New data on dinophilid neurogenesis: a variation of a common pattern

The structure and development of the nervous system in Lophotrochozoa species is of the most important questions for comparative neurobiology. During the last the number of comprehensive studies on the development of serotonergic and FMRFamidergic systems skyrocketing. However, the detailed research of the earliest of Polychaeta neurogenesis is still sparce. Polychaeta is a huge taxon within Lophotrochozoa. Its representatives are widely used as model systems in developmental and physiological investigations. Dinophilidae is a unique Polychaeta group. Its representatives combine morphological traits of different lophotrochozoan taxa. Moreover, adult dinophilids demonstrate morphological similarity to a trochophore larva. This similarity may be associated with either archaic origin of this group or neoteny. The main goal of our study is to provide a detailed description of the earliest events in Dinophilus neurogenesis. These data might improve our understanding of Polychaeta development and evolution. Our work analyzes the earliest events in neurogenesis of both species by immunochemical staining with pan-neural marker (anti-acetylated α-tubulin antibodies) as well as with antibodies to specic neuronal molecules (5-HT, and FMRFamide). Our results demonstrate that the early neurogenesis is rather similar in two Dinophilus species, but has signicant difference from what was revealed in other Lophotrochozoa.

Although many lophotrochozoans exhibit general bauplan of the nervous system, some animals have peculiar characteristics in the larval nervous system; for example, some structures might be completely lost or highly elaborated. The data obtained on the previously neglected taxa (Phoronida, Sipuncula, Acoela, Entoprocta, etc.) not only show intriguing neuromorphological patterns, but help to elucidate the origin and phylogenetic position of some questionable groups. For example, the neural systems of phoronids and brachiopods are shown to have a number of similarities with deuterostomian neural systems [69][70][71]83], challenging molecular phylogeny view considering phoronids and brachiopods as Protostomia [69]. Likewise, similarities between the neurogenesis of creeping entoproct larvae and molluscs propose a monophyletic assemblage of Mollusca and Entoprocta [92,95,96]. The data on Acoela neural organization support separation of this group from Platyhelminthes and allow to consider Acoela as the most primitive living Bilateria [50]. Segmental organization of the central nervous system at the larval stages of Sipuncula and Echiura (worms lacking any trace of metamerism) demonstrates the segmental origin of Sipuncula [51,52] and Echiura [36][37][38].
Genus Dinophilus includes only two species: Dinophilus gyrociliatus [77] and Dinophilus taeniatus [31]. Our work analyzes the earliest events in neurogenesis of both species by immunochemical staining with pan-neural marker (anti-acetylated α-tubulin antibodies) as well as with antibodies to speci c neuronal molecules (5-HT, and FMRFamide). Our results demonstrate that the early neurogenesis is rather similar in two Dinophilus species, but has signi cant difference from what was revealed in other Lophotrochozoa.

Materials And Methods
Culture maintaining. The culture of Dinophilus gyrociliatus was established from the Mediterranean Sea, Napoli Zoological station (Italy). In laboratory the animals were reared in small plastic aquaria with arti cial seawater (33‰ salinity) at 21º C without aeration. Worms were fed with homogenized frozen Urtica leaves once a week. Water was changed every day in order to collect all developmental stages of embryos. D. gyrociliatus cocoons contained 1-9 large (female) embryos and several small (dwarf males) embryos. In our work we studied only female (large) embryos of D. gyrociliatus, so we use the term "embryo" here in relation to female embryos only.
The work on Dinophilus taeniatus was conducted during summer seasons at the White Sea, Pertsov White Sea biological station (Russia; 66º34'N, 33º08'E). The worms were collected in a subtidal during a low tide and were then kept in small tanks without aeration and in six-well plates at 10 °C in ltered seawater. The worms were fed with diatomic Phaeodactilum sp. and Pseudinicshea delicatissima every other day. Water was changed every time before feeding. The cocoons of D. taeniatus contained up to 30 embryos the same size of both sexes.
