Neuronal regulated ire-1-dependent mRNA decay controls germline differentiation in Caenorhabditis elegans
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Evaluation Summary:
The authors of this manuscript previously showed that ER stress, and in particular the ER stress sensor Ire1, regulates transdifferentiation in C. elegans, leading to the ectopic differentiation of germline cells. In this follow-up manuscript, the authors present several lines of evidence supporting the idea that Ire1 modules these effects through degrading a novel mRNA substrate flp6. The authors identify the neurons and neuromodulators that affect accumulation of abnormal germline cells. The reviewers agreed that the discovery that flp6 is a regulated Ire-1-dependent decay target in C. elegans, and the demonstration of a non-cell-autonomous effect of Ire1 activity, are novel and likely to be of interest to a broad readership. However, more evidence is required to support some of the main conclusions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in Caenorhabditis elegans demonstrate that knockout of the germline-specific translation repressor gld-1 causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that endoplasmic reticulum (ER) stress enhances this phenotype to suppress germline tumor progression(Levi-Ferber et al., 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated inositol requiring enzyme-1 (IRE-1)-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuit’s integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation and demonstrate that regulated Ire1-dependent decay can affect animal physiology by controlling a specific neuronal circuit.
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Author Response:
Reviewer #1 (Public Review):
In this manuscript, Levi-Ferber et al use C elegans to study how germline cells maintain pluripotency and avoid GED (germline ectopic differentiation) before fertilization. The authors previously showed that activation of the ER stress sensor Ire1 (but not its major downstream target Xbp1) enhances GED, and here they explore the mechanism of this effect.
The authors convincingly – and surprisingly – show that the Ire1-mediated GED increase results not from Ire1 activity in the germline but in the nervous system, specifically in certain sensory neurons. Worms lacking a specific neuropeptide (FLP-6) or a particular neuron that produces this peptide (ASE) also displayed increased GED. Although FLP-6 deficiency did not induce ER stress, ER stress did lead to a reduction of FLP-6 transcript …
Author Response:
Reviewer #1 (Public Review):
In this manuscript, Levi-Ferber et al use C elegans to study how germline cells maintain pluripotency and avoid GED (germline ectopic differentiation) before fertilization. The authors previously showed that activation of the ER stress sensor Ire1 (but not its major downstream target Xbp1) enhances GED, and here they explore the mechanism of this effect.
The authors convincingly – and surprisingly – show that the Ire1-mediated GED increase results not from Ire1 activity in the germline but in the nervous system, specifically in certain sensory neurons. Worms lacking a specific neuropeptide (FLP-6) or a particular neuron that produces this peptide (ASE) also displayed increased GED. Although FLP-6 deficiency did not induce ER stress, ER stress did lead to a reduction of FLP-6 transcript (and protein) levels in an Ire1-dependent manner, suggesting this RNA is a target of Regulated Ire1-dependent decay (RIDD). The authors then go on to map out the signaling cascade that begins with FLP6 reduction in ASE by Ire1 and is transmitted to the gonad via an ASE-AIY-HSN circuit, including serotonin produced by HYE.
This paper is quite interesting and for the most part the data are very convincing and support the model. The demonstration that Ire1 and the ER stress response have non-cell autonomous effects is of particular interest, and is very well supported here. The description of this circuit linking particular neurons and signaling molecules to gonad pluripotency is also very strong.
A weakness of the paper is the link between RIDD of FLP6 and the disruption of this circuit. The data presented do clearly support the model. However, additional information would strengthen this considerably. The authors show that FLP6 mRNA levels are reduced in Ire1+ but not Ire-/- animals subjected to ER stress. They also show that GED results from the nuclease activity of Ire1 in the ASE; and that loss of FLP6 can also induce a similar effect. However, they do not show as clearly that Ire1's effects on GED are mediated primarily through FLP6.
We significantly strengthened the link between RIDD of FLP6 and the disruption of this circuit, as detailed above in the response to the essential revisions. We have also added experiments to show that that ire-1's effects on GED are mediated primarily through FLP-6. This has been achieved by generating CRISPR-designed worms harboring silent mutations in the flp-6 gene that disrupt the sequence and structure of the predicted cleavage site while preserving the CDS. We find that this mutation stabilizes the flp-6 transcript under ER stress conditions and protects from ER stress-induced GED in otherwise WT animals.
