The doublesex and mab-3 related transcription factor 1 (dmrt1) plays a crucial role in metazoan sexual differentiation. This gene, or its paralogs, independently became triggers for sex determination several times, including in the tetraploid African clawed frog Xenopus laevis. To explore functional evolution of this gene, we generated knockout lines of each of two dmrt1 homeologs in X. laevis and an ortholog in the closely related diploid Western clawed frog X. tropicalis. Our findings evidence sex-specific functional evolution following duplication by allotetraploidization in an ancestor of X. laevis. In females, dmrt1 was essential for fertility and oogenesis in the Xenopus ancestor, but this important function was lost (subfunctionalized) in one X. laevis homeolog (dmrt1.S) after allotetraploidization. In males - in sharp contrast - dmrt1 was not essential for fertility and spermatogenesis in the Xenopus ancestor, but this essentiality was acquired (neofunctionalized) in the other X. laevis homeolog (dmrt1.L) after allotetraploidization. Transcriptomic analysis of the mesonephros/gonad complex during sexual differentiation identifies distinctive patterns of dysregulation in male and female knockouts of dmrt1.L and dmrt1.S relative to same-sex wildtype siblings, including possible autocatalysis of dmrt1.L and activation of the female-determining gene dm-w. Previous work demonstrates that dm-w was recently derived from partial gene duplication of dmrt1.S - a gene that our analysis demonstrates is non-essential in both sexes. Thus, in X. laevis, a developmental system was pushed past a "tipping point" to a novel state where sexual differentiation is now orchestrated by a sex-specific duplicate of a dispensable gene.

Biology Department Life Sciences Building McMaster University Hamilton Canada

Department of Cell Biology Faculty of Science Charles University Prague Czech Republic

Eugene Bell Center for Regenerative Biology and Tissue Engineering and National Xenopus Resource Marine Biological Laboratory Woods Hole Massachusetts United States of America

The School of Biological Sciences University of Aberdeen Aberdeen United Kingdom

Zobrazit více v PubMed

Raymond CS, Shamu CE, Shen MM, Seifert KJ, Hirsch B, Hodgkin J, et al. Evidence for evolutionary conservation of sex-determining genes. Nature. 1998;391(6668):691–5. doi: 10.1038/35618 PubMed DOI

Raymond CS, Kettlewell JR, Hirsch B, Bardwell VJ, Zarkower D. Expression of PubMed DOI

Raymond CS, Murphy MW, O’Sullivan MG, Bardwell VJ, Zarkower D. PubMed DOI PMC

Murphy MW, Zarkower D, Bardwell VJ. Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA. BMC Mol Biol. 2007;8:58. doi: 10.1186/1471-2199-8-58 PubMed DOI PMC

Bellefroid EJ, Leclère L, Saulnier A, Keruzore M, Sirakov M, Vervoort M, et al. Expanding roles for the evolutionarily conserved PubMed DOI PMC

Murphy MW, Lee JK, Rojo S, Gearhart MD, Kurahashi K, Banerjee S, et al. An ancient protein-DNA interaction underlying metazoan sex determination. Nat Struct Mol Biol. 2015;22(6):442–51. doi: 10.1038/nsmb.3032 PubMed DOI PMC

Zarkower D, Murphy MW. DMRT1: An Ancient Sexual Regulator Required for Human Gonadogenesis. Sex Dev. 2022;16(2–3):112–25. doi: 10.1159/000518272 PubMed DOI PMC

Lindeman RE, Murphy MW, Agrimson KS, Gewiss RL, Bardwell VJ, Gearhart MD, et al. The conserved sex regulator PubMed DOI PMC

Dai S, Qi S, Wei X, Liu X, Li Y, Zhou X, et al. Germline sexual fate is determined by the antagonistic action of PubMed DOI

Huang S, Ye L, Chen H. Sex determination and maintenance: the role of PubMed DOI PMC

Ungewitter EK, Yao HH-C. How to make a gonad: cellular mechanisms governing formation of the testes and ovaries. Sex Dev. 2013;7(1–3):7–20. doi: 10.1159/000338612 PubMed DOI PMC

Matson CK, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D. PubMed DOI PMC

Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, et al. Somatic sex reprogramming of adult ovaries to testes by PubMed DOI

Bertho S, Herpin A, Schartl M, Guiguen Y. Lessons from an unusual vertebrate sex-determining gene. Philos Trans R Soc Lond B Biol Sci. 2021;376(1832):20200092. doi: 10.1098/rstb.2020.0092 PubMed DOI PMC

