Owenia fusiformis – a basally branching annelid suitable for studying ancestral features of annelid neural development
© The Author(s). 2016
Received: 23 November 2015
Accepted: 23 May 2016
Published: 16 June 2016
Comparative investigations on bilaterian neurogenesis shed light on conserved developmental mechanisms across taxa. With respect to annelids, most studies focus on taxa deeply nested within the annelid tree, while investigations on early branching groups are almost lacking. According to recent phylogenomic data on annelid evolution Oweniidae represent one of the basally branching annelid clades. Oweniids are thought to exhibit several plesiomorphic characters, but are scarcely studied - a fact that might be caused by the unique morphology and unusual metamorphosis of the mitraria larva, which seems to be hardly comparable to other annelid larva. In our study, we compare the development of oweniid neuroarchitecture with that of other annelids aimed to figure out whether oweniids may represent suitable study subjects to unravel ancestral patterns of annelid neural development. Our study provides the first data on nervous system development in basally branching annelids.
Based on histology, electron microscopy and immunohistochemical investigations we show that development and metamorphosis of the mitraria larva has many parallels to other annelids irrespective of the drastic changes in body shape during metamorphosis. Such significant changes ensuing metamorphosis are mainly from diminution of a huge larval blastocoel and not from major restructuring of body organization. The larval nervous system features a prominent apical organ formed by flask-shaped perikarya and circumesophageal connectives that interconnect the apical and trunk nervous systems, in addition to serially arranged clusters of perikarya showing 5-HT-LIR in the ventral nerve cord, and lateral nerves. Both 5-HT-LIR and FMRFamide-LIR are present in a distinct nerve ring underlying the equatorial ciliary band. The connections arising from these cells innervate the circumesophageal connectives as well as the larval brain via dorsal and ventral neurites. Notably, no distinct somata with 5-HT -LIR in the apical organ are detectable in the larval stages of Owenia.
Most of the larval neural elements including parts of the apical organ are preserved during metamorphosis and contribute to the juvenile nervous system.
Our studies in Owenia fusiformis strongly support that early branching annelids are comparable to other annelids with regard to larval neuroanatomy and formation of the juvenile nervous system. Therefore, Owenia fusiformis turns out to be a valuable study subject for comparative investigations and unravelling ancestral processes in neural development in Annelida and Bilateria in general.
The structure and formation of metazoan nervous systems are topics with long scientific histories, and that relate to basic questions of animal evolution and development. Numerous recent studies have broadened the knowledge on neuroanatomical plasticity and neural patterning mechanisms in several invertebrate groups [1–5]. A major focus of these studies has been to resolve the origin of bilaterian brain structures and trunk nerve cords, and to clarify whether they evolved out of orthogon-like versus net-like nervous systems [4, 6, 7]. In many of these studies annelids have been an important group as they have provided deep insights into the molecular patterning of nervous system development [2, 8, 9], neural circuitry  as well as molecular characteristics and functions of specific sensory cells or brain areas in protostomes other than arthropods or nematodes [11, 12]. This has had a significant impact on the general understanding of animal nervous system evolution and function. However, phylogenomic analyses of annelids indicate that thoroughly investigated species such as Platynereis dumerilii and Capitella teleta are deeply nested at different positions within the annelid tree [13–16], which coincide with different sets of traits relating to the molecular control of neural development. Further, neuroanatomy and the course of neurogenesis have been shown to be variable in annelids. Based on comparative histological and immunohistochemical studies special attention was given to brain complexity and organization, trunk nervous system architecture and centralization, sensory systems and direction of differentiation and maintenance of larval neuronal elements, in the adults [9, 17–31]. Comparative data on nervous system development of the basal-most branching annelid taxa are scarce and are purely based on old histological investigations [32, 33]. Thus, generation of conclusive data is important to provide a basis for studying the organization and developmental patterning of the ancestral annelid nervous system as well as the emergence of nervous system variety and complexity within the group. Oweniids, which occupy the basal-most branch of the annelid tree in recent analyses [14–16] are known to exhibit characters often considered to represent an ancestral condition. Some of these characters are monociliated epidermal cells [34, 35], nephridia similar to those of deuterostomes , and a rather simple organized intraepithelial nervous system [37, 38]. Furthermore, certain oweniid species occur in high abundance in the intertidal, and high quantities of larvae can easily be cultured in the lab - an attractive feature for subjects of molecular and developmental studies. However, oweniids have an enigmatic type of larva - the mitraria, which, in contrast to other annelids, undergoes a rather catastrophic metamorphosis [32, 39]. In this study we generated immunohistochemical and histological data to analyze the neuroanatomy of the oweniid Owenia fusiformis Delle Chiaje, 1844 from early larva through metamorphosis until the juvenile stage. Our main focus was on whether the larval nervous system is comparable to that of other annelids and whether the main parts of the central nervous system are maintained throughout metamorphosis.
