Functional characterisation of neuropeptides that act as ligands for both calcitonin-type and pigment-dispersing factor-type receptors in a deuterostome

Reviewer #1 (Public review):

Summary:

The manuscript characterizes a functional peptidergic system in the echinoderm Apostichopus japonicus that is related to the widely conserved family of calcitonin/diuretic hormone 31 (CT/DH31) peptides in bilaterian animals. In vitro analysis of receptor-ligand interactions, using multiple receptor activation assays, identifies three cognate receptors for two CT-like peptides in the sea cucumber, which stimulate cAMP, calcium, and ERK signaling. Only one of these receptors is closely related to the family of calcitonin and calcitonin-like receptors (CTR/CLR) in bilaterian animals, whereas two other receptors cluster with invertebrate pigment dispersing factor receptors (PDFRs). In addition, this study sheds light on the transcript expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, in situ hybridization, pharmacological experiments on body wall muscle and intestine preparations, and peptide injection and RNAi knockdown experiments. This reveals a conserved function of CT-like peptides as muscle relaxants and hints at a potential role as growth regulators in A. japonicus.

Strengths:

This work combines both in vitro and in vivo functional assays to identify a CT-like peptidergic system in an economically relevant echinoderm species, the sea cucumber A. japonicus. A major strength of the study is that it identifies three G protein-coupled receptors for AjCT-like peptides, one related to the CTR/CLR family and two related to the PDFR family. A similar finding was previously reported for the CT-related peptide DH31 in Drosophila melanogaster that activates both CT-type and PDF-type receptors. Here, the authors expand this observation to a deuterostomian animal, which suggests that receptor promiscuity is a more general feature of the CT/DH31 peptide family and that CT/DH31-like peptides may activate both CT-type and PDF-type receptors in other animals as well.

Besides the identification of receptor-ligand pairs, the downstream signaling pathways of AjCT receptors have been characterized, highlighting broad effects on cAMP, calcium, and ERK signaling. Functional characterization of the CT-related peptide system in heterologous cells is complemented with ex vivo and in vivo experiments. First, peptide injection and RNAi knockdown experiments establish transcriptional regulation of all three identified receptors in response to changing AjCT peptide levels. Second, ex vivo experiments reveal a conserved role for the two CT-like peptides as muscle relaxants, which have differential effects on body wall muscle and intestine preparations. Finally, peptide injection studies suggest a putative role for one of the two CT-like peptides (AjCT2) in growth regulation.

Weaknesses:

Analysis of transcript expression is limited to the CT-peptide encoding gene, while no gene expression analysis was attempted for the three identified receptors. Differences in the activation of downstream signaling pathways between the three receptors are also questionable due to unclarities in the statistical analysis and variation in the control and experimental data in heterologous assays. Together, this makes it difficult to propose a mechanism underlying differences in the functions of the two CT-like peptides in muscle control and growth regulation.

The authors also suggest a putative orexigenic role for the CT-like peptidergic system in feeding behavior. This effect is not well supported by the experimental data provided, as no detailed analysis of feeding behavior was carried out (only indirect measurements were performed that could be influenced by other peptidergic effects, such as on muscle relaxation) and no statistically significant differences were reported in these assays.

Overall, details regarding statistical analyses are not (clearly) specified in the manuscript, and there are several instances where statements are not supported by literature evidence.

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor but also activate the two PDF receptors, ex vivo. They also explore secondary messenger pathways that are recruited following receptor activation. They determine the source of CT1 and CT2 using qPCR and in situ hybridization and finally test the effects of these peptides on tissue contractions, feeding, and growth. This study provides solid evidence that CT1 and CT2 act as ligands for calcitonin receptors; however, evidence supporting cross-talk between CT peptides and PDF receptors is only based on ex vivo experiments.

Strengths:

This is the first study to report the pharmacological characterization of CT receptors in an echinoderm. Multiple lines of evidence in cell culture (receptor internalization and secondary messenger pathways) support this conclusion.

Weaknesses:

The authors claim that A. japonicus CTs activate "PDF" receptors and suggest that this cross-talk is evolutionarily ancient since a similar phenomenon also exists in the fly Drosophila melanogaster. These conclusions are not fully supported for several reasons. The authors perform phylogenetic analysis to show that the two "PDF" receptors form an independent clade. This clade is sister to the clade comprising CT receptors. This phylogenetic analysis suffers from several issues. Firstly, the phylogenies lack bootstrap support. Secondly, the resolution of the phylogeny is poor because representative members from diverse phyla have not been included. For instance, insect or other protostomian PDF receptors have not been included so how can the authors distinguish between "PDF" receptors or another group of CT receptors? Thirdly, no in vivo evidence has been presented to support that CT can activate "PDF" receptors in vivo.

