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Conserved genes act as modifiers of invertebrate SMN loss of function defects.

Dimitriadi M, Sleigh JN, Walker A, Chang HC, Sen A, Kalloo G, Harris J, Barsby T, Walsh MB, Satterlee JS, Li C, Van Vactor D, Artavanis-Tsakonas S, Hart AC - PLoS Genet. (2010)

Bottom Line: Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model.Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects.Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Brown University, Providence, Rhode Island, USA.

ABSTRACT
Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

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Survival and average length of Cesmn-1(lf) animals is decreased.Homozygous loss of Cesmn-1(lf) results in slower growth and most animals die during larval stages. Animals that reach the adult stage are short-lived and sterile. A) Image of age-matched heterozygous hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) and homozygous Cesmn-1(lf) individuals (below). Scale bar indicates 150 microns. B) To test the impact of candidate modifier genes on growth rates and size, a C. elegans growth assay was established. +/Cesmn-1(lf) animals were collected as unhatched eggs, reared for 5 days/2 generations on standard C. elegans plates upon RNAi bacterial strains, as illustrated in the flow chart. Bacterial cultures expressed dsRNA corresponding to the gene of interest for RNAi knockdown; bacterial cultures containing vector with no insert were used as a control (‘empty’). C) Using an automated system, the length and fluorescence of animals was determined [51]; the smallest larvae in each culture were indistinguishable from debris and were excluded from the analysis as illustrated in the graph. Genotypes were discriminated by GFP fluorescence and length was determined as ‘time-of-flight’ through the laser chamber; each dot in the graph represents an individual animal. The disparate growth/survival rates of Cesmn-1(lf) and +/Cesmn-1(lf) animals alters the percentage of ‘large’ animals in these mixed stage cultures. As illustrated graphically, +/Cesmn-1(lf) and Cesmn-1(lf) animals can be sorted into blue and red boxes, respectively; animals designated as ‘large’ fall into shaded boxes. The fraction of large animals was calculated (# animals in shaded box/# animals in both shaded and unshaded box for each genotype) and is reported as % large with standard error of the mean (S.E.M.); significance was determined by Mann-Whitney U two-tailed test with p≤0.05. At least three independent cultures of more than 200 animals were scored for each targeted gene.
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pgen-1001172-g001: Survival and average length of Cesmn-1(lf) animals is decreased.Homozygous loss of Cesmn-1(lf) results in slower growth and most animals die during larval stages. Animals that reach the adult stage are short-lived and sterile. A) Image of age-matched heterozygous hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) and homozygous Cesmn-1(lf) individuals (below). Scale bar indicates 150 microns. B) To test the impact of candidate modifier genes on growth rates and size, a C. elegans growth assay was established. +/Cesmn-1(lf) animals were collected as unhatched eggs, reared for 5 days/2 generations on standard C. elegans plates upon RNAi bacterial strains, as illustrated in the flow chart. Bacterial cultures expressed dsRNA corresponding to the gene of interest for RNAi knockdown; bacterial cultures containing vector with no insert were used as a control (‘empty’). C) Using an automated system, the length and fluorescence of animals was determined [51]; the smallest larvae in each culture were indistinguishable from debris and were excluded from the analysis as illustrated in the graph. Genotypes were discriminated by GFP fluorescence and length was determined as ‘time-of-flight’ through the laser chamber; each dot in the graph represents an individual animal. The disparate growth/survival rates of Cesmn-1(lf) and +/Cesmn-1(lf) animals alters the percentage of ‘large’ animals in these mixed stage cultures. As illustrated graphically, +/Cesmn-1(lf) and Cesmn-1(lf) animals can be sorted into blue and red boxes, respectively; animals designated as ‘large’ fall into shaded boxes. The fraction of large animals was calculated (# animals in shaded box/# animals in both shaded and unshaded box for each genotype) and is reported as % large with standard error of the mean (S.E.M.); significance was determined by Mann-Whitney U two-tailed test with p≤0.05. At least three independent cultures of more than 200 animals were scored for each targeted gene.

Mentions: Although complete loss of SMN function causes lethality, C. elegans that are homozygous mutant for Cesmn-1(lf) can survive for several days due to partial maternal rescue. It has been suggested that +/Cesmn-1(lf) hermaphrodites load sufficient Cesmn-1 maternal protein and/or perhaps mRNA into oocytes to support development through embryogenesis and early larval stages [44]. Accordingly, homozygous Cesmn-1(lf) larvae initially resemble wild type animals. Eventually maternally-loaded Cesmn-1 product is lost; Cesmn-1(lf) animals grow more slowly than +/Cesmn-1(lf) siblings, are shorter, sterile, and most Cesmn-1(lf) animals die before reaching adulthood (Figure 1A). Combined, these defects decrease the average size of the Cesmn-1(lf) population versus control animals; decreased average population size will be referred to herein as a growth defect. This growth defect was harnessed in an automated assay to identify Cesmn-1(lf) modifier genes in a genome-wide screen.


Conserved genes act as modifiers of invertebrate SMN loss of function defects.

