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Molecular-genetic mapping of zebrafish mutants with variable phenotypic penetrance.

Jain RA, Wolman MA, Schmidt LA, Burgess HA, Granato M - PLoS ONE (2011)

Bottom Line: Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders.Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task.Using a 'phenotyping by segregation' strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders. Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task. Using a 'phenotyping by segregation' strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes.

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Related in: MedlinePlus

A ‘phenotyping by segregation’ strategy to map the variably penetrant houdini mutation.(A) Mapping zebrafish mutants with weak or variable penetrance using a ‘phenotyping by segregation’ strategy, where F3 phenotypic segregation is used to validate homozygous F2 mutants. 1) A standard mapcross is generated using a mutant carrier G0 and a polymorphic wildtype G0 [35]. 2) Heterozygous carrier F1s are isolated and incrossed to generate F2 larvae. 3) F2 larvae at the top 15% of the phenotypic range of the clutch are raised to adulthood as potential mutants, alongside an equal number of siblings (from the bottom 15% of the phenotypic range of the clutch) as controls. 4) Genomic DNA is taken from each raised F2 individual, and F2s are then randomly incrossed. 5) F2 pairs producing clutches with a greater frequency of phenotypic outliers than a control F1 heterozygous incross are next individually backcrossed to a known F1 heterozygote. Any raised F2 individual which again produced a clutch with a greater frequency of phenotypic outliers than the control F1 heterozygote incross is deemed a “validated” mutant, and is used for subsequent bulked segregant mapping. (B-C) Distributions of SLC startle responsiveness to weak subthreshold acoustic stimuli in 5 dpf larval progeny of a houdini heterozygote and a wildtype TLF adult (B) and two heterozygous houdini carriers in the same genetic background (C). Responsiveness was measured over 20 weak “subthreshold” acoustic stimuli. The mean %SLC+2SD was set as the hypersensitivity threshold for each experiment, 42% in this example. If >15% of a clutch performed above the hypersensitivity threshold for the experiment (in red), both parents were considered to carry the recessive houdini mutation.
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pone-0026510-g001: A ‘phenotyping by segregation’ strategy to map the variably penetrant houdini mutation.(A) Mapping zebrafish mutants with weak or variable penetrance using a ‘phenotyping by segregation’ strategy, where F3 phenotypic segregation is used to validate homozygous F2 mutants. 1) A standard mapcross is generated using a mutant carrier G0 and a polymorphic wildtype G0 [35]. 2) Heterozygous carrier F1s are isolated and incrossed to generate F2 larvae. 3) F2 larvae at the top 15% of the phenotypic range of the clutch are raised to adulthood as potential mutants, alongside an equal number of siblings (from the bottom 15% of the phenotypic range of the clutch) as controls. 4) Genomic DNA is taken from each raised F2 individual, and F2s are then randomly incrossed. 5) F2 pairs producing clutches with a greater frequency of phenotypic outliers than a control F1 heterozygous incross are next individually backcrossed to a known F1 heterozygote. Any raised F2 individual which again produced a clutch with a greater frequency of phenotypic outliers than the control F1 heterozygote incross is deemed a “validated” mutant, and is used for subsequent bulked segregant mapping. (B-C) Distributions of SLC startle responsiveness to weak subthreshold acoustic stimuli in 5 dpf larval progeny of a houdini heterozygote and a wildtype TLF adult (B) and two heterozygous houdini carriers in the same genetic background (C). Responsiveness was measured over 20 weak “subthreshold” acoustic stimuli. The mean %SLC+2SD was set as the hypersensitivity threshold for each experiment, 42% in this example. If >15% of a clutch performed above the hypersensitivity threshold for the experiment (in red), both parents were considered to carry the recessive houdini mutation.

Mentions: Zebrafish are rapidly proving to be an excellent model system in which to genetically dissect a wide variety of motor and cognitive behaviors and disease endophenotypes [10], [11], [12], [13]. Unbiased forward genetic screens for behavioral mutants have been successfully performed, and an extensive array of mutants have been isolated and cloned via their neuromorphological defects during development [14], [15], [16], [17], [18]. However, mapping and molecularly identifying mutants purely based on their behavioral phenotype in the absence of a visible anatomical defect has been much more challenging, and relatively few have been mapped and cloned in this fashion [17], [19], [20], [21], [22], [23]. To identify genetic factors regulating acoustic startle responsiveness that may be relevant to neuropsychiatric disorders, we previously reported a forward genetic screen of ENU-mutagenized zebrafish larvae at 5 days post-fertilization (5 dpf) for mutants with subtle defects in the sensitivity and gating of the larval acoustic startle response [18]. Many of these mutants, including the hypersensitive mutant houdini, were morphologically normal and initial attempts at standard bulked segregant mapping failed, likely since the overlapping phenotypic variance of mutant and wildtype individuals led to misclassification of siblings as mutants (see below). To overcome this misclassification at the larval stage, we adopted a ‘phenotyping by segregation’ strategy to map the houdini mutant, broadly applicable to mutants with variable phenotypic expressivity and penetrance (Figure 1A).


Molecular-genetic mapping of zebrafish mutants with variable phenotypic penetrance.

