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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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Loss of PLS3 orthologs enhances SMN loss of function defects in invertebrates.Previous studies in vertebrates suggested that PLS3 orthologs in C. elegans and Drosophila might modify SMN loss of function defects [37]. A) A genetic interaction was found between the Drosophila PLS3 ortholog (Fimbrin or Fim) and DmSmn using previously described DmSmn RNAi knockdown lines [26] and Fim loss of function alleles. Percentage early or late larval lethality is reported. Ubiquitous RNAi knockdown of DmSmn using the tubulinGAL4 (TubGAL4) driver results in pupal death; modifier genes alter the percentage of animals that die at early versus late pupal stages (day 7 versus day 9 [26]). Loss of Fim function significantly increased the percentage of animals that died as early pupae (p≤0.05 by Chi-square analysis); Fim is an enhancer of DmSmn loss of function growth/survival defects. At least 100 animals of each genotype were scored in four replicates for the lethality assay. No significant variation was observed between control and experimental crosses with each independent trial. B) The Drosophila d02114 allele likely eliminates Fim function and in homozygous animals modestly perturbs NMJ morphology at larval muscle 4. Fimd02114 enhances DmSmn loss of function NMJ defects consistent with studies in vertebrates [37]. All strains carry the ubiquitous TubGAL4 driver. Significance of p≤0.05 versus single mutant strains was determined by ANOVA and is indicated with asterisk; S.E.M. is shown. In representative images, red corresponds to anti-Discs Large (DLG), green is anti-synaptotagmin and blue is DAPI; scale bar indicates 15 microns.
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pgen-1001172-g005: Loss of PLS3 orthologs enhances SMN loss of function defects in invertebrates.Previous studies in vertebrates suggested that PLS3 orthologs in C. elegans and Drosophila might modify SMN loss of function defects [37]. A) A genetic interaction was found between the Drosophila PLS3 ortholog (Fimbrin or Fim) and DmSmn using previously described DmSmn RNAi knockdown lines [26] and Fim loss of function alleles. Percentage early or late larval lethality is reported. Ubiquitous RNAi knockdown of DmSmn using the tubulinGAL4 (TubGAL4) driver results in pupal death; modifier genes alter the percentage of animals that die at early versus late pupal stages (day 7 versus day 9 [26]). Loss of Fim function significantly increased the percentage of animals that died as early pupae (p≤0.05 by Chi-square analysis); Fim is an enhancer of DmSmn loss of function growth/survival defects. At least 100 animals of each genotype were scored in four replicates for the lethality assay. No significant variation was observed between control and experimental crosses with each independent trial. B) The Drosophila d02114 allele likely eliminates Fim function and in homozygous animals modestly perturbs NMJ morphology at larval muscle 4. Fimd02114 enhances DmSmn loss of function NMJ defects consistent with studies in vertebrates [37]. All strains carry the ubiquitous TubGAL4 driver. Significance of p≤0.05 versus single mutant strains was determined by ANOVA and is indicated with asterisk; S.E.M. is shown. In representative images, red corresponds to anti-Discs Large (DLG), green is anti-synaptotagmin and blue is DAPI; scale bar indicates 15 microns.

Mentions: First, we determined if Fimbrin (Fim), the Drosophila ortholog of PLS3, modifies DmSmn loss of function defects in growth and NMJ assays. It has been shown that RNAi knockdown of DmSmn (DmSmn RNAi) results in 44% lethality in early pupal stages with 56% lethality at late pupal stages [26]. Loss of Fim alone does not cause larval or pupal lethality (data not shown). Three Fim loss of function alleles were crossed into the DmSmn(RNAi) background and each accelerated death compared to DmSmn(RNAi) control animals (Figure 5A).


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)

Loss of PLS3 orthologs enhances SMN loss of function defects in invertebrates.Previous studies in vertebrates suggested that PLS3 orthologs in C. elegans and Drosophila might modify SMN loss of function defects [37]. A) A genetic interaction was found between the Drosophila PLS3 ortholog (Fimbrin or Fim) and DmSmn using previously described DmSmn RNAi knockdown lines [26] and Fim loss of function alleles. Percentage early or late larval lethality is reported. Ubiquitous RNAi knockdown of DmSmn using the tubulinGAL4 (TubGAL4) driver results in pupal death; modifier genes alter the percentage of animals that die at early versus late pupal stages (day 7 versus day 9 [26]). Loss of Fim function significantly increased the percentage of animals that died as early pupae (p≤0.05 by Chi-square analysis); Fim is an enhancer of DmSmn loss of function growth/survival defects. At least 100 animals of each genotype were scored in four replicates for the lethality assay. No significant variation was observed between control and experimental crosses with each independent trial. B) The Drosophila d02114 allele likely eliminates Fim function and in homozygous animals modestly perturbs NMJ morphology at larval muscle 4. Fimd02114 enhances DmSmn loss of function NMJ defects consistent with studies in vertebrates [37]. All strains carry the ubiquitous TubGAL4 driver. Significance of p≤0.05 versus single mutant strains was determined by ANOVA and is indicated with asterisk; S.E.M. is shown. In representative images, red corresponds to anti-Discs Large (DLG), green is anti-synaptotagmin and blue is DAPI; scale bar indicates 15 microns.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965752&req=5

pgen-1001172-g005: Loss of PLS3 orthologs enhances SMN loss of function defects in invertebrates.Previous studies in vertebrates suggested that PLS3 orthologs in C. elegans and Drosophila might modify SMN loss of function defects [37]. A) A genetic interaction was found between the Drosophila PLS3 ortholog (Fimbrin or Fim) and DmSmn using previously described DmSmn RNAi knockdown lines [26] and Fim loss of function alleles. Percentage early or late larval lethality is reported. Ubiquitous RNAi knockdown of DmSmn using the tubulinGAL4 (TubGAL4) driver results in pupal death; modifier genes alter the percentage of animals that die at early versus late pupal stages (day 7 versus day 9 [26]). Loss of Fim function significantly increased the percentage of animals that died as early pupae (p≤0.05 by Chi-square analysis); Fim is an enhancer of DmSmn loss of function growth/survival defects. At least 100 animals of each genotype were scored in four replicates for the lethality assay. No significant variation was observed between control and experimental crosses with each independent trial. B) The Drosophila d02114 allele likely eliminates Fim function and in homozygous animals modestly perturbs NMJ morphology at larval muscle 4. Fimd02114 enhances DmSmn loss of function NMJ defects consistent with studies in vertebrates [37]. All strains carry the ubiquitous TubGAL4 driver. Significance of p≤0.05 versus single mutant strains was determined by ANOVA and is indicated with asterisk; S.E.M. is shown. In representative images, red corresponds to anti-Discs Large (DLG), green is anti-synaptotagmin and blue is DAPI; scale bar indicates 15 microns.
Mentions: First, we determined if Fimbrin (Fim), the Drosophila ortholog of PLS3, modifies DmSmn loss of function defects in growth and NMJ assays. It has been shown that RNAi knockdown of DmSmn (DmSmn RNAi) results in 44% lethality in early pupal stages with 56% lethality at late pupal stages [26]. Loss of Fim alone does not cause larval or pupal lethality (data not shown). Three Fim loss of function alleles were crossed into the DmSmn(RNAi) background and each accelerated death compared to DmSmn(RNAi) control animals (Figure 5A).

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