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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

houdini mutants verified by F3 segregation show molecular linkage to chromosome 5.(A) The pool of F2 mutants validated by F3 segregation (“Adult Mut Pool”), shows strong linkage to the z22250 and z14591 alleles on chromosome 5 of the mutant G0 grandparent. Very little linkage to the G0 mutant alleles is evident using a pool of unvalidated mutants selected only by F2 larval behavior (“Larval Mut Pool”), or control F2 larval siblings (“Larval Sib Pool”) with these same markers. (B) Individuals composing the validated adult F2 mutant pool. 11/12 individuals are homozygous for the mutant z22250 allele, while 8/12 individuals are homozygous for the mutant z14591 allele. Individual #8 is homozygous for the mutant z22250 and wildtype z14591 alleles, indicating both the maternal and paternal copies of the chromosome underwent meiotic recombination between the houdini mutation and z14591. (C) 12 representative individuals from the unvalidated larval F2 mutant pool. The F2 individuals raised or pooled all performed above the hypersensitivity threshold and furthermore were among the most responsive 15% of their clutch. 5/12 larvae contain wildtype z22250 and z14591 alleles (marked with X) and are likely not homozygous mutants.
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pone-0026510-g003: houdini mutants verified by F3 segregation show molecular linkage to chromosome 5.(A) The pool of F2 mutants validated by F3 segregation (“Adult Mut Pool”), shows strong linkage to the z22250 and z14591 alleles on chromosome 5 of the mutant G0 grandparent. Very little linkage to the G0 mutant alleles is evident using a pool of unvalidated mutants selected only by F2 larval behavior (“Larval Mut Pool”), or control F2 larval siblings (“Larval Sib Pool”) with these same markers. (B) Individuals composing the validated adult F2 mutant pool. 11/12 individuals are homozygous for the mutant z22250 allele, while 8/12 individuals are homozygous for the mutant z14591 allele. Individual #8 is homozygous for the mutant z22250 and wildtype z14591 alleles, indicating both the maternal and paternal copies of the chromosome underwent meiotic recombination between the houdini mutation and z14591. (C) 12 representative individuals from the unvalidated larval F2 mutant pool. The F2 individuals raised or pooled all performed above the hypersensitivity threshold and furthermore were among the most responsive 15% of their clutch. 5/12 larvae contain wildtype z22250 and z14591 alleles (marked with X) and are likely not homozygous mutants.

Mentions: To molecularly link houdini to a genomic region, we used pooled DNA from 12 F2 mutants validated by the ‘phenotyping by segregation’ strategy to look for linkage to SSLP markers. The “validated” F2 mutant pool showed strong linkage to the z22250 and z14591 markers on chromosome 5, whereas both G0 grandparent alleles were represented in the F2 sibling pool (Figure 3A). To confirm linkage of houdini to these two SSLP markers, the individuals comprising the adult F2 pools were tested (Figure 3B). 11/12 individuals were homozygous for the mutant z22250 allele, while 8/12 individuals were homozygous for the mutant z14591 allele. The presence of the wildtype allele in any mutant individual indicates that individual is either a houdini homozygote in which meiotic recombination occurred between the wildtype and mutant chromosomes in an F1 parent, or a houdini heterozygote which was misclassified based on its behavior. As these two markers have been mapped as 25.5 cM apart (i.e. showing a 25.5% meiotic recombination frequency), the observed frequency of segregation of z22250 and z14591 alleles (6/24 meioses, or a 25.0% recombination frequency) is consistent with classifying these as recombination events. Importantly, the individual heterozygous at the z22250 locus (#6) was homozygous for the z14591 mutant allele, and the individuals carrying the wildtype z14591 allele (#3,4,7,8) were all homozygous for the z22250 mutant allele, suggesting that these individuals are all houdini mutants carrying recombinant chromosomes, rather than misclassified individuals. These data additionally suggest that these markers are on opposite sides of the houdini mutation. In contrast to the homozygous frequencies observed in validated F2 adult mutant individuals, only 2/49 and 9/49 F2 adult wildtype sibling individuals were homozygous for the mutant alleles of z22250 and z14591, respectively (data not shown). Furthermore, 39/49 of these sibling individuals contained wildtype alleles of both markers, indicating they are likely heterozygous or homozygous for the wildtype houdini locus (data not shown). As a result, the F2 adult sibling pool did not show any enrichment of the G0 mutant alleles for these markers, even showing a slight enrichment of the wildtype allele in the case of the z14591 marker (Figure 3A).


