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The RHO-1 RhoGTPase modulates fertility and multiple behaviors in adult C. elegans.

McMullan R, Nurrish SJ - PLoS ONE (2011)

Bottom Line: Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity.Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology.Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours.

View Article: PubMed Central - PubMed

Affiliation: MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Department of Neuroscience, Physiology and Pharmacology, University College, London, United Kingdom.

ABSTRACT
The Rho family of small GTPases are essential during early embryonic development making it difficult to study their functions in adult animals. Using inducible transgenes expressing either a constitutively active version of the single C. elegans Rho ortholog, RHO-1, or an inhibitor of endogenous Rho (C3 transferase), we demonstrate multiple defects caused by altering Rho signaling in adult C. elegans. Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity. Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology. Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours. The multiple post-developmental roles for Rho in C. elegans demonstrate that RhoA signaling pathways continue to be used post-developmentally and the resulting phenotypes provide an opportunity to further study post-developmental Rho signaling pathways using genetic screens.

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Heat shock-inducible RHO-1 is a model for post-developmental Rho signaling.(A–C). In the absence of heat shock mixed stage populations of nzIs1 (B) or nzEx4 (C) animals were indistinguishable from wild type (A) and were viable and fertile. (D–J). Approximately 200 L1 stage animals were transferred to assay plates and either, heat shocked immediately at 33°C, or maintained at 20°C as controls. For hsC3 transferase (nzEx4) assays 50 L1 animals carrying the GFP injection marker were selected. Following heat shock animals were maintained at 20°C and animals that had reached adulthood after 5 days were visualized. No adult animals could be observed when hsRHO-1(G14V) (nzIs1) (H and J) or hsC3 transferase (nzEx4) (I and J) were expressed at the L1 stage indicating that expression of these transgenes led to developmental arrest. Arrested animals are indicated with arrows in H and I. Adult animals could clearly be observed after 3 days in the absence of heat shock (D–F and J) or in wild type controls (G and J). Vulva of adult animals indicated with an arrowhead in D–G. (K, L and M). The cholinergic motor neurons of wild type animals (J) or animals expressing neuronal RHO-1(G14V) (nzIs34 and nzIs33) (K and L) were labeled with p.unc-17::gfp. In some transgenic lines (nzIs33) (M) the commissures that normally extend from the ventral to the dorsal nerve cord branched prematurely extending a process towards the posterior of the animal (arrow) indicating that expression of nRHO-1(G14V) results in neuronal pathfinding defects.
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pone-0017265-g001: Heat shock-inducible RHO-1 is a model for post-developmental Rho signaling.(A–C). In the absence of heat shock mixed stage populations of nzIs1 (B) or nzEx4 (C) animals were indistinguishable from wild type (A) and were viable and fertile. (D–J). Approximately 200 L1 stage animals were transferred to assay plates and either, heat shocked immediately at 33°C, or maintained at 20°C as controls. For hsC3 transferase (nzEx4) assays 50 L1 animals carrying the GFP injection marker were selected. Following heat shock animals were maintained at 20°C and animals that had reached adulthood after 5 days were visualized. No adult animals could be observed when hsRHO-1(G14V) (nzIs1) (H and J) or hsC3 transferase (nzEx4) (I and J) were expressed at the L1 stage indicating that expression of these transgenes led to developmental arrest. Arrested animals are indicated with arrows in H and I. Adult animals could clearly be observed after 3 days in the absence of heat shock (D–F and J) or in wild type controls (G and J). Vulva of adult animals indicated with an arrowhead in D–G. (K, L and M). The cholinergic motor neurons of wild type animals (J) or animals expressing neuronal RHO-1(G14V) (nzIs34 and nzIs33) (K and L) were labeled with p.unc-17::gfp. In some transgenic lines (nzIs33) (M) the commissures that normally extend from the ventral to the dorsal nerve cord branched prematurely extending a process towards the posterior of the animal (arrow) indicating that expression of nRHO-1(G14V) results in neuronal pathfinding defects.

Mentions: Mutations that either increase or decrease RHO-1 signaling result in lethality [3]. In support of this we found that a recently isolated strain carrying a deletion in rho-1 (rho-1(ok2418)) is homozygous lethal (data not shown). To confirm that our transgenic animals were a good model for the post-developmental effects of RHO-1 we looked to see if altering RHO-1 signaling in adults using our heat shock-inducible transgenes could bypass this lethality. In the absence of heat shock animals that carried transgenes expressing constitutively active RHO-1(G14V) or the RHO-1 inhibitor C3 transferase from the heat shock, or cholinergic specific unc-17 promoter, were viable, fertile adults demonstrating that RHO-1 signaling in these animals was not changed sufficiently to cause developmental lethality (Figure 1A–C).


