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First published online August 8, 2008
Journal of Experimental Biology 211, 2551-2558 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018507
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Induced cell proliferation in putative haematopoietic tissues of the sea star, Asterias rubens (L.)

Kristina Holm1, Samuel Dupont2, Helen Sköld2, Anna Stenius2, Michael Thorndyke3 and Bodil Hernroth3,*

1 Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Göteborg, Sweden
2 Institute of Marine Ecology, Kristineberg 566, SE-450 34 Fiskebäckskil, Sweden
3 The Royal Swedish Academy of Sciences, Kristineberg 566, SE-450 34 Fiskebäckskil, Sweden


Figure 1
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  		Fig. 1. (A) Mean numbers (± s.e.m.) of total coelomocyte counts (106 ml–1) of A. rubens determined 4h (N=8) and 24h (N=10) post-injection (p.i.) of either 100 µl CMFSS buffer (Control), lipopolysaccharide (LPS) or concanavalin A (ConA). (B) A comparison between the different treatments showed significant differences between LPS and Control, both at 4 and 24h, and between LPS and ConA at 4h p.i.

 

Figure 2
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  		Fig. 2. Images showing Texas Red labelled anti-BrdU in tissue sections of (A) coelomic epithelium and (B) axial organ, indicating that cell proliferation occurred during the 4h of BrdU incubation, as described in Materials and methods. (C) Corresponding tissue section of pyloric caeca (negative control).

 

Figure 3
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  		Fig. 3. The percentage of cells (± s.e.m.) from cell monolayers of Tiedemann body, coelomic epithelium, axial organ, pyloric caeca and coelomocytes that show FITC-labelled anti-BrdU 4h post injection of either CMFSS buffer (Cont) or ConA (N=6). The numbers of labelled cells indicate the rate of cell division during 3h of BrdU incubation, as described in Materials and methods. P-values show significant increases of proliferation in coelomic epithelium and axial organ compared with controls.

 

Figure 4
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  		Fig. 4 (A) The percentage of cells (± s.e.m.) from cell monolayers of Tiedemann body (TB), coelomic epithelium (CE), axial organ (AO), pyloric caeca (PC) and coelomocytes (CC) that show FITC-labelled anti-BrdU during a time course (4, 16 and 24h) post-injection of LPS or CMFSS buffer (N=4). The numbers of labelled cells indicate the rate of cell division during 3h of BrdU incubation, as described in Materials and methods. Proliferation in pyloric caeca and coelomocytes was not affected, but in the Tiedemann body, coelomic epithelium and axial organ, proliferation had already increased at 4h. The corresponding organs of the animals pre-treated with CMFSS (control) also showed an increase in proliferation but the response came later. (B) Table presenting the differences between the mean values of proliferation and the P-values when comparing the initial proliferation of the different organs of LPS-treated and Control animals and between the results obtained at 4 and 24h p.i.

 

Figure 5
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  		Fig. 5. A comparison of the protein composition between different organs, revealed by gel electrophoretic separation. Proteins were extracted from coelomic epithelia (lane 1), axial organ (lane 2), coelomocytes (lane 3), pyloric caeca (lane 4) and Tiedemann body (lane 5). Note the similar protein composition pattern between coelomocytes, coelomic epithelia and Tiedemann body; the axial organ showed a similar pattern but a higher density of some protein bands. Pyloric caeca showed the most different protein pattern compared with coelomocytes. Protein molecular masses, as approximated from markers, are shown in kDa on the left.

 

Figure 6
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  		Fig. 6. Confocal microscope pictures showing coelomocytes and cells from coelomic epithelium explants of A. rubens and FITC-labelled yeast (green). (A–D) Different shapes of cells released by coelomic epithelium explant. (E,F) Different shapes of phagocytes in the coelomic fluid. Cells in B show high similarity to the phagocytes shown in E, and D shows high similarity to F. (G,H) Evidence of phagocytosis (G shows cells from the explant; H shows a cell from coelomic fluid). (I) Cluster of phagocytes at high density. Scale bars, 25 µm. (Supplementary material includes movies on phagocytic behaviour of cells from coelomocytes and from coelomic epithelium.)

 

Figure 7
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  		Fig. 7. Confocal microscope pictures of coelomocytes from coelomic fluid and cells from coelomic epithelium explants of A. rubens taken after 30 min incubation with FITC-labelled yeast (green). (A,C,E) Coelomocytes in different dilutions in filtered seawater (FSW) (A, low; C, medium; E, high density). High density is the cell density of undiluted coelomic fluid. (B,D) Cells from coelomic epithelium (explant in FSW). In A and B there is no clear network formation; in C and D there is evidence of network formation; in E there is clear network formation. Scale bars, 50 µm.

 

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