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Cell kinetics during regeneration in the sponge Halisarca caerulea: how local is the response to tissue damage?

Alexander BE, Achlatis M, Osinga R, van der Geest HG, Cleutjens JP, Schutte B, de Goeij JM - PeerJ (2015)

Bottom Line: We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h).There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges.The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam , Amsterdam , The Netherlands ; Porifarma B.V. , Ede , The Netherlands.

ABSTRACT
Sponges have a remarkable capacity to rapidly regenerate in response to wound infliction. In addition, sponges rapidly renew their filter systems (choanocytes) to maintain a healthy population of cells. This study describes the cell kinetics of choanocytes in the encrusting reef sponge Halisarca caerulea during early regeneration (0-8 h) following experimental wound infliction. Subsequently, we investigated the spatial relationship between regeneration and cell proliferation over a six-day period directly adjacent to the wound, 1 cm, and 3 cm from the wound. Cell proliferation was determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h). The percentage of proliferative choanocytes increased over time in all areas and after six days of regeneration choanocyte proliferation rates were comparable to steady-state tissue. Tissue areas farther from the wound had higher rates of choanocyte proliferation than areas closer to the wound, indicating that more resources are demanded from tissue in the immediate vicinity of the wound. There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges. Our data suggest that the production of collagen-rich wound tissue is a key process in tissue regeneration for H. caerulea, and helps to rapidly occupy the bare substratum exposed by the wound. Regeneration and choanocyte renewal are competing and negatively correlated life-history traits, both essential to the survival of sponges. The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.

No MeSH data available.


Regenerative tissue of H. caerulea.(A) Cross-section through BrdU- and hematoxylin-stained sponge tissue two days after wound infliction, showing regenerative tissue at the site of the wound, the location of mesohyl tracts containing cells, which were occasionally observed close to the wound, and an area away from the wound containing choanocyte chambers. Choanocyte chambers appeared 250 ± 8.9 µm from the edge of the wound tissue. (B) BrdU immunohistochemistry of a regenerative sponge labeled with BrdU for 6 h. Brdu-positive cells (brown-stained nuclei) were absent from regenerative wound tissue, and cells located in mesohyl tracts were BrdU-negative (blue-stained nuclei, white arrows). Tissue areas away from the wound that had retained their structural integrity contained BrdU-positive choanocytes (black arrows) and occasionally BrdU-positive mesohyl cells (black arrow heads). (C) Picrosirius red staining showed a higher density of collagen in regenerative wound tissue compared to areas farther from the wound. High densities of collagen could also be seen surrounding tracts in the mesohyl containing cells (white arrows). (D) Visualization of picrosirius red staining under cross polarized light revealed thin (green) and thick (orange) collagen fibers in all tissue areas.
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fig-2: Regenerative tissue of H. caerulea.(A) Cross-section through BrdU- and hematoxylin-stained sponge tissue two days after wound infliction, showing regenerative tissue at the site of the wound, the location of mesohyl tracts containing cells, which were occasionally observed close to the wound, and an area away from the wound containing choanocyte chambers. Choanocyte chambers appeared 250 ± 8.9 µm from the edge of the wound tissue. (B) BrdU immunohistochemistry of a regenerative sponge labeled with BrdU for 6 h. Brdu-positive cells (brown-stained nuclei) were absent from regenerative wound tissue, and cells located in mesohyl tracts were BrdU-negative (blue-stained nuclei, white arrows). Tissue areas away from the wound that had retained their structural integrity contained BrdU-positive choanocytes (black arrows) and occasionally BrdU-positive mesohyl cells (black arrow heads). (C) Picrosirius red staining showed a higher density of collagen in regenerative wound tissue compared to areas farther from the wound. High densities of collagen could also be seen surrounding tracts in the mesohyl containing cells (white arrows). (D) Visualization of picrosirius red staining under cross polarized light revealed thin (green) and thick (orange) collagen fibers in all tissue areas.

