Stable Transgenesis in the Marine Annelid Platynereis Dumerilii Sheds New Light on Photoreceptor Evolution

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Stable transgenesis in the marine annelid Platynereis dumerilii sheds new light on photoreceptor evolution

Benjamin Backﬁsch1, Vinoth Babu Veedin Rajan1, Ruth M. Fischer, Claudia Lohs2, Enrique Arboleda3, Kristin Tessmar-Raible, and Florian Raible4

Max F. Perutz Laboratories, Department of Microbiology, Immunobiology, and Genetics, University of Vienna, A-1030 Vienna, Austria

Edited by Eric H. Davidson, California Institute of Technology, Pasadena, CA, and approved November 2, 2012 (received for review June 18, 2012) Research in eye evolution has mostly focused on eyes residing in the body plans, and may, where present, still retain functions that head. In contrast, noncephalic light sensors are far less understood complement cephalic PRCs (9). and rather regarded as evolutionary innovations. We established The discovery of noncephalic photoreceptive organs is facili- stable transgenesis in the annelid Platynereis, a reference species tated by the presence of associated pigment spots. Unpigmented for evolutionary and developmental comparisons. EGFP controlled PRCs might therefore be abundant, but are less likely to be dis- by cis-regulatory elements of r-opsin, a characteristic marker for covered by morphological or ultrastructural techniques (10). In rhabdomeric photoreceptors, faithfully recapitulates known r-opsin contrast, molecular markers should be able to detect such unpig- expression in the adult eyes, and marks a pair of pigment-associated mented photoreceptive structures, and thereby provide insight into frontolateral eyelets in the brain. Unexpectedly, transgenic animals their abundance and localization. Together with covalently bound revealed an additional series of photoreceptors in the ventral nerve retinal, Opsin-type G protein-coupled receptors serve as main cord as well as photoreceptors that are located in each pair of the light sensors in animal photoreceptors. Rhabdomeric Opsins (r- segmental dorsal appendages (notopodia) and project into the ven- Opsins) are an ancient group of Opsins particularly widespread tral nerve cord. Consistent with a photosensory function of these among invertebrates, typically expressed in larval PRCs and/or noncephalic cells, decapitated animals display a clear photoavoid- cephalic eyes (reviewed in ref. 11). Notable exceptions are the ance response. Molecular analysis of the receptors suggests that expression of r-opsin orthologs in sea urchin tube feet (6, 12) and they differentiate independent of pax6, a gene involved in early the photoreceptors of the Hesse and Joseph cells (13) whose eye development of many metazoans, and that the ventral cells function remains enigmatic. may share origins with the Hesse organs in the amphioxus neural The paired-homeodomain transcription factor Pax6 is a key tube. Finally, expression analysis of opn4×-2 and opn4m-2, two gene for the development of cephalic eyes in both Drosophila and zebrafish orthologs of Platynereis r-opsin, reveals that these genes chordates (reviewed in ref. 11, also see ref. 14), even though the share expression in the neuromasts, known mechanoreceptors of question of whether Pax6 has an ancient or recent role in direct the lateral line peripheral nervous system. Together, this establishes regulation of opsin genes remains debated (15, 16). There are also, that noncephalic photoreceptors are more widespread than as- however, pax6-independent PRCs. For instance, Hesse cells were sumed, and may even reflect more ancient aspects of sensory sys- found void of amphioxus pax6 (17). The fact that Hesse cells do not tems. Our study marks significant advance for the understanding of express pax6 has been interpreted as evidence for a recent origin of photoreceptor cell (PRC) evolution and development and for Platy- these noncephalic PRCs (17). However, in the absence of molec- nereis as a functional lophotrochozoan model system. ular data for a larger spectrum of noncephalic PRCs, it remains unclear if the absence of pax6 from these cells might, alternatively, regulation | transposon | polychaete | worm represent a common feature of noncephalic PRCs. Here, we report the establishment and analysis of a stable r-opsin:: ur view on animal photoreception is dominated by the anal- egfp strain that marks rhabdomeric PRCs throughout the lifetime of Oysis of pigmented cephalic eyes, which are prominent in most the marine annelid Platynereis dumerilii, a key reference species for branches of animal evolution (1). However, eyes and other pho- eye and brain development and evolution (11, 18–22). The strain toreceptive cells are also present outside the brains of animals. highlights adult eyes and their projections, as well as a set of eyelets Among bilaterians, examples include the early Cambrian Lobo- in the frontolateral adult brain. Moreover, we discover a series of podian fossil Microdictyon sinicum, a probable ancestor of extant pax6-negative noncephalic PRCs in the dorsal appendages (noto- arthropods, that displays segmentally arranged compound eyes pods) as well as the ventral trunk that allow comparisons with the above each of its leg pairs (2). Similarly, Opheliid worms of the noncephalic visual organs of other polychaetes. By their position in genus Polyophthalmus, or the Sabellid polychaete Branchiomma the medial posterior neuraxis and by gene expression, the ventral carry segmental ocelli on, or in close vicinity to, their appendages (reviewed in ref. 3). Another polychaete genus, Eunice (including Palolo worms) shows segmentally arranged, midventral eyespots Author contributions: K.T.-R. and F.R. designed research; B.B., V.B.V.R., R.M.F., and E.A. performed research; C.L. contributed new reagents/analytic tools; B.B., V.B.V.R., R.M.F., (1, 4). Drosophila was recently demonstrated to possess photore- K.T.-R., and F.R. analyzed data; B.B. established Platynereis transgenesis; V.B.V.R. discov- ceptive cells in the larval body wall that mediate a photoavoidance ered Platynereis noncephalic photoreceptors; and K.T.-R. and F.R. wrote the paper. response (5). Sea urchins possess photoreceptor cells (PRCs) in The authors declare no conflict of interest. their tube feet (6). Finally, the basal chordate amphioxus displays This article is a PNAS Direct Submission. BIOLOGY a series of prototypical visual organs (each consisting of a PRC with