Using this approach we aim to elucidate the oweniid neural development, discuss the ontogeny of adult neuronal precursors, and shed light on the metamorphosis of the remarkable mitraria. Our study points out the potential of Owenia fusiformis, one of the basal-most annelid groups, to serve as a valuable model for studying the development, ancestral features, and evolution of the annelid nervous system.
Results and discussion
General development of the mitraria
Reared at 18 °C, the larvae start swimming approximately 24 h after fertilization. At this stage a circumferential band of cilia, the chaetal sacs containing two pairs of short chaetae, an early apical tuft, a mouth and an anus are well developed. Within 48 h post fertilization, the larval episphere is enlarged, the chaetae are elongated, and the larvae start to feed. In the next 3–7 days the characteristic bell-shaped mitraria is almost fully developed featuring the well-developed chaetal sacs with numerous chaetae, a dense ciliated band running around the whole equatorial midline, a through gut, with discernable mouth, esophagus, midgut, hindgut and anus, and a well-developed apical tuft. Solely the eyespots are still missing at this time and appear approximately at 14 dpf (days post fertilization). Posterior of the circumferential ciliary band, the larval hyposphere elongates and forms a worm-shaped appendage. Furthermore, the esophagus and intestine become well developed, and the mid- and hindgut tissue drastically increase in length. At 21 dpf larvae start to metamorphose. This stage is characterized by collapse and oral assimilation of the bell-shaped anterior part of the larva and simultaneous posterior extension of the trunk. Subsequently the chaetae fall off and the chaetal sacs invaginate. Although the body shape changes drastically, the whole metamorphosis can end in just a few minutes.
Annelids in general show a variety of larval types and developmental modes , however, oweniid development seems to stand out due to the conspicuous bell-like shape of the larva as well as due to the abrupt and drastic changes in body shape during metamorphosis. In order to better understand the changes in body architecture during metamorphosis of O. fusiformis, we firstly investigated the internal anatomy of the free swimming mitraria by serial semi-thin sections, followed by 3D reconstruction and transmission electron microscopy (TEM).
Relative to Owenia, clear differences are apparent with respect to the development of certain nemertines, which feature a so-called pilidium larva - the only kind of lophotrochozoan larva that superficially resembles the oweniid mitraria - due to a likewise bell-shaped envelope. Notably, nemertines represent a taxon with the potential of being the annelid sister group [14–16]. In contrast to other nemertines and also to oweniids, body organization of nemertines with a pilidium changes drastically during metamorphosis. In pilidium larvae, the whole anlage of the juvenile forms as a distinct part of the larva. During metamorphosis the juvenile anlage erupts from and subsequently devours the entire remaining larval tissue. Nothing similar is observable in Owenia. The main part of the larval tissue gives rise to the juvenile and only part of the epidermis invaginates into the mouth. Although both the oweniid mitraria and the nemertean represent large bell-shaped swimming stages, which differ in shape considerably from the juveniles, the pillidium and the oweniid mitraria have to be regarded as convergently evolved spiralian larval types [41–43].