Reviewer #2 (Public Review):
Levi-Ferber and colleagues showed in their previous paper that ER stress regulates germline transdifferentiation in a way that is IRE-1 dependent, but XBP-1 independent. An open question at that time was how IRE-1 activation could mediate this signaling. The authors present several experiments in this manuscript that support the idea that neuronal Ire-1 can cell non-autonomously control germline differentiation through regulation of the neuropeptide FLP-6. Mechanistically, the authors characterize that FLP-6 is a target of IRE-1 RIDD activity. This is the first demonstration of RIDD in C. elegans, an important finding given that no RIDD targets have yet been identified in this organism. Using a wide range of mutants, the authors were also able to identify a neuronal circuit that can control the germline ectopic differentiation (GED) phenotype, involving the sensory neuron ASE, the interneuron AIY, and the motor neuron HSN. The data presented in the manuscript are sound, the mapping of a pivotal three-neuron circuit is impressive, and the findings are likely to be of high interest to a broad readership. However, some more evidence is required to support some of the conclusions made, in particular the characterization of flp-6 as a substrate for RIDD.
We significantly strengthened the link between RIDD of FLP6 and the disruption of this circuit.
Reviewer #3 (Public Review):
In a previous study, the authors had shown that germline tumors that accumulate in the C. elegans gonad because of the lack the RNA binding translational repressor GLD-1, have an increased propensity to differentiate and express somatic proteins in response to ER stress induced by tunicamycin or the absence of the TRK kinase protein tfg-1 (a process the authors call GED). Using this as a model, here, the authors investigate the mechanisms by which the abnormal nuclei accumulate in the tumorous gonad of glp-1 animals by manipulating genes in the soma and germline.
The key message of this paper is, then, the identification of neurons and neuromodulators that suppress or enhance this accumulation of abnormal germline cells in the glp-1 germline. While the results of this analysis could potentially provide an interesting advance, the validity of the many of the conclusions are difficult to evaluate because of limitations posed by the experimental methods and ambiguity in defining the GED.
Weaknesses:
A key issue is the identity of the abnormal germline cells that accumulate in glp-1 gonads. Modulation of the neuronal circuits examined (FLP-6, serotonin, cholinergic) change the germline, alter ovulation rates, modulate somatic gonad contraction rates etc. in wild-type animals. The effects of these circuits on a glp-1 germline are not known, but some of the same effects are likely to continue even if germ cells turned tumorous. Therefore, how neurons and neuromodulators alter the accumulation of abnormal cells in the gonad may or may not be surprising or novel, based on what is actually happening to these cells (the phenotype scored as GED). However, this is unclear as all the abnormal effects on the germline are assessed using DAPI at some steady state. Therefore, GED (ectopic differentiation) needs to be better demonstrated separate from the simple accumulation of abnormal nuclei, which could happen for a number of different reasons.
Because depletion of gld-1 prevents the transition of the mitotic germline into oocytes, this creates a more simplified gonad for analysis, devoid of oocytes and embryos, in which all the cells within the gonad should be mitotic germline. Thus, the relative homogeneity imposed in the gld-1 gonads simplifies the analysis of the nature of the aberrant nuclei and rule out the possibility that these are endomitotic oocytes or their derivatives. Nevertheless, to further demonstrate that the disruption of the proposed neuronal circuit results in the accumulation of ectopically differentiated cells in the gonad, we directly assessed expression of somatic markers within the gonad, under similar conditions. These include neuronal over-expression of ire-1 as well as mutations that disrupt the ASE-AIY-HSN neuronal circuit by impairing the relevant neurons or by interfering/repairing their ability to communicate via specific neurotransmitters and neuropeptides (Fig 1D, Fig 2F, Fig 5D, Fig 6A).
Strengths:
One strength of this paper is the identification of the neuropeptide FLP-6 as a suppressor of GED and a possible RIDD target. However, there is insufficient analysis conducted to fully support this claim.
We provide more in-vivo and in-vitro evidence demonstrating that flp-6 is a target of RIDD. In-vitro: We confirmed that the in vitro cleavage assay results in cleavage products of the expected sizes, and that mutation of the predicted cleavage site prevents degradation of the flp-6 RNA. In-vivo: We now show that flp-6 RNA levels are reduced under different ER stress conditions in an ire-1-dependent xbp-1 independent manner. We show that over-expression of IRE-1 in ASE is sufficient to reduce flp-6 transcript levels. We show that CRISPR-designed worms harboring silent mutations in the flp-6 gene that disrupt the predicted cleavage site while preserving the CDS protect the stability of the transcript under ER stress conditions and protect the animals from ER stress-induced germline differentiation.
