Specific sensory neurons and insulin-like peptides modulate food type-dependent oogenesis and fertilization in Caenorhabditis elegans
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This work presents useful and potentially valuable findings on how food signals may influence reproduction in the nematode C. elegans. In the current manuscript, the evidence in support of the authors' model is incomplete, and additional experimental data is needed to buttress the authors' conclusions.
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Abstract
An animal’s responses to environmental cues are critical for its reproductive program. Thus, a mechanism that allows the animal to sense and adjust to its environment should make for a more efficient reproductive physiology. Here, we demonstrate that in Caenorhabditis elegans specific sensory neurons influence onset of oogenesis through insulin signaling in response to food-derived cues. The chemosensory neurons ASJ modulate oogenesis onset through the insulin-like peptide (ILP) INS-6. In contrast, other sensory neurons, the olfactory neurons AWA, regulate food type-dependent differences in C. elegans fertilization rates, but not onset of oogenesis. AWA modulates fertilization rates at least partly in parallel to insulin receptor signaling, since the insulin receptor DAF-2 regulates fertilization independently of food type, which requires ILPs other than INS-6. Together our findings suggest that optimal reproduction requires the integration of diverse food-derived inputs through multiple neuronal signals acting on the C. elegans germline.
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Author Response
Reviewer #1 (Public Review):
In this manuscript the authors perform a detailed analysis of the impact of food type on reproduction in C. elegans. They find that, in comparison with the standard OP50 strain of E. coli that is ubiquitously used to maintain C. elegans in the laboratory setting, the CS180 strain results in a reduction in the number of progeny that may be a consequence of an early transition from spermatogenesis to oogenesis that reduces total sperm number. They also find that the rate of oocyte fertilization is increased in animals fed CS180 vs. OP50. Using mutants and laser ablations, the authors show that, whereas the insulin-like peptide INS-6 acts in the ASJ sensory neurons to mediate the food type effect on total progeny and early oogenesis, the increased fertilization rate phenotype does not require …
Author Response
Reviewer #1 (Public Review):
In this manuscript the authors perform a detailed analysis of the impact of food type on reproduction in C. elegans. They find that, in comparison with the standard OP50 strain of E. coli that is ubiquitously used to maintain C. elegans in the laboratory setting, the CS180 strain results in a reduction in the number of progeny that may be a consequence of an early transition from spermatogenesis to oogenesis that reduces total sperm number. They also find that the rate of oocyte fertilization is increased in animals fed CS180 vs. OP50. Using mutants and laser ablations, the authors show that, whereas the insulin-like peptide INS-6 acts in the ASJ sensory neurons to mediate the food type effect on total progeny and early oogenesis, the increased fertilization rate phenotype does not require ASJ or insulin-like signaling and instead requires the AWA olfactory neurons.
The major strengths of the manuscript are the establishment of INS-6 as a link between food type and reproduction and the detail and rigor with which the experiments were executed. The results presented generally support the authors' model. This role of insulin-like signaling in connecting food type and reproduction makes it a plausible target for evolutionary forces that may have shaped insulin-like signaling in invertebrates. As such, this work contributes broadly to our understanding of how insulin signaling may have evolved prior to the emergence of vertebrates.
We thank the Reviewer for these nice comments.
A weakness of the work is the epistasis analysis of insulin-like pathway components, which is incomplete and at times difficult to interpret.
We conducted an epistasis analysis between ins-6 and daf-16 with regard to early oogenesis onset on the CS180 diet. Through recombination of lin-41::GFP with the daf-16 deletion mutation on chromosome I, we showed that daf-16 mutants exhibit early oogenesis at mid L4 on CS180 (Figure 5C and F), which is unlike the ins-6 deletion (null) mutants or the reduction-offunction mutations in daf-2. Both ins-6 and daf-2 mutants exhibit delayed oogenesis on CS180 (Figure 5B, D, and F). Interestingly, the delayed oogenesis phenotype of ins-6 null mutants was not rescued by loss of daf-16, suggesting that wild-type ins-6 promotes early oogenesis independent of daf-16 (Figure 5F). This is reminiscent of the Arur lab’s findings, where daf-2 promotes germline meiotic progression independent of daf-16 in response to food availability (Lopez et al., Dev Cell 2013, vol 27, pp 227-240).
Reviewer #2 (Public Review):
The manuscript by Mishra et al. examines the modulation of the nervous system by different bacterial food to influence reproductive phenotypes-specifically onset of oogenesis, fertilization rate, and progeny production. Defining how animal reproduction could be modulated by bacterial food cues through neuroendocrine signaling is a fascinating subject of study for which C. elegans is well-suited. However, the overall scope of the current study is limited, and some of the central data do not provide compelling evidence for the authors' underlying hypothesis and model.
