN: 294971, Task leader)

* 2020-2025 111 Project (China, Grant No. D20007, Partner)

## Functional Organigram of my scientific Environment (INRA)

### - Laboratory:

<figure><img src="image_2.png" /></figure>
- Team:

<figure><img src="image_3.png" /></figure>

Functional diagram of my work interactions with my colleagues at INRA/LPGP

Within the "Fish Physiology and Genomics Institute" (LPGP), I belong to the "Sex Oogenesis and behaviours" (SOCs) team. There I have developed a thematic dealing with functional genomics approaches for studying sex determination (SD). In addition to students, I directly supervise one assistant engineer (Alexandra Depincé). Within the SOCs team I daily interact and collaborate with Yann Guiguen (evolution of sex determination through genomic approaches) and Julien Bobe (functional genomics applied to oocyte development). Within the laboratory I developed several collaborations mainly focusing on genome editing in salmonids and single cell transcriptomics (Pierre-Yves Rescan).

At INRA I am mainly collaborating with Iban Seiliez (INRA, NuMeA, see page 54). That long-term and fruitful collaboration, not centred on sex determination but rather on autophagy, nevertheless allows us to get fundings and students in co-supervision (two PhD students so far), and... good publications [see papers #37, #46 and #47 in the publication list]. I also collaborate with Gael LeTrionnaire at INRA for setting up genome editing in insects [see papers #34, #41 and BC#5]. Last, I also like to collaborate with Eric Paillhoux (INRA, BDR, ENVA). Working on mammalian species we always use that opportunity for conducting comparative approaches in order to confer a higher visibility to our respective studies (we wrote several ANR grants together) [see papers #34, #39, #40].
- Ongoing international collaborations:

<figure><img src="image_4.png" /></figure>

Since I have been mainly working abroad, I have also developed an international network of reliable collaborations.

**Manfred Schartl** is the person with who I have been working for more than 10 years on sex determination, first as postdoc and then progressively independently. During that period, I could settle down and fine tune my own thematic, studying the evolution and plasticity of the underlying gene regulatory networks controlling and regulating sex determination (SD) in fish. Still, Manfred is my main collaborator and we regularly write grant applications together [see papers #5, #9, #13 to #18, #20 to #31, #33 to #36, #38 to #40, #42 to #45, #48 and #49]. **Minoru Tanaka** is also working on medaka SD, and we have been collaborating in a concerted manner for many years. Hence, we do not only share transgenic lines and molecular tools, but also data and information. Our collaborations have already resulted in common publications [see papers #18, #21, #28 and #29, and #42]. Currently, both of us have interest in autophagy and sexual development in medaka, topic for which we share mutant medaka lines. More recently I have been working with **Yusuke Takehana**, who is a young professor working on evolution of sex determination systems in the *Oryzias* lineage [see paper #45]. Thanks to Yusuke, I can get access to valuable genetic resources (different medaka species and populations). With the help of **Dagmar Wilhelm**, Institute for Molecular Bioscience, University of Queensland, Australia, we are also considering mammalian models, still having in mind a comparative and evolutionary view of sex determination mechanisms. This comparative approach will be extended to many aspects of our projects (pronephric contribution, gene regulatory network evolution, TGF-β...) using the mouse model in order to confer a higher scientific visibility to our research [see papers #36 and #40]. Dagmar is nowadays a very reliable collaborator. **Thomas Müller** (Julius-von-Sachs Institute, Germany) is a structural biologist/protein chemist internationally recognized in the field of TGFβ/BMP. Thomas usually brings support to our projects (sex determination, TGF-β and autophagy) giving his expertise on protein chemistry and structural biology to explore ligand/receptors and protein/protein interactions using molecular modelling, experimental structure analysis (X-ray crystallography) and protein chemistry. We know each other very well and have been collaborating already [see paper #39]. He will again collaborate with us in the future, especially with the TGF-β (ANR grant submitted) and autophagy projects (see below). **Lazaro Centanin/Jochen Wittbrodt** are good friends of mine also working on medaka. We regularly share mutant and transgenic (brainbow) lines and develop new technics together [see paper #36]. **Rolf Edvardsen/Anna Wargelius** are Norwegian colleagues I met during my PhD time in Bergen. We now collaborate to adapt technical protocols (KO, KI, allelic replacement...) from medaka to salmon and therefore wrote several successful grants together.
Publications:

50 publications, 5 book chapters; h-index: 24 (Thomson Reuters).

1. **Herpin, A.**, Favrel, P. & Cunningham, C. (2002) Gene structure and expression of cg-ALR1, a type I activin-like receptor from the bivalve mollusc Crassostrea gigas, *Gene*. **301**, 21-30. (IF=2.778)

2. **Herpin, A.**, Badariotti, F., Rodet, F. & Favrel, P. (2004) Molecular characterization of a new leucine-rich repeat-containing G protein-coupled receptor from a bivalve mollusc: evolutionary implications, *Biochim Biophys Acta*. **1680**, 137-44. (IF=3.810)

3. **Herpin, A.**, Lelong, C. and Favrel, P. (2004) Transforming growth factor-beta-related proteins: an ancestral and widespread superfamily of cytokines in metazoans, *Dev Comp Immunol*. **28**, 461-85. (IF=2.652)

4. Huvet, A., **Herpin, A.**, Degremont, L., Labreuche, Y., Samain, J. F. and Cunningham, C. (2004) The identification of genes from the oyster Crassostrea gigas that are differentially expressed in progeny exhibiting opposed susceptibility to summer mortality, *Gene*. **343**, 211-20. (IF=2.705)

5. Kluver, N., Kondo, M., **Herpin, A.**, Mitani, H. and Schartl, M. (2005) Divergent expression patterns of Sox9 duplicates in teleosts indicate a lineage specific subfunctionalization, *Dev Genes Evol*. **215**, 297-305. (IF=2.549)

6. **Herpin, A.**, Lelong, C., Becker, T., Rosa, F. M., Favrel, P. and Cunningham, C. (2005) Structural and functional evidences for a type 1 TGF-beta sensu stricto receptor in the lophotrochozoan Crassostrea gigas suggest conserved molecular mechanisms controlling mesodermal patterning across bilateria, *Mech Dev*. **122**, 695-705. (IF=3.838)

7. **Herpin, A.**, Lelong, C., Becker, T., Rosa, F., Favrel, P. and Cunningham, C. (2005) Structural and functional evidence for a singular repertoire of BMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests a shared ancestral BMP/activin pathway, *Fehs J*. **272**, 3424-40. (IF=3.415)

8. **(Herpin, A., Gueguen, Y.) equal contribution**, Aumelas, A., Garnier, J., Fievet, J., Escoubas, J. M., Bulet, P., Gonzalez, M., Lelong, C., Favrel, P. and Bachere, E. (2006) Characterization of a defensin from the oyster crassostrea gigas: Recombinant production, folding, solution structure, antimicrobial activities and gene expression, *J Biol Chem*. **281**, 313-323. (IF=5.808)

9. **Herpin, A.**, Rohr, S., Riedel, D., Kluever, N., Raz, E., and Schartl, M. (2007) Specification of primordial germ cells in medaka (Oryzias latipes), *BMC Dev Biol*. **7**, 3. (Highly accessed, top 10 most viewed articles of all time in 2009, 2010 and 2011). (IF=3.337)

10. **Herpin, A.**, Lelong, C., Becker, T., Favrel, P., and Cunningham, C. (2007) A tolloid homologue from the pacific oyster Crassostrea gigas, *Gene Expr Patterns*. **7**, 700-708. (IF=2.238)

11. **Herpin, A.** (2007) BMP signaling pathways, *FEBS J*. **274**, 2959. (IF=3.396) * Cover picture of the issue.*

12. **Herpin, A.** and Cunningham, C. (2007) Cross-talk between the Bone Morphogenetic Protein pathway and other major signaling pathways results in tightly regulated cell-specific outcomes, *FEBS J*. **274**, 2977-85. (IF=3.396)

* Top-Cited Paper Award.

13. Hornung, U., **Herpin, A.**, and Schartl, M. (2007). Expression of the male determining gene dmrt1bY and its autosomal coorthologue dmrt1a in Medaka, *Sex Dev*. **3**, 197-206. (IF=1.000)

14. **Herpin, A.**, Schindler, D., Kraiss, A., Hornung, U., Winkler, C. and Schartl, M. (2007) Inhibition of germ cell proliferation by the medaka male determining gene Dmrt1bY, *BMC Dev Biol*. **7**, 99. (IF=3.337) (Highly accessed).

15. **Herpin, A.**, Fischer, P., Liedtke, D., Kluever, N., Neuner, C., Raz, E. and Schartl, M. (2008) Sequential SDF1a and b – induced mobility guides Medaka PGC migration, *Dev Biol*. **320**, 319-327. (IF=4.416)

* Cover picture of the issue.

16. **Herpin, A.**, and Schartl, M. (2008) Regulatory putsches make new ways of determining sexual development, *EMBO reports*. **9**(10) 966-8. (IF=7.099)

17. Kluever, N., **Herpin, A.**, Braasch, I., Driessle, J. and Schartl, M. (2009) Regulatory back-up circuit of Medaka Wt1 co-orthologs ensures PGC maintenance, *Dev Biol*. **325**(1):179-88. (IF=4.379)

18. **Herpin, A.**, Nakamura, S., Wagner, T., Tanaka, M. and Schartl, M. (2009) A highly conserved mRNA sequence motif directs differential gonadal regulation of mRNA during gonad development, *Nucleic Acids Research*. **37**(5):1510-1520. (IF=7.479)

19. Le Quere, H., **Herpin, A.**, Huvet, A., Lelong, C. and Favrel, P. (2009) Structural and functional characterization of an Activin type II receptor from the pacific oyster Crassostrea gigas, *Gene*. **436**, 1-2. (IF=2.416)

20. **Herpin, A.** and Schartl, M. (2009) Molecular mechanisms of sex determination and evolution of the Y chromosome: insights from the medakafish (*Oryzias latipes*), *Molecular and Cellular Endocrinology* **306**(1-2):51-58. (IF=3.503)

21. **Herpin, A.**, Braasch, I., Kraeusling, M., Schmidt, C., Thoma, E. C., Nakamura, S., Tanaka, M. and Schartl, M. (2010) Transcriptional rewiring of the sex determining *dmrt1* gene duplicate by transposable elements, *PLoS Genetics*. **6**(2). (IF=9.543)

22. Research highlights: Male regulator switched (2010), *Nature*. **463** (1003), doi:10.1038/4631003c. (IF=36.101)

23. **Herpin, A.** and Schartl, M. (2011) Vertebrate sex determination: challenging the hierarchy, *FEBS journal*. **278**(7):1001. (IF=3.790) * Cover picture of the issue.*

24. **Herpin, A.** and Schartl, M. (2011) Dmrt1 at the crossroad: a widespread and central class of sexual development factors in fish, *FEBS journal*. **278**(7):1010-1019. (IF=3.790) * Top-Cited Paper Award.*

25. Thoma, E. C., Wagner, T. U., Weber, I. P., **Herpin, A.**, Fischer, A. and Schartl, M. (2011) Ectopic expression of transcription factors enables directed differentiation of a Medaka spermatogonial cell line, *Stem Cells Dev*. **20**(8): 1425-1438. (IF=4.459)
26. He, X., Yan, Y-L., Eberhart, J. K., **Herpin, A.**, Wagner, T. U., Schartl, M., and Postlethwait, J. H. (2011) miR-196 regulates axial patterning and pectoral appendage initiation, **Dev Biol.** 357(2): 463-477. (IF=4.069)

27. **Herpin, A.** and Schartl, M. (2011) Sex determination: switch and suppress, **Curr Biol.** 21(17): R656-659. (IF=9.647)

28. **Herpin, A.**, Adolfi, M., Nicol, B., Hinzmann, M., Schmidt, C., Klughammer, J., Engel, M., Tanaka, M., Guiguen, Y. and Schartl, M. (2013) Divergent expression regulation of gonad development genes in medaka shows incomplete conservation of the downstream regulatory network of vertebrate sex determination, **Mol Biol Evol.** 30(10): 2328-46. (IF=14.308)

29. Nishimura, T., **Herpin, A.**, Kimura, T., Hara, I., Kawasaki, T., Nakamura, S., Yamamoto, Y., Saito, T. L., Yoshimura, J., Morishita, S., Tsukahara, T., Kobayashi, S., Naruse, K., Shigenobu, S., Sakai, N., Schartl, M. and Tanaka, M. (2013) Cell autonomous sexual identity of germ cells by sex chromosomes prior to the gonadal formation in medaka, **Development.**141(17):3363-9. (IF=6.273)

30. **Herpin, A.**, Englberger, E., Zehner, M., Wacker, R., Gessler, M. and Schartl, M. (2015) Defective autophagy through *epg5* mutation results in germ plasm and mitochondria reduction failure during spermatogenesis, **FASEB J.** 29(10)4145-61. (IF=5.299)

31. **Herpin, A.**, Schartl, M. (2015) Plasticity of the gene regulatory networks controlling sex determination: of masters, slaves, usual suspects, newcomers and usurpators, **EMBO reports.** 16(10):1260-74. (IF=7.739)

32. Zhang, X., Guan, G., Li, M., Zhu, F., Liu, Q., Naruse, K., **Herpin, A.**, Li, J., Nagahama, Y. and Hong, Y. (2016) Autosomal *gsdf* acts as a male sex initiator in the fish medaka, *Sci Rep.* 6, 19738. (IF=4.259)

33. Pan, Q., Anderson, J., Bertho, S., **Herpin, A.**, Wilson, C., Postlethwait, J.H., Schartl, M., and Guiguen, Y. (2016) Vertebrate sex-determining genes play musical chairs, **C R Biol** 339, 258-262. (IF=0.731)

34. Bertho, S., Pasquier, J., Pan, Q., Le Trionnaire, G., Bobe, J., Postlethwait, J.H., Pailhoux, E., Schartl, M., **Herpin, A.**, and Guiguen, Y. (2016) Foxl2 and Its Relatives Are Evolutionary Conserved Players in Gonadal Sex Differentiation, **Sex Dev** 10, 111-129. (IF=1.972)

35. Adolfi, M.C., **Herpin, A.**, Regensburger, M., Sacquegno, J., Waxman, J.S., and Schartl, M. (2016) Retinoic acid and meiosis induction in adult versus embryonic gonads of medaka. **Sci Rep** 6, 34281. (IF=4.259)

36. Schartl, M., Schories, S., Wakamatsu, Y., Nagao, Y., Hashimoto, H., Bertin, C., Mourot, B., Schmidt, C., Wilhelm, D., Centanin, L., Guiguen, Y. and **Herpin, A.** (2018) Sox5 is involved in germ cell regulation and sex determination in medaka following co-option of nested transposable elements, **BMC Biology** 16(1):16.

*Included into the cross journal collection "In the Light of Evolution" (BMC Biology, BMC Evolutionary Biology and Biology Direct).* (IF=6.723)

37. (**Herpin, A.**, Lescat, L.), Mourot, B., Véron, V., Guiguen, Y., Bobe, J. and Seiliez, I. (2018) CMA restricted to mammals and birds: myth or reality? **Autophagy** 20, 1-4. (IF=11.059)

38. Regneri, J., Klotz, B., Wilde, B., Kottler, V., Hausmann, M., Kneitz, S., Regensburger, M., Maurus, K., Götz, R., Lu, Y., Walter, R., **Herpin, A.** and Schartl, M. (2018) Analysis of the putative tumor suppressor gene *cdkn2ab* in pigment cells and melanoma of *Xiphophorus* and medaka, *Pigment cell and melanoma research* doi: 10.1111/pcmr.12729 (IF=4.172)

39. Bertho, S., **Herpin, A.**, Jouanno, E., Yano, A., Nicol, B., Müller, T., Pannetier, M., Pailhoux, E., Miwa, M., Yoshizaki, G., Schartl, M. and Guiguen, Y. (2018) The unusual rainbow trout sex determination gene hijacked the canonical vertebrate gonadal differentiation pathway, **PNAS** 115(50):12781-12786. (IF=9.58)

40. **Herpin, A.**, Schmidt, C., Kneitz, S., Gobé, C., Regensburger, M., Le Cam, A., Montfort, J., Adolfi, M.C., Lillesaar, C., Wilhelm, D., Kraeusling, M., Mourot, B., Porcon, B., Pannetier, M., Pailhoux, E., Ettwiller, L., Dolle, D., Guiguen, Y. and Schartl, M. (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 Biology** *Apr 4;17(4):e3000185* (IF=7.076)

41. Le Trionnaire, G., Tanguy, S., Hudaverdian, F., Gleonnec, F., Richard, G., Cayrol, B., Monsion, B., Pichon, E., Deshoux, M., Webster, C., Uzest, M., (Tagu, D. and **Herpin, A.**) (2019) An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid, **Insect Biochemistry and Molecular Biology** Jul; 110:34-44; doi: 10.1016/j.ibmb.2019.04.016. (IF=3.827)

42. Adolfi, M. C., Fisher, P., **Herpin, A.**, Regensburger, M., Kikuchi, M., Tanaka, M. and Schartl, M. (2019) Increase of cortisol levels after temperature stress activates *dmrt1a* causing female-to-male sex reversal and reduced germ cell number in medaka, **Molecular Reproduction and Development Oct; 86(10):1405-1417; doi: 10.1002/mrd.23177.** (IF=2.823)

43. Pan, Q., Feron, R., Yano, A., Guyomard, R., Jouanno, E., Vigouroux, E., Wen, M., Busnel, JM., Bobe, J., Concordet, JP., Parinello, H., Journot, L., Klopp, C., Luuch, J., Roques, C., Postlethwait, J., Schartl, M., **Herpin, A.** and Guiguen, Y. (2019) Identification of the master sex determining gene in the Northern pike (*Esox Lucius*) reveals restricted sex chromosome differentiation, **PLoS genetics** Aug 22;15(8):e1008013. (IF=5.174)

