Fig 1.
Oscarella lobularis habitus and simplified diagrams of its histological organization.
A. Oscarella lobularis in vivo and in situ. B. Close view of a lobe before operation. Diagram of ectosome structure and cell composition before injury (C) and after injury (D). ect—ectosome. See inset for cell legends. Scale bars: A—5 cm, B—5 mm. l—lobes, os—oscula.
Fig 2.
The morphology of intact Oscarella lobularis.
A. Semithin section of upper part of sponge. B. SEM of an exopinacocytes. C. SEM of an endopinacocytes. D. TEM of a choanocytes. E. SEM of an apopyle with apopylar cells. F. TEM of type 1 and type 2 vacuolar cells. b—symbiotic bacteria, bm—basal membrane, ca—canal cavity, cc—choanocyte chamber, ec—exhalant canal, enp—endopinacocytes, exp—exopinacocytes, f—flagellum, ic—inhalant canal, m—mesohyl, mv—microvilli, n—nucleus, o—ostium, ph—phagosome, ps—pseudopodia, v—vacuole, v1—vacuolar cell type 1, v2—vacuolar cell type 2. Scale bars: A—50 μm, B—10 μm; C—5 μm; D—2 μm; E—5 μm; F—3 μm.
Fig 3.
A. Semithin section of injured sponge. Box shows the magnified area displayed in panel C. B. SEM of the wound surface. C. Semithin section of a wound. D. SEM of the marginal zone of a wound and intact exopinacoderm. E. Semithin section showing the condensation of the extracellular matrix of mesohyl, collagen fibers and concentration of symbiotic bacteria in the wound, and choanocytes having migrated inside chamber cavities (arrowhead). F. SEM of intact exopinacocytes of marginal zones that have begun preparation for migration in the direction of the wound surfaces. This preparation is expressed in changes in the form of cells: from flat to oval and pseudopodia formation, directed toward the wound surface. cc—choanocyte chamber, d—debris; da—damaged area, exp—exopinacocyte, iz—intact zone, ps—pseudopodia. Scale bars: A— 200 μm, B—250 μm; C—100 μm; D—25 μm, E—50 μm; F—2 μm.
Fig 4.
Diagram of type 1 and 2 vacuolar cell dynamics during Oscarella lobularis ectosome regeneration.
Fig 5.
A. SEM of the wound surface. B. SEM of wound surface covered by ECM layer. C. SEM of the apopyles with apopylar cells and choanocyte chamber remaining on the wound surface. D. Semithin section of wound surface with spread choanocyte chamber (scc). SEM (E) and semithin (F) section of single dedifferentiated choanocytes (arrowhead), that appeared on wound surfaces. G, H. TEM of choanocytes labeled with Indian ink (arrows) beginning of dedifferetntiation at the wound surface. ap—apopyle, b—symbiotic bacteria, cc—choanocyte chamber, enp—endopinacocytes, ECM—extracellular matrix, mv—microvilli, ph—phagosome, n—nucleus, scc—sectioned choanocyte chamber. Scale bars: A—25 μm, B, C, D—15 μm; E, F—5 μm, G—100 μm, H—50 μm.
Fig 6.
SEM images of the wound surface and new exopinacoderm after 24 hours of regeneration.
A. A wound surface. B. A wound surface and marginal zone (mz). C, D. The new exopinacoderm with the cell "streams" (cells are artificially coloured to accentuate the "streams"). exp—exopinacocytes. Scale bars: A—D—20 μm.
Fig 7.
A. Semithin section of a wound surface during an epithelization by exopinacocytes, showing some small areas at the surface with no epithelium. B. TEM image of a wound surface covered with a dense ECM layer. C. TEM of the exopinacocytes of new exopinacoderm. D. TEM of the exopinacocyte that phagocytes a particle of cell debris at the sponge surface. E. TEM of the endopinacocytes of adjacent aquiferous system canals to the regenerate, which are migrating at the wound surface. F. Semithin section of the regenerate with new exopinacoderm and intact basement membrane (arrowhead) underlining the migrating sheet of endopinacoderm. b—symbiotic bacteria; cc—choanocyte chamber, ECM—extracellular matrix, enp—endopinacocytes, exp—exopinacocytes, gx—glycocalyx; m—mesohyl, o—ostium, ph—phagosome, v1—vacuolar cells type 1, v2—vacuolar cells type 2. Scale bars: A—50 μm, B—20 μm; C—5 μm; D, E—2 μm; F—50 μm.
Fig 8.
Transdifferentiation of the choanocytes and successive steps of choanocyte layer transdifferentiation into a new exopinacoderm.
