Development of the paired imaginal discs

The results of this study confirm classical reports that the typical pilidium larva possesses three pairs of imaginal discs which develop as ectodermal invaginations -- the cephalic discs, the trunk discs, and the cerebral organ discs. This study disagrees with the only other detailed report on the order of appearance and the origin of these discs in pilidium larvae [23], and confirms Schmidt's report for Cerebratulus marginatus[24, 25]. Salensky, who relied solely on planktonic samples reported that all three pairs of imaginal discs appear simultaneously [[23]: p. 21]. Schmidt [24, 25] reared pilidia of C. marginatus from fertilization to the point of formation of almost all of the juvenile rudiments and reported that the discs appear in the following sequence: cephalic discs first, followed by the trunk discs, and finally, the cerebral organ discs. The present study shows that in M. alaskensis, the appearance of the paired imaginal discs follows the same order: the cephalic discs appear after about one week of development, followed by the trunk discs at two weeks, and the cerebral organ discs at about three weeks of development at ambient sea temperature.

My unpublished observations of development in another Northwest Pacific coast species Cerebratulus cf. marginatus (which is likely not the same species as Schmidt's C. marginatus from the Gulf of Naples, Italy), as well as in numerous pilidia belonging to different species captured in plankton in coastal waters of Washington and Oregon suggest that they all follow the same sequence of appearance of imaginal discs. Although the development of the juvenile was not the focus of his study, Cantell's observations of pilidium larvae of Lineus albocinctus, Lineus bilineatus, Micrura purpurea, and several unidentified pilidial morphotypes collected from plankton [12] suggest the same order of imaginal disc formation as described by Schmidt [24, 25] and in this study. It is easy to see, however, that if one is relying solely on the larvae obtained from plankton, as opposed to rearing them in the lab, one might be misled into thinking that all three pairs of imaginal discs appear simultaneously, if intermediate stages of development did not present themselves to the collector.

According to Salensky [23], all three pairs of imaginal discs develop as invaginations of subumbrellar pilidial epidermis, i.e. below the larval ciliated band, or in other words -- from the hyposphere. I document here for M. alaskensis that while the trunk discs and the cerebral organ discs indeed invaginate from the subumbrellar epidermis, the cephalic discs are derived from the umbrellar epidermis, i.e. above the larval ciliated band, or from the episphere (Figure 13). This particular observation is in agreement with the known cell lineage of the cephalic discs, which are derived from the first-quartet micromeres in pilidium larvae of Cerebratulus lacteus [[32], reviewed in [33]]. The cell lineage of the trunk discs and the cerebral organ discs remains to be determined using long-term lineage markers, but their position suggests that they are produced by the progeny of the second-quartet or the third-quartet micromeres.

Figure 13 Origin of the paired pilidial imaginal discs with respect to the larval ciliated band. This simplified diagram depicts a pilidium larva as a sphere, with the apical tuft pointing up and the subequatorial larval ciliated band represented by a dash line. The cephalic imaginal discs (cd) invaginate from the larval episphere, i.e. above the larval ciliated band, and have been shown experimentally to be derived from the first-quartet micromeres (1q cells). The trunk discs (td) and the cerebral organ discs (cod) invaginate from the hyposphere, i.e. below the larval ciliated band, and therefore are derived either from the second-quartet (2q cells), or the third-quartet micromeres (3q cells). Full size image

The proboscis rudiment in pilidial development

According to textbooks, the juvenile inside the pilidium larva develops from seven imaginal discs: the paired cephalic discs, paired trunk discs, paired cerebral organ discs, and an unpaired dorsal disc [1, 9, 30]. This is largely based on the most detailed published study of development of the juvenile inside the pilidium larva by Salensky [23]. Eight imaginal discs, including a separate proboscis rudiment are described in development of Desor's larva [26–29]. According to Salensky [23], the proboscis of the pilidium larva develops from the fused cephalic discs, and a separate proboscis rudiment is lacking. Interestingly, Bürger [22] and Schmidt [24, 25] described a separate proboscis rudiment in development of the pilidium larva, but this observation was not confirmed by Salensky [23] and is ignored in the more recent literature.

