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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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Mutation of tp53 rescues the female-to-male sex-reversal phenotype of fancl mutants by reducing germ cell apoptosis.(A) The distribution of individuals of different tp53 genotypes among fancl−/− homozygous mutant progeny (n = 44) from an in-cross of double heterozygotes (fancl+/−;tp53+/−) is shown in a bar graph representing the number of females (purple bar) and males (green bars) distributed according to their tp53 genotypes (wild type, heterozygous or homozygous mutant). Rescue of female-to-male sex reversal was observed exclusively in fancl−/− mutant homozygotes that were also homozygous for the tp53 mutation (n = 15): 11 fancl−/−;tp53−/− animals developed as females and 4 developed as males. No rescue was observed in fancl mutants that were either wild-type (n = 8; fancl−/−;tp53+/+) or heterozygous for the tp53 mutation (n = 21; fancl−/−;tp53+/−), which all developed as males. Total numbers of animals (n) are indicated on the graph per each sex in each genotype. (B,C) Hematoxylin and eosin staining of gonad sections of wild-type female (fancl+/+;tp53+/+, B) and rescued female doubly homozygous mutant (fancl−/−;tp53−/−, C) at adult stage, revealed the presence of morphologically normal ovaries in the rescued fancl−/−;tp53−/− females. Ovaries of both genotypes had oocytes at different stages of development (i.e.: IB, II, III, IV). Scale bar: 0.1 mm (B,C). (D,E,F) tp53 mutation reduces germ cell apoptosis in fancl mutants at 25 dpf. Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of fancl homozygous mutants simultaneously homozygous wild-type (D) or homozygous mutant for tp53 (E) at 25 dpf. Dashed lines outline gonad boundaries (D,E). (F) Bar graph representing the average number of Caspase-3-positive germ cells in fancl−/−;tp53+/+ (n = 5) and fancl−/−;tp53−/− (n = 5) at 25 dpf. Results showed that the average number of apoptotic germ cells was approximately three fold lower in doubly homozygous mutant animals (fancl−/−;tp53−/−; x– = 30±56) than their fancl−/− mutant siblings that were wild-type for tp53 (fancl−/−;tp53+/+; x– = 105±71). This result shows that tp53 mutation decreased the number of apoptotic germ cells in fancl mutants at 25 dpf and demonstrates that the abnormal increase in germ cell apoptosis in fancl mutants that compromised the survival of developing oocytes was the mechanism responsible for the female-to-male sex reversal.
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pgen-1001034-g007: Mutation of tp53 rescues the female-to-male sex-reversal phenotype of fancl mutants by reducing germ cell apoptosis.(A) The distribution of individuals of different tp53 genotypes among fancl−/− homozygous mutant progeny (n = 44) from an in-cross of double heterozygotes (fancl+/−;tp53+/−) is shown in a bar graph representing the number of females (purple bar) and males (green bars) distributed according to their tp53 genotypes (wild type, heterozygous or homozygous mutant). Rescue of female-to-male sex reversal was observed exclusively in fancl−/− mutant homozygotes that were also homozygous for the tp53 mutation (n = 15): 11 fancl−/−;tp53−/− animals developed as females and 4 developed as males. No rescue was observed in fancl mutants that were either wild-type (n = 8; fancl−/−;tp53+/+) or heterozygous for the tp53 mutation (n = 21; fancl−/−;tp53+/−), which all developed as males. Total numbers of animals (n) are indicated on the graph per each sex in each genotype. (B,C) Hematoxylin and eosin staining of gonad sections of wild-type female (fancl+/+;tp53+/+, B) and rescued female doubly homozygous mutant (fancl−/−;tp53−/−, C) at adult stage, revealed the presence of morphologically normal ovaries in the rescued fancl−/−;tp53−/− females. Ovaries of both genotypes had oocytes at different stages of development (i.e.: IB, II, III, IV). Scale bar: 0.1 mm (B,C). (D,E,F) tp53 mutation reduces germ cell apoptosis in fancl mutants at 25 dpf. Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of fancl homozygous mutants simultaneously homozygous wild-type (D) or homozygous mutant for tp53 (E) at 25 dpf. Dashed lines outline gonad boundaries (D,E). (F) Bar graph representing the average number of Caspase-3-positive germ cells in fancl−/−;tp53+/+ (n = 5) and fancl−/−;tp53−/− (n = 5) at 25 dpf. Results showed that the average number of apoptotic germ cells was approximately three fold lower in doubly homozygous mutant animals (fancl−/−;tp53−/−; x– = 30±56) than their fancl−/− mutant siblings that were wild-type for tp53 (fancl−/−;tp53+/+; x– = 105±71). This result shows that tp53 mutation decreased the number of apoptotic germ cells in fancl mutants at 25 dpf and demonstrates that the abnormal increase in germ cell apoptosis in fancl mutants that compromised the survival of developing oocytes was the mechanism responsible for the female-to-male sex reversal.