Both Dinophilus species laid cocoons regularly on a regular base. The cocoons were gently removed from the aquaria using glass Pasteur pipette and placed into 30 mm Petri dishes with ltered sea water. The embryos were mechanically extracted from the cocoon and fertilization envelope with tiny needles. Then they were tested under a microscope in order to select the only embryos without any visible damages for further immunocytochemical procedures.
Cell nuclei were stained with DAPI (0,25 µg/ml). After 3 × 10 min washing in PBS, the specimens were mounted on glass slides in 70% glycerol for microscopic analysis and image acquisition.
Confocal scanning microscopes TCS-SPE, TCS-SP5 (Leica Microsystems, Germany) and Nikon A1 (Nikon, Japan) equipped with the appropriate set of lasers, lters, and detectors were used for the detailed study of the specimens. Stacks of optical sections taken with 0.3-0.5 µm intervals were processed with Leica LAS AF (Leica Microsystems, Germany) and Image J (NIH, USA) to obtain two-dimensional images. For each certain preparation the optimal number of stacks was selected in order to demonstrate the structures of interest. To compose a picture, optical sections were projected into one image and then imported into the Adobe Photoshop CC; the only parameters to be changed were brightness and contrast.

Relative staging
Duration of embryonic development in Dinophilus gyrociliatus differs from that of Dinophilus taeniatus signi cantly. D. gyrociliatus embryos hatch after 5 days after oviposition whereas D. taeniatus embryos hatch approximately two weeks after oviposition. External ciliary structures appear sequentially in anterior-to-posterior direction both species. Each stage has speci c ciliary pattern. We used external ciliation as marker for staging of D. taeniatus and D. gyrociliatus embryos.
It's noticeable that during the course of development Dinophilus embryos curve with it's ventral ciliary eld outside (Fig. 1). Thus in our Figures we have photos of curved embryos, not round or oval-shaped, especially at late stages.
In both studied species the rst cilia are visualized after stomadeum emergence when embryos are at a gastrula stage. These cilia appear in the epishere above the stomadeum and form a prototroch ( Fig.1 a).
Then prototroch develops a circular band in ventro-dorsal direction. Prototroch extends to the dorsal side without forming a complete ring.
After the prototroch is developed, ventral ciliary eld appeares at the midline of the ventral hyposphere beneath the mouse opening. This ventral ciliary eld elongates in rostro-caudal direction ( Fig.1 b). During the further embryogenesis the ventral ciliary eld becomes wider and bushier, and its cilia become longer.
After that, another ciliary band (acrotroch) appears in an episphere above the prototroch. Acrotroch also extends to the dorsal side and forms an incomplete ring. In hyposphere ciliated transversal stripes emerge under the prototroch on both sides of ventral ciliary eld. In the course of development these ciliary stripes fuse at the dorsal side forming ciliary bands ot the hypospere ( Fig.1 c). The number of these ciliary bands increases in rostro-caudal direction, reaching ve by the moment of hatching ( Fig.1 c).
Thus, we suggest the following postgastrulation embryonic developmental stages in Dinophilus: prototroch, ventral ciliary eld, and ciliary bands stages ( Fig. 1 a -c). The name of the stage refers to the moment of a certain structure formation (i.e. the stage of prototroch means that the prototroch begins to develop, but no other ciliary structures have yet been formed, etc.) ( Table 1). We described below the general structure of a nervous system as well as the distribution of speci c neurons at these stages during early embryogenesis of D. taeniatus and D. gyrociliatus. In both D. gyrociliatus and D. taeniatus the earliest neurons develop during the prototroch stage. These earliest nerve elements construct the scaffold for nervous system. In both species rst neurons demonstrate no 5-HT-LIR and no FMRFa-LIR (Fig. 2).
