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A role for Ras in inhibiting circular foraging behavior as revealed by a new method for time and cell-specific RNAi.

Hamakawa M, Uozumi T, Ueda N, Iino Y, Hirotsu T - BMC Biol. (2015)

Bottom Line: The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior.Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion.We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 812-8581, Japan. beachriver14@gmail.com.

ABSTRACT

Background: The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. In particular, RNAi is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible RNAi or cell-specific RNAi. However, conventional RNAi is insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions.

Results: Here, we investigated the Ras gene for its role in exploratory behavior in C. elegans. We found that, under poor environmental conditions, mutations in the Ras-MAPK signaling pathway lead to circular locomotion instead of normal exploratory foraging. Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion. We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons. To investigate the temporal- and cell-specific profiles of the functions of Ras, we developed a new RNAi method that enables simultaneous time- and cell-specific knockdown. In this method, one RNA strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded RNA in the target cell when heat shock is induced. This technique revealed that control of GLR-1 localization in RMD neurons requires Ras at the adult stage. Further, we demonstrated the application of this method to other genes.

Conclusions: We have established a new RNAi method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the Ras-MAPK pathway in the control of exploratory behavior under poor environmental conditions.

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Related in: MedlinePlus

Confirmation of the effect of the novel method, time- and cell-specific RNAi (T.C.RNAi). (A) The average recovery ratio of GFP intensity of AWC neurons in animals without RNAi constructs, animals with only gcy-10p::gfp(s), gcy-10p::gfp(as), hsp16-2::gfp(s), hsp16-2::gfp(as), animals with both gcy-10p::gfp(s) and hsp16-2::gfp(as) or both hsp16-2::gfp(s) and gcy-10p::gfp(as) after heat-shocked or control condition. (n ≥9 animals). (B) The average recovery ratio of GFP intensity of AWB and AWC neurons in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥10 animals). (C) Representative images of gcy-10p::GFP in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) before (left panel, intact) and after (center panel, photobleach) photobleaching and after heat-shocked condition (right panel, heat shock). Arrow heads indicate AWB neurons, and arrows indicate AWC neurons. Scale bars = 10 μm. (D) The average recovery ratio of GFP intensity of anterior or lateral protrusions of vulva in animals without RNAi constructs or animals with myo-3p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥14 protrusions of vulva). (E) Chemotaxis to isoamyl alcohol (1:1000) in wild-type animals, odr-3 mutants and wild-type animals with odr-3 T.C.RNAi (n ≥4 assays). AWC::(s) and hsp::(as) mean gcy-10p::odr-3(s) and hsp16-2::odr-3(as). Error bars represent SEM and asterisks indicate significant differences (*P <0.05, **P <0.01, Dunnett’s test or Student’s t-test). SEM, standard error of the mean.
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Fig4: Confirmation of the effect of the novel method, time- and cell-specific RNAi (T.C.RNAi). (A) The average recovery ratio of GFP intensity of AWC neurons in animals without RNAi constructs, animals with only gcy-10p::gfp(s), gcy-10p::gfp(as), hsp16-2::gfp(s), hsp16-2::gfp(as), animals with both gcy-10p::gfp(s) and hsp16-2::gfp(as) or both hsp16-2::gfp(s) and gcy-10p::gfp(as) after heat-shocked or control condition. (n ≥9 animals). (B) The average recovery ratio of GFP intensity of AWB and AWC neurons in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥10 animals). (C) Representative images of gcy-10p::GFP in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) before (left panel, intact) and after (center panel, photobleach) photobleaching and after heat-shocked condition (right panel, heat shock). Arrow heads indicate AWB neurons, and arrows indicate AWC neurons. Scale bars = 10 μm. (D) The average recovery ratio of GFP intensity of anterior or lateral protrusions of vulva in animals without RNAi constructs or animals with myo-3p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥14 protrusions of vulva). (E) Chemotaxis to isoamyl alcohol (1:1000) in wild-type animals, odr-3 mutants and wild-type animals with odr-3 T.C.RNAi (n ≥4 assays). AWC::(s) and hsp::(as) mean gcy-10p::odr-3(s) and hsp16-2::odr-3(as). Error bars represent SEM and asterisks indicate significant differences (*P <0.05, **P <0.01, Dunnett’s test or Student’s t-test). SEM, standard error of the mean.

Mentions: We first analyzed the effect of GFP T.C.RNAi in AWC sensory neurons. GFP was expressed by the gcy-10 promoter which drives the expression in AWC, AWB and I1 [49] and monitored the GFP intensity in cell bodies of AWC. In adult animals expressing both gcy-10::gfp(s) and hsp::gfp(as), the recovery ratio of GFP intensity in AWC significantly decreased after the heat shock compared to that after the mock treatment (Figure 4A). The expression of double-stranded RNA by the reciprocally exchanged promoter also induced GFP knockdown after the heat shock (Figure 4A). The GFP intensity in AWC was normally recovered after the heat shock in animals without expression of the RNAi constructs, suggesting the decrease of the recovery ratio was not due to an influence of heat shock on the gcy-10 promoter itself (Figure 4A). We also confirmed that expression of only a single RNA strand driven by a heat shock promoter or a cell-specific promoter could not decrease the recovery ratio (Figure 4A).Figure 4


A role for Ras in inhibiting circular foraging behavior as revealed by a new method for time and cell-specific RNAi.