Mentions: All slides were examined under a light microscope (Olympus BH-2) and photographs were taken using an Olympus DP70 camera (Olympus, Tokyo, Japan). To investigate the cell kinetics of the choanocyte population directly next to the wound (the wound tissue contained no choanocyte chambers, Fig. 2A) in early regenerative tissue, percentages of BrdU-positive choanocytes were determined at each time point: t = 0, 0.5, 1.5, 2, 6, 8 h. Three histological sections were made from each tissue sample, each approximately 100 µm apart (Fig. 1A). From each section at least 400 choanocytes were counted making a total of at least 1,200 (3 sections × 400 cells) choanocytes counted per sponge. The ‘one population model’ (Nowakowski, Lewin & Miller, 1989; De Goeij et al., 2009) was used to estimate the growth fraction (GF; percentage of choanocytes involved in proliferation), duration of the cell cycle (Tc), labeling index (LI; percentage of choanocytes in S-phase), and the duration of the S-phase (Ts) for choanocytes in early regenerative tissue. Parameters (mean ± 95% confidence intervals) were estimated from the model according to an iterated least squares fit of the data using the following specified initial conditions: (1)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle f(t)=G F\times (t+T s)/T c,\hspace{1em}\text{for}&&\displaystyle \end{eqnarray*}\end{document}ft=GF×t+Ts/Tc,for(2)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle t\leq {T}_{c}-{T}_{s},\hspace{1em}\text{and}&&\displaystyle \end{eqnarray*}\end{document}t≤Tc−Ts,and(3)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle f(t)=\mathit{GF},\hspace{1em}\text{for}&&\displaystyle \end{eqnarray*}\end{document}ft=GF,for(4)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle t\geqslant {T}_{c}-{T}_{s}.&&\displaystyle \end{eqnarray*}\end{document}t⩾Tc−Ts. Data on the cell kinetics of the choanocyte population directly adjacent to the wound in early regenerative tissue were compared to choanocyte kinetics of steady-state sponges measured by De Goeij and colleagues in 2009, as this is the only study describing detailed cell cycle parameters in H. caerulea.


Cell kinetics during regeneration in the sponge Halisarca caerulea: how local is the response to tissue damage?

Alexander BE, Achlatis M, Osinga R, van der Geest HG, Cleutjens JP, Schutte B, de Goeij JM - PeerJ (2015)