The results of the present study clearly show that in the pilidium larva of Micrura alaskensis there are eight separate juvenile rudiments, including an unpaired proboscis rudiment, which confirms earlier reports by Bürger for an unidentified species [22] and Schmidt for Cerebratulus marginatus[24, 25]. Because the proboscis rudiment is relatively small and is evident as a separate entity from the cephalic discs only for a short period of time, it is possible that Salensky and other investigators relying as they did on planktonic samples instead of culturing larvae, did not have the opportunity to observe and confirm its presence.

The proboscis rudiment in M. alaskensis does not develop as an invagination, contrary to Bürger's report [22]. It is not clear whether it arises by delamination from the pilidial epidermis, or from a cluster of mesodermal cells associated with the pilidial epidermis. In the first case the proboscis rudiment would be derived from the first-quartet micromeres. In the latter it would most likely originate from the 4d cell, the spiralian mesentoblast. The 4d cell gives rise to a population of scattered mesenchyme cells in the pilidium of Cerebratulus lacteus[32], including a small cluster of cells in vicinity of the cephalic discs, i.e. where proboscis rudiment would be developing later (Figure 7d in [32]). It appears very likely that the bilayered proboscis bud described here (Figure 6E) has a dual origin, so that its inner part ("the arm") is derived from the fused cephalic discs, as proposed by Salensky [23], while the outer part ("the sleeve") from the separate proboscis rudiment described here. Conceivably, the separate rudiment is derived from the 4d cell and gives rise to the muscle layers of the proboscis and the rhynchocoel, while the portion derived from the cephalic discs gives rise to the glandular epidermis of the proboscis. This would make sense in the context of a now widely accepted hypothesis that the nemertean rhynchocoel is homologous to the annelid and mollusk coeloms [34], which are also derived from the 4d cell. A study utilizing long-lasting cell lineage markers is necessary to determine which is the case.

Another piece of evidence that the proboscis is not derived solely from the cephalic discs, as suggested by Salensky [23], comes from an incidental observation of development of one particular cohort of pilidium larvae reared in the lab, which exhibited numerous abnormalities in juvenile, but not larval, development [Maslakova unpublished]. In many of these pilidia one or several juvenile rudiments were missing, while others appeared to be unaffected. Some larvae had all of the discs, including both of the cephalic discs, but were missing the proboscis. This suggests that even if cephalic discs normally contribute to the proboscis, it is possible that they may need some sort of inductive signal from the unpaired proboscis rudiment in order to do so. It would be possible to test whether the anterior unpaired rudiment observed in this study is essential to the formation of the proboscis by experimentally destroying it (e.g. by laser ablation) in otherwise normally developing pilidium larvae.

Origin of the dorsal rudiment

Although textbooks often describe all of the juvenile rudiments as invaginations of larval epidermis, the results of this study suggest that only the paired imaginal discs develop as distinct invaginations, whereas the proboscis rudiment and the dorsal rudiment do not. Salensky [23] reported that the unpaired dorsal disc develops via delamination from the pilidial dorsal epidermis. The present study is inconclusive as to whether the dorsal disc is of mesodermal origin (i.e. likely derived from the 4d cell) or is in fact an epidermal derivative (i.e. likely derived from one of the first-quartet micromeres).

Nephridial rudiments and the juvenile foregut

Several earlier studies describe a pair of esophageal pouches during juvenile development inside Desor's larva [26] and pilidium larvae [22, 23] as rudiments of the nephridia (although Nusbaum and Oxner [27] disagree with this interpretation). Bürger [22] reported that the two pouches completely separate from the esophageal cavity of the pilidium larva, fuse with the trunk discs, then branch like fingers of a glove, and that the nephridiopores develop after metamorphosis. Salensky [23] reported two pouches in a similar position (between the cerebral organ discs and the larval stomach), but interpreted them as being derived from the subumbrellar epidermis near esophagus, rather than the esophageal wall. He insisted on this particular distinction, because he believed that the pilidial esophagus is of endodermal origin, and the nephridia must be derived from the ectoderm. Histologically, however, there is no difference between the subumbrellar epidermis and esophageal epidermis; one gradually transitions into the other. Both are composed of squamous cells, and both are likely ingested by the juvenile during pilidial metamorphosis. Moreover, cell lineage analysis in the pilidium larva of Cerebratulus lacteus[32] shows that the larval esophagus is derived from the second-quartet and third-quartet micromeres, i.e. is of ectodermal origin. More importantly, however, Salensky [23] did not observe these pouches closing off from the esophagus, even in the latest stages of juvenile formation inside the pilidium larva, nor did he observe any branching.