Data deposition: The sequences reported in this paper have been deposited in the Gen- DEVELOPMENTAL an associated pigment cell) that are located along the ventral Bank database (accession nos. KC109635, KC109636, and KC109637). neural tube and are commonly referred to as organs of Hesse or 1B.B. and V.B.V.R. contributed equally to this work. pigmented ocelli (7). The existence of noncephalic visual organs 2Present address: Institut für Molekulare Immunologie, Helmholtz Zentrum München, observed in these and other taxa could represent independent D-81377 Munich, Germany. evolutionary events, e.g., regulatory mutations affecting genetic 3Present address: Council on International Educational Exchange Research Station Bon- master switches like the homeodomain protein Pax6, which can aire, Kralendijk, Bonaire, Dutch Caribbean. cause the development of ectopic eyes when misexpressed in 4To whom correspondence should be addressed. E-mail: ﬂ[email protected]. ﬂ Drosophila (8). Alternatively, these segmental organs could re ect This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ancient sites of photoreception that predated the cephalization of 1073/pnas.1209657109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1209657109 PNAS | January 2, 2013 | vol. 110 | no. 1 | 193–198 Downloaded by guest on October 5, 2021 cells even resemble the Hesse organs of amphioxus. Such a deep relationship supports the existence of a previously unnoticed ancient type of PRCs in bilaterians. Finally, we uncover that even in a vertebrate model, the zebrafish, two orthologs of Platynereis r-opsin are specifically expressed in peripheral sensory cells. As these are the neuromasts of the lateral line, well-known mechanoreceptive cells, this finding also supports the hypothesis that mechanic and photic senses are linked. Results Generation of a Stable Transgenic Strain Driving EGFP Expression Under the Control of the r-opsin Locus. To produce a transgenic strain for r-opsin–positive cells, we took advantage of the Tc1/ mariner-type element Mos1 (23). A recombineering approach was used to insert a cassette containing enhanced green fluorescent protein (egfp) coding sequence into a previously identified bac- terial artificial chromosome carrying the Platynereis r-opsin locus (24). An 8-kb piece of the recombineered locus was then amplified and cloned into a modified Mos1 target vector to give rise to the reporter construct pMos{r-opsin::egfp}frkt890 (Fig. S1). Coinjection of plasmid DNA and synthetically produced mos1 mRNA into Platynereis zygotes yielded transient transgenic animals that were raised to adults and crossed against wild-type animals. The strain analyzed in this study is referred to as r-opsin::egfpvbci2 and derives from a single carrier. The stability of the fluorescent signal upon inheritance of the construct (meanwhile to the third generation) indicates that Mos1-mediated insertions in the Platy- nereis genome are stably transmitted and remain accessible to the transcriptional machinery.

