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

Xiao Cong; Huachen Liu; Lihua Liu; Nayeli Escudero Castelán; Kite GE Jones; Michaela Egertová; Maurice R Elphick; Muyan Chen

doi:10.7554/eLife.101799.3

Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.

Reviewing Editor
Gáspár Jékely
Heidelberg University, Heidelberg, Germany
Senior Editor
Albert Cardona
University of Cambridge, Cambridge, United Kingdom

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 clusters within 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 expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, immunohistochemistry, 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 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, revealing broad and in some cases receptor-specific 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 and knockdown experiments uncover a growth-promoting role for one CT-like peptide (AjCT2). Injection of AjCT2 at high concentration, or long-term knockdown of the AjCT precursor, affects diverse growth-related parameters including weight gain rate, specific growth rate, and transcript levels of growth-regulating transcription factors. The authors also reveal a growth-promoting function for the PDFR-like receptor AjPDFR2, suggesting that this receptor mediates the effects of AjCT2 on growth.

Weaknesses:

Expression of CT-like peptides was investigated both at transcript and protein level, but insight into the expression of the three peptide receptors is limited. This makes it difficult to understand the mechanism underlying the (different) functions of the two CT-like peptides in vivo. The authors identify differences in signal transduction cascades activated by each peptide, which might underpin distinct functions, but these differences were established only in heterologous cells.

The authors show overlapping phenotypes for a long-term knockdown of the AjCT precursor and the AjPDFR2 receptor, suggesting that the growth-regulating functions of AjCT2 are mediated by this receptor pathway. However, it remains unclear whether this mechanism underpins the growth-regulating function of AjCT2, until further in vivo evidence for this ligand-receptor interaction is presented. For example, the authors could investigate whether knockdown of AjPDFR2 attenuates the effects of AjCT2 peptide injection. In addition, a functional PDF system in this species remains uncharacterized, and a potential role of PDF-like peptides in growth regulation has not yet been investigated in A. japonicus. Therefore, it also remains unclear whether the ability of CT-like peptides to activate PDFRs is an evolutionary ancient property of this peptide family or whether this is an example of convergent evolution in some protostomian (Drosophila) and deuterostomian (sea cucumber) species.

https://doi.org/10.7554/eLife.101799.3.sa2

Reviewer #2 (Public review):

Summary:

The authors show that A. japonicus calcitonins (AjCT1 and AjCT2) activate not only the calcitonin/calcitonin-like receptor, but they 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 weak.

Strengths:

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

https://doi.org/10.7554/eLife.101799.3.sa1

Author response:

The following is the authors’ response to the previous 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 clusters within 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 expression and in vivo functions of CT-like peptides in A. japonicus, by quantitative real-time PCR, immunohistochemistry, 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 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, revealing broad and in some cases receptor-specific 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 and knockdown experiments uncover a growth-promoting role for one CT-like peptide (AjCT2). Injection of AjCT2 at high concentration, or long-term knockdown of the AjCT precursor, affects diverse growth-related parameters including weight gain rate, specific growth rate, and transcript levels of growth-regulating transcription factors. The authors also reveal a growth-promoting function for the PDFR-like receptor AjPDFR2, suggesting that this receptor mediates the effects of AjCT2 on growth.

Weaknesses:

The authors present a more detailed phylogenetic analysis in the revised version, including a larger number of species. But some clusters in the analysis are not well supported because they have only low bootstrap values. This makes it difficult to interpret the clustering in some parts of the tree.

Thank you for the reviewer’s comments. In response, we have produced a new phylogenetic analysis using the maximum likelihood method. This was done by Nayeli Escudero Castelán and Kite Jones in the Elphick group at QMUL and therefore they have been added as co-authors of this paper. The new phylogenetic tree (Figure 2, line 206) includes broad taxonomic sampling of CT-type receptors and PDF-type receptors. CRH-type receptors, which are also members of the secretin-type GPCR sub-family, have been included as an outgroup to root the tree. In the previous version the much more distantly related vasopressin/oxytocin-type receptors, which are rhodopsin-type GPCRs, were included as an outgroup. Furthermore, VIP-type receptors were also included in the previous tree but these have been omitted from the new tree because VIP receptor orthologs only occur in vertebrates and therefore they are not representative of a bilaterian GPCR family. The new tree shows high bootstrap support for key clades, notably achieving a bootstrap value of 100 for a clade comprising both deuterostomian and protostomian PDF receptors. This provides important evidence that the A. japonicus PDF-type receptors characterised in this study (AjPDFR1, AjPDFR2) are co-orthologs of the PDF-type receptor that has been characterised previously in Drosophila. Similarly, there is strong bootstrap support (100) for a clade comprising CT/DH31-type receptors and, importantly, the CT-type receptor characterised in this study (AjCTR) is positioned in a branch of this clade that comprises deuterostomian CT-type receptors (with bootstrap support of 100). Details of methods employed to produce the new receptor tree are included in lines 727-739. The new phylogenetic tree is shown below and has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183).

References:

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Gorn A H, Lin H Y, Yamin M, et al. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. The Journal of clinical investigation, 1992, 90(5), 1726–1735. doi: 10.1172/JCI116046.

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McLatchie L M, Fraser N J, Main M J, et al. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 1998, 393(6683): 333-339. doi: 10.1038/30666.

