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Sex reversal in zebrafish fancl mutants is caused by Tp53-mediated germ cell apoptosis.

Rodríguez-Marí A, Cañestro C, Bremiller RA, Nguyen-Johnson A, Asakawa K, Kawakami K, Postlethwait JH - PLoS Genet. (2010)

Bottom Line: Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival.Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females.Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America.

ABSTRACT
The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.

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The absence of females in fancl homozygous mutants is due to sex reversal.The bar graph represents percentages of expected (ex, grey bars) and observed (ob, black bars) females and males among 211 progeny from a cross of fancl heterozygous females (fancl+/−) to fancl homozygous mutant males (fancl−/−). Total numbers (n) and percentages (%) of animals in each category are indicated on the graph. The expected ratio of female heterozygotes to male heterozygotes to female homozygous mutants to male homozygous mutants is 1∶1∶1∶1, but we observed a ratio of about 1∶1∶0∶2 (46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males). This result rules out the hypothesis that homozygous mutant females die, but is predicted by the hypothesis that homozygous mutants that otherwise would have become females develop instead as males.
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pgen-1001034-g002: The absence of females in fancl homozygous mutants is due to sex reversal.The bar graph represents percentages of expected (ex, grey bars) and observed (ob, black bars) females and males among 211 progeny from a cross of fancl heterozygous females (fancl+/−) to fancl homozygous mutant males (fancl−/−). Total numbers (n) and percentages (%) of animals in each category are indicated on the graph. The expected ratio of female heterozygotes to male heterozygotes to female homozygous mutants to male homozygous mutants is 1∶1∶1∶1, but we observed a ratio of about 1∶1∶0∶2 (46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males). This result rules out the hypothesis that homozygous mutant females die, but is predicted by the hypothesis that homozygous mutants that otherwise would have become females develop instead as males.

Mentions: To characterize the fancl HG10A phenotype, we crossed fancl +/HG10A heterozygotes (called fancl+/− below), and after genotyping the progeny by PCR, observed that all fancl HG10A/HG10A homozygous mutants (called fancl−/− below) developed exclusively as males, even though their wild-type and heterozygous siblings developed about as many females as males. Two alternative hypotheses could explain the lack of homozygous fancl mutant females: female-specific lethality or female-to-male sex reversal. To distinguish between these two hypotheses, we crossed female fancl+/− heterozygotes to male fancl−/− homozygotes. We raised 211 progeny to adulthood, determined their phenotypic sex according to sexually dimorphic characters including the color of the anal fin and body shape, and finally scored their fancl genotypes by PCR. Under normal conditions, this cross should give 50% heterozygotes (about half of which should be female), and 50% homozygous mutants (about half of which should be female), expecting a 1∶1∶1∶1 ratio of heterozygous females to heterozygous males to homozygous mutant females to homozygous mutant males. The fancl female death hypothesis predicts a 1∶1∶0∶1 ratio, or 66% heterozygotes and 33% homozygous mutants, but the sex reversal hypothesis, predicts a 1∶1∶0∶2 ratio, or equal proportions (50%∶50%) of homozygous mutants (all male) and heterozygotes (males plus females). Resulting genotypes revealed 46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males, which showed that about half of the progeny were fancl homozygous mutants (103/211, 49%) and the other half were heterozygous for the fancl mutation (108/211, 51%) (Figure 2). These results had strong statistical support (chi-square likelihood ratio  = 0.794, p-value >0.1), thus ruling out the hypothesis that homozygous fancl mutant females died. Results, however, were consistent with the hypothesis that animals that otherwise would have become females developed as males due to female-to-male sex reversal. Sex distributions within each genotype confirmed our previous observations that all fancl homozygous mutants developed as males (n = 103, 100%), and while approximately half of fancl heterozygous siblings developed as males (n = 62, 57%), the other half developed as females (n = 46, 43%) (Figure 2). These scores showed strong statistical support for the hypothesis that fancl mutants experienced female-to-male sex reversal (chi-square likelihood ratio  = 73.946, p-value<0.0001). To exclude the possibility that some of the fancl mutants could have ovaries despite their external male phenotypic characters, we dissected the gonads of adult fancl homozygous mutants (n = 45), heterozygous females (n = 11) and heterozygous males (n = 29). In all cases, we found a perfect match between external sexual characters and gonadal sex. These results ruled out the possibility that fancl mutants masqueraded as males externally while having female gonads. We conclude that the HG10A Tol2 insertion into fancl induced a female-to-male sex reversal phenotype in zebrafish.


Sex reversal in zebrafish fancl mutants is caused by Tp53-mediated germ cell apoptosis.