Robust Labeling of Adult Eye Photoreceptors and Neuronal Projections Throughout the Lifetime of the Animal. To assess if the regulatory sequence included in the r-opsin::egfpvbci2 strain was sufficient, we first determined if known sites of r-opsin expression showed fluorescent signal in transgenic animals. Endogenous r-opsin is prominently expressed during differentiation of the adult eyes around 2 d of development (18). Consistent with this, we detect EGFP fluorescence in the bilateral pairs of adult eye primordial at 2 d of development. The adult eye primordia can be easily detected by light microscopy due to the developing shading pigments (Fig. S2 A–C). As expected (18), in contrast to the adult eye primordia, the larval eyes do not show expression of the reporter construct at 2 d of development (Fig. S2 A–C). When we assessed fluorescence at later stages of development, we found that EGFP expression in the adult eyes was maintained throughout development (Fig. 1A) and even persisted in mature fl Fig. 1. Labeling of cephalic rhabdomeric photoreceptor cells (PRCs) by the animals (Fig. 1B). Notably, uorescence was not only detected stable pMos{rops::egfp}vbci2 strain. (A) At 14 d of development, EGFP demarcates in the cell bodies, but was also well observable in the neuronal the adult eye photoreceptors (asterisks) and their neuronal projections (green projections connecting the adult eyes toward the medial brain (Fig. arrowheads) as well as a newly identified set of frontolateral eyelets (green 1 A and B). This attests to the suitability of transgenic Platynereis arrows). (B) EGFP expression in adult eye PRCs (asterisks) and their projections strains for detailed cell-morphological analyses. The robust labeling (green arrowheads) in a mature transgenic animal after spawning. White arrows of adult eye PRCs and their projections represents a major advance indicate autofluorescent iridophore pigments. (C and D) The frontolateral eyelet over previous attempts to track the projections of the PRCs (25). PRCs (green arrow) are frequently found to be associated with pigment cells (blue arrowhead) (A) dorsal view, anterior to the top; (B) frontal view, dorsal to Labeling of Frontolateral Eyelets in the Anterior Brain. The persistent the top; (C and D) lateral views, anterior to the left. ae, adult eyes. expression of EGFP in the adult eye PRCs extends the previously described expression of r-opsin in the emerging adult eyes (18) to all previously characterized larval PRCs (18). We also detected EGFP postlarval development. In addition, a careful analysis of postlarval or r-opsin in the same region in 40-segmented worms, indicating r-opsin::egfpvbci2 animals revealed additional sites of EGFP fluorescence. On the one hand, we detected bilateral pairs of cells an- that frontolateral eyelets persist until premature adult stages. In fl terior to the adult eyes (Fig. 1A). These anterior EGFP-positive sexually mature animals, neither EGFP uorescence nor r-opsin cells are accompanied by pigment cells, indicating that they are able expression is detectable in the anterior head, indicating that the to sense directional light (Fig. 1 C and D). We refer to this assembly frontolateral eyelets either change their photopigments or become of PRCs and pigment cells as frontolateral eyelets. From previous dispensable upon maturation. studies, it remains unclear if cells in this region are remnants of the larval eye (26) or if larval eyes are reduced after the second day of Identification of Noncephalic Photoreceptors in the Notopods and the development (25). We observe, however, that the PRCs of the Ventral Trunk. When investigating juveniles and adult worms, we frontolateral eyelets express pax6 (Fig. S3), reminiscent of the found that r-opsin::egfpvbci2 animals showed additional EGFP

