A Go-type opsin mediates the shadow reflex in the annelid Platynereis dumerilii

Abstract Background The presence of photoreceptive molecules outside the eye is widespread among animals, yet their functions in the periphery are less well understood. Marine organisms, such as annelid worms, exhibit a ‘shadow reflex’, a defensive withdrawal behaviour triggered by a decrease in illumination. Herein, we examine the cellular and molecular underpinnings of this response, identifying a role for a photoreceptor molecule of the Go-opsin class in the shadow response of the marine bristle worm Platynereis dumerilii. Results We found Pdu-Go-opsin1 expression in single specialised cells located in adult Platynereis head and trunk appendages, known as cirri. Using gene knock-out technology and ablation approaches, we show that the presence of Go-opsin1 and the cirri is necessary for the shadow reflex. Consistently, quantification of the shadow reflex reveals a chromatic dependence upon light of approximately 500 nm in wavelength, matching the photoexcitation characteristics of the Platynereis Go-opsin1. However, the loss of Go-opsin1 does not abolish the shadow reflex completely, suggesting the existence of a compensatory mechanism, possibly acting through a ciliary-type opsin, Pdu-c-opsin2, with a Lambdamax of approximately 490 nm. Conclusions We show that a Go-opsin is necessary for the shadow reflex in a marine annelid, describing a functional example for a peripherally expressed photoreceptor, and suggesting that, in different species, distinct opsins contribute to varying degrees to the shadow reflex

HSPIR: a manually annotated heat shock protein information resource

Heat shock protein information resource (HSPIR) is a concerted database of six major heat shock proteins (HSPs), namely, Hsp70, Hsp40, Hsp60, Hsp90, Hsp100 and small HSP. The HSPs are essential for the survival of all living organisms, as they protect the conformations of proteins on exposure to various stress conditions. They are a highly conserved group of proteins involved in diverse physiological functions, including de novo folding, disaggregation and protein trafficking. Moreover, their critical role in the control of disease progression made them a prime target of research. Presently, limited information is available on HSPs in reference to their identification and structural classification across genera. To that extent, HSPIR provides manually curated information on sequence, structure, classification, ontology, domain organization, localization and possible biological functions extracted from UniProt, GenBank, Protein Data Bank and the literature. The database offers interactive search with incorporated tools, which enhances the analysis. HSPIR is a reliable resource for researchers exploring structure, function and evolution of HSPs

Metronidazole induces apoptosis in transgenically labeled cells.

<p>(A-L) Head sections of mtz treated (A-D), DMSO treated (E-H) and untreated (I-L) premature adult <i>r-opsin1::eGFP-f2A-ntr</i> worms (eye PRCs and projections, green) processed for terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) detection (red). (A-D) Apoptosis was detected in PRCs exposed to 25mM mtz after 28 hrs incubation, whereas transgenic animals treated with DMSO alone (E-H) or transgenic untreated animals (I-L), did not show staining above background. ae: adult eye. Scale bars C: 50µm; D: 15µm.</p

Construction and expression of the <i>r-opsin1::egfp-f2a-ntr transgene</i>.

<p>(A) Schematized generation of the <i>r-opsin1::egfp-f2a-ntr</i> construct. The <i>egfp-f2a-ntr</i> cassette was recombined into the <i>Platynereis </i><i>r-opsin1</i> locus by homologous recombination. 8kbps of the surrounding genomic locus plus cassette were PCR amplified and subcloned into the mariner transposon vector used for transgenesis. (B-D) Co-expression of <i>egfp</i> and <i>ntr</i> in the adult eye photoreceptors of <i>r-opsin1::egfp-f2a-ntr</i> stable transgenic worms. (B) eGFP fluorescence demarcating the adult eye photoreceptors and their projections in stable <i>r-opsin1::egfp-f2a-ntr</i> transgenic worms. (C) Expression of <i>nitroreductase</i> (red) in the same cell type as visualized by whole mount in situ hybridization (WMISH) using <i>nitroreductase</i> antisense riboprobe. (D) Co-staining with <i>nitroreductase</i> (detected in red) and <i>egfp</i> (detected in blue) riboprobes results in purple color, indicative of faithful co-expression of both genes in the adult eye photoreceptors. ae- adult eyes; arrowheads point at expressing cells; arrow points at axonal projection of PRCs; asterisks- head pigment cells which show autofluorescence in the channel used for eGFP documentation. Scale bar: 20µm.</p

Conditional and Specific Cell Ablation in the Marine Annelid <i>Platynereis dumerilii</i>