Peichel CL, McCann SR, Ross JA, Naftaly AFS, Urton JR, Cech JN, et al. Assembly of the threespine stickleback Y chromosome reveals convergent signatures of sex chromosome evolution. Genome Biol. 2020;21(1):177. doi: 10.1186/s13059-020-02097-x PubMed DOI PMC

Pan Q, Feron R, Yano A, Guyomard R, Jouanno E, Vigouroux E, et al. Identification of the master sex determining gene in Northern pike ( PubMed DOI PMC

Myosho T, Otake H, Masuyama H, Matsuda M, Kuroki Y, Fujiyama A, et al. Tracing the emergence of a novel sex-determining gene in medaka, PubMed DOI PMC

Takehana Y, Matsuda M, Myosho T, Suster ML, Kawakami K, Shin-I T, et al. Co-option of PubMed DOI

Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, et al. A W-linked DM-domain gene, PubMed DOI PMC

Smith CA, Roeszler KN, Ohnesorg T, Cummins DM, Farlie PG, Doran TJ, et al. The avian Z-linked gene PubMed DOI

Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, et al. PubMed DOI

Ioannidis J, Taylor G, Zhao D, Liu L, Idoko-Akoh A, Gong D, et al. Primary sex determination in birds depends on PubMed DOI PMC

Cauret CMS, Jordan DC, Kukoly LM, Burton SR, Anele EU, Kwiecien JM, et al. Functional dissection and assembly of a small, newly evolved, W chromosome-specific genomic region of the African clawed frog PubMed DOI PMC

Bewick AJ, Anderson DW, Evans BJ. Evolution of the closely related, sex-related genes PubMed DOI

Evans BJ, Carter TF, Greenbaum E, Gvoždík V, Kelley DB, McLaughlin PJ, et al. Genetics, Morphology, Advertisement Calls, and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog ( PubMed DOI PMC

Hayashi S, Suda K, Fujimura F, Fujikawa M, Tamura K, Tsukamoto D, et al. Neofunctionalization of a Noncoding Portion of a DNA Transposon in the Coding Region of the Chimerical Sex-Determining Gene PubMed DOI PMC

Hayashi S, Tamura K, Tsukamoto D, Ogita Y, Takamatsu N, Ito M. Promoter generation for the chimeric sex-determining gene PubMed DOI

Cauret CMS, Gansauge M-T, Tupper AS, Furman BLS, Knytl M, Song X-Y, et al. Developmental Systems Drift and the Drivers of Sex Chromosome Evolution. Mol Biol Evol. 2020;37(3):799–810. doi: 10.1093/molbev/msz268 PubMed DOI

Evans BJ, Gvoždík V, Knytl M, Cauret CMS, Herrel A, Greenbaum E, et al. Rapid Sex Chromosome Turnover in African Clawed Frogs ( PubMed DOI PMC

Furman BLS, Evans BJ. Sequential Turnovers of Sex Chromosomes in African Clawed Frogs ( PubMed DOI PMC

Evans BJ, Gansauge M-T, Stanley EL, Furman BLS, Cauret CMS, Ofori-Boateng C, et al. PubMed DOI PMC

Evans BJ. Genome evolution and speciation genetics of clawed frogs ( PubMed DOI

Yoshimoto S, Ikeda N, Izutsu Y, Shiba T, Takamatsu N, Ito M. Opposite roles of DMRT1 and its W-linked paralogue, PubMed DOI

Mawaribuchi S, Musashijima M, Wada M, Izutsu Y, Kurakata E, Park MK, et al. Molecular Evolution of Two Distinct PubMed DOI

Cohen J. Statistical power analysis for the behavioral sciences. New York: Routledge Academic; 2013.

Session AM, Uno Y, Kwon T, Chapman JA, Toyoda A, Takahashi S, et al. Genome evolution in the allotetraploid frog PubMed DOI PMC

Lin Y-T, Capel B. Cell fate commitment during mammalian sex determination. Curr Opin Genet Dev. 2015;32:144–52. doi: 10.1016/j.gde.2015.03.003 PubMed DOI PMC

Jeng S-R, Wu G-C, Yueh W-S, Kuo S-F, Dufour S, Chang C-F. PubMed DOI

Johnsen H, Seppola M, Torgersen JS, Delghandi M, Andersen Ø. Sexually dimorphic expression of PubMed DOI