Rodríguez-Marí A, Cañestro C, Bremiller RA, Nguyen-Johnson A, Asakawa K, Kawakami K, Postlethwait JH - PLoS Genet. (2010)

The absence of females in fancl homozygous mutants is due to sex reversal.The bar graph represents percentages of expected (ex, grey bars) and observed (ob, black bars) females and males among 211 progeny from a cross of fancl heterozygous females (fancl+/−) to fancl homozygous mutant males (fancl−/−). Total numbers (n) and percentages (%) of animals in each category are indicated on the graph. The expected ratio of female heterozygotes to male heterozygotes to female homozygous mutants to male homozygous mutants is 1∶1∶1∶1, but we observed a ratio of about 1∶1∶0∶2 (46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males). This result rules out the hypothesis that homozygous mutant females die, but is predicted by the hypothesis that homozygous mutants that otherwise would have become females develop instead as males.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908690&req=5

pgen-1001034-g002: The absence of females in fancl homozygous mutants is due to sex reversal.The bar graph represents percentages of expected (ex, grey bars) and observed (ob, black bars) females and males among 211 progeny from a cross of fancl heterozygous females (fancl+/−) to fancl homozygous mutant males (fancl−/−). Total numbers (n) and percentages (%) of animals in each category are indicated on the graph. The expected ratio of female heterozygotes to male heterozygotes to female homozygous mutants to male homozygous mutants is 1∶1∶1∶1, but we observed a ratio of about 1∶1∶0∶2 (46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males). This result rules out the hypothesis that homozygous mutant females die, but is predicted by the hypothesis that homozygous mutants that otherwise would have become females develop instead as males.
Mentions: To characterize the fancl HG10A phenotype, we crossed fancl +/HG10A heterozygotes (called fancl+/− below), and after genotyping the progeny by PCR, observed that all fancl HG10A/HG10A homozygous mutants (called fancl−/− below) developed exclusively as males, even though their wild-type and heterozygous siblings developed about as many females as males. Two alternative hypotheses could explain the lack of homozygous fancl mutant females: female-specific lethality or female-to-male sex reversal. To distinguish between these two hypotheses, we crossed female fancl+/− heterozygotes to male fancl−/− homozygotes. We raised 211 progeny to adulthood, determined their phenotypic sex according to sexually dimorphic characters including the color of the anal fin and body shape, and finally scored their fancl genotypes by PCR. Under normal conditions, this cross should give 50% heterozygotes (about half of which should be female), and 50% homozygous mutants (about half of which should be female), expecting a 1∶1∶1∶1 ratio of heterozygous females to heterozygous males to homozygous mutant females to homozygous mutant males. The fancl female death hypothesis predicts a 1∶1∶0∶1 ratio, or 66% heterozygotes and 33% homozygous mutants, but the sex reversal hypothesis, predicts a 1∶1∶0∶2 ratio, or equal proportions (50%∶50%) of homozygous mutants (all male) and heterozygotes (males plus females). Resulting genotypes revealed 46 fancl+/− females: 62 fancl+/− males: 0 fancl−/− females: 103 fancl−/− males, which showed that about half of the progeny were fancl homozygous mutants (103/211, 49%) and the other half were heterozygous for the fancl mutation (108/211, 51%) (Figure 2). These results had strong statistical support (chi-square likelihood ratio  = 0.794, p-value >0.1), thus ruling out the hypothesis that homozygous fancl mutant females died. Results, however, were consistent with the hypothesis that animals that otherwise would have become females developed as males due to female-to-male sex reversal. Sex distributions within each genotype confirmed our previous observations that all fancl homozygous mutants developed as males (n = 103, 100%), and while approximately half of fancl heterozygous siblings developed as males (n = 62, 57%), the other half developed as females (n = 46, 43%) (Figure 2). These scores showed strong statistical support for the hypothesis that fancl mutants experienced female-to-male sex reversal (chi-square likelihood ratio  = 73.946, p-value<0.0001). To exclude the possibility that some of the fancl mutants could have ovaries despite their external male phenotypic characters, we dissected the gonads of adult fancl homozygous mutants (n = 45), heterozygous females (n = 11) and heterozygous males (n = 29). In all cases, we found a perfect match between external sexual characters and gonadal sex. These results ruled out the possibility that fancl mutants masqueraded as males externally while having female gonads. We conclude that the HG10A Tol2 insertion into fancl induced a female-to-male sex reversal phenotype in zebrafish.

Bottom Line: Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival.Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females.Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America.

ABSTRACT
The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53) mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.

Show MeSH
Related in: MedlinePlus