I observed a pair of thick-walled rounded esophageal pouches sandwiched between the cerebral organs and the larval stomach in advanced developmental stages (e.g. "complete proboscis" and "hood") of M. alaskensis (Figure 12, Additional files 6 and 7 -- Movies 6 and 7). Similar to Salensky [23], I did not observe these pouches closing off from the esophagus, or their distal ends branching. Judging from their position and morphology, it seems unlikely that they represent rudiments of nephridia (see also [27]). A more likely explanation is that they form the juvenile foregut, while the larval stomach gives rise to the juvenile midgut. In adult pilidiophorans, these two regions of the digestive tract are well differentiated histologically, and the transition between the two is very distinct. The adult foregut comprises a muscular, thick-walled tube of densely ciliated, glandular, and typically deeply folded epithelium. It appears unlikely that it could be derived from the larval esophagus, or the larval stomach, as previously suggested ([23] and references therein). I observed a distinctly bipartite digestive tract in newly metamorphosed juveniles of M. alaskensis with a thick-walled foregut positioned between the cerebral organs and the midgut, where the paired pouches used to be (data not shown). Upon request from one of the reviewers I am including a diagram, which shows relative position of the juvenile foregut rudiments to the various parts of the larval digestive system (Figure 14).

Figure 14 Relative position of the juvenile foregut rudiments in the pilidial digestive tract. The lower drawing represents the pilidial digestive tract as if it were straightened along the path of a hypothetical food particle (pink line in each), which passes: 1 - larval ciliated band, 2 - larval esophagus, 3 - esophageal pouches (paired rudiments of the juvenile foregut), 4 - ciliated ridges in the larval esophagus, 5 - stomach. Full size image

At the same time, I have also observed what appears to be a pair of protonephridia -- one on each side of the developing juvenile, in the immediate vicinity of the paired esophageal pouches described above, but not physically connected to them. These organs, provisionally referred to as the nephridia, are sandwiched between the cerebral organs and the stomach on two sides, and the esophageal pouches and the juvenile body wall on two other sides. Each nephridium has a branched distal portion, and at least one efferent duct leading through the body wall to the nephridiopore on the lateral side of the developing juvenile in advanced stages of M. alaskensis (Figure 12, Additional file 7). The position of these rudiments corresponds to that of protonephridia in adult nemerteans (restricted to the foregut region in most species). Because the nephridial rudiments are located in close proximity to the foregut rudiments, and because Bürger's study [22] was based on regular histological sections which are typically 7-8 μm thick (compared to 1-μm-thick confocal sections in this study), he might have been mislead into thinking that a) the nephridial rudiments are connected to the esophageal pouches, b) these pouches are closed off from the esophagus, and c) the nephridial openings develop only after metamorphosis. Future studies utilizing TEM of ultra-thin sections of advanced developmental stages of pilidium larvae should be able to confirm the nature of these provisional nephridia.

Larval and juvenile nervous systems

The results of this study largely confirm previous reports on the structure of the pilidial larval and juvenile nervous systems based on classical histological methods [22, 23], TEM [31] and immunohistochemistry and fluorescent microscopy [35].

Salensky [23] believed that the central nervous system of the juvenile (cerebral ganglia and lateral nerve cords) is derived from the cephalic imaginal discs, that there are no rudiments of the nervous system in the trunk discs, and that the lateral nerve cords invade the tissue of the trunk discs after fusion with the cephalic discs. Bürger [22] believed that the central nervous system is derived from both the cephalic imaginal discs and the trunk discs and that the juvenile nervous system develops at about the time when the cephalic and the trunk discs fuse with each other. According to Bürger [22] the cephalic discs give rise to the dorsal cerebral ganglia, while the trunk discs give rise to the ventral cerebral ganglia and the lateral nerve cords. The results of this study cannot confirm or disconfirm either hypothesis. It does appear very likely that at least some portion of the cerebral ganglia originates from the cephalic discs. However, a long-term lineage tracing is necessary to determine with confidence which imaginal discs give rise to various parts of the juvenile nervous system.