Owenia fusiformis - a basally branching annelid suitable for studying ancestral features of annelid neural development

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Annelida; Evolution; Immunohistochemistry; Nervous system; Neuroanatomy; clsm.

Fig. 1

Schematic overview of the normal development of Owenia fusiformis. Different developmental stages used in this study are shown in schematic drawings (upper part) and photographs (lower part). The timescale indicates the age of different larval stages according to the emergence of distinct morphological features. Stages are shown in dpf (days post fertilization). Note that the development is somewhat asynchronous and only approximately 80–90 % of the larvae of a certain age exhibit the same developmental stage. ao, apical organ; at, apical tuft; cb, ciliary band; ch, chaetae; cs, chaetal sac; ep, episphere; es, esophagus; ey, eye; mg, midgut; mo, mouth opening

Fig. 2

Whole mounts, histological cross-sections and electron microscopical images of O. fusiformis at 7 dpf. Light microscopic images of entire larvae (a), toluidin-blue stained cross-sections (b, d, e) and electron microscopy (c, f). Note that the sectional planes of b, d, e are indicated by red lines in a. There are three planes. The position of the electron microscopical detail is indicated by red rectangles. a Early developmental stage 7 dpf. Ventral (including the mouth opening) is left, apical is up. The animal exhibits a well-developed ciliary band (cb), a distinct esophagus (es) and a defined midgut (mg). b In cross-sections of the esophagus region the prominent blastocoel is visible, which occupies most of the space within the larval episphere. The inset shows the esophageal region (es) and the position of the epidermis (ep) in higher magnification. c The outer epidermis (ep) is composed of a monolayer of epidermal cells and a prominent cuticle (cu). The underlying basal lamina (bl) borders directly borders the large blastocoel (bc) being a primary body cavity. The inset in (c) shows a higher magnification of the basal lamina (bl). d A cross-section in the plane of the apical organ (ao) reveals the presence of distinct muscle bundles (mu) and the circumesophageal connective (cc) framing the esophagus (es). The inset shows the the apical organ (ao) in higher magnification. e The blastocoel (bc) occupies most of the larva. The inset in (e) shows the epidermis (ep) lateral to the apical organ. f The central tissue is covered by a basal lamina directly bordering the internal blastocoel (bc). The inset in (f) shows a higher magnification of the basal lamina (bl). an, anus; ao, apical organ; bc, blastocoel; bl, basal lamina; cb, ciliary band; cc, circumesophageal connective; ci, cilium; cu, cuticle; ep, epidermis; es, esophagus;; hg, hindgut; mg, midgut; mu, muscle bundle; ne, nerve; nu, nucleus; tr, trunk region. Scale bars = 100 μm (b, d, e), 1 μm (c, f) and 500 nm (inset in c and f). The scale bars only refer to the main images in b, d and e, not to the insets

