General observations on the anatomy of Syllis malaquini and the distribution of S-phase cells
The anatomy of Syllis malaquini is comparable to that of other syllids [39, 49, 59, 63] (Fig. 1a). The anterior region is characterized by a relatively cephalized prostomium (head, asegmental part) with eyes and antennae, and a peristomium (considered herein as the presetigerous segment following Heacox [64]) that bears two pairs of cirri. The body has a variable number of segments with cirri and parapodia that bear compound chaetae, and ends in a pygidium (tail, asegmental part). Running from anterior to posterior, the foregut of Syllis malaquini presents a tooth-bearing pharynx that extends to the first 6–8 segments; a proventricle, an organ that contains large muscular cells and two anterior plates, and that extends through 4–9 segments (see Fig. 4b in [49]); and a ventricle from which two ventricular caeca emerge (Fig. 1a). The intestine has two parts, recognized here by comparative anatomy as described for other syllids by Williams [65], Claparède [66] and Malaquin [39]. The anterior and medium portion of the intestine is identified as the glandular or secretory intestine (“l’intestin glandulaire, secretant”; sensu Malaquin [39]); and the posterior portion (last 7–9 segments of the body) is identified as the rectal intestine (“l’intestin rectal ou urinaire”; sensu Malaquin [39]) that contains urinary concretions (“concrétions urinaires”; sensu Malaquin [39]) located in two lateral grooves of the intestine walls. These urinary concretions can be seen as dark globules under the microscope (Fig. 1b/stage 5).
Uncut animals were stained with EdU/BrdU-pulse to observe proliferating regions of S. malaquini’s body in non-experimental conditions (see Methods). Our results showed that S-phase cells are irregularly distributed in the anterior and midbody, being located in the prostomial appendages and dorsal parapodial cirri of anterior segments (Fig. 2a, b). Additionally, S-phase cells of the midbody were located in the ventral midline, cirri, and digestive tube (Additional files 1a–f and 2a–c). In contrast, the posterior body showed a prominent accumulation of S-phase cells in the growing segments and segment addition zone (SAZ, where new segments are generated [67]), with an overall average of 2.5x more S-phase cells than in the anterior body (n = 6 specimens). The observed animals presented a 2:7 anterior-to-posterior labelled cells ratio in the EdU-labelled specimens (n = 3), and 4:9 ratio in the BrdU-labelled specimens (n = 3) (Fig. 2c, d, Additional file 2a–c). Last, similar to what was observed at the anterior ends, S-phase cells were seen in the ventral midline of the posterior ends (Additional file 2b).
Description of Syllis malaquini regeneration
In order to observe cell proliferation and blastema formation during regeneration, and the effect of proventricle absence in amputees of S. malaquini, we performed bisection-based experiments at different body levels: L1, L2, L3 and L2 + L3 (Fig. 1a; see Methods). In addition, in order to provide a more detailed description of our results, we divided the regeneration process in five developmental stages (Fig. 1b): 1) wound closure; 2) blastema development; 3) blastema differentiation, when the prostomium or pygidium appear; 4) resegmentation; 5) growth and restoration of body functions (the digestive tube is completely restored and the animals are able to feed). The mortality rate in our experiments was null.
Anterior regeneration
In all experiments, the specimens were able to completely restore the lost anterior body, although with few differences in pace (Figs. 3, 4, 5, and 6, Additional files 3 and 4). The main difference was that while in experiment L1 specimens accomplished regeneration around 10–12 dpa, amputees of experiments L2, L3 and L2 + L3 only reached stage 5 after 14 dpa (days post-amputation).
