García-Arrarás JE et al. (2011), Cell dedifferentiation and epithelial to mesenc...

ECB-ART-42204

BMC Dev Biol 2011 Oct 17;11:61. doi: 10.1186/1471-213X-11-61.

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Cell dedifferentiation and epithelial to mesenchymal transitions during intestinal regeneration in H. glaberrima.

García-Arrarás JE , Valentín-Tirado G , Flores JE , Rosa RJ , Rivera-Cruz A , San Miguel-Ruiz JE , Tossas K .

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BACKGROUND: Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima. RESULTS: We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes. CONCLUSIONS: Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.

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Genes referenced: LOC100887844 LOC115919910 LOC115925415 srpl

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	Figure 2. Quantification of the area of the intestinal rudiment during the process of intestinal regeneration. The area encompassed by the thickening of the mesentery was measured in transverse sections. The main growth of the structure begins 3 days after eviscerations. At 10 days a lumen has formed in all animals, thus the black bar at 10 days denotes only the area of tissue (does not include the lumen area), while the gray bar represents the total area of the intestine (including the area encompassed by the lumen). Each point represents the mean percentage ± S.E. of at least 3 animals. *p < 0.05.
	Figure 3. Longitudinal sections of normal uneviscerated large intestine, showing Meso-1 immunoreactivity. (A) Meso-1 labels the cells of the coelomic epithelium (CE) and the muscle layer (ML) (green). Cells of the connective tissue (CT) are not labeled. (B) The same section stained with rhodamine-labelled phalloidin only labels the muscle layer. (C) DAPI-labeled nuclei. (D) The colored overlay of the triple labeled section clearly shows that the Meso-1 antibody labels both mesothelium components while the phalloidin labeling is restricted to the muscle tissue. Bar = 25 μm
	Figure 4. Meso-1 labeling of the regenerating intestine (Days 1-5 of regeneration). Meso-1 labeling (green) and nuclei DAPI stain (red) show the process of ingression and the concomitant changes in the rudiment during regeneration. (A-E) The mass of cells at the distal tip of the rudiment is not observed at 1-dpe (A), becomes noticeable at 3-dpe (B) and increases in the subsequent days (C-E). Transverse sections of (F) Toluidene blue staining of 5-dpe intestine, shows the thickening of the mesenterial tip that forms the intestinal rudiment and differential staining of the coelomic epithelium (CE) and connective tissue (CT) compartments. (G) A similar section labeled with Meso-1 antibody highlights the ingressing cells. (H-I) Two additional examples of ingressing cells at 5-dpe, one is an invagination of the coelomic epithelium (H) while in the other, cells can be observed moving from the rudiment tip into the connective tissue (I). Figure H shows regions in the coelomic epithelia (bracket) that are thicker when compared to those in the 1-dpe mesentery (see bracket in Figure 4A). At higher magnification (J) some Meso-1 labeled ingressing cells show an elongated nuclei and cellular morphology (arrows) while other cells within the connective tissue are not labeled (arrowhead). (K) At the lateral side of the rudiment, an isolated ingressing cell (arrow) can be observed. Bar = (A) 70 μ m (B-E) 100 μ m (F-G) 65 μ m, (H-I) 30 μ m (J) 13 μ m (K) 20 μ m. All sections are from 5dpe animals except A (1dpe) and B (3-dpe).


	Figure 7. Quantification of cell density in the regenerating rudiment. The number of DAPI stained nuclei per μ m2 was measured in tissue sections of regenerating rudiments of animals at 3-, 5-, and 7-days post evisceration. The connective tissue of the rudiment was subdivided into 3 different parts (tip, midsection and proximal). Insert shows an example of a rudiment with the areas that were measured. Each point represents the mean ± S.E. of at least three animals. Statistical analyses were done by comparing the density of cells in the proximal section to those on other areas of the rudiment at the same stage. Different from proximal p < 0.05, *p < 0.01.

	Figure 9. Patterns of cell proliferation in regenerating intestinal rudiments. Sections were labeled with an antibody against BrdU (green) and DAPI (red) to determine cell proliferation in the intestinal rudiment at (A) 2-dpe and (B) 5-dpe. Actively dividing cells were mainly observed in the coelomