Cellular migration, transition and interaction during regeneration of the sponge Hymeniacidon heliophila

Abstract

Sponges have a high capacity for regeneration and this process improves biomass production in some species, thus contributing to a solution for the biomass supply problem for biotechnological applications. The aim of this work is to characterize the dynamics of cell behavior during the initial stages of sponge regeneration, using bright-field microscopy, confocal microscopy and SEM. We focused on the first 20 h of regeneration, during which blastema formation and epithelium initialization occur. An innovative sponge organotypic culture of the regenerating internal region is described and investigated by confocal microscopy, cell transplantation and vital staining. Cell-cell interaction and cell density are shown to affect events in morphogenesis such as epithelial/mesenchymal and mesenchymal/epithelial transitions as well as distinct cell movements required for regeneration. Extracellular matrix was organized according to the morphogenetic process observed, with evidence for cell-signaling instructions and remodeling. These data and the method of organotypic culture described here provide support for the development of viable sponge biomass production.

Fig 1. Histological sections from a natural living Hymeniacidon heliophila.

A) 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) 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 (*).

Fig 2. Schematic representation of the methodological steps for the sponge regeneration model.

A) Naturally occurring Hymeniacidon heliophila at the intertidal zone of the collecting point. B) Explant is collected and a sharp blade is used for a transverse cut in relation to the erect chimneys, fistules and uneven digitations. C) Two apposed coverslips are introduced into the cut. D) 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. E, F) 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). G) 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.

Fig 3. Organotypic culture at the initial stage (T0).

A) Endosomal tissue observed by low magnification showing mineral skeleton, spicules, and distinct regions with varied tissue thickness on the coverslips. 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). C) A representative field of the organotypic culture stained by DAPI to show the choanocyte chambers (arrowheads) in a region of high cell density. D) 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. E) 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).

Fig 4. Fine structure of an organotypic culture at the initial stage (T0), visualized by SEM.

A) Basopinacocytes (green) and mesenchymal cells (purple) with filopodia. B) Detailed structure of a basopinacocyte (green) showing anchoring fibers at the basopinacoderm (arrowheads). C) Endopinacocyte forming endopinacoderm (yellow). Spicules are shown in blue. D) Endosomal tissue with spicules (blue), round (light purple) and fusiform (dark purple) cells with mesenchymal phenotype with filopodium and isolated pinacocyte (yellow). E) Endosomal region dominated by fusiform mesenchymal cells (purple). Spicules are in blue. F) Basopinacocytes with filopodia (arrowheads) and some cells with mesenchymal phenotype over them. A long, thick collagen fiber (red) links two cells with mesenchymal phenotype (purple) and a spicule (blue). Basopinacocytes are represented in green. G) Mesh of thin collagen fibers (red) surrounding round and fusiform mesenchyme-like cells (purple).

Fig 5. Cell movement during regeneration.

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 S1 Fig).

Fig 6. Individual mesenchyme-like cell migration in low cell density.

Serial images from time-lapse video microscopy of the initial regeneration stage showing low density of cells with mesenchymal phenotype individually migrating for to form clusters. The white rectangle indicates a representative area in each image frame. The tip of the spicule inside the white square is the center of the indicated cluster. The regeneration time is shown in each stage (see S2 Fig).

Fig 7. Initial flow of small groups of mesenchyme-like cells.

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 S3 Fig).

Fig 8. Tissue displacement in high density of mesenchyme-like cells.

A-H) Serial images from time-lapse video microscopy of the initial regeneration stage showing high number of cells with spherical (nonaligned) morphology that are progressively pushed by fusiform mesenchymal cells forming a tissue stream (red). Spicules are identified by different colors to show their displacement and alignment inside the stream of cells with mesenchymal phenotype. The regeneration time is shown in each stage. I) Micrograph of a 20-h culture stained by Picrosirius to show the aligned organization of the collagen fibers inside the cell stream region (see S4 Fig).

Fig 9. Tissue morphology at the initial stage of regeneration (T9hrs).

A) Magnification of the area with cell stream marked at the inset image on the top left. Fusiform cells predominate in regions with cell stream. Filopodia are observed in all cells (white arrowheads). A representative area with collagen network of thin interlaced collagen fibrils organized into firm tracts is indicated by a red arrow. B) Magnification of the area marked at the inset image on the top left. Tissue with spherical cells predominating (white arrows) and some individualized epithelial cells (red arrowheads indicating a representative structure) and fusiform cells (yellow arrows for some of them). Filopodia are observed in almost all cells. C) 3D network of thin interlaced collagen fibrils organized into firm tracts (white arrows), that surround epithelial and mesenchyme-like cells. D) Long extracellular fibers (white arrowhead) with mesenchyme-like cell aggregation, debris (white arrows) and no epithelial cells.

Fig 10. Persistence of some choanocyte chambers during the initial stage of regeneration.

A-H) Serial images from time-lapse video microscopy of the initial regeneration stage taken from 2 focal plans. Each focal plane is shown in a column. Some representative choanocyte chambers are colored to show persistence after 11.7 h. The regeneration time is indicated for each stage. I-M) Serial images from time-lapse video microscopy of the initial regeneration stage of the endosomal tissue stained with vital staining Hoechst. The arrows indicate disorganization of choanocyte chambers after 3.5 h, and maintenance of the close contact among the choanocytes. The regeneration time is shown in each stage (see S5and S6 Figs).

Fig 11. Basoepithelial to mesenchymal transition.

A-E) 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 A) is visible at the initial stage. White circles illustrate the progressive increase of gaps in the basoepithelial layer. F-K) 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 F) is visible at the initial stage (F) but is no longer visible at the final stage (K, gray area). Mesenchyme-like cell streams are formed at the final recording stage. The regeneration time is shown in each stage (see S7 and S8 Figs).

Fig 12. Basopinacocytes remain basal after 4 h mesenchymal transition.

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 S9 Fig).

Fig 13. Evidence for basopinacocytes transition from a 9-h regeneration culture observed by SEM and Picrosirius staining.

A) 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) Picrosirius staining of the same field shown in “A”. C) Merged image from “A” and “B” showing the collagen nature of the basopinacocyte footprint (arrowheads). D) Putative field with epithelial-mesenchymal transition. Preserved basopinacocyte is shown in green and putative pinacocyte converting to mesenchymal morphology is colored in purple.

Fig 14. Superficial epithelialization process.

A) A continuous epithelial layer of adjacent basopinacoderm (green) and superficial-covering pinacoderm (orange) was formed after 9 h of regeneration. Spicules are shown in blue. B) Nine-h regenerating culture showing incomplete superficial epithelialization. C) Three-day regenerating culture showing complete superficial epithelialization. D) Three-day regenerating culture showing complete superficial epithelialization and porocytes forming ostia (arrows). The observed gaps between the pinacocytes could be due to artifacts of fixation (B-D).

Fig 15. Time-lapse video microscopy with addition of dissociated cells.

Dissociated cells were added over a basopinacoderm layer (A). Yellow arrows indicate representative basopinacocyte nuclei. B-H) 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 S10 Fig).

Fig 16. Dissociated cells integrate into the regenerating tissue and fewer clusters are formed.

A) 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) Cell clusters with spherical morphology (black) were formed in regions without previous regenerating tissue. C) 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. D) SEM of a cell cluster formed in a region without regenerating tissue. E) Representative micrograph of 20-h regeneration after addition of dissociated cells showing advanced epithelialization and absence of fusiform cells. F) Higher magnification of a field shown in E.