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The Esg Gene Is Involved in Nicotine Sensitivity in Drosophila melanogaster.

Sanchez-Díaz I, Rosales-Bravo F, Reyes-Taboada JL, Covarrubias AA, Narvaez-Padilla V, Reynaud E - PLoS ONE (2015)

Bottom Line: Two fly lines, L4 and L70, whose HRT was significantly longer than control´s were identified.In this work, we demonstrate that esg loss of function induces nicotine sensitivity possibly by altering development of sensory organs and neurons in the medial section of the thoracoabdominal ganglion.The ectopic expression of the miR-310c also induces nicotine sensitivity by lowering Esg levels thus disrupting sensory organs and possibly to the modulation of other miR-310c targets.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad, 2001, Apartado Postal, 510-3, Cuernavaca 62210, México.

ABSTRACT
In humans, there is a strong correlation between sensitivity to substances of abuse and addiction risk. This differential tolerance to drugs has a strong genetic component. The identification of human genetic factors that alter drug tolerance has been a difficult task. For this reason and taking advantage of the fact that Drosophila responds similarly to humans to many drugs, and that genetically it has a high degree of homology (sharing at least 70% of genes known to be involved in human genetic diseases), we looked for genes in Drosophila that altered their nicotine sensitivity. We developed an instantaneous nicotine vaporization technique that exposed flies in a reproducible way. The amount of nicotine sufficient to "knock out" half of control flies for 30 minutes was determined and this parameter was defined as Half Recovery Time (HRT). Two fly lines, L4 and L70, whose HRT was significantly longer than control´s were identified. The L4 insertion is a loss of function allele of the transcriptional factor escargot (esg), whereas L70 insertion causes miss-expression of the microRNA cluster miR-310-311-312-313 (miR-310c). In this work, we demonstrate that esg loss of function induces nicotine sensitivity possibly by altering development of sensory organs and neurons in the medial section of the thoracoabdominal ganglion. The ectopic expression of the miR-310c also induces nicotine sensitivity by lowering Esg levels thus disrupting sensory organs and possibly to the modulation of other miR-310c targets.

No MeSH data available.


Related in: MedlinePlus

Temporally controlled expression of the miR-310c in adult flies phenocopies L70 mutant line.(A) The HRT of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly longer only when it is at the GAL80 permissive temperature (28°C). Embryos were reared at 18°C for sixteen days, just before eclosion pupae were transferred to 28°C and maintained at this temperature for 3 days before assaying, controls were kept at 18°C, n ≥ 100. (B) Survival of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly reduced. Restricted mean life of Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c genotype is significantly reduced when maintained in standard cornmeal food supplemented with 0.5 mg/ml nicotine at the GAL80 permissive temperature n ≥ 100 flies, n = 3 in all experiments, ** = P ≤ 0.001, ns = not significant.
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pone.0133956.g006: Temporally controlled expression of the miR-310c in adult flies phenocopies L70 mutant line.(A) The HRT of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly longer only when it is at the GAL80 permissive temperature (28°C). Embryos were reared at 18°C for sixteen days, just before eclosion pupae were transferred to 28°C and maintained at this temperature for 3 days before assaying, controls were kept at 18°C, n ≥ 100. (B) Survival of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly reduced. Restricted mean life of Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c genotype is significantly reduced when maintained in standard cornmeal food supplemented with 0.5 mg/ml nicotine at the GAL80 permissive temperature n ≥ 100 flies, n = 3 in all experiments, ** = P ≤ 0.001, ns = not significant.

Mentions: To demonstrate that the ectopic expression of the miR-310c is enough to enhance the nicotine-sensitivity, we used the transgenic UAS-DsRed53-mir-310-313 line driven by a battery of GAL4 drivers of different strengths and directed to different target organs; however, no driver was able to direct the expression of the miR-310c in such a way that it phenocopies L70 (S1 Table). A possible explanation for this result could be that the miR-310c expression was not directed to the correct tissues, time and/or levels of expression. We attempted to drive the expression of the miR-310c by strong constitutive GAL4 drivers such as the actin promoter but we only confirmed that constitutive strong expression of the microRNAs is lethal as previously reported (S1 Table) [15]. To overcome this issue, we decided to use the GAL4/GAL80ts system in order to control the time at which the miR-310c was expressed. For this, we induced the miR-310c expression by incubating flies at 28°C, the permissive temperature, at different times during development using the Act5c-GAL4;tub-Gal80ts driver. Under these conditions, flies were viable (60%) only when the expression of the miR-310c was induced after sixteen days of development at 18°C when they are just at the end of pupation or after eclosion. Flies expressing miR-310c became as sensitive to nicotine as the L70 line after 72 hrs of induction (Fig 6A). Also, these flies are more sensitive to nicotine-containing food thus fully phenocoping L70 (Fig 6B).


The Esg Gene Is Involved in Nicotine Sensitivity in Drosophila melanogaster.