Development of early brain precursors and the anterior stomatogastric nervous system
Development of the early mitraria (1 dpf- 7 dpf)
Development of the late mitraria (14 dpf and older)
In later stages (14 dpf) the intensity of the aTub-LIR, 5-HT-LIR and FMRFamide-LIR increases along the circumesophageal connective, in the apical organ and within the esophageal tissue (Figs. 3g, h, 5, 6 and Additional file 5). Although additional FMRFamidergic somata are detectable in the apical organ, no distinct somata with 5-HT-LIR occur close to the apical tuft (Figs. 3g and 5). The circumesophageal connective splits into a dorsal and ventral root, both showing aTub-LIR. The ventral root furthermore provides a subdivision into two parts, a ventral and a dorsal one (Figs. 3h, 5 and 6). Unlike the dorsal root which exhibits both 5-HT-LIR and FMRFamide-LIR, the ventral root mainly shows aTub-LIR, and only the apical part before the subdivision in the ventral and dorsal part of the ventral root shows FMRFamide-LIR (Figs. 3g, h, 5 and 6). As shown for the previous stage, the stomatogastric nervous system at 14 dpf reveals clear 5-HT-LIR of the ventral most esophageal tissue, distinct anterior somata showing 5-HT-LIR and a prominent midline nerve with branching nerves running towards the ventral side (Figs. 3g and 6). A novel feature of this developmental stage is the appearance of FMRFamide-LIR in the ventral-most esophageal tissue (Figs. 3h and 5).
Shortly after metamorphosis (28 dpf) the dorsal root of the circumesophageal connective, consisting of a dorsal and a ventral part, still shows strong aTub-LIR and FMRFamide-LIR (Figs. 4e–g and 5). Furthermore, prominent somata appear, situated posterior to the mouth opening at the transition from the circumesophageal connective to the ventral nerve cord (Figs. 4e–g and 5). Forming a dense assemblage of perikarya, these FMRFamide-LIR somata frame the anterior end of the ventral nerve cord laterally (Figs. 4f and 5). Additionally, a distinct mass of FMRFamide-LIR somata is present dorsally, posterior to the circumesophageal connective (Figs. 4f and 5) and presumably belongs to the stomatogastric nervous system. 5-HT-LIR is present mainly in the dorsal and ventral root of the circumesophageal connective, although all subdivisions show some staining (Figs. 4h and 6). Notably, the ventral root consists of a dorsal and ventral part and frames the mouth opening which is only visible via anti-5-HT-staining. Furthermore several 5-HT-LIR somata are visible in close proximity to both major roots of the circumesophageal connective (Figs. 4h and 6).
The exhibited immunoreactivity of the esophageal nervous system is similar to the previous stage in regard to shape and composition (Figs. 4g, 5 and 6). But in contrast to earlier stages, the whole esophageal nervous system is inverted (together with the esophagus) and points inside the animal (Figs. 4g and 6). In earlier stages the immunoreactivity in the nerve fibers and perikarya underlies the esophagus and is detectable anterior to the circumesophageal connective which results in a distinct staining of the esophageal nerve with branching nerves and numerous antero-ventral oriented somata (see Fig. 4a and b). After metamorphosis the entire meshwork of fibers and somata that formerly underlied the larval esophagus anterior to the circumesophageal connective, is flapped backwards and can be detected posterior to the circumesophageal connective (Fig. 4g and h). This remarkable change in position and orientation of neural tissue is in line with the above described oral uptake of epidermal tissue during metamorphosis. We assume that this transformation goes along with a posteriorly directed folding of the esophageal nerve tissue. To which extent the uptake of epidermal tissue undergoes apoptosis, as assumed by Wilson , or whether it is reorganized into stomatogastric epidermis, needs further investigation.
At the former position of the larval apical organ, remnants of the FMRFamide-LIR nerve ring and FMRFamide-LIR somata are still present at the apical end of the dorsal circumesophageal root (Fig. 4f). Thus, an integration of apical organ cells into the adult brain can be verified. To what extent these cells become part of the adult nervous system cannot be determined by the present investigation. So far, investigations on different protostome lineages indicate a partial loss of the apical organ during or shortly after metamorphosis. Whether apical cells become part of the adult brain is still questionable [52–54]. Instead, investigations in C. teleta and P. dumerilii reveal retention of many larval neuronal structures in post-metamorphic stages, and therefore support the hypothesis of at least partial inclusion of apical larval somata into the adult nervous system [9, 22, 55]. Further detailed investigations on basally branching annelid taxa like O. fusiformis, including cell fate studies, would clarify this issue.