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Reviewer #3 (Public Review):
In a previous study, the authors had shown that germline tumors that accumulate in the C. elegans gonad because of the lack the RNA binding translational repressor GLD-1, have an increased propensity to differentiate and express somatic proteins in response to ER stress induced by tunicamycin or the absence of the TRK kinase protein tfg-1 (a process the authors call GED). Using this as a model, here, the authors investigate the mechanisms by which the abnormal nuclei accumulate in the tumorous gonad of glp-1 animals by manipulating genes in the soma and germline.
The key message of this paper is, then, the identification of neurons and neuromodulators that suppress or enhance this accumulation of abnormal germline cells in the glp-1 germline. While the results of this analysis could potentially provide an …
Reviewer #3 (Public Review):
In a previous study, the authors had shown that germline tumors that accumulate in the C. elegans gonad because of the lack the RNA binding translational repressor GLD-1, have an increased propensity to differentiate and express somatic proteins in response to ER stress induced by tunicamycin or the absence of the TRK kinase protein tfg-1 (a process the authors call GED). Using this as a model, here, the authors investigate the mechanisms by which the abnormal nuclei accumulate in the tumorous gonad of glp-1 animals by manipulating genes in the soma and germline.
The key message of this paper is, then, the identification of neurons and neuromodulators that suppress or enhance this accumulation of abnormal germline cells in the glp-1 germline. While the results of this analysis could potentially provide an interesting advance, the validity of the many of the conclusions are difficult to evaluate because of limitations posed by the experimental methods and ambiguity in defining the GED.
Weaknesses:
A key issue is the identity of the abnormal germline cells that accumulate in glp-1 gonads. Modulation of the neuronal circuits examined (FLP-6, serotonin, cholinergic) change the germline, alter ovulation rates, modulate somatic gonad contraction rates etc. in wild-type animals. The effects of these circuits on a glp-1 germline are not known, but some of the same effects are likely to continue even if germ cells turned tumorous. Therefore, how neurons and neuromodulators alter the accumulation of abnormal cells in the gonad may or may not be surprising or novel, based on what is actually happening to these cells (the phenotype scored as GED). However, this is unclear as all the abnormal effects on the germline are assessed using DAPI at some steady state. Therefore, GED (ectopic differentiation) needs to be better demonstrated separate from the simple accumulation of abnormal nuclei, which could happen for a number of different reasons.
Strengths:
One strength of this paper is the identification of the neuropeptide FLP-6 as a suppressor of GED and a possible RIDD target. However, there is insufficient analysis conducted to fully support this claim.
-
Reviewer #2 (Public Review):
Levi-Ferber and colleagues showed in their previous paper that ER stress regulates germline transdifferentiation in a way that is IRE-1 dependent, but XBP-1 independent. An open question at that time was how IRE-1 activation could mediate this signaling. The authors present several experiments in this manuscript that support the idea that neuronal Ire-1 can cell non-autonomously control germline differentiation through regulation of the neuropeptide FLP-6. Mechanistically, the authors characterize that FLP-6 is a target of IRE-1 RIDD activity. This is the first demonstration of RIDD in C. elegans, an important finding given that no RIDD targets have yet been identified in this organism. Using a wide range of mutants, the authors were also able to identify a neuronal circuit that can control the germline …
Reviewer #2 (Public Review):
Levi-Ferber and colleagues showed in their previous paper that ER stress regulates germline transdifferentiation in a way that is IRE-1 dependent, but XBP-1 independent. An open question at that time was how IRE-1 activation could mediate this signaling. The authors present several experiments in this manuscript that support the idea that neuronal Ire-1 can cell non-autonomously control germline differentiation through regulation of the neuropeptide FLP-6. Mechanistically, the authors characterize that FLP-6 is a target of IRE-1 RIDD activity. This is the first demonstration of RIDD in C. elegans, an important finding given that no RIDD targets have yet been identified in this organism. Using a wide range of mutants, the authors were also able to identify a neuronal circuit that can control the germline ectopic differentiation (GED) phenotype, involving the sensory neuron ASE, the interneuron AIY, and the motor neuron HSN. The data presented in the manuscript are sound, the mapping of a pivotal three-neuron circuit is impressive, and the findings are likely to be of high interest to a broad readership. However, some more evidence is required to support some of the conclusions made, in particular the characterization of flp-6 as a substrate for RIDD.
