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Experimental selection for Drosophila survival in extremely high O2 environments.

Zhao HW, Zhou D, Nizet V, Haddad GG - PLoS ONE (2010)

Bottom Line: Although oxidative stress is deleterious to mammals, the mechanisms underlying oxidant susceptibility or tolerance remain to be elucidated.In this study, through a long-term laboratory selection over many generations, we generated a Drosophila melanogaster strain that can live and reproduce in very high O(2) environments (90% O(2)), a lethal condition to naïve flies.Gene expression profiling revealed that 227 genes were significantly altered in expression and two third of these genes were down-regulated.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Although oxidative stress is deleterious to mammals, the mechanisms underlying oxidant susceptibility or tolerance remain to be elucidated. In this study, through a long-term laboratory selection over many generations, we generated a Drosophila melanogaster strain that can live and reproduce in very high O(2) environments (90% O(2)), a lethal condition to naïve flies. We demonstrated that tolerance to hyperoxia was heritable in these flies and that these hyperoxia-selected flies exhibited phenotypic differences from naïve flies, such as a larger body size and increased weight by 20%. Gene expression profiling revealed that 227 genes were significantly altered in expression and two third of these genes were down-regulated. Using a mutant screen strategy, we studied the role of some altered genes (up- or down-regulated in the microarrays) by testing the survival of available corresponding P-element or UAS construct lines under hyperoxic conditions. We report that down-regulation of several candidate genes including Tropomyosin 1, Glycerol 3 phosphate dehydrogenase, CG33129, and UGP as well as up-regulation of Diptericin and Attacin conferred tolerance to severe hyperoxia. In conclusion, we identified several genes that were not only altered in hyperoxia-selected flies but we also prove that these play an important role in hyperoxia survival. Thus our study provides a molecular basis for understanding the mechanisms of hyperoxia tolerance.

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Related in: MedlinePlus

Role of single genes in hyperoxia tolerance.A) Embryos from 47 P-element insertion lines were collected and exposed to 90% O2 and eclosion rate was determined. Embryos from five P-element insertion lines that were able to develop into adult stage and their percent eclosion rate were shown in Fig. 4A. B) Gene expression changes in the microarrays and P-element lines, which were determined by real time PCR, were summarized in Fig. 4B. C) Adult flies with P-element insertion f07745 and without P-element insertion by precise excision (Ex f07745) were exposed to 90% O2 and percent survival rate was determined. Flies with precise excision were more sensitive to 90% O2 than flies with P-element insertion (p<0.01). D) Embryos from da-Gal4>UAS-Dpt and actin-Gal4>UAS-Dpt flies and their control lines were exposed to 90% O2 and eclosion rate was determined. Flies with overexpression of Dpt had a significantly higher eclosion rate in hyperoxia as compared to the controls (p<0.001). The percent eclosion was also determined in da-Gal4>UAS-DptRNAi flies and control flies as above. E) Survival of da-Gal4>UAS-Att flies in hyperoxia was determined as above, flies with overexpression of Att had a significantly higher eclosion rate than control flies (p<0.05). F) Overexpression of Dpt and Att in the da-Gal4>UAS-AMP flies was confirmed by real time PCR. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.
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pone-0011701-g004: Role of single genes in hyperoxia tolerance.A) Embryos from 47 P-element insertion lines were collected and exposed to 90% O2 and eclosion rate was determined. Embryos from five P-element insertion lines that were able to develop into adult stage and their percent eclosion rate were shown in Fig. 4A. B) Gene expression changes in the microarrays and P-element lines, which were determined by real time PCR, were summarized in Fig. 4B. C) Adult flies with P-element insertion f07745 and without P-element insertion by precise excision (Ex f07745) were exposed to 90% O2 and percent survival rate was determined. Flies with precise excision were more sensitive to 90% O2 than flies with P-element insertion (p<0.01). D) Embryos from da-Gal4>UAS-Dpt and actin-Gal4>UAS-Dpt flies and their control lines were exposed to 90% O2 and eclosion rate was determined. Flies with overexpression of Dpt had a significantly higher eclosion rate in hyperoxia as compared to the controls (p<0.001). The percent eclosion was also determined in da-Gal4>UAS-DptRNAi flies and control flies as above. E) Survival of da-Gal4>UAS-Att flies in hyperoxia was determined as above, flies with overexpression of Att had a significantly higher eclosion rate than control flies (p<0.05). F) Overexpression of Dpt and Att in the da-Gal4>UAS-AMP flies was confirmed by real time PCR. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