In D. gyrociliatus the rst neuron appears at the periphery of anterior part of the embryo, above the mouth (Fig. 2 a, d, Fig. 8 a-a''). This cell has two lateral processes, growing on the ventral side along the prototroch (Fig. 2 a, Fig. 8 a-a''). A bit later, the projects of the rst cell develop the basic structures of the nervous system: neuropil in the episphere, two ventrolateral bundles, two commissures and two lateral bundles ( Fig. 2. b, e). At the level of the rst commissure another pair of neurons differenciates (Fig. 2 b). Their projects extend along the scaffold formed by the rst cell ( Fig. 2 b, e). Thus, the scaffold of the nervous system is build by projects of the only one cell (Fig 2. b, e, d).
In D. taeniatus one of the rst neurons appears at the apical of anterior part of the embryo (Fig. 2 f), the other neuron is at the caudal part on the dorsal side ( Fig. 2 f, g, l). Anterior cell develops lots of processes towards the head neuropile. This cell is transitory and disappears a bit later at the prototroch stage (Fig 2  g, h). The projects of the anterior cell form the basic structures of the nervous system: thin neuropil in the episphere, two ventro-lateral bundles, rst commissure and two lateral bundles ( Fig. 2. g, j, k). At the level of rst commissure additional neurons appear (Fig. 2 g, j, k). Thus, the scaffold of the nervous system is build by projects of the only one cell (Fig 2. g-k). Caudal cell develops two lateral processes, but they are short and still on the dorsal side (Fig 2 f, l).

Neurogenesis during the ventral ciliary eld stage
During ventral ciliary eld stage rst 5-HT-LIR and FMRFa-LIR elements differentiate, but they form only minor part of the whole nervous system in both species D. gyrociliatus and D. taeniatus.
D. gyrociliatus embryos demonstrate quite developed nervous system at the ventral ciliary eld stage. The rst nerve cell is still above the mouth opening, it becomes bigger (Fig. 3 a, Fig. 4 a, Fig. 5). Neuropile in the episphere contains more processes, become more dense and prominent (Fig. 3 a, Fig. 4 a, Fig. 5). No neurons are detectable in neuropil region (Fig. 3 a, Fig. 4 a, c, e, Fig. 5 b-d). The main parts of nervous system are detectable: neuropil, ventro-lateral, medial and lateral bundles, commissures (Fig. 3 a, Fig. 4 a, Fig. 5). On the ventral side two ventrolateral and lateral bundles are detectable, they contain more processes (Fig. 3 a, Fig. 4 a, Fig. 5). Additional ventral medial bundle extends in rostro-caudal direction ( Fig. 3 a, Fig. 4 a, Fig. 5). At the caudal region ventral and medial bundles extend to the dorsal side of an embryo (Fig. 3 c, e, f). The caudal cell appears on the dorsal side of an embryo ( Fig. 3 f, Fig. 4 f ), its' processes grow towards ventro-lateral bundles and join them.
First 5-HT-LIR neurons develop at the level of the rst commissure (Fig. 3 b). These neurons do not have any cilia (Fig. 3 b, c, e). The processes of these neurons extend to the main nervous structures: ventrolateral, medial bundles and head neuropil (Fig. 3 b, c, e).
First FMRFa-LIR detectable in the anterior cell above the mouth opening ( Fig. 4 b-e, Fig. 5 a-c, Fig. 8 Moreover, the processes of the cell also FMRFa-LIR (Fig. 4 b-e). Thus, FMRFa-LIR bundles detectable in the ventro-lateral, medial and lateral nerve bundles (Fig. 4b, c, Fig. 5 d, e). It's noticeable that, the rst cell has an FMRFa-LIR extension towards the surface and sensory cilia (Fig. 4 b, c, f, Fig. 5  D. taeniatus embryos also demonstrate quite developed nervous system at the ventral ciliary eld stage (Fig. 3g). The rst cell is not detectable, thus we propose it's transient. Neuropile in the episphere contains more processes, become more dense and prominent (Fig. 3 g, i, j). No neurons are detectable in its region ( Fig. 3 g, i, j). The main parts of nervous system are detectable: neuropil, ventro-lateral, medial and lateral bundles, commissures ( Fig. 3 g, h). Multiple projects extend to the surface from neuropile (Fig. i, j, k ). On the ventral side two ventro-lateral and lateral bundles are detectable, they contain more processes (Fig. 3  g, h, i ,k, l). At the caudal region ventral and medial bundles extend to the dorsal side and meet the caudal cell projects (Fig. 3 i, l). No prototroch circular nerve is visible (Fig. 3).