Innervation of the ciliary band
Thus, the presence and the characteristics of an underlying ciliary ring nerve and the comparable connection to the circumesophageal connectives indicate homology of the equatorial ciliary band of O. fusiformis with the prototroch of other annelids. Further comparative investigations like cell lineage studies on the origin of the ciliary band may be interesting in this context.
Beneath the prominent nerve fibers, the equatorial ciliary band possesses numerous unique somata with 5-HT-LIR and FMRFamide-LIR located in the entire ciliary band (Figs. 5, 7c and e). They may represent sensory organs, but detailed investigations are missing so far. After metamorphosis the ciliary band and the underlying nerve ring with the distinct somata disappear.
Trunk nervous system
Development of the late mitraria (14 dpf and older)
Another remarkable feature of the investigated post-metamorphic animals is the presence of serially arranged clusters of somata that show 5-HT-LIR and which are suggestive of ganglion-like assemblages, and numerous branching lateral nerves with 5-HT-LIR/ aTub-LIR arranged in a similar serial pattern (Figs. 9a–c, f and 6). Notably, additional lateral nerves can be observed, which show only aTub-LIR and do not originate from the somata clusters showing 5-HT-LIR (Fig. 9b and e). The FMRFamide-LIR somata are limited to the anterior end of the ventral cord and do not exhibit seriality (Fig. 9d; 5). The observed serial patterns of neural structures reflect the external body segmentation of the juvenile worms and resemble the conditions known for most other annelids including C. teleta and P. dumerilii [9, 22, 26, 27]. Whereas no such serial clusters of somata are described for adult Owenia fusiformis [34, 37], immunohistochemical investigations in Galathowenia oculata revealed the presence of comparable structures only in few posterior-most segments of adult worms . The early serial patterns of neural structures within the entire ventral nerve cord as described for Owenia fusiformis are not detectable in later ontogenetic stages, a situation that is well described for annelid taxa without adult external body segmentation, such as sipunculans, echiurans or myzostomids [17, 21, 62, 63]. Thus, the lack of ventral nerve cord ganglia in adult Owenia fusiformis differs from the situation in other annelids, but the serial arrangement of at least certain neural structures in juvenile stages as described above might be well comparable with the nervous system architecture known from other taxa.
Our analyses of neurogenesis in different developmental stages of Owenia fusiformis obviously show that the metamorphosis of the enigmatic mitraria larva to the juvenile is not that catastrophic as previously thought. Instead, an antero-posterior development of neural structures showing prominent 5-HT-LIR, FMRFamide-LIR and aTub-LIR is detectable, and adult nervous system precursors are present in early stages of development. The drastic changes in body shape during metamorphosis occur mainly by diminution of the larval blastocoel and rearrangement of the detached epidermis of the bell-shaped swimming larva. Our investigations on the development of the nervous system reveal also many similarities to other annelids throughout all developmental stages. Thus, adult precursors are present in early stages and juveniles. The same developmental stages exhibit a prominent apical organ formed by flask-shaped perikarya, early development of circumesophageal connectives interconnecting apical and trunk nervous system, and the serially arranged clusters of somata displaying 5-HT-LIR in the ventral nerve cord in addition to the serial lateral nerves (at least in early juveniles). These highly comparable features are also known among basally branching and deeply nested annelid groups and are described for other Lophotrochozoa such as mollusks. It will be an interesting to study, whether all neuronal somata are clustered and whether they form ganglion-like structures during early development in basally branching annelids as in Owenia fusiformis. However, we are not in position to infer this from our current data. We observed a high number of lateral nerves showing only aTub-LIR not following a serial pattern, but the position of the respective somata remains unclear. Furthermore, the presence of an equatorial ciliary band including a distinct underlying nerve ring possessing 5-HT-LIR and FMRFamide-LIR and its connection to the circumesophageal connectives are features well known for annelid larvae. Notable differences include the absence of 5-HT-LIR in early larvae, the lack of a first prominent 5-HT-LIR at the posterior pole, an equatorial ciliary band that bears numerous distinct putative sensory cell clusters and the serial clusters of immunoreactive somata of the juvenile nervous system that disappear in older stages. Such annelid features are only known for Owenia fusiformis so far, but should be examined in other basally branching annelid groups, such as Magelonidae. Such comparative investigations are necessary for revealing the polarity of annelid trait evolution. With its comparatively simple brain consisting of relatively few somata (we here refer to the roots of the circumesophageal connectives as ‘brain’, because no other distinct brain elements were detected so far) and intraepidermal trunk nervous system, which bears distinct differences between juveniles and adults in terms of centralization and somata organization. Therefore, O. fusiformis offers great potential as a study object for unraveling the architecture and neural patterning mechanisms of the ancestral annelid nervous system. A deeper knowledge of these issues could yield further important insights into bilaterian developmental topics.