Fig. 3

Innervation of apical organ and esophageal region in early developmental stages of O. fusiformis (1 dpf - 14 dpf). FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue). Confocal z-projections and volume renderings of FMRFamide-LIR (D, E, H (inset)). Ventral is left and apical is up in all images, except (d) which shows a ventral view. Stages are given in days post fertilization (dpf). The shape of the esophagus is indicated by white lines. a Early stages exhibit weak FMRFamide-LIR in the apical organ (ao) (white dotted circle). The inset shows the apical organ (ao) (white dotted circle) in higher magnification. Note that the staining in the chaetal sacs (cs) and the chaetae (ch) is due to autofluorescence. The yellow signal along the ciliary band (cb) is caused by unspecific staining and differed between the investigated specimens. The inset shows the FMRFamide-LIR underlying the apical organ (white dotted circle) in higher magnification. b, c At 2 dpf the mitraria shows strong 5-HT-LIR in the circumesophageal connective (cc) and in the tissue underlying the esophagus (es). No 5-HT-LIR is visible underlying the apical tuft (at). Note that the purple dots surrounding the apical tuft (at) are labeling artifacts. Position of the apical tuft (at) is indicated by sketch. Note that the 5-HT-LIR in the posterior larva is restricted to the midgut and the chaetal sacs. d At 7 dpf the apical circumesophageal connective (cc) shows distinct FMRFamide-LIR. Contour of the apical nerve ring (ar) is denoted (red dotted line), prominent perikarya are marked (red asterisk). e Lateral view of previous stage. Contour of the apical ring (ar) and the circumesophageal connective (cc) is denoted (dotted red line), prominent perikaryon is marked (red asterisk). f 5-HT-LIR at 7 dpf is present in the circumesophageal connective (cc), an esophageal nerve (en) with branching nerve fibers (eb) and the tissue underlying the esophagus (es). Note distinct 5-HT-LIR in at least 1 perikaryon (arrow). The connection of the latter perikarya with the esophageal nerve (en) is indicated by white dots. The inset shows the 5-HT-LIR (now grey) within the tissue underlying the esophagus in higher resolution. Notably, the signal underlying the esophageal region may also contain some unspecific staining due to the possible presence of gland cells in the esophagus. g At 14 dpf the 5-HT-LIR (and aTub-LIR) increases, but the pattern is comparable to the previous stage. No 5-HT-LIR is detectable apically (lack of signal indicated by red dotted circle). The inset shows a higher magnification of the tissue underlying the apical tuft. The purple dots surrounding the region of interest are labeling artifacts. Note that the overlap of the 5-HT-LIR and the aTub-LIR causes a white color in some parts of the shown image. The esophageal branching nerves are not shown in the image. h FMRFamide-LIR at 14 dpf is restricted to the apical ventral (vr) and dorsal root (dr) of the circumesophageal connective, the apical nerve ring and apical perikarya, and detectable in the tissue underlying the esophagus (white dotted circle). Inset shows volume rendering of FMRFamide-LIR. Position of the apical tuft (at) is indicated by sketch. Position of perikarya (red asterisks), apical nerve ring (ar, red dotted line) and roots of the circumesophageal connective (vr, dr, red dotted line) is indicated. Both roots of the ventral circumesophageal connective (vpvr, dpvr) are detectable by tubulin staining (dotted red line). Note that the overlap of the FMRFamide-LIR and the aTub-LIR causes a white color in some parts of the shown image. The inset shows the same volume rendered FMRFamide-LIR. The shape of neural structures is implemented by red dotted lines. ao, apical organ; ar, apical nerve ring; at, apical tuft; cb, ciliary band; cc, circumesophageal connective; ch, chaetae; cs, chaetal sac; dr, dorsal root of cc; dpvr, dorsal part of vr; eb, branches of the esophageal nerve; en, esophageal nerve; es, esophagus; gl, gland; mg, midgut; vr, ventral root of cc; vpvr, ventral part of vr. Scale bars = 30 μm (a), 100 μm (b), 15 μm (d, e), 50 μm (f-h). The scale bars only refer to the main images, not to the insets. c represents a more detailed picture of (b) and therefore has no scale bar

