Development of the Nervous System of Carinina ochracea (Palaeonemer-tea, Nemertea)

Abstract

The various clades of Lophotrochozoa possess highly disparate adult morphologies. Most of them, including Nemertea (ribbon worms), are postulated to develop via a pelagic larva of the trochophora type, which is regarded as plesiomorphic in Lophotrochozoa. With respect to the nervous system, the trochophora larva displays a set of stereotypic features, including an apical organ and trochal neurites, both of which are lost at the onset of metamorphosis. In the investigated larvae of Nemertea, the nervous system is somewhat divergent from the postulated hypothetical trochophore-like pattern. Moreover, no detailed data is available for the "hidden" trochophore larva, the hypothetical ancestral larval type of palaeonemertean species. Therefore, the development of the nervous system in the larva of Carinina ochracea, a basally branching palaeonemertean species, was studied by means of immunofluorescence and confocal laserscanning microscopy. Like in the other investigated nemertean larvae, the prospective adult central nervous system in C. ochracea develops in an anterior to posterior direction, as an anterior brain with paired longitudinal nerve cords. Thus, development of the adult nervous system in Nemertea is largely congruent with currently accepted hypotheses of nervous system development in Spiralia. In early development, transitory apical, serotonin-like immunoreactive flask-shaped cells are initially present, but the trochal neurites that have been considered as pivotal to lophotrochozoan development, are absent. In the light of the above stated hypothesis, trochal neurites have to be interpreted as reduced in Nemertea. On the other hand, due to the unsettled systematic status of Palaeonemertea, more comparative data are desirable to answer the remaining questions regarding the evolution of nervous system development in Nemertea.

Fig 1. Z-Projections of immuno-stained specimens of Carinina ochracea.

Orientation indicated by compass (d, dorsal; l, left; r, right; v, ventral); apical is up. A and B: larva three days post-fertilization (3-dpf). A: Acetylated α-tubulin-like immunoreactivity (tub-lir) (γ: 0.89). Tub-lir has the strongest signals in the epidermal cilia (ci), the cilia of the midgut (mg) and the nephridial tubules (ne). Neuronal tub-lir signals outline the brain-ring (br), the developing lateral nerve cords (nc) and the peripheral plexus (pp). B: Synapsin-like immunoreactivity (syn-lir) (γ: 0.82). Syn-lir marks the developing neuropil of the brain-ring (br), especially the ventral part and the lateral nerve cords (nc). Additionally, the peripheral plexus (pp) displays syn-lir. Note the strong unspecific signals of large epidermal mucus gland cells (gc) partly extruded at fixation. C and D: Combined syn-lir and serotonin-like immunoreactivity (5HT-lir). C: Larva at 5-dpf (γ: 0.89 (5HT-lir), γ: 0.88 (syn-lir)). Syn-lir is visible in the brain-ring neuropil (br) the lateral nerve cords (nc), the peripheral plexus (pp), and the dorsal nerve (dn). Additionally, the first signals of the oral ring neurite bundle (or) are detectable. Note the unspecific signals of epidermal mucus gland cells (gc). The 5HT-lir neurons associated with the ventral (bv) and dorsal (bd) brain-ring (br) are distinct, as are the 5HT-lir neurites of the brain-ring. The peripheral plexus (pp) and the apical signals (as) are displaying 5HT-lir. Along the 5HT-lir neurite signals of the lateral nerve cords (nc) the first paired associated neurons (ln) can be seen. A weak dorso-median 5HT-lir signal indicates the neurites of the dorsal nerve (dn). D: Larva at 10-dpf (γ: 0.89 (5HT-lir), γ: 0.88 (syn-lir)). Syn-lir is still weaker in the dorsal-most part of the brain-ring neuropil (br) compared to the ventral part and the lateral nerve cords (nc). The peripheral plexus (pp) and the anteriorly elongated dorsal nerve (dn) display syn-lir. The oral ring neurite bundle (or) has become more prominent and paired pre-oral neurite bundles (po), connecting to the ventral portion of the brain-ring, are visible. Note the unspecific signals of epidermal mucus gland cells (gc) and the weaker signal of the left lateral nerve cord (nc) due to signal attenuation. The 5HT-lir neurons associated with the brain-ring (bv and bd) and the lateral nerve cords (ln) have increased in numbers. The 5HT-lir dorsal nerve signal (dn) extends anteriorly beyond the brain-ring neurites. Apical 5HT-lir signals (as) are numerous but comparably weak.