44. Feron, R., Zahm, M., Cabau, C., Klopp, C., Roques, C., Bouchez, O., Eché, C., Valière, S., Donnadieu, C., Haffray, P., Bestin, A., Morvezen, R., Acloque, H., Euclide, P., Wen, M., Jouano, E., Schartl, M., Postlethwait, J., Schraidt, C., Christie, M., Larson, W., **Herpin, A.** and Guiguen, Y. (2020) Characterization of a Y-specific duplication/insertion of the anti-Müllerian hormone type II receptor gene based on a chromosome-scale genome assembly of yellow perch, *Perca flavescens*, **Molecular Ecology Resources** doi: 10.1111/1755-0998.13133. (2017 IF: 7.049)

45. Takehana, Y., Zahm, M., Cabau, C., Klopp, C., Roques, C., Bouchez, O., Donnadieu, C., Barrachina, C., Journot, L., Kawaguchi, M., Yasumasu, S., Ansai, S., Naruse, K., Inoue, K., Shinzato, C., Schartl, M., Guiguen, Y. and **Herpin, A.** (2020) Genome sequence of the euryhaline Javafish medaka, *Oryzias javanicus*: a small aquarium fish model for studies on adaptation to salinity, **G3 Genes Genomes Genetics** doi: 10.1534/g3.119.400725. (2017 IF: 2.742)

46. Lescat, L., Véron, V., Mourot, B., Dias, K., Riera, N., Beaumatin, F., Panserat, S., Guiguen, Y., Bobe, J., **Herpin, A.** and Seiliez, I. (2020) Chaperon-Mediated Autophagy in the light of evolution: insight from fish, **Molecular Biology and Evolution**, https://doi.org/10.1093/molbev/msaa127. (2018 IF: 14.797)

47. **Herpin, A.**, Lescat, L., Bobe, J., Jenny, A. and Seiliez, I. (2020) Lighting Chaperone-Mediated_Autophagy (CMA)
evolution with an ancient LAMP: the existence of a functional CMA activity in fish, *Autophagy* Aug 10:1-3. doi: 10.1080/15548627.2020.1797344. (2018 IF: 11,059)

48. Wen, M., Feron, R., Pan, Q., Guguin, J., Jouanno, E., **Herpin, A.**, Klopp, C., Cabau, C., Zahm, M., Parrinello, H., Journot, L., Burgess, S., Omori, Y., Postlethwait, J., Schartl, M. and Guiguen, Y. (2020) Sex chromosome and sex locus characterization in goldfish, *Carassius auratus* (Linnaeus, 1758), **BMC genomics**, Aug 11;21(1):552. doi: 10.1186/s12864-020-06959-3. (2017 IF: 3.730)

49. **(Herpin, A.**, Rafati, N., Chen, J.), Petterson, M., Han, F., Feng, C., Wallerman, O., Rubin, CJ., Péron, S., Cocco, A., Larsson, M., Trötschel, C., Poetsch, A., Korsching, K., Bönigk, W., Körschen, HG., Berg, F., Folkvord, A., Kaupp, UB., Schartl, M. and Andersson, L. (2020) Reconstruction of the birth of an unexpected male sex chromosome in Atlantic herring, **PNAS** (2018 IF=9.58)

50. Imarazene, B., Beille, S., Jouanno, E., Branthonne, A., Thermes, V., Thomas, M., **Herpin, A.**, Retaux, S., Guiguen, Y. (2020) Histoligical and molecular characterization of gonadal differentiation in Pachon cavefish Astyanax mexicanus, **Sex. Dev.** (2018 IF= 2.25)

51. Pan, Q., Kay, T., Depincé, A., Adolfi, M., Schartl, M., Guiguen, Y. and **Herpin, A.** (2021) Evolution of master sex determiners: TGF-β signalling pathways at the crossroads, **Phil. Trans. R. Soc. B.** (2018 IF= 6.139).

(submitted, under review, in revision)

1. Klionsky, D., (...), A., **Herpin**, (...), and Z., Zhang. (2020) Guidelines for the use and interpretation of assays for monitoring Autophagy, (in revision in *Autophagy*, (2018 IF: 11,059))

2. Bertho, S., **Herpin, A.**, Schartl, M. and Guiguen, Y. (2020) Lessons from an unusual vertebrate sex-determining gene, (in revision, *Phil. Trans. R. Soc. B.* (2018 IF= 6.139)).

3. Adolfi, M., **Herpin, A.** and Schartl, M. (2020) The replaceable master of sex determination: bottom-up hypothesis revisited, (in revision, *Phil. Trans. R. Soc. B.* (2018 IF= 6.139)).

4. Pan, Q., Feron, R., Jouanno, E., Darras, H., **Herpin, A.**, Koop, B., Rondeau, E., (...) Schartl, M., Postlethwait, J. and Guiguen, Y. (2020) The rise and fall of the ancient northern pike master sex determining gene, (under review, *E-life* (2018 IF= 7.08)).

5. Feron, R., Pan, Q., Wen, M., Imarazene, B., Jouanno, E., Anderson, J., Herpin, A., Journot, L., (...) Postlethwait, J., Schartl, M. and Guiguen, Y. (2020) RADSex: a computational workflow to study sex determination using Restriction Site-Associated DNA Sequencing data, (In revision, Molecular Ecology Resources (2018 IF=6.286)).

6. **Herpin, A.**, Schartl, M., Depincé, A., Guiguen, Y., Bobe, J., Hua-Van, A., Hayman, E., Octavera, A., Yoshizaki, G., Nichols, K., Goetz, G. and Luckenbach, A. (2020) Allelic diversification after transposable element exaptation into its proximal regulatory region promoted Gsdf as the master sex determining gene of sablefish *Anoplopoma fimbria*, (to be submitted to Genome Biology)

**Publications in 32 different journals:**

Autophagy, BMC Biology, BMC Developmental Biology, BMC Genomics, Biochim Biophys Acta, Comptes Rendus Biologie, Current Biology, Development, Developmental and Comparative Immunology, Development Genes and Evolution, Developmental Biology, EMBO reports, FASEB Journal, FEBS Journal, G3 Genes Genomes Genetics, Gene, Gene Expression Patterns, Insect Biochemistry and Molecular Biology, Journal of Biological Chemistry, Mechanisms of Development, Molecular Biology and Evolution, Molecular and Cellular Endocrinology, Molecular Reproduction and Development, Molecular Ecology Resources, Nature, Nucleic Acids Research, Philosophical Transactions of the Royal Society B, PLoS Biology, PLoS Genetics, Pigment cell and melanoma research, PNAS, Sexual Development, Scientific Reports, Stem Cells Dev.

Book Chapters

1. Guiguen, Y., Fostier, A., and **Herpin, A.** (2017) **Book chapter**: Sex determination and differentiation in fish: genetic, genomic and endocrine aspects in “Sex control in aquaculture” (Wang, H.P., F. Piferrer & S.L Chen Ed.). ISBN: 978-1-119-12726-0. Wiley-Blackwell. DOI:10.1002/9781119127291. (cover picture)

2. Guiguen, Y., Bertho, S., **Herpin, A.**, and Fostier, A. (2017) **Book chapter**: Sex determination and control in salmonidae in “Sex control in aquaculture” (Wang, H.P., F. Piferrer & S.L Chen Ed.). ISBN: 978-1-119-12726-0. Wiley-Blackwell. DOI:10.1002/9781119127291.

3. Quiaowei, P., Guiguen, Y., and **Herpin, A.** (2017) **Book chapter**: Evolution of sex determining genes in fish. Encyclopedia of Reproduction, Second Edition. Elsevier.

4. Schartl, M. and **Herpin, A.** (2017) **Book chapter**: Sex determination systems and sex determiners in vertebrates. Encyclopedia of Reproduction, Second Edition. Elsevier.

5. Bertin, C. and **Herpin, A.** (2018) **Book chapter**: “Analyse de la fonction d’un gène par mutagénèse ou transgénèse chez les poisons” dans “Principes des techniques de biologie moléculaire et génomique”. Editions QUAE. ISBN 978-2-7592-2885-0.
Cover Pictures & Highlights

<figure><img src="image_5.png" /></figure>

Sex Control in Aquaculture
By Hanping Wang, Francesc Piferer, Songlin Chen, Zhi-Gang Shen

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>
# I- Experience and Current Research Work

## 1- Past and Current Scientific Research Career

I have received my undergraduate degree in cellular biology from the University of Caen (France, Laboratory of Biology and Marine Biotechnologies / IFREMER), working on the early development of one of the world's most economically important bivalve species for aquaculture: The Pacific oyster (*Crassostrea gigas*).

To acquire a broader expertise, and as a follow up of this work, I have then joined the Sars International Centre for Marine Molecular Biology / EMBL in Bergen (Norway) where I have obtained my PhD degree (jointly between the Universities of Caen and Bergen) in 2003. Intending at gaining insight into the mechanisms triggering early development, reproduction, fertility and immunity, the subject of my PhD was dealing with the characterization of the morphogenetic roles of oyster TGF-β superfamily receptors during early development as well as their involvement as cytokine receptors. To circumvent experimental limitations inherent to the oyster model, for functional analyses, I have set up and used the zebrafish as a “reporter organism” [see papers #1 to #4, #6 to #8, #10 to #12 and #19].

Thanks to the experience gathered with fish model organisms (zebrafish, salmon, hagfish) during my PhD, I have then taken the opportunity to join the group of Pr. M. Schartl at the University of Wuerzburg (Germany, Physiological Chemistry), one of the leading teams working on fish sex determination. There, over more than ten years, I have been studying the different aspects of molecular sex determination and gonad formation and differentiation using the medaka fish (*Oryzias latipes*) as a main model organism [see papers #5, #9, #13 to #15, #16 and #17, #21, #25, #28 and #29, and #32]. During that period, I have settle down my thematic and established my own group. I then got interest in studying, on a larger scale, the evolution and plasticity of the underlying gene regulatory networks controlling and regulating such mechanisms in metazoans. Using functional genomic approaches, this also brought us to face, challenge and discuss the current evolutionary concepts for the diversity of sex determining mechanisms and evolution of sex chromosomes [see papers #16, #22 to #24, #27 and #28 and #31].

Being willing to pursue my career on that specific thematic for which I gained a kind of expertise in the field, in 2015 I have joined as a full-time researcher the INRA Fish Physiology and Genomics Laboratory (France, UR1037, LPGP, PHASE). There, in line with my past scientific background and still keeping the same “editorial policy”, I have developed cutting edge techniques of genome editing (CRISPR-engineered KO and KI, conditional KI...) and other genomic (high throughput genome sequencing, in-vivo ChIP-seq) and functional genomic (cell lineage tracing, topological single cell transcriptomiques) approaches in fish to better tackle our current/long standing concerns [see papers #33 to #49].

In the following I will first describe the current concerns in the vertebrate sex determination field, and how I have been trying to address them as a guiding thread for my research using teleost fish. Then, I will present the context and positioning of my research at INRAE and discuss the main results. Finally, after a general discussion, I will give some perspectives to illustrate the way forward I would like to conduct my research in the future.
## 2- Sex Determination in Vertebrates: Current Concerns

Sexual dimorphism is one of the most pervasive and diverse features of animal morphology, physiology, and behaviour. Despite the generality of the phenomenon itself, the mechanisms how sex is determined are very different among various organismic groups, have evolved repeatedly and independently across metazoans, and the underlying molecular pathways can change quickly during evolution [see papers #23, #27, #31 and BC#1 to BC#4]. Even within closely related groups of organisms for which the development of gonads on the morphological, histological and cell biological level is undistinguishable, the molecular control and the regulation of the factors involved in sex determination and gonad differentiation can be substantially different [see paper #28]. The biological meaning of the high molecular plasticity of an otherwise common developmental program is unknown. While phylogenetic studies suggest that the downstream "business" ends of sex-determining pathways do tend to be more stable than the triggering mechanisms at the top (1, 2), how similar are the downstream gene networks that respond to these triggers and actually do the work? In other words, did sex determination (SD) arise several times with different regulatory mechanisms slowly converging by recruiting conserved key effectors, or is it an ancient process with little surviving evidence of ancestral genes? This question becomes truly meaningful in the light of the recent identification of related sexual regulators in different phyla indicating that at least some aspects of the sexual core regulation might be anciently co-opted or preserved [see papers #16, #24, #27, #31]. In the recent years, however, some common themes in the development of sex-specific traits in different animal lineages have started to emerge. Central to this stream has been the discovery of the DMRT1 factors, one of the most ancestral and widespread family of transcription factors involved in the molecular sex-determination cascades over phyla (3).

In stark contrast to birds and mammals, where the whole group has the same sex determination mechanisms with only enigmatic exceptions, but similar to the situation in amphibians and reptiles, the diversity of sex determination mechanisms is especially obvious in fish, where within groups of closely related species a wide spectrum of different systems can be found (4). Within teleosts, for instance in the poeciliid fish, which include guppies, mollies, platyfish and swordtails, there are reports on temperature-dependent SD (TSD) and various forms of genetic sex determination (GSD), ranging from polyfactorial SD to female and male heterogamety, multiple sex chromosomes and autosomal modifiers. Even within the same species several SD mechanisms can occur (5). The coexistence of two or more of these systems has also been reported within the same genus, like for instance, the XY/XX and ZW/ZZ genotypes that are found in different *Oreochromis* species (6) or even within the same species like in some platyfish populations (5).

## 3- Research Strategy: Animal Model and Scientific Value

For approaching numbers of questions dealing with sex determination / differentiation from either comparative evolutionary or functional point of views, I have mainly used the medaka fish, *Oryzias latipes*, as a model species. The medaka is a small freshwater fish species that lives in the small rivers and rice fields of East Asia. During the last decade, it became a widely used laboratory fish for developmental and biomedical research, comparable to the well-known zebrafish with which the medaka shares many advantages and characteristics. Medaka offers valuable genomic resources and the latest most advanced transgenic technologies, including genome editing, that are available for functional studies (7). With respect to sex determination, and in contrast to zebrafish, for which sex determination genetics and mechanisms still remain obscure, the medaka has a XX/XY genetic sex determination system with homomorphic sex chromosomes and the
master male sex determination gene (*dmrt1bY*) is known and has been characterized (8, 9). Interestingly this *dmrt1bY* gene is however absent from closely related species (10). *De facto* relying on different master sex determining genes, these evolutionary switches offer interesting opportunities for studying how transitions among sex-determining mechanisms can occur in closely related species of the same genus (Figure 1)...

<figure><img src="image_8.png" /><figcaption>**Figure 1. Phylogenetic relationships and sex determination systems in *Oryzias* fish species.**</figcaption></figure>

Medaka fish (*Oryzias* genus) show an amazing diversity regarding to their sex determination systems and sex chromosomes, providing an excellent model group for investigating the molecular mechanisms underlying the rapid turnover of sex chromosomes. They possess both XY and ZW systems, and their sex chromosomes differ from one species to another (11, 12).