194 | www.pnas.org/cgi/doi/10.1073/pnas.1209657109 Backfisch et al. Downloaded by guest on October 5, 2021 fluorescence outside the brain. On the one hand, we detected projection pattern of these notopodial cells. First, they project a very regular pattern of PRCs located in the worms’ appendages. toward the base of the parapodia (Fig. 2B). From there, they follow The cell bodies are located in the ventral lobe (“upper lip”) of the the anterior branch of segmental nerve II into a more posterior dorsal parapodia (notopods), in a region free of visible pigments direction, before turning more anteriorly, and joining nerve I (Fig. (Fig. 2 A–C, “np”). These cells are already present when we in- 2C). Inside the ventral nerve cord (VNC), the projections from vestigate young adults (seven to eight segments), but exclude the each side of the worm follow a distinct longitudinal fiber (Fig. 2 C first two pairs of appendages that are known to correspond to the and D). As we do not observe commissures between these fibers, last two pairs of larval parapodia (27). We therefore conclude that we assume that the projections remain ipsilateral and hence might they are restricted to postlarval segments. Analysis of fixed animals transmit information on lateral light conditions even to adjacent stained with an anti-EGFP serum, and counterstained with anti- segments, possibly influencing segmental motor microcircuits (28). acetylated tubulin allowed us to determine the highly stereotypical Whereas notopodial PRCs are part of the peripheral nervous system, we also detect EGFP-positive cells in the ventral midline of r-opsin::egfpvbci2 worms, associated with the trunk central nervous system. We refer to these cells as midventral cells (Fig. 2 A and D, “mv”). In contrast to the notopodial cells, these PRCs are more sparse and irregularly spaced with respect to the visible segment boundaries. Based on the analysis of fixed specimens stained with anti-GFP sera, these midventral PRCs appear to project along a midventral fiber separate from the lateral fibers harboring the projections of the notopodial cells. Finally, we also find EGFP- positive cells located at the base of the parapodia that we refer to as ventrolateral cells (Fig. 2A, “vl”). These cells share properties of both the midventral cells and the notopodial cells: they are similar to the midventral cells concerning both their diameter and their sparse and more irregular occurrence along the anteroposterior axis of a given parapodium. On the other hand, they join the tra- jectories of the notopodial PRCs, sending neuronal projections along the lateral fibers. Neither midventral nor ventrolateral cells are associated with visible pigmentation. Both the localization of these cells in the medial VNC and the projection along the neuraxis (Fig. 2D) makes these cells similar to the PRCs described in the abdominal eyespots described for the Palolo worm Eunice (4).

Platynereis Trunk Shows a Robust Photoavoidance Response. In Eunice, posterior segments are constricted upon maturation, and therefore the abdominal eyespots might be involved in autonomous phototaxis of the tail pieces. To our knowledge, however, photosensory functions for the Platynereis trunk have not yet been reported. To assess this question, we took advantage of the fact that worms sur- vive decapitation, and—apart from entering maturation—remain functional (e.g., ref. 29). To assess if these decapitated tails responded to light, we shone bright light onto an area close to the tail tip of dim-light–adapted trunk pieces and measured the movement of the specimens over 30 s, in comparison with specimens that remained in dim light. These experiments revealed that light trig- gers a robust photoavoidance response in the Platynereis trunk (Fig. 3), compatible with the idea that one or more of the described noncephalic receptors is involved in photoavoidance. Notably, according to current sequence data, Platynereis does not possess an ortholog of Gr28b, the receptor mediating larval photoavoidance in Drosophila (5).