Fig 2. Z-Projections of immuno-stained specimens of Carinina ochracea.

Serotonin-like immunoreactivity (5HT-lir). A: gastrula stage one day post-fertilization (1-dpf) (γ: 0.84). Two apical neurons (apical median: an1, medio-ventral: an2) and one caudal neuron (cn1) display 5HT-lir. Note the neurite connecting cn1 and an2 (arrowhead). B: Post-gastrula stage larva at 1-dpf (γ: 0.77). Apically, only the medio-ventral neuron (an2) is detectable. Caudally a second, more dorsally located neuron displays 5HT-lir (cn2). A pair of longitudinal 5HT-lir neurite-like signals connects cn1 and an2 (arrowheads). C: Larva at 2-dpf (γ: 0.9). The first-formed 5HT-lir neurons show weak and patchy signals. Posterior of an2, the 5HT-lir brain-ring neurites (br) are seen, along with ventrally located neurons displaying 5HT-lir (bv). The dorso-median neurite signal is only visible in its anterior extension (arrow). Note the paired dorsally located 5HT-lir neurons (pn), possibly contributing to the forming peripheral plexus. D: Larva at 3-dpf (γ: 0.9). The first-formed signals have ceased to display 5HT-lir. Dorsally and ventrally located 5HT-lir neurons (bd, bv) are visible associated with the 5HT-lir brain-ring neurites (br). A 5HT-lir peripheral plexus (pp) has formed in the vicinity of the peripheral neurons (pn). At the apical pole of the larva, several weak 5HT-lir signals (as) are present.

Fig 3. Schematic representation of nervous system development of Carinina ochracea.

Serotonin-like (5HT-lir) and FMRFamide-like (RFa-lir) immunoreactivity are color coded in magenta and green respectively; the general nervous system architecture as outlined by synapsin-like immunoreactivity (syn-lir) is shown in grey. All larval stages are drawn to scale with apical tuft (at), epidermal cilia, fronto-lateral epidermal invaginations (fi), midgut (mg), mouth opening (mo), and the pigmented larval eye (pe) serving as landmarks (cilia of the frontal epidermal invaginations, the stomodaeum, the midgut, and epidermal cilia in the aspect of view omitted), apical is up. A and B: left: lateral view from right, right: ventral view. Transitory neuronal/neuritic elements are indicated by stippling/dotted lines. A: Post-gastrula stage larva at one day post-fertilization (1-dpf). B: Larva at 2-dpf. C: Larva at 3-dpf. From left to right: lateral view from right, ventral view, dorsal view, lateral view from left. Since the nervous system is roughly symmetrical, only one side is shown (left: 5HT-lir, right: RFa-lir). For clarity, the peripheral plexus is not shown. Further explanations, see text.–bd, dorsal brain neuron; br, brain-ring; bv, vental brain neuron; an1, 1^st apical median (transitory) neuron; an2, 2^nd apical medio-ventral (transitory) neuron; nc, lateral nerve cord; cn1, 1^st caudal median (transitory) neuron; cn2, 2^nd caudal medio-dorsal (transitory) neuron; nm, dorso-median neurite mass.

Fig 4. Z-Projections of immuno-stained specimens of Carinina ochracea.

Orientation indicated by compass (d, dorsal; l, left; r, right; v, ventral); apical is up. A and B: serotonin-like immunoreactivity (5HT-lir). A: Larva at four days post-fertilization (4-dpf) (γ: 0.9). The number of neurites and neurons of the brain, displaying 5HT-lir, have increased both ventrally (bv) and dorsally (bd). From the ventral portion of the brain-ring neurite bundle (br) the first, weak 5HT-lir neurites of the lateral nerve cords (nc) are detectable. 5HT-lir signals of the peripheral plexus (pp) are visible as well as numerous 5HT-lir apical signals (as). Note the weak unspecific signals of epidermal mucus gland cells (gc) B: Larva at 7-dpf (γ: 0.69). The neurites of the brain-ring (br) and the associated brain neurons (bv and bd) have further increased in number. The 5HT-lir dorsal neurite signals (dn) and neurite signals of the lateral nerve cords (nc) are extended posteriorly. More anteriorly located neurons (ln), associated with the lateral nerve cords, display 5HT-lir. 5HT-lir is also visible in the peripheral plexus (pp), the apical signals (as), and, albeit weak and unspecific, in some epidermal mucus gland cells (gc). C and D: FMRFamide-like immunoreactivity (RFa-lir). C: Larva at 2-dpf (γ: 0.96). The first RFa-lir signal detectable is a neurite-like mass (nm) located dorso-medially, anterior of the mouth opening. D: Larva at 3-dpf (γ: 1). The first pair of large, dorsal, RFa-lir neuron signals (bd) is visible. Their neurite signals project into the dorsal neurite-like mass that is continuous with the first RFa-lir neurites of the developing brain-ring (br). Note the unspecific signals of epidermal mucus gland cells (gc).