Hamakawa M, Uozumi T, Ueda N, Iino Y, Hirotsu T - BMC Biol. (2015)

Confirmation of the effect of the novel method, time- and cell-specific RNAi (T.C.RNAi). (A) The average recovery ratio of GFP intensity of AWC neurons in animals without RNAi constructs, animals with only gcy-10p::gfp(s), gcy-10p::gfp(as), hsp16-2::gfp(s), hsp16-2::gfp(as), animals with both gcy-10p::gfp(s) and hsp16-2::gfp(as) or both hsp16-2::gfp(s) and gcy-10p::gfp(as) after heat-shocked or control condition. (n ≥9 animals). (B) The average recovery ratio of GFP intensity of AWB and AWC neurons in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥10 animals). (C) Representative images of gcy-10p::GFP in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) before (left panel, intact) and after (center panel, photobleach) photobleaching and after heat-shocked condition (right panel, heat shock). Arrow heads indicate AWB neurons, and arrows indicate AWC neurons. Scale bars = 10 μm. (D) The average recovery ratio of GFP intensity of anterior or lateral protrusions of vulva in animals without RNAi constructs or animals with myo-3p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥14 protrusions of vulva). (E) Chemotaxis to isoamyl alcohol (1:1000) in wild-type animals, odr-3 mutants and wild-type animals with odr-3 T.C.RNAi (n ≥4 assays). AWC::(s) and hsp::(as) mean gcy-10p::odr-3(s) and hsp16-2::odr-3(as). Error bars represent SEM and asterisks indicate significant differences (*P <0.05, **P <0.01, Dunnett’s test or Student’s t-test). SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4321700&req=5

Fig4: Confirmation of the effect of the novel method, time- and cell-specific RNAi (T.C.RNAi). (A) The average recovery ratio of GFP intensity of AWC neurons in animals without RNAi constructs, animals with only gcy-10p::gfp(s), gcy-10p::gfp(as), hsp16-2::gfp(s), hsp16-2::gfp(as), animals with both gcy-10p::gfp(s) and hsp16-2::gfp(as) or both hsp16-2::gfp(s) and gcy-10p::gfp(as) after heat-shocked or control condition. (n ≥9 animals). (B) The average recovery ratio of GFP intensity of AWB and AWC neurons in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥10 animals). (C) Representative images of gcy-10p::GFP in animals with srd-17p::gfp(s) and hsp16-2::gfp(as) before (left panel, intact) and after (center panel, photobleach) photobleaching and after heat-shocked condition (right panel, heat shock). Arrow heads indicate AWB neurons, and arrows indicate AWC neurons. Scale bars = 10 μm. (D) The average recovery ratio of GFP intensity of anterior or lateral protrusions of vulva in animals without RNAi constructs or animals with myo-3p::gfp(s) and hsp16-2::gfp(as) after heat-shocked or control condition (n ≥14 protrusions of vulva). (E) Chemotaxis to isoamyl alcohol (1:1000) in wild-type animals, odr-3 mutants and wild-type animals with odr-3 T.C.RNAi (n ≥4 assays). AWC::(s) and hsp::(as) mean gcy-10p::odr-3(s) and hsp16-2::odr-3(as). Error bars represent SEM and asterisks indicate significant differences (*P <0.05, **P <0.01, Dunnett’s test or Student’s t-test). SEM, standard error of the mean.
Mentions: We first analyzed the effect of GFP T.C.RNAi in AWC sensory neurons. GFP was expressed by the gcy-10 promoter which drives the expression in AWC, AWB and I1 [49] and monitored the GFP intensity in cell bodies of AWC. In adult animals expressing both gcy-10::gfp(s) and hsp::gfp(as), the recovery ratio of GFP intensity in AWC significantly decreased after the heat shock compared to that after the mock treatment (Figure 4A). The expression of double-stranded RNA by the reciprocally exchanged promoter also induced GFP knockdown after the heat shock (Figure 4A). The GFP intensity in AWC was normally recovered after the heat shock in animals without expression of the RNAi constructs, suggesting the decrease of the recovery ratio was not due to an influence of heat shock on the gcy-10 promoter itself (Figure 4A). We also confirmed that expression of only a single RNA strand driven by a heat shock promoter or a cell-specific promoter could not decrease the recovery ratio (Figure 4A).Figure 4

Bottom Line: The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior.Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion.We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, 812-8581, Japan. beachriver14@gmail.com.

ABSTRACT

Background: The nematode worm Caenorhabditis elegans, in which loss-of-function mutants and RNA interference (RNAi) models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. In particular, RNAi is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible RNAi or cell-specific RNAi. However, conventional RNAi is insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions.

Results: Here, we investigated the Ras gene for its role in exploratory behavior in C. elegans. We found that, under poor environmental conditions, mutations in the Ras-MAPK signaling pathway lead to circular locomotion instead of normal exploratory foraging. Spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: IL1, OLQ, and RMD, and we found that Ras functions in this neural circuit to modulate the direction of locomotion. We further observed that Ras plays an essential role in the regulation of GLR-1 glutamate receptor localization in RMD neurons. To investigate the temporal- and cell-specific profiles of the functions of Ras, we developed a new RNAi method that enables simultaneous time- and cell-specific knockdown. In this method, one RNA strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded RNA in the target cell when heat shock is induced. This technique revealed that control of GLR-1 localization in RMD neurons requires Ras at the adult stage. Further, we demonstrated the application of this method to other genes.

Conclusions: We have established a new RNAi method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the Ras-MAPK pathway in the control of exploratory behavior under poor environmental conditions.

Show MeSH
Related in: MedlinePlus