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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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Identification of homozygous houdini mutants using ‘phenotyping by segregation.’(A-D) Representative distributions larval responsiveness within F2 and F3 clutches, with hypersensitive larvae displayed in red. Larval behavior was stimulated and analyzed as in Figure 1. (A) A wildtype (WIK) incross produced larvae with a meannSLC responsiveness of 8.9±13.5% (mean±SD). The hypersensitivity threshold was set at 36% SLC responsiveness (mean + 2SD), where 2/30 (6.7%) wildtype larvae were classified as hypersensitive. (B) A F1 houdini heterozygous incross produced 9/45 (20%) hypersensitive larvae. (C) An incross of raised houdini F2 mutants produced 24/32 (75%) hypersensitive larvae. (D) A backcross of a raised F2 mutant from (C) and a known F1 heterozygote produced 16/32 (50%) hypersensitive larvae. This cross was performed on a separate occasion where the hypersensitivity threshold was set to 43% based on the wildtype (WIK) control.
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pone-0026510-g002: Identification of homozygous houdini mutants using ‘phenotyping by segregation.’(A-D) Representative distributions larval responsiveness within F2 and F3 clutches, with hypersensitive larvae displayed in red. Larval behavior was stimulated and analyzed as in Figure 1. (A) A wildtype (WIK) incross produced larvae with a meannSLC responsiveness of 8.9±13.5% (mean±SD). The hypersensitivity threshold was set at 36% SLC responsiveness (mean + 2SD), where 2/30 (6.7%) wildtype larvae were classified as hypersensitive. (B) A F1 houdini heterozygous incross produced 9/45 (20%) hypersensitive larvae. (C) An incross of raised houdini F2 mutants produced 24/32 (75%) hypersensitive larvae. (D) A backcross of a raised F2 mutant from (C) and a known F1 heterozygote produced 16/32 (50%) hypersensitive larvae. This cross was performed on a separate occasion where the hypersensitivity threshold was set to 43% based on the wildtype (WIK) control.

Mentions: To map the houdini mutation to a chromosomal region, we crossed a houdini carrier to a polymorphic wildtype strain (WIK), classified F2 larvae as putative mutants or wildtype siblings based on startle sensitivity at 5 dpf, then raised mutant and wildtype sibling F2 larvae separately to adulthood (Figure 1A) [28]. To evaluate homozygosity of the adult F2s, potential mutant F2 individuals were first incrossed, and the distribution of F3 larval responsiveness was compared to known wildtype and F1 heterozygous incross clutches. Second, potential adult F2 mutants were backcrossed to known F1 heterozygotes, again comparing the larval responsiveness distribution to wildtype and F1 heterozygous incrosses. Theoretically, crosses of two houdini homozygotes should produce 100% hypersensitive F3 progeny, a homozygote and a heterozygote should produce 50% hyper-responsive progeny, and two heterozygotes should produce 25% hypersensitive progeny. However, given the variable penetrance of the houdini phenotype, we expected that even a clutch of 100% houdini homozygous larvae would still show a responsiveness distribution partially overlapping the normal range of wildtype responsiveness. Therefore, we always incrossed potential F2 houdini homozygotes alongside F1×F1 heterozygous crosses and F1×WIK outcrosses, classifying the F2 pair as potential homozygotes or heterozygotes only if their clutch contained a significantly higher fraction of hypersensitive larvae than the known F1×F1 cross (Figure 2).


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

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

Identification of homozygous houdini mutants using ‘phenotyping by segregation.’(A-D) Representative distributions larval responsiveness within F2 and F3 clutches, with hypersensitive larvae displayed in red. Larval behavior was stimulated and analyzed as in Figure 1. (A) A wildtype (WIK) incross produced larvae with a meannSLC responsiveness of 8.9±13.5% (mean±SD). The hypersensitivity threshold was set at 36% SLC responsiveness (mean + 2SD), where 2/30 (6.7%) wildtype larvae were classified as hypersensitive. (B) A F1 houdini heterozygous incross produced 9/45 (20%) hypersensitive larvae. (C) An incross of raised houdini F2 mutants produced 24/32 (75%) hypersensitive larvae. (D) A backcross of a raised F2 mutant from (C) and a known F1 heterozygote produced 16/32 (50%) hypersensitive larvae. This cross was performed on a separate occasion where the hypersensitivity threshold was set to 43% based on the wildtype (WIK) control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198425&req=5

pone-0026510-g002: Identification of homozygous houdini mutants using ‘phenotyping by segregation.’(A-D) Representative distributions larval responsiveness within F2 and F3 clutches, with hypersensitive larvae displayed in red. Larval behavior was stimulated and analyzed as in Figure 1. (A) A wildtype (WIK) incross produced larvae with a meannSLC responsiveness of 8.9±13.5% (mean±SD). The hypersensitivity threshold was set at 36% SLC responsiveness (mean + 2SD), where 2/30 (6.7%) wildtype larvae were classified as hypersensitive. (B) A F1 houdini heterozygous incross produced 9/45 (20%) hypersensitive larvae. (C) An incross of raised houdini F2 mutants produced 24/32 (75%) hypersensitive larvae. (D) A backcross of a raised F2 mutant from (C) and a known F1 heterozygote produced 16/32 (50%) hypersensitive larvae. This cross was performed on a separate occasion where the hypersensitivity threshold was set to 43% based on the wildtype (WIK) control.
Mentions: To map the houdini mutation to a chromosomal region, we crossed a houdini carrier to a polymorphic wildtype strain (WIK), classified F2 larvae as putative mutants or wildtype siblings based on startle sensitivity at 5 dpf, then raised mutant and wildtype sibling F2 larvae separately to adulthood (Figure 1A) [28]. To evaluate homozygosity of the adult F2s, potential mutant F2 individuals were first incrossed, and the distribution of F3 larval responsiveness was compared to known wildtype and F1 heterozygous incross clutches. Second, potential adult F2 mutants were backcrossed to known F1 heterozygotes, again comparing the larval responsiveness distribution to wildtype and F1 heterozygous incrosses. Theoretically, crosses of two houdini homozygotes should produce 100% hypersensitive F3 progeny, a homozygote and a heterozygote should produce 50% hyper-responsive progeny, and two heterozygotes should produce 25% hypersensitive progeny. However, given the variable penetrance of the houdini phenotype, we expected that even a clutch of 100% houdini homozygous larvae would still show a responsiveness distribution partially overlapping the normal range of wildtype responsiveness. Therefore, we always incrossed potential F2 houdini homozygotes alongside F1×F1 heterozygous crosses and F1×WIK outcrosses, classifying the F2 pair as potential homozygotes or heterozygotes only if their clutch contained a significantly higher fraction of hypersensitive larvae than the known F1×F1 cross (Figure 2).

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