Sexual cell-fate reprogramming in the ovary by DMRT1

Abstract

Transcription factors related to the insect sex-determination gene doublesex (DMRT proteins) control sex determination and/or sexual differentiation in diverse metazoans and are implicated in transitions between sex-determining mechanisms during vertebrate evolution [1]. In mice, Dmrt1 is required for male gonadal differentiation in somatic cells and germ cells [2-4]. DMRT1 also maintains male gonadal sex: its loss, even in adults, can trigger sexual cell-fate reprogramming in which male Sertoli cells transdifferentiate into their female equivalents-granulosa cells-and testicular tissue reorganizes to a more ovarian morphology [5]. Here we use a conditional Dmrt1 transgene to show that Dmrt1 is not only necessary but also sufficient to specify male cell identity in the mouse gonad. DMRT1 expression in the ovary silenced the female sex-maintenance gene Foxl2 and reprogrammed juvenile and adult granulosa cells into Sertoli-like cells, triggering formation of structures resembling male seminiferous tubules. DMRT1 can silence Foxl2 even in the absence of the testis-determining genes Sox8 and Sox9. mRNA profiling found that DMRT1 activates many testicular genes and downregulates ovarian genes and single-cell RNA sequencing in transdifferentiating cells identified dynamically expressed candidate mediators of this process. Strongly upregulated genes were highly enriched on chromosome X, consistent with sexually antagonistic functions. This study provides an in vivo example of single-gene reprogramming of cell sexual identity. Our findings suggest a reconsideration of mechanisms involved in human disorders of sex development (DSDs) and empirically support evolutionary models in which loss or gain of Dmrt1 function promotes establishment of new vertebrate sex-determination systems.

Figure 1. Ectopic DMRT1 in the ovary causes granulosa cell to Sertoli-like cell differentiation

(A) Schematic diagram of conditional DMRT1 expression transgene CAG-Stop-Dmrt1-Gfp, which is transcribed to express DMRT1 and GFP upon Cre-mediated deletion of a floxed “STOP” cassette (for additional detail see Figure S1A). (B-D) Immunofluorescence (IF) of gonads from 8-10 week old mice showing that activation of CAG-Dmrt1-Gfp in somatic cells of the fetal ovary by Sf1-Cre activates DMRT1, silencing the ovarian granulosa cell transcription factor FOXL2. Dashed boxes indicate areas shown in higher magnification insets. (E-G) IF of adult gonads showing that activation of CAG-Dmrt1-Gfp also activates the Sertoli cell determinant SOX9 and the Sertoli cell differentiation factor GATA1. Dashed boxes indicate areas shown in higher magnification insets. (H-M) Hematoxylin and Eosin (H&E) stained sections of adult testes, ovaries, and CAG-Dmrt1-Gfp expressing ovaries, at low and high magnification (dashed boxes indicate magnified areas shown in K-M). Ovaries expressing DMRT1 show tubule-like morphology typical of testes, with polarized Sertoli-like cells lining the periphery and extending cytoplasmic veils into a central lumen. Scale bars: 100 μm (B-D, H-J); 40 μm (E-G); 20 μm (K-M). See also Figure S1.

Figure 2. DMRT1 expression triggers postnatal granulosa cell transdifferentiation

(A-C) Activation of CAG-Dmrt1-Gfp in the fetal gonad. Confocal images of whole mount IF on E13.5 gonads showing normal expression of DMRT1 in testis (A) and ovarian germ cells (B) and activation of CAG-Dmrt1-Gfp in ovarian somatic cells (C) as indicated by cytoplasmic GFP (example is shown in higher magnification inset). Dispersed green cells lacking DMRT1 in wild type gonads are autofluorescent cells of unknown type. (D-F) SOX9 expression in the fetal gonad. IF showing that SOX9 is strongly expressed in pre-Sertoli cells of wild type testes at E13.5 (D) but is not detected in wild type fetal ovaries (E) or CAG-Stop-Dmrt1-Gfp;Sf1-Cre transgenic ovaries (F). (G-I) Postnatal expression of SOX9 and FOXL2. IF showing that wild type testes at P10 express SOX9 and not FOXL2 (G), wild type ovaries express FOXL2 and not SOX9 (H), and CAG-Stop-Dmrt1-Gfp;Sf1-Cre transgenic ovaries have cells expressing each protein (I), indicating the onset of transdifferentiation. (J-O) Transdifferentiation in the adult ovary. (J-L) Control tamoxifen-injected ovaries from adults carrying CAG-Stop-Dmrt1-Gfp but lacking a Cre transgene do not express DMRT1 or GFP (J), but ovaries from animals also containing UBC-CreERT2 or Hsd17β1-Cre have cells expressing both proteins (K,L). (M-O) Somatic cells from control adult ovaries express FOXL2 but not SOX9 (L), whereas animals with UBC-CreERT2 or Hsd17β1-Cre have cells expressing each protein. (SOX9 IF in adult oocytes is thought to be a non-specific antibody artifact). Scale bars: 40 μm (A-I); 100 μm (J-O). See also figure S2.

Figure 3. DMRT1 masculinizes the ovarian transcriptome

(A) Heat map comparing mRNA expression in adult wild type testis and ovary with CAG--Dmrt1-Gfp;Sf1-Cre ovaries. Columns are from RNA-seq of two gonads (rep1, rep 2) of each genotype. Genes differentially expressed in wild type ovary and DMRT1-expressing ovary (>4-fold ; p<0.05, Table S1A) are shown in rows that are sorted based on high expression in the wild type ovary (top) to high expression in the testis (bottom). Each gene was normalized to a range of −2 (violet) to +2 (green). (B) Scatter plot comparing gene expression in adult Foxl2 conditionally mutant ovaries (data from [7]) and CAG-Stop-Dmrt1-Gfp;Sf1-Cre ovaries. Blue indicates mRNAs with 4-fold or greater expression in wild-type testis vs wild-type ovary and red indicates those with 4-fold or greater expression in wild-type ovary vs wild-type testis. Grey indicates mRNAs not differing significantly between testis and ovary. Identities of some of the most strongly affected mRNAs are indicated. Triangles denote X-linked genes and blue and pink boxes highlight mRNAs strongly up or down-regulated, respectively, in CAG-Dmrt1-Gfp expressing ovaries but not in Foxl2 mutant ovaries. (C-H) IF showing that P17 CAG-Stop-Dmrt1-Gfp;Sf1-Cre transgenic ovaries have a mix of GFP+ cells expressing DMRT1, SOX9, or FOXL2. Scale bars: 40 μm (C,E,G); 20 μm (D,F,H). (I) Expression levels (FPKM) of select mRNAs in single cells from P17 CAG-Stop-Dmrt1-Gfp;Sf1-Cre transgenic ovaries, ordered by pseudotime along the x-axis. See also Figure S3 and Table S1.

Figure 4. DMRT1 can silence FOXL2 without SOX8 and SOX9

(A-C) CAG-Stop-Dmrt1-Gfp can silence FOXL2 in Sox9 conditional mutant ovaries. IF showing that SOX9 is expressed in Sertoli cells of wild-type testes and in Sertoli-like cells of control conditional Sox9/+ DMRT1-expressing ovaries. FOXL2 is almost completely silenced in DMRT1-expressing ovaries conditionally deleted for one (B) or both (C) copies of Sox9 with Sf1-Cre. (D-G) CAG-Dmrt1-Gfp can activate SOX8 in SOX9 mutant granulosa cells. IF showing that SOX8 is expressed in Sertoli cells in wild-type adult testes (D) and is not detectable in wild type adult ovaries (E). CAG-Dmrt1-Gfp can activate SOX8 in ovaries conditionally deleted for one copy (F) or both copies (G) of Sox9 in somatic cells using Sf1-Cre. (H-J) CAG-Dmrt1-Gfp can activate the mature Sertoli cell marker GATA1 in Sox9 mutant granulosa cells. IF showing that GATA1 is not expressed in wild type adult ovaries (H) but is expressed in ovaries conditionally deleted for one (I) or two (J) copies of Sox9 in somatic cells using Sf1-Cre. (K-M) DMRT1 silences FOXL2 in granulosa cells lacking both Sox8 and Sox9. IF showing that Sox8;Sox9 double mutant ovaries have normal FOXL2 expression, normal morphology and lack DMRT1 (K). Activation of CAG-Dmrt1-Gfp in ovaries heterozygous for Sox8 and Sox9 (L) or homozygous mutant for both genes in somatic cells (M) can induce DMRT1 expression and silence FOXL2. Scale bars: 40 μm (A-D,F-J); 20 μm (E,K-M)