The RHO-1 RhoGTPase modulates fertility and multiple behaviors in adult C. elegans.

McMullan R, Nurrish SJ - PLoS ONE (2011)

Heat shock-inducible RHO-1 is a model for post-developmental Rho signaling.(A–C). In the absence of heat shock mixed stage populations of nzIs1 (B) or nzEx4 (C) animals were indistinguishable from wild type (A) and were viable and fertile. (D–J). Approximately 200 L1 stage animals were transferred to assay plates and either, heat shocked immediately at 33°C, or maintained at 20°C as controls. For hsC3 transferase (nzEx4) assays 50 L1 animals carrying the GFP injection marker were selected. Following heat shock animals were maintained at 20°C and animals that had reached adulthood after 5 days were visualized. No adult animals could be observed when hsRHO-1(G14V) (nzIs1) (H and J) or hsC3 transferase (nzEx4) (I and J) were expressed at the L1 stage indicating that expression of these transgenes led to developmental arrest. Arrested animals are indicated with arrows in H and I. Adult animals could clearly be observed after 3 days in the absence of heat shock (D–F and J) or in wild type controls (G and J). Vulva of adult animals indicated with an arrowhead in D–G. (K, L and M). The cholinergic motor neurons of wild type animals (J) or animals expressing neuronal RHO-1(G14V) (nzIs34 and nzIs33) (K and L) were labeled with p.unc-17::gfp. In some transgenic lines (nzIs33) (M) the commissures that normally extend from the ventral to the dorsal nerve cord branched prematurely extending a process towards the posterior of the animal (arrow) indicating that expression of nRHO-1(G14V) results in neuronal pathfinding defects.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046162&req=5

pone-0017265-g001: Heat shock-inducible RHO-1 is a model for post-developmental Rho signaling.(A–C). In the absence of heat shock mixed stage populations of nzIs1 (B) or nzEx4 (C) animals were indistinguishable from wild type (A) and were viable and fertile. (D–J). Approximately 200 L1 stage animals were transferred to assay plates and either, heat shocked immediately at 33°C, or maintained at 20°C as controls. For hsC3 transferase (nzEx4) assays 50 L1 animals carrying the GFP injection marker were selected. Following heat shock animals were maintained at 20°C and animals that had reached adulthood after 5 days were visualized. No adult animals could be observed when hsRHO-1(G14V) (nzIs1) (H and J) or hsC3 transferase (nzEx4) (I and J) were expressed at the L1 stage indicating that expression of these transgenes led to developmental arrest. Arrested animals are indicated with arrows in H and I. Adult animals could clearly be observed after 3 days in the absence of heat shock (D–F and J) or in wild type controls (G and J). Vulva of adult animals indicated with an arrowhead in D–G. (K, L and M). The cholinergic motor neurons of wild type animals (J) or animals expressing neuronal RHO-1(G14V) (nzIs34 and nzIs33) (K and L) were labeled with p.unc-17::gfp. In some transgenic lines (nzIs33) (M) the commissures that normally extend from the ventral to the dorsal nerve cord branched prematurely extending a process towards the posterior of the animal (arrow) indicating that expression of nRHO-1(G14V) results in neuronal pathfinding defects.
Mentions: Mutations that either increase or decrease RHO-1 signaling result in lethality [3]. In support of this we found that a recently isolated strain carrying a deletion in rho-1 (rho-1(ok2418)) is homozygous lethal (data not shown). To confirm that our transgenic animals were a good model for the post-developmental effects of RHO-1 we looked to see if altering RHO-1 signaling in adults using our heat shock-inducible transgenes could bypass this lethality. In the absence of heat shock animals that carried transgenes expressing constitutively active RHO-1(G14V) or the RHO-1 inhibitor C3 transferase from the heat shock, or cholinergic specific unc-17 promoter, were viable, fertile adults demonstrating that RHO-1 signaling in these animals was not changed sufficiently to cause developmental lethality (Figure 1A–C).

Bottom Line: Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity.Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology.Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours.

View Article: PubMed Central - PubMed

Affiliation: MRC Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Department of Neuroscience, Physiology and Pharmacology, University College, London, United Kingdom.

ABSTRACT
The Rho family of small GTPases are essential during early embryonic development making it difficult to study their functions in adult animals. Using inducible transgenes expressing either a constitutively active version of the single C. elegans Rho ortholog, RHO-1, or an inhibitor of endogenous Rho (C3 transferase), we demonstrate multiple defects caused by altering Rho signaling in adult C. elegans. Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity. Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology. Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours. The multiple post-developmental roles for Rho in C. elegans demonstrate that RhoA signaling pathways continue to be used post-developmentally and the resulting phenotypes provide an opportunity to further study post-developmental Rho signaling pathways using genetic screens.

Show MeSH
Related in: MedlinePlus