<div><p>The marine annelid Platynereis dumerilii has become a model system for evo-devo, neurobiology and marine biology. The functional assessment of its cell types, however, has so far been very limited. Here we report on the establishment of a generally applicable, cell type specific ablation technique to overcome this restriction. Using a transgenic strain expressing the bacterial enzyme nitroreductase (ntr) under the control of the worm’s r-opsin1 locus, we show that the demarcated photoreceptor cells can be specifically ablated by the addition of the prodrug metronidazole (mtz). TUNEL staining indicates that ntr expressing cells undergo apoptotic cell death. As we used a transgenic strain co-expressing ntr with enhanced green fluorescent protein (egfp) coding sequence, we were able to validate the ablation of photoreceptors not only in fixed tissue, using r-opsin1 riboprobes, but also by monitoring eGFP+ cells in live animals. The specificity of the ablation was demonstrated by the normal presence of the eye pigment cells, as well as of neuronal markers expressed in other cells of the brain, such as phc2, tyrosine hydroxylase and brn1/2/4. Additional analyses of the position of DAPI stained nuclei, the brain’s overall neuronal scaffold, as well as the positions and projections of serotonergic neurons further confirmed that mtz treatment did not induce general abnormalities in the worm’s brain. As the prodrug is administered by adding it to the water, targeted ablation of specific cell types can be achieved throughout the life of the animal. We show that ablation conditions need to be adjusted to the size of the worms, likely due to differences in the penetration of the prodrug, and establish ablation conditions for worms containing 10 to 55 segments. Our results establish mtz/ntr mediated conditional cell ablation as a powerful functional tool in Platynereis.</p> </div

Loss of <i>r-opsin1+</i> cells by metronidazole treatment.

<p>Whole mount <i>in </i><i>situ</i> hybridization with a riboprobe specific for <i>Platynereis </i><i>r-opsin1</i> on immature adult <i>Platynereis</i> worms (10-20 segments). (A,C,E) DMSO controls, (B,D,F) metronidazole treated worms (12mM, 48hrs). (A,B) Dorsal views on heads (anterior down), focused on the position of the lateral frontal eyelets (arrows). (C-F) Ventral views (anterior down) on immature adult worm tails (C,E) treated with DMSO, (D,F) treated with mtz. Arrows point at the position of the peripheral <i>r-opsin1+</i> photoreceptor cells. Black ‘needle-like’ structures in C-F are aciculae (bristles). Scale bar: 30µm.</p

Metronidazole treatment specifically ablates <i>ntr</i> expressing cells, without affecting other brain cells.

<p>(A-F) Metronidazole treatment has no effect on non-PRC marker genes. Comparative analysis of expression patterns of the neuronal marker genes <i>prohormone </i><i>convertase2/phc2</i> (arrowheads in A-C) and <i>tyrosine </i><i>hydroxylase/th</i> (arrowheads in D-F) in untreated (A,A’,D,D’), DMSO treated (B,B’,E,E’) and metronidazole treated (C,C’,F,F’) animals. Each set of panels compares non-transgenic control animals (left) and <i>r-opsin1::eGFP-f2A-ntr</i> transgenic animals. Neuronal marker genes are detected in blue, <i>r-opsin1</i> were detected with FastRed substrate (red). In panels (A’-F’), FastRed is visualized using fluorescence microscopy. Scale bar: 50µm. (G,H) Quantification of cell numbers in untreated, DMSO treated and mtz treated animals. Individual eGFP fluorescent PRCs (green bars) were counted in live transgenic worms (same animals were counted before and after treatment). <i>phc2</i> (G) and <i>th</i> (H) expressing cells (black bars) were determined by counting all cells that showed complete cellular outlines in WMISH analyses of animals fixed after the respective experiment. Data represent means ± S.E.M. (n=10 worms for each experiment). ****<i>p</i><0.0001; ns. - no statistically significant differences. The two-tailed paired student <i>t</i>-test was used for statistical analyses.</p

Characterization of cephalic and non-cephalic sensory cell types provides insight into joint photo- and mechanoreceptor evolution

Rhabdomeric Opsins (r-Opsins) are light-sensors in cephalic eye photoreceptors, but also function in additional sensory organs. This has prompted questions on the evolutionary relationship of these cell types, and if ancient r-Opsins cells were non-photosensory. Our profiling of cephalic and non-cephalic r-opsin1-expressing cells of the marine bristleworm Platynereis dumerilii reveals shared and distinct features. Non-cephalic cells possess a full set of phototransduction components, but also a mechanosensory signature. We determine that Pdu-r-Opsin1 is a Gαq-coupled blue-light receptor. Profiling of cells from r-opsin1 mutants versus wild-types, and a comparison under different light conditions reveals that in the non-cephalic cells, light – mediated by r-Opsin1 – adjusts the expression level of a calcium transporter relevant for auditory mechanosensation in vertebrates. We establish a deep learning-based quantitative behavioral analysis for animal trunk movements, and identify a light-and r-Opsin-1-dependent fine-tuning of the worm’s undulatory movements in headless trunks, which are known to require mechanosensory feedback.Our results suggest an evolutionary concept in which r-Opsins act as ancient, light-dependent modulators of mechanosensation, and suggest that light-independent mechanosensory roles of r-Opsins likely evolved secondarily