First 5-HT-LIR neurons in D. taeniatus embryo also develop at the level of the rst commissure (Fig. 3 h, i,  k). These neurons do not have any cilia (Fig. 3 k). They send processes to the main nervous structures: ventro-lateral bundles and commissure (Fig. 3 i, k, l).
No FMRFa-LIR is detectable in D. taeniatus at this stage.

Neurogenesis during ciliary bands stage
During ciliary bands stage rst 5-HT-LIR neurons become detectable in the epishere of an embryo in both D. gyrociliatus and D. taeniatus (Fig. 6). First FMRFa-LIR elements differentiate in D. taeniatus embryos (Fig. 7 h-k). 5-HT-LIR and FMRFa-LIR still form only minor part of the whole nervous system in both species D. gyrociliatus and D. taeniatus.
In D. gyrociliatus embryos nervous system revealed by tubulin immunostaining becomes more obvious and prominent. The main structures: neuropil, ventro-lateral, medial and lateral bundles contain more nerve bers (Fig 6 a, Fig. 7 a). Additional commissures develop in rostro-caudal direction (Fig 6 a, Fig. 7  a). Additional circular bundles develop (ciliary bands innervation) (Fig 6 a, Fig. 7 a ).
First 5-HT-LIR neurons appear in the head region of an embryo (Fig. 6 c, d). These four cells are located on the dorsal side of the head region just under the head neuropil ( Fig. 6 c, d ). Their processes intersect and go through the head neuropil ( Fig. 6 c, d ). Additional 5-HT-LIR neurons appear on the ventral side of the embryo (Fig. 6 e).
FMRfa-LIR elements in D. gyrociliatus become more prominent and easier to detect. The rst cell (is still here!) and its multiple processes in the neuropil, ventro-lateral, medial and lateral bundles (Fig. 7 b-e). Also some thin projects are detectable in the neuropil, but they form only small part of the neuropil (Fig.7 b-e ).
D. taeniatus embryos nervous system revealed by tubulin immunostaining also becomes more complex.
First 5-HT-LIR neurons in D. taeniatus embryos in the head region appear (Fig. 6 g, h, i). These four cells are located on the dorsal side of the head region just under the head neuropil, which is similar to D. gyrociliatus (Fig. 6 c, d ). Their processes intersect and go through the head neuropil (Fig. 6 c, h). Additional 5-HT-LIR neurons appear on the ventral side of the embryo (Fig. 6 e, j). Also 5-HT-LIR reveals media-ventral bundles on the ventral side (Fig. 6 b, e j).
First FMRFa-LIR neurons in D. taeniatus embryos develop at apical top of the head region and send their processes tho the neuropil (Fig. 7 h-k). At the caudal part of the embryo a single FMRFa-LIR cell with a short process differentiates in close proximity to the gut (Fig. h-k).

Discussion
We described the development of the nerve elements at the early embryogenesis of two dinophilid species: Dinophilus taeniatus and Dinophilus gyrociliatus. Neurogenesis in both species begins with the differentiation of the peripheral pioneering neurons. The scaffold of the central nervous system at rst is visualized with only a pan-neural marker (anti-alpha-tubulin antibody); the immunoreaction to speci c neuronal markers (anti-5-HT, and FMRFa-antibodies) develops later when the pattern of the central nervous system has already been established. We further discuss and compare early neurogenesis of dinophilids with that of other known lophotrochozoans. Data on the earlierst events in neurodevelopment are thus on high demand from both, a developmental and an evolutionary point of view.