Mentions: To investigate which individual loci with differential expression have an impact on hyperoxia tolerance and in sustaining survival in hyperoxia in the SO2A, a mutant screen strategy was applied. We first focused on the genes that had single P-element insertion lines in the Bloomington Stock Center, with the assumption that some such insertions would mimic the up- or down-regulation seen in the microarray data. Forty-seven P-element insertion lines (Table S3) were used in this study for 26 down-regulated genes and 13 up-regulated genes that were significantly altered in the microarrays. We tested the survival of all P-element lines in 90% O2 along with yw and w serving as controls. Embryos from most P-element lines and control flies stopped development and died at the larval stages. In contrast, embryos from five P-element lines EY12089, f07745 and BG02672, EY13829 and EY01534 were able to develop into the adult stage in extreme high oxygen environments and their eclosion rates were between 30% and about 60% (Fig. 4A). These P-element lines have insertions within or around genes including Tropomyosin 1 (Tm1), CG33129, UGP, and Glycerol 3 phosphate dehydrogenase (Gpdh) respectively. The effect of P-element insertion was determined by real time PCR and these results demonstrated that these genes were down-regulated in P-element lines, which was consistent with the microarray data (Fig. 4B), suggesting that the down-regulation of these genes in SO2A flies played a role in sustaining survival in hyperoxia. The results of their survival were interesting since these genes were singularly down-regulated in each of the flies. To further confirm that an enhanced survival observed in these P-element lines was due to the down-regulation of these genes caused by P-element insertion, we excised one of the P-element allele f07745 from gene CG33129. We found that adult flies with precise excision (Ex f07745) were much more sensitive to hyperoxia and had a significant shorter life span than flies with P-element insertion (Fig. 4C, p<0.01) and embryos from this precise excision line could not develop into adult flies in 90% O2 (data not shown), further demonstrating the important role of this particular gene, as an example, in oxidant stress.


Experimental selection for Drosophila survival in extremely high O2 environments.

Zhao HW, Zhou D, Nizet V, Haddad GG - PLoS ONE (2010)

Role of single genes in hyperoxia tolerance.A) Embryos from 47 P-element insertion lines were collected and exposed to 90% O2 and eclosion rate was determined. Embryos from five P-element insertion lines that were able to develop into adult stage and their percent eclosion rate were shown in Fig. 4A. B) Gene expression changes in the microarrays and P-element lines, which were determined by real time PCR, were summarized in Fig. 4B. C) Adult flies with P-element insertion f07745 and without P-element insertion by precise excision (Ex f07745) were exposed to 90% O2 and percent survival rate was determined. Flies with precise excision were more sensitive to 90% O2 than flies with P-element insertion (p<0.01). D) Embryos from da-Gal4>UAS-Dpt and actin-Gal4>UAS-Dpt flies and their control lines were exposed to 90% O2 and eclosion rate was determined. Flies with overexpression of Dpt had a significantly higher eclosion rate in hyperoxia as compared to the controls (p<0.001). The percent eclosion was also determined in da-Gal4>UAS-DptRNAi flies and control flies as above. E) Survival of da-Gal4>UAS-Att flies in hyperoxia was determined as above, flies with overexpression of Att had a significantly higher eclosion rate than control flies (p<0.05). F) Overexpression of Dpt and Att in the da-Gal4>UAS-AMP flies was confirmed by real time PCR. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2909141&req=5