Fig. 4

Innervation of apical organ and esophageal region in late developmental stages of O. fusiformis (21 dpf - 28 dpf). FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue). Confocal z-projections and volume renderings of 5-HT-LIR (a; a, inset) (except of (h) where 5-HT-LIR is shown in grey), FMRFamide-LIR (c, d, f) and aTub-LIR (e, inset). Ventral is left and apical is up in all images, except of (b) which shows a view from dorsal and (h) where the specimen is shown in antero-dorsal view with the anterior part down. Stages are given in days post fertilization (dpf). The shape of the esophagus is indicated by white lines. a At 21 dpf 5-HT-LIR is visible in the circumesophageal connective (cc) and its roots (vr, dr), within the esophageal nerve (en, dotted line) including all branching fibers and the tissue underlying the esophagus. Position of the apical tuft (at) is indicated by sketch. The inset shows absence of 5-HT-LIR in the apical region (indicated by white arrow heads). b The 5-HT-LIR frames the entire esophagus. Position of the esophageal nerve (en) is indicated by the dotted line. Note that -LIR is absent in the region of the apical organ. c Although both roots of the circumesophageal connective (cc) are detectable via anti-tubulin staining at 21 dpf, FMRFamide-LIR is restricted to the dorsal root (dr) including prominent lateral perikarya (arrow head), the apical nerve ring (red dotted line in inset), and the distinct apical perikarya (red asterisks). The inset shows the FMRFamide-LIR in the same view without background staining. The position of the apical ring is implicated by red dots. The distinct apical perikarya are marked (red asterisks). d A lateral view reveals prominent FMRFamide-LIR in the tissue underlying the esophagus (es) (red dotted circle). The inset shows the dorsal root of circumesophageal connective (dr) and the tissue underlying the esophagus (es) (red dotted circle) in confocal z-projection (with the same scale). Note the prominent lateral (arrow head) and apical perikarya (red asterisks). e At 28 dpf, approximately 1 week after metamorphosis, FMRFamide-LIR is can be detected within the dorsal root of the circumesophageal connective (dr), in the ventral nerve cord (vn) and numerous antero-ventral perikarya. Inset shows the FMRFamide-LIR of the prominent dorsal and ventral part of the dorsal root of the circumesophageal connective (vpdr, dpdr). Note that the overlap of the FMRFamide-LIR and the aTub-LIR causes a white color in some parts of the shown image. The inset shows only the FMRFamide-LIR. f FMRFamide-LIR reveals presence of the apical perikarya in juveniles (red asterisk) and shows a prominent dorsal assemblage of perikarya (arrow head). g 5-HT-LIR is exhibited within both roots (and the dorsal and ventral part of dr) of the circumesophageal connective (vpdr, dpdr, vr) and the tissue underlying the esophagus (dotted white line). Note that the pattern of -LIR in this tissue is invaginated compared to pre-metamorphic stages. h An antero-dorsal view reveals the presence of both parts of both circumesophageal roots (vpvr, dpvr, vpdr, dpdr) and perikarya (white asterisks) with 5-HT-LIR close to the roots. The tissue underlying the esophagus is detectable (dotted white line). Note that the pattern of -LIR in this tissue is invaginated compared to pre-metamorphic stages. at, apical tuft; cc, circumesophageal connective; dpdr, dorsal part of dr; vpdr, ventral part of dr; dpvr, dorsal part of vr; dr, dorsal root of cc; en, esophageal nerve; es, esophagus; gl, gland; vpvr, ventral part of vr; vr, ventral root of cc. Scale bars = 50 μm (a–e) and 30 μm (h). The scale bars only refer to the main images, not to the insets

Fig. 5

Schematic overview showing the pattern of FMRFamide-LIR and aTub-LIR during the development of O. fusiformis. Note that main neural patterns and the position of the digestive tract are color coded. Stages are given in days post fertilization (dpf). Ventral is left and apical is up in all images, except of the 28 dpf stage, where ventral is down. The inset shows a ventral view of the 28 dpf stage. The schemes presented here are simplified to support an understanding of the complex morphology. acn, apical ciliary ring nerve; an, anus; ao, apical organ; at, apical tuft; cb, ciliary band; cc, circumesophageal connective; ch, chaetae; cs, chaetal sac; dpdr, dorsal part of the dorsal root of cc; dpvr, dorsal part of the ventral root of cc; dr, dorsal root of cc; en, esophageal nerve; ep, episphere; es, esophagus; fn, FMRFamidergic neuron; hg, hindgut; ln, lateral nerve; lo, longitudinal nerve; mg, midgut; mo, mouth opening; pcn, posterior ciliary ring nerve; vn, ventral nerve cord; vpdr, ventral part of the dorsal root of cc; vpvr, ventral part of the ventral root of cc; vr, ventral root; 1–5, numbers referring to the specific nerves of vn

Fig. 6

Schematic overview showing the pattern of 5-HT-LIR and aTub-LIR during the development of O. fusiformis. Note that main neural patterns and the position of the digestive tract are color coded. Stages are given in days post fertilization (dpf). Ventral is left and apical is up in all images, the inset shows a ventral view of the 28 dpf stage. The schemes presented here are simplified to support an understanding of the complex morphology. acn, apical ciliary ring nerve; an, anus; ao, apical organ; at, apical tuft; cb, ciliary band; cc, circumesophageal connective; ch, chaetae; cs, chaetal sac; dpdr, dorsal part of the dorsal root of cc; dpvr, dorsal part of the ventral root of cc; dr, dorsal root of cc; en, esophageal nerve; ep, episphere; es, esophagus; hg, hindgut; ln, lateral nerve; lo, longitudinal nerve; mg, midgut; mo, mouth opening; pcn, posterior ciliary ring nerve; sn, serotonergic neuron; vn, ventral nerve cord; vpdr, ventral part of the dorsal root of cc; vpvr, ventral part of the ventral root of cc; vr, ventral root; 1–5, numbers referring to the specific nerves of vn

