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Proc Natl Acad Sci U S A. Jan 2, 2013; 110(1): 193–198.
Published online Dec 17, 2012. doi:  10.1073/pnas.1209657109
PMCID: PMC3538230
Developmental Biology

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

Abstract

Research in eye evolution has mostly focused on eyes residing in the head. In contrast, noncephalic light sensors are far less understood and rather regarded as evolutionary innovations. We established stable transgenesis in the annelid Platynereis, a reference species for evolutionary and developmental comparisons. EGFP controlled by cis-regulatory elements of r-opsin, a characteristic marker for rhabdomeric photoreceptors, faithfully recapitulates known r-opsin expression in the adult eyes, and marks a pair of pigment-associated frontolateral eyelets in the brain. Unexpectedly, transgenic animals revealed an additional series of photoreceptors in the ventral nerve cord as well as photoreceptors that are located in each pair of the segmental dorsal appendages (notopodia) and project into the ventral nerve cord. Consistent with a photosensory function of these noncephalic cells, decapitated animals display a clear photoavoidance response. Molecular analysis of the receptors suggests that they differentiate independent of pax6, a gene involved in early eye development of many metazoans, and that the ventral cells may share origins with the Hesse organs in the amphioxus neural tube. Finally, expression analysis of opn4×-2 and opn4m-2, two zebrafish orthologs of Platynereis r-opsin, reveals that these genes share expression in the neuromasts, known mechanoreceptors of the lateral line peripheral nervous system. Together, this establishes that noncephalic photoreceptors are more widespread than assumed, and may even reflect more ancient aspects of sensory systems. Our study marks significant advance for the understanding of photoreceptor cell (PRC) evolution and development and for Platynereis as a functional lophotrochozoan model system.

Keywords: regulation, transposon, polychaete, worm

Our view on animal photoreception is dominated by the analysis of pigmented cephalic eyes, which are prominent in most branches of animal evolution (1). However, eyes and other photoreceptive cells are also present outside the brains of animals. Among bilaterians, examples include the early Cambrian Lobopodian fossil Microdictyon sinicum, a probable ancestor of extant arthropods, that displays segmentally arranged compound eyes above each of its leg pairs (2). Similarly, Opheliid worms of the genus Polyophthalmus, or the Sabellid polychaete Branchiomma 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 (1, 4). Drosophila was recently demonstrated to possess photoreceptive cells in the larval body wall that mediate a photoavoidance response (5). Sea urchins possess photoreceptor cells (PRCs) in their tube feet (6). Finally, the basal chordate amphioxus displays a series of prototypical visual organs (each consisting of a PRC with an associated pigment cell) that are located along the ventral neural tube and are commonly referred to as organs of Hesse or pigmented ocelli (7). The existence of noncephalic visual organs observed in these and other taxa could represent independent evolutionary events, e.g., regulatory mutations affecting genetic master switches like the homeodomain protein Pax6, which can cause the development of ectopic eyes when misexpressed in Drosophila (8). Alternatively, these segmental organs could reflect ancient sites of photoreception that predated the cephalization of body plans, and may, where present, still retain functions that complement cephalic PRCs (9).

The discovery of noncephalic photoreceptive organs is facilitated by the presence of associated pigment spots. Unpigmented PRCs might therefore be abundant, but are less likely to be discovered by morphological or ultrastructural techniques (10). In contrast, molecular markers should be able to detect such unpigmented photoreceptive structures, and thereby provide insight into their abundance and localization. Together with covalently bound retinal, Opsin-type G protein-coupled receptors serve as main light sensors in animal photoreceptors. Rhabdomeric Opsins (r-Opsins) are an ancient group of Opsins particularly widespread among invertebrates, typically expressed in larval PRCs and/or cephalic eyes (reviewed in ref. 11). Notable exceptions are the expression of r-opsin orthologs in sea urchin tube feet (6, 12) and the photoreceptors of the Hesse and Joseph cells (13) whose function remains enigmatic.

The paired-homeodomain transcription factor Pax6 is a key gene for the development of cephalic eyes in both Drosophila and chordates (reviewed in ref. 11, also see ref. 14), even though the question of whether Pax6 has an ancient or recent role in direct regulation of opsin genes remains debated (15, 16). There are also, however, pax6-independent PRCs. For instance, Hesse cells were found void of amphioxus pax6 (17). The fact that Hesse cells do not express pax6 has been interpreted as evidence for a recent origin of these noncephalic PRCs (17). However, in the absence of molecular data for a larger spectrum of noncephalic PRCs, it remains unclear if the absence of pax6 from these cells might, alternatively, represent a common feature of noncephalic PRCs.