pone-0011701-g004: Role of single genes in hyperoxia tolerance.A) Embryos from 47 P-element insertion lines were collected and exposed to 90% O2 and eclosion rate was determined. Embryos from five P-element insertion lines that were able to develop into adult stage and their percent eclosion rate were shown in Fig. 4A. B) Gene expression changes in the microarrays and P-element lines, which were determined by real time PCR, were summarized in Fig. 4B. C) Adult flies with P-element insertion f07745 and without P-element insertion by precise excision (Ex f07745) were exposed to 90% O2 and percent survival rate was determined. Flies with precise excision were more sensitive to 90% O2 than flies with P-element insertion (p<0.01). D) Embryos from da-Gal4>UAS-Dpt and actin-Gal4>UAS-Dpt flies and their control lines were exposed to 90% O2 and eclosion rate was determined. Flies with overexpression of Dpt had a significantly higher eclosion rate in hyperoxia as compared to the controls (p<0.001). The percent eclosion was also determined in da-Gal4>UAS-DptRNAi flies and control flies as above. E) Survival of da-Gal4>UAS-Att flies in hyperoxia was determined as above, flies with overexpression of Att had a significantly higher eclosion rate than control flies (p<0.05). F) Overexpression of Dpt and Att in the da-Gal4>UAS-AMP flies was confirmed by real time PCR. * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.
Mentions: To investigate which individual loci with differential expression have an impact on hyperoxia tolerance and in sustaining survival in hyperoxia in the SO2A, a mutant screen strategy was applied. We first focused on the genes that had single P-element insertion lines in the Bloomington Stock Center, with the assumption that some such insertions would mimic the up- or down-regulation seen in the microarray data. Forty-seven P-element insertion lines (Table S3) were used in this study for 26 down-regulated genes and 13 up-regulated genes that were significantly altered in the microarrays. We tested the survival of all P-element lines in 90% O2 along with yw and w serving as controls. Embryos from most P-element lines and control flies stopped development and died at the larval stages. In contrast, embryos from five P-element lines EY12089, f07745 and BG02672, EY13829 and EY01534 were able to develop into the adult stage in extreme high oxygen environments and their eclosion rates were between 30% and about 60% (Fig. 4A). These P-element lines have insertions within or around genes including Tropomyosin 1 (Tm1), CG33129, UGP, and Glycerol 3 phosphate dehydrogenase (Gpdh) respectively. The effect of P-element insertion was determined by real time PCR and these results demonstrated that these genes were down-regulated in P-element lines, which was consistent with the microarray data (Fig. 4B), suggesting that the down-regulation of these genes in SO2A flies played a role in sustaining survival in hyperoxia. The results of their survival were interesting since these genes were singularly down-regulated in each of the flies. To further confirm that an enhanced survival observed in these P-element lines was due to the down-regulation of these genes caused by P-element insertion, we excised one of the P-element allele f07745 from gene CG33129. We found that adult flies with precise excision (Ex f07745) were much more sensitive to hyperoxia and had a significant shorter life span than flies with P-element insertion (Fig. 4C, p<0.01) and embryos from this precise excision line could not develop into adult flies in 90% O2 (data not shown), further demonstrating the important role of this particular gene, as an example, in oxidant stress.

Bottom Line: Although oxidative stress is deleterious to mammals, the mechanisms underlying oxidant susceptibility or tolerance remain to be elucidated.In this study, through a long-term laboratory selection over many generations, we generated a Drosophila melanogaster strain that can live and reproduce in very high O(2) environments (90% O(2)), a lethal condition to naïve flies.Gene expression profiling revealed that 227 genes were significantly altered in expression and two third of these genes were down-regulated.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Medicine, Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Although oxidative stress is deleterious to mammals, the mechanisms underlying oxidant susceptibility or tolerance remain to be elucidated. In this study, through a long-term laboratory selection over many generations, we generated a Drosophila melanogaster strain that can live and reproduce in very high O(2) environments (90% O(2)), a lethal condition to naïve flies. We demonstrated that tolerance to hyperoxia was heritable in these flies and that these hyperoxia-selected flies exhibited phenotypic differences from naïve flies, such as a larger body size and increased weight by 20%. Gene expression profiling revealed that 227 genes were significantly altered in expression and two third of these genes were down-regulated. Using a mutant screen strategy, we studied the role of some altered genes (up- or down-regulated in the microarrays) by testing the survival of available corresponding P-element or UAS construct lines under hyperoxic conditions. We report that down-regulation of several candidate genes including Tropomyosin 1, Glycerol 3 phosphate dehydrogenase, CG33129, and UGP as well as up-regulation of Diptericin and Attacin conferred tolerance to severe hyperoxia. In conclusion, we identified several genes that were not only altered in hyperoxia-selected flies but we also prove that these play an important role in hyperoxia survival. Thus our study provides a molecular basis for understanding the mechanisms of hyperoxia tolerance.

Show MeSH
Related in: MedlinePlus