Animal culture and fixation
Adult specimens of Owenia fusiformis were collected in Saint-Efflam during summer 2013–15 (Brittany/ France), transferred to Bergen (Norway) and reared in a tempered sea-water cycle. After artificial fertilization in filtered sea water (FSW) the developmental stages were reared at 18 °C in glass flasks containing 1 l FSW. The culture was aerated, set under strict diurnal rhythm (14:10 – light: dark) and fed with a mix of unicellular algae (Isochrysis). Water was changed regularly.
Prior to fixation different larval stages were anaesthetized using 7 % MgCl2 in FSW. The larvae were then fixed in 4 % paraformaldehyde (PFA) in 1x phosphate buffered saline with Tween (1x PBS: 0.05 M PB / 0.3 M NaCl / 0.1 % Tween20) for 2 h at 4 °C. After fixing, the animals were rinsed in 1x PBS several times and stored in 1x PBS containing 0.05 % NaN3 at 4 °C until usage.
For semi-thin sections the specimens were fixed in 2.5 % glutaraldehyde in 1x PBS (0.05 M PB, 0,3 M NaCl) for 1 h at 4 °C and subsequently washed several times with PBS buffer over the next 24 h on 4 °C before stored in 1x PBS containing 0.05 % NaN3 at 4 °C until usage. For postfixation specimens were treated with 2 % Osmium tetroxide in 1x PBS for 20–40 min at 4 °C and subsequently dehydrated in a graded series of acetone/PBS at room temperature, transferred to propylenoxide and finally embedded using TAAB Araldite 502/812 Kit according to manufacturer’s recommendations.
Semi-thin sections were made from specimens at 7 dpf with a Leica EM UC7 ultramicrotome using a Diatome ultra jumbo diamond knife. Sections were transferred to glass slides, stained with toluidine blue (1 % toluidine blue, 1 % sodium tetraborate and 20 % sucrose) and mounted with Depex. Light microscopic images were taken with a Zeiss Imager Z2 microscope and a Zeiss Axiocam 506 color camera. Images were processed and image stacks were registered with Fiji. The final panels were designed using Adobe (San Jose, CA, USA) Photoshop CC and Illustrator CC.
For electron microscopy animals were treated as described in . Accordingly, they were fixed in 2.5 % glutaraldehyde/ 0.1 M sodium cacodylate/ 0.24 M NaCl and subsequently post-fixed in 1 % OsO4/ 0.1 M sodium cacodylate/ 0.24 M NaCl. Specimens were then en bloc stained for 30 min in 2 % OsO4/ 1.5 % potassium ferricyanide/ 0.1 M sodium cacodylate followed by incubation in 2 % aqueous uranyl acetate for 30 min. Dehydration of the samples was performed gradually in ethanol series and then with propylene oxide. All steps were done at room temperature. Following embedding (using the TAAB Araldite 502/812 kit), ultrathin sections (70 nm) were cut with a Leica EM UC7 and counterstained with 2 % uranyl acetate and lead citrate. Images were acquired on a JEOL 1011 transmission electron microscope equipped with an Olympus MORADA camera. The final panels were prepared using Adobe (San Jose, CA, USA) Photoshop CC and Illustrator CC.