Here, we report the establishment and analysis of a stable r-opsin::egfp strain that marks rhabdomeric PRCs throughout the lifetime of the marine annelid Platynereis dumerilii, a key reference species for eye and brain development and evolution (11, 1822). The strain highlights adult eyes and their projections, as well as a set of eyelets in the frontolateral adult brain. Moreover, we discover a series of pax6-negative noncephalic PRCs in the dorsal appendages (notopods) as well as the ventral trunk that allow comparisons with the noncephalic visual organs of other polychaetes. By their position in the medial posterior neuraxis and by gene expression, the ventral 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 bacterial 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 Platynereis 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 AC). 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 AC).

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 animals (Fig. 1B). Notably, fluorescence was not only detected in the cell bodies, but was also well observable in the neuronal projections connecting the adult eyes toward the medial brain (Fig. 1 A and B). This attests to the suitability of transgenic Platynereis strains for detailed cell-morphological analyses. The robust labeling of adult eye PRCs and their projections represents a major advance over previous attempts to track the projections of the PRCs (25).

Fig. 1.
Labeling of cephalic rhabdomeric photoreceptor cells (PRCs) by the stable pMos{rops::egfp}vbci2 strain. (A) At 14 d of development, EGFP demarcates the adult eye photoreceptors (asterisks) and their neuronal projections (green arrowheads) as well as a ...

Labeling of Frontolateral Eyelets in the Anterior Brain.

The persistent expression of EGFP in the adult eye PRCs extends the previously described expression of r-opsin in the emerging adult eyes (18) to all postlarval development. In addition, a careful analysis of postlarval r-opsin::egfpvbci2 animals revealed additional sites of EGFP fluorescence. On the one hand, we detected bilateral pairs of cells anterior to the adult eyes (Fig. 1A). These anterior EGFP-positive cells are accompanied by pigment cells, indicating that they are able to sense directional light (Fig. 1 C and D). We refer to this assembly of PRCs and pigment cells as frontolateral eyelets. From previous 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 development (25). We observe, however, that the PRCs of the frontolateral eyelets express pax6 (Fig. S3), reminiscent of the previously characterized larval PRCs (18). We also detected EGFP or r-opsin in the same region in 40-segmented worms, indicating that frontolateral eyelets persist until premature adult stages. In sexually mature animals, neither EGFP fluorescence nor r-opsin expression is detectable in the anterior head, indicating that the frontolateral eyelets either change their photopigments or become dispensable upon maturation.

Identification of Noncephalic Photoreceptors in the Notopods and the Ventral Trunk.

When investigating juveniles and adult worms, we found that r-opsin::egfpvbci2 animals showed additional EGFP fluorescence outside the brain. On the one hand, we detected a very regular pattern of PRCs located in the worms’ appendages. The cell bodies are located in the ventral lobe (“upper lip”) of the dorsal parapodia (notopods), in a region free of visible pigments (Fig. 2 AC, “np”). These cells are already present when we investigate young adults (seven to eight segments), but exclude the first two pairs of appendages that are known to correspond to the last two pairs of larval parapodia (27). We therefore conclude that they are restricted to postlarval segments. Analysis of fixed animals stained with an anti-EGFP serum, and counterstained with antiacetylated tubulin allowed us to determine the highly stereotypical projection pattern of these notopodial cells. First, they project toward the base of the parapodia (Fig. 2B). From there, they follow the anterior branch of segmental nerve II into a more posterior direction, before turning more anteriorly, and joining nerve I (Fig. 2C). Inside the ventral nerve cord (VNC), the projections from each side of the worm follow a distinct longitudinal fiber (Fig. 2 C and D). As we do not observe commissures between these fibers, we assume that the projections remain ipsilateral and hence might transmit information on lateral light conditions even to adjacent segments, possibly influencing segmental motor microcircuits (28).

Fig. 2.
The pMos{rops::egfp}vbci2 strain reveals a complex set of unpigmented noncephalic photoreceptors. (A and A′) Schematic drawing of four segments of the ventral Platynereis trunk (A) and a view onto a parapodium (A′). Rectangles and labels ...

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 trajectories 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 survive 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 triggers 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).

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 ...

Noncephalic Photoreceptors Possess a Gq-Type Alpha Subunit and Develop Independent of pax6.

To extend our insight into the molecular characteristics of the newly identified PRCs, we next investigated if r-opsin–positive cells coexpressed other molecular markers. For this, we first established a robust double in situ hybridization protocol that allows reliable codetection of two riboprobes in adults, a stage that is rather problematic for the existing fluorescent detection method (30).