Noncephalic Photoreceptors Possess a Gq-Type Alpha Subunit and Develop Independent of pax6. To extend our insight into the mo- ﬁ vbci2 lecular characteristics of the newly identi ed PRCs, we next in- Fig. 2. The pMos{rops::egfp} strain reveals a complex set of unpigmented – noncephalic photoreceptors. (A and A′) Schematic drawing of four segments of vestigated if r-opsin positive cells coexpressed other molecular ′ markers. For this, we ﬁrst established a robust double in situ hy-

the ventral Platynereis trunk (A) and a view onto a parapodium (A ). Rectangles BIOLOGY and labels indicate regions and orientation of A′ and B–D; green circles indicate bridization protocol that allows reliable codetection of two ribo- location of noncephalic PRCs, green lines demarcate typical projection trajec- probes in adults, a stage that is rather problematic for the existing DEVELOPMENTAL tories. (B–D) Representative examples of noncephalic PRCs (green) visualized by fluorescent detection method (30). EGFP fluorescence in living animals (B and D) or by anti-GFP immunohisto- We hypothesized that r-opsin–positive cells, if functioning as chemistry (C); arrows indicate cell bodies and arrowheads projections. (B and C) photoreceptors, should express a suitable G alpha protein for pho- Notopodial cell and its projection along nerves II (II) and I (I) into a longitudinal totransduction. Therefore, we cloned the ortholog of Gq, a specific fiber (lof) in the ventral nerve cord; t demarcates turning point away from nerve II; nervous system highlighted by antiacetylated tubulin staining (red). (D)Mid- G alpha subunit commonly associated with r-Opsin-based PRCs and ventral cell in the ventral nerve cord. (A and D) Ventral views, anterior to the required for signal transduction (11). Two-color in situ hybridization left; (A′ and B) frontal views dorsal to the top; (C) ventral view, anterior to the with riboprobes against r-opsin and gq revealed a perfect coex- top. np, notopodial cell; mv, midventral cell; vl, ventrolateral cell. pression of both genes, both in the ventral trunk and the notopodia

Backﬁsch et al. PNAS | January 2, 2013 | vol. 110 | no. 1 | 195 Downloaded by guest on October 5, 2021 Fig. 3. The Platynereis trunk shows an autonomous photoavoidance response. (A, C, and D) Schematic outline of light avoidance assay. Decapitated premature worms adapted to dim light were either left in the same conditions (dark) or received a bright light stimulus (light) close to their tail tip (area circled in C). Thirty seconds after starting the experiment, the distance (d) that the illuminated segments had moved was determined. (B) Results of measurements on 20 animals. ****P < 0.0001 in a two-tailed paired t test. Error bars indicate SEM. (Scale bar in C and D, 1 mm.)

