Distinct roles of cyp11a1 and cyp11c1 in zebrafish gastrulation movements and gonad differentiation
Yonghua Sun*1, Ru Zhang1,2, Qifeng Zhang1,2, Mudan He1, Ding Ye1
1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
In our previous study, we have obtained the maternal zygotic mutant of zebrafish nanog (MZnanog). MZnanog showed strong defects in cytoskeleton and gastrulation movements. RNA-Seq analysis showed that cyp11a1, coding an enzyme that converts cholesterol to pregnenolone (P5), was dramatically downregulated in MZnanog embryos. Interestingly, overexpression of cyp11a1 or treatment of P5 could largely rescue the defects of microtubule assembly and gastrulation movements in MZnanog embryos. Further study revealed that cyp11a1 is the direct transcriptional target of nanog. We generated knockout mutants of cyp11a1 by CRISPR/Cas9 technology, but to our surprise, MZcyp11a1 did not show any developmental defects. We further proved that cyp11a2 was transcriptionally activated in MZcyp11a1 oocytes and embryos, and the transcriptional activation of cyp11a2 compensated the genetic loss of cyp11a1. On the other hand, we isolated zebrafish juvenile ovary and juvenile testis at different stages and conducted RNA-Seq analysis. We found that cyp11c1, encoding an enzyme that is critical for the synthesis of cortisol and androgen, was one of the most differentially expressed genes. The cyp11c1 mutant (cyp11c1(-/-)) displayed adrenal hyperplasia. When the cyp11c1(-/-) mutants reached adulthood, the males showed female secondary sexual characteristics, development defects of leyding cells and failure of spermiation, and the mutant females showed failure of ovulation. Further study showed that when compared with testis differentiation process in wildtype juveniles, the cyp11c1(-/-) juveniles displayed prolonged oogonia apoptosis and delayed “juvenile ovary-to-testis” transition. However, these early defects did not affect the future differentiation of ovary or testis, and the maturation of female and male gametes. Our study revealed that the regulation of cytoskeleton formation and gastrulation movements by Nanog is mediated by cyp11a1, and the oogonia apoptosis in juvenile testis and leyding cell development is regulated by cyp11c1.
Keywords：cyp11a1, cyp11c1, genome editing, genetic compensation, gonad differentiation
Yonghua Sun*1, Ru Zhang1,2, Qifeng Zhang1,2, Mudan He1, Ding Ye1
1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
In our previous study, we have obtained the maternal zygotic mutant of zebrafish nanog (MZnanog). MZnanog showed strong defects in cytoskeleton and gastrulation movements. RNA-Seq analysis showed that cyp11a1, coding an enzyme that converts cholesterol to pregnenolone (P5), was dramatically downregulated in MZnanog embryos. Interestingly, overexpression of cyp11a1 or treatment of P5 could largely rescue the defects of microtubule assembly and gastrulation movements in MZnanog embryos. Further study revealed that cyp11a1 is the direct transcriptional target of nanog. We generated knockout mutants of cyp11a1 by CRISPR/Cas9 technology, but to our surprise, MZcyp11a1 did not show any developmental defects. We further proved that cyp11a2 was transcriptionally activated in MZcyp11a1 oocytes and embryos, and the transcriptional activation of cyp11a2 compensated the genetic loss of cyp11a1. On the other hand, we isolated zebrafish juvenile ovary and juvenile testis at different stages and conducted RNA-Seq analysis. We found that cyp11c1, encoding an enzyme that is critical for the synthesis of cortisol and androgen, was one of the most differentially expressed genes. The cyp11c1 mutant (cyp11c1(-/-)) displayed adrenal hyperplasia. When the cyp11c1(-/-) mutants reached adulthood, the males showed female secondary sexual characteristics, development defects of leyding cells and failure of spermiation, and the mutant females showed failure of ovulation. Further study showed that when compared with testis differentiation process in wildtype juveniles, the cyp11c1(-/-) juveniles displayed prolonged oogonia apoptosis and delayed “juvenile ovary-to-testis” transition. However, these early defects did not affect the future differentiation of ovary or testis, and the maturation of female and male gametes. Our study revealed that the regulation of cytoskeleton formation and gastrulation movements by Nanog is mediated by cyp11a1, and the oogonia apoptosis in juvenile testis and leyding cell development is regulated by cyp11c1.
Keywords：cyp11a1, cyp11c1, genome editing, genetic compensation, gonad differentiation