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  1. Reviewer #4 (Public Review):

    Using a transgenic line of Platynereis, in which GFP is expressed under the control of cis-regulatory elements for r-opsin, the study isolates r-opsin expressing cells from the head (eye photoreceptors) and trunk region (a population of segmentally repeated r-opsin expressing cells associated with the parapodia) by FACS. Subsequent RNA-Seq establishes that both populations of cells express genes for all components of the rhabdomeric phototransduction cascade, while the population of trunk sensory cells additionally expresses genes encoding proteins involved in mechanosensation. Using heterologous expression in a mammalian cell line, it is shown that the Platynereis r-opsin responds to blue light via coupling to Gαq suggesting that it mediates photoresponses via a canonical rhabdomeric phototransduction cascade. Transcriptomic analysis of an r-opsin mutant created by TALEN mediated gene editing then reveals that expression levels of the mechanosensory Atp2b channel are modulated by protracted exposure to blue light, a response abolished in the mutant. Behavioral analysis further suggests that undulatory movements of the worms are equally altered under these illumination conditions. Taken together this suggests that the r-opsin expressing trunk sensory cells act as both photo- and mechanoreceptors and that their mechanosensory properties are modulated in response to light. In combining the transcriptomic analysis of cell types with experimental studies of gene function and behavioral analyses, this study provides exciting new insights into the evolution of sensory cells. Several prior studies have found co-expression of photosensory and mechanosensory proteins in sensory cells of various bilaterians, and comparative studies suggested that photo- and mechanosensory cells may share a common evolutionary origin. However, the current study goes far beyond these findings in establishing a direct functional link between photo-and mechanosensation in a population of sensory cells suggesting that these sensory cells function as multimodal cells and that their mechanosensory properties are altered in response to light. Furthermore, the behavioral data (based on a novel machine-learning based tool of analysing the animals' movement) suggest that these cells have a behaviorally relevant function. Because r-opsin was found to be expressed in mechanoreceptors not only in lophotrochozoans (including Platynereis) but also in ecdysozoans and vertebrates (although functional studies are lacking here) and r-opsins belong to a large family of opsins, almost all of which are responsive to light, the present study suggests that r-opsins may have an ancestral bilaterian role in modulating mechanosensory function in response to light (in addition to their purely photosensory role in the photoreceptors of the eyes). Light-independent functions of r-opsin as recently revealed in Drosophila may, thus, be secondarily derived.

    The study is very carefully conducted and well presented. The only minor flaw is that in its present form, the discussion of the evolutionary implications of the finding lacks in clarity and specificity. The authors here often refer ambiguously to an "ancient" or "ancestral" role of r-opsins without specifying the lineage referred to (ancestral for lophotrochozoans? bilaterians? eumetazoans? metazoans?). The discussion should, therefore be revised with an explicit phylogenetic framework in mind.

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  2. Reviewer #3 (Public Review):

    Opsin proteins are ancient light-sensitive molecules found in photoreceptor cells throughout the animal kingdom. Recent discoveries including those made in the current paper have revealed that besides r-opsins, some classes of photoreceptor cell also express genes that are found in mechanosensory cells, and that r-opsins have both light-dependent and light-independent effects on mechanical force transduction or motion. A question remains as to whether or not: 1) a protosensory cell of animals existed which contained both photoreceptor and mechanoreceptor-like features and, 2) whether the original function of opsin included light-dependent mechanosensory features? The authors consider three competing hypotheses for the cellular evolution of photoreceptor and mechanosensory function. Two of the hypotheses envision either photo- or mechanosensory function for opsins evolving first, the third imagines them evolving simultaneously. The authors note that the majority of what we know about rhabdomeric opsins comes from studying the eye photoreceptors of the fruit fly, Drosophila melanogaster. But might this kind of photoreceptor have functions that are derived compared to the ancestral photoreceptor cell? To investigate this question, the authors turn to the non-model system, Platynereis dumerilii, which has both head and non-head photoreceptors. Here the authors use 1) a fluorescent cell sorting method to perform RNA profiling of eye and trunk photoreceptor cells of a mutant marine worm and find evidence of co-expression of photo- and mechanosensory genes in photoreceptor cells. They also compare the genes that are expressed in Platyneris photoreceptors with genes expressed in Drosophila JO (hearing organ in flies), Zebrafish lateral lines and mouse IEH (inner ear hair) cells, and again they find some commonly-expressed genes. 2) The authors use cell culture to express the opsin, demonstrate that it interacts with G-alphaq, and that it's peak sensitivity is in the blue range. 3) They use in situ hybridization to validate the RNA-seq and detect select enriched transcripts in the photoreceptor tissues. 4) They use a new method, which should be widely useful to other researchers, to detect undulation behavior of the opsin mutant vs. wildtype worms and show that the mutant worm behavior is perturbed in altered light cycles. Taken together, the authors suggest that an ancient light-dependent function of opsin was linked to mechanosensation and that light-independent mechanosensory functions of opsins evolved secondarily. The interpretation is somewhat reasonable given the available data but does not yet entirely rule out other possibilities (see below).

