Histological sections from a natural living <i>Hymeniacidon heliophila</i>.

<p><b>A)</b> Representative structures are indicated in 7 μm-sections stained with hematoxylin-eosin showing the choanosome with random organization of choanocyte chambers (*), aquifer channels (ac) and sparse collagen fascicles with associated fusiform aligned cells (cf). The inset shows a choanocyte chamber sectioned at the center with an apparent choanocyte flagellum (arrowhead) toward the center of the chamber. Arrows point to fragments of spicules that were generated after the sectioning of the sponge samples. <b>B)</b> Picrosirius staining revealing the ubiquitous presence of collagen with sparse collagen fascicles (cf) in the endosomal region. A bright red staining was also observed bordering the aquifer channels (ac) and choanocyte chambers (*).</p

Evidence for basopinacocytes transition from a 9-h regeneration culture observed by SEM and Picrosirius staining.

<p><b>A)</b> Differential contrast photomicrography of an endosomal region with cells with mesenchymal phenotype (outlined in black). Basopinacocytes already converted and migrated as mesenchymal cells after 9 h in culture (arrowheads point to the remaining collagen footprints). <b>B)</b> Picrosirius staining of the same field shown in “<b>A</b>”. <b>C)</b> Merged image from “<b>A</b>” and “<b>B</b>” showing the collagen nature of the basopinacocyte footprint (arrowheads). <b>D)</b> Putative field with epithelial-mesenchymal transition. Preserved basopinacocyte is shown in green and putative pinacocyte converting to mesenchymal morphology is colored in purple.</p

Cell movement during regeneration.

<p>Serial images from time-lapse video microscopy of the initial regeneration stage showing contraction of the organotypic culture. The area of the sponge tissue at the initial stage was outlined and projected onto the image at the final stage (black line in E) to show the tissue contraction. The regeneration time is shown in each stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178350#pone.0178350.s001" target="_blank">S1 Fig</a>).</p

Initial flow of small groups of mesenchyme-like cells.

<p>Serial images from time-lapse video microscopy of the initial regeneration stage showing the formation of small groups of cells with mesenchymal phenotype streaming (arrows) toward the tissue with higher cell density. The regeneration time is shown in each stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178350#pone.0178350.s003" target="_blank">S3 Fig</a>).</p

Dissociated cells integrate into the regenerating tissue and fewer clusters are formed.

<p><b>A)</b> Differential contrast micrograph of a 20-h culture with addition of dissociated cells over a regenerating tissue. The cell-dense regions (dark gray and black) are not spherical. These high-cell-density areas were formed from previous regenerating tissue, indicating less cell aggregation. <b>B)</b> Cell clusters with spherical morphology (black) were formed in regions without previous regenerating tissue. <b>C)</b> SEM of a 20-h culture with addition of dissociated cells over a regenerating tissue. No spherical cell clusters were observed in this representative figure. <b>D)</b> SEM of a cell cluster formed in a region without regenerating tissue. <b>E)</b> Representative micrograph of 20-h regeneration after addition of dissociated cells showing advanced epithelialization and absence of fusiform cells. <b>F)</b> Higher magnification of a field shown in <b>E</b>.</p

Basopinacocytes remain basal after 4 h mesenchymal transition.

<p>Serial images of Hoescht-stained nuclei in two different focuses, red for basoepithelial layer and green for superficial mesenchymal stream. Four basopinacocyte nuclei were manually tracked, and their route labeled (A-I) with the corresponding color to show cell displacement. Most cells with red nuclei converted to mesenchymal morphology (as it is supported by their displacement) without change of optical layer (red-to-green). The regeneration time is shown in each stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178350#pone.0178350.s009" target="_blank">S9 Fig</a>).</p

Time-lapse video microscopy with addition of dissociated cells.

<p>Dissociated cells were added over a basopinacoderm layer (<b>A</b>). Yellow arrows indicate representative basopinacocyte nuclei. <b>B-H</b>) Two initial cell clusters from incomplete dissociation are identified (yellow outlines). Cells of these two clusters dispersed progressively, as evidenced by the enlargement of marked areas and lightening color, from black to gray. The regeneration time is shown in each stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178350#pone.0178350.s010" target="_blank">S10 Fig</a>).</p

Organotypic culture at the initial stage (T0).

<p><b>A)</b> Endosomal tissue observed by low magnification showing mineral skeleton, spicules, and distinct regions with varied tissue thickness on the coverslips. <b>B)</b> DIC of a representative field showing the basopinacoderm (gray at the center) and two flanking regions with high density of cells with mesenchymal phenotype (dark) and mineral spicules (blue). <b>C)</b> A representative field of the organotypic culture stained by DAPI to show the choanocyte chambers (arrowheads) in a region of high cell density. <b>D)</b> Representative field of the organotypic culture observed by SEM, showing a high density of mesenchymal cells and mineral spicules (blue) at the center and the basopinacoderm with some cells with mesenchymal phenotype and spicules at the border. <b>E)</b> Collagen fibers stained by Picrosirius in a high-density region of an organotypic culture. The adhesion spot of each basopinacocyte is present in the background (indicated by arrowheads).</p

Basoepithelial to mesenchymal transition.

<p><b>A-E)</b> Serial images (T0-T9 hrs) from time-lapse video microscopy of the basoepithelial to mesenchymal transition in a basopinacocyte-rich region. The homogeneous distribution of the basopinacocyte nuclei (white arrows in <b>A</b>) is visible at the initial stage. White circles illustrate the progressive increase of gaps in the basoepithelial layer. <b>F-K)</b> Serial images from time-lapse video microscopy of basoepithelial to mesenchymal transition in a basopinacocyte (green) and cells with mesenchymal phenotype (purple)-rich region. The homogeneous distribution of the basopinacocyte nuclei (white arrows in <b>F</b>) is visible at the initial stage (<b>F</b>) but is no longer visible at the final stage (<b>K</b>, gray area). Mesenchyme-like cell streams are formed at the final recording stage. The regeneration time is shown in each stage (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178350#pone.0178350.s007" target="_blank">S7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178350#pone.0178350.s008" target="_blank">S8</a> Figs).</p

Schematic representation of the methodological steps for the sponge regeneration model.

<p><b>A)</b> Naturally occurring <i>Hymeniacidon heliophila</i> at the intertidal zone of the collecting point. <b>B)</b> Explant is collected and a sharp blade is used for a transverse cut in relation to the erect chimneys, fistules and uneven digitations. <b>C)</b> Two apposed coverslips are introduced into the cut. <b>D)</b> This sandwich of sponge and coverslips was suspended for at least four days on a piece of Styrofoam floating in the bay close to the original collecting point. <b>E, F)</b> At the lab, the explant is opened and the two coverslips are removed from inside with internal sponge tissue attached over the area in previous contact. This was considered time 0 for regeneration, representing the initial stage and the normal histomorphology. The two areas over the coverslips with different color intensity represent basopinacoderm epithelial cell layer (light color) and dense regions with mesenchymal/amoeboid/spherical cells (dark color) and spicules (black lines). <b>G)</b> One coverslip is immersed in a Petri dish with fresh seawater (50 ml), mounted with a thin glass bottom for observation by inverted optical microscopy. Time-lapse video microscopy was recorded for the maximum time of 16.5 hours, with 1 photo every 10 min. Alternatively, regenerating tissue over the coverslips was investigated by SEM and histochemistry.</p