A. Semithin section of wound surface with sectioned choanocyte chamber (scc). B. SEM of spread choanocyte chamber showing the beginning of choanocytes transdifferentiation into exopinacocytes. C. SEM of spread choanocyte chamber showing intermediate stage of choanocytes transdifferentiation within the exopinacocytes. D. SEM of spread choanocyte chamber showing the last stage of choanocytes transdifferentiation within the exopinacocytes. E. TEM of transdifferentiated choanocytes after labeling in Indian ink (arrows). F. TEM of new exopinacocyte with particles of Indian ink (arrow). cc—choanocyte chambers, exp—exopinacocytes, f—flagella, n—nucleus. Scale bars: A—25 μm; B—D—10 μm; E, F—2 μm.
Fig 9.
Diagram of transdifferentiation of the choanocytes (A) and successive steps of choanocyte layer transdifferentiation into a new exopinacoderm (B-D).
See Fig 1 inset for the legends of (B). bm—basal membrane, f—flagellum, mv—microvilli, n—nucleus.
Fig 10.
24 hours of regeneration and new ostia formation.
A, B. SEM images of small "rosettes" of exopinacocytes on a regenerate surface (cells are artificially coloured to accentuate). C, D. Semithin sections of different stages of ostia formation by exopinacoderm invagination (arrowhead). E. TEM image of exopinacoderm invagination. F. TEM of new ostia connected with a choanocyte chamber. cc—choanocyte chambers, exp—exopinacocytes, ic—inhalant canal, m—mesohyl, nic—new inhalant canal, o—ostia, v1—type 1vacuolar cells, v2—type 2 vacuolar cells. Scale bars: A, B—10 μm; C, D—25 μm, E, F—10 μm.
Fig 11.
SEM (A) and semithin section (B) of new functioning regenerate (exopinacoderm and choanoderm). C. SEM image of new exopinacoderm with ostia. D, E. Semithin section of regenerated ectosome with functioning ostia. F, TEM of a new exopinacocyte with Indian ink particles (arrow) inside of phagosome. G. TEM of a new endopinacocyte with Indian ink particles (arrow) inside of phagosome. bm—basement membrane, cc—choanocyte chambers, ec—exhalant canal, exp—exopinacocytes, ic—inhalant canal, n—nucleus, o—ostia, ph—phagosome, v—vacuole. Scale bars: A, B—150 μm; C—20 μm; D, E—10 μm; F, G—2 μm.
Fig 12.
A. SEM of the regenerate and intact marginal zone. B. SEM of a new exopinacoderm. C. SEM of new exopinacoderm with the ostia and exopinacocytes, covered with a layer of glycocalyx. D. Semithin section of regenerated ectosome. cc—choanocyte chambers, ec—exhalant canal, exp—exopinacocytes, ic—inhalant canal, mz—intact marginal zone, o—ostia. Scale bars: A—50 μm, B—20 μm; C—10 μm; D—100 μm.
Fig 13.
DNA synthesis in unwounded Oscarella lobularis (6 hours incubation with EdU); Z-stacks of confocal sections.
A. DNA synthesis in choanocytes. B. DNA synthesis in exopinacocytes. Insert: part of the exopinacoderm—labeled nucleus marked with arrowheads. EdU is red, DNA is green, tubulin is blue. cc—choanocyte chamber. Scale bar—15 μm.
Fig 14.
DNA synthesis in Oscarella lobularis during regeneration (stage I, 6 hours); confocal sections of the same specimen at different levels. Left panels—EdU, right panels—combined colors.
a, diagram of confocal sections of sponge. A, B: wound surface with crossed aquiferous canals at the level A-B (depth 5 μm). C, D: cross section of wounded sponge at level C-D (depth 30 μm). E, F: deep part of sponge body at level E-F (depth 75 μm). EdU is red, DNA is green. Arrowheads indicate labeled nuclei of choanocytes, arrows indicate labeled nuclei of pinacocytes, choanocyte chambers outlined at panels E-F. ac—aquiferous canal. Scale bars: A, B—15 μm, C, D—30 μm; E, F—30 μm.
Fig 15.
DNA synthesis in unwounded Oscarella lobularis (A, B) and during regeneration (C, D—stage II—12h; E, F—stage III—48h); stacks of confocal sections (3D, view at artificial transverse section).
Wound surface or exopinacoderm is at the top. EdU is red, DNA is green. Arrowheads indicate aquiferous canal, choanocyte chambers highlighted by dotted line. Scale bars—20 μm, thickness of the Z-stacks 70 μm.
Fig 16.
A. Diagram of the spreading of the layer of intact exopinacocytes, surrounding a wound during reparative regeneration of the ectosome in Oscarella lobularis to form new exopinacoderm. B. Diagram of the spreading of the layers of intact endopinacocytes from adjacent aquiferous system canals to form new exopinacoderm.