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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 Drosophila Cbp20 or FMRF enhances DmSmn loss of function neuromuscular defects.For two C. elegans modifier genes, loss of function alleles were available for orthologous Drosophila genes: Cbp20 and FMRF [68]–[70]. Loss of either Drosophila gene enhanced the NMJ defects of DmSmn animals. The number of synaptic boutons in the A2 segment of muscles 6 and 7 was counted in third instar larvae (visualized using Discs large and synaptotagmin immunoreactivity, shown in red and green, respectively). Homozygous loss of DmSmn in DmSmnf1109/DmSmn73Ao animals dramatically decreases bouton number but loss of one copy of DmSmn, Cbp20 or FMRF had little effect. Loss of one copy of either Cbp20 or FMRF in animals heterozygous for either DmSmn allele significantly decreased bouton numbers. This nonallelic noncomplementation suggests a strong genetic interaction between these two conserved modifier genes and DmSmn. Scale bar indicates 10 microns in representative images; S.E.M. is shown in graph; all transheterozygous combinations are significantly different from the corresponding single heterozygous controls with p≤0.05 by ANOVA as indicated with asterisk.
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pgen-1001172-g006: Loss of Drosophila Cbp20 or FMRF enhances DmSmn loss of function neuromuscular defects.For two C. elegans modifier genes, loss of function alleles were available for orthologous Drosophila genes: Cbp20 and FMRF [68]–[70]. Loss of either Drosophila gene enhanced the NMJ defects of DmSmn animals. The number of synaptic boutons in the A2 segment of muscles 6 and 7 was counted in third instar larvae (visualized using Discs large and synaptotagmin immunoreactivity, shown in red and green, respectively). Homozygous loss of DmSmn in DmSmnf1109/DmSmn73Ao animals dramatically decreases bouton number but loss of one copy of DmSmn, Cbp20 or FMRF had little effect. Loss of one copy of either Cbp20 or FMRF in animals heterozygous for either DmSmn allele significantly decreased bouton numbers. This nonallelic noncomplementation suggests a strong genetic interaction between these two conserved modifier genes and DmSmn. Scale bar indicates 10 microns in representative images; S.E.M. is shown in graph; all transheterozygous combinations are significantly different from the corresponding single heterozygous controls with p≤0.05 by ANOVA as indicated with asterisk.

Mentions: Heterozygous loss of DmSmn function in +/Smn73Ao or +/Smnf01109 animals had no significant effect on bouton number as expected, Smn73Ao/Smnf01109 animals had dramatically decreased bouton numbers (Figure 6). Loss of one copy of Cbc20 or Fmrf modestly decreased synaptic bouton number compared to control animals. However, simultaneous loss of one copy of DmSmn and one copy of either modifier gene resulted in further synaptic bouton loss (Figure 6). The genetic interaction in trans-heterozygous animals is consistent with a strong genetic interaction between Smn and the two modifier genes. We were unable to obtain classical alleles of the grk-2 Drosophila ortholog. We conclude that Cbp20 and FMRFamide are conserved invertebrate enhancers of Smn loss of function defects and that this genetic interaction is conserved across species.


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 Drosophila Cbp20 or FMRF enhances DmSmn loss of function neuromuscular defects.For two C. elegans modifier genes, loss of function alleles were available for orthologous Drosophila genes: Cbp20 and FMRF [68]–[70]. Loss of either Drosophila gene enhanced the NMJ defects of DmSmn animals. The number of synaptic boutons in the A2 segment of muscles 6 and 7 was counted in third instar larvae (visualized using Discs large and synaptotagmin immunoreactivity, shown in red and green, respectively). Homozygous loss of DmSmn in DmSmnf1109/DmSmn73Ao animals dramatically decreases bouton number but loss of one copy of DmSmn, Cbp20 or FMRF had little effect. Loss of one copy of either Cbp20 or FMRF in animals heterozygous for either DmSmn allele significantly decreased bouton numbers. This nonallelic noncomplementation suggests a strong genetic interaction between these two conserved modifier genes and DmSmn. Scale bar indicates 10 microns in representative images; S.E.M. is shown in graph; all transheterozygous combinations are significantly different from the corresponding single heterozygous controls with p≤0.05 by ANOVA as indicated with asterisk.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965752&req=5

pgen-1001172-g006: Loss of Drosophila Cbp20 or FMRF enhances DmSmn loss of function neuromuscular defects.For two C. elegans modifier genes, loss of function alleles were available for orthologous Drosophila genes: Cbp20 and FMRF [68]–[70]. Loss of either Drosophila gene enhanced the NMJ defects of DmSmn animals. The number of synaptic boutons in the A2 segment of muscles 6 and 7 was counted in third instar larvae (visualized using Discs large and synaptotagmin immunoreactivity, shown in red and green, respectively). Homozygous loss of DmSmn in DmSmnf1109/DmSmn73Ao animals dramatically decreases bouton number but loss of one copy of DmSmn, Cbp20 or FMRF had little effect. Loss of one copy of either Cbp20 or FMRF in animals heterozygous for either DmSmn allele significantly decreased bouton numbers. This nonallelic noncomplementation suggests a strong genetic interaction between these two conserved modifier genes and DmSmn. Scale bar indicates 10 microns in representative images; S.E.M. is shown in graph; all transheterozygous combinations are significantly different from the corresponding single heterozygous controls with p≤0.05 by ANOVA as indicated with asterisk.
Mentions: Heterozygous loss of DmSmn function in +/Smn73Ao or +/Smnf01109 animals had no significant effect on bouton number as expected, Smn73Ao/Smnf01109 animals had dramatically decreased bouton numbers (Figure 6). Loss of one copy of Cbc20 or Fmrf modestly decreased synaptic bouton number compared to control animals. However, simultaneous loss of one copy of DmSmn and one copy of either modifier gene resulted in further synaptic bouton loss (Figure 6). The genetic interaction in trans-heterozygous animals is consistent with a strong genetic interaction between Smn and the two modifier genes. We were unable to obtain classical alleles of the grk-2 Drosophila ortholog. We conclude that Cbp20 and FMRFamide are conserved invertebrate enhancers of Smn loss of function defects and that this genetic interaction is conserved across species.

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