Immunohistochemistry and confocal laser scanning microscopy
Anatomical details of developmental stages of Owenia fusiformis Delle Chiaje, 1844, were revealed in whole animal preparations using standard immunohistochemical staining protocols and a range of well-established antisera as neural markers. Every staining was carried out using at least 25–30 specimens of each stage. Although the specificities of the employed antibodies have all been established in numerous invertebrates (details below), we cannot fully exclude that a given antiserum may bind to a related antigen in the investigated specimens. We hence refer to observed labelled profiles as exhibiting (antigen-) like immunoreactivity (−LIR). Negative controls were obtained by omitting the primary antibody in order to check for antibody specificity and yielded no fluorescence signal. For immunohistochemistry specimens were rinsed 2 x 5 min in PTW (PBS with 0.1 % Tween 20) at RT (room temperature) and transferred into 10 μg proteinase K/ml PTW for 2–3.5 min depending on the developmental stage (24hpf-3dpf = 90s; 7 dpf = 2 min; 14–21 dpf = 2,5 min; after metamorphosis = 3,5 min). After 2 short rinses in glycine (2 mg glycine/ml PTW), and 3x 5 min washes in PTW, the specimens were re-fixed using 4 % PFA in PBS containing 0.1 % Tween for 20 min at RT. Subsequently the developmental stages were rinsed 2x5 min in PTW, 2x5 min in THT (0,1 M TrisCl, 0,1 % Tween) and blocked for 1-2 h in 5 % sheep serum in THT according to the protocol of Conzelmann and Jekely (2012) . The primary antibodies, polyclonal rabbit anti-serotonin (INCSTAR, Stillwater, USA, dilution 1:500), monoclonal mouse anti-acetylated α-tubulin (clone 6-11B-1, Sigma-Aldrich, St. Louis, USA, dilution 1:500), and polyclonal rabbit anti-FMRFamide (ImmunoStar Inc., Hudson, USA, dilution 1:1000) were applied for 24–72 h in THT containing 5 % sheep serum at 4 °C. Specimens were then rinsed 2x 10 min in 1 M NaCl in THT, 5x 30 min in THT and incubated subsequently with secondary fluorochrome conjugated antibodies (goat anti-rabbit Alexa Fluor 488, Invitrogen, USA, dilution 1:500; goat anti-mouse Alexa Fluor 633, ANASPEC, Fremont, USA, dilution 1:500) in THT containing 5 % sheep serum for 24 h at 4 °C. Subsequently, the samples were washed 6x 30 min in THT, stained with DAPI for 10–15 min (5 mg/ml stock solution, working solution: 2 μl in 1 ml THT – final concentration 10 μg/ml) and washed 2x 5 min in THT. For clsm-analyses samples were mounted on glass slides using 90 % glycerol/10 % 10x PBS containing DABCO. Specimens were analyzed with the confocal laser-scanning microscope Leica TCS STED (Leica Microsystems, Wetzlar, Germany). Confocal image stacks were processed with Leica AS AF v2.3.5 (Leica Microsystems), ImageJ and Imaris 6.3.1 (Bitplane AG, Zurich, Switzerland). The final panels were designed using Adobe (San Jose, CA, USA) Photoshop CC and Illustrator CC.
5-HT, 5-Hydroxytryptamin (Serotonin); aTub, acetylated-α-tubulin; dpf, days post fertilization; FMRFamide, the neuropeptide Phe-Met-Arg-Phe; −LIR, −like immunoreactivity
We thank the staff of the ‘Station Biologique de Roscoff’ for providing facilities and support of sampling and sending animals. We thank Kenneth M. Halanych, Suman Kumar and three anonymous reviewers for helpful comments on an earlier version of this manuscript. We also thank Egil S. Erichsen for technical help with electron microscopic imaging.
This research was funded by the Sars Centre core budget and CH was financed by a personal research fellowship from the DFG (HE 7224/1-1).
Availability of data and materials
Z-stacks (Additional file 1, Additional file 2, Additional file 3, Additional file 4, Additional file 5, Additional file 6, Additional file 7) of the nervous system stainings showing relevant developmental stages are deposited online. The additional files supporting the results of this article are available in the Dryad digital repository ; http://dx.doi.org/10.5061/dryad.m8k24/ .
CH, OV and HH designed the experiments. CH and HH wrote the manuscript. CH and OV were responsible for larval rearing and fixation and performed immunostaining and subsequent imaging at the clsm. IK and HH performed the histological and electron microscopical work. All authors analysed the data, discussed the results, read, commented on and approved the final version of the manuscript.
The authors declare that they don’t have any competing interests.
Consent for publication
Ethics approval and consent to participate
No permits or authorizations were required to collect the animals used in this study.
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