Schwartz J, Réalis-Doyelle E, Dubos M P, et al. Characterization of an evolutionarily conserved calcitonin signaling system in a lophotrochozoan, the Pacific oyster (Crassostrea gigas). Journal of Experimental Biology, 2019, 222(13): jeb201319. doi: 10.1242/jeb.201319.

Sekiguchi T, Kuwasako K, Ogasawara M, et al. Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insights into the molecular and functional evolution in chordates. Journal of Biological Chemistry, 2016, 291(5): 2345-2356. doi: 10.1074/jbc.M115.664003.

Expression of CT-like peptides was investigated both at transcript and protein level, but insight into the expression of the three peptide receptors is limited. This makes it difficult to understand the mechanism underlying the (different) functions of the two CT-like peptides in vivo. The authors identify differences in signal transduction cascades activated by each peptide, which might underpin distinct functions, but these differences were established only in heterologous cells.

We appreciate the reviewer's insightful comments. Regarding expression of CT-like peptide receptors, we have quantitatively analyzed the mRNA expression levels of the three receptors in key tissues using qRT-PCR (Figure 6, line 319) and receptor expression exhibits significant tissue-specific differences. Combined with the heterologous expression assays and In vivo functional validation, we believe our findings have provided clear mechanistic insights into the functional divergence of the two CT-like peptides. Investigation of the expression of the three receptor proteins in A. japonicus would require generation of specific antibodies, which was beyond the scope of this study. Furthermore, immunohistochemical visualization of neuropeptide receptor expression in other invertebrates has not been reported widely, which likely reflects technical difficulties in generation of antibodies that can be used to specifically detect receptor proteins that are typically expressed a low level in comparison to the neuropeptides that act as their ligands.

We acknowledge that investigating signal transduction cascades in heterologous cells (rather than native A. japonicus cells) is a limitation. However, as a non-model organism, A. japonicus currently lacks established cell lines for such research. Therefore, using heterologous cells was the most feasible approach to examine the differential signaling cascades activated by the peptides through the three receptors. Importantly, our in vivo experiments demonstrated that long-term knockdown of either the AjCT precursor or AjPDFR2 resulted in similar and significant growth defects. The phenotypic consistency strongly suggests that AjCT2 and AjPDFR2 function within the same signaling pathway, with AjPDFR2 serving as the key receptor functionally activated by AjCT2.

The authors show overlapping phenotypes for a long-term knockdown of the AjCT precursor and the AjPDFR2 receptor, suggesting that the growth-regulating functions of AjCT2 are mediated by this receptor pathway. However, it remains unclear whether this mechanism underpins the growth-regulating function of AjCT2, until further in vivo evidence for this ligand-receptor interaction is presented. For example, the authors could investigate whether knockdown of AjPDFR2 attenuates the effects of AjCT2 peptide injection. In addition, a functional PDF system in this species remains uncharacterized, and a potential role of PDF-like peptides in growth regulation has not yet been investigated in A. japonicus. Therefore, it also remains unclear whether the ability of CT-like peptides to activate PDFRs is an evolutionary ancient property of this peptide family or whether this is an example of convergent evolution in some protostomian (Drosophila) and deuterostomian (sea cucumber) species.

Thank you for the reviewer’s insightful comments and constructive questions. We acknowledge the request for more direct evidence to demonstrate how AjCT2 functions in vivo through AjPDFR2. However, long-term knockdown of the AjCT precursor and AjPDFR2 both resulted in identical and significant growth defect phenotypes. The high phenotypic consistency, combined with the activation effect of AjCT2 on AjPDFR2 in heterologous cells, strongly suggests that they function within the same signaling pathway, with AjPDFR2 serving as the key receptor functionally activated by AjCT2. While exogenous peptide injection combined with receptor knockdown is a classic method for verifying receptor activation, phenotypic overlap itself is widely accepted in genetic research as robust evidence for pathway association (Shafer and Taghert, 2009; Van Sinay et al., 2017). A. japonicus is a non-model organism with a 3-month aestivation period in summer followed shortly by winter hibernation. During these periods, we are unable to conduct in vivo experiments. Any single experimental suggestion from reviewers could potentially require one more year of research and we have already conducted an additional year of research, in response to reviewer feedback, since submitting the original manuscript. We hope therefore that these challenges associated with working with aquatic invertebrate non-model organisms is recognized by the reviewers.

We fully agree that the functional PDF/PDFR system in A. japonicus and its potential role in growth regulation remain uncharacterized. Currently, the precursors of the PDF-type neuropeptide in echinoderms remain unidentified, which precludes clear pharmacological characterization of the two receptors. While further exploration of echinoderm PDF-type neuropeptides is still needed, our phylogenetic analysis-conducted using the maximum likelihood method with optimized parameters and rigorous sequence curation-demonstrates that the deuterostomian PDFRs (including AjPDFR1 and AjPDFR2) are positioned in a clade with the well-characterized protostomian PDFR clades with extremely high bootstrap support (value=100). Therefore, these two receptors in A. japonicus clearly belong to the PDF receptor family and our findings clearly indicate that the ability of CT-like peptides to activate PDFRs is either an evolutionarily ancient and conserved property or has arisen independently in different lineages. Details of methods employed to produce the new receptor tree are included in line 727-739. The new phylogenetic tree is shown below and has been incorporated into the revised manuscript (Figure 2, line 206). The description of new phylogenetic tree has also been modified accordingly in the revised manuscript (line 169-183).