(Fig. 4 A and I and Table S1). This is consistent with these cells being pax6, around 80% of the r-opsin–positive cells of developing functional PRCs. parapodia are found directly adjacent to this domain (Fig. 4F Next, we investigated if these cells also expressed the transcrip- and Fig. S4C). This close correlation, as well as the faint staining tional regulator pax6. In contrast to the frontolateral eyelets (Fig. of the emerging PRCs with the pax2/5/8 riboprobe (Fig. 4F and S3), pax6 appears to be absent from notopodial PRCs, and only Fig. S4C), let us suggest that the r-opsin cells are differentiating demarcates few cells in the ventral adult trunk, typically close to the outofapax2/5/8-positive territory. parapodial bases, that showed no significant overlap with r-opsin Brn3 is a POU homeobox transcription factor involved in sen- (Fig. 4B and Table S1). To assess if pax6 might be transiently sory neuron development. Similar to pax6, Platynereis brn3 (Fig. expressed during development of these PRCs, we turned toward the S4C) is expressed in—segmentally clustered—longitudinal col- analysis of 8-d-old posterior tail regenerates that have been estab- umns along the regenerated VNC (Fig. S4D), as well as in distinct lished as a highly suitable system for studying gene expression during cells in the developing parapodia (Fig. 4G). When we first observe thetimecourseofsegmentformationandprovideaccesstopara- r-opsin–positive cells in young parapodia, they are typically ac- podia of different developmental stages of development (31). In line companied by brn3-positive cells (Fig. 4G, Right). A tight coupling with the spatial arrangement observed during larval development between brn3- and r-opsin–positive cells is also observed for the (20), pax6 is expressed in longitudinal columns along the regenerated adult parapodia (Fig. 4H and Table S1). Together, this supports ventral nervous system (Fig. S4A). However, we do not detect the notion that these two cells share a common developmental coexpression with r-opsin in the ventral trunk (Fig. 4D). Moreover, precursor and/or are functionally connected. although we observe a highly stereotypical expression of pax6 in the Although these coexpression data still await to be extended to basal–posterior aspect of each developing notopod, r-opsin is other genes, they already provide insight into the “molecular fin- detected in anterior–distal cells in the same specimens, clearly dis- gerprint” (34) of noncephalic PRCs. At least for the parapodial tinct from pax6 (Fig. 4E, Fig. S4A,andTable S1). This establishes PRCs, whose stereotypic occurrence facilitates analyses about that even during their early development, noncephalic PRCs do not spatiotemporal expression similarities, we conclude that r-opsin express pax6. cells differentiate from a territory expressing pax2/5/8, but not pax6, and that they are tightly linked with the expression of dach Correlation of Noncephalic PRCs with dach, pax2/5/8, and brn3 and brn3. Gq is clearly coexpressed in all noncephalic PRCs. Expression. Next, we assessed if there are other transcription factors correlating with the development of noncephalic r-opsin Zebrafish r-opsin Orthologs Are Expressed Outside the Brain in cells in the regenerates. Dach was previously only reported for Mechanoreceptors of the Lateral Line. The tight association of Pla- its expression in Platynereis mushroom bodies (32), but in a re- tynereis r-opsin cells with brn3 is reminiscent of the zebrafish, where lated polychaete, Neanthes arenaceodentata,italsoprominently a brn3c-driven egfp transgene is expressed in the retinal ganglion labels cells in the VNC and at the base of each parapodium (33). cells (35), a cell type known to express functional zebrafish r-opsin In adult Platynereis specimens, dach is weakly detectable in orthologs, including opn4x-2 and opn4m-2 (36). However, brn3c dispersed cells of the ventral trunk. In the regenerates, dach has also specifically demarcates the neuromasts, mechanosensory cells a more prominent, complex expression pattern comprising cells of the lateral line, neurons of the peripheral nervous system (35). in the VNC and parapodia (Fig. S4B). In the ventral trunk, Given the specificity of this expression, we investigated if there amajorityofr-opsin cells coexpress dach (Table S1). In the were further similarities between the neuromasts and retinal gan- parapodia, in close to 90% of the analyzed cases, an r-opsin cell glion cells. Indeed, riboprobes against opn4x-2 and opn4m-2 both is immediately neighbored by a basally adjacent dach-positive show distinct expression of both genes in the neuromasts (Fig. 4 J–L), cell (Fig. S4B). establishing that r-opsin orthologs also occur in noncephalic sen- Platynereis Pax2/5/8 is an ortholog of Drosophila Sv/Spa and sory cells in vertebrates. vertebrate Pax2, Pax5, and Pax8 proteins. Pax2/5/8 transcripts are specifically detected in the epithelial cell layer of regener- Molecular Similarities Between Noncephalic Photoreceptors. Our ating segments (Fig. S4C). In contrast to the case observed for observations on the presence and molecular characteristics of