...or in fish in general (Figure 2).
<table><caption>Figure 2. Currently known or suspected master sex determining genes and sex determination systems in vertebrates.</caption><thead><tr><th>Organism</th><th>Master Sex-Determining Gene</th><th>Sex-Determination System</th><th>Ancestral MSD gene</th><th>Establishment</th></tr></thead><tbody><tr><td>Therian mammals</td><td>SRY</td><td>XY</td><td>SOX3</td><td>Allelic Diversification</td></tr><tr><td>Monotremes / Platypus (Ornithorhynchus anatinus)</td><td>AMH</td><td>Multiple different X and Y chromosomes</td><td>AMH</td><td>Chromosome fusion and allelic diversification</td></tr><tr><td>Birds</td><td>DMRT1</td><td>ZW</td><td>Dmrt1</td><td>Allelic Diversification</td></tr><tr><td>African clawed frog (Xenopus laevis)</td><td>DM-W</td><td>ZW</td><td>dmrt1</td><td>Gene Duplication (&amp; truncation)</td></tr><tr><td>Atlantic herring (Clupea harengus)</td><td>bmpr1bbY</td><td>XY</td><td>bmpr1bb</td><td>Gene Duplication (&amp; truncation)</td></tr><tr><td>Characiformes species</td><td>gdf6-bY</td><td>XY</td><td>gdf6</td><td>Gene Duplication</td></tr><tr><td>European pike (Esox lucius)</td><td>amhbY</td><td>XY</td><td>amh</td><td>Gene Duplication</td></tr><tr><td>Rainbow trout (Oncorhynchus mykiss)</td><td>sdY</td><td>XY</td><td>irf9</td><td>Gene Duplication</td></tr><tr><td>Seriola fishes</td><td>hsd17b1</td><td>ZW</td><td>hsd17b1</td><td>Allelic Diversification</td></tr><tr><td>Chinese tongue sole (Cynoglossus semilaevis)</td><td>dmrt1</td><td>ZW</td><td>dmrt1</td><td>Allelic Diversification</td></tr><tr><td>Nile tilapia (Oreochromis niloticus)</td><td>amhY</td><td>XY</td><td>amh</td><td>Gene Duplication</td></tr><tr><td>Medaka (Oryzias latipes)</td><td>dmrt1bY</td><td>XY</td><td>dmrt1</td><td>Gene Duplication</td></tr><tr><td>Luzon rice fish (Oryzias luzonensis)</td><td>gsdf-Y</td><td>XY</td><td>gsdf</td><td>Allelic diversification</td></tr><tr><td>Indian rice fish (Oryzias dancena)</td><td>sox3-Y</td><td>XY</td><td>sox3</td><td>Allelic Diversification</td></tr><tr><td>Pejerrey (Odontesthes hatcheri) and other Odontesthes species</td><td>amhY</td><td>XY</td><td>amh</td><td>Gene Duplication</td></tr><tr><td>Turquoise Killifish (Nothobranchius furzeri)</td><td>gdf6-Y</td><td>XY</td><td>gdf6</td><td>Allelic Diversification</td></tr><tr><td>Yellow perch (Perca flavescens)</td><td>amhr2bY</td><td>XY</td><td>amhr2</td><td>Gene Duplication (&amp; truncation)</td></tr><tr><td>Stickleback (Gasterosterus aculeatus)</td><td>amhY</td><td>XY</td><td>amh</td><td>Gene Duplication</td></tr><tr><td>Lingcod (Ophiodon elongatus)</td><td>amhbY</td><td>XY</td><td>amh</td><td>Gene Duplication</td></tr><tr><td>Sablefish (Anoplopoma fimbria)</td><td>gsdf-Y</td><td>XY</td><td>gsdf</td><td>Allelic Diversification</td></tr><tr><td>Torafugu (Takifugu rubripes)</td><td>amhr2-Y</td><td>XY</td><td>amhr2</td><td>Allelic Diversification</td></tr></tbody></table>

Currently known and documented master sex determining (MSD) genes in vertebrates. Of special interest, MSD genes belonging to the TGF-β are highlighted in red (see perspectives). MSD genes that arose after allelic diversification or gene duplication are highlighted in green and blue respectively.
In that particular context of functional and comparative evolution and using the medaka fish as a model organism of reference, my past and current research projects on sex determination / sex differentiation in fish are articulated around two main general questions and subsequent precise research objectives:

**I. How stable are SD mechanisms in evolutionary terms? And what is the evolutionary meaning of the high variability of sex determination mechanisms?**

On these aspects, I have focused my research mainly on the sex determination function of *dmrt1bY* in medaka and also on the comparative functional analysis of other master sex-determining genes (*SdY*, *Amh*, *AmhR2*, *BMPR1*, *Gsdf*, *Gdf6*)...) in different fish (*Rainbow trout*, *Northern pike*, *Killifish*, *Atlantic Herring*, *Goldfish*, *Java medaka*, *Yellow perch*, *Cave fish...*)

**II. Are different SD mechanisms triggering the same, related or totally specific molecular pathways during the process of the fate differentiation of the gonad anlage?**

To address that precise point, I have directed my research toward the characterization of the gene regulatory network triggering sex differentiation with further interest on the germ line/soma interactions and gonadogenesis in medaka and in fish in general.

Being the guidelines for conducting my research, my contributions in addressing these two major questions and objectives in the field of sex determination are further developed in the following.

**3.1- *Sex determination evolution and the central role of the dmrt1 gene in fish.***

The occurrence of two different sexes and consequently the necessity to make a developmental decision for an embryo to become male or female (the so-called sex-determination process) and the further differentiation of the whole organism into two distinct phenotypes, are common throughout the animal, plant and fungi kingdoms. While developmental cascades are generally headed by highly conserved universal master regulators that univocally determine the developmental fate of a cell lineage to a tissue or organ during embryogenesis, all the evidence suggests that sex determination might disobey the conventional rules of evolutionary conservation [see papers #16, #22 and #23, #27 and #28]. Hence, decades of genetic studies have led to the global picture that the gene-regulatory cascades triggering sexual differentiation from worms and *Drosophila* to mammals bear little resemblance to each other. The remarkable diversity of ‘master sex-determining genes’ at the top of the genetic hierarchies now seems obvious, while downstream components surprisingly appear to be evolutionarily more conserved and tend to converge upon the regulation of few central common effectors [see paper #16]. Hence, a comparative view on genetic sex determination mechanisms led to the paradigm that “master change, slaves remain” (2). A well-known example illustrating this paradigm is the SRY gene – the master sex-determining gene of mammals- that has not been detected outside of the placental mammals. Conversely SRY subordinated genes (Sox9 [see paper #28], Wt1 [see paper #17], Dmrt1, AMH, SF1, FoxL2...) or signalling pathways (TGF-β, Wnt4/β-catenin, Hedgehog [see paper #28]) have homologues involved in gonadogenesis or gonadal differentiation in a much broader spectrum of species, including non-vertebrates and even protostomes lacking Sry (see Figure 3).
<figure><img src="image_9.png" /><figcaption>Figure 3. Gene-regulatory network of gonadal sex induction and maintenance in vertebrates.</figcaption></figure>

Schematic representation of main interactions within the regulatory network. In gonadal fate determination of mammals, Sry initiates activation of the male pathway (blue) through up-regulation of Sox9. Dmrt1 is not only important for keeping the male pathway on but also in suppressing the two female networks (red). These two female networks involve Foxl2 as well as the Wnt/β-catenin signalling pathways. Maintenance of gonadal identity in the differentiated gonads is a result of the cross-inhibition activities of Dmrt1 and Foxl2. A critical equilibrium between these conflicting pathways underlies the bi-potentiality of the gonadal somatic cells. Tipping the balance into one direction or the other will regulate the gonadal fate as a consequence of the activation of the male or female pathways. Solid lines define negative regulations. Dashed lines designate positive regulations. Beside the Sry ancestor Sox3 and Dmrt1, other genes (pink) can become the master sex-determining genes by similarly impacting on the seesaw between the male and female programme [see paper #31].
In contrast to this relatively good conservation at the bottom of the sex determination cascade, examples show that the master regulator is not necessarily elected -co-opted-, like SRY in mammals, from the outside to perform a new sex-determining function, but rather frequently recruited as a member of the existing cascade, usurping the position at the top [**see paper #16**]. Indeed, independent duplication events [**see papers #16**] or dosage effect led, for example, Dmrt1, AMH/AMHR or GSDF genes to take over the leadership as master sex determining gene in Medaka, *Xenopus*, Chicken or Pejerrey and *Fugu* or *Oryzias luzonensis* respectively ([**see paper #20**] and **Figure 2**). Beyond vertebrates, and definitively illustrating an evolutionary convergence driving that scheme, a similar duplication event was also described in the honeybee (3) (**Figure 4**, [**see paper #16**]).

<figure><img src="image_10.png" /><figcaption>**Figure 4.** Sex-determining cascades in the worm, insects and medaka fish.</figcaption></figure>

Molecular pathways leading to the formation of the gonad in *Caenorhabditis elegans*, *Drosophila melanogaster*, *Apis mellifera* and *Oryzias latipes*. Conserved doublesex/male abnormal 3/dsx and mab-3 related transcription factor 1 (dmrt1) or Drosophila transformer (tra)-like homologues are indicated with light-brown and light-blue boxes, respectively; duplicated factors in the cascades are also highlighted (brown and blue boxes, respectively). Note that tra of C. elegans is not related to tra of Drosophila. csd, complementary sex determiner; dmd3, doublesex/mab-3 domain family member 3; dsx, doublesex; fem, feminizer; her-1, hermaphroditization of XO-1; sdc, sex determination and dosage compensation defective; sx1, sex lethal; xol, XO lethal. [**see paper #31**].

Additionally, it is now emerging that the primary sex-determining decision is not final but has to be constantly affirmed life-long by suppressing the opposing sex determination programs, thereby challenging our basic notions of the function and evolution of the sex-determining pathways and later on of the gonadal maintenance [**see papers #26 and #28**]. Interestingly, in addition to the primary function as sex determining gene in the above-mentioned species –and probably in others as well- Dmrt1 factors have been pointed out to have an even more universal evolutionary widespread role in male gonadal maintenance (Figures 3 and 4) [**see papers #22, #23 and #26**].
Among the few highly evolutionary conserved downstream effector genes of the genetic sex determining cascades, a gene family involved in sex differentiation in organisms as phylogenetically divergent as *Caenorhabditis elegans*, *Drosophila*, frogs, fish, birds, mammals and corals is the *dmrt* gene family [**see papers #22 and #23**]. The Dmrt group of molecules is characterized by a conserved DNA-binding motif known as the DM (Doublesex and Mab-3 related) domain (3). Dmrts were originally described to play important roles during sex determination in flies and worms by regulating several aspects of somatic sexual dimorphism (13). They were also reported to be able to substitute for each other across species, indicating that their function is possibly interchangeable and that sex determination in invertebrates might rely on conserved molecules. Interestingly, recent studies have linked DM domain genes to primary sexual differentiation in many other species including medaka fish, frog and chicken, providing valuable entry points for understanding the control of sexual differentiation and evolution of regulatory networks in diverse animals [**see papers #22 and #23**]. But certainly, the deep interest of Dmrt1 in the field of sex determination in fish first came with the discovery -in my former laboratory in Germany- of a dmrt1 homologue on the Y-chromosome of the medaka fish: *dmrt1bY* (9). From then on, genetic evidences have suggested that the *dmrt1* gene is an important regulator of male development in vertebrates ([**see paper #24**] for review). In humans, haploinsufficiency of the genomic region that includes *DMRT1* and its paralogs *DMRT2* and *DMRT3* leads to XY male to female sex reversal (14). In chicken and other avian species *Dmrt1* is located on the Z chromosome, but absent from W, making it an excellent candidate for the male sex-determining gene of birds (15). Finally in frog it has been shown that DM-W, a W-linked truncated Dmrt1 homolog gene antagonizing the Dmrt1 transcriptional activity, participated in primary ovary development ((16) and [**see paper #23**]).

As mentioned above, the medaka male sex-determining gene has been identified in my former laboratory, making the medaka the first vertebrate species outside mammals where such gene has been described and characterized as being necessary and sufficient for directing testes development. With respect to its biochemical function, *Dmrt1bY*, but also the other *Dmrt1s* in fish, appear to act as transcription factors (Figure 5). This is evident from the nuclear localization of *Dmrt1* fusion proteins [**see papers #13 and #14**] and studies showing direct effects of *dmrt1* on reporter gene expression as well as binding to cognate motif in electric mobility shift assays [**see paper #21**]. Finally, linking the earliest sexual dimorphic trait to its expression dynamic, we could show *Dmrt1bY* to be possibly responsible for the male-specific primordial germ cell mitotic arrest [**see paper #9**] (Figure 5). Indeed, after cell transfection and FACS analyses, additional *in vivo* functional evidences showed that expression of *Dmrt1bY* leads to negative regulation of male primordial germ cell proliferation (G2/M arrest cell cycle) prior to sex differentiation at the sex determination stage [**see paper #9**], suggesting that in XY medaka males, *Dmrt1bY*-driven primordial germ cell number regulation, as well as determination of pre-Sertoli cells, is the primary event by which the whole gonad (germ-line and soma) would be specified through a directional cross-talk from pre-Sertoli and Sertoli cells with the primordial germ cells [**see paper #9**] (Figure 5).
<figure><img src="image_11.png" /><figcaption>**Figure 5.** Medaka *dmrt1a/dmrt1bY* regulations and functions.</figcaption></figure>

Grey arrows illustrate the different levels for which active *dmrt1a/dmrt1bY* regulation mechanisms could be shown. **Transcriptional regulation:** the feedback autoregulation of *dmrt1bY* promoter activity and transregulation by its paralogue *Dmrt1a* is a key mechanism of *dmrt1bY* transcriptional tuning. **Post-transcriptional regulation:** a highly conserved cis-regulatory motif directs differential gonadal synexpression of *dmrt1* transcripts during gonadal development. **Post-translational regulation:** *Dmrt1a* and *Dmrt1bY* have a short half-life and consequently a high turnover. **Functions:** *Dmrt1bY* inhibition of germ cell proliferation might be part of its known male determining function. [see papers #14, #18, #21, and #24].
## 3.2- Characterization of the molecular network regulating gonad differentiation in medaka.

Medaka gonad is formed by the coordinated development of two different cell lineages: the germ cells and the somatic gonadal mesoderm surrounding the germ cells. Using mRNA microinjection in one-cell staged embryos we were able to *in vivo* monitor medaka primordial germ cells (PGCs) specification and migration dynamics during gonadal formation [see paper #9]. Hence, confocal and electronic microscopic analyses revealed that PGCs can be first recognized as early as gastrulation stage. At that stage PGCs are aligned bilaterally along the trunk until somitogenesis completes and then finally migrate posteriorly in the ventrolateral region of the developing hindgut [see paper #9]. Together with the lateral plate mesoderm where they now lie, PGCs then move to the future gonadal region at the dorsal region of the hindgut where they combine to form a single undifferentiated gonadal primordium. Interestingly carrying on over-expression (capped mRNA) or transient knock-down (morpholino) experiments *via* microinjections we have additionally shown that the observed PGC migration dynamic was tightly regulated by cytokines interactions (Sdf1a/b and Cxcr4, (Figure 6)) [see paper #15] and that their maintenance was under the control of a regulatory back-up circuit mediated by Wt1 co-orthologs [see paper #17]. Shortly before hatching the germ cells of the female gonadal primordium actively proliferate and undergo meiosis while in male gonads, correlating with the male-specific onset of Dmrt1bY expression in the gonadal anlage, the PGCs remain quiescent [see paper #14]. Ten days later the first somatic gonadal dimorphisms are apparent with the formation of the acinus (the seminiferous tubule precursor) and the follicles in gonads of male and female respectively. Interestingly, during the process of gonadal differentiation in medaka, germ line soma interactions and cross talk have been shown to be of primary importance since germ cell depleted gonads develop as male while conversely an excessive number of germ cells lead to the development of a female-type gonad [see paper #14].

<figure><img src="image_12.png" /><figcaption>**Figure 6.** PGC migration in Medaka is the consequence of alternation of active OlaSDF1a/b-induced mobility.</figcaption></figure>

During the process of early PGC migration (Step I), CXCR4 and Sdf1a are absolutely required for proper PGC migration. During neurulation/early somitogenesis (Step II), PGC migration is dependant of somatic movements, while arrest of PGCs in the lateral plate mesoderm along the embryo body is due to SDF1a action. During late PGC migration (step III), the role of Sdf1b is becoming predominant while the function of Sdf1a and CXCR4 appears to be much less important. The recent description of the sequential action of the CXCR4 and CXCR7 receptors in regulating PGC migration in Medaka (17), indicates that a CXCR7-dependent process would act after the CXCR4-dependent bilateral alignment of PGC and causes drift of PGC to the gonadal area. [see papers #9 and #15].
The medaka male sex-determining gene is a duplicated version of the *dmrt1* gene. This duplicated copy was designated *dmrt1bY*. The ancestral copy, which is located on an autosome, was consequently named *dmrt1a*. The duplicated fragment, consisting of *dmrt1* and three neighbouring genes, was inserted into another chromosome while its homologue became the X ([see paper #20] and Figure 7). This duplication event occurred approximately 5-10 million years ago in the lineage leading to the medaka. Compared to the human Y, which is more than 200 million years old, the medaka Y is very young and allows studying the initial events of Y-chromosome evolution.

<figure><img src="image_13.png" /><figcaption>**Figure 7.** Origin and evolution of the sex chromosomes in medaka.</figcaption></figure>

(a) A segment on linkage group (LG) 9 containing the *dmrt1* gene and neighbouring genes including the paralogs *dmrt3* and 2 is duplicated. (b) The duplicated segment is inserted into one of the chromosomes of LG1. This chromosome is the proto Y while its homologue becomes the proto X. (c) Due to lack of recombination between the duplicated fragment on the Y and the X genes not involved in sex determination degenerate and transposable elements and repetitive sequences accumulate. [see paper #20].

Of special interest is the question how a gene, *dmrt1* for instance, which is placed downstream in the majority of the sex determination cascades (see Figure 4), was brought about to take over the position and function of the gene at the top in medaka. So far, using phylogenetics we found that the *Dmrt1* duplicated copy on the Y-chromosome (*Dmrt1bY*), which became the male determining gene, diverged considerably in the 5' flanking region from its ancestral gene, *dmrt1a* [see paper #21]. Further on, functional promoter analyses in different transfected cell lines (luciferase and promoter bashing assays) and *in vivo* via transgenic reporter fish, revealed that a transposable element (called "Izanagi"), inserted in *dmrt1bY* promoter shortly after the gene duplication event, brought in 'ready to use' transcription factor binding sites including a *dmrt1* target site [see paper #21]. Finally, using electromobility shift assay (EMSA) and *in vivo* chromatin immunoprecipitation (*in vivo* ChIP) we have demonstrated that the insertion of the "Izanagi" element results in a feedback (*cis*-) downregulation of *dmrt1bY* after the sex determination period and (*trans*-) downregulation of the sex determination gene by its autosomal ancestor, *dmrt1a*, in adult testes [see papers #21 and #13]. This new transcriptional regulatory element, nested within *Dmrt1bY* promoter, allowed neo-functionalization of the sex chromosomal copy and guaranteed the survival of the autosomal copy by a possible sub-functionalization in Sertoli cells of adult testes ([see papers #20, #21, #23 and #24] and Figure 8). This is a first mosaic stone of the evolutionary history how *dmrt1bY* became the master sex regulatory gene, but certainly much more has to be explained to understand this process in detail.
<figure><img src="image_14.png" /><figcaption>**Figure 8.** Transcriptional rewiring of the sex determining *dmrt1* gene duplicate by transposable elements.</figcaption></figure>

(A) Comparative analysis of the dmrt1bY promoter and its transcription factor binding sites. The analysed promoter region of dmrt1bY in comparison to its dmrt1a paralog. Length differences between the promoters are based on regions I–IV, of which three (I, II, IV) have been added to the dmrt1bY promoter after duplication, while two others have been lost from the dmrt1bY promoter (KIAA0172 region V) or from the dmrt1a promoter (MHCLp region III), respectively. Bold dashed lines indicate cutting sites for transcriptional regulation analyses. Region I contains a putative Izanagi DNA transposons (repeat 1), into which a Rex1 element (repeat 2) was inserted secondarily. The upstream part of split repeat 1 (repeat 1b) in the dmrt1bY promoter contains multiple Sox5 and Pax2 binding sites as well as a Dmrt1 binding site. Dark green indicates coding sequence of genes and pseudogenes, light green indicates their untranslated regions.

