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The histone demethylase KDM6B regulates temperature-dependent sex determination in a turtle species

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Science  11 May 2018:
Vol. 360, Issue 6389, pp. 645-648
DOI: 10.1126/science.aap8328
  • Fig. 1 Kdm6b exhibits a temperature-dependent sexually dimorphic expression pattern in early gonads of T. scripta.

    (A and B) Results from qRT-PCR analysis of Kdm6b in (A) whole embryos at stage 12 and different embryonic tissues at stages 13 and 14 and (B) embryonic gonads at stages 15 to 20. Analyses were conducted at both 26° and 32°C. Expression was normalized to Gapdh. The relative expression levels, both measured at 26°C, in the brain at stage 14 and the gonad at stage 19 were defined as 1.0 in (A) and (B), respectively. St, stage; Em, embryo; Li, liver; He, heart; Br, Brain; GMC, gonad-mesonephros complex. (C) Coimmunofluorescence of KDM6B (green) and CTNNB1 (β-catenin, red) in gonadal sections of stage 16 embryos at 26° and 32°C. The panels at right are higher-magnification views of the boxed areas in the middle panels. The dotted circle outlines a germ cell. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 50 μm. (D) Time course response of Kdm6b expression to temperature shifts in vivo at each stage from 16 to 19. Gonads were dissected for qRT-PCR analysis. Results were normalized to Gapdh, and the expression level in stage 16 gonads at female-producing temperature (32°C) was defined as 1. Data in (A), (B), and (D) are means ± SD, n = 3 biological replicates. **, ##P < 0.01; ***, ###P < 0.001.

  • Fig. 2 Knockdown of Kdm6b at 26°C leads to male-to-female sex reversal in T. scripta.

    (A) Representative images of the gonad-mesonephros complexes from 26°C control, 26°C loss-of-function mutants (Kdm6b-RNAi#1 and Kdm6b-RNAi#2), and 32°C control embryos at stage 25. Gd, gonad (outlined by yellow dotted lines). Scale bars, 1 mm. (B) Sex reversal ratio (percentage of ovaries) of gonads with Kdm6b-RNAi#1 and Kdm6b-RNAi#2 at 26°C. Gonadal sex was determined by morphological analysis of gonads and the SOX9 stain. GFP, green fluorescent protein. (C) Coimmunofluorescence of SOX9 and CTNNB1 (β-catenin) in gonadal sections of 26°C control embryos, three examples of embryos at 26°C after Kdm6b knockdown, and 32°C control embryos at stages 21 and 25. (D) VASA and CTNNB1 delineate the distribution pattern of germ cells in Kdm6b-deficient gonads at stages 21 and 25.

  • Fig. 3 Knockdown of Kdm6b abolishes the expression of Dmrt1, but the male pathway can be rescued by overexpression of Dmrt1.

    (A) Results of qRT-PCR analysis of Dmrt1 in gonads from 26°C control embryos, 26°C Kdm6b-RNAi#1 embryos, and 26°C Kdm6b-RNAi#2 embryos at stages 15 to 19. After normalization to Gapdh, the relative expression level in stage 18 gonads at 26°C was defined as 1.0. Data are means ± SD, n = 3 biological replicates. **P < 0.01; ***P < 0.001. (B) Immunofluorescence of DMRT1 with CTNNB1 in stage 15 gonadal sections from 26°C control, 26°C Kdm6b-RNAi#1, and 32°C control embryos. Scale bars, 50 μm. (C) Two representative light microscopy images of 26°C Kdm6b-RNAi#1 gonads overexpressing Dmrt1 (Dmrt1-OE) at stage 25. Scale bars, 1 mm. (D and E) Immunofluorescence analysis for SOX9 (D) and VASA (E) with CTNNB1 in stage 25 Kdm6b-RNAi#1 gonads at 26°C after forced ectopic expression of Dmrt1. Scale bars, 50 μm.

  • Fig. 4 Kdm6b directly regulates H3K27 demethylation at the Dmrt1 locus.

    (A) Immunofluorescence of H3K27me3 with CTNNB1 in gonadal sections of 26°C control, 26°C Kdm6b-RNAi#1, and 32°C control embryos at stage 21. The dotted circles indicate germ cells. Scale bars, 50 μm. (B) Quantitative enrichment of KDM6B at the promoter region of Dmrt1 in 26°C control, 32°C control, and 26°C Kdm6b-deficient gonads at stages 15 and 16, as determined by ChIP-qPCR analysis. Signals are shown as a percentage of the input. IgG, immunoglobulin G. (C and D) Results of ChIP-qPCR assays with antibodies specific for H3K27me3 (C) and pan-H3 (D) at the promoter of Dmrt1 in 26°C control, 32°C control, and 26°C Kdm6b-deficient gonads at stages 15 and 16. Data in (B) to (D) are means ± SD, n = 3 biological replicates. *P < 0.05; **P < 0.01.

Supplementary Materials

  • The histone demethylase KDM6B regulates temperature-dependent sex determination in a turtle species

    Chutian Ge, Jian Ye, Ceri Weber, Wei Sun, Haiyan Zhang, Yingjie Zhou, Cheng Cai, Guoying Qian, Blanche Capel

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    • Materials and Methods 
    • Figs. S1 to S18
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