Regenerative tissue of H. caerulea.(A) Cross-section through BrdU- and hematoxylin-stained sponge tissue two days after wound infliction, showing regenerative tissue at the site of the wound, the location of mesohyl tracts containing cells, which were occasionally observed close to the wound, and an area away from the wound containing choanocyte chambers. Choanocyte chambers appeared 250 ± 8.9 µm from the edge of the wound tissue. (B) BrdU immunohistochemistry of a regenerative sponge labeled with BrdU for 6 h. Brdu-positive cells (brown-stained nuclei) were absent from regenerative wound tissue, and cells located in mesohyl tracts were BrdU-negative (blue-stained nuclei, white arrows). Tissue areas away from the wound that had retained their structural integrity contained BrdU-positive choanocytes (black arrows) and occasionally BrdU-positive mesohyl cells (black arrow heads). (C) Picrosirius red staining showed a higher density of collagen in regenerative wound tissue compared to areas farther from the wound. High densities of collagen could also be seen surrounding tracts in the mesohyl containing cells (white arrows). (D) Visualization of picrosirius red staining under cross polarized light revealed thin (green) and thick (orange) collagen fibers in all tissue areas.
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fig-2: Regenerative tissue of H. caerulea.(A) Cross-section through BrdU- and hematoxylin-stained sponge tissue two days after wound infliction, showing regenerative tissue at the site of the wound, the location of mesohyl tracts containing cells, which were occasionally observed close to the wound, and an area away from the wound containing choanocyte chambers. Choanocyte chambers appeared 250 ± 8.9 µm from the edge of the wound tissue. (B) BrdU immunohistochemistry of a regenerative sponge labeled with BrdU for 6 h. Brdu-positive cells (brown-stained nuclei) were absent from regenerative wound tissue, and cells located in mesohyl tracts were BrdU-negative (blue-stained nuclei, white arrows). Tissue areas away from the wound that had retained their structural integrity contained BrdU-positive choanocytes (black arrows) and occasionally BrdU-positive mesohyl cells (black arrow heads). (C) Picrosirius red staining showed a higher density of collagen in regenerative wound tissue compared to areas farther from the wound. High densities of collagen could also be seen surrounding tracts in the mesohyl containing cells (white arrows). (D) Visualization of picrosirius red staining under cross polarized light revealed thin (green) and thick (orange) collagen fibers in all tissue areas.
Mentions: All slides were examined under a light microscope (Olympus BH-2) and photographs were taken using an Olympus DP70 camera (Olympus, Tokyo, Japan). To investigate the cell kinetics of the choanocyte population directly next to the wound (the wound tissue contained no choanocyte chambers, Fig. 2A) in early regenerative tissue, percentages of BrdU-positive choanocytes were determined at each time point: t = 0, 0.5, 1.5, 2, 6, 8 h. Three histological sections were made from each tissue sample, each approximately 100 µm apart (Fig. 1A). From each section at least 400 choanocytes were counted making a total of at least 1,200 (3 sections × 400 cells) choanocytes counted per sponge. The ‘one population model’ (Nowakowski, Lewin & Miller, 1989; De Goeij et al., 2009) was used to estimate the growth fraction (GF; percentage of choanocytes involved in proliferation), duration of the cell cycle (Tc), labeling index (LI; percentage of choanocytes in S-phase), and the duration of the S-phase (Ts) for choanocytes in early regenerative tissue. Parameters (mean ± 95% confidence intervals) were estimated from the model according to an iterated least squares fit of the data using the following specified initial conditions: (1)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle f(t)=G F\times (t+T s)/T c,\hspace{1em}\text{for}&&\displaystyle \end{eqnarray*}\end{document}ft=GF×t+Ts/Tc,for(2)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle t\leq {T}_{c}-{T}_{s},\hspace{1em}\text{and}&&\displaystyle \end{eqnarray*}\end{document}t≤Tc−Ts,and(3)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle f(t)=\mathit{GF},\hspace{1em}\text{for}&&\displaystyle \end{eqnarray*}\end{document}ft=GF,for(4)\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{upgreek}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}}{}\begin{eqnarray*} \displaystyle t\geqslant {T}_{c}-{T}_{s}.&&\displaystyle \end{eqnarray*}\end{document}t⩾Tc−Ts. Data on the cell kinetics of the choanocyte population directly adjacent to the wound in early regenerative tissue were compared to choanocyte kinetics of steady-state sponges measured by De Goeij and colleagues in 2009, as this is the only study describing detailed cell cycle parameters in H. caerulea.

Bottom Line: We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h).There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges.The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Aquatic Ecology and Ecotoxicology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam , Amsterdam , The Netherlands ; Porifarma B.V. , Ede , The Netherlands.

ABSTRACT
Sponges have a remarkable capacity to rapidly regenerate in response to wound infliction. In addition, sponges rapidly renew their filter systems (choanocytes) to maintain a healthy population of cells. This study describes the cell kinetics of choanocytes in the encrusting reef sponge Halisarca caerulea during early regeneration (0-8 h) following experimental wound infliction. Subsequently, we investigated the spatial relationship between regeneration and cell proliferation over a six-day period directly adjacent to the wound, 1 cm, and 3 cm from the wound. Cell proliferation was determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). We demonstrate that during early regeneration, the growth fraction of the choanocytes (i.e., the percentage of proliferative cells) adjacent to the wound is reduced (7.0 ± 2.5%) compared to steady-state, undamaged tissue (46.6 ± 2.6%), while the length of the cell cycle remained short (5.6 ± 3.4 h). The percentage of proliferative choanocytes increased over time in all areas and after six days of regeneration choanocyte proliferation rates were comparable to steady-state tissue. Tissue areas farther from the wound had higher rates of choanocyte proliferation than areas closer to the wound, indicating that more resources are demanded from tissue in the immediate vicinity of the wound. There was no difference in the number of proliferative mesohyl cells in regenerative sponges compared to steady-state sponges. Our data suggest that the production of collagen-rich wound tissue is a key process in tissue regeneration for H. caerulea, and helps to rapidly occupy the bare substratum exposed by the wound. Regeneration and choanocyte renewal are competing and negatively correlated life-history traits, both essential to the survival of sponges. The efficient allocation of limited resources to these life-history traits has enabled the ecological success and diversification of sponges.

No MeSH data available.