- Two strains of E. coli are examined, the standard C. elegans bacterial food strain OP50 and an E. coli strain that Alcedo and colleagues have previously characterized to influence aging and longevity through nervous system modulation. While the authors determine that differences in LPS structure present between the strains does not account for the food-dependent effects, there is little further insight regarding the bacterial features that contribute to the observed differences in reproductive physiology. Moreover, at least two of the phenotypes examined-total progeny and fertilization rate-are known to be affected by bacterial food quality and may be affected by bacteria in many ways, so the description of these phenotypes is somewhat less compelling than the study of the onset of oogenesis.
Our study focused on how specific sensory neurons mediate the effects of different bacterial diets on three different aspects of C. elegans reproductive physiology—total progeny, oogenesis onset and fertilization rates. We examined the effects of three different bacteria, E. coli OP50, CS180 and CS2429, on these three phenotypes and the effects of two Serratia marcescens strains, Db11 and Db1140, on oogenesis onset. Of these five bacteria, only CS180 and its derivative CS2429, promote early C. elegans oogenesis.
In the revised manuscript, we included the effects of a fourth E. coli strain, the K-12 HT115 on total progeny (Figure 2—supplement 1), oogenesis onset (Figure 2E) and fertilization rates (Figure 2F). We found that HT115 does not elicit the same response as CS180 on oogenesis onset and fertilization rates. Thus, the oogenic-inducing and fertilization-enhancing cue(s) appear to be specific to CS180 and its derivative CS2429. We started characterizing the potential nature of these CS180-derived cue(s). So far, we found that these cues are unlikely to be free, small metabolites, since they were lost upon filtration of the CS180-conditioned LB media through a nylon membrane that has a pore size of 0.45 µm (Figure 2G and H). While we agree with the Reviewer that the identification of these cues are important, we believe that it is beyond the scope of this manuscript.
More importantly, we showed that the sensory neuron ASJ does modulate the timing of oogenesis and that this involves the insulin-like peptide ins-6 (please see our responses to the Essential Revisions section and Figures 5 and 6). We also showed that ASJ (Figure 7G and K) or ins-6 (Figure 8D) does not affect the food type-dependent fertilization rates, which are modulated by a different sensory neuron, the olfactory neuron AWA (Figure 7J and K). AWA in turn has no effect on the timing of oogenesis (Figure 7L). Thus, this manuscript links specific sensory neurons and insulin-like peptides to distinct aspects of oocyte biology, which we believe is a significant advance in the field of reproductive biology.
- The onset of oogenesis phenotype, using the lin-41::GFP reporter, seems more specific and tractable, and the authors nicely decouple this phenotype from the total progeny and fertilization rate phenotypes through experiments that shift animals to different bacterial food at specific developmental stages.
We thank the Reviewer for this comment.
However, as it stands, the data regarding the role of ins-6 and ASJ in modulating this phenotype, and the model that exposure to CS180 bacterial food causes a change in the ASJ expression of ins-6, which is sufficient to promote the earlier onset of oogenesis at the mid-L4 stage, seems somewhat incomplete and have some inconsistencies to be addressed.
a) The ins-6 mutant phenotype is rescued by genome ins-6 and partially rescued by ins-6 expressed under and ASJ-specific promoter. The lack of rescue from an ASI promoter is puzzling given the secreted nature of ins-6.
We address this in Essential Revisions, point 3. Briefly, we disagree that this is puzzling, since several labs have already shown that there are functional differences between the INS-6 produced from ASI versus the INS-6 produced from ASJ, using different experimental approaches (Chen et al., 2013; Tang et al., 2023; and this work). Indeed, the cell-specific activities of a secreted signal is not limited to INS-6, but has also been described for other secreted peptides, such as INS-1 (Kodama et al., 2006; Tomioka et al., 2006; Takeishi et al., eLife 2020, vol 9, e61167. Thus, the interesting question is why functional differences exist between the INS-6 peptides from the two neurons. This is a fascinating question, but beyond the scope of this manuscript.
b) The ins-6 mutant phenotype with regard to delaying the early expression of lin-41::GFP on CS180 appears weaker than the daf-2 mutant phenotype. This is difficult to reconcile with what is known about the relative strength of the daf-2 mutant alleles relative to ins-6 for a wide range of phenotypes.