-
Reviewer #1 (Public Review):
In this manuscript, Levi-Ferber et al use C elegans to study how germline cells maintain pluripotency and avoid GED (germline ectopic differentiation) before fertilization. The authors previously showed that activation of the ER stress sensor Ire1 (but not its major downstream target Xbp1) enhances GED, and here they explore the mechanism of this effect.
The authors convincingly – and surprisingly – show that the Ire1-mediated GED increase results not from Ire1 activity in the germline but in the nervous system, specifically in certain sensory neurons. Worms lacking a specific neuropeptide (FLP-6) or a particular neuron that produces this peptide (ASE) also displayed increased GED. Although FLP-6 deficiency did not induce ER stress, ER stress did lead to a reduction of FLP-6 transcript (and protein) levels …
Reviewer #1 (Public Review):
In this manuscript, Levi-Ferber et al use C elegans to study how germline cells maintain pluripotency and avoid GED (germline ectopic differentiation) before fertilization. The authors previously showed that activation of the ER stress sensor Ire1 (but not its major downstream target Xbp1) enhances GED, and here they explore the mechanism of this effect.
The authors convincingly – and surprisingly – show that the Ire1-mediated GED increase results not from Ire1 activity in the germline but in the nervous system, specifically in certain sensory neurons. Worms lacking a specific neuropeptide (FLP-6) or a particular neuron that produces this peptide (ASE) also displayed increased GED. Although FLP-6 deficiency did not induce ER stress, ER stress did lead to a reduction of FLP-6 transcript (and protein) levels in an Ire1-dependent manner, suggesting this RNA is a target of Regulated Ire1-dependent decay (RIDD). The authors then go on to map out the signaling cascade that begins with FLP6 reduction in ASE by Ire1 and is transmitted to the gonad via an ASE-AIY-HSN circuit, including serotonin produced by HYE.
This paper is quite interesting and for the most part the data are very convincing and support the model. The demonstration that Ire1 and the ER stress response have non-cell autonomous effects is of particular interest, and is very well supported here. The description of this circuit linking particular neurons and signaling molecules to gonad pluripotency is also very strong.
A weakness of the paper is the link between RIDD of FLP6 and the disruption of this circuit. The data presented do clearly support the model. However, additional information would strengthen this considerably. The authors show that FLP6 mRNA levels are reduced in Ire1+ but not Ire-/- animals subjected to ER stress. They also show that GED results from the nuclease activity of Ire1 in the ASE; and that loss of FLP6 can also induce a similar effect. However, they do not show as clearly that Ire1's effects on GED are mediated primarily through FLP6.
-
Evaluation Summary:
The authors of this manuscript previously showed that ER stress, and in particular the ER stress sensor Ire1, regulates transdifferentiation in C. elegans, leading to the ectopic differentiation of germline cells. In this follow-up manuscript, the authors present several lines of evidence supporting the idea that Ire1 modules these effects through degrading a novel mRNA substrate flp6. The authors identify the neurons and neuromodulators that affect accumulation of abnormal germline cells. The reviewers agreed that the discovery that flp6 is a regulated Ire-1-dependent decay target in C. elegans, and the demonstration of a non-cell-autonomous effect of Ire1 activity, are novel and likely to be of interest to a broad readership. However, more evidence is required to support some of the main conclusions.
(This preprint …
Evaluation Summary:
The authors of this manuscript previously showed that ER stress, and in particular the ER stress sensor Ire1, regulates transdifferentiation in C. elegans, leading to the ectopic differentiation of germline cells. In this follow-up manuscript, the authors present several lines of evidence supporting the idea that Ire1 modules these effects through degrading a novel mRNA substrate flp6. The authors identify the neurons and neuromodulators that affect accumulation of abnormal germline cells. The reviewers agreed that the discovery that flp6 is a regulated Ire-1-dependent decay target in C. elegans, and the demonstration of a non-cell-autonomous effect of Ire1 activity, are novel and likely to be of interest to a broad readership. However, more evidence is required to support some of the main conclusions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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