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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 Tol2 insertion HG10A disrupts fancl transcripts.(A) Zebrafish fancl gene structure showing the Tol2 insertion in exon 12 and the position of primer pairs used for RT-PCR experiments (arrows, F1-R1; F2-R2). Numbered boxes represent exons and dashed boxes indicate untranslated regions. (B) Schematic representation from exon 11 to 13 of the wild-type fancl transcript (1.WT) and fancl mutant transcripts (2.fanclTol2 and 3.fanclDTol2). The PHD finger domain is highlighted in grey. The Tol2 insertion is shown in black and an arrowhead points to its insertion site in the amino acid sequence in B.2. Predicted protein sequences are shown; the highly conserved Cys and His residues are underlined and the critical Trp is double underlined. Asterisks represent premature stop codons. (C) RT-PCR using as template cDNA of adult testes shows that the 232 bp band containing the intact PHD domain in wild types (amplified by F2-R2 primers) is absent from fancl mutants. The smaller band (174 bp) amplified in fancl mutants corresponds to the fanclDTol2 transcript in B.3. Abbreviations: M, DNA-Marker.
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pgen-1001034-g001: The Tol2 insertion HG10A disrupts fancl transcripts.(A) Zebrafish fancl gene structure showing the Tol2 insertion in exon 12 and the position of primer pairs used for RT-PCR experiments (arrows, F1-R1; F2-R2). Numbered boxes represent exons and dashed boxes indicate untranslated regions. (B) Schematic representation from exon 11 to 13 of the wild-type fancl transcript (1.WT) and fancl mutant transcripts (2.fanclTol2 and 3.fanclDTol2). The PHD finger domain is highlighted in grey. The Tol2 insertion is shown in black and an arrowhead points to its insertion site in the amino acid sequence in B.2. Predicted protein sequences are shown; the highly conserved Cys and His residues are underlined and the critical Trp is double underlined. Asterisks represent premature stop codons. (C) RT-PCR using as template cDNA of adult testes shows that the 232 bp band containing the intact PHD domain in wild types (amplified by F2-R2 primers) is absent from fancl mutants. The smaller band (174 bp) amplified in fancl mutants corresponds to the fanclDTol2 transcript in B.3. Abbreviations: M, DNA-Marker.

Mentions: A zebrafish fancl mutant (allele HG10A, accession number AB353980) was generated by insertional mutagenesis in a Tol2 transposon-mediated enhancer trap screen [42]. Cloning and sequencing of genomic DNA surrounding the insertion revealed that the Tol2 construct was inserted into exon 12 of fancl, thereby disrupting the coding region of the PHD finger domain (Figure 1A and 1B), which is essential for Fancl function [39].


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 Tol2 insertion HG10A disrupts fancl transcripts.(A) Zebrafish fancl gene structure showing the Tol2 insertion in exon 12 and the position of primer pairs used for RT-PCR experiments (arrows, F1-R1; F2-R2). Numbered boxes represent exons and dashed boxes indicate untranslated regions. (B) Schematic representation from exon 11 to 13 of the wild-type fancl transcript (1.WT) and fancl mutant transcripts (2.fanclTol2 and 3.fanclDTol2). The PHD finger domain is highlighted in grey. The Tol2 insertion is shown in black and an arrowhead points to its insertion site in the amino acid sequence in B.2. Predicted protein sequences are shown; the highly conserved Cys and His residues are underlined and the critical Trp is double underlined. Asterisks represent premature stop codons. (C) RT-PCR using as template cDNA of adult testes shows that the 232 bp band containing the intact PHD domain in wild types (amplified by F2-R2 primers) is absent from fancl mutants. The smaller band (174 bp) amplified in fancl mutants corresponds to the fanclDTol2 transcript in B.3. Abbreviations: M, DNA-Marker.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001034-g001: The Tol2 insertion HG10A disrupts fancl transcripts.(A) Zebrafish fancl gene structure showing the Tol2 insertion in exon 12 and the position of primer pairs used for RT-PCR experiments (arrows, F1-R1; F2-R2). Numbered boxes represent exons and dashed boxes indicate untranslated regions. (B) Schematic representation from exon 11 to 13 of the wild-type fancl transcript (1.WT) and fancl mutant transcripts (2.fanclTol2 and 3.fanclDTol2). The PHD finger domain is highlighted in grey. The Tol2 insertion is shown in black and an arrowhead points to its insertion site in the amino acid sequence in B.2. Predicted protein sequences are shown; the highly conserved Cys and His residues are underlined and the critical Trp is double underlined. Asterisks represent premature stop codons. (C) RT-PCR using as template cDNA of adult testes shows that the 232 bp band containing the intact PHD domain in wild types (amplified by F2-R2 primers) is absent from fancl mutants. The smaller band (174 bp) amplified in fancl mutants corresponds to the fanclDTol2 transcript in B.3. Abbreviations: M, DNA-Marker.
Mentions: A zebrafish fancl mutant (allele HG10A, accession number AB353980) was generated by insertional mutagenesis in a Tol2 transposon-mediated enhancer trap screen [42]. Cloning and sequencing of genomic DNA surrounding the insertion revealed that the Tol2 construct was inserted into exon 12 of fancl, thereby disrupting the coding region of the PHD finger domain (Figure 1A and 1B), which is essential for Fancl function [39].

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