Fig. 7

The innervation of the ciliary band in the mitraria of O. fusiformis. FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue). Confocal z-projections and volume renderings of aTub-LIR (b, e), FMRFamide-LIR (c) and 5-HT-LIR (e). Ventral is left and apical is up in all images, except (c) and (e) which show a view from ventral. Stages are given in days post fertilization (dpf). a The mitraria exhibits two distinct neurites forming the apical and posterior ciliary ring nerves (acn, pcn) (inset), and three pairs of peripheral nerves (pn1–3) running from apical towards ventral (pn1, pn2) and lateral (pn 3), and innervating the ciliary ring nerves (cn). Furthermore, a single prominent dorsal nerve (dn) runs from apical to the posterior ciliary ring nerve (pcn). The inset shows a higher magnification of the ciliary ring nerves. b The peripheral nerves (pn1–3) and the dorsal nerve (dn) interconnect the apical part of the developing embryo with the ciliary ring nerves (cn). c FMRFamide-LIR in the ciliary ring nerves is restricted to the apical ciliary ring nerve (acn), which also bears numerous FMRFamide-LIR perikarya. The inset shows a confocal z-projection of the same area in same orientation. Only the apical ciliary ring nerve (acn) is visible. This pattern is visible throughout the entire ring nerve also in stages 7 dpf - 21 pdf. d The peripheral nerves (pn1–3) and the dorsal nerve branch off from the circumesophageal connective (cc) at the apical pole of the larva. e 5-HT-LIR in the ring nerve is restricted to the posterior ciliary ring nerve (pcn) and mainly detected close to the esophagus (es). Note the perikarya showing 5-HT-LIR along the anterior ciliary ring nerve (white asterisks). The inset shows a confocal z-projection of the same area in same orientation. Only the posterior ciliary ring nerve (pcn) is visible. The staining shown here appeared to be fragmentary when looking at the entire ring nerve in stages 7 dpf - 21 pdf. acn, apical ciliary ring nerve; at, apical tuft; bn, bell nerve; cc, circumesophageal connective; cn, ciliary ring nerve; dn, dorsal nerve; en, esophageal nerve; es, esophagus; mg, midgut; pcn, posterior ciliary ring nerve; pn1–3, peripheral nerves 1–3. Scale bars = 50 μm. The scale bars only refer to the main images (a, b, d), not to the insets. Note that (c) and (e) are without scale

Fig. 8

Development of the trunk nervous system in different pre-metamorphic developmental stages of O. fusiformis (14 dpf–21 dpf). FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue). Confocal z-projections and volume renderings of FMRFamide-LIR (b) and aTub-LIR (b). Ventral is left and apical is up in (a) and (b), in (c) ventral is down and apical is left, (e) and (f) which show a view from apical with the ventral side left (e) and down (f). d shows a view from latero-apical with the ventral side left. Stages are given in days post fertilization (dpf). The shape of the esophagus (and intestine) is indicated by white lines. The position of somata clusters showing 5-HT-LIR is indicated with white dotted circles. a 5-HT-LIR of the trunk nervous system is detectable earliest at 14 dpf. The ventral nerve cord (vn) is represented by four neurites including numerous serial perikarya (inset). Note that the overlap of the 5-HT-LIR and the aTub-LIR causes a white color of the circumesophageal connective (cc). The inset shows an apical view of the ventral nerve cord (vn). b FMRFamide-LIR at 14 dpf is restricted to the two inner parallel neurites and four pairs of perikarya. c At 21 dpf, shortly prior to metamorphosis, the 5-HT-LIR of the ventral nerve cord (vn) is detectable within distinct neurites, serially arranged clusters of somata (white dotted circle) and serial lateral nerves (ln) showing 5-HT-LIR and branching off from the serotonergic cell clusters. Notably, numerous longitudinal nerves (lo) are visible in pre-metamorphic stages. The specimens represent conditions later on observable in juveniles as well. aTub-LIR was hardly detectable due to a dense ciliation of the entire specimen. d A lateral view at 21 dpf reveals several clusters of somata exhibiting 5-HT-LIR. Several lateral nerves (ln) branch off from the ventral nerve cord (vn) at the position of the cell clusters. Note that the posterior part of the ventral cord (vn) is bent under the anterior part and the esophageal region (es) due to the shape of the mitraria. The dotted square indicates the region that is shown in (f) with higher magnification. e An apical view at 21 dpf reveals the presence the dorsal and the ventral parts of the dorsal root of the connective (vpdr, dpdr) and the ventral root of the connective (vr). Furthermore, distinct clusters of serotonergic neurons (sn) along the ventral nerve cord (vn) are detectable. Prominent lateral nerves (ln) branch off from these clusters. The posterior part of the ventral cord (vn) is bent under the anterior one and therefore not visible. f A higher magnification of the posterior part of the ventral nerve cord (vn) at 21 dpf reveals the presence of 4 distinct nerves showing 5-HT-LIR (numbered 1–4). The location of the posterior ventral nerve cord (vn) in the entire animal is shown in (d) and marked with the white dotted square. at, apical tuft; cc, circumesophageal connective; cs, chaetal sac; dpdr, dorsal part of the dorsal root of cc; en, esophageal nerve; es, esophagus; gl, gland; hg, hindgut; ln, lateral nerve; lo, longitudinal nerve; mg, midgut; vn, ventral nerve cord; vpdr, ventral part of the dorsal root of cc;vr, ventral root of the cc. Sale bars = 100 μm (a), 30 μm (b), 50 μm (c–e) and 20 μm (f). The scale bars only refer to the main images, not to the insets