We hypothesized that r-opsin–positive cells, if functioning as photoreceptors, should express a suitable G alpha protein for phototransduction. Therefore, we cloned the ortholog of Gq, a specific G alpha subunit commonly associated with r-Opsin-based PRCs and required for signal transduction (11). Two-color in situ hybridization with riboprobes against r-opsin and gq revealed a perfect coexpression of both genes, both in the ventral trunk and the notopodia (Fig. 4 A and I and Table S1). This is consistent with these cells being functional PRCs.

Fig. 4.
Molecular specification of Platynereis noncephalic photoreceptors and presence of r-opsin orthologs in the zebrafish neuromasts. (AI) Codetection of Platynereis r-opsin (red arrowheads) and indicated transcripts (purple arrowheads) by double ...

Next, we investigated if these cells also expressed the transcriptional regulator pax6. In contrast to the frontolateral eyelets (Fig. S3), pax6 appears to be absent from notopodial PRCs, and only demarcates few cells in the ventral adult trunk, typically close to the parapodial bases, that showed no significant overlap with r-opsin (Fig. 4B and Table S1). To assess if pax6 might be transiently expressed during development of these PRCs, we turned toward the analysis of 8-d-old posterior tail regenerates that have been established as a highly suitable system for studying gene expression during the time course of segment formation and provide access to parapodia of different developmental stages of development (31). In line with the spatial arrangement observed during larval development (20), pax6 is expressed in longitudinal columns along the regenerated ventral nervous system (Fig. S4A). However, we do not detect coexpression with r-opsin in the ventral trunk (Fig. 4D). Moreover, although we observe a highly stereotypical expression of pax6 in the basal–posterior aspect of each developing notopod, r-opsin is detected in anterior–distal cells in the same specimens, clearly distinct from pax6 (Fig. 4E, Fig. S4A, and Table S1). This establishes that even during their early development, noncephalic PRCs do not express pax6.

Correlation of Noncephalic PRCs with dach, pax2/5/8, and brn3 Expression.

Next, we assessed if there are other transcription factors correlating with the development of noncephalic r-opsin cells in the regenerates. Dach was previously only reported for its expression in Platynereis mushroom bodies (32), but in a related polychaete, Neanthes arenaceodentata, it also prominently labels cells in the VNC and at the base of each parapodium (33). In adult Platynereis specimens, dach is weakly detectable in dispersed cells of the ventral trunk. In the regenerates, dach has a more prominent, complex expression pattern comprising cells in the VNC and parapodia (Fig. S4B). In the ventral trunk, a majority of r-opsin cells coexpress dach (Table S1). In the parapodia, in close to 90% of the analyzed cases, an r-opsin cell is immediately neighbored by a basally adjacent dach-positive cell (Fig. S4B).

Platynereis Pax2/5/8 is an ortholog of Drosophila Sv/Spa and vertebrate Pax2, Pax5, and Pax8 proteins. Pax2/5/8 transcripts are specifically detected in the epithelial cell layer of regenerating segments (Fig. S4C). In contrast to the case observed for pax6, around 80% of the r-opsin–positive cells of developing parapodia are found directly adjacent to this domain (Fig. 4F and Fig. S4C). This close correlation, as well as the faint staining of the emerging PRCs with the pax2/5/8 riboprobe (Fig. 4F and Fig. S4C), let us suggest that the r-opsin cells are differentiating out of a pax2/5/8-positive territory.

Brn3 is a POU homeobox transcription factor involved in sensory neuron development. Similar to pax6, Platynereis brn3 (Fig. S4C) is expressed in—segmentally clustered—longitudinal columns along the regenerated VNC (Fig. S4D), as well as in distinct cells in the developing parapodia (Fig. 4G). When we first observe r-opsin–positive cells in young parapodia, they are typically accompanied by brn3-positive cells (Fig. 4G, Right). A tight coupling between brn3- and r-opsin–positive cells is also observed for the adult parapodia (Fig. 4H and Table S1). Together, this supports the notion that these two cells share a common developmental precursor and/or are functionally connected.

Although these coexpression data still await to be extended to other genes, they already provide insight into the “molecular fingerprint” (34) of noncephalic PRCs. At least for the parapodial PRCs, whose stereotypic occurrence facilitates analyses about spatiotemporal expression similarities, we conclude that r-opsin cells differentiate from a territory expressing pax2/5/8, but not pax6, and that they are tightly linked with the expression of dach and brn3. Gq is clearly coexpressed in all noncephalic PRCs.

Zebrafish r-opsin Orthologs Are Expressed Outside the Brain in Mechanoreceptors of the Lateral Line.