Krentz AD, Murphy MW, Sarver AL, Griswold MD, Bardwell VJ, Zarkower D. PubMed DOI PMC

Webster KA, Schach U, Ordaz A, Steinfeld JS, Draper BW, Siegfried KR. PubMed DOI PMC

Veitia RA, Nunes M, Quintana-Murci L, Rappaport R, Thibaud E, Jaubert F, et al. Swyer syndrome and 46,XY partial gonadal dysgenesis associated with 9p deletions in the absence of monosomy-9p syndrome. Am J Hum Genet. 1998;63(3):901–5. doi: 10.1086/302023 PubMed DOI PMC

Li M-H, Yang H-H, Li M-R, Sun Y-L, Jiang X-L, Xie Q-P, et al. Antagonistic roles of PubMed DOI

Shi J-F, Li Y-K, Ren K, Xie Y-J, Yin W-D, Mo Z-C. Characterization of cholesterol metabolism in Sertoli cells and spermatogenesis (Review). Mol Med Rep. 2018;17(1):705–13. doi: 10.3892/mmr.2017.8000 PubMed DOI PMC

Ni F-D, Hao S-L, Yang W-X. Multiple signaling pathways in Sertoli cells: recent findings in spermatogenesis. Cell Death Dis. 2019;10(8):541. doi: 10.1038/s41419-019-1782-z PubMed DOI PMC

Jamnongjit M, Hammes SR. Oocyte maturation: the coming of age of a germ cell. Semin Reprod Med. 2005;23(3):234–41. doi: 10.1055/s-2005-872451 PubMed DOI PMC

McKeegan PJ, Sturmey RG. The role of fatty acids in oocyte and early embryo development. Reprod Fertil Dev. 2011;24(1):59–67. doi: 10.1071/RD11907 PubMed DOI

Zhang Q, Zhu L, Li H, Chen Q, Li N, Li J, et al. Insights and progress on the biosynthesis, metabolism, and physiological functions of gamma-aminobutyric acid (GABA): a review. PeerJ. 2024;12:e18712. doi: 10.7717/peerj.18712 PubMed DOI PMC

Fan C, Cowgill J, Howard RJ, Lindahl E. Divergent mechanisms of steroid inhibition in the human ρ1 GABAA receptor. Nat Commun. 2024;15(1):7795. doi: 10.1038/s41467-024-51904-7 PubMed DOI PMC

Miller WL. Steroid hormone synthesis in mitochondria. Mol Cell Endocrinol. 2013;379(1–2):62–73. doi: 10.1016/j.mce.2013.04.014 PubMed DOI

Park Y-J, Pang M-G. Mitochondrial Functionality in Male Fertility: From Spermatogenesis to Fertilization. Antioxidants (Basel). 2021;10(1):98. doi: 10.3390/antiox10010098 PubMed DOI PMC

Hiort O. The differential role of androgens in early human sex development. BMC Med. 2013;11:152. doi: 10.1186/1741-7015-11-152 PubMed DOI PMC

Eldredge N, Gould SJ. Punctuated equilibria: an alternative to phyletic gradualism. Models in Paleobiology. 1972. p. 82–115.

Casanova EL, Konkel MK. The developmental gene hypothesis for punctuated equilibrium: combined roles of developmental regulatory genes and transposable elements. Bioessays. 2020;42(2):e1900173. doi: 10.1002/bies.201900173 PubMed DOI PMC

Bakhtin Y, Katsnelson MI, Wolf YI, Koonin EV. Evolution in the weak-mutation limit: Stasis periods punctuated by fast transitions between saddle points on the fitness landscape. Proc Natl Acad Sci U S A. 2021;118(4):e2015665118. doi: 10.1073/pnas.2015665118 PubMed DOI PMC

Perrin N. Random sex determination: When developmental noise tips the sex balance. Bioessays. 2016;38(12):1218–26. doi: 10.1002/bies.201600093 PubMed DOI

Castelli MA, Georges A, Cherryh C, Rosauer DF, Sarre SD, Contador‐Kelsall I, et al. Evolving thermal thresholds explain the distribution of temperature sex reversal in an Australian dragon lizard. Diversity and Distributions. 2020;27(3):427–38. doi: 10.1111/ddi.13203 DOI

Vicoso B. Molecular and evolutionary dynamics of animal sex-chromosome turnover. Nat Ecol Evol. 2019;3(12):1632–41. doi: 10.1038/s41559-019-1050-8 PubMed DOI

