Stimulatory and inhibitory G-protein signaling relays drive cAMP accumulation for timely metamorphosis in the chordate Ciona

Reviewer #1 (Public Review):

Summary:
In this manuscript, the authors use gene functional analysis, pharmacology and live imaging to develop a proposed model of diverse G protein family signalling that takes place in the papillae during the ascidian Ciona larval adhesion to regulate the timing of initiation of the morphological changes of metamorphosis. Their experiments provide solid evidence that antagonistic G protein signalling regulates cAMP levels in the papillae, which provides a threshold for triggering metamorphosis that is reflective of a larva keeping a strong and sustained level of contact with a substrate for a minimum period of approximately half an hour. The authors discuss their reasoning and address different specific aspects of their proposed timing mechanism to provide a logical flow to the manuscript. The results are nicely linked to
the ecology of Ciona larval settlement and will be of interest to developmental biologists, neurobiologists, molecular biologists, marine biologists as well as provide information relevant to antifouling and aquaculture sectors.

First, they knock down the G proteins Gaq and Gas to show that these genes are important for Ciona larval metamorphosis. They then provide evidence that the Gaq protein acts through a Ca2+ pathway mediated by phospholipase C and inositol triphosphate by showing that inositol phosphate and phospholipase C gene knockdown also inhibits metamorphosis, while overexpression of Gaq or phospholipase C allows larvae to undergo metamorphosis even in the absence of their mechanosensory cue, which is deprived by removing the posterior half of the tail and culturing the larvae on agar-coated dishes. The authors used calcium imaging which is a genetically encoded fluorescent calcium sensor to show that Gq knockdown larvae lack a Ca2+ spike in their papillae after mechanostimulation, confirming that Gaq acts through a Ca2+ pathway. Similarly the authors show that overexpression of Gas also enables larvae to metamorphose in the absence of mechanostimulation, suggesting a role for both Gaq and Gas in this process.

To confirm that Gas acts through cAMP signalling, the authors use pharmacological treatment or overexpression of a photoactivating adenylate cyclase to increase cAMP, and show that this also enables larvae to metamorphose in the absence of mechanostimulation, but only
when their adhesive papillae are still present. Transcriptome data indicate that both Gs and Gq pathway genes are expressed in the adhesive papillae of the Ciona larva. One missing detail seems to be the need for evidence that cAMP is elevated in the papillae directly as a result of Gs activation. The authors use a fluorescent cAMP indicator, Pink Flamindo, to show that cAMP increases in the papillae upon adhesion to a substrate. Complementary to this, larvae that fail to undergo metamorphosis lack a cAMP increase in papillae. However, it is unclear whether the measured larvae that failed to undergo metamorphosis were wildtype or Gas knockdown larvae. If they were Gas knockdown larvae, this could provide evidence that cAMP does act downstream of the Gas activation.

The authors then provide evidence that GABA signalling within the papillae is acting downstream of the G proteins to induce metamorphosis. Transcriptome data shows that the genes for the GABA-producing enzyme, and for GABAb receptors, are both expressed in papillae. Pharmacological experiments show that GABA induces metamorphosis in the absence of mechanosensory cues, but only in larvae that retain their papillae. To show that GABA signalling within the papillae, rather than from the brain of the larva is important, the authors also demonstrate that anterior segments of larvae lacking the brain can also be stimulated to metamorphose by GABA, and show changes in gene expression caused by GABA.

The authors then use a combination of pharmacology and knockdown experiments in the presence or absence of mechanosensory cues to show that Gq/Ca2+ signalling acts upstream of Gs/cAMP signalling. As the elevation of cAMP by pharmacology or photoactivating adenylate cyclase rescued GABA pathway mutant larvae, the Gq and Gs pathways were concluded to be downstream of GABA signaling. However, GABA treatment could still induce Gaq- and Gas-knockdown larvae to metamorphose, suggesting an alternative pathway to metamorphosis, which the authors deduce to be through a third G protein, Gai. They identify an unusual Gai protein that based on transcriptome data is strongly expressed in the papillae. Gai knockdown larvae fail to metamorphose but are rescued by GABA treatment, which can be explained by a potential additional Gai protein being still present (transcriptome evidence suggests this although it is not further confirmed experimentally, for example by hybridization, immunohistochemistry, fluorescent labelling, or knockdown). The authors then use overexpression and knockdown experiments to show that the Gai protein acts through Gβγi complex to activate phospholipase C. Their experiments also indicate a potential for a complementary or compensatory role for Gai and Gaq signalling through Gβγi. By inhibiting the potassium channel GIRK through knockdown, and the MAPK pathway gene MEK1/2 by pharmacology, the authors also establish a role for these in their proposed model of signalling, allowing GABA and cAMP to compensate or interact with each other.

