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Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development.

Bratic I, Hench J, Henriksson J, Antebi A, Bürglin TR, Trifunovic A - Nucleic Acids Res. (2009)

Bottom Line: Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo.Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli.Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Division of Metabolic Diseases, Novum, Karolinska Institutet, Stockholm SE-141 86, Sweden.

ABSTRACT
A number of studies showed that the development and the lifespan of Caenorhabditis elegans is dependent on mitochondrial function. In this study, we addressed the role of mitochondrial DNA levels and mtDNA maintenance in development of C. elegans by analyzing deletion mutants for mitochondrial polymerase gamma (polg-1(ok1548)). Surprisingly, even though previous studies in other model organisms showed necessity of polymerase gamma for embryonic development, homozygous polg-1(ok1548) mutants had normal development and reached adulthood without any morphological defects. However, polg-1 deficient animals have a seriously compromised gonadal function as a result of severe mitochondrial depletion, leading to sterility and shortened lifespan. Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo. Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli. Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

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Lifespan analysis of polg-1(ok1548) animals. (A) Lifespan of polg-1(ok1548) mutant was shown in comparison with DR2078, wild-type (N2) and polg-1(ok1548/+) animals. (B) Lifespan analysis of polg-1(ok1548), HIS-72::mCherry, with HIS-72::mCherry and polg-1(ok1548) animals used as controls. (C) Pie charts present percentage of animals that developed vulva protrusion during their lifespan. (D) mtDNA copy number in polg-1(ok1548), HIS-72::mCherry (black bars) and HIS-72::mCherry (squared bars) animals. The steady-state mtDNA levels are measured at day 1 (D1), day 8 (D8) and day 16 (D16). Values represent the mtDNA copy number per animal. The error bars represent the SD. Asterisks show statistical significance (Student's t-test, ***P < 0.001).
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Figure 5: Lifespan analysis of polg-1(ok1548) animals. (A) Lifespan of polg-1(ok1548) mutant was shown in comparison with DR2078, wild-type (N2) and polg-1(ok1548/+) animals. (B) Lifespan analysis of polg-1(ok1548), HIS-72::mCherry, with HIS-72::mCherry and polg-1(ok1548) animals used as controls. (C) Pie charts present percentage of animals that developed vulva protrusion during their lifespan. (D) mtDNA copy number in polg-1(ok1548), HIS-72::mCherry (black bars) and HIS-72::mCherry (squared bars) animals. The steady-state mtDNA levels are measured at day 1 (D1), day 8 (D8) and day 16 (D16). Values represent the mtDNA copy number per animal. The error bars represent the SD. Asterisks show statistical significance (Student's t-test, ***P < 0.001).

Mentions: All polg-1(ok158) animals die after nearly complete gonadal and intestinal protrusion, impairing the movements of the animal (Figure S4A). The animals died within 1 or 2 days after protrusion, most likely due to starvation. The protrusion phenotype starts to occur around the sixth day of adulthood. The mean lifespan of polg-1 deficient animals was 10 ± 3 days, with a maximum of 15.3 days (Figure 5A, Table 4). Heterozygous animals carrying the balancer chromosome as well as animals homozygous for the balancer showed the same life span as N2 (Figure 5A, Table 4). However, previous RNAi-based genome-wide screening analysis did not reveal any phenotypes caused by downregulation of the polg-1 gene (16–18). We believe that RNAi over just one generation failed to decrease mtDNA levels under the critical threshold for obvious mitochondrial dysfunction. Therefore, we performed RNAi analysis by continuous feeding over several generations. The third generation of worms develops the same phenotype as polg-1(ok158) mutants, with gonad or intestinal protrusion and their progeny undergoes embryonic arrest after 72 h (Figure SB).Figure 5.


Mitochondrial DNA level, but not active replicase, is essential for Caenorhabditis elegans development.

