Transdifferentiation is a driving force of regeneration in Halisarca dujardini (Demospongiae, Porifera)

Sponge materials

Sponges Halisarca dujardini Johnston (Demospongiae, Chondrosida) were collected in the White Sea (Chupa Inlet, Kandalaksha Bay) in June–July 2010–2012 and in April of 2010 and March of 2011 near island Sotra in the North Sea (Bergen, Norway). Sponges were collected with their algal substrate and maintained in aquariums with sea water at +12 °C in the Marine Station of the Zoological Institute RAS (Kartesh, White Sea) and in the Sars International Center for Marine Molecular Biology (Bergen, Norway).

Field study permissions

No specific permissions were required for these locations because the study was done outside of the national park, private land or protected area. We confirm that the field studies did not involve endangered or protected species.

Surgical procedures

Manual dissections were performed with the aid of a stereomicroscope and use of Castroviejo scissors and microscalpels. For each experiment, a portion of apical ectosome (superficial part of the sponge) was removed, along with a directly underlying section of the aquiferous system (choanocyte chambers and canals). The depth of excision varied slightly between the operations, from 150 to 500 µm. The osculum (exhalant opening) remained intact in all cases. Wounded sponges were maintained in 40 mm Petri dishes with 0.22 µm-filtered sea water replaced daily. Six individuals were observed for each time point at various intervals until regeneration was complete. Timing was started from 0 h (wounding); regeneration was monitored under a dissecting microscope and specimens were fixed at 3, 6, 12, 24, 48 and 72 h after excision.

Light and electron microscopy

For microscopic investigation sponges were fixed in a solution composed of one volume of 25% glutaraldehyde, four volumes of 0.2 M cacodylate buffer and five volumes of filtered seawater for 2 h and post-fixed in 2% OsO₄ in seawater at room temperature for 2 h. After fixation, samples were washed in 0.2 M cacodylate buffer and distilled water successively, and finally dehydrated through a graded ethanol series. For semi thin sections and transmission electron microscopy (TEM) specimens were embedded in Araldite resin. Semi thin sections (1 µm in thickness) were cut on a Reichert Jung ultramicrotome equipped with a diamond knife “Micro Star” 45°, then stained with toluidine blue, and observed in a WILD M20 microscope. The digital photos were made on a Leica microscope DMLB with the system of photo capture Evolution LC color. Ultrathin sections (60–80 nm) were cut with a Leica UCT ultramicrotome equipped with a diamond knife Drukkert 45°. Ultrathin sections, contrasted with uranyl acetate, were observed under a Zeiss-1000 transmission electron microscope (TEM). For scanning electron microscopy (SEM), fixed specimens were critical-point-dried, sputter-coated with gold-palladium, and observed under a Hitachi S 570 SEM.

Cell proliferation investigation

For labeling of newly synthesized DNA we used 5-Ethynyl-2′-deoxyuridine (EdU; Molecular Probes), which is incorporated into genomic DNA during S-phase (Salic & Mitchison, 2008). A series of experiments with different concentrations of EdU and incubation times was performed to establish optimal conditions of labeling. In most cases, incubation time between addition of EdU and fixation was 6 h although images with longer incubation are also presented. EdU was added 6 h before the end of the experiment; thus, in the case of a 6 h of regeneration, EdU was added immediately after surgery, and in experiments with 12 h of regeneration—6 h after surgery and 6 h before fixation. This experimental setup resulted in labeling of DNA synthesized during the last 6 h of regeneration in each experiment. Stock solution of EdU in DMSO (500 mM) was added to filtered sea water with sponges to a final concentration of 800 µM. Six sponges were used for each stage of regeneration. Non-operated sponges, placed at the same time in the seawater with EdU as the operated experimental sponges, served as experimental controls. Sponges cultivated in sea water without EdU served as negative technical controls.

Specimens at different stages of regeneration and immediately after wounding were fixed in 4% paraformaldehyde in PBS (phosphate buffered saline with pH 7.4) for 1 h followed by washing in PBS. Fixed and rinsed specimens were blocked in 5% normal sheep serum/0.05% Tween-20 in PBS during 1 h at room temperature. Specimens were treated with the Click-iT^® EdU Alexa Fluor^® 488 Imaging Kit (Molecular Probes, Carlsbad, California, USA) according to manufacturer instructions, and then incubated in mouse anti-tubulin antibody (Sigma-Aldrich, Seelze, Germany) at 4 °C overnight. Specimens were then rinsed three times in blocking buffer during 3–5 h at while rotating and incubated in Alexa Fluor^® labeled anti-mouse secondary antibody (Molecular Probes, Carlsbad, California, USA). After 12 h of incubation in the secondary antibody, specimens were rinsed and the DNA was stained with TO-PRO^® -3 (Molecular Probes, Carlsbad, California, USA) at concentration 1 µM in PBS during 2 h. Rinsed samples were mounted in DABCO-glycerol and images obtained using a multiphoton confocal microscope Leica TCS SP5 MP with white laser WLL. Pictures processed with LAS AF Lite (Leica Microsystems, Wetzlar, Germany) and ImageJ software (http://imagej.nih.gov/ij/).