Sanchez-Díaz I, Rosales-Bravo F, Reyes-Taboada JL, Covarrubias AA, Narvaez-Padilla V, Reynaud E - PLoS ONE (2015)

Temporally controlled expression of the miR-310c in adult flies phenocopies L70 mutant line.(A) The HRT of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly longer only when it is at the GAL80 permissive temperature (28°C). Embryos were reared at 18°C for sixteen days, just before eclosion pupae were transferred to 28°C and maintained at this temperature for 3 days before assaying, controls were kept at 18°C, n ≥ 100. (B) Survival of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly reduced. Restricted mean life of Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c genotype is significantly reduced when maintained in standard cornmeal food supplemented with 0.5 mg/ml nicotine at the GAL80 permissive temperature n ≥ 100 flies, n = 3 in all experiments, ** = P ≤ 0.001, ns = not significant.
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Related In: Results  -  Collection

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pone.0133956.g006: Temporally controlled expression of the miR-310c in adult flies phenocopies L70 mutant line.(A) The HRT of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly longer only when it is at the GAL80 permissive temperature (28°C). Embryos were reared at 18°C for sixteen days, just before eclosion pupae were transferred to 28°C and maintained at this temperature for 3 days before assaying, controls were kept at 18°C, n ≥ 100. (B) Survival of the line Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c is significantly reduced. Restricted mean life of Act5c-GAL4;Tub-Gal80ts>UAS-miR-310c genotype is significantly reduced when maintained in standard cornmeal food supplemented with 0.5 mg/ml nicotine at the GAL80 permissive temperature n ≥ 100 flies, n = 3 in all experiments, ** = P ≤ 0.001, ns = not significant.
Mentions: To demonstrate that the ectopic expression of the miR-310c is enough to enhance the nicotine-sensitivity, we used the transgenic UAS-DsRed53-mir-310-313 line driven by a battery of GAL4 drivers of different strengths and directed to different target organs; however, no driver was able to direct the expression of the miR-310c in such a way that it phenocopies L70 (S1 Table). A possible explanation for this result could be that the miR-310c expression was not directed to the correct tissues, time and/or levels of expression. We attempted to drive the expression of the miR-310c by strong constitutive GAL4 drivers such as the actin promoter but we only confirmed that constitutive strong expression of the microRNAs is lethal as previously reported (S1 Table) [15]. To overcome this issue, we decided to use the GAL4/GAL80ts system in order to control the time at which the miR-310c was expressed. For this, we induced the miR-310c expression by incubating flies at 28°C, the permissive temperature, at different times during development using the Act5c-GAL4;tub-Gal80ts driver. Under these conditions, flies were viable (60%) only when the expression of the miR-310c was induced after sixteen days of development at 18°C when they are just at the end of pupation or after eclosion. Flies expressing miR-310c became as sensitive to nicotine as the L70 line after 72 hrs of induction (Fig 6A). Also, these flies are more sensitive to nicotine-containing food thus fully phenocoping L70 (Fig 6B).

Bottom Line: Two fly lines, L4 and L70, whose HRT was significantly longer than control´s were identified.In this work, we demonstrate that esg loss of function induces nicotine sensitivity possibly by altering development of sensory organs and neurons in the medial section of the thoracoabdominal ganglion.The ectopic expression of the miR-310c also induces nicotine sensitivity by lowering Esg levels thus disrupting sensory organs and possibly to the modulation of other miR-310c targets.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad, 2001, Apartado Postal, 510-3, Cuernavaca 62210, México.

ABSTRACT
In humans, there is a strong correlation between sensitivity to substances of abuse and addiction risk. This differential tolerance to drugs has a strong genetic component. The identification of human genetic factors that alter drug tolerance has been a difficult task. For this reason and taking advantage of the fact that Drosophila responds similarly to humans to many drugs, and that genetically it has a high degree of homology (sharing at least 70% of genes known to be involved in human genetic diseases), we looked for genes in Drosophila that altered their nicotine sensitivity. We developed an instantaneous nicotine vaporization technique that exposed flies in a reproducible way. The amount of nicotine sufficient to "knock out" half of control flies for 30 minutes was determined and this parameter was defined as Half Recovery Time (HRT). Two fly lines, L4 and L70, whose HRT was significantly longer than control´s were identified. The L4 insertion is a loss of function allele of the transcriptional factor escargot (esg), whereas L70 insertion causes miss-expression of the microRNA cluster miR-310-311-312-313 (miR-310c). In this work, we demonstrate that esg loss of function induces nicotine sensitivity possibly by altering development of sensory organs and neurons in the medial section of the thoracoabdominal ganglion. The ectopic expression of the miR-310c also induces nicotine sensitivity by lowering Esg levels thus disrupting sensory organs and possibly to the modulation of other miR-310c targets.

No MeSH data available.


Related in: MedlinePlus