    This paper is a tour-de-force and a really impressive collection of experiments which examines the function of r-opsin in Platyneris. There's lots of innovation here from the use of fluorescent cell sorting and cell-specific RNA-Seq on a non-model system to the deep-learning based approach to examining behavior. Overall, the authors' interpretation of their data seems reasonable however I do believe a even stronger case could be made that what we are talking about is shared ancestry vs. recent recruitment if the authors made phylogenetic trees of the numerous TRE genes that are enriched between Drosophila JO and mouse IEH cells. If a significant number of these genes were true orthologs vs. paralogs across all three species then this would provide stronger evidence of an ancient light-dependent mechanosensory function for r-opsin. GO enrichment terms, while intriguing and suggestive, don't go far enough into the weeds. Also, I think the estimate of there being only 12 genes involved in making a photoreceptor cell able to detect light is probably an underestimate, as this ignores, for example, the understudied molecular machinery required for chromophore metabolism and transport. At the very least, the work should help inspire vigorous debate between vision and auditory neuroscience communities (which do not usually converse with one another) to more carefully consider the ways in which their systems overlap and why.

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  3. Reviewer #2 (Public Review):

    Rhabdomeric Opsins (r-Opsins) are well known for their role in photon detection by photosensory cells which are commonly found in eyes. However, r-Opsin expression has also been detected in non-photosensory cells (e.g., mechanosensors), but their function(s) in these other sensory cells is less well understood. To explore the function of r-Opsins outside the context of an eye/head (non-cephalic function) as well as to investigate the potential evolutionary path by which sensory systems that rely on r-Opsins have evolved, Revilla-i-Domingo et al. have investigated gene expression in two distinct subsets of r-Opsin expressing cells in the marine bristle worm Platynereis dumerilii : EP (eye photoreceptor) and TRE (trunk r-opsin1 expressing) cells. The authors also generate two Pdu-r-Opsin1 mutant strains in order to investigate how the loss of r-Opsin function affects gene expression and behavior.

    The question of what role r-Opsins play outside of photoreceptors is an interesting one that remains poorly understood. In this manuscript, the authors demonstrate a powerful protocol for FACS sorting and sequencing different cell populations from an important evolutionary model organism.

    The transcriptomic analysis presented here demonstrates that both the cephalic EP cells and the non-cephalic TRE cells express components of the photosensory transduction pathway. This observation, together with heterologous cell expression data presented demonstrating sensitivity of Pdu-r-Opsin1 to blue light, suggests that both EP and TRE cells are likely to be light sensitive. The authors also suggest that they observe "mechanosensory signatures" in the transcriptomes, which, together with the analysis of undulatory movements in headless animals, lead them to suggst that r-Opsin in TRE cells functions as an evolutionarily conserved light-dependent modulator of mechanosensation, a conclusion that is not well-supported by the data presented.

    Overall, many of the conclusions drawn from the transcriptome data are inferential and based on weak evidence. Key limitations are listed below:

    1. The apparent overlap between the phototransduction and mechanosensory systems has already been shown (in Drosophila for instance) and the current work adds limited information to this story, and what is added is weakened by the absence of functional and physiological analyses. This is particularly true for supporting the claims of mechanosensory signatures in these cells. For example, genes whose expression is suggested in the text as being indicative of a mechanosensory function (glass and waterwitch) are, in fact, expressed in multiple sensory cell types. Glass (gl) is a transcription factor best known for regulating the expression of phototransduction proteins in photoreceptors. The function of waterwitch (wtrw) is not fully understood, but it is broadly expressed in sensory cells in Drosophila. It would be more compelling if mechanotransduction channels like Piezo and NompC were expressed in the TREs, but there is no mention of this.

