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

Jason Ioannidis, Gunes Taylor, Debiao Zhao, Long Liu, Alewo Idoko-Akoh, Daoqing Gong, Robin Lovell-Badge, Silvana Guioli, Mike McGrew, Michael Clinton

Preprint posted on September 19, 2020 https://www.biorxiv.org/content/10.1101/2020.09.18.303040v1

Crispy chicken has never looked so sexy. CRISPR-Cas9 DMRT1 knock out induced gonadal sex reversal in chickens

Selected by Martin Estermann

Background:

The embryonic gonad has the unique ability to differentiate into two completely different organs: testis or ovary. Gonadal differentiation is induced by a genetic or environmental switch that activates one pathway and represses the other, making both developmental processes mutually exclusive. Mammals have XX/XY sex chromosome system, where Y chromosome and specifically the gene Sry is known to be the master switch that induces the testicular developmental pathway. Birds, instead, have the ZZ/ZW system: males have a ZZ sex chromosome constitution, while females are ZW. Because a Sry homologous gene is absent in the avian genome, the exact mechanism of sex determination in birds has yet to be definitively resolved. There are two main theories about this process:

1) There is a dominant acting female factor on the W chromosome, similar to the Sry on the Y chromosome, which drives gonadal differentiation into the ovarian pathway.

2) Because the dosage compensation mechanism known in mammals and responsible for X chromosome inactivation is absent, double dosage expression of male determining genes located on the Z chromosome would drive the testicular pathway.

The candidate sex determinant under the Z-dosage hypothesis is DMRT1. In ovo manipulation studies showed that knocking down DMRT1 leads to feminisation of the genetically male (ZZ) gonad and that overexpression of DMRT1 leads to masculinisation of the genetically female (ZW) gonad. Until now, no feminizing factor responsible for inducing the ovarian program was found on the W chromosome, reinforcing the Z dosage theory.

In addition, it is known that hormones influence avian gonadal sex differentiation. Estrogen plays a central role in ovarian differentiation and influences the outcome of the gonadal differentiation into testis or ovaries, overcoming the genetic information. In ovo injection of estradiol or the estrogen synthesis inhibitor fadrozole (FAD) are sufficient to cause feminization and masculinization of the gonads, respectively.

Recent studies of gynandromorph birds, male/female chimeras that display bilateral asymmetry (half male half female), strongly suggest that the avian non-gonadal tissues are dependent on the nature of the cells comprising the individual tissue rather than being imposed by the type of gonad formed. These analyses led to the conclusion that male and female chicken somatic cells possess a cell-autonomous sex identity (CASI).

To elucidate the role of DMRT1 dosage in chicken gonadal sex determination, the authors knocked out DMRT1 using CRISPR-Cas9. The results show that avian gonadal sex fate is dependent on DMRT1 dosage and that DMRT1 is essential for testicular differentiation. This is consistent with previous knock down and overexpression results, reinforcing the Z (DMRT1) dosage theory. Furthermore, the development of secondary sexual characteristics of non-reproductive tissues in birds is independent of gonadal sex or DMRT1, strengthening the cell-autonomous sex identity (CASI) theory.

Key findings:

1) DMRT1 knock out resulted in gonadal feminization

DMRT1 was knocked out in cultured chicken primordial germ cells using CRISPR-Cas9, resulting in single allele mutations (ZD+ZD-). Later these heterozygous germ cells were injected into a surrogate chicken embryo lacking germ cells to generate the G0 founders. Male G0 were crossed with wild type females (ZW) to obtain a segregated G1: ZD+ZD+ (wildtype homozygous male), ZD+W (wildtype hemizygous female), ZD+ZD- (DMRT1 heterozygous knock out male) and ZD-W (DMRT1 hemizygous female).

Male (SOX9 green) and female (FOXL2 female) supporting cell markers were evaluated by immunofluorescence in E13.5 gonads (Fig. 1A). ZD+ZD- gonads resembled female control ovaries (ZD+W) expressing the female marker FOXL2 (red) but no SOX9 (testicular marker, green). In addition, germ cells (white) were located in the cortical region instead of the medullary testicular cords, consistent with the ovarian phenotype. ZD-W females also resembled an ovary but were smaller than the control.