After bisection, the wound was closed by muscular contraction within two hours in specimens cut at L1 (in which the proventricle usually protrudes outside the body and has to be retracted back inside; Fig. 3i), and immediately closed in specimens cut at L2, L3 and L2 + L3 (Figs. 4i, 5i, 6 i). Somes specimens cut at L2 and L2+L3 showed the ventricle squeezed by the wound muscular contraction after 1 dpa (Fig. 4j, 6j). The wound was completely healed (stage 1) after 1–2 dpa in all experiments (Figs. 3j, 4k, 5j, 6k). Next, a blastema developed from 2–3 dpa (stage 2; Figs. 3k, l, 4 k, l, 5 k, l, 6 k, l; see also stainings in Figs. 3c, d, 4c, d, 5c, d, 6c, d). Stages 3 and 4 (blastema differentiation and resegmentation) started simultaneously after 4 dpa (L2, L3) or 5 dpa (L1, L2 + L3) (Figs. 3m, n, Fig. 4m, n, 5m, n, 6m, n). Resegmentation seemed to slow down while amputees enlarged their newly generated appendages and differentiated the foregut. Once the mouth appeared (after 6–7 dpa), segment addition continued from the zone close to the amputation site with a clear anterior-to-posterior developmental gradient (around 10–14 dpa, Figs. 3o, p; 4o, p, 5o, r, 6o, r). When six to eight segments had been regenerated (around 10–14 dpa, Figs. 3q, r, 4q, r, 5q, r, 6q, r), segment addition was definitively interrupted. Then, amputees advanced to stage 5, when they enlarged the newly formed appendages and completed the differentiation of the digestive tube to make it functional. Amputees cut at L1 regenerated a new pharynx around 10–12 dpa (Fig. 3q). Meanwhile, in the other experiments, the lost digestive organs were completely differentiated after 14–20 dpa. Amputees cut at L2 regenerated the pharynx and the proventricle after 14 dpa (Fig. 4r). Amputees cut at level L3 regenerated the pharynx, proventricle, and ventricle (with caeca) after 14 dpa (Fig. 5r). Amputees cut at L2 + L3 completed stage 5 after 15–20 dpa. Last, after 35 dpa, specimens of all experiments had almost reached the original body width (Additional file 3a–d). The regenerated pharynx and proventricle were morphologically similar to the original ones, even bearing the pharyngeal tooth and the proventricular plates (Fig. 7a). Thus, the examined specimens re-established digestive functions and were able to feed and grow again. By the end of the experiments, all specimens had been able to regrow six to eight new segments (n = 12, three specimens per experiment). No signs of stolonization were observed in the posterior end of any amputees at any time during the experiments.
Posterior regeneration
Posterior regeneration was characterized by the regeneration of the pygidium, SAZ, and the lost parts of the digestive tube. The regenerated segments arise from the SAZ and have an anterior-to-posterior developmental gradient. All specimens were able to regenerate the posterior body during the observed time of experimentation (35 dpa), although with some differences in the pace and extent of regeneration (Figs. 3, 4, 5, and 6, Additional files 3 and 4). In this case, despite amputees in experiments L2, L3 and L2 + L3 reaching stage 5 within 10–14 dpa, the wound closure (stage 2) in L2 was delayed, probably because proventricle retraction was a prerequisite. Additionally, in specimens cut at L1, regeneration was delayed by at least 21 days, and resulted in the regrow of up to three segments, while up to eight segments were regenerated in the other experiments. This difference was probably due to the need to regenerate all post-pharyngeal digestive structures (proventricle, ventricle, secretory and rectal intestine).
Specimens cut at L1 stretched the segments close to the wound within 1–2 h after bisection (Fig. 3i’). Wound healing was completed after 1 dpa (stage 1, Fig. 3j’) and a small blastema developed during 2–5 dpa (stage 2, Figs. 3k’–n′; see also stainings in Figs. 3c’, d′). The pygidium appeared after 6 dpa (stage 3, Fig. 3o’). The anal opening was developed (7–10 dpa, Figs. 3p’–q’) and the SAZ was re-established within 10–14 dpa, when two segments were clearly visible (stage 4, Fig. 3r’). Stage 5 had been reached after 20 dpa, and the proventricle, ventricle, and intestine were completely differentiated only after 35 dpa. At this stage, the specimens had regenerated two or three segments, within which the proventricle and the intestine were squeezed (Additional file 3a’).
When animals were amputated at L2, the wound remained open with the proventricle protruding outside the body for some time (ranging from half an hour to more than 24 h) (Figs. 4i’, j’). After this time, the proventricle was retracted by muscular action. The wound was completely healed at 2 dpa (stage 1, Fig. 4k’) and a small blastema started to appear from 3–4 dpa (stage 2, Figs. 4l’, m’; see also stainings in Figs. 4d’, e’). After 5 dpa, the pygidium was regenerated (stage 3, Fig. 4n’). First signs of resegmentation were seen around 6–7 dpa (stage 4, Figs. 4o’, p′). However, segmentation was slightly delayed in some specimens as can be observed in Figs. 4p′. After 10–14 dpa, the rectal intestine was recognized by the presence of urinary concretions (stage 5, Fig. 4q’, r’). Finally, the animals regenerated up to eight segments after 35 dpa and the first regenerated segment (the closest to the amputation site) almost reached the width of the stock body segments (Additional file 3b’).
