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Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

Caruana G, Farlie PG, Hart AH, Bagheri-Fam S, Wallace MJ, Dobbie MS, Gordon CT, Miller KA, Whittle B, Abud HE, Arkell RM, Cole TJ, Harley VR, Smyth IM, Bertram JF - PLoS ONE (2013)

Bottom Line: ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads.Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia. georgina.caruana@monash.edu

ABSTRACT

Background: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).

Methodology/principal findings: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.

Conclusions/significance: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

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Related in: MedlinePlus

Testis cord defects in line 12BCC-20 at E13.5.Immunofluorescence staining for the Sertoli cell marker AMH (red, cytoplasm) on testis sections from unaffected (XY control) and affected (XY mutant) embryos. In affected embryos, testis cords are less organized when compared to unaffected embryos. Insets: Gross-examination of testes from unaffected and affected embryos. In affected embryos, testes are smaller and testis cords are barely detectable. Arrows indicate testis cords. All scale bars are 50 µm.
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pone-0055429-g005: Testis cord defects in line 12BCC-20 at E13.5.Immunofluorescence staining for the Sertoli cell marker AMH (red, cytoplasm) on testis sections from unaffected (XY control) and affected (XY mutant) embryos. In affected embryos, testis cords are less organized when compared to unaffected embryos. Insets: Gross-examination of testes from unaffected and affected embryos. In affected embryos, testes are smaller and testis cords are barely detectable. Arrows indicate testis cords. All scale bars are 50 µm.

Mentions: At E11.5, in the mouse, the bipotenital gonad commits to being either male (testis) or female (ovary) [43]. By E13.5, testes can be easily distinguished from ovaries by the presence of testis cords and their bigger size. In this screen we were interested in identifying mutants in which this decision had been disrupted resulting in an ovotestis phenotype. In addition, we were interested in mutations that may disrupt testis size, shape and testis cord formation. In line 12BCC-20, male G3 embryos with smaller testes were identified at E13.5 (7/36 embryos from affected litters (19.4%)) (Figure 5, insets). Moreover, while testis cords were clearly visible in unaffected gonads by gross examination, in affected gonads they were barely detectable (Figure 5, arrows in insets). Immunofluorescence staining for the Sertoli cell marker, AMH, revealed that in affected embryos the testis cords were less were less clearly defined when compared to unaffected embryos (arrows in Figure 5). The embryos with testis cord abnormalities were genotyped for chromosomal sex by PCR. All embryos had an XY karyotype; thus they did not display XX gonadal sex reversal. Unfortunately, the gonad anomalies were not detected in the second generation males possibly due to a weakly penetrant phenotype and the reduced contribution of the C57BL/6 mouse strain. C57BL/6 mice are exquisitely sensitive to testis development defects due to a higher gonadal expression of a female transcriptome relative to other mouse strains [44].


Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

Caruana G, Farlie PG, Hart AH, Bagheri-Fam S, Wallace MJ, Dobbie MS, Gordon CT, Miller KA, Whittle B, Abud HE, Arkell RM, Cole TJ, Harley VR, Smyth IM, Bertram JF - PLoS ONE (2013)

Testis cord defects in line 12BCC-20 at E13.5.Immunofluorescence staining for the Sertoli cell marker AMH (red, cytoplasm) on testis sections from unaffected (XY control) and affected (XY mutant) embryos. In affected embryos, testis cords are less organized when compared to unaffected embryos. Insets: Gross-examination of testes from unaffected and affected embryos. In affected embryos, testes are smaller and testis cords are barely detectable. Arrows indicate testis cords. All scale bars are 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585849&req=5

pone-0055429-g005: Testis cord defects in line 12BCC-20 at E13.5.Immunofluorescence staining for the Sertoli cell marker AMH (red, cytoplasm) on testis sections from unaffected (XY control) and affected (XY mutant) embryos. In affected embryos, testis cords are less organized when compared to unaffected embryos. Insets: Gross-examination of testes from unaffected and affected embryos. In affected embryos, testes are smaller and testis cords are barely detectable. Arrows indicate testis cords. All scale bars are 50 µm.
Mentions: At E11.5, in the mouse, the bipotenital gonad commits to being either male (testis) or female (ovary) [43]. By E13.5, testes can be easily distinguished from ovaries by the presence of testis cords and their bigger size. In this screen we were interested in identifying mutants in which this decision had been disrupted resulting in an ovotestis phenotype. In addition, we were interested in mutations that may disrupt testis size, shape and testis cord formation. In line 12BCC-20, male G3 embryos with smaller testes were identified at E13.5 (7/36 embryos from affected litters (19.4%)) (Figure 5, insets). Moreover, while testis cords were clearly visible in unaffected gonads by gross examination, in affected gonads they were barely detectable (Figure 5, arrows in insets). Immunofluorescence staining for the Sertoli cell marker, AMH, revealed that in affected embryos the testis cords were less were less clearly defined when compared to unaffected embryos (arrows in Figure 5). The embryos with testis cord abnormalities were genotyped for chromosomal sex by PCR. All embryos had an XY karyotype; thus they did not display XX gonadal sex reversal. Unfortunately, the gonad anomalies were not detected in the second generation males possibly due to a weakly penetrant phenotype and the reduced contribution of the C57BL/6 mouse strain. C57BL/6 mice are exquisitely sensitive to testis development defects due to a higher gonadal expression of a female transcriptome relative to other mouse strains [44].

Bottom Line: ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads.Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia. georgina.caruana@monash.edu

ABSTRACT

Background: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).

Methodology/principal findings: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.

Conclusions/significance: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

Show MeSH
Related in: MedlinePlus