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The avian Z-linked gene DMRT1 is required for male sex determination in the chicken

Abstract

Sex in birds is chromosomally based, as in mammals, but the sex chromosomes are different and the mechanism of avian sex determination has been a long-standing mystery1,2,3. In the chicken and all other birds, the homogametic sex is male (ZZ) and the heterogametic sex is female (ZW). Two hypotheses have been proposed for the mechanism of avian sex determination. The W (female) chromosome may carry a dominant-acting ovary determinant4,5,6. Alternatively, the dosage of a Z-linked gene may mediate sex determination, two doses being required for male development (ZZ)7,8. A strong candidate avian sex-determinant under the dosage hypothesis is the conserved Z-linked gene, DMRT1 (doublesex and mab-3-related transcription factor 1)9,10,11. Here we used RNA interference (RNAi) to knock down DMRT1 in early chicken embryos. Reduction of DMRT1 protein expression in ovo leads to feminization of the embryonic gonads in genetically male (ZZ) embryos. Affected males show partial sex reversal, characterized by feminization of the gonads. The feminized left gonad shows female-like histology, disorganized testis cords and a decline in the testicular marker, SOX9. The ovarian marker, aromatase, is ectopically activated. The feminized right gonad shows a more variable loss of DMRT1 and ectopic aromatase activation, suggesting differential sensitivity to DMRT1 between left and right gonads. Germ cells also show a female pattern of distribution in the feminized male gonads. These results indicate that DMRT1 is required for testis determination in the chicken. Our data support the Z dosage hypothesis for avian sex determination.

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Figure 1: Knockdown of DMRT1 expression in vitro , using RCASBP(B) virus to deliver miRNA or shRNA against DMRT1.
Figure 2: Feminization of male gonads following knockdown of DMRT1.
Figure 3: Feminization of male chicken embryos following knockdown of DMRT1.
Figure 4: Downregulation of SOX9 in male gonads following DMRT1 knockdown.
Figure 5: Ectopic expression of female markers in male gonads following DMRT1 knockdown.

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Acknowledgements

We thank C. Tabin for providing the RCASBP retroviral vectors, and X.-J. Yang for advice on viral preparation. We also thank M. Clinton and H. Sang for methods of manipulating early chicken embryos, S. White for statistical advice and P. Farmer for the histology. P. Koopman provided comments on the manuscript. This work was supported by an Australian Research Council (ARC) DP grant held by C.A.S. and A.H.S., and an NH&MRC Program grant jointly held by A.H.S.

Author Contributions C.A.S. and A.H.S. initiated the project and supervised the study. C.A.S., K.N.R., T.O. and A.H.S. conceived and designed the experiments. P.G.F. designed the pRmiR vector, whereas D.M.C. and T.J.D. designed the RCASBP(B)GFP.U6.shRNA343 and associated shuttle vectors. C.A.S., K.N.R., T.O. and D.M.C. performed the experiments. C.A.S., K.N.R., T.O. and A.H.S. analysed the data and prepared the manuscript. All authors approved the final manuscript.

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Correspondence to Craig A. Smith.

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Smith, C., Roeszler, K., Ohnesorg, T. et al. The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. Nature 461, 267–271 (2009). https://doi.org/10.1038/nature08298

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