Specimens cut at L3 closed the wound by muscular contraction immediately after bisection (Fig. 5i’), and completed stages 1 and 2 after 1 dpa (Fig. 5j’). The pygidium and two anal cirri could be distinguished at 2–3 dpa (stage 3, Fig. 5k’, l’). After 4 dpa, the amputees exhibited the first regenerated segment (stage 4, Fig. 5m’), and up to three segments were added in following days (stage 4, Figs. 5n’–p’). After 10–14 dpa, specimens reached stage 5, as recognized by the presence of urinary concretions in the rectal intestine (stage 5, Fig. 5q’, r‘). Stage 5 lasted at least until 35 dpa, when the animals had regenerated up to eight segments (Additional file 3C’).
After bisection, specimens cut at L2 + L3 close the wound by muscular contraction similarly to specimens cut at L3 (Fig. 6i’). The amputees completed stage 1 after 1 dpa (Fig. 6j’). The blastema developed from 2–3 dpa, (stage 2, Fig. 6k’, l’; see also staining in Fig. 6c’) and the pygidium appeared after 4–6 dpa (stage 3, Fig. 6m’–o′). Resegmentation started at 7–8 dpa (stage 4, Fig. 6p’). Around 10–14 dpa, the animals had regenerated up to four segments (Fig. 6q’–r’) and the intestine was completely restored after 14–20 dpa (stage 5). By the last day of observation, the animals had regenerated up to seven segments (Additional file 3d’).
Blastema development and cellular proliferation
In order to describe proliferation, S-phase cells were labelled using the thymidine analogues 5-ethynyl-2′-deoxyuridine (EdU) and 5-bromo-2′-deoxyuridine (BrdU). Those thymidine analogues are incorporated by annelid S-phase cells, as shown in previous studies [17, 23, 37, 68]. We used an EdU (pulse-chase)/Brdu (pulse) approach that allowed us to track cells that were in S-phase before amputation (labelled with EdU) and during regeneration (labelled with BrdU) [17, 69, 70].
Anterior regeneration
Despite the different cutting levels, cellular dynamics of S-phase cells during anterior regeneration were similar among all experiments (Figs. 3, 4, 5, and 6). An accumulation of BrdU pulsed cells on the border of the wound was first seen after 2 dpa, during blastema development (stage 2, Figs. 3c, 4c, 5c, 6c). S-phase cells labelled before bisection (EdU chased cells) were distributed in certain parts of the body of all amputees, mainly on the base of the dorsal cirri, epidermis, in the digestive tube, and in the posterior body. Interestingly, no EdU chased cells contributed to the development of the blastema; i.e. all S-phase cells in the blastema were BrdU pulsed ones, entering S-phase only after bisection (Figs. 3a–h, 4a–h, 5a–h, 6a–h and 7c). Following comparative data generated with other annelids and syllids, two different types of BrdU pulsed cells could be recognized in the blastema based on the shape of their nuclei [27, 28, 37]. Epidermal cells had elongated nuclei (Fig. 7c) and were located on the border of the blastema; endodermal cells had spherical nuclei (Fig. 7c) and were distributed in the inner blastema (gut region). Proliferation persisted at stage 5, during enlargement of the regenerates (14 dpa) (Figs. 3h, 4h, 5h, 6h).
Posterior regeneration
Similar to the anterior regeneration results, the blastema of posteriorly-regenerating individuals was seen as an accumulation of BrdU pulsed cells (Figs. 3a'–h', 4a'–h', 5a'–h', 6a'–h'). EdU chased cells were few and distributed in some parts of the dorsal cirri and in the ventral midline in amputees of all experiments. Notably, among all fixed posteriorly regenerating amputees (n = 96), double-labelled cells were only found in the digestive tube of two of the three amputees cut at L2 (stage 2, 2 dpa, Fig. 7d), which means that cells labelled with EdU before cutting were proliferating during and at the site of regeneration. However, double labelled cells were not found in later stages of posterior regeneration at L2. Remarkably, 1:2 of specimens cut at L3 and 1:7 of specimens cut at L2 + L3 showed the original part of the intestine prominently occupied by EdU chased cells (Figs. 5b’, d′, 6d’), while the regenerated part was occupied only by BrdU pulsed ones (Figs. 5e’–h′, 6e’–h′). Finally, during stage 5 (10–14 dpa), amputees showed BrdU-labelled S-phase cells in the regenerated segments and in the SAZ (Figs. 3g’, h′, 4g’, h′, 5g’, h′, 6g’, h′).