The source of CT which mediates the effects on longitudinal muscles and intestine is unclear. Is it autocrine or paracrine signaling by CT from the same tissue or is it long-range hormonal signaling?

Pharmacology experiments showing the effects of CT1 and CT2 on ACh-induced contractions were performed. Sample traces have been provided but no traces with ACh alone have been included. How long do ACh-induced contractions persist? These controls are necessary to differentiate between the eventual decay of ACh effects and relaxation induced by CT1 and CT2. The traces also do not reflect the results portrayed in dose-response curves. For instance, in Figure 6B, maximum relaxation is reported for 10-6M. Yet, the trace hardly shows any difference before and after the addition of 10-6M peptide. The maximum effect in the trace appears to be after the addition of 10-8M peptide.

I am unsure how differences in wet mass indicate feeding and growth differences since no justification has been provided. Couldn't wet mass also be influenced by differences in osmotic balance, a key function of calcitonin-like peptides in protostomian invertebrates? The statistical comparisons have not been included in Figure 7B.

While the authors succeeded in knocking down CT, the physiological effects of reduced CT signaling were not examined.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The manuscript characterizes a functional peptidergic system in the echinoderm Apostichopus japonicus that is related to the widely conserved family of calcitonin/diuretic hormone 31 (CT/DH31) peptides in bilaterian animals. In vitro analysis of receptor-ligand interactions, using multiple receptor activation assays, identifies three cognate receptors for two CT-like peptides in the sea cucumber, which stimulate cAMP, calcium, and ERK signaling. Only one of these receptors is closely related to the family of calcitonin and calcitonin-like receptors (CTR/CLR) in bilaterian animals, whereas two other receptors cluster with invertebrate pigment dispersing factor receptors (PDFRs). In addition, this study sheds light on the transcript expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, in situ hybridization, pharmacological experiments on body wall muscle and intestine preparations, and peptide injection and RNAi knockdown experiments. This reveals a conserved function of CT-like peptides as muscle relaxants and hints at a potential role as growth regulators in A. japonicus.

Strengths:

This work combines both in vitro and in vivo functional assays to identify a CT-like peptidergic system in an economically relevant echinoderm species, the sea cucumber A. japonicus. A major strength of the study is that it identifies three G protein-coupled receptors for AjCT-like peptides, one related to the CTR/CLR family and two related to the PDFR family. A similar finding was previously reported for the CT-related peptide DH31 in Drosophila melanogaster that activates both CT-type and PDF-type receptors. Here, the authors expand this observation to a deuterostomian animal, which suggests that receptor promiscuity is a more general feature of the CT/DH31 peptide family and that CT/DH31-like peptides may activate both CT-type and PDF-type receptors in other animals as well.

Besides the identification of receptor-ligand pairs, the downstream signaling pathways of AjCT receptors have been characterized, highlighting broad effects on cAMP, calcium, and ERK signaling. Functional characterization of the CT-related peptide system in heterologous cells is complemented with ex vivo and in vivo experiments. First, peptide injection and RNAi knockdown experiments establish transcriptional regulation of all three identified receptors in response to changing AjCT peptide levels. Second, ex vivo experiments reveal a conserved role for the two CT-like peptides as muscle relaxants, which have differential effects on body wall muscle and intestine preparations. Finally, peptide injection studies suggest a putative role for one of the two CT-like peptides (AjCT2) in growth regulation.

Weaknesses:

(1) Analysis of transcript expression is limited to the CT-peptide encoding gene, while no gene expression analysis was attempted for the three identified receptors. Differences in the activation of downstream signaling pathways between the three receptors are also questionable due to unclarities in the statistical analysis and variation in the control and experimental data in heterologous assays. Together, this makes it difficult to propose a mechanism underlying differences in the functions of the two CT-like peptides in muscle control and growth regulation.

Thank you for the reviewer’s comment, we will supplement the expression analysis for the three identified receptors. Actually, we did all the statistical tests for all the experiments, and maybe the form of marking is a bit messy, so sorry for the confusion and we will uniform them and include all this information both in the figure legends and the methods section. And for the variation in the control and experimental data, because every control is transfected with different receptors or uses cells from different batches, that is why the control conditions in different experiments have little bit variation.

(2) The authors also suggest a putative orexigenic role for the CT-like peptidergic system in feeding behavior. This effect is not well supported by the experimental data provided, as no detailed analysis of feeding behavior was carried out (only indirect measurements were performed that could be influenced by other peptidergic effects, such as on muscle relaxation) and no statistically significant differences were reported in these assays.