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

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

houdini mutants verified by F3 segregation show molecular linkage to chromosome 5.(A) The pool of F2 mutants validated by F3 segregation (“Adult Mut Pool”), shows strong linkage to the z22250 and z14591 alleles on chromosome 5 of the mutant G0 grandparent. Very little linkage to the G0 mutant alleles is evident using a pool of unvalidated mutants selected only by F2 larval behavior (“Larval Mut Pool”), or control F2 larval siblings (“Larval Sib Pool”) with these same markers. (B) Individuals composing the validated adult F2 mutant pool. 11/12 individuals are homozygous for the mutant z22250 allele, while 8/12 individuals are homozygous for the mutant z14591 allele. Individual #8 is homozygous for the mutant z22250 and wildtype z14591 alleles, indicating both the maternal and paternal copies of the chromosome underwent meiotic recombination between the houdini mutation and z14591. (C) 12 representative individuals from the unvalidated larval F2 mutant pool. The F2 individuals raised or pooled all performed above the hypersensitivity threshold and furthermore were among the most responsive 15% of their clutch. 5/12 larvae contain wildtype z22250 and z14591 alleles (marked with X) and are likely not homozygous mutants.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198425&req=5

pone-0026510-g003: houdini mutants verified by F3 segregation show molecular linkage to chromosome 5.(A) The pool of F2 mutants validated by F3 segregation (“Adult Mut Pool”), shows strong linkage to the z22250 and z14591 alleles on chromosome 5 of the mutant G0 grandparent. Very little linkage to the G0 mutant alleles is evident using a pool of unvalidated mutants selected only by F2 larval behavior (“Larval Mut Pool”), or control F2 larval siblings (“Larval Sib Pool”) with these same markers. (B) Individuals composing the validated adult F2 mutant pool. 11/12 individuals are homozygous for the mutant z22250 allele, while 8/12 individuals are homozygous for the mutant z14591 allele. Individual #8 is homozygous for the mutant z22250 and wildtype z14591 alleles, indicating both the maternal and paternal copies of the chromosome underwent meiotic recombination between the houdini mutation and z14591. (C) 12 representative individuals from the unvalidated larval F2 mutant pool. The F2 individuals raised or pooled all performed above the hypersensitivity threshold and furthermore were among the most responsive 15% of their clutch. 5/12 larvae contain wildtype z22250 and z14591 alleles (marked with X) and are likely not homozygous mutants.
Mentions: To molecularly link houdini to a genomic region, we used pooled DNA from 12 F2 mutants validated by the ‘phenotyping by segregation’ strategy to look for linkage to SSLP markers. The “validated” F2 mutant pool showed strong linkage to the z22250 and z14591 markers on chromosome 5, whereas both G0 grandparent alleles were represented in the F2 sibling pool (Figure 3A). To confirm linkage of houdini to these two SSLP markers, the individuals comprising the adult F2 pools were tested (Figure 3B). 11/12 individuals were homozygous for the mutant z22250 allele, while 8/12 individuals were homozygous for the mutant z14591 allele. The presence of the wildtype allele in any mutant individual indicates that individual is either a houdini homozygote in which meiotic recombination occurred between the wildtype and mutant chromosomes in an F1 parent, or a houdini heterozygote which was misclassified based on its behavior. As these two markers have been mapped as 25.5 cM apart (i.e. showing a 25.5% meiotic recombination frequency), the observed frequency of segregation of z22250 and z14591 alleles (6/24 meioses, or a 25.0% recombination frequency) is consistent with classifying these as recombination events. Importantly, the individual heterozygous at the z22250 locus (#6) was homozygous for the z14591 mutant allele, and the individuals carrying the wildtype z14591 allele (#3,4,7,8) were all homozygous for the z22250 mutant allele, suggesting that these individuals are all houdini mutants carrying recombinant chromosomes, rather than misclassified individuals. These data additionally suggest that these markers are on opposite sides of the houdini mutation. In contrast to the homozygous frequencies observed in validated F2 adult mutant individuals, only 2/49 and 9/49 F2 adult wildtype sibling individuals were homozygous for the mutant alleles of z22250 and z14591, respectively (data not shown). Furthermore, 39/49 of these sibling individuals contained wildtype alleles of both markers, indicating they are likely heterozygous or homozygous for the wildtype houdini locus (data not shown). As a result, the F2 adult sibling pool did not show any enrichment of the G0 mutant alleles for these markers, even showing a slight enrichment of the wildtype allele in the case of the z14591 marker (Figure 3A).

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