Anatomy and cytology of Halisarca dujardini

Halisarca dujardini is a predominantly shallow water, encrusting sponge growing up to 0.5–4 cm in diameter with thickness of 0.5–12 mm (Fig. 1A). Inorganic and organic skeletons are absent, and the surface is smooth. The body is composed of the peripheral ectosome and the internal choanosome (Figs. 1B and 1C; see also cartoon representation on Fig. 2). The ectosomal region is up to 27 µm thick and consists of three layers: (1) external parts of T-shaped exopinacocytes (Fig. 1D) which are covered an acellular mucous cuticle; (2) layer containing collagen fibrils and rare spherulous cells; and (3) the inner layer, consisting of condensed collagen fibrils and the cell bodies of exopinacocytes. The choanosome (Fig. 1B) makes up the greatest volume of the sponge body and is composed of choanocyte chambers (built of choanocytes the only cell type which is flagellated, Fig. 1C), aquiferous canals (built of endopinacocytes, Fig. 1E) and the mesohyl. Populations of free cells in the mesohyl of H. dujardini include: archaeocytes, lophocytes, spherulous cells, granular cells, microgranular cells, and vacuolar cells (Ereskovsky et al., 2011) (Figs. 1E–1H). Neither specialized cell junctions, nor basement membranes could be identified in association with any of the cell types.

Description of regeneration

The observed regeneration processes in Halisarca dujardini can be subdivided into three stages: I—formation of “regenerative plug” (0–12 h), II—wound healing and formation of a “blastema” (12–36 h), and III—restoration of ectosome and choanosome (36–72 h).

Stage I—formation of a “regenerative plug”

Sections of ectosome together with the directly adjacent choanosome were surgically removed at the beginning of the experiment (Fig. 2). Immediately after the injury, the wound surface retracts, leaving the surface of the intact ectosome protruding around the edges of the wound (Figs. 3A and 3B). The ectosome and the upper areas of choanosome are destroyed (Fig. 3C). However, the structure of the aquiferous system in the deeper zone of choanosome is preserved. The wound surface is covered with exudate and cell debris. A number of amoeboid cells can be identified among the extracellular matrix fibers (ECM) (Figs. 3A–3D). Choanocyte chambers and aquiferous canals in the damaged zone disintegrate: cells of these structures lose contacts with adjacent cells and change their shape from trapeziform (choanocytes) and flat (endopinacocytes) to spherical or amoeboid (Figs. 3E–3G). These changes mark the beginning of dedifferentiation (followed by transdifferentiation, see next section), which is accompanied by migration of the cells into the mesohyl. The de- and trans-differentiating cells can be tracked thanks to preservation of their characteristic organelles. The natural label of dedifferentiated choanocytes is the flagellar apparatus (basal body and accessory centriole situated next to the nucleus), which remains in the cell. The collar of microvilli is reduced and disappears, and the flagellum resorbs (Fig. 3E), although some dedifferentiated choanocytes keep their flagella up to the last stage of transdifferentiation into an exopinacocyte (see: Fig. 9H). Endopinacocytes, which are flat in their intact state, assume spherical to amoeboid shapes and can be identified by their small anucleolated nuclei.

During the initial six hours after injury, the “regenerative plug” forms at the wound area. It consists of exudate (mucus), bacteria and cell debris, and includes dedifferentiated pinacocytes, choanocytes, spherulous and granular cells of the mesohyl (Figs. 3A and 3D). In the wound area, vacuolar cells, phagocytes and archaeocytes can also be found. With exception of the spherulous cells, all cells are filled with phagosomes, testifying to their involvement in active phagocytosis of the cell debris (Figs. 3E–3G).