Strengths:
The strength of this paper is the meticulous and extensive experiments, which are carefully designed to be able to precisely target specific genes in the putative signalling pathway to build step by step a complex model that can demonstrate how metamorphosis of the ascidian larva is timed so as to only undergo metamorphosis when strongly attached to a
suitable substrate. The unique possibility of inhibiting mechanosensory-induced metamorphosis by removing some of the tail and smoothing the attachment substrate allows the authors to investigate potential effects on both activation and inhibition of metamorphosis, and to confirm that specific signalling pathways are clearly downstream of the initial
mechanosensory stimulation. The study is also clear about which aspects of the model still remain unknown, such as which ligands and receptors may be responsible for the binding and activation of Gaq and Gas. Experiments testing metamorphosis of just the anterior region of the larvae nicely demonstrates the need for signalling in the region of the papillae, as do experiments where the papillae are removed, which then block metamorphosis in treatments that would otherwise stimulate it. The final model is a nice end point and makes a clear summary of how the extensive experiments all fit together into a cohesive potential signalling network, which can be built upon in the future.

Weaknesses:
The paper has few weaknesses, however the main difficulty it poses is that due to the sheer number of precise experiments carried out and the complexity of the interwoven signalling pathways, it quickly becomes very difficult to follow exactly what is going on when and why or to keep track of the story as it develops. To improve this, an initial section in the results could be included showing a summary of the known G proteins in Ciona, their types and potential downstream signalling or upstream receptors, where known, and their expression levels in papillae. This could be in the form of a table and/or include the phylogenetic tree from the supplementary data. This would help clarify why the study first focuses on Gaq and Gas, and only later looks at Gai. This could be supplemented by a schematic workflow giving an overview of the experimental process of the study. A second minor weakness (understandable as the focus of the study is metamorphosis induced by mechanosensory stimulation) is that the study does not take into account any potential role for other types of sensory modalities (light, chemicals) that may also feed into the regulation of Ciona larval metamorphosis. This aspect would be interesting to discuss in light of the recent paper suggesting that some sensory cells in the Ciona adhesive papillae are polymodal and detect both chemicals and mechanical stimuli (Hoyer et al. 2024 Current Biology 34(6): 1168 - 1182).

Reviewer #2 (Public Review):

Summary:
This work aims to characterize the neural signaling cascade underlying the initiation of metamorphosis in Ciona larvae. Combining gene-specific functional analyses, pharmacological experiments, and live imaging approaches, the authors identify the molecular players downstream of GABA to initiate Ciona metamorphosis. The results of this study may serve as a useful framework for future research on animal metamorphosis.

Strengths:
The authors did a great job in connecting their experiments with previous findings on Ciona metamorphosis. Taking advantage of the Ciona model system, they meticulously conducted genetic manipulation and pharmacological experiments to test the epistatic relationships among the signaling players controlling the initiation of Ciona metamorphosis.

Weaknesses:
The causal relationship between cAMP accumulation and the initiation of metamorphosis was not clearly demonstrated by the life-imaging observation with the fluorescent cAMP indicator (Pink Flamindo). It is a pity that this experiment was only conducted using normal larvae to compare those who underwent metamorphosis versus those who failed to initiate metamorphosis. This approach should be applied to some of the genetic manipulation and pharmacological experiments, to strengthen their main thesis on the "cAMP timer" mechanism.
On several occasions, the interpretation of the results seems to be imprecise and may lead to misunderstanding. This should be improved by rewriting the descriptions of those results and carefully comparing the differences in results from various treatments and experiments.

Author response:

We would like to thank all reviewers for their valuable comments that help us to improve our manuscript. We will make the following modifications in the revised manuscript:

(1) To reduce the complexity of the experiments we carried out, we will summarize trimeric G proteins in Ciona in the first paragraph of the Result section and explain how we focused on Gas and Gaq in the initial phase of this study.

(2) As the reviewer 1 suggested, the polymodal roles of papilla neurons are interesting. We will add a discussion regarding this aspect. The sentences will be like the following:

“The recent study (Hoyer et al., 2024) provided several lines of evidence suggesting that papilla neurons can serve as the sensors of several chemicals in addition to the mechanical stimuli. This finding and our model seem mutually related because these chemicals could modify Ca2+ and cAMP signaling. The use of G protein signaling may allow Ciona to reflect various environmental stimuli to initiate metamorphosis in the appropriate situation, both mechanically and chemically.”

(3) As both reviewers suggested, imaging cAMP on the backgrounds of some G protein knockdowns and pharmacological treatments is important, and we will carry out some of these experiments.

(4) According to reviewer 2's comment, we will carefully modify the text about interpreting the results so that the descriptions suitably reflect the results.