Jain RA, Wolman MA, Schmidt LA, Burgess HA, Granato M - PLoS ONE (2011)

A ‘phenotyping by segregation’ strategy to map the variably penetrant houdini mutation.(A) Mapping zebrafish mutants with weak or variable penetrance using a ‘phenotyping by segregation’ strategy, where F3 phenotypic segregation is used to validate homozygous F2 mutants. 1) A standard mapcross is generated using a mutant carrier G0 and a polymorphic wildtype G0 [35]. 2) Heterozygous carrier F1s are isolated and incrossed to generate F2 larvae. 3) F2 larvae at the top 15% of the phenotypic range of the clutch are raised to adulthood as potential mutants, alongside an equal number of siblings (from the bottom 15% of the phenotypic range of the clutch) as controls. 4) Genomic DNA is taken from each raised F2 individual, and F2s are then randomly incrossed. 5) F2 pairs producing clutches with a greater frequency of phenotypic outliers than a control F1 heterozygous incross are next individually backcrossed to a known F1 heterozygote. Any raised F2 individual which again produced a clutch with a greater frequency of phenotypic outliers than the control F1 heterozygote incross is deemed a “validated” mutant, and is used for subsequent bulked segregant mapping. (B-C) Distributions of SLC startle responsiveness to weak subthreshold acoustic stimuli in 5 dpf larval progeny of a houdini heterozygote and a wildtype TLF adult (B) and two heterozygous houdini carriers in the same genetic background (C). Responsiveness was measured over 20 weak “subthreshold” acoustic stimuli. The mean %SLC+2SD was set as the hypersensitivity threshold for each experiment, 42% in this example. If >15% of a clutch performed above the hypersensitivity threshold for the experiment (in red), both parents were considered to carry the recessive houdini mutation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198425&req=5

pone-0026510-g001: A ‘phenotyping by segregation’ strategy to map the variably penetrant houdini mutation.(A) Mapping zebrafish mutants with weak or variable penetrance using a ‘phenotyping by segregation’ strategy, where F3 phenotypic segregation is used to validate homozygous F2 mutants. 1) A standard mapcross is generated using a mutant carrier G0 and a polymorphic wildtype G0 [35]. 2) Heterozygous carrier F1s are isolated and incrossed to generate F2 larvae. 3) F2 larvae at the top 15% of the phenotypic range of the clutch are raised to adulthood as potential mutants, alongside an equal number of siblings (from the bottom 15% of the phenotypic range of the clutch) as controls. 4) Genomic DNA is taken from each raised F2 individual, and F2s are then randomly incrossed. 5) F2 pairs producing clutches with a greater frequency of phenotypic outliers than a control F1 heterozygous incross are next individually backcrossed to a known F1 heterozygote. Any raised F2 individual which again produced a clutch with a greater frequency of phenotypic outliers than the control F1 heterozygote incross is deemed a “validated” mutant, and is used for subsequent bulked segregant mapping. (B-C) Distributions of SLC startle responsiveness to weak subthreshold acoustic stimuli in 5 dpf larval progeny of a houdini heterozygote and a wildtype TLF adult (B) and two heterozygous houdini carriers in the same genetic background (C). Responsiveness was measured over 20 weak “subthreshold” acoustic stimuli. The mean %SLC+2SD was set as the hypersensitivity threshold for each experiment, 42% in this example. If >15% of a clutch performed above the hypersensitivity threshold for the experiment (in red), both parents were considered to carry the recessive houdini mutation.
Mentions: Zebrafish are rapidly proving to be an excellent model system in which to genetically dissect a wide variety of motor and cognitive behaviors and disease endophenotypes [10], [11], [12], [13]. Unbiased forward genetic screens for behavioral mutants have been successfully performed, and an extensive array of mutants have been isolated and cloned via their neuromorphological defects during development [14], [15], [16], [17], [18]. However, mapping and molecularly identifying mutants purely based on their behavioral phenotype in the absence of a visible anatomical defect has been much more challenging, and relatively few have been mapped and cloned in this fashion [17], [19], [20], [21], [22], [23]. To identify genetic factors regulating acoustic startle responsiveness that may be relevant to neuropsychiatric disorders, we previously reported a forward genetic screen of ENU-mutagenized zebrafish larvae at 5 days post-fertilization (5 dpf) for mutants with subtle defects in the sensitivity and gating of the larval acoustic startle response [18]. Many of these mutants, including the hypersensitive mutant houdini, were morphologically normal and initial attempts at standard bulked segregant mapping failed, likely since the overlapping phenotypic variance of mutant and wildtype individuals led to misclassification of siblings as mutants (see below). To overcome this misclassification at the larval stage, we adopted a ‘phenotyping by segregation’ strategy to map the houdini mutant, broadly applicable to mutants with variable phenotypic expressivity and penetrance (Figure 1A).

Bottom Line: Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders.Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task.Using a 'phenotyping by segregation' strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Forward genetic screens in vertebrates are powerful tools to generate models relevant to human diseases, including neuropsychiatric disorders. Variability in phenotypic penetrance and expressivity is common in these disorders and behavioral mutant models, making their molecular-genetic mapping a formidable task. Using a 'phenotyping by segregation' strategy, we molecularly map the hypersensitive zebrafish houdini mutant despite its variable phenotypic penetrance, providing a generally applicable strategy to map zebrafish mutants with subtle phenotypes.

Show MeSH
Related in: MedlinePlus