(B) Model for feedback and cross-regulation of the medaka dmrt1 paralogs. During sex determination stages only dmrt1bY is expressed and dmrt1a is off. Hence, the sex determining function of dmrt1bY is exerted. In adult testes, both paralogs are expressed notably in Sertoli cells, but the auto-repression of the dmrt1bY promoter by its own gene product and the cross-regulation by Dmrt1a lead to a predominant expression of dmrt1a compared to dmrt1bY (approx. 50-fold) [see paper #21].
To further understand the function of *Dmrt1bY* in orchestrating such a complex gene regulatory network, I have then driven my research toward the identification of potential targets genes and regulated pathways downstream of the induction of *Dmrt1bY*. Hence, monitoring *in vivo* gene expression and interactions by transgenic fluorescent reporter fish lines, using *in vivo* tissue-chromatin immunoprecipitation and *in vitro* gene regulation assays, I already started to outline the genetic framework in which *Dmrt1bY* is interacting during medaka gonadal formation and maintenance [**see papers #27 and #29**] (Figure 9). The results of these analyses revealed several concordances but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signalling pathways ([**see paper #28**], Figure 9). For example, unlike their mammalian counterparts, none of the different medaka gonadal marker genes analysed (*r-spo-1*, *fst* or *ptch-2*) were detected in germ cells at any time of gonadal development. This major inconsistency between mammals and fish certainly reflects intrinsically divergent modes of germ cell commitment and interaction between germ and somatic cells, possibly accounting for a higher sexual plasticity of germ cells in fish. In this context, our data reflect a profound reorganization of parts of the fish gonadal regulatory network compared to mammals. While some components like *DMRT1*, *SOX9*, *FOXL2* and pathways such as Hedgehog or R-spo1/Wnt/Fst of the gonadal gene regulatory network are conserved on the DNA sequence level across phyla, their functions, regulation and interplays might be considerably different (see Figure 9 and [**papers #27 and #29**]).

<figure><img src="image_15.png" /><figcaption>**Figure 9.** The *Dmrt1bY/Dmrt1a* gene regulatory networks during gonadal formation in medaka.</figcaption></figure>

Interaction scheme of the possible *Dmrt1bY/Dmrt1a*-triggered regulations of the Hedgehog and Wnt4 pathways during gonadal formation in medaka. Solid red arrows indicate for both *Dmrt1bY* and *Dmrt1a* positive regulation while dashed lines indicate sex-specific regulations. The green dashed line indicates a *Dmrt1a*, female-specific, indirect positive regulation favoring the expression of *follistatin* while the blue dashed line reports a *Dmrt1bY*, male-specific, indirect repression of *patched-2* transcription. For the *Dmrt1bY/Dmrt1a*-triggered transcriptional regulation of *R-spondin1*, depending on the cellular contexts, the involvement of a sex-specific co-factor is proposed [**see paper #28**].
Interestingly, examining early gene expression and behaviour of just-specified germ cells, we could show that medaka germ cells exhibit sexually different characters before the formation of the somatic gonadal primordium. Into that direction *in vitro* culture analysis additionally revealed that XY germ cells behave differently from XX germ cells with respect to their mitotic activity. Again, our results challenge the traditional view of the initial acquisition of sexual identity in the somatic cells and provide insights into the evolutional and developmental processes on cell-autonomous sexual identity at cellular levels [**see paper #29**]. In addition to the genes mentioned above, examination of Foxl2 protein distribution in the adult medaka ovary revealed a new sub-population of theca cells, where ovarian-type *aromatase* transcriptional regulation appears to be independent of Foxl2 (Figure 10).

<figure><img src="image_16.png" /><figcaption>**Figure 10.** Schematic representation of granulosa and theca cell populations in mouse and medaka.</figcaption></figure>

In mammals, granulosa cells are the only cell type with steroidogenic activity, expressing both *foxl2* and *aromatase*. Aromatase expression is directly induced by *foxl2*. In medaka, like in mammals, granulosa cells express both *Foxl2* and *aromatase*. Examination of *Foxl2* protein distribution in the medaka ovary revealed a new subpopulation of theca cells expressing *Foxl2* [**see paper #28**].

As a result, we could show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought and emphasize the importance of the cellular context on modulating these regulations in Vertebrates.
1- Context and positioning of our current research work at INRAE

In stark contrast to the traditional ovine, bovine or porcine animal productions, most of the aquaculture fish species, due to the recent expansion of fisheries, are only slightly domesticated. While genetic improvement, as it is in other more mature forms of animal productions, could be also considered as a conventional contributing factor in developing efficient fish farming, it has only recently been applied to just a handful of species (carp, catfish, trout and tilapia). Whereas additionally the number of fish species suitable for aquaculture is steadily increasing worldwide, nearly all farmed aquatic species are still very similar to their wild relatives. As a matter of fact, most fish did/do not benefit from a deep basic understanding of their biology, often precluding development of a rational and sustainable aquaculture. Therefore, efforts should converge towards an improvement of production efficiency (growth, disease resistance, fertility...) and adaptation to new contexts (fluctuating environment, pollution, density...). Improvement in that direction, in addition to the high potential of “classical” methods, should also take advantage of the development of genomic technologies and biotechnologies, first to make the best use of molecular pedigrees and in a long-term vision to improve the knowledge in the genetic and biological bases of traits as well as in directly selecting on the genotype. Biotechnologies certainly provide powerful tools for sustainable development of aquaculture, fisheries, as well as food industry. Beside the “hot-seller” idea of universally increasing fish growth rates, more reasonable and need-driven genetic biotechnologies are already importantly contributing but also significantly challenging aquaculture and fisheries development. For instance, chromosome sex manipulations to induce polyploidy (triploidy and tetraploidy) and uniparental chromosome inheritance (gynogenesis and androgenesis) are important in the improvement of fish breeding as they provide a rapid approach for gonadal sterilization, sex control improvement of hybrid viability and clonation. But also, the development of embryonic stem cell technologies, cryopreservation, manipulation and transplantation of fish germ cells would allow the creation of broodstock systems in which target species can be produced from surrogate or allogenic parents. Undeniably the benefits offered by these new technologies couldn't be fulfilled without constant commitment for basic research.

Control of sex is an important issue of modern aquaculture as it allows the mass production of either all-female or all-male populations of fish that are often economically more interesting to breed than normal mixed-sex populations [see BC #2]. In salmonid species for instance, all-female populations are often preferred because males have the propensity to mature precociously, resulting in reduced growth rates, lower food conversion efficiency, lesser flesh quality and a high sensitivity to pathological problems. In other fish species, sex control can either i) facilitate broodstock management, for instance by optimizing the ratio between males and females in hermaphrodite species, ii) prevent uncontrolled reproduction that favours energy investment into the gonad instead of body growth like in the tilapias or iii) allow the production of a sex specific product like the caviar in female sturgeon fish. A better knowledge on fish sex determination and sex differentiation mechanisms is then a pre-requisite towards a more rational and efficient control. This is especially essential for fish as the number of aquaculture fish species is important and diversification is still a current challenge in many countries. Akin to sex determination systems, genetic sex determinants and even their downstream regulations are not well conserved (see part I), the transfer of a sex-control technique from one species to another is often problematic. Even in species in which biotechnologies are available, and allowing mass production of all-male or all-female populations, a better knowledge of sex-determination and sex-differentiation mechanisms would still allow an evolution of the current biotechnologies
towards more sustainability. This could be done, for instance in salmonids, by finding alternative methodology for the production of neo-males that are currently obtained by (massive) steroid hormone treatment [see BC #2].

Consequently, my project at INRAE was built into that perspective of bringing in and developing, in parallel to the existing fish models already well-established at the INRAE/LPGP laboratory for studying sex differentiation, a new -complementary- model for studying gonadal differentiation and development in order to bypass some limitations inherent to the salmonid models. These limitations are mainly related to the size and generation time of salmonids that prevent any development of easy and affordable cell lineage tracing, transgenesis, targeted inactivation and other genetic manipulations that are now needed to develop deep functional genomic approaches that are absolutely mandatory to gain further insight into the regulatory mechanisms of sex determination in fish. Medaka is certainly one of the best-understood fish model organisms regarding to early gonadal induction, differentiation and maintenance (morphogenesis, sex determination and differentiation, oogenesis and spermatogenesis (see part I). Its sex determination system relies on a simple male heterogametic system (XX/XY) and its master sex-determining gene (*dmrt1bY*) has been characterized. This species is also eligible for the most cutting-edge tools in terms of transgenesis (BAC clones recombination, meganuclease and transposon-mediated fluorescent reporters...), RNA injection, *in vivo* ChIP, *in vivo* inducible systems (on/off) after recombination, genome editing (TALENS, CRISPR), stem cells (embryonic, spermatogonia, and embryoid bodies) and imaging (live confocal imaging).

Hence, in that particular context of functional and comparative evolution and genomics, and using the medaka fish as a model organism of reference, my current research projects on sex determination / sex differentiation in fish are interdependently constructed around few main general questions that constitute my research objectives:

(i) **Functions and Evolution of Master Sex Determining Genes and Systems**

(ii) **Functional Analysis of the Gene Regulatory Networks Underlying Sex Determination and Differentiation.**

(iii) **Gonadal Morphogenesis and Plasticity**

(iv) **Research and Development: Genome Editing / Single Cell Resolution**

**[v] Autophagy in Fish**

**(i) Functions and evolution of master sex determining genes and systems: cases studies**

The high diversity of sex determination systems seen in teleost fish is linked to the high turnover rate of their sex chromosomes (Figure 2 [see BC #1, #3, #4]). As a result, most fish sex chromosomes are considered to be relatively young and are often described as being homomorphics, i.e., with little differentiation and no cytological difference. Sex chromosomes turnover is usually connected with the emergence of new master SD genes (4, 18). Indeed, in therian mammals and birds, master SD genes are highly conserved with the quasi-exclusive usage of SRY for mammals and DMRT1 for birds. But, a high diversity of SD genes (Figure 2) has been found in the past two decades in many teleost fish and this has greatly expanded our understanding of the SD mechanisms and SD evolution. Despite this great diversity, most of these newly
discovered fish master SD genes are classical “usual suspects” [see paper #31] that fall into three independent protein families implicated in the “canonical” vertebrate sex differentiation gene network, namely the Sry-related HMG box (SOX), the Doublesex and mab-3 related transcription factors (DMRT) and the transforming growth factors (TGF-β) family. Their recruitments as master SD genes do not follow any obvious phylogenetic relationship and are more likely to be independent and repeated events during teleost evolution (Figure 11).

<figure><img src="image_17.png" /><figcaption>Figure 11. Diversity and evolution of sex determination systems and master sex-determining genes in vertebrates.</figcaption></figure>

In contrast to therian mammals and birds, the other vertebrate groups including reptiles, amphibians and fish, exhibit plasticity of sex determination mechanisms (XX/XY, multiple X or Y, ZZ/ZW, multiple Z or W, autosomal modifiers and environmentally induced sex determination) and of master sex-determining genes.

Only one known exception to this “usual suspects” rule is the salmonid SD gene, sdY (sex determining region on the Y chromosome, (19)), which has derived from the duplication of an immune-related gene unsuspected to be involved in the sex differentiation pathway. The birth of a new master SD genes can start with a duplication of an autosomal gene that will acquire a new SD function leading to new sex chromosomes. On the other hand, it can begin with allelic diversification from a step-wise diversification of two alleles of the future sex chromosome pair. One allele then becomes the sex determiner, while the other allele either retains a non-SD function or favours the development of the opposite sex (20). The gene duplication and allelic diversification mechanisms have both been found in teleost fish to generate new master SD genes and drive the turnover of sex chromosomes (Figure 12). In the following part of this manuscript, I will review the cases of master sex determination genes in teleost fish we have been working with.
<figure><img src="image_18.png" /><figcaption>Figure 12. Mechanisms accounting for the emergence of new master sex-determining genes after either gene duplication (examples medaka and frog) or allelic diversification (examples torafugu, birds and tongue sole).</figcaption></figure>

(A) The medaka *dmrt1* gene, together with neighbouring genes (including *dmrt2* and 3), first underwent local gene duplication. This whole duplicated segment was then inserted into another chromosome, creating a proto Y-chromosome. Because of non-homology of the insert to the proto-X recombination is suppressed and this part of the proto Y segment degenerated. Transposable elements and repetitive sequences accumulated, resulting in transcriptional rewiring of the *dmrt1bY* gene and acquisition of an early gonadal expression pattern compatible with a master sex-determining function. Further on, point mutations accounted for neo-functionalization and specialization of the *dmrt1bY* gene [see papers #16, #18, #21 and #22]. (B) In the pufferfish as a result of allelic diversification two versions of the *amh* receptor II exist differing only by one aminoacid located in the kinase domain (H384D). This hypomorphic mutation conferring lower receptor activity is encoded on the X-chromosome. Quantitative variations in Amh signalling in females (homozygous for the hypomorphic *amhrII* allele) versus males (heterozygous for the wild type and hypomorphic alleles), account for male gonadal development (Reproduced from (21)). (C) Birds and the tongue sole and have a ZW female heterogametic system. A single loss of function event in the *dmrt1* gene occurred (W allele). This resulted in ZW individuals having only one copy of the *dmrt1* gene while ZZ individuals harbour two copies (red stars indicate functional *dmrt1* genes). Here a dosage effect leads to either female (ZW, 1 “dose” of *dmrt1*) or male (ZZ, 2 “doses” of *dmrt1*) gonadal development respectively (15, 22). (D) Sex determination in the frog *Xenopus* laevis is female heterogamety. Similar to medaka the frog *dmrt1* gene underwent gene duplication and translocation. Additionally, truncation of the *dmrt1* gene occurred, generating a dominant negative version (DM-W). The DM-W protein product from the W-chromosome antagonizes the action of the wild type *dmrt1* gene on the Z-chromosome, leading to female gonadal development. ZZ individuals having two copies of the *dmrt1* gene (and no DM-W) develop as males (16). Red and blue stars indicate functional and dominant-negative version of the *dmrt1* gene respectively.
(ia) *Dmrt1bY* the medaka master sex determining gene: function and evolution.

The *dmrt1* gene is a transcriptional factor belonging to the evolutionarily well-conserved DM (Doublesex and Mad-3) domain DMRT gene family, which is involved in sexual development in many phylogenetically distant groups from worms to mammals [**see papers #23 and #24, #31, and BC03**]. In teleost fish, sexually dimorphic expression of *dmrt1* has been correlated with gonadal identity in both gonochoristic and hermaphroditic fish species [**see paper #24】].

*Dmrt1bY*, was the first non-mammalian SD gene found in vertebrates and it is a duplicated copy of autosomal *dmrt1/dmrt1a* gene found in two ricefish species of the genus *Oryzias*: the Japanese medaka (*O. latipes*) and the Malabar ricefish (*O. curvinotus*), both having an XX/XY sex determination system (8, 9) (Figure 1). *Dmrt1bY* arose about 5–10 million years ago from a local duplication of an autosomal fragment encompassing the *dmrt1* gene (Figure 7). Consequently, this pair of X and Y chromosomes are very young in comparison with the chromosome pair found in therian mammals, and remain homomorphic [**see paper #20】].

The expression pattern of the medaka *dmrt1bY* is typical of a master SD gene with an early and transient expression during gonadal primordium differentiation, and low persistence in the adult gonads [**see paper #13**]. Around the time of hatching *dmrt1bY* is first expressed in the nuclei of the somatic undifferentiated cells surrounding the primordial germ cells (PGC) and later on exclusively in the Sertoli cells in males [**see papers #13, #18 and #29**] (Figure 13). In comparison, the autosomal *dmrt1/dmrt1a* is expressed much later at 20 days after hatching in males and shows a testis-specific expression pattern in adult males with 50 times higher expression than that of *dmrt1bY* [**see paper #13**].
<figure><img src="image_19.png" /><figcaption>Figure 13. Detection of dmrt1bY expression in germ cells and Sertoli cells of dmrt1bY-eGFP reporter transgenic medaka at hatching and adult stages.</figcaption></figure>

(A-C) Dmrt1bY expression is first detected in clustered germ cells located within the lateral plate mesoderm at stage 28. Expression is co-localized with the fluorescence of the germ cell-specific marker (mCherry:nos-3'UTR). (D-F) Germ cell-specific expression is observed until stages 30-31. (G-L) By stage 33, while germ cell expression becomes weaker (G-I), additional dmrt1bY expression is detected in the somatic cells directly surrounding the germ cells (J-L). (M-O) In adult testes dmrt1bY expression is restricted to Sertoli cells.
Concomitantly to the acquisition of a dominant position within the sex-determining network, we have previously shown that *dmrt1bY* was subjected to a profound rearrangement of its regulatory landscape brought about by exaptation of a “ready-to-use” cis-regulatory element from a transposable element (TE). This element, called *Izanagi*, recruits *Dmrt1bY* and *Dmrt1a* proteins to turn off the *dmrt1bY* gene after it has fulfilled its function as the primary male SD gene [**see paper #21**] (Figure 8).

Interestingly we have further reported that TE-mediated transcriptional rewiring can reach an unexpected level of complexity that exceeds this simple feedback regulation. Indeed, we found that another TE, *Rex1*, has jumped into *Izanagi* [**see paper #36**]. 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 have then demonstrated 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* (Figure 14). 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* [**see paper #36**].