    2. The suggestion that the TRE cells share similarity with the mechanosensitive mammalian inner ear is provocative, but lacks strong support. For instance, physiological characterization of the response properties of these sensory cells or identification of anatomical similarities analogous to the stereocilia upon which hair cell mechanosensitivity is based would greatly increase plausibility of this claim. Particularly for a species that diverged from mice and flies many hundreds of millions of years ago, speculation based largely on transcriptome analysis is risky. Careful validation is required as identified genes might not share a conserved function with their assigned orthologs in mice and Drosophila.

    3. The current analysis lacks sufficient power to make compelling claims with regard to potential ancestral protosensory cells. The investigators are examining a single species of marine worm and doing so without detailed anatomical and functional studies of the r-Opsin-expressing cells in the worm.

    4. The behavioral experiments require more functional data to interpret unambiguously. The data indicate that r-opsin1 is required for light to surpress the undulation of decapitated worms. Does this mean that the TREs are photosensors whose activity inhibits locomotion or that the TREs are light-sensitive mechanosensors ?

    5. It is assumed that the TREs constitute a homogenous cell population, but this is not demonstrated. This means that the TREs could be a mixed population (for example, distinct sets of photosensors and mechanosensors) and some of the TRE-expressed genes identified could be expressed in different specific subset of TREs.

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  4. Reviewer #1 (Public Review):

    Strengths and Weaknesses. The authors did quite a lot to establish gene expression and function of the annelid's trunk cells and compare them to photoreceptors of the annelid's eye. They isolated the cells with FACS and characterized gene expression in detail, they knocked down r-opsin with TALEN in the trunk and found a significant difference in a crawling response, and they express the opsin in cell culture to confirm wavelength and G-protein sensitivity. As a potential link between light sensitivity and mechano-sensitivity, they report r-opsin function and light intensity influence expression of atp2b2, a gene that modulates neuronal sensitivity in other organisms. Wavelength and G-protein activation data are valuable because I can think of few or no other organisms in the entire group of lophotrochozoan animals, where this level of experimental manipulation could be done. In short, a strength of this manuscript is the detailed characterization of the trunk receptor cells, which express r-opsins. The authors have brought much evidence to the claim that these TRE cells have both light and mechano-sensitive gene expression and function. Based on these findings in an annelid worm, I believe the paper is a significant advance, and of interest to a broad audience by adding to a growing set of discoveries of similar hybrid sensory cells.

    If a hybrid mechano/photo-receptor is indeed an ancient cell type in bilaterians, this would bring many evolutionary implications for sensory biology. However, in these evolutionary interpretations is where I find a weakness of the manuscript. Namely, with only a handful of species shown thus far to have the hybrid cell type - and many differences in detail about these cell types in different organisms - we can not yet make firm conclusions about whether the multi-functional cells were ancestral. I believe other interpretations are equally valid (and still interesting) and should be given more consideration. Namely, it seems possible that photo- and mechan- sensory processes "joined forces" (e.g. through separate co-option events) in new cell-types, multiple times during evolution. The current manuscript loosely indicates ancestral multi-functionality is more parsimonious. However, no detail is given about that. I suppose the authors mean a single origin of hybrid cell types requires fewer evolutionary transitions than multiple origins. However, such a parsimony count does not count the transitions requiring loss of phototransduction in mouse hearing and do not count transitions to loss of mechanosensitivity in eye photoreceptor cells.

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  5. Evaluation Summary:

    This manuscript presents an investigation of receptors in the trunk of Platynereis annelids that express genes involved in both photoreception (e.g. r-Opsin) and mechanosensation. This is particularly interesting in light of other work in model organisms like flies that uncovered broadly similar results. The authors compare gene expression of fly Johnston Organ cells and mouse hearing cells to the worm receptors. Because Platynereis is distantly related to flies and mice, the authors suggest this "hybrid" sensory receptor could be very old and homologous across many animals. The question of what role r-Opsins play outside of photoreceptors is an interesting one that remains poorly understood.

    (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. Reviewer #1 and Reviewer #4 agreed to share their names with the authors.)

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