Fig 5. Schematic representation of nervous system development of Carinina ochracea.

Serotonin-like (5HT-lir) and FMRFamide-like (RFa-lir) immunoreactivity are color coded in magenta and green respectively; the general nervous system architecture as outlined by synapsin-like immunoreactivity (syn-lir) is shown in grey. All larval stages are drawn to scale with apical tuft (at), epidermal cilia, fronto-lateral epidermal invaginations (fi), midgut (mg), mouth opening (mo), and the pigmented larval eye (pe) serving as landmarks (cilia of the frontal epidermal invaginations, the stomodaeum, the midgut, and epidermal cilia in the aspect of view omitted), apical is up. For clarity, the peripheral plexus is not shown. A and B: from left to right: lateral view from right, ventral view, dorsal view, lateral view from left. Since the nervous system is roughly symmetrical only one side is shown (left: 5HT-lir, right: RFa-lir). A: Larva at five days post-fertilization (5-dpf). B: Larva at 7-dpf. Further explanations, see text.–bd, dorsal brain neuron; br, brain-ring; bv, vental brain neuron; ln, lateral nerve cord neuron; dl, dorso-lateral neuron; dn, dorsal nerve; nc, lateral nerve cord; or, oral ring neurite bundle; po, pre-oral neurite bundle; so, sub-oral neurite; vl, ventrolateral neuron.

Fig 6. Z-Projections of immuno-stained specimens of Carinina ochracea.

Orientation indicated by compass (d, dorsal; l, left; r, right; v, ventral); apical is up. FMRFamide-like immunoreactivity (RFa-lir). A: Larva at four days post-fertilization (4-dpf) (γ: 0.93). The neurites of the brain have become more numerous and form a closed ring (br). There are comparably large dorsal RFa-lir neurons (bd) associated with the brain-ring. From the ventral part of the brain-ring the first RFa-lir neurites of the lateral nerve cords (nc) extend posteriorly. Around the mouth opening RFa-lir neurite signals (or) start to form along with weak RFa-lir pre-oral neurite signals (po) connecting the oral ring neurite with the ventral part of the brain-ring. Note the putative growth cone signals associated with the left lateral nerve cord (arrow) B: Larva at 5-dpf (γ: 0.87). The RFa-lir neurites of the brain-ring (br) and the associated dorsal neurons (bd) have increased in number. The same is true for the oral ring neurites (or) and the neurites of the lateral nerve cords (nc) that are also considerably extended posteriorly. A pair of RFa-lir sub-oral neurites (so) connects the posterior of the oral ring with paired dorsolateral neurons (dl). C: Larva at 7-dpf (γ: 1). The RFa-lir neurites of the ventral part of the brain (br) are arranged in two concentrated bundles (arrowheads) and several, putatively individual neurites. The RFa-lir neurites of the oral ring (or) have become considerably stronger and the neurites of the lateral nerve cords (nc) extend further posteriorly. In their vicinity and proximal of them paired ventro-lateral neurons (vl) display RFa-lir. The RFa-lir pre-oral neurite bundle (po) that is connecting the ventral part of the brain-ring with the oral ring has become more distinct. The RFa-lir neurons associated with the dorsal part of the brain (bd) show a conspicuous, lobate shape. D: Larva at 10-dpf (γ: 1). The RFa-lir neurites in the brain-ring (br), and the oral ring neurite bundle (or) have increased in number and have become more distinct. The neurites of the lateral nerve cords (nc) seem to remain unchanged. Additional neurons displaying RFa-lir are found associated with the dorsal part of the brain-ring (bd). An additional pair of dorso-lateral neurons (dl) anterior of the first pair is detectable associated with a branch of the suboral neurite bundle (arrowhead).–gc, mucus gland cells.