Showing that *sox5* was recruited 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 ancestral SD function of *Sox5* in regulating germ-cell number and, in consequence, gonadal identity.

Interestingly, the finding that a preformed transcription factor binding site contributed by the *Rex1* transposon modulates 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 (23), *Rex1* is the second such event found in the same promoter. It will be interesting to analyse whether the other repeats present in the *dmrt1bY*, but not in the promoter 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 (3), 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. 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.
<figure><img src="image_20.png" /><figcaption>**Figure 14.** Comparative analysis of *sox5* and *dmrt1bY* expression dynamics during gonadal primordium formation.</figcaption></figure>

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,k) 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.
(ib) *sdY*: an unusual and conserved SD gene in Salmonids.

The recent discovery of a new type of SD gene in rainbow trout (*Oncorhynchus mykiss*) demonstrated an unexpected flexibility of the system in vertebrate SD (19). This gene, called sdY, came to the spotlight as it was found to be expressed only in embryonic testes and was also tightly linked to the sex-locus on the Y chromosome. It was further confirmed to be the trout SD gene based on functional proofs showing that sdY is by itself necessary and sufficient to induce testicular differentiation (19). One of the multiple unusual characteristics of sdY is that it evolved from the duplication of an immune-related gene, irf9 (interferon related factor 9). Compared to its ancestral protein, sdY lost the N-terminal DNA-binding domain, but preserved its C-terminal protein-protein interaction domain. Another interesting feature is that sdY is sex-linked in most salmonid species, indicating that it is a conserved SD gene in this teleost group. Yet, the SEX locus containing sdY is not located in the same chromosome in different salmonids species (24), suggesting a rapid turnover of sex chromosome while preserving what seems to be a jumping master SD gene (25).

Our additional studies strongly suggested that SdY could mediate its sex-determining effects through protein-protein interactions. Indeed, the search for potential interacting partners uncovered that SdY is interacting with the forkhead box L2 (Foxl2) protein [see paper #39], a transcription factor well-known for its crucial role in ovarian differentiation [see paper #34]. By binding to Foxl2, SdY is able to prevent the Foxl2 and Nr5a1 (Nuclear Receptor Subfamily 5 Group A Member 1) synergistic activation of the aromatase (cyp19a1a) gene, blocking the positive feedback loop of regulation required for synthesis of the feminizing estrogenic steroids (26) in the early differentiating gonads. Thus, we could show that SdY is not a “genuine” male-promoting factor as, despite being a male MSD gene it acts as an anti-female factor by blocking a default female differentiation regulatory network, and thereby allowing testicular differentiation to proceed. Hence, SdY presents a paradigmatic case that un-related genes outside the “usual suspects” are able to acquire *de novo* sex determining functions in teleosts. These results additionally suggest that the evolution of unusual vertebrate master sex determination genes recruited from outside the classical pathway -like sdY- is nevertheless strongly constrained by their ability to interact with the canonical gonadal differentiation pathway (27) (Figure 3).

(ic) *Esocidae* species challenge the fitness advantage and sexually antagonistic models.

Classical models of sex chromosome evolution postulate that there first occurs an expansion of regions with reduced recombination near the MSD gene, and that the loss of recombination subsequently results in the gradual decay of the whole sex chromosome (28, 29). These models are based on the highly degenerated sex chromosomes of model species, and the universality of these models has recently been called into question by new findings in non-model species we have been looking at. For example, we could show that the MSD gene in Northern pike (Amhby) arose > 65 My ago [see paper #43] and that the surrounding Y-specific region remains restricted in size and without other protein coding genes (Figure 15). Furthermore, no male beneficial/female antagonistic genes have been found in this tiny SD region [see paper #43]. Interestingly, we pointed out that this very restricted sex-differentiated region encompassing *amhby* is a conserved feature of all Y-chromosomes in several species of this clade, illustrating that SD systems can be stable for over 50 million years [see paper #43, and Pan *et al.* submitted]. These examples temper the “need” for highly degenerated sex-chromosomes as ‘evolutionary traps’ for supposedly preventing the turnover of SD systems (4).
<figure><img src="image_21.png" /><figcaption>**Figure 15.** Phylogenetic analysis of amh homologs from the Esociformes revealed an ancient origin of *amhby*.

(A) Phylogeny tree of amh-coding sequences built with the maximum likelihood method. Bootstrap values are given on each node of the tree. The amha ortholog cluster is highlighted with blue background and the amhby ortholog cluster is highlighted with red background. (B) Species phylogeny of the Esociformes. The three putative sex determination transitions are shown by a black star. The presence of pre-duplication amh, amha, and amhby along with its sex-linkage is represented by coloured dots at the end of each branch. The earliest duplication timing of amh is denoted by a black arrow at the root of the Esocidae lineage.</figcaption></figure>

*(id) The “improbable” sex determination in the Atlantic herring*

Classically, the different sex determination triggers have been identified to be components
of either the genome itself (genetic sex determination, GSD) or come from the environment
(environmental sex determination, ESD). Adaptive hypotheses prevail in explaining the evolution
of the various sex determining triggers, which finally will guarantee a stable sex ratio of the
population. For ESD according to the Charnov-Bull model (30) an environmental trigger, e.g. the
incubation temperature of the developing embryo, is favoured when the developmental
environment differentially influences male *versus* female fitness, as for instance shown in the
Jacky dragon (23). GSD is generally explained to be stabilized (if we except the just above-
described situation in esociforms) through the evolution of sex chromosomes, when sexual
selection links the genetic sex determiner to a *locus* that is beneficial to the same sex or even
antagonistic to the opposite sex (31–35). Recently, a third general mechanism has been proposed,
namely random sex determination (RSD) (36). Perrin (36) recognized that many ESD and GSD
systems are not always perfectly reflecting the command of the trigger, when for instance in an
ESD system at a transitional temperature range males and females are produced, or when GSD
species without an obvious reason generate individuals which have the phenotype of the sex that
is opposing their genotypes. This indicated stochastic processes resulting from developmental
noise (36). Extrapolating this reasoning, a model was proposed that sex as a bistable equilibrium
(the male and female phenotype) can be triggered by any random process which, from the
undifferentiated state, tips the balance to the one or the other direction. If the population size is
large enough RSD would guarantee an equal sex ratio as well (Figure 16).
<figure><img src="image_22.png" /><figcaption>**Figure 16.** Sex as a bistable equilibrium.</figcaption></figure>

Genomes are fundamentally bipotential, encoding developmental landscapes (black line) that allow for either male (left) or female (right) development. From an undifferentiated larval state (dark ball in unstable equilibrium), initial triggers tip the balance in one or the other direction (arrows), after which mutually antagonistic gene pathways ensure convergence toward fully fledged male or female phenotypes (36).

We have initially chosen to look at sex determination in the Atlantic herring (*Clupea harengus*) because this fish has characteristics that would allow RSD to act. Indeed, it is one of the most abundant vertebrates on earth (swarms of up to 4 billion individuals) with massive spawns (up to 200,000 eggs spawned per female), it has no intraspecific social interactions or a structured environment that would provide triggers for ESD in its natural range. There is no obvious sexual morphological dimorphism or courtship behaviour that would indicate sexual selection. And, neither the karyotype nor the male and female linkage map (28) indicated sex chromosomes. Despite, we found SD in the Atlantic herring is ruled by a strong genetic determiner: a truncated BMPR1BBY receptor carried by a well differentiated sex chromosome. Interestingly, the herring Y-chromosome should be considered as a minimal differentiated sex chromosome: the male specific region of the Y contains just three genes – a testis-determining gene from the TGF-β signalling pathway and two other male beneficial genes [see paper #49] (Figure 17).

<figure><img src="image_23.png" /><figcaption>**Figure 17.** Reconstruction of the evolution of sex chromosomes in Atlantic herring.</figcaption></figure>

The evolution of ChrY occurred in four steps, but the exact order of steps 2 to 4 is not yet known. 1) Duplication and translocation of BMPR1BB from Chr21 to Chr8. 2) Duplication and translocation of CATSPER3 within Chr8/ChrY. 3) Incorporation of CATSPERG in ChrY and loss from ChrX. 4) CATSPER3A becomes pseudogenized or evolved a new function.

Hence our work revealed that RSD processes might be sporadically resilient, but not perfectly random. Thus, even minimal deviations from optimal sex ratios might initiate evolutionary driftage towards GSD, particularly when population sizes are large and/or natural selection is effective.
Obviously, the evolution of genetic sex determination mechanisms is closely linked to the evolution of sex chromosomes. The discovery of multiple new master SD genes in teleosts provided invaluable insights on the evolution of SD and the processes of the establishment of new sex chromosomes. Before the identification of the first fish master SD gene, models of sex chromosome evolution predicted that SD genes should evolve from a stepwise allelic diversification process starting from a single gene on an autosome. After fixation of a sex-specific allele in that newly formed SD gene, recombination reduction around that new sex locus in the proto-sex chromosome pair would give rise to classical heteromorphic sex chromosomes. Some newly described fish SD genes have evolved thanks to this allelic diversification mechanism, for instance sox3Y (37) and gsdfY (11) in some medaka species and amhr2 in Takifugu (21). But this mechanism did not lead to strongly differentiated heteromorphic sex chromosomes in these teleost species. This phenomenon can probably be explained if we consider that these SD genes are very young in term of evolution.

But the conservation of a single missense SNP in amhr2 acting as the sole signal to initiate sex determination (Figure 12) in at least three Takifugu species that diverged over 10 million years ago, shows that recombination reduction around a new sex locus in the proto-sex chromosome is *de facto* not necessary to fix a new SD gene.

The characterization of new teleost SD genes (Figure 2) also emphasized novel mechanisms for sex chromosome and SD gene evolution as many fish SD genes are the result of a duplication/insertion event. In these cases, a genomic area containing an ancestral gene is duplicated into a different chromosome that will initiate the formation of a new sex chromosome after subsequent functional and/or regulatory changes of the proto SD gene. This mechanism of gene duplication leading to sex chromosome formation first came in light for dmrt1bY in Japanese medaka, and has also been found to generate other teleost- and vertebrates- master SD genes (like in medaka, rainbow trout, European pike, Atlantic herring, Mexican tetra and Yellow perch, for the fish species we have been working with; [see papers #20, #39, #43, #44, #49]). In addition, thanks to their high turnover rate, teleost sex chromosomes also provide many different models to study the process of sex chromosome formation, like for instance in the medaka [see papers #20, #36, #40] or Atlantic herring [see paper #49].

## (ii) Functional analysis of the gene regulatory networks underlying sex determination and differentiation.

While many recent studies dealing with sex determination/differentiation in fish have been focusing on the quest for new master sex determining genes, the genetic architecture of such a complex phenotypic trait as sex cannot be simply limited -or restricted- to the action of unique and totipotent master sex determining triggers. Then, what happens when “masters change”? The classical view of sexual development suggests that not much would change downstream, since “slaves remain” (3). Potentially a new master would pop up at the top from a postulated conserved downstream gene regulatory network, possibly slightly adjusting (bottom-up theory (1, 38)). However, accumulating evidence, notably gathered within teleost fish species, indicates that the phenotypic expression of sex is a rather plastic trait, relying on a complex and unstable equilibrium of a constantly adjusting network of regulatory interactions ([see papers #28 and #31] for review). Hence, the main emerging idea is that sex determination gene regulatory cascades should no longer be seen as simply hierarchical but, rather, as a regulatory network or, even more, as connections of interdependent regulatory networks.
To explore more onto that direction, and because the amazing diversity of sex triggers in
fishes emphasizes the many options possible at the sex determination stage (and possibly beyond)
to switch and supervise over the destiny of the gonad, we have additionally been focusing our
researches on the functional analysis of the gene regulatory network(s) underlying sex
determination and differentiation. For that, we mainly used (i) medaka, which MSD gene arose
after gene duplication, and (ii) sablefish, which MSD gene arose after allelic diversification. Our
main idea is now to progress towards a comprehensive and comparative view of the evolution of
sex determining genes and regulatory networks in relation to the emergence or turnover of master
sex determining genes.

(iia) *The dmrt1bY regulatory network of medaka*

Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation
of gonad development of vertebrates [**see paper #24**].

In medaka, a functional duplicate of the autosomal dmrt1a gene on the Y chromosome—dmrt1bY
—became the master regulator of male sex determination (8, 9). This duplication event went along
with the acquisition of a tightly timely and spatially controlled gonadal expression pattern. Indeed,
dmrt1bY mRNA expression is very dynamic, occurring first in the PGCs prior to morphological
somatic sex differentiation and then quickly switches to an exclusive Sertoli cell localisation [**see
papers #21, #29, #36**]. Importantly, dmrt1bY is expressed in PGCs of male embryos much before
its expression in the pre-Sertoli cells at the sex determination stage [**see papers #29 and #36**].
This early PGC expression is necessary for the later onset of dmrt1bY expression in the pre-Sertoli
cells at the sex-determination stage of male development [**see papers #36**]. There, the level of
dmrt1bY mRNA needs to reach a certain threshold to exert the sex-determining function (Figures
13 and 14). This suggested that medaka germ cells exhibit sexually different characters before the
formation of the somatic gonadal primordium depending on dynamic and tightly timely regulated
mechanisms of transcriptional/post-transcriptional regulations [**see papers #18 and #29**]. We had
previously shown that transcriptional rewiring was brought about by exaptation of two
transposable elements, *Izanagi* and *Rex1*, co-opted to act as silencers (see above in this
manuscript, and [**see papers #21 and #40**]). These turn off the somatic and the germ cell-specific
expressions of the dmrt1bY gene. Thus far, two factors, dmrt1 itself [**see papers #21**] and sox5
[**see papers #36**], were identified, which turn off dmrt1bY expression after it has fulfilled its
function in the early developing gonad.

Further on we identified a *cis*-regulatory 11-bp motif in the 3' UTR of dmrt1bY called the "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 [**see papers #18 and #40**]. The D3Ubox 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 *Bactocera oleae*), (Figure 18).
<figure><img src="image_24.png" /><figcaption>**Figure 18. Conservation of the D3U-box motif from ecdysozoans to mammals.**</figcaption></figure>

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 [see paper #40].

Both, phylogenetic conservation together with the additional presence of the D3U-box in several germ cell-specific transcripts 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. Further bioinformatics analyses and literature searches revealed that the D3U-box motif is a putative target for two RNA-binding proteins, namely cug-bp (39, 40) and bsf (also known as lrppc in mammals (41–43). 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 [see paper #40]. Additionally, our results suggest that the observed regulation of dmrt1bY transcript abundance is likely to be the result of a differential binding of the two RNA-binding proteins (Ol-bsf and Olcug-bp1) with antagonistic properties, *trans*-regulating RNA stability *via* the D3U-box (Figure 19), [see paper #40].
<figure><img src="image_25.png" /><figcaption>**Figure 19. Model for D3U-box-mediated mRNA regulation.**</figcaption></figure>

Overall and in addition to a cytoplasmic localization of Ol-bsf, Ol-bsf and O-cug-bps might mutually antagonize toward the access to the D3U-box, resulting in either stabilisation (more Ol-bsf binding) or destabilisation (more Ol-cug-bp binding) of the transcripts harbouring the D3U-box.

Hence, using complementary approaches, our data suggests that the D3U-box motif is— depending on the cellular context—targeted by two 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 on the preservation of the D3U-box motif.

Altogether the data we could gather in medaka allows to decipher and reconstruct the different evolutionary steps that were necessary to establish –and preserve- *dmrt1bY* ahead of the gonadal gene regulatory network after duplication (see **Figure 20**).
<figure><img src="image_26.png" /><figcaption>**Figure 20. How Dmrt1bY was born in medaka?**</figcaption></figure>

This scheme depicts the different evolutionary steps that were necessary (and sufficient) to establish and preserve *dmrt1bY* ahead of the gonadal gene regulatory network in medaka. First a *duplication/insertion* of a gene (dmrt1) from the sex-determination pathway (maintenance) occurred. In a second step the region of insertion (proto Y) was kind of “corrupted” by *insertion of transposable elements*. For instance, in medaka this resulted in a *transcriptional rewiring* of the duplicated copy ((dmrt1bY) that led (*i*) to the acquisition of a new pattern of expression compatible with gonadal induction and (*ii*) to be rapidly off or under the control of sex-differentiating genes (like dmrt1a or Sox5). In parallel a *specific mechanism of post-transcriptional* set up, resulting in a tightly timely and spatially controlled gonadal expression pattern. Finally *sub/neo-functionalization* together with co-evolution of the downstream gene regulatory network processes developed.
(iib) Allelic diversification after transposable element exaptation into its proximal regulatory region promoted Gsdf as the master sex determining gene of sablefish (Anoplopoma fimbria)

New MSD genes primarily emanate from one of two evolutionarily conserved processes: (i) sporadic gene duplication and insertion, followed by sub- and/or neo-functionalization, or (ii) allelic diversification of a pre-existing *locus* (20) [see BC #1 and BC#4] (Figure 12). However, the molecular changes that allow new MSD genes to exert a novel function are not well understood, aside from those of a few model species.

In the marine teleost, sablefish (*Anoplopoma fimbria*), complementary genomic and genetic studies recently led to the identification of a sex *locus* on the Y-chromosome (44). Further characterization of this *locus* resulted in identification of the transforming growth factor β (TGF-β) gene, *gonadal soma-derived factor (gsdf)*, as the main candidate for fulfilling the MSD function. The presence of different X- and Y-chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification.