Bratic I, Hench J, Henriksson J, Antebi A, Bürglin TR, Trifunovic A - Nucleic Acids Res. (2009)

Lifespan analysis of polg-1(ok1548) animals. (A) Lifespan of polg-1(ok1548) mutant was shown in comparison with DR2078, wild-type (N2) and polg-1(ok1548/+) animals. (B) Lifespan analysis of polg-1(ok1548), HIS-72::mCherry, with HIS-72::mCherry and polg-1(ok1548) animals used as controls. (C) Pie charts present percentage of animals that developed vulva protrusion during their lifespan. (D) mtDNA copy number in polg-1(ok1548), HIS-72::mCherry (black bars) and HIS-72::mCherry (squared bars) animals. The steady-state mtDNA levels are measured at day 1 (D1), day 8 (D8) and day 16 (D16). Values represent the mtDNA copy number per animal. The error bars represent the SD. Asterisks show statistical significance (Student's t-test, ***P < 0.001).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2665216&req=5

Figure 5: Lifespan analysis of polg-1(ok1548) animals. (A) Lifespan of polg-1(ok1548) mutant was shown in comparison with DR2078, wild-type (N2) and polg-1(ok1548/+) animals. (B) Lifespan analysis of polg-1(ok1548), HIS-72::mCherry, with HIS-72::mCherry and polg-1(ok1548) animals used as controls. (C) Pie charts present percentage of animals that developed vulva protrusion during their lifespan. (D) mtDNA copy number in polg-1(ok1548), HIS-72::mCherry (black bars) and HIS-72::mCherry (squared bars) animals. The steady-state mtDNA levels are measured at day 1 (D1), day 8 (D8) and day 16 (D16). Values represent the mtDNA copy number per animal. The error bars represent the SD. Asterisks show statistical significance (Student's t-test, ***P < 0.001).
Mentions: All polg-1(ok158) animals die after nearly complete gonadal and intestinal protrusion, impairing the movements of the animal (Figure S4A). The animals died within 1 or 2 days after protrusion, most likely due to starvation. The protrusion phenotype starts to occur around the sixth day of adulthood. The mean lifespan of polg-1 deficient animals was 10 ± 3 days, with a maximum of 15.3 days (Figure 5A, Table 4). Heterozygous animals carrying the balancer chromosome as well as animals homozygous for the balancer showed the same life span as N2 (Figure 5A, Table 4). However, previous RNAi-based genome-wide screening analysis did not reveal any phenotypes caused by downregulation of the polg-1 gene (16–18). We believe that RNAi over just one generation failed to decrease mtDNA levels under the critical threshold for obvious mitochondrial dysfunction. Therefore, we performed RNAi analysis by continuous feeding over several generations. The third generation of worms develops the same phenotype as polg-1(ok158) mutants, with gonad or intestinal protrusion and their progeny undergoes embryonic arrest after 72 h (Figure SB).Figure 5.

Bottom Line: Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo.Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli.Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Division of Metabolic Diseases, Novum, Karolinska Institutet, Stockholm SE-141 86, Sweden.

ABSTRACT
A number of studies showed that the development and the lifespan of Caenorhabditis elegans is dependent on mitochondrial function. In this study, we addressed the role of mitochondrial DNA levels and mtDNA maintenance in development of C. elegans by analyzing deletion mutants for mitochondrial polymerase gamma (polg-1(ok1548)). Surprisingly, even though previous studies in other model organisms showed necessity of polymerase gamma for embryonic development, homozygous polg-1(ok1548) mutants had normal development and reached adulthood without any morphological defects. However, polg-1 deficient animals have a seriously compromised gonadal function as a result of severe mitochondrial depletion, leading to sterility and shortened lifespan. Our results indicate that the gonad is the primary site of mtDNA replication, whilst the mtDNA of adult somatic tissues mainly stems from the developing embryo. Furthermore, we show that the mtDNA copy number shows great plasticity as it can be almost tripled as a response to the environmental stimuli. Finally, we show that the mtDNA copy number is an essential limiting factor for the worm development and therefore, a number of mechanisms set to maintain mtDNA levels exist, ensuring a normal development of C. elegans even in the absence of the mitochondrial replicase.

Show MeSH
Related in: MedlinePlus