External ciliation -a key structure to de ne a stage.
External ciliation pattern is a reliable key to distinguish developmental stages during embryogenesis in polychaetas [8]. Despite the fact that duration of embryogenesis is different in D. taeniatus and D. gyrociliatus (14 and 5 days, respectively), the mode of differentiation of external ciliary structures is the same in both species: prototroch appears the rst followed by ventral ciliary eld and then by ciliary bands. Thus, these ciliary structures (along with absolute time scale) can be used as temporal landmarks to allow comparison of developmental stages in two Dinophilus species with different duration of embryogenesis.
Though dinophilids have direct development, their embryonic stages are somewhat similar to free swimming trochophores. The prototroch stage matches well with the early trochophore stage since the last is characterized by the presence of the prototroch, the rst ciliary structure to develop in Spiralia [27,28,10,60,48,53,21]. Moreover, recent analysis of gene expression con rmed the homology of the indirect-developing annelid larval prototroch and the ciliary band of adult D. gyrociliatus [ 43,45] developing from the prototroch [44]. The ventral ciliary eld stage matches middle trochophore stage (or just trochophore) [60]. The stage of ciliary bands corresponds to late trochophore stage if to draw a parallel between a ciliary band and metatroch [39,21].
Neurogenesis inDinophilus-a variation of general template.
Early neurogenesis in vast number of lophotrochozoans often begins with the differentiation of pioneer neurons. These are transient 5-HT or FMRFamide IR cells located peripherally, the processes of which guide the differentiation of the central nervous system [14, 15, 17-19, 83-90, 11, 79]. Dinophilids demonstrate peripheral pioneering neurons at the anterior and posterior pole of the embryo at the early trochophore stage like other lophotrochozoans ( Fig. 9-12). However, the perikarya of the rst differentiated neurons are detected with anti-alpha tubulin antibodies only (Fig. 9). Their processes constitute the anlagen of the central nervous system: a head neuropil and the ventral neural cords. Later on, these rst processes will be joined by numerous newly differentiated nervous elements ( Fig. 9-12); the number of neural bers increases drastically during the development so the central nervous system at the late trochophore stage is very similar to that of juveniles [62,63,75,23,[43][44][45]. Therefore, the rst detected neurons in dinophilids are the rst elements of the central nervous system and certainly are not the typical pioneer neurons. This assumes that the pathways orchestrating differentiation of the central nervous system might be different in dinophilids and other lophotrochozoans. Moreover, the anterior pioneer neuron in D. gyrociliatus is not transient and seems to be present through all life long (Fig. 8). This may be regarded as paedomorphic feature in D. gyrociliatus.
Development of speci c neural elements.
The earliest pioneer neurons develop at the stage of early trochophorein both Dinophilus species.
In both Dinophilus species the rst neural cells with speci c IR differentiate at middle or late trochophore stages. The rst 5-HT immunopositive cells are found close to the rst commissure at the middle trochophore stage (Fig. 3). Later on, an additional four 5-HT IR cells emerges within the head neuropil at the late trochophore stage (Fig. 6). Thus, 5-HT IR is localized to the central nervous system and is similar in two dinophilid trochophores; moreover, it is also reminiscent of that in other polychaeta larvae where 5-HT IR perikarya are located at the level of the rst commissure as well as in a group of cells in the head neuropil [21,61,96]. The rst FMRFamide IR cells were found in a head region in D. taeniatus and D. gyrociliatus at the late and middle trochophore stages, respectively (Fig. 4, Fig. 5, Fig. 7). The early FMRFa IR nervous system is different in the two species. A large solitary FMRFamide IR cell found just above the mouth opening in D. gyrociliatus trochophore resembles a cell, described in polychaete trochophores Capitella teleta [61] and Phyllodoce maculata [89]. However, in C. teleta and P. maculata, this cell is not the rst differentiated FMRFamidergic neuron as is in D. gyrociliatus. Moreover, a similar cell was revealed in front of the cerebral ganglion in the sipunculan Phascolosoma agassizii [50,51]. D. taeniatus embryos develop a couple of FMRFa IR neurons closer to the rostral pole (right above the head neuropil), which are similar to two FMRFa immunopositive cells observed in a head region of another sipunculan Phascolion strombus [93].