In sablefish, *gsdfY*, in contrast to its X-variant counterpart, is specifically expressed in male (XY-genotype) fry earlier than any other male or female sex-related genes and prior to both molecular and morphological sexual differentiation of the gonads (45). Comparative analysis of the *gsdfX* and *gsdfY* expression patterns clearly showed that *gsdf-Y*, which is expressed much earlier than *gsdfX*, experienced transcriptional rewiring during the process of allelic diversification that ultimately gave rise to the Y- and X- chromosomes of sablefish.

Such acquisition of a new transcriptional context represented by a different spatio-temporal expression pattern, compatible with a sex-determining function, seems to be the main prerequisite in the process of establishment and fixation of a new MSD gene. In the two sister species, *Oryzias latipes* and *O. dancena*, and in mammals, either *dmrt1* or *sox3* genes respectively, were subjected to profound transcriptional rewiring for establishing either *dmrt1bY* (*O. latipes*; duplication / insertion, (46–48) (see Figure 12)), *sox3Y* (*O. dancena*; allelic diversification, (37)) or *SRY* (most mammals; allelic diversification, (49)) as master sex-determining genes respectively (see (50) for review, and Figure 21).
<figure><img src="image_27.png" /><figcaption>**Figure 21.** Independent evolution of SOX3 genes toward a master sex-determining function in mice and Indian rice fish.</figcaption></figure>

While SRY appears to be restricted to the therian mammals, evidence accumulates that SOX3 has independently been
recruited as a “precursor” of master sexdetermining genes also outside mammals. Hence, although not a priori
destined to have a direct function during sex determination, common mechanisms of evolution seem to be repeatedly
employed. Given that SOX3 is not generally expressed during gonadal induction or during gonadal development, the
first step toward a sexdetermining function is a transcriptional rewiring in order to acquire a timed pattern of
expression compatible with sex determination. Such transcriptional rewiring, although not unique to SOX3 (see
Dmrt1bY in medaka fish for example, (Figure 12)), generally involves either fusion of the gene to new promoters or
insertions of transposable elements into their pre-existing promoter, bringing in cis-regulatory elements compatible
with the timing of gonadal induction. Interestingly and surprisingly, it seems that at least in mice and rice fish, this
step alone was sufficient to endow SOX3 with a sex-determining function. Usually, the transcriptional rewiring steps
seem to be accompanied by neo-functionalization or functional specialization processes. These include specialization
of the protein activity itself in therian mammals (adapted from reference (49)) or more surprisingly adaptation of the
downstream gene-regulatory network (target genes) in the Indian rice fish.

In sablefish, while a unique missense mutation between the two *gsdf* variants does not appear to
drastically impact their physiological activity with regard to downstream activation of Smad's,
any processes of functional divergence of the variants after allelic diversification might be
reasonably excluded. Uniquely, the newly acquired MSD function of the *gsdfY* gene seems to be
entirely ascribable to its new pattern of expression.

Into that direction we reported that the high expression of the gsdfY allelic copy during gonadal
differentiation is largely imputable to a Y-specific insert derived from a TE of the hAT family
incorporated into its promoter (Y-specific insert in Figure 22). Gsdf is an important downstream
component of the male sex determination regulatory network, which in sablefish, like in *Oryzias luzonensis* (11) acquired the role of the MSD. Interestingly, co-option of this TE within the “neo”-gsdfY promoter was likely sufficient for transforming and elevating a protein acting downstream in the sex determination network to a MSD gene expressed at the right time and right place.

<figure><img src="image_28.png" /><figcaption>**Figure 22. Comparative analysis of the gsdfX and gsdfY promoters.**</figcaption></figure>

The analysed promoter region of *gsdfX* in comparison to its *gsdfY* paralog. Length differences between the two *gsdfX* and *gsdfY* promoters are due to Y- and X- specific regions of which unique Y- (935 bp) and X- (412 bp) specific inserts have been respectively added and lost concomitantly during the allelic diversification event. The Y-specific insert is made of a transposable element of the hAT family.

Deciphering the mechanism by which the *neo-gsdfY* is transcriptionally controlled, we could further show that the hAT-type TE facilitated early up-regulation of *gsdf-Y* expression by Dmrt1 and Wt1 (Figure 23), two key genes of the canonical gonadal gene regulatory network, preventing any expression pattern redundancy between the two *gsdf* allelic copies, that therefore might constitute a reasonable evolutionary way for preserving both *gsdf* gene copies from any purification /degeneration processes after allelic diversification. Altogether our results demonstrate that allelic diversification of the *gsdf* gene gave rise to the sex determination system in sablefish. Importantly, the MSD function of *gsdfY* was not attained by acquisition of a new function of the protein itself, *via* amino acid changes, but rather through to the acquisition of elements in the promotor region resulting in a unique expression profile, which relocated *gsdfY* to the most upstream position in the sex-determining network.
<figure><img src="image_29.png" /><figcaption>Figure 23. hAT-mediated male-specific transcriptional regulation of gsdfY promoter by Dmrt1 and Wt1.</figcaption></figure>

(A) The hAT element (Y-specific insert) induces modulation of transcriptional activity of a thymidine kinase minimal promoter upon transient transfection of Dmrt1 or Wt1(-KTS). (B) Model for gsdfX and gsdfY transcriptional modulation by Dmrt1 and Wt1.

Evolution of new sex determination genes by allelic diversification has often intuitively been associated with gradual processes that proceed slowly over evolutionary timescales (34, 51). We found that in *A. fimbria*, allelic diversification of a sex determination gene initiated by the exaptation of a TE led to complete transcriptional rewiring of the allele on the proto-Y chromosome. This provides a unique functional example of a *bona fide* punctual process as an efficient alternative to the phyletic gradualism model (52) for the molecular evolution of a master sex determining gene.
(iii) Germline/soma interactions, gonadal morphogenesis and plasticity

(iiia) *The autosomal gsdf gene acts as a male sex initiator in medaka fish.*

As much as possible, we always have been trying to correlate/connect regulatory
mechanisms and evolution with physiology. It is then not surprising that we also got interest for
gonadal morphogenesis, notably *via* studying the function of the genes for which we studied
regulation. I think this is, in the field, our specificity (strength?) in the way that we are able to
report comprehensive “stories” about evolution of master sex determining genes, blending
genomic plasticity, phylogeny, transcriptional and post-transcriptional regulations, and up to the
physiology *in fine*.

Gonadal development is a multi-step process including sex determination, initiation, differentiation, maintenance, and culminating with the production of sperm or eggs for germline transmission. Like seen before, a hallmark of genetic mechanisms underlying sex determination and development is that they show a remarkable diversity and do not follow any evolutionary trajectories (Figure 11). For example, even master sex-determining genes or sex determiners (SDs) so far identified in different animal taxa (Figures 1 and 2) show considerable differences in sequence and activity of their products. The primary role of an SD is to determine the initial sex by triggering testicular or ovarian differentiation of a sexually bi-potential gonad. Hence, the presence of a SD determines the genetic sex, whereas the onset of gonadal differentiation towards a testis or an ovary delineates primary sex initiation. SDs act at the top of hierarchical networks to control sex differentiation. For example, sry in mammals initiates testicular differentiation through activating its direct target sox9 (Figure 3). Notably, we have previously shown that the networks downstream of SDs also vary enormously from one animal to another (Figure 9) [see paper #28]. The enormous diversity of genetic sex determination mechanisms is a long-standing mystery and also a major challenge for understanding sex development. Fish have sex-determination mechanisms ranging from environmental to different modes of genetic determination and thus provide a paradigm for studying sex plasticity and development. Particularly, medaka fish is the first vertebrate that showed crossing-over between X and Y chromosomes (53), induction of sex reversal (54), and most importantly, offered the first vertebrate SD besides the mammalian sry, namely dmrt1bY (8, 9). Most recent studies have revealed female germ stem cell markers capable of making intrinsic sperm-egg fate decision in medaka (55). It is known that dmrt1bY activates dmrt1, which in turn maintains testicular differentiation, as dmrt1 mutation causes male-to-female sex reversal after the initiation of testicular differentiation (56). However, beside mediating a mitotic arrest of PGCs in males prior to testes differentiation [see paper #14], how dmrt1bY exerts its primary role in male decision via triggering testicular differentiation remains unknown.

Paradoxically, there are several cases where dmrt1bY is dispensable for maleness (9, 57), which
points to the presence of autosomal essential gene(s) for male sex initiation in medaka (57, 58).
Recently, the gsdf gene has emerged as a novel sex-related factor in several distantly related fish
species (including in the sablefish, like discussed above). This gene (59) encodes the gonadal
soma derived factor, which belongs to the transforming growth factor-β superfamily (60). In
medaka, gsdf is located on chromosome 12 and is predominantly expressed in the Sertoli cells
and granulosa cells in mature gonads (61).

Beside suggesting its potential role as an endogenous inducer of gonadal development, our studies have additionally provided sufficient evidence to firmly established gsdf as a male sex initiator acting downstream of dmrt1bY in medaka, thus serving as a prime candidate for the searched autosomal gene essential for maleness [see paper #32]: -First, gsdf addition is sufficient for masculinization in the absence of dmrt1bY, and gsdf disruption causes feminization without compromising dmrt1bY expression, pointing to its hypostasis to dmrt1bY in action. -Second,
feminization upon gsdf disruption results from the initiation of ovarian differentiation despite the presence of dmrt1bY, demonstrating that the earliest and perhaps the primary role of gsdf is to initiate testicular differentiation, which cannot be replaced by dmrt1bY. -Finally, the fact that dmrt1bY protein binds to the gsdf promoter and activates gsdf transcription convincingly reveals that gsdf mechanistically acts downstream of dmrt1bY (Figure 24, [see paper #32]).

<figure><img src="image_30.png" /><figcaption>Figure 24. Sex determination and its evolution in Oryzias.</figcaption></figure>

(A) The core cascade controlling sex development in medaka. The black horizontal line depicts developmental day(s) post fertilization (dpf) or hatching (dph). Shown are key developmental events including testis initiation (TI), testis differentiation (TD) and testis formation (TF). Horizontal boxes depict developmental RNA expression stages of dmrt1bY, gsdf and dmrt1. Arrows depict transcriptional activation of gsdf and dmrt1 by dmrt1bY. Indicated are the primary roles for gsdf in testis initiation and differentiation and for dmrt1 in testis maintenance. (B) Hypothetical evolution of sex determiners in the genus Oryzias. gsdf may easily become a sex determiner (namely gsdfY in *O. luzonensis*) via acquiring proper temporospatial expression (bent arrow) or preferentially recruit gsdf-regulating genes as new sex determiners (namely dmrt1bY in *O. latipes* and sox3Y in *O. dancena*). Shown are potential or putative binding sites (black) for Dmrt1, Sox3 and an unknown transcription factor in the prototype gsdf promoter. These binding sites may be used (orange) by transcription factors such as Dmrt1 and Sox3 for activation (horizontal arrows) of proper temporospatial gsdf expression to initiate testicular differentiation [see paper #32].

Additionally, gsdf disruption causes the alteration of global gene expression and dmrt1 down-regulation in adult gonads, suggesting the potential involvement of gsdf in subsequent processes such as male sex maintenance besides acting as a male sex initiator (Figure 24, [see paper #32]).

### (iiib) Of the pronephric contribution to the differentiating gonads in fish (focus on medaka).

Still aiming at deciphering how gonad morphogenesis is induced and sets up after the different master sex determining genes have fulfilled their function(s), we are currently addressing the question of a possible contribution of the pronephros in the differentiation of the teleost fish gonads ([see paper #17] and ongoing work). Although so far not investigated in fish, this question is of prime interest since pronephric contribution is a major component of gonadal differentiation in other vertebrates (62, 63).

Critical to our understanding of sex-determination processes are studies investigating the origin and development of cells involved in the formation of the primordial gonad. Despite the fact that different determinants and plastic gene regulatory networks [see papers #28 and #33] trigger gonadal formation amongst vertebrates, the adult gonads are however very similar in morphology,
cellular organization and physiology. In contrast to the ambiguities and complexities of the developing gonad in mammals and birds, the histological structures present during the formation of the gonadal primordium are relatively simple in medaka fish. The gonad of medaka is formed by the coordinated development of two different cell lineages: the germ cells and the somatic gonadal mesoderm surrounding the germ cells. Once specified, PGCs remain closely associated with endodermal tissues and migrate *via* the dorsal gut mesentery to the region of presumptive gonad (Figure 6 and [see papers #9 and #15]). Somatic cells of the presumptive gonad have an embryological origin distinct from PGCs. The external somatic layer of the developing gonad is derived from the genital ridge epithelium, but some somatic cells are probably also derived by the invasion of mesenchyme (similar to the primitive sex chords in mammals, (64)). However, unlike mammals, no clear indication of medullary tissue can be found in the teleost gonad. Prior to differentiation, all somatic cells appear to be derived from a cortex epithelial layer, and are similar in presumptive males and females (64). Following migration of PGCs into the germinal ridge, cell division occurs to form oogonia and spermatogonia, and differentiation and migration of somatic cells is initiated concomitant with this process ([see papers #9 and #15]. Shortly before hatching, at the time of expression of *dmrt1bY* in the male gonad primordium (Figure 14), the germ cells in the female gonad actively proliferate and undergo meiosis, while this is not observed in male gonads (65). It is only ten days later that the first somatic gonadal dimorphisms are apparent with the formation of the acinus (the seminiferous tubule precursor) and the follicles in gonads of male and female respectively (65). Interestingly, ovarian cords within the germinal epithelia of medaka ovaries have been recently characterized. These cords, composed of somatic *Sox9b*-expressing cells and mitotic *nos2*-expressing oogonia continually give rise to germ cells and form a stem cell niche within the ovary referred to as germinal cradle (66). These cradles, containing germline stem cells contribute to the production of fertile eggs and are reminiscent of the germanium of the *Drosophila* ovary, hence implying fundamental processes governing oogenesis across animal species to be conserved.

In mammals, anlagen of gonads, or genital ridges, are formed as two thickenings of coelomic epithelium protruding into the coelomic cavity and situated near the mesonephros, laterally to the dorsal mesentery. In most species, the genital ridges initially consist of somatic cells derived exclusively from coelomic epithelium, and later primordial germ cells (PGCs) invade this preformed gonadal mesoderm. Cells that delaminate from the coelomic epithelium seem to provide one source of cells for the growing genital ridges, while recruitment of underlying cells from the mesonephros to the epithelial population also augments the cell population in the gonadal primordium in males (62). Later, as the male gonad differentiates into a testis, the mesonephric duct develops into the Wolffian duct, and is also thought to contribute to the rete testis and, in the female to the rete ovarii. During that process of mammalian gonad differentiation, mesonephric cells make a substantial contribution to the structure of the ovary or testis itself (62). Hence in mammals a mesonephric origin for both Sertoli cells and interstitial cells, including peritubular myoid and Leydig cells, has been first postulated and later clearly demonstrated (63). Nevertheless, the source of medullar cell precursors in gonads remains a controversial aspect of vertebrate gonadal development.

Interestingly, in amphibians, while the mainstream studies report the origin of medullary cells from proliferating coelomic epithelium surrounding the gonad, suggesting that cells of the gonadal medulla are equivalent to Sertoli cells in the testis and follicular cells in the ovary (55–58), others point to the mesonephric blastema or mesonephric tubules as the origin of medullary cells (71–76), or even the interrenal blastema, constituting the primordium of interrenal glands homologous to the cortex of the adrenal glands in mammals (77, 78). While teleost gonads have many features in common with those of other vertebrates, the general understanding is that teleost gonads only originate from one primordium, the cortex or peritoneal wall (79). Hence, unlike other vertebrates,
the early gonad does not -or would not- have any equivalent of the medulla that is derived from
the interrenal or mesonephric blastema. However, some of our data using BAC transgenic
fluorescent reporter medaka lines challenge this view and seriously point to a possible pronephric
contribution to the gonad in medaka and consequently to other teleosts as well.

Indeed, while analysing the early expression patterns of an array of specific gonadal markers
(BAC transgenic reporter lines for Dmrt1bY, Dmrt1a, Follistatin, Aromatase, Bicoid stability
factor...) we observed that all these markers, prior to be expressed in the forming gonads, were
firstly expressed within the pronephros (co-expression with Cdh17 a pronephros only marker,
Figure 25) at hatching stage when the gonad is forming. Later on, from hatching stage and up to
twenty days post hatching, the expression of these markers was progressively lost within the
pronephros while on the other hand gradually rising in the forming somatic gonad. Therefore, the
question relative to a possible pronephric contribution to the gonad formation conserved across
vertebrates was legitimately addressed. In other words, does this apparent switch in expression of
our markers between pronephros and gonad during the course of early gonadal formation result
from two independent dynamics of expression or from actively migrating cells from the
pronephros towards the forming gonad (Figure 25)?
<figure><img src="image_31.png" /><figcaption>Figure 25. Examples of gonad-specific markers firstly expressed in the pronephric precursors during early primordial gonadal induction.</figcaption></figure>

(A to I) dmrt1bY expression. (J to L) follistatin expression. (M to O) aromatase expression.
Ol-Cdh17 (medaka Cadherin 17), red fluorescence is a specific marker of the pronephros (80).
To address the question of a possible active pronephric contribution to the gonad in medaka and more generally in teleost fish, we next made use of the “brainbow” system to perform *in vivo* specific cell lineage tracing of pronephric precursor cells. **Figure 26** gives an overview of the method that was already successfully used in mice (81) and already adapted for that precise application in zebrafish (82) and medaka (83).