Lenormand T, Fyon F, Sun E, Roze D. Sex chromosome degeneration by regulatory evolution. Curr Biol. 2020;30(15):3001-3006.e5. doi: 10.1016/j.cub.2020.05.052 PubMed DOI

Adolfi MC, Herpin A, Schartl M. The replaceable master of sex determination: bottom-up hypothesis revisited. Philos Trans R Soc Lond B Biol Sci. 2021;376(1832):20200090. doi: 10.1098/rstb.2020.0090 PubMed DOI PMC

Koopman P, Münsterberg A, Capel B, Vivian N, Lovell-Badge R. Expression of a candidate sex-determining gene during mouse testis differentiation. Nature. 1990;348(6300):450–2. doi: 10.1038/348450a0 PubMed DOI

Pan Q, Kay T, Depincé A, Adolfi M, Schartl M, Guiguen Y, et al. Evolution of master sex determiners: PubMed DOI PMC

Nakayama T, Fish MB, Fisher M, Oomen-Hajagos J, Thomsen GH, Grainger RM. Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in PubMed DOI PMC

Fares MA. The origins of mutational robustness. Trends Genet. 2015;31(7):373–81. doi: 10.1016/j.tig.2015.04.008 PubMed DOI

Wiechmann AF, Wirsig-Wiechmann CR. Color atlas of

Shaidani NI, McNamara S, Wlizla M, Horb ME. Obtaining PubMed PMC

Zahn N, Levin M, Adams DS. The Zahn drawings: new illustrations of PubMed DOI PMC

Nieuwkoop PD, Faber J. Normal table of

Karimi K, Fortriede JD, Lotay VS, Burns KA, Wang DZ, Fisher ME, et al. Xenbase: a genomic, epigenomic and transcriptomic model organism database. Nucleic Acids Res. 2018;46(D1):D861–8. doi: 10.1093/nar/gkx936 PubMed DOI PMC

Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016;34(5):525–7. doi: 10.1038/nbt.3519 PubMed DOI

Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. doi: 10.1186/s13059-014-0550-8 PubMed DOI PMC

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–402. doi: 10.1093/nar/25.17.3389 PubMed DOI PMC

Piprek RP, Damulewicz M, Kloc M, Kubiak JZ. Transcriptome analysis identifies genes involved in sex determination and development of PubMed DOI

Mawaribuchi S, Ikeda N, Fujitani K, Ito Y, Onuma Y, Komiya T, et al. Cell-mass structures expressing the aromatase gene Cyp19a1 lead to ovarian cavities in PubMed DOI

Briggs JA, Weinreb C, Wagner DE, Megason S, Peshkin L, Kirschner MW, et al. The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. Science. 2018;360(6392):eaar5780. doi: 10.1126/science.aar5780 PubMed DOI PMC

Thumfart KM, Mansuy IM. What are Sertoli cells? Historical, methodological, and functional aspects. Andrology. 2023;11(5):849–59. doi: 10.1111/andr.13386 PubMed DOI

Penrad-Mobayed M, Perrin C, L’Hôte D, Contremoulins V, Lepesant J-A, Boizet-Bonhoure B, et al. A role for PubMed DOI PMC

Nguyen TMX, Vegrichtova M, Tlapakova T, Krulova M, Krylov V. Epithelial-Mesenchymal Transition Promotes the Differentiation Potential of PubMed DOI PMC

Matson CK, Murphy MW, Griswold MD, Yoshida S, Bardwell VJ, Zarkower D. The mammalian doublesex homolog PubMed DOI PMC

Saba R, Wu Q, Saga Y. CYP26B1 promotes male germ cell differentiation by suppressing PubMed DOI

Komiya T, Itoh K, Ikenishi K, Furusawa M. Isolation and characterization of a novel gene of the DEAD box protein family which is specifically expressed in germ cells of PubMed DOI

Shimaoka K, Mukumoto Y, Tanigawa Y, Komiya T. PubMed DOI

Duffin K, Bayne RAL, Childs AJ, Collins C, Anderson RA. The forkhead transcription factor PubMed DOI PMC

Jakob S, Lovell-Badge R. Sex determination and the control of PubMed DOI

Kyrönlahti A, Euler R, Bielinska M, Schoeller EL, Moley KH, Toppari J, et al. GATA4 regulates Sertoli cell function and fertility in adult male mice. Mol Cell Endocrinol. 2011;333(1):85–95. doi: 10.1016/j.mce.2010.12.019 PubMed DOI PMC