Fig. 9

Development of the trunk nervous system in post-metamorphic juveniles of O. fusiformis (~28 dpf). FMRFamide-LIR (yellow) and 5-HT-LIR (purple), aTub-LIR (cyan), DNA (dark blue) (except for inset (C) where DNA staining is shown in grey). Confocal z-projections. Anterior is left in all images, except of (c) where anterior is up. a and b are lateral views, (c–e) are views from ventral, (f) is a view from dorsal. Stages are given in days post fertilization (dpf). The shape of the esophagus (and intestine) is indicated by white lines. The position of somata clusters showing 5-HT-LIR is indicated with white dotted rectangles. a Shortly after metamorphosis, at 28 dpf, 5-HT-LIR is present in the ventral nerve cord (vn), in numerous lateral nerves (ln) and prominent longitudinal nerves (lo). b Using the same specimen and orientation as in (A), the aTub-LIR reveals numerous lateral nerves (ln). c A ventral view at 28 dpf reveals serial clusters of somata (with 5-HT-LIR) and lateral nerves branching off from the ventral cord (vn) at the position of the cell clusters. The inset shows the region of the 2nd cluster of somata possessing 5-HT-LIR (marked with white asterisk in (C)) stained with DAPI. Notably, DNA staining does not show any cluster. d The ventral nerve cord (vn) is represented by two parallel neurites and a median nerve with FMRFamide-LIR (see left inset, nerves are labeled 1–3) and two outer parallel nerves only showing aTub-LIR (see right inset, nerves are labeled 4 and 5). At least nerves 2 and 3 arise in 5-HT staining as well (not shown). Nerve 1 is visible in some 5-HT stainings only. Notably, no serial clusters of somata showing FMRFamide-LIR are detectable along the ventral cord (vn). e aTub-LIR at 28 dpf reveals a pair of lateral longitudinal nerves (arrow head) running besides the main cord, and numerous lateral nerves (ln) leading towards the periphery. f 5-HT staining at 28 dpf shows serially arranged lateral nerves (ln) and numerous lateral perikarya. Furthermore, longitudinal nerves (lo) are running in antero-posterior direction. Note that the overlap of the 5-HT-LIR and the aTub-LIR causes a white color of the dorsal root of the circumesophaegal connective (dr). at, apical tuft; cc, circumesophageal connective; ch, chaetae; dr, dorsal root of cc; dpdr, dorsal part of the dorsal root of cc; dpvr, dorsal part of the ventral root of cc; en, esophageal nerve; es, esophagus; gl, gland; hg, hindgut; ln, lateral nerve; lo, longitudinal nerve; mg, midgut; sn, serotonergic neuron; vn, ventral nerve cord; vpdr, ventral part of the dorsal root of cc. Sale bars = 100 μm (A, B, D, E, F) and 50 μm (C). The scale bars only refer to the main images, not to the insets