<figure><img src="image_32.png" /><figcaption>Figure 26. The multicolour “Brainbow” system: stochastic recombination using Cre-mediated excisions and inversions.</figcaption></figure>

The brainbow system uses random Cre/Lox recombination to create varied combinations of red, blue and green fluorescent proteins in each cell. The differences in colour allow users to follow multiple cells, regardless of how closely they are positioned. For cell lineage analysis Cre and brainbow cassette promoter specific driven expressions allow to mark neighbouring clones with distinct colours for further tracing during development. (A) The *Cre/LoxP* system. Cre recombinase catalyses recombination between LoxP sites. Depending on LoxP site orientations Cre expression triggers intermediate DNA fragment excision or inversion. (B) The “Brainbow” system. The *Brainbow 2.1* construct contains two tandem invertible DNA segments. Inversion (i-iii) and excision (iv, v) recombination events create four expression possibilities. In medaka we developed that system together with L. Centanin (EMBL, Eidelberg, Germany, see collaborations).

Briefly, a first transgenic line specifically expressing the Cre recombinase in the pronephros at hatching stage has been created. Such a line expresses a fluorescent Cre-recombinase driven by Cdh17 promoter, and show specific expression in the pronephros only throughout development. In a second step, this Cdh17:Cre line was crossed with another line expressing the “brainbow” cassette ubiquitously (ubiquitin promoter, (Figure 27)). After Tamoxifen administration (in order to nuclear localize the recombinase) and subsequent induced specific recombination exclusively in the pronephric cells, cell tracing is activated (recombination and fluorescent switch, Figure 27). It is then possible to live track any migration or commitment of the pronephric precursor cells during development.
<figure><img src="image_33.png" /><figcaption>**Figure 27.** Cre-mediated excisions and inversions in medaka.</figcaption></figure>

Example of strategy for tracking the contribution of pronephric progenitor cells to the adult gonad in medaka: first a reporter line expresses the “brainbow” cassette driven by an ubiquitin promoter (Red expression). A second line expresses an inducible Cre recombinase driven by Cdh17 promoter (pronephros-specific). After Tamoxifen treatment and translocation of the Cre to the nucleus, the recombination will occur, hence switching from red to green expression. Cell lineage tracing of the subset of cells that recombined is now possible.

Interestingly, following specific recombination (cytoplasmic red to nuclear green colour switching) in the pronephric cells at hatching stage, we were able, as early as 8 days post hatching and up to adulthood, to identify these very same cells in gonads of both sexes (Figure 28). This strongly accredits an early and effective pronephric field contribution to the gonad in medaka.
<figure><img src="image_34.png" /><figcaption>Figure 28. Cell lineage of pronephric cell in medaka.</figcaption></figure>

Cell tracking of recombined pronephric cells at hatching stage allowed to identify cells of pronephric origin in gonads of both sexes as early as 8 dph (A and B), in adult ovaries (C), as well as in adult testes (D).

For the functional part of this study two complementary approaches will be conducted. The first one relies on pronephros development interference *via* morpholino knockdown experiments. For these experiments expression of Cdh17, a gene we have shown to be essential for the pronephros formation, will be morpholino-knockdown with the perspective of interfering with gonadal development in case of an effective pronephric field contribution. In that case, gonadal development will be conjointly monitored through dmrt1bY expression (fluorescent transgenic line). The second one will rely on mapping single cell transcriptomes with the tissue context of the pronephros (Visium Spatial Gene Expression, 10X Genomics) in order to potentially identify different cell subtypes/populations/lineages within the pronephros at hatching stage.
[iv] Autophagy in fish

Few years ago, we published a paper entitled "Defective autophagy through epg5 mutation results in germ plasm and mitochondria failure during spermatogenesis" [see paper #30]. When I came to INRAE, I was contacted by a colleague who wanted to benefit of our experience in transgenesis and genome editing (...and autophagy) for his own project dealing with autophagy in fish (Iban Seiliez, INRA nutrition métabolisme et aquaculture, NuMeA). What was at first initiated as a punctual co-operation turned out to be a more serious and real collaboration. We then got fundings for it, students (two PhDs), and now, although this topic is not my core business, I am having pleasure at doing it. Following are some lines to contextualize that (recreational) work.

Chaperone-mediated autophagy (CMA) is an intracellular catabolic pathway that mediates the degradation of specific soluble proteins within lysosomes. Because CMA defects are associated to several human pathologies, including neurodegenerative diseases, cancers and immune disorders, research efforts over the past years have been undertaken to study that essential cellular function. Accordingly, recent findings emphasized the fundamental role of CMA for regulating numerous cellular functions including cellular energetics, transcriptional programs, cell death, and cell survival mechanisms or DNA repair.

Briefly, during CMA, cytosolic proteins containing a KFERQ-like motif are first recognized by HSPA8/HSC70 (heat shock protein family A [Hsp70] member 8) and co-chaperones. The substrate-chaperone complex then docks at the lysosomal membrane through specific binding to the cytosolic tail of LAMP2A (lysosomal associated membrane protein 2A). LAMP2A then organizes into a multimeric complex that allows the substrate to translocate across the lysosomal membrane where it is degraded by acid hydrolases. LAMP2A is one of the three variants (namely, LAMP2A, LAMP2B and LAMP2 C) that originate from alternative splicing of the LAMP2 gene. All three splice variants share a common lumenal domain and only differ by their cytosolic and transmembrane regions. CMA activity has been directly correlated to the amount of LAMP2A (but not those of LAMP2B and LAMP2 C) at the lysosomal membrane. As such, LAMP2A has been described as being the limiting component for CMA activity.

Until recently, the absence of any clearly identifiable LAMP2A protein outside of the mammalian and bird species raised concerns about the presence of CMA in other vertebrate lineages. However, using Basic Local Alignment Search Tools (BLASTs) against different expression databases of ray-finned fish species, we were able to identify several expressed sequence tags (ESTs) displaying high homology with mammalian LAMP2A (Figure 30 and [paper #37]). This suggested that this protein possibly appeared much earlier during evolution than initially thought, and provided the grounds for looking at the existence of a "genuine" CMA function in fish.

We then provided evidence in this direction. Next, we demonstrated that the LAMP2 gene and its structure containing the three alternatively spliced exons (B, A and C) encoding the transmembrane domain and cytoplasmic tail specific to each isoform (LAMP2B, LAMP2A and LAMP2 C, respectively), is definitively not restricted to mammals or birds, but is also present in the genome of different fish species (Figure 30). In contrast, no homologous sequence was found in invertebrate species, suggesting that CMA is indeed restricted to vertebrates. We could then show that the splice variant lamp2a is expressed from the earliest stages of development as well as in several adult tissues of medaka, supporting the idea that fish might exhibit CMA activity - or at least a CMA-like process- [see paper #37]. In order to firmly establish whether or not CMA exists in fish, a medaka fibroblast cell line was transfected with the photoactivable KFERQ-PA-mCherry construct, which has proven to be a reliable reporter for tracking and measuring CMA activity in mammalian cells [see paper #46]. Results clearly showed that, upon long-term starvation, this CMA reporter accumulates in characteristic *puncta* that co-localize with lysosomes and/or late endosomes, and that specific knockdown of lamp2a results in a significant
loss of these *puncta*, thereby providing functional evidence for the existence of CMA activity in fish. Finally, to address the physiological role of Lamp2a in fish, a medaka knockout for the splice variant lamp2a was generated. These KO fish display severe alterations in carbohydrate and fat metabolism, similar to what has been observed in the liver of mice deficient for CMA. These results further demonstrated that the CMA function is definitively not restricted to mammals and birds [see paper #46].

Overall, these findings open up new and exciting perspectives to approach (or differently appreciate) CMA under a novel angle. For instance, the relative sequence variability observed within the different functional domains of LAMP2A between phylogenetically distant species will certainly be informative for identifying evolutionarily conserved, or species-dependent, key residues necessary for the structure function relationship of this protein (Figure 30). Beyond these perspectives on the structure-function relationship of LAMP2A, these new findings also emphasize the interest of teleost fish, which diverged from the tetrapod lineage early during vertebrate evolution, as attractive and unique models at the functional interface between invertebrates (assumed to lack any CMA activity, but relying on an endosomal microautophagy [eMI]-like system for targeting KFERQ-like-motif-containing proteins) and mammals (making use of both eMI and CMA functions), for studying the interplay between these two related pathways.

<figure><img src="image_35.png" /><figcaption>**Figure 30.** Sequence variability within different functional domains of LAMP2A between phylogenetically distant species.</figcaption></figure>

(A) Schematic representation of selected LAMP2As. (B) Sequence alignment of the boxed region. Three positively charged amino acids (in blue) necessary for the binding of substrate proteins are present in fish sequences. However, whereas two glycine (G) residues (in red) located within the transmembrane (TM) region are essential for the multimerization of LAMP2A in rodents, only one G is found in that region in some fish species, including medaka. A GYXXF sequence at the C terminus (in green) is required for targeting LAMP2A to the lysosomal membrane. Although conserved in most teleosts, the divergence of that motif in zebrafish, encoding an additional asparagine residue (N, purple), raises a question about the ability of this species to perform CMA, and certainly deserves special attention.

Hence, much more than challenging the currently tetrapod-centred paradigm, our studies [see papers #30, #37, #46 and #47] tackle the urgent need of considering complementary and powerful alternative genetic models, for approaching the entirety of that fundamental catabolic process from an evolutionary perspective (like for sex determination I would say).
## 2- Discussion (evolution of master sex determining genes)

More and more, sex determination is now appreciated as a developmental perspective. As a matter of fact, sex determination is not anymore seen as one hierarchical cascade with the master SD gene on the top, but as a complex network with different types of genetic and environmental factors influencing cell proliferation and hormone levels to push sexual development over either a male or female threshold (84, 85) [see papers #28, #31, #33].

Definitely, studies on the teleost SD support this view in which each node of the SD gene network has the potential of becoming the major-effect *loci*, not necessarily being on the top of the cascade. Archetype of this hypothesis would be amhr2 in Takifugu that modulates transduction efficiency of an upstream ligand signal, i.e., AMH acting on germ cell proliferation or regulating steroid synthesis activity (21), to direct male or female development. Nevertheless, many open questions still remain concerning teleost SD and probably most important, why teleost fish use so many SD genes and SD systems with such a high sex chromosomes turnover rate?

Rapid turnover of sex chromosomes is thought as a way to escape the decaying fate of sex chromosomes, assuming that the lack of recombination around the sex *locus* will lead to the accumulation of deleterious mutations. Alternatively, sexual antagonistic selection has also been put forward to explain the expansion of the sex *locus* and the accumulation of sex specific genes near it. Sex ratio selection provides a scenario that a new master SD gene could rise in frequency when it confers a more balanced sex ratio in the population. Other mechanisms such as random genetic drift and pleiotropic selection have also been used to explain the rise of new SD systems. Another hypothesis, explaining the lack of sex chromosome turnover in mammals and birds, proposes that these highly heteromorphic sex chromosomes might be acting as evolutionary traps that would stabilize SD genes for long span of evolutionary time (4). Interestingly, SD in many teleost species with a genetic SD system can still be influenced by environmental cues like for instance in the Nile tilapia (86), the half-smooth tongue sole (87), and medaka [see paper #42]. Transition stages between GSD and TSD are also well illustrated in the case of pejerrey species that conserves a SD gene (amhY) that is used alone in *O. hatcheri* and used in combination with temperature in the closely related species *O. bonariensis* (88). These transition stages could potentially facilitate the turnover of SD systems, leading to the emergence of new SD genes.

It is now clear that the phenotypic expression of sex translates from either genetic triggers, environmental triggers, endocrine triggers or a blend of all. The main emerging idea is that sex determination gene regulatory cascades should no longer be seen as simply hierarchical but, rather, as a regulatory network or, even more, as connections of interdependent regulatory networks. It also seems that the sex determination case should now be treated more like a developmental perspective, rather than a simple one-way top-down differentiation process (neither genetically nor physiologically). Indeed, the process of gonadal differentiation and maintenance is highly plastic lifelong, with formed gonads able to transdifferentiate after what was thought to be “final” differentiation (Figure 29).
<figure><img src="image_36.png" /><figcaption>**Figure 29.** Sex differentiation as a threshold phenotype relying on fine regulations of plastic gene regulatory networks.</figcaption></figure>

It is obvious that the phenotypic expression of sex cannot be seen any longer as a simple one-way top down differentiation process under the action of a unique and totipotent master sex determining trigger (left). Sex determination should, rather, be seen through the prism of a developmental perspective as the emanation of either genetic, environmental, endocrine triggers, or a blend of all, acting among interconnected gene regulatory networks (right).

Sex determination seen as a hierarchized cascade also led to the view that master sex determining genes were necessarily up-recruited from the pre-existing sex cascade (duplication, transcriptional rewiring and sub-functionalization), and then added to the top. Further on, but still as a variation on the theme, one could say that such up-recruitment is not necessary: the gene can stay at its place in the cascade and just become more powerful. But these views are rather mechanistic. Indeed, the underlying mechanisms are always the same: gene duplication, transcriptional rewiring, neo-/sub-functionalization (coding change, truncation ...), and specialization. The translation of these mechanisms into physiology opens many more options for evolution (presence or absence does not make it all). Hence, mechanistically speaking, it might be much “easier” to find a gene already known for being able to influence gene regulatory network(s), although others, essentially any other gene, could do the same function. Sdy in salmonids, a gene *a priori* not primarily involved in sex determination, differentiation or maintenance, does not play any physiological role on its own but, rather, provokes a slight bend into the gonadal gene regulatory network [see paper #39]. This is enough for doing the job. Indeed, for such bending, maybe some signalling pathway components are better at doing it – for instance, as seen with the emergence of the TGF-β signalling pathway (Figure 2, and perspective part of this manuscript). The existence of such intricate and plastic regulatory networks has drastically changed our traditional perception of a standard linear developmental process for initiating and developing either a male or a female gonad and now opens up fascinating questions for future research (Figure 29).
## 3- Perspectives

### (i) TGF-β molecules challenge the paradigms.

#### (ia) Emergence of the TGF-β signalling molecules ahead of the sex determining networks in fish.

To date, more than twenty different vertebrate MSD genes have been identified on different sex chromosomes of mammals, birds, frogs and fish (Figure 2). Interestingly, six of these genes are transcription factors (Dmrt1- or Sox3- related) and thirteen others belong to the TGF-β signalling pathway (Amh, Amhr2, Bmpr1b, Gsdf and Gdf6). This pattern suggests that only a limited group of factors/signalling pathways are prone to become top regulators, while other well-characterized and indispensable components of sex-determining pathways –e.g. Sox9 or Foxl2- have apparently not been recruited as master regulators in any species studied so far. Moreover, while TGF-β members are clearly subordinate in the mammalian sex-regulatory network, they have independently and recurrently made it to the top in fish (Figure 2). Few years ago, we were among the first to describe and pinpoint the major role of Amh signalling during fish gonadal induction [see paper #17]. The biological importance of TGF-β members is unfortunately contrasted by the lack of information on how such signalling(s) is/are elicited and physiologically integrated during gonadal induction and development. Additionally, such a profusion of master regulators from TGF-β pathway-related members draws attention to the evolutionary meaning of this convergent evolution. Thus, outstanding issues remain unanswered:

**1- What is the evolutionary meaning of this recurrent convergent evolution toward re-establishing TGF-β signaling pathways for controlling sex-determination in fish?**

**2- How are the respective sex determining function(s) of TGF-β signalling molecules (Amh, Gsdf and Gdf6) accomplished during fish gonadal induction? Do they converge to a general "TGF-β hub" that connects and integrates them all, or do they remain independent of each other?**

**3- How do(es) that gonadal TGF-β regulatory network(s) interact with the canonical gonadal gene regulatory network?**

To address these questions, our project -for the next years- will be articulated around three main objectives that are detailed and contextualized below.

#### (ib) A central role for gonadal TGF-β signalling?

The TGF-β family of cell signalling active polypeptides have attracted much attention because of their ability, from nematodes to mammals, to control cellular functions that regulate embryonic development and tissue homeostasis. TGF-β molecules can be subdivided into (i) TGF-β sensu stricto, bone morphogenetic proteins (BMP), (iii) activins and most growth and differentiation factors (GDFs), and (iv) distant members of the TGFβ family not fitting into one of the other three subgroups. Despite the tremendous diversity of physiological responses that this family elicits, an astonishingly “simple” system is the core of this signalling pathway. Canonical TGF-β family members have initially been shown to transmit signals through heteromeric complexes consisting of type I and type II serine/ threonine kinase receptors. Ligand binding induces the formation of a heteromeric ternary complex, which is competent to initiate intracellular signal transduction. In this complex, the constitutively active serine/threonine kinase domain of type II receptors
transphosphorylate type I receptors. Activated type I receptors in turn activate specific receptor substrates (Smads) organized in multisubunit complexes that move into the nucleus to regulate transcription of target genes (see **Figure 31** and **[papers #3, #11 and #12]** for review). Interestingly an emerging concept is that the distinctive activities of TGF-β family growth factors (GFs) are not only determined as simply explained by signalling type I and II receptors, which can show varying degrees of promiscuity for GFs-, but also by other proteins binding either intracellularly to TGFβ receptors or when interacting in the extracellular space, e.g. soluble TGFβ/BMP antagonists or membrane-located co-receptors, can function as activating or inactivating modulator protein. They thereby greatly diversify signalling activity by adding another layer of signalling [**see papers #11 and #12**]. They do not only control whether GFs reach their receptors on cells, but also whether additional components are present within GF-receptor complexes (89). Thus, molecular recognition in the TGF-β family is not singularly achieved by GF-receptor interactions, but by a

<figure><img src="image_37.png" /></figure>

network of interactions with multiple partners (89).

<caption>**Figure 31.** Cell signalling pathway of TGF-β superfamily of ligands.</caption>

(1) Ligands bind and interact with constitutively phosphorylated dimers of type II receptors. (2) Heterotetramers form by recruitment of dimers of type I receptors. (3) Transphosphorylation of type I receptors by type II receptors occurs.