At 12 h after injury, the wound surface is completely covered with exudate and ECM (Figs. 4A and 4B), with minor amount of debris still detectable at the surface. Underlying choanosome areas are disorganized (Figs. 4C–4F). Intact exopinacocytes, surrounding the wound, change the shape of their flat cytoplasmic outgrowths, and the peripheral parts of adjacent cells become separated (Fig. 4G). Reorganization of the ectosome begins with restoration of its middle layer containing interlaced collagen fibrils organized into firm tracts (Fig. 4A).

Stage II—wound healing and blastema formation

24 h after wounding, the ectosome is composed of a well-developed dense collagen layer, which is comparable to that of the intact ectosome (Fig. 5A compare with Figs. 8A and 8B).

Archaeocytes, which are toti- or pluripotent cells of demosponges (Funayama, 2013), and the dedifferentiated cells (choanocytes, pinacocytes) accumulate beneath the wound surface (Fig. 5B). This accumulation of cells strikingly resembles blastemas formed during regeneration of other animals. In line with this similarity, the new exopinacoderm (ectosome) and aquiferous system (choanosome) structures form from the cells of the blastema.

Differentiation of exopinacocytes is individual, and begins with cells migrating from the blastema towards the wound surface. Upon reaching the wound, the cells flatten, assuming positions parallel to the surface (Figs. 5C and 5D). Ultrastructural characters demonstrate that the migrating cells include archaeocytes, dedifferentiated choanocytes (Figs. 5E and 5F), endopinacocytes and exopinacocytes and newer other mesohyl cells types. Then, these cells begin to transform into T-shaped exopinacocytes, characteristic of the adult sponge: most of the cytoplasm of the cell and the nucleus are inside the ectosome. A fine cytoplasmic bridge is retained between the apical plate and the immersed part of the cell (Fig. 6). The apical parts of new exopinacocyte extend into large polygonal plates.

At 24 h after injury, the part of choanosome which is underlying the wound remains anarchized, presenting as an accumulation of individual cells rather than continuous epithelium (Fig. 5G). These cells include many phagosomes (Fig. 5H); some cell debris remains visible in the area.

Stage III—restoration of ectosome and choanosome

At 48 h after injury, many newly differentiated exopinacocytes are present at the wound surface, but a continuous exopinacoderm has not yet formed (Fig. 7A). Numerous small cytoplasm microvilli develop at the apical surface of the newly formed exopinacocytes, showing that these cells are motile (Fig. 7B). Cell bodies of these differentiated exopinacocytes are concentrated in the middle layer of the ectosome (Figs. 7C and 7D).

During regeneration of the choanosome, the aquiferous canals and choanocyte chambers form by association of previously disaggregated endopinacocytes and choanocytes, respectively (Figs. 7D and 7E). Cells of each type contact each other and connect by interdigitations (Fig. 7F). Individual choanocytes form groups of cells, forming structures less compact than in the intact choanocyte chamber (Figs. 7G and 7H). Some mesohyl cells still include phagosomes.

Scar-free wound healing is complete approximately 72 h after the injury (Fig. 8). At this point, the new exopinacoderm is identical to the intact one (Figs. 8A and 8B). External flat cytoplasmic parts of exopinacocytes connect with each other by interdigitations. Their apical surfaces are covered with a glycocalyx. All mesohyl cells, as well as the choanocytes and endopinacocytes, are free of the phagosomes and have morphology typical of the intact area (Fig. 8D).

Cell proliferation during regeneration

Cells of intact sponges actively incorporate EdU, and after 6 h, labeling nuclei of numerous cells—mainly choanocytes—become marked (Figs. 9A and 9B). Interestingly, long exposure to EdU (20 h and more) results in labeling of the cytoplasm of granular cells, while nuclei of these cells remain unlabeled (Figs. 9C and 9D). This phenomenon has been observed in all sponge samples submitted to long term exposure to EdU (e.g., Fig. 9C).

Many of the choanocytes migrating towards the wound surface after 6 h of regeneration have labeled nuclei (Figs. 9C, 9E and 9F). However, at 12 h after wounding, the labeled cells are absent at the wound surface and underlying mesohyl, although many choanocytes in the deeper chambers are labeled (Fig. 9G). This suggests that choanocytes migrating towards the wound surface to form new exopinacoderm stop their DNA synthesis, while choanocytes in the deeper chambers (even though disorganized) continue to synthesize DNA.

During the reorganization of the aquiferous system between 24 and 48 h following wounding, labeled choanocytes and mesohyl cells are randomly distributed around the wound (Fig. 9H). Thus, at this stage no local proliferation at the wound surface or in deeper layers of mesohyl can be detected. Some choanocytes with their remaining flagella can be found between the newly differentiated exopinacocytes at the wound surface (Fig. 9H).