Pioneer neurons and their role in lophotrochozoans.
Insights in pioneer neurons of lophotrochozoans are mostly based on immunochemical study of different members. Pioneer neurons are considered the rst nervous cells to be developed. Their bers grow and construct the "sceleton" of future nervous system. Later in development other neurons use this "sceleton" to navigate their ber growth. Pioneer neurons are often considered to be transient and disappear soon after constructing the "sceleton".
According to existing data these neurons are either 5-HT immunopositive or FMRFa-immunopositive.
The direction of neurogenesis can be rostro-caudal or vice versa. Matching and analyzing the common modes of neurogenesis in annelids and molluscs proposes that, despite of some inclinations, the rang of development in the formation of the nervous system are usually in the following way: peripheral pioneering neurons appear and their processes build a scaffold upon which the de nitive nervous system will be constructed. Then develops larval nervous system, and after that the de nitive nerve system appears along the pattern navigated by the pioneer neurons. After all the larval and pioneer neurons die or partly combine with the de nitive nervous system.
Our results demonstrate that in terms of immunoreactivity Dinophillids' pioneer neurons are different from most lophotrochozoans.
This means, that not only 5-HT or FMRFa play important role in axon guidance during neurogenesis. Additional research are need to nd the diversity of neurogenic transmitters.
Anterior cell inD. gyrociliatus-a special case.
The rst neuron in D. gyrociliatus appears at the stage of early trochophore at the antrerior extreme of the embryo at the midline above the prototroch and it's visualised only with anti-tubulin antibodies. No other neurons were visualised at this time by anti-tubulin immunostaining thus proposing this cell to be the rst neuron. The cell has a short apical neurite and several surface cilia (Fig. 3, Fig. 8, Fig. 9-12) and two bers, which run towards the prototroch on the ventral side.
At the stage of middle trochophore, this cell and its' bers reveal with anti-FMRFa antibodies. Its' bers are present in all crucial nervous system elements: neuropil, ventro-lateral, medial and lateral cords and spread in posterior direction.
At the stage of late trochophore this cell and its' bers visualized with antitubulin and anti-FMRFa antibodies. It becomes more conspicuous and noticable.
This cell is visible even after hatching at the stages of juvenile, adult and even senior individual. It's still revealed with anti-tubulin and anti-FMRFa antibodies only. It has characteristic morphological traits and position at the periphery above the prototroch and still has surface cilia.
1. The cell bears cilia at all stages, has short anterior surface ber and two basal bers, running towards the prototroch 2. This cell is located at the anterior extreme of the organism between surface epithelium and head neuropil.
3. This cell is revealed with tubulin and FMRFa immunostaining.
Thus we propose that D. gyrociliatus pioneer neuron is not transient. D. taeniatus do not demonstrate such type of cell. At the stage of early trochophore there is an anterior peripheral cell, revealed with antitubulin antibodies only, but this cell has no cilia and located at the apical pole of the epishere, it has multiple bers extenting towards the future neuropil. In D. taeniatus anterior cell is not sensory, due to lacking of cilia and no surface extensions. This cell is transient and disappears just after neuropil and ventro-lateral cords are constructed.
The fact that D. gyrociliatus pioneer neurons present through all life span and D. taeniatus pioneer neurons are transient may be treated as paedomorphic feature.