There are evidence in the literature that the ins-6 mutant phenotype will not look exactly like that of daf-2 (Chen et al., 2013; Cornils et al., Development 2011, vol 138, pp1183-93; Fernandes de Abreu et al., PLoS Genet 2014, vol 10, e1004225). The DAF-2 insulin-like receptor is predicted to bind multiple insulin-like peptides (Pierce et al., Genes Dev 2001, vol 15, pp 672-686), some of which can act antagonistic to DAF-2 function (Pierce et al., 2001; Cornils et al., 2011; Chen et al., 2013; Fernandes de Abreu et al., 2014). Thus, the oogenic effects of the reduction-offunction mutations in daf-2 are likely the sum of multiple insulin-like peptides, some of which might also delay oogenesis. This could explain why the manipulation of an individual insulin-like peptide, INS-6, which could bind DAF-2 to promote oogenesis, does not closely resemble the phenotype of daf-2 mutants.
c) The daf-16 loss-of-function phenotype and suppression of daf-2 and ins-6 mutant phenotypes are not shown for the lin-41::GFP expression phenotype.
We address this in the Public Review comments of Reviewer 1. Briefly, we focused on the epistasis analysis between ins-6 and daf-16 and showed that ins-6 promotes early oogenesis independent of daf-16.
d) The modest difference in ins-6p::mCherry expression in the ASJ neurons (Figure 5D) make the idea that this difference causes onset of oogenesis somewhat implausible.
We disagree that this change is modest and that the oogenic effect of such a change is implausible.
First, the change in ins-6p::mCherry expression in ASJ on CS180 is comparable to other physiologically-important expression changes that have been reported for other genes (for example, Entchev et al., eLife 2015, vol 4, 4:e06259, for the tryptophan hydroxylase tph-1 and the TGF-β daf-7; and Tataridas-Pallas et al, PLoS Genet 2021, vol 17, e1009358, for the neuronally expressed NRF transcription factor skn-1b). Second, it is worth noting that we were using a single-copy reporter for ins-6 expression, where detected changes will be smaller but should be closer to physiological responses. It is possible that multiple-copy reporters will give larger changes, but that would be further from a physiological response. Third, the change in ins-6p::mCherry expression is comparable in scale to the ins-6 mutant phenotype. Our results showed that the 35% increase in ASJ expression of ins-6 is due to food type (Figure 6A; mean fluorescence on OP50 = 1526 + 94; mean fluorescence on CS180 = 2056 + 104). This change in magnitude is similar to the loss of lin-41::GFP expression in mid L4 of ins-6 mutants versus controls. About 30% to 43% of control worms express lin-41::GFP, whereas 0% of ins-6 mutants express the same reporter at mid L4 on CS180 (Figure 5 and its associated supplement).
e) The strain carrying an genetic ablation of ASJ appears to have a markedly different baseline of kinetics of lin-41::GFP expression (even at lethargus, less than half of the animals appear to express lin-41::GFP). Given this phenotype, it seems difficult to draw conclusions about bacterial food-dependent effects on expression of lin-41::GFP. Additional characterization corroborating timing of oogenesis independent of the lin-41::GFP marker may be helpful, but something seems amiss.
We address this in Essential Revisions, point 4. Briefly, we disagree that the kinetics of lin-41::GFP expression in ASJ-ablated animals is puzzling, compared to the kinetics observed in insulin signaling mutants. Besides ins-6, ASJ expresses multiple signals (Taylor et al., 2021), some of which might also regulate the multiple functions of oogenic lin-41::GFP. Thus, it should not be surprising that loss of ASJ will have a markedly different effect on oogenesis than the loss of ins-6.
Reviewer #3 (Public Review):
I very much enjoyed reading this paper by Shashwat Mishra and team from Joy Alcedo's and from Queelim Ch'ng's laboratories dissecting how sensory signals regulate reproduction in worms. The mechanisms by which sensory inputs affect the function of the germline, the balance between growth and differentiation within this tissue, are of broad interest not only to those interested in reproduction and differentiation, but also to those interested in the mechanisms of plasticity that enable organisms to adjust to changing environmental conditions. These mechanisms are only now beginning to be characterized. Here the focus is on the role of insulin signals expressed in sensory neurons. This work builds on previous findings by the Alcedo lab that sensory perception of bacterial-type dependent signals regulates C. elegans lifespan. Here their focus is on the effects on reproduction, and on the communication of that information by insulin-like signals.
We thank the Reviewer for these nice comments.