Dimitriadi M, Sleigh JN, Walker A, Chang HC, Sen A, Kalloo G, Harris J, Barsby T, Walsh MB, Satterlee JS, Li C, Van Vactor D, Artavanis-Tsakonas S, Hart AC - PLoS Genet. (2010)

Survival and average length of Cesmn-1(lf) animals is decreased.Homozygous loss of Cesmn-1(lf) results in slower growth and most animals die during larval stages. Animals that reach the adult stage are short-lived and sterile. A) Image of age-matched heterozygous hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) and homozygous Cesmn-1(lf) individuals (below). Scale bar indicates 150 microns. B) To test the impact of candidate modifier genes on growth rates and size, a C. elegans growth assay was established. +/Cesmn-1(lf) animals were collected as unhatched eggs, reared for 5 days/2 generations on standard C. elegans plates upon RNAi bacterial strains, as illustrated in the flow chart. Bacterial cultures expressed dsRNA corresponding to the gene of interest for RNAi knockdown; bacterial cultures containing vector with no insert were used as a control (‘empty’). C) Using an automated system, the length and fluorescence of animals was determined [51]; the smallest larvae in each culture were indistinguishable from debris and were excluded from the analysis as illustrated in the graph. Genotypes were discriminated by GFP fluorescence and length was determined as ‘time-of-flight’ through the laser chamber; each dot in the graph represents an individual animal. The disparate growth/survival rates of Cesmn-1(lf) and +/Cesmn-1(lf) animals alters the percentage of ‘large’ animals in these mixed stage cultures. As illustrated graphically, +/Cesmn-1(lf) and Cesmn-1(lf) animals can be sorted into blue and red boxes, respectively; animals designated as ‘large’ fall into shaded boxes. The fraction of large animals was calculated (# animals in shaded box/# animals in both shaded and unshaded box for each genotype) and is reported as % large with standard error of the mean (S.E.M.); significance was determined by Mann-Whitney U two-tailed test with p≤0.05. At least three independent cultures of more than 200 animals were scored for each targeted gene.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965752&req=5

pgen-1001172-g001: Survival and average length of Cesmn-1(lf) animals is decreased.Homozygous loss of Cesmn-1(lf) results in slower growth and most animals die during larval stages. Animals that reach the adult stage are short-lived and sterile. A) Image of age-matched heterozygous hT2(lethal)[myo-2p::GFP]/Cesmn-1(lf) (+/Cesmn-1(lf), described in text) and homozygous Cesmn-1(lf) individuals (below). Scale bar indicates 150 microns. B) To test the impact of candidate modifier genes on growth rates and size, a C. elegans growth assay was established. +/Cesmn-1(lf) animals were collected as unhatched eggs, reared for 5 days/2 generations on standard C. elegans plates upon RNAi bacterial strains, as illustrated in the flow chart. Bacterial cultures expressed dsRNA corresponding to the gene of interest for RNAi knockdown; bacterial cultures containing vector with no insert were used as a control (‘empty’). C) Using an automated system, the length and fluorescence of animals was determined [51]; the smallest larvae in each culture were indistinguishable from debris and were excluded from the analysis as illustrated in the graph. Genotypes were discriminated by GFP fluorescence and length was determined as ‘time-of-flight’ through the laser chamber; each dot in the graph represents an individual animal. The disparate growth/survival rates of Cesmn-1(lf) and +/Cesmn-1(lf) animals alters the percentage of ‘large’ animals in these mixed stage cultures. As illustrated graphically, +/Cesmn-1(lf) and Cesmn-1(lf) animals can be sorted into blue and red boxes, respectively; animals designated as ‘large’ fall into shaded boxes. The fraction of large animals was calculated (# animals in shaded box/# animals in both shaded and unshaded box for each genotype) and is reported as % large with standard error of the mean (S.E.M.); significance was determined by Mann-Whitney U two-tailed test with p≤0.05. At least three independent cultures of more than 200 animals were scored for each targeted gene.
Mentions: Although complete loss of SMN function causes lethality, C. elegans that are homozygous mutant for Cesmn-1(lf) can survive for several days due to partial maternal rescue. It has been suggested that +/Cesmn-1(lf) hermaphrodites load sufficient Cesmn-1 maternal protein and/or perhaps mRNA into oocytes to support development through embryogenesis and early larval stages [44]. Accordingly, homozygous Cesmn-1(lf) larvae initially resemble wild type animals. Eventually maternally-loaded Cesmn-1 product is lost; Cesmn-1(lf) animals grow more slowly than +/Cesmn-1(lf) siblings, are shorter, sterile, and most Cesmn-1(lf) animals die before reaching adulthood (Figure 1A). Combined, these defects decrease the average size of the Cesmn-1(lf) population versus control animals; decreased average population size will be referred to herein as a growth defect. This growth defect was harnessed in an automated assay to identify Cesmn-1(lf) modifier genes in a genome-wide screen.

Bottom Line: Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model.Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects.Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Brown University, Providence, Rhode Island, USA.

ABSTRACT
Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the molecular pathways critical for SMA pathology remain elusive. We have used genetic approaches in invertebrate models to identify conserved SMN loss of function modifier genes. Drosophila melanogaster and Caenorhabditis elegans each have a single gene encoding a protein orthologous to human SMN; diminished function of these invertebrate genes causes lethality and neuromuscular defects. To find genes that modulate SMN function defects across species, two approaches were used. First, a genome-wide RNAi screen for C. elegans SMN modifier genes was undertaken, yielding four genes. Second, we tested the conservation of modifier gene function across species; genes identified in one invertebrate model were tested for function in the other invertebrate model. Drosophila orthologs of two genes, which were identified originally in C. elegans, modified Drosophila SMN loss of function defects. C. elegans orthologs of twelve genes, which were originally identified in a previous Drosophila screen, modified C. elegans SMN loss of function defects. Bioinformatic analysis of the conserved, cross-species, modifier genes suggests that conserved cellular pathways, specifically endocytosis and mRNA regulation, act as critical genetic modifiers of SMN loss of function defects across species.

Show MeSH
Related in: MedlinePlus