The tight association of Platynereis r-opsin cells with brn3 is reminiscent of the zebrafish, where a brn3c-driven egfp transgene is expressed in the retinal ganglion cells (35), a cell type known to express functional zebrafish r-opsin orthologs, including opn4x-2 and opn4m-2 (36). However, brn3c also specifically demarcates the neuromasts, mechanosensory cells of the lateral line, neurons of the peripheral nervous system (35). Given the specificity of this expression, we investigated if there were further similarities between the neuromasts and retinal ganglion cells. Indeed, riboprobes against opn4x-2 and opn4m-2 both show distinct expression of both genes in the neuromasts (Fig. 4 J–L), establishing that r-opsin orthologs also occur in noncephalic sensory cells in vertebrates.

Molecular Similarities Between Noncephalic Photoreceptors.

Our observations on the presence and molecular characteristics of noncephalic PRCs impact on our understanding of photoreceptor evolution on two time scales. First, the observed noncephalic receptors can be compared with visual organs present in other species within the same groups. For instance, the unpigmented midventral PRCs of Platynereis resemble pigment-associated PRCs of the midventral eyespots of Eunicids (1). The segmental visual organs of the Opheliid Polyophthalmus qingdaoensis localize anteriorly to the parapodia, ventral to the lateral grooves (3). As we observe ventrolateral PRCs at the parapodial bases of Platynereis, it is likely that the PRCs in the Platynereis ventral trunk are correlates of the more regularly distributed segmental visual organs of Opheliids or Sabellids. Such regular, segmental sets of PRCs may even represent the evolutionary ground state that could have been secondarily reduced or concentrated into specific segments. This scenario is compatible with the fact that 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 (4042). 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 Platynereis (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, but is specified 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, in the mechanoreceptors of the fly’s auditory sense, the Johnston’s organ (47), a tissue known to depend on a fly pax2/5/8 ortholog (48). The co-occurrence of mechanosensory and photosensory molecules in the same cell may reflect ancient conditions in a multimodal, “protosensory” cell, or light-independent functions of Opsins (47, 4951) (Fig. S5). Our discovery of opn4x-2 and opn4m-2 in the neuromast, and the fact that the respective proteins are known to be functional PRCs (36), suggests that the fish neuromasts will be an excellent system to further test this hypothesis and address Opsin function in mechanosensory cells also in a deuterostome model species.

Perspective for Platynereis Transgenesis.

Platynereis has emerged as a prominent reference species for the annelid taxon and the large 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 instance of their transcriptomes, or the growth of their projections in normal development and upon regeneration. Secondly, targeted ablation of labeled cells from stable transgenic carriers will critically extend the functional toolkit for Platynereis that has been restricted to chemical interference and laser-assisted ablation of morphologically tractable cells (21). Such assays will allow assessment of the requirement of the identified PRCs for photoavoidance, hormonal activity, or the entrainment of endogenous oscillators (52, 53). Stable transgenesis in Platynereis therefore helps to address open biological questions in both developmental and evolutionary biology, as well as chronobiology.

Methods

Generation of Transgenic Strain.

A translational fusion of the endogenous r-opsin coding sequence and egfp was generated by BAC recombineering. The construct was subsequently amplified and introduced into the I-SceI sites of a modified Mos1-transposon vector. The resulting plasmid was introduced along with synthetic mos1 mRNA into Platynereis zygotes by microinjection to generate carriers (SI Methods). Animal research and transgenic work followed applicable legislation, as also approved by the MFPL committee for biological safety (session on Feb 19, 2009).

Probes, Stainings, and Imaging.

For gene references, gene orthology, and cloning, see SI Methods. Whole-mount in situ hybridizations of zebrafish and Platynereis larvae as well as immunohistochemistry were performed according to established protocols. For adult worms, a robust two-color detection protocol was established (details in SI Methods). For imaging of live animals, Platynereis juvenile or adult worms were paralyzed by adding dropwise 1 M MgCl2 into sea water until no movement of the animals occurred. Details on microscopy are specified in SI Methods.

Supplementary Material

Supporting Information:

Acknowledgments

We thank all members of the K.T.-R and F.R. laboratories for stimulating discussions and help with worm cultures, Detlev Arendt and Graham Warren for continuous support, the Max F. Perutz Laboratories animal care for maintenance of all Platynereis animals used for microinjection experiments in this study, Charlotte Nikbakht and Filip Nikic (University of Vienna) for outstanding librarian services, Ina Arnone (Stazione Zoologica, 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 Ernst Wimmer (University of Göttingen) for sharing recombineering reagents and the pMos plasmid modified in this study, and two anonymous referees for constructive criticism. Our work was supported by funds from the Max F. Perutz Laboratories/University of Vienna (to F.R. and K.T.-R.), the Austrian Science Fund (FWF): AY0041321, and Human Frontier Science Program (HFSP) Research Grant RGY0082/2010 (to K.T.-R.). The research leading to these results has received funding from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement 260304 (to F.R.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. KC109635, KC109636, and KC109637).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1209657109/-/DCSupplemental.

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