Thank you for the reviewer’s comment. Actually, we did all the statistical tests for all the experiments, the mass of remaining bait and the excrement were added in Figure 7A-figure supplement 1 and we will conduct additional behavioral experiments to explore the changes in feeding behavior of A. japonicus after injection of CT-type neuropeptides to support the role of CT-like peptidergic system in the regulation of feeding behavior, as I mentioned above, maybe the form of marking is a bit messy, so sorry for the confusion and we will uniform them and include all this information both in the figure legends and the methods section. And also we will supplement the experiments to further support our claim by assessing the feeding and growth factors after knocking down the CTP encoding genes.

(3) Overall, details regarding statistical analyses are not (clearly) specified in the manuscript, and there are several instances where statements are not supported by literature evidence.

Again, actually, we did all the statistical tests for all the experiments, as I mentioned above, maybe the form of marking is a bit messy, so sorry for the confusion and we will uniform them and include all this information both in the figure legends and the methods section. And we will also supplement more experiments and add more literature evidence to support our statements.

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor but also activate the two PDF receptors, ex vivo. They also explore secondary messenger pathways that are recruited following receptor activation. They determine the source of CT1 and CT2 using qPCR and in situ hybridization and finally test the effects of these peptides on tissue contractions, feeding, and growth. This study provides solid evidence that CT1 and CT2 act as ligands for calcitonin receptors; however, evidence supporting cross-talk between CT peptides and PDF receptors is only based on ex vivo experiments.

Strengths:

This is the first study to report the pharmacological characterization of CT receptors in an echinoderm. Multiple lines of evidence in cell culture (receptor internalization and secondary messenger pathways) support this conclusion.

Weaknesses:

(1) The authors claim that A. japonicus CTs activate "PDF" receptors and suggest that this cross-talk is evolutionarily ancient since a similar phenomenon also exists in the fly Drosophila melanogaster. These conclusions are not fully supported for several reasons. The authors perform phylogenetic analysis to show that the two "PDF" receptors form an independent clade. This clade is sister to the clade comprising CT receptors. This phylogenetic analysis suffers from several issues. Firstly, the phylogenies lack bootstrap support. Secondly, the resolution of the phylogeny is poor because representative members from diverse phyla have not been included. For instance, insect or other protostomian PDF receptors have not been included so how can the authors distinguish between "PDF" receptors or another group of CT receptors? Thirdly, no in vivo evidence has been presented to support that CT can activate "PDF" receptors in vivo.

We do agree with the reviewer that the cross-talk between CTs and PDFRs is not so solid based on our current study.

So firstly, we will re-do the phylogenetic analyses as the reviewer suggested and mark the bootstrap value, then we will supplement more experiments (like PDFR knockdown) to further confirm that CT can activate “PDF” receptors in vivo.

(2) The source of CT which mediates the effects on longitudinal muscles and intestine is unclear. Is it autocrine or paracrine signaling by CT from the same tissue or is it long-range hormonal signaling?

Thank you for the reviewer’s comment, actually we have done in situ and immunohistochemical experiments for CTP and CT in different tissues, we just did not put them in our current manuscript, we will add them in the revised version.

(3) Pharmacology experiments showing the effects of CT1 and CT2 on ACh-induced contractions were performed. Sample traces have been provided but no traces with ACh alone have been included. How long do ACh-induced contractions persist? These controls are necessary to differentiate between the eventual decay of ACh effects and relaxation induced by CT1 and CT2. The traces also do not reflect the results portrayed in dose-response curves. For instance, in Figure 6B, maximum relaxation is reported for 10-6M. Yet, the trace hardly shows any difference before and after the addition of 10-6M peptide. The maximum effect in the trace appears to be after the addition of 10-8M peptide.

Thank you for the reviewer’s comment, we will provide the trace of contraction caused by ACh alone. In Figure 6B, the trace represents successive treatments of neuropeptides at different concentrations, which represents a cumulative effect. Therefore, the corresponding receptors may become desensitized when high concentration of peptide is finally applied. Actually, we examined the pharmacological effects of CT2 at 10-6M concentrations, which exhibited the maximum relaxation, and we will provide this trace.

(4) I am unsure how differences in wet mass indicate feeding and growth differences since no justification has been provided. Couldn't wet mass also be influenced by differences in osmotic balance, a key function of calcitonin-like peptides in protostomian invertebrates? The statistical comparisons have not been included in Figure 7B.

Thank you for the reviewer’s comment, we will analyze the weight gain rate, growth rate and feeding rate of A. japonicus to explain the difference of feeding and growth between injection group and control group. And we can confirm that wet mass is not influenced by differences in osmotic balance, we will put our supporting evidence in supplementary files in revised manuscript and we did not find the key function of calcitonin-like peptides observed in protostomian invertebrates. And we will include the statistical comparisons in Figure 7B.

(5) While the authors succeeded in knocking down CT, the physiological effects of reduced CT signaling were not examined.

Thank you for the reviewer’s insightful suggestion, we will supplement the experiments about the physiological effects after knocking down CT.