Male (AMH and SOX9) and female markers (FOXL2 and Aromatase) were measured in E8.5 gonads by qRT-PCR (Fig. 1B). These confirmed lack of male markers in ZD+ZD- and the upregulation of female markers, in similar levels to the female controls (ZD+W). Taken together, these results indicate that a single dose of DMRT1 results in an ovarian phenotype, as shown in ZD+ZD- samples. Furthermore, double dose of DMRT1 is required for testicular formation. In addition, the lack of DMRT1 is not necessary for the gonadal formation but might be important for gonadal size (ZD-W).

Fig. 1. DMRT1 knock out results in ovarian differentiation. (A) SOX9, FOXL2, and VASA (PGC) immunofluorescence in wildtype and DMRT1 mutant E13.5 gonadal samples. (B) Relative expression levels of male (SOX9 and AMH) and female (FOXL2 and Aromatase) markers by qRT-PCR in E8.5 gonads. Bars represent mean ± SD. Different letters specify statistically significant groups, P < 0.05 (Preprint Fig. 2f and 2g).

2) DMRT1 knock out does not affect secondary sex characteristics

Surprisingly, despite ZD+ZD- birds having ovaries instead of testes, 24 weeks old ZD+ZD- birds had secondary sex characteristics of a male. These birds had greater muscle mass and bone density than females, larger combs and wattles and they developed leg spurs like the male counterparts (Fig 2). These results suggest that these typical male secondary sexual characteristics are independent of the gonadal type (ovary or testis), reinforcing the idea of a cell-autonomous sex identity (CASI).

Fig. 2. Phenotyping of adult DMRT1 mutants. Physical appearance of wild type and of DMRT1-mutant birds at 24 weeks of age. (Preprint Fig. 4a). 
3) DMRT1 is required in female to male sex reversal experiments by estrogen synthesis inhibition

It is known that estrogens play a key role in gonadal differentiation in chickens. To assess the effects of blocking estrogen synthesis on gonadal development in DMRT1 mutants, the authors exposed ZD+ZD-, ZD-W and female control (ZD+W) E2.5 embryos to fadrozole (FAD). Gonads were collected at E13.5 and female (Aromatase) and male (AMH and SOX9) markers were evaluated by immunofluorescence (Fig 3). Interestingly, ZD+ZD-gonads exposed to FAD expressed both male (SOX9) and female (Aromatase) markers, but no AMH, partially recovering the gonadal sex reversal caused by DMRT1 knock out. These results indicate that despite the absence of estrogens, DMRT1 is essential for proper testicular development and differentiation.

Fig. 3. Effect of FAD treatment in DMRT1 knockout embryos. E13.5 left gonadal Immunofluorescence of male (SOX9 and AMH) and female (FOXL2) markers in FAD treated, wildtype or DMRT1 knockout embryos. (Preprint Fig. 5b).

Why I choose this paper:

As a person who works in chicken gonadal development and sex differentiation, I can completely relate to the findings of this preprint.  Although DMRT1 knock down and overexpression in the chicken gonads already confirmed a key role of DMRT1 in chicken gonadal differentiation, DMRT1 knock out was never performed before. Monoallelic mutation of DMRT1 allowed for a precise evaluation of the DMRT1 dosage effect, something that was not possible to achieve in knock down or overexpression experiments. These findings clearly demonstrate the DMRT1-dependent dosage-based mechanism of sex-determination in birds.

Furthermore, the authors used a novel and efficient technology to generate knockout birds, by genetically modifying primordial germ cells and injecting them to a surrogate chicken lacking germ cells. This technology allowed a whole-body knockout in birds and has the potentiality to be extended to any existing gene. I believe this technology will revolutionize avian transgenics.

Future directions / questions for the authors:

  • You showed that DMRT1 knock out was not sufficient to regulate the sexual differentiation of non-gonadal tissues. What do you think is the factor that regulates the cell-autonomous sex identity? Do you consider the cells sense the whole Z or W chromosomes? Or it is a specific gene in the sex chromosomes, other than DMRT1?
  • It is interesting that in ZD+ZD- FAD treated gonads you see half of the gonad expressing male (SOX9) or female (Aromatase) markers, instead of a complete masculinization of the gonad. Is there any other factor that could be explaining this incomplete, half and half, phenotype?

Tags: avian, bird, phenotype, poultry, sex, transgenic

Posted on: 14th October 2020

doi: https://doi.org/10.1242/prelights.25187

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