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Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation

View Article: PubMed Central - PubMed

ABSTRACT

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.

No MeSH data available.


Expression of GFP in the gonads of the transgenic line Tg(vas:EGFP).The embryos/offspring were obtained by crossing the WT fish with either female (tg/tg) or male (tg/tg) transgenic fish. The GFP signal was observed under fluorescent microscope from 24 hpf to 30 dpf (12 individuals each time).
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f3: Expression of GFP in the gonads of the transgenic line Tg(vas:EGFP).The embryos/offspring were obtained by crossing the WT fish with either female (tg/tg) or male (tg/tg) transgenic fish. The GFP signal was observed under fluorescent microscope from 24 hpf to 30 dpf (12 individuals each time).

Mentions: By introducing the vas:EGFP reporter to the aromatase mutant, we were able to trace the early gonadal development in the mutant fish [Tg(vas:EGFP);cyp19a1a−/−], allowing accurate sampling for histological analysis. As shown in Fig. 3, the female adult transgenic fish (tg/tg) showed strong GFP signal in the ovary under fluorescent stereomicroscope. In contrast, the testis from the transgenic male showed much weaker GFP signal, which could barely be seen through the body wall without dissection. This feature is useful for gender identification, especially before sexual maturation. As reported previously54, we observed that the heterozygous offspring (tg/w) from homozygous transgenic female and wild type male showed GFP signal in the germ cells throughout embryonic and post-hatching development including primordial germ cells (PGCs), probably due to the maternal mRNA specifically localized in the germ cells; however, those from homozygous transgenic male and wild type female had no signal in the germ cells until 8 dpf when some individuals (4 out of 12; 4/12) started to show signal in the gonads. This is likely due to the lack of an essential element in the transgene promoter for expression in the embryonic germ cells54. Our observation differed from the previous reports that the zygotic expression of the transgene GFP started in most of the individuals around 16–24 dpf 26 or 21–23 dpf 56, respectively. At 10 dpf, all juveniles from either crossing combination exhibited visible GFP signal in the undifferentiated gonads with equal signal intensity. This lasted until gonadal differentiation around 25 dpf when the GFP signal became intensified in some fish that were destined to become females; however, in those that later developed into males, the signal remained unchanged or even decreased (Fig. 3). The timing of GFP signal divergence agreed well with that reported by Krovel and Olsen56. This provides a useful marker for assessing the onset and extent of gonadal differentiation.


Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation
Expression of GFP in the gonads of the transgenic line Tg(vas:EGFP).The embryos/offspring were obtained by crossing the WT fish with either female (tg/tg) or male (tg/tg) transgenic fish. The GFP signal was observed under fluorescent microscope from 24 hpf to 30 dpf (12 individuals each time).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120357&req=5

f3: Expression of GFP in the gonads of the transgenic line Tg(vas:EGFP).The embryos/offspring were obtained by crossing the WT fish with either female (tg/tg) or male (tg/tg) transgenic fish. The GFP signal was observed under fluorescent microscope from 24 hpf to 30 dpf (12 individuals each time).
Mentions: By introducing the vas:EGFP reporter to the aromatase mutant, we were able to trace the early gonadal development in the mutant fish [Tg(vas:EGFP);cyp19a1a−/−], allowing accurate sampling for histological analysis. As shown in Fig. 3, the female adult transgenic fish (tg/tg) showed strong GFP signal in the ovary under fluorescent stereomicroscope. In contrast, the testis from the transgenic male showed much weaker GFP signal, which could barely be seen through the body wall without dissection. This feature is useful for gender identification, especially before sexual maturation. As reported previously54, we observed that the heterozygous offspring (tg/w) from homozygous transgenic female and wild type male showed GFP signal in the germ cells throughout embryonic and post-hatching development including primordial germ cells (PGCs), probably due to the maternal mRNA specifically localized in the germ cells; however, those from homozygous transgenic male and wild type female had no signal in the germ cells until 8 dpf when some individuals (4 out of 12; 4/12) started to show signal in the gonads. This is likely due to the lack of an essential element in the transgene promoter for expression in the embryonic germ cells54. Our observation differed from the previous reports that the zygotic expression of the transgene GFP started in most of the individuals around 16–24 dpf 26 or 21–23 dpf 56, respectively. At 10 dpf, all juveniles from either crossing combination exhibited visible GFP signal in the undifferentiated gonads with equal signal intensity. This lasted until gonadal differentiation around 25 dpf when the GFP signal became intensified in some fish that were destined to become females; however, in those that later developed into males, the signal remained unchanged or even decreased (Fig. 3). The timing of GFP signal divergence agreed well with that reported by Krovel and Olsen56. This provides a useful marker for assessing the onset and extent of gonadal differentiation.

View Article: PubMed Central - PubMed

ABSTRACT

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.

No MeSH data available.