Worms have a huge family of 40 insulin-like genes, which the Alcedo and Ch'ng labs have been studying for many years. The paper starts with the interesting premise that the brood size of the worms is food type dependent. The authors show that this is due to effects on the timing of the onset of oogenesis during larval development (which constrains the size of the pool of sperm available for subsequent oocyte fertilization) as well as on effects on the rate of oocyte fertilization during adulthood. Using clever timing for food switching, they show that the effects on oogenesis onset and on fertilization rate are separable. In addition, these effects did not appear to be merely the outcome of indirect effects of food ingestion, but were, instead, at least in part, due to the perception of environmental information by specific sensory neurons. Using mutants affecting transduction of sensory information in specific neurons and genetic ablation of specific neurons, the authors show that the onset of oogenesis and the rate of reproduction were controlled by different sensory neurons, ASJ and AWA, respectively. One of these neurons, ASJ, transmitted environmental information via the ins-6 neuropeptide.
Altogether, the paper advances our understanding of how environmental determinants influence reproduction.
We thank the Reviewer for these nice comments.
-
eLife assessment
This work presents useful and potentially valuable findings on how food signals may influence reproduction in the nematode C. elegans. In the current manuscript, the evidence in support of the authors' model is incomplete, and additional experimental data is needed to buttress the authors' conclusions.
-
Reviewer #1 (Public Review):
In this manuscript the authors perform a detailed analysis of the impact of food type on reproduction in C. elegans. They find that, in comparison with the standard OP50 strain of E. coli that is ubiquitously used to maintain C. elegans in the laboratory setting, the CS180 strain results in a reduction in the number of progeny that may be a consequence of an early transition from spermatogenesis to oogenesis that reduces total sperm number. They also find that the rate of oocyte fertilization is increased in animals fed CS180 vs. OP50. Using mutants and laser ablations, the authors show that, whereas the insulin-like peptide INS-6 acts in the ASJ sensory neurons to mediate the food type effect on total progeny and early oogenesis, the increased fertilization rate phenotype does not require ASJ or …
Reviewer #1 (Public Review):
In this manuscript the authors perform a detailed analysis of the impact of food type on reproduction in C. elegans. They find that, in comparison with the standard OP50 strain of E. coli that is ubiquitously used to maintain C. elegans in the laboratory setting, the CS180 strain results in a reduction in the number of progeny that may be a consequence of an early transition from spermatogenesis to oogenesis that reduces total sperm number. They also find that the rate of oocyte fertilization is increased in animals fed CS180 vs. OP50. Using mutants and laser ablations, the authors show that, whereas the insulin-like peptide INS-6 acts in the ASJ sensory neurons to mediate the food type effect on total progeny and early oogenesis, the increased fertilization rate phenotype does not require ASJ or insulin-like signaling and instead requires the AWA olfactory neurons.
The major strengths of the manuscript are the establishment of INS-6 as a link between food type and reproduction and the detail and rigor with which the experiments were executed. The results presented generally support the authors' model. This role of insulin-like signaling in connecting food type and reproduction makes it a plausible target for evolutionary forces that may have shaped insulin-like signaling in invertebrates. As such, this work contributes broadly to our understanding of how insulin signaling may have evolved prior to the emergence of vertebrates.
A weakness of the work is the epistasis analysis of insulin-like pathway components, which is incomplete and at times difficult to interpret.
-
Reviewer #2 (Public Review):
The manuscript by Mishra et al. examines the modulation of the nervous system by different bacterial food to influence reproductive phenotypes-specifically onset of oogenesis, fertilization rate, and progeny production. Defining how animal reproduction could be modulated by bacterial food cues through neuroendocrine signaling is a fascinating subject of study for which C. elegans is well-suited. However, the overall scope of the current study is limited, and some of the central data do not provide compelling evidence for the authors' underlying hypothesis and model.
1. Two strains of E. coli are examined, the standard C. elegans bacterial food strain OP50 and an E. coli strain that Alcedo and colleagues have previously characterized to influence aging and longevity through nervous system modulation. While …
Reviewer #2 (Public Review):
The manuscript by Mishra et al. examines the modulation of the nervous system by different bacterial food to influence reproductive phenotypes-specifically onset of oogenesis, fertilization rate, and progeny production. Defining how animal reproduction could be modulated by bacterial food cues through neuroendocrine signaling is a fascinating subject of study for which C. elegans is well-suited. However, the overall scope of the current study is limited, and some of the central data do not provide compelling evidence for the authors' underlying hypothesis and model.
1. Two strains of E. coli are examined, the standard C. elegans bacterial food strain OP50 and an E. coli strain that Alcedo and colleagues have previously characterized to influence aging and longevity through nervous system modulation. While the authors determine that differences in LPS structure present between the strains does not account for the food-dependent effects, there is little further insight regarding the bacterial features that contribute to the observed differences in reproductive physiology. Moreover, at least two of the phenotypes examined-total progeny and fertilization rate-are known to be affected by bacterial food quality and may be affected by bacteria in many ways, so the description of these phenotypes is somewhat less compelling than the study of the onset of oogenesis.