(4) Smads are recruited and phosphorylated by type I receptors, and then move from the cytoplasm to the nucleus where they act as transcription factors.

With respect to gonad development, the following factors are of special interest when investigating the TGF-β signalling pathway:

**AMH.** The anti-Müllerian hormone (Amh) is a distant member of the TGF-β family belonging rather to the fourth subgroup which has limited homology to members of the activin/inhibin and *sensu stricto* TGFβs. In mammals *Amh* plays a major role for the regression of the Müllerian duct-forming part of the female reproductive tract during male embryo development (90, 91). Nevertheless, it is not required for mouse testis development although apparently playing a central role in testis formation in non-mammalian species. For instance, in chicken embryonic gonads, Amh is expressed much higher in males and is predicted in birds to be responsible for organizing the early testis structures (92). Fish do not have a Müllerian duct, but have an *Amh* homolog, as we showed for the first time (93). Although being clearly a subordinate member of the sex regulatory network in mammals, the Amh/Amh-receptor system has made it to the top in several species (Figure 32).

In zebrafish (*Danio rerio*), a major model for vertebrate development and human health, the genetic basis of SD is still unknown. Interestingly, being expressed during the gonadal
differentiation period Amh has been shown to be implicated in zebrafish testis formation (94). Nevertheless, to date the presence of the bona-fide receptor (Amhr 2) could not be detected in the genome of zebrafish and other Cyprinids. This indicates that Amh signalling in zebrafish must employ a so far unknown or uncharacterized type of receptor. Certainly, this opens up questions regarding the exclusive specificity of the Amh with respect to “its own” type II receptor in zebrafish and cyprinids, but also in other fish species.

**GSDF.** Gonadal soma-derived factor (Gsdf) is another growth factor displaying key features of the TGF-β family (see Figure 32). During evolution, it has been lost in tetrapods (95), and its biochemical function is by far not well studied. It is assumed to have a role in male gonad development due to its exclusive expression in the early differentiating testis of all fish analysed so far. Besides its proposed role in the downstream regulatory network (96), Gsdf has made it up to the top in *Oryzias luzonensis* (12) (a sister species to medaka) and most likely also in the sablefish (44) and Herpin et al. in prep). However, the receptors and Smads through which Gsdf elicits signalling are still completely unknown (Figure 32). Interestingly, its peculiar and composite nature, being most homologous to inhibins, but harbouring a C-terminal LEFTY/DAN-like extension and on the other hand lacking any α-helix type I receptors epitopes (personal data) make of Gsdf a real biochemical challenge for deciphering its likely atypical signalling modalities.

**BMP/GDF.** In the mouse embryo, several independent BMP signals are necessary for proper PGC induction: the primary induction of PGCs at the posterior proximal epiblast is driven by BMP4 (97), whereas the number of PGCs is guided by BMP2, BMP4 and BMP8b in a synergistic action (98). Hence, although not apparently involved during the early events of SD, the BMP signalling pathway is implicated during mammalian germ cell specification and gametogenesis (99). Through participation in the turquoise killifish (*Nothobranchius*) genome project, a well-established fish model for aging research we have recently shown that *Gdf6/Bmp13* is likely to be the master sex-determining gene of this species (100). Nevertheless, what the downstream targets of that signalling route are during fish sex-determination are still completely unknown (an ongoing collaboration has been set up with C. Englert (University of Jena, Germany)).

### (ic) TGF-β signalling specificity

One most important open question is how signalling specificity is determined? Specificity of BMP signalling relies critically on the particular combination of type I and II receptors, as well as on the identity of the specific ligand associated with the active receptor complex [see paper #3]. It also involves the differential usage of several substrates (Smads), which are distinct between activated forms of different type I receptors (Figure 32, (101)). What is not so straightforward, however, is how such a simple core-system coordinates, integrates and elicits a plethora of physiological outcomes in a tightly regulated cell-specific context. Binding versatility and flexible receptor oligomerization patterns may lead in some cases to cross-talk between the TGF-β ligand family pathways. It could possibly result in direct competition at the membrane through shared receptors or at the level of transcription factors. This represents a first level of complexity that leads to the multiplicity of biological end-points compared to the relatively low number of primary interacting participants (ligands, receptors and Smads, [see papers #3, #11 and #12]). A second and more complex level of integration can be attributed to the ability of Smads to act as signalling platforms through interactions with a variety of different proteins. Thus, the composition of Smad transcriptional complexes and the resulting physiological activities are necessarily driven and modulated in a cell-specific fashion. An emerging concept is that the response of a given cell to
extrinsic signals relies not only on the effect of single, isolated pathways, but also more on the integration of multiple signals from a plethora of cross-talking pathways [see paper #12].

<figure><img src="image_38.png" /><figcaption>Figure 32. Phylogenetic relationship of paralogous TGF-β, GDF, BMP, AMH, and GSDF ligands, as well as characterized receptors and signalling pathways for individual ligands.</figcaption></figure>

To draw near these questions, more information on the molecular mechanisms of TGF-β signalling involved during gonadal development is required. To this end, our perspectives aim at deciphering the importance of TGF-β signalling for SD and maintenance. We want to explore the nodes that create specificity in the signalling process, the ligand/receptor and receptor/Smad levels. Using the medaka fish as model organism, we will employ functional genomics approaches combining classical comparative investigations together with cutting edge methods for functional investigation. We will develop transgenic approaches and tools for testing, in vivo, the responsiveness dynamics of the different TGF-β signalling pathways at a cellular resolution. Synergistically, in silico modelling of predictable ligand/receptor interaction will be conducted thanks to our collaborations (Thomas Müller, see collaborators), in order to address our 3 main objectives:
**Objective 1:** Signalling specificity and crosstalk of different gonadal TGF-β signal transducing factors (Amh/AmhR2/Gsdf/GDF6) and inferred structure/function relationship.

This objective aims at connecting each ligands/receptor pairs (Amh/AmhR2, GDF6 and Gsdf with identified partners) to SMAD (1, 2, 3, 5 and 8) activation *via* phosphorylation. Specifically testing the relative degrees of phosphorylation for each Smads upon various ligand/receptors interactions will additionally be informative in terms of crosstalk and relative specificity between different TGF-β signalling pathways (see Gal4_UAS system in Figure 33). This will notably also allow us to tell whether (or not) a general gonadal TGF-β “hub” centralizes and converts signalling(s) from different ligand/receptor combinations into a common downstream outcome (similar combinations of Smads’ activation).

<figure><img src="image_39.png" /><figcaption>Figure 33.</figcaption></figure>

**Upper:** Gal-4/UAS reporter system for TGF-β signalling to detect specific Smad activation.

Our own system, based on a Gal4 transcriptional activator coupled with the transactivation domains of the different Smads (1, 2, 3, 5 and 8) and an UAS-Luc reporter expressing a luciferase will be employed in different medaka cell lines.

**Lower:** UAS/Gal4-mediated smad phosphorylation assay of medaka Gdf6a/b signal through phosphorylation of Smads 2, 3, and 8. Our system has been tested for sensitivity and specificity. For example, preliminary data using this system shows that GDF6a/b, in a cell-specific context, “signal” through phosphorylation of smads 2 and 3 and to a lesser extent through smad8.

This system was initially developed by Sedes *et al.* (102).
**Objective 2: Sex-determining function of TGF-β pathway/signalling during sex-determination and maintenance in medaka and selected representative fish species.**

Facing the lack of data on how TGF-β signalling is elicited and physiologically integrated during gonadal induction, development and maintenance in fish (and beyond), we first want to identify the components that are active during sexual development.

For that purpose, first, detailed comparative expression patterns analysis of Amh, AmhR2, Gsdf, and Gdf6 but also all other TGF-β-related components (including receptors) will be conducted in key phylogenetically fish species (23 ray-finned fish, including 2 holosteans, and 21 teleost species). Blast-based searches on our in-house developed PhyloFish database (http://phylofish.sigenae.org, (103)) providing consistent and exhaustive gene expression data from 23 different ray-finned fish species will be perform to determine the spatial and temporal collinearity expression relationship of these TGF-β components during SD stages and in adult gonads (see **Figure 34** for preliminary data in medaka). This is a prerequisite for assigning and validating functional and physiologically relevant interactions between TGF-β ligands/receptors/smads.

<figure><img src="image_40.png" /><figcaption>**Figure 34.** Expressional analysis of different TGF-β components shows collinearity of expression of certain members in medaka gonads.</figcaption></figure>

Next, in order to better picture how the whole gonadal gene regulatory network is affected by TGFβ signalling, we will perform RNA-seq after TGFβ modulation in different medaka cell lines (spermatogonia and embryonic) transfected with either Amh, Gsdf or Gdf6. In parallel, proliferation assays will be performed.

Functional assessment will then be also performed *in vivo* for Amh, Gdf6a and b. For this specific purpose, “knock out” medaka lines for Amh, Gdf6a and b have already been produced (CRISPR/Cas9) and will be analysed (on histological and transcriptomical levels) for deviations in the development of gonads and for possible sex-reversal. This will complement our knowledge previously generated for the role of Gsdf in male gonadal sexual differentiation initiation [see paper #32].
**Objective 3: Integration of the TGF-β signalling pathway(s) within the canonical sex-determination gene regulatory network.**

In tilapia (104) and medaka (96), evidences suggest that Gsdf is a direct effector of DMRT1 and DMRT1bY respectively. In order to assign and estimate the importance of the different gonadal TGF-β signaling pathways within the canonical SD gene regulatory network we will investigate the regulation of the different essential TGF-β pathways components by DMRT1bY, the master sex-determining factor of medaka. We will determine the different relationships and transactivating properties among these factors by *in vivo* Chromatin Immuno-Precipitation of an *in vivo* tagged version of DMRT1bY (*in vivo* ChIP-seq). To this end we will make use of our already established transgenic reporter fish expressing a tagged version of Dmrt1bY (CRISPR/Cas9 mediated KI) and assess its binding to specific promoter region of TGF-β signalling factors involved in sexual development. We have validated this method in several publications (48, 96, 105), (Figure 35).

<figure><img src="image_41.png" /><figcaption>**Figure 35.** Western blot and fluorescent validation for expression of the *in vivo*-tagged Dmrt1bY gene; and set up conditions for DNA sonication (ChIP-seq).</figcaption></figure>

The pleiotropic nature of the TGF-β signalling pathway (combining signalling molecules, ligands, receptors, effectors, and transcription factors) makes this pathway particularly capable of re-structuring and fine-tuning intricate networks of gene regulation. While TGF-β family members clearly play a crucial role in integrating a plethora of signals, and have been functionally and genomically proven to be MSD genes, environmental factors can still override them and cause sex reversal both in the lab and in the wild. This plasticity in species with clear sex chromosomes and a GSD system emphasizes that sex should no longer be viewed as a rigid and pre-determined path from genotype to phenotype, but rather as a multilayer reaction norm resulting from developmental noise, and which can be contingently modulated or totally ruled by genetic factors. In this perspective, sex is the net product of a variety of environmental, genomic and epigenomic, and stochastic determinants. The master genetic trigger therefore has to cope with influences from factors which may disturb its action directly, or disturb the downstream action. TGF-β, with its tight-knit regulatory network, and surplus copies in teleosts, is probably especially suitable for the production of a phenotype as plastic as sex.
(ii) Physiological readout of the neo-functionalization of the medaka *Dmrt1* paralogs (*Dmrt1a* vs *Dmrt1bY*)

Like said before, in the medaka fish a duplicated copy of *dmrt1*, designated *dmrt1bY*, on the Y chromosome was shown to be the master regulator of male development. Further on we found that the *dmrt1bY* gene has acquired a new feedback downregulation of its expression [see papers #21 and #36]. Additionally, the autosomal *dmrt1a* gene is also able to regulate transcription of its duplicated paralog by binding to a unique target *Dmrt1* site nested within the *dmrt1bY* proximal promoter region [see paper #21]. We could trace back this novel regulatory element to a highly conserved sequence within a new type of TE that inserted into the upstream region of *dmrt1bY* shortly after the duplication event (Figures 8 and 20). This new transcriptional regulatory element allowed neo-functionalization of the sex chromosomal copy and guaranteed the survival of the autosomal copy by a possible sub-functionalization in Sertoli cells of adult testes. In the particular case of *dmrt1bY*, this contributed to create new hierarchies of sex-determining genes (Figure 20). Of special interest is the question, how a gene, *dmrt1* for instance, which is placed downstream in the sex determination cascade, can take over the *position* and *function* of the gene at the top. In other words, we now want to know whether -or not- the two *dmrt1* paralogs have acquired different functions after duplication. Interestingly few lines of evidences suggest that it is indeed the case (Figures 36 to 38):

(i) Single aminoacid changes between dmrt1 paralogs from *O. latipes* and *curvinotus* within either the DM DNA-binding or transactivation domains suggest functional neo-functionalization (Figure 36).

<figure><img src="image_42.png" /><figcaption>Figure 36. Sequence comparison of the *dmrt1* paralog genes (*dmrt1a* and *dmrt1bY*) in *O. latipes* and *O. curvinotus*.</figcaption></figure>
(ii) Positive selection is apparent between the two paralogs (Figure 37).

<figure><img src="image_43.png" /><figcaption>**Figure 37. Evolution of the dmrt1 paralogs after duplication.**</figcaption></figure>

-Higher rate of synonymous substitution in Dmrt1bY than in Dmrt1a (higher mutation rate in Dmrt1bY than in Dmrt1a). –Higher rate of non-synonymous substitution in Dmrt1bY than in Dmrt1a (elevation of mutation rate and change in natural selection). –dn/ds ratios up to 3.25 for the DM DNA-binding domain (provide evidence for positive selection). See also (106).

(iii) Dmrt1a and Dmrt1bY differentially activate target genes and bind to different targets (Figure 38).

<figure><img src="image_44.png" /><figcaption>**Figure 38. Activation and binding of target genes by the Dmrt1 paralogs.**</figcaption></figure>

While *in vitro* experiments show that Dmrt1a activates transcription of Sox5, on the other hand Dmrt1bY represses Sox5 transcription. *In vivo* ChIP (chromatin immunoprecipitation) reveals that Dmrt1a binds to Sox5 promoter, while Dmrt1bY does not.
Hence, using sequences analyses, phylogeny, promoter activity, ChIP-PCR and interspecific hybrids (107), all the evidence indeed suggests that the function of the *dmrt1* paralogs diverged after duplication.

To decipher the physiological readout of that functional divergence between the two paralogs, *in vivo* Chromatin ImmunoPrecipitation, sequencing (*in vivo* ChIP-seq), and Co-ImmunoPrecipitation (Co-IP) will be employed to isolate target genes and co-factors interacting with the *Dmrt1* paralogs. We will hence get a clearer picture of the *Dmrt1bY*-aheaded medaka gonadal regulatory network.

Because no specific antibodies could be produced to specifically recognize *Dmrt1a* or *Dmrt1bY* (which sequences are nevertheless too similar), we have tagged both paralogs *in vivo* using genome editing (Knock-in, Figure 39).

<figure><img src="image_45.png" /><figcaption>**Figure 39.** *In vivo* tagging of the two *Dmrt1* paralogs.</figcaption></figure>

Using guide RNAs specific of either *dmrt1* paralogs together with the *Cas9* enzyme, we were able to specifically remove the last exon (exon5) of *Dmrt1a* and *Dmrt1bY*. Thanks to micro-homology repair mechanism acting on provided homologous DNA fragment encompassing exons 5 fused to either FLAG- or HA- tags, we could *in vivo* tag both *dmrt1* paralogs. Detection of either *Dmrt1* paralogs was verified with specific antibodies (anti-HA or anti-FLAG) by mean of western-blotting or immunofluorescence.

After ChIP-seq, bioinformatic analysis should then give us an extensive list of genes, for which *Dmrt1s* are binding to their corresponding promoters. Validation of the data will be done by real time RT-PCR from the cell lines and *in-vivo* from different stages of male gonad development as well as adult testes (and ovary for control). Further on, interactions and transcriptional regulation will be tested in different cell lines. Genes proven to be critical in the genetic network governed by *dmrt1bY* will be targeted for functional analyses by genome editing (Knock-out).

Protein partners of both *Dmrt1* paralogs will be isolated after Co-Imunoprecipitation and mass spectrometry analysis.

Eventually, knowing how *Dmrt1a/Dmrt1bY* function and target genes evolved after duplication will certainly give us the most comprehensive view how a master sex determining gene is spawn, evolve and maintained in a Vertebrate.
(iii) Of the pronephric contribution to the differentiating gonads in fish...at a cellular resolution.

As a follow-up of our pronephric field contribution to the gonad story (see Figures 25 to 28), and considering the fact that cell lineage tracing experiments strongly suggested that, indeed very early on during development, cells from the pronephros contribute to the gonads, we now want to focus our research on the molecular mechanisms underlying this phenomenon, but at a cellular level/resolution. For that purpose, our approach will rely on mapping single cell transcriptomes within the pronephros tissue context (Visium Spatial Gene Expression System, 10X Genomics, Figure 40).

<figure><img src="image_46.png" /><figcaption>**Figure 40. Spatial gene expression system workflow.**</figcaption></figure>

Each slide contains four capture areas with approximately 5000 barcoded spots, which in turn contain millions of spatially-barcoded capture oligonucleotides. From cryosections slices, tissue mRNAs are released and binds to the barcoded oligos, enabling capture of gene expression information. Overlaid histology together with gene expression information allows transcriptomic information to each cells/group of cells keeping tissue topology.

Hence, we will be able to identify different cell subtypes/populations/lineages within the pronephros at hatching stage and decipher which sub-lineage(s) (according to its/their transcriptomic signatures) indeed contribute to the gonads in medaka.
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