Mentions: The hypothesis that the female-to-male sex reversal of fancl mutants is caused by increased germ cell apoptosis predicts that blocking apoptotic pathways should rescue the sex reversal phenotype. Because tumor protein Tp53 (alias p53) is an important activator of apoptosis [54], we can inhibit apoptosis in fancl mutants by introducing a tp53 mutation into the fancl mutant line. To generate double mutants, we crossed a zebrafish female carrier of the hypomorphic mutation tp53M214K [55] to a male homozygous fancl mutant, identified double heterozygotes (fancl+/HG10A;tp53+/M214K called fancl+/−;tp53+/− below) among F1 progeny by PCR, and in-crossed double heterozygotes to obtain an F2 population containing double homozygous mutants. Among the F2 raised to adulthood, 44/171 (25.7%), or about a quarter, were fancl−/− homozygous mutants. Among these 44 fancl−/− homozygous mutants, 15 were also tp53−/− homozygous mutants, from which 11 developed as females and four as males (Figure 7A). All of the fancl homozygous mutant siblings (n = 29) that were either homozygous wild type for tp53+/+ (n = 8) or heterozygous for the tp53+/− mutation (n = 21) developed exclusively as males (Figure 7A). This result shows that the female-to-male sex reversal phenotype characteristic of fancl mutants was rescued in fancl−/−;tp53−/− doubly homozygous mutants (Figure 7A). The sex-ratio scores observed in the three genotypes showed strong statistical support (chi-square likelihood ratio  = 32.088, p-value<0.0001) for the hypothesis that the presence of females in fancl−/−;tp53−/− double mutants and the absence of females in the other tp53 genotypes (fancl−/−;tp53+/− and fancl−/−;tp53+/+) is linked to the tp53 genotype. Histological analyses of fancl−/−;tp53−/− females corroborated the conclusion that external female sex characteristics were accompanied by ovaries filled with normal oocytes at all stages of development similar to fancl+/+; tp53+/+ wild-type female siblings (Figure 7B and 7C).


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)

Mutation of tp53 rescues the female-to-male sex-reversal phenotype of fancl mutants by reducing germ cell apoptosis.(A) The distribution of individuals of different tp53 genotypes among fancl−/− homozygous mutant progeny (n = 44) from an in-cross of double heterozygotes (fancl+/−;tp53+/−) is shown in a bar graph representing the number of females (purple bar) and males (green bars) distributed according to their tp53 genotypes (wild type, heterozygous or homozygous mutant). Rescue of female-to-male sex reversal was observed exclusively in fancl−/− mutant homozygotes that were also homozygous for the tp53 mutation (n = 15): 11 fancl−/−;tp53−/− animals developed as females and 4 developed as males. No rescue was observed in fancl mutants that were either wild-type (n = 8; fancl−/−;tp53+/+) or heterozygous for the tp53 mutation (n = 21; fancl−/−;tp53+/−), which all developed as males. Total numbers of animals (n) are indicated on the graph per each sex in each genotype. (B,C) Hematoxylin and eosin staining of gonad sections of wild-type female (fancl+/+;tp53+/+, B) and rescued female doubly homozygous mutant (fancl−/−;tp53−/−, C) at adult stage, revealed the presence of morphologically normal ovaries in the rescued fancl−/−;tp53−/− females. Ovaries of both genotypes had oocytes at different stages of development (i.e.: IB, II, III, IV). Scale bar: 0.1 mm (B,C). (D,E,F) tp53 mutation reduces germ cell apoptosis in fancl mutants at 25 dpf. Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of fancl homozygous mutants simultaneously homozygous wild-type (D) or homozygous mutant for tp53 (E) at 25 dpf. Dashed lines outline gonad boundaries (D,E). (F) Bar graph representing the average number of Caspase-3-positive germ cells in fancl−/−;tp53+/+ (n = 5) and fancl−/−;tp53−/− (n = 5) at 25 dpf. Results showed that the average number of apoptotic germ cells was approximately three fold lower in doubly homozygous mutant animals (fancl−/−;tp53−/−; x– = 30±56) than their fancl−/− mutant siblings that were wild-type for tp53 (fancl−/−;tp53+/+; x– = 105±71). This result shows that tp53 mutation decreased the number of apoptotic germ cells in fancl mutants at 25 dpf and demonstrates that the abnormal increase in germ cell apoptosis in fancl mutants that compromised the survival of developing oocytes was the mechanism responsible for the female-to-male sex reversal.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908690&req=5