As was mentioned above, polychaetes Capitella teleta [62], Phyllodoce maculata [90] and sipunculan Phascolosoma agassizii [51,52] posess similar FMRFa-IR cell. But these cells do not rst in course of neurogenesis of these organisms. There is no data on the ontogeny of these cells, nobody knows wherether they transient or not.
Thus additional invesigations and experiments reqiured to clarify the role of this non-transient pioneer neuron in nervous system ontogeny.
Funding: The reported study was funded by RFBR, project number 19-3460040.

Conclusion
We revealed that the early neurogenesis of dinophilids demostrate the same pattern as most Polychaeta and Lophotrochozoans. The rst nerve elements located at the anterior and posterior poles of an embryo, their bers construct the scaffold of future nervous system. Nervous system develops in rostro-caudal direction in both dinophilid species. We propose that such mode of neurogenesis with peripheral pioneer cells is ancestral for Annelids. First nervous cells do not 5-HT-immunopositive or FMRFa-immunopositive, which is different from most described lophotrochozoans. Anterior cell in D. taeniatus is transient, while in D. gyrociliatus anterior cell is different in morphology and detectable during the whole life span. This fact may be considered as one more paedomorphic trait in D. gyrociliatus. In both dinophilids the rst 5-HTimmunopositive and FMRFa-immunopositive cells appear after the main scaffold of nervous system is preformed, and their bers are only small part of the main bundles. These data add to our knowledge of how diverse the early development in Lophotrochozoa could be and to what extent evolutionary changes might affect such conservative processes like early neurogenesis.

Declarations Funding
The reported study was funded by RFBR, project number 19-3460040, which is gratefully acknowledged.  bundles are being developed (g, i). In D.gyrociliatus the anterior cell is detectable when the nervous system scaffold is being constructed (b, d). At the cross of ventrolateral bundles and rst commissure additional neurons appear (arrows). In D. gyrociliatus ventrolateral bundles demontrate growth cones at the caudal part of an embryo (arrows). In D. taeniatus ventrolateral bundles also demonstrate growth cones (arrows). Abbreviations: pt -prototroch, n-nephridium, np-neuropile, c-commissure. Scale bars: a-c, f-h 25 μm; d,e 15 μm; i-l 10 μm.
Red-acetylated tubulin immunoreactivity; green-5-HT-like immunoreactivity; blue-DAPI. Upper part-Dinophilus gyrociliatus; Lower part-Dinophilus taeniatus. The scaffold of nervous system is developed in Figure 5 FMRFa-immunoreactive structures development at the ventral ciliary eld stage in Dinophilus gyrociliatus. Red-acetylated tubulin immunoreactivity; cyan-FMRFa-like immunoreactivity. The only cell body is located at the peryphery of the nervous system, it has surface cilia (a) just above the prototroch.
The cell has several processes, growing within the ventrolateral bundles and prototroch nerve (b, c, d).
Scale bar 25 μm.  Thin processes of these cells included into the neuropile. A bit later, additional FMRFa-IR cells appear in the head region (j) and their processes follow the ventral nerve bundles ( k). Scale bars: a-c, g-i20 μm; d-f 10 μm, j, k 15 μm.

Figure 8
The ontogeny of anterior cell in D. gyrociliatus from the prototroch stage to senior specimen. Redacetylated tubulin immunoreactivity;cyan-FMRFa-like immunoreactivity. The anterior cell is shown in frontal, apical and lateral projections through all developmental stages. At the prototroch stage (a) the cell appears, it's located above the prototroch, and bears cilia (arrow) on its' surface and do not demostrate FMRFa-IR. At the ventral ciliary eld stage the cell becomes FMRFa-IR ( b). Later this cell becomes more prominent and obvious (c, d, f) and it's obvious that cell is not a part of CNS, it's located sepate from the neuropil (d).Even at the stage of senior individual this cell is noticable ( g) and it's still prominent and contains cilia on its' surface. Scale bar: a-c, f-g 5 μm; d 40 μm.