2. The onset of oogenesis phenotype, using the lin-41::GFP reporter, seems more specific and tractable, and the authors nicely decouple this phenotype from the total progeny and fertilization rate phenotypes through experiments that shift animals to different bacterial food at specific developmental stages. However, as it stands, the data regarding the role of ins-6 and ASJ in modulating this phenotype, and the model that exposure to CS180 bacterial food causes a change in the ASJ expression of ins-6, which is sufficient to promote the earlier onset of oogenesis at the mid-L4 stage, seems somewhat incomplete and have some inconsistencies to be addressed.
a. The ins-6 mutant phenotype is rescued by genome ins-6 and partially rescued by ins-6 expressed under and ASJ-specific promoter. The lack of rescue from an ASI promoter is puzzling given the secreted nature of ins-6.
b. The ins-6 mutant phenotype with regard to delaying the early expression of lin-41::GFP on CS180 appears weaker than the daf-2 mutant phenotype. This is difficult to reconcile with what is known about the relative strength of the daf-2 mutant alleles relative to ins-6 for a wide range of phenotypes.
c. The daf-16 loss-of-function phenotype and suppression of daf-2 and ins-6 mutant phenotypes are not shown for the lin-41::GFP expression phenotype.
d. The modest difference in ins-6p::mCherry expression in the ASJ neurons (Figure 5D) make the idea that this difference causes onset of oogenesis somewhat implausible.
e. The strain carrying an genetic ablation of ASJ appears to have a markedly different baseline of kinetics of lin-41::GFP expression (even at lethargus, less than half of the animals appear to express lin-41::GFP). Given this phenotype, it seems difficult to draw conclusions about bacterial food-dependent effects on expression of lin-41::GFP. Additional characterization corroborating timing of oogenesis independent of the lin-41::GFP marker may be helpful, but something seems amiss.
-
Reviewer #3 (Public Review):
I very much enjoyed reading this paper by Shashwat Mishra and team from Joy Alcedo's and from Queelim Ch'ng's laboratories dissecting how sensory signals regulate reproduction in worms. The mechanisms by which sensory inputs affect the function of the germline, the balance between growth and differentiation within this tissue, are of broad interest not only to those interested in reproduction and differentiation, but also to those interested in the mechanisms of plasticity that enable organisms to adjust to changing environmental conditions. These mechanisms are only now beginning to be characterized. Here the focus is on the role of insulin signals expressed in sensory neurons. This work builds on previous findings by the Alcedo lab that sensory perception of bacterial-type dependent signals regulates C. …
Reviewer #3 (Public Review):
I very much enjoyed reading this paper by Shashwat Mishra and team from Joy Alcedo's and from Queelim Ch'ng's laboratories dissecting how sensory signals regulate reproduction in worms. The mechanisms by which sensory inputs affect the function of the germline, the balance between growth and differentiation within this tissue, are of broad interest not only to those interested in reproduction and differentiation, but also to those interested in the mechanisms of plasticity that enable organisms to adjust to changing environmental conditions. These mechanisms are only now beginning to be characterized. Here the focus is on the role of insulin signals expressed in sensory neurons. This work builds on previous findings by the Alcedo lab that sensory perception of bacterial-type dependent signals regulates C. elegans lifespan. Here their focus is on the effects on reproduction, and on the communication of that information by insulin-like signals.
Worms have a huge family of 40 insulin-like genes, which the Alcedo and Ch'ng labs have been studying for many years. The paper starts with the interesting premise that the brood size of the worms is food type dependent. The authors show that this is due to effects on the timing of the onset of oogenesis during larval development (which constrains the size of the pool of sperm available for subsequent oocyte fertilization) as well as on effects on the rate of oocyte fertilization during adulthood. Using clever timing for food switching, they show that the effects on oogenesis onset and on fertilization rate are separable. In addition, these effects did not appear to be merely the outcome of indirect effects of food ingestion, but were, instead, at least in part, due to the perception of environmental information by specific sensory neurons. Using mutants affecting transduction of sensory information in specific neurons and genetic ablation of specific neurons, the authors show that the onset of oogenesis and the rate of reproduction were controlled by different sensory neurons, ASJ and AWA, respectively. One of these neurons, ASJ, transmitted environmental information via the ins-6 neuropeptide.
Altogether, the paper advances our understanding of how environmental determinants influence reproduction.
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