pgen-1001034-g007: Mutation of tp53 rescues the female-to-male sex-reversal phenotype of fancl mutants by reducing germ cell apoptosis.(A) The distribution of individuals of different tp53 genotypes among fancl−/− homozygous mutant progeny (n = 44) from an in-cross of double heterozygotes (fancl+/−;tp53+/−) is shown in a bar graph representing the number of females (purple bar) and males (green bars) distributed according to their tp53 genotypes (wild type, heterozygous or homozygous mutant). Rescue of female-to-male sex reversal was observed exclusively in fancl−/− mutant homozygotes that were also homozygous for the tp53 mutation (n = 15): 11 fancl−/−;tp53−/− animals developed as females and 4 developed as males. No rescue was observed in fancl mutants that were either wild-type (n = 8; fancl−/−;tp53+/+) or heterozygous for the tp53 mutation (n = 21; fancl−/−;tp53+/−), which all developed as males. Total numbers of animals (n) are indicated on the graph per each sex in each genotype. (B,C) Hematoxylin and eosin staining of gonad sections of wild-type female (fancl+/+;tp53+/+, B) and rescued female doubly homozygous mutant (fancl−/−;tp53−/−, C) at adult stage, revealed the presence of morphologically normal ovaries in the rescued fancl−/−;tp53−/− females. Ovaries of both genotypes had oocytes at different stages of development (i.e.: IB, II, III, IV). Scale bar: 0.1 mm (B,C). (D,E,F) tp53 mutation reduces germ cell apoptosis in fancl mutants at 25 dpf. Immunodetection of apoptosis by anti-active Caspase-3 in paraffin sections of gonads of fancl homozygous mutants simultaneously homozygous wild-type (D) or homozygous mutant for tp53 (E) at 25 dpf. Dashed lines outline gonad boundaries (D,E). (F) Bar graph representing the average number of Caspase-3-positive germ cells in fancl−/−;tp53+/+ (n = 5) and fancl−/−;tp53−/− (n = 5) at 25 dpf. Results showed that the average number of apoptotic germ cells was approximately three fold lower in doubly homozygous mutant animals (fancl−/−;tp53−/−; x– = 30±56) than their fancl−/− mutant siblings that were wild-type for tp53 (fancl−/−;tp53+/+; x– = 105±71). This result shows that tp53 mutation decreased the number of apoptotic germ cells in fancl mutants at 25 dpf and demonstrates that the abnormal increase in germ cell apoptosis in fancl mutants that compromised the survival of developing oocytes was the mechanism responsible for the female-to-male sex reversal.
Mentions: The hypothesis that the female-to-male sex reversal of fancl mutants is caused by increased germ cell apoptosis predicts that blocking apoptotic pathways should rescue the sex reversal phenotype. Because tumor protein Tp53 (alias p53) is an important activator of apoptosis [54], we can inhibit apoptosis in fancl mutants by introducing a tp53 mutation into the fancl mutant line. To generate double mutants, we crossed a zebrafish female carrier of the hypomorphic mutation tp53M214K [55] to a male homozygous fancl mutant, identified double heterozygotes (fancl+/HG10A;tp53+/M214K called fancl+/−;tp53+/− below) among F1 progeny by PCR, and in-crossed double heterozygotes to obtain an F2 population containing double homozygous mutants. Among the F2 raised to adulthood, 44/171 (25.7%), or about a quarter, were fancl−/− homozygous mutants. Among these 44 fancl−/− homozygous mutants, 15 were also tp53−/− homozygous mutants, from which 11 developed as females and four as males (Figure 7A). All of the fancl homozygous mutant siblings (n = 29) that were either homozygous wild type for tp53+/+ (n = 8) or heterozygous for the tp53+/− mutation (n = 21) developed exclusively as males (Figure 7A). This result shows that the female-to-male sex reversal phenotype characteristic of fancl mutants was rescued in fancl−/−;tp53−/− doubly homozygous mutants (Figure 7A). The sex-ratio scores observed in the three genotypes showed strong statistical support (chi-square likelihood ratio  = 32.088, p-value<0.0001) for the hypothesis that the presence of females in fancl−/−;tp53−/− double mutants and the absence of females in the other tp53 genotypes (fancl−/−;tp53+/− and fancl−/−;tp53+/+) is linked to the tp53 genotype. Histological analyses of fancl−/−;tp53−/− females corroborated the conclusion that external female sex characteristics were accompanied by ovaries filled with normal oocytes at all stages of development similar to fancl+/+; tp53+/+ wild-type female siblings (Figure 7B and 7C).

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