196 | www.pnas.org/cgi/doi/10.1073/pnas.1209657109 Backfisch et al. Downloaded by guest on October 5, 2021 varying numbers of segmental visual organs are observed in both Opheliid and Sabellid representatives (e.g., refs. 1, 37). Second, we note various molecular commonalities that link noncephalic PRCs in Platynereis and cephalochordates. These comprise the expression of orthologous Opsins and G alpha subunits (this study and ref. 13), as well as the absence of Pax6 (this study and ref. 17). Instead of Pax6, Pax2/5/8 correlates with the early phases of PRC development in both systems (this study and ref. 38). Likewise, the regulator dach associates with differentiated noncephalic PRCs (this study and refs. 38, 39). As illustrated in Fig. S5, these data are compatible with the notion that noncephalic PRCs are specified by a regulatory system that is related to, but distinct from the Pax6- dependent Pax-Six-Eya-Dach regulatory network (PESDN) used in the specification of cephalic eyes in a broad panel of species (40–42). Following the homology concept of Remane (43), the position along the midline of the trunk central nervous system is equivalent between Platynereis and amphioxus (Fig. S5A), suggesting that these similarities might even reflect common ancestry. It remains to be seen if further research into noncephalic PRCs will support this hypothesis, or will rather argue for independent cooption of an existing regulatory network in two lineages. Likewise, the notion of a parallel, Pax6-idependent PESDN could also be useful to assess the origin of Pax6-independent cephalic eyes, as they occur in Pla- tynereis (18), Limulus (44), or planarians (45).

Opsins in Mechanoreceptors: Ancient Feature? Our discovery that two orthologous opsins are specifically expressed in the teleost peripheral nervous system indicates that noncephalic PRCs are even more widespread than anticipated. Whereas a detailed molecular characterization of these cells is still lacking, it is notable that the lateral line primordium does also not depend on pax6,butisspecified by orthologs of the pax2/5/8 gene (reviewed in ref. 46). In contrast to the trunk central nervous system, the lateral line has no obvious equivalent in protostomes, precluding direct comparisons between cell types. However, the detection of the two r-opsin orthologs in a mechanosensory organ is reminiscent of a recent report on the requirement of two r-Opsin orthologs, Rh5 and Rh6,inthe mechanoreceptors of the fly’s auditory sense, the Johnston’sorgan (47),atissueknowntodependonafly pax2/5/8 ortholog (48). The co-occurrence of mechanosensory and photosensory molecules in Fig. 4. Molecular specification of Platynereis noncephalic photoreceptors the same cell may reflect ancient conditions in a multimodal, “pro- and presence of r-opsin orthologs in the zebrafish neuromasts. (A–I) Code- tosensory” cell, or light-independent functions of Opsins (47, 49–51) tection of Platynereis r-opsin (red arrowheads) and indicated transcripts (Fig. S5). Our discovery of opn4x-2 and opn4m-2 in the neuromast, (purple arrowheads) by double in situ hybridization. Coexpression is in- and the fact that the respective proteins are known to be functional – – dicated by black arrows. (A C) Expression in the adult ventral trunk. (D G) PRCs (36), suggests that the fish neuromasts will be an excellent Expression in 8-d tail regenerates (trunk and parapodia). (H and I) Expression in adult parapodia. (J–L) Expression of opn4x-2 and opn4m-2 in the zebrafish system to further test this hypothesis and address Opsin function in neuromast. (J) Live image of neuromast cells (green arrowheads) in the mechanosensory cells also in a deuterostome model species. brn3c::egfp strain 6 d after fertilization; (K and L) corresponding labeling of neuromast cells (purple arrowheads) with the opn4x-2 and opn4m-2 ribop- Perspective for Platynereis Transgenesis. Platynereis has emerged as robes. (A–G) Ventral views, anterior to the left; (H and I) frontal views, dorsal a prominent reference species for the annelid taxon and the large to the top. (J–L) Lateral views, anterior to the left. See text for details. superphylum of lophotrochozoans. Our work opens up this model for research on two levels: First, stable transgenic strains provide an entry point into the in-depth characterization of the labeled cell types, for noncephalic PRCs impact on our understanding of photorecep- instance of their transcriptomes, or the growth of their projections in tor evolution on two time scales. First, the observed noncephalic normal development and upon regeneration. Secondly, targeted receptors can be compared with visual organs present in other ablation of labeled cells from stable transgenic carriers will critically species within the same groups. For instance, the unpigmented extend the functional toolkit for Platynereis that has been restricted to midventral PRCs of Platynereis resemble pigment-associated chemical interference and laser-assisted ablation of morphologically BIOLOGY PRCs of the midventral eyespots of Eunicids (1). The segmental tractable cells (21). Such assays will allow assessment of the re- DEVELOPMENTAL visual organs of the Opheliid Polyophthalmus qingdaoensis lo- quirement of the identified PRCs for photoavoidance, hormonal calize anteriorly to the parapodia, ventral to the lateral grooves activity, or the entrainment of endogenous oscillators (52, 53). Stable (3). As we observe ventrolateral PRCs at the parapodial bases of transgenesis in Platynereis therefore helps to address open biological Platynereis, it is likely that the PRCs in the Platynereis ventral questions in both developmental and evolutionary biology, as well as trunk are correlates of the more regularly distributed segmental chronobiology. visual organs of Opheliids or Sabellids. Such regular, segmental sets of PRCs may even represent the evolutionary ground state Methods that could have been secondarily reduced or concentrated into Generation of Transgenic Strain. A translational fusion of the endogenous r- specific segments. This scenario is compatible with the fact that opsin coding sequence and egfp was generated by BAC recombineering. The

Backfisch et al. PNAS | January 2, 2013 | vol. 110 | no. 1 | 197 Downloaded by guest on October 5, 2021 construct was subsequently amplified and introduced into the I-SceI sites of ACKNOWLEDGMENTS. We thank all members of the K.T.-R and F.R. labora- amodified Mos1-transposon vector. The resulting plasmid was introduced tories for stimulating discussions and help with worm cultures, Detlev Arendt and Graham Warren for continuous support, the Max F. Perutz Laboratories along with synthetic mos1 mRNA into Platynereis zygotes by microinjection to animal care for maintenance of all Platynereis animals used for microinjection generate carriers (SI Methods). Animal research and transgenic work followed experiments in this study, Charlotte Nikbakht and Filip Nikic (University of applicable legislation, as also approved by the MFPL committee for biological Vienna) for outstanding librarian services, Ina Arnone (Stazione Zoologica, safety (session on Feb 19, 2009). Naples) for providing access to original literature, Guido Krieten (Alfred Wegener Institute, Bremerhaven) for supply of sea water, Mihail Sarov (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden) and Probes, Stainings, and Imaging. For gene references, gene orthology, and Ernst Wimmer (University of Göttingen) for sharing recombineering reagents fi cloning, see SI Methods. Whole-mount in situ hybridizations of zebra sh and and the pMos plasmid modified in this study, and two anonymous referees Platynereis larvae as well as immunohistochemistry were performed for constructive criticism. Our work was supported by funds from the Max F. according to established protocols. For adult worms, a robust two-color Perutz Laboratories/University of Vienna (to F.R. and K.T.-R.), the Austrian detection protocol was established (details in SI Methods). For imaging of Science Fund (FWF): AY0041321, and Human Frontier Science Program (HFSP) Research Grant RGY0082/2010 (to K.T.-R.). The research leading to these live animals, Platynereis juvenile or adult worms were paralyzed by adding results has received funding from the European Research Council under the dropwise 1 M MgCl2 into sea water until no movement of the animals oc- European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC curred. Details on microscopy are specified in SI Methods. Grant Agreement 260304 (to F.R.).

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