Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics

Abstract

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Keywords: chicken embryo; gonadal development; ovary differentiation; sex determination; testis differentiation.

Fig. 1.

Genome editing of DMRT1 mutations and genetic crosses. (A) Diagram of the DMRT1 locus in ZZ wild-type and edited ZZ PGC clones carrying a synonymous mutation and a loss of function mutation. The DM DNA binding domain is shown in blue. Details of the Sanger sequencing traces and resulting nucleotide sequences are shown. Nucleotide changes are shown in red. The nonsynonymous change introduced in one allele generates a stop codon and a frameshift in the sequence, resulting in a predicted 69-amino acid (aa) truncated protein, which lacks part of the DNA binding domain. (B) Diagram illustrating the overall technical approach and the mating used to produce DMRT1-mutant offspring.

Fig. 2.

Gonadal development in DMRT1-mutant embryos. Gross morphology of gonads (A) and Müllerian ducts (B) in Z^D+Z^D+ and Z^D+W embryos and Z^D+Z^D-and Z^D-W DMRT1-mutant embryos (n = 3–7 embryos per genotype). Immuno-sections from right (R) and left (L) gonads from E13.5 wild-type and DMRT1-mutant embryos (C–F). Expression of DMRT1, aromatase (AROM) and AMH (C, E) and SOX9, FOXL2, and of PGC-specific marker (VASA) (D, F). A minimum of three embryos of each genotype were examined. Arrows indicate gonads in A and Müllerian ducts in B. Asterisks indicate Wolffian ducts in B. c, cortex; m, medulla. (G) Relative gene expression of DMRT1 and of testis and ovary markers in gonads of E8.5 wild-type and DMRT1-mutant embryos. Individual expression levels were calculated relative to levels in Z^D+Z^D+. Five replicates on pools of two gonads per genotype. Bars represent mean ± SD. Different letters specify statistically significant groups, P < 0.05.

Fig. 3.

Effect of DMRT1 loss on follicular development. (A) FOXL2 and γH2AX expression in germ cells of gonads from wild-type and DMRT1-mutant embryos at E13.5 and E17.5 of development (n = 3 embryos per genotype). (B) Analysis of gonads of wild-type and DMRT1-mutant birds at 5 wk posthatch (n = 1–2 per genotype). Sections were stained with either H&E or for testis or ovary-specific markers (FOXL2, AROM, SOX9, and DMRT1).

Fig. 4.

Phenotyping of adult DMRT1 mutants. (A) Physical appearance of wild-type and of DMRT1-mutant birds at 24 wk. (B) Body weight of wild-type and DMRT1-mutant birds. Asterisks indicate a statistically significant difference in body weight between each of the ZZ genotypes (Z^D+Z^D-, Z^D+Z^D+) and each of the ZW genotypes (Z^D+W, Z^D-W), on days 120 and 192 (n = 5–8 per genotype). P < .05.

Fig. 5.

Expression of testis and ovary markers in gonads of fadrozole (FAD)-treated E13.5 embryos. Left gonads are shown. (A) IHC of DMRT1, FOXL2, and PGC-marker (VASA). (B) IHC of aromatase, SOX9, and AMH. FAD, Fadrozole-treated. Representative of three embryos per genotype.

Fig. 6.

Overview of sex determination in chickens. (A) Outcomes resulting from different combinations of DMRT1 and E₂. (B and C) Schematics illustrating regulation of gene networks that define male and female reproductive systems (DMRT1: ++/+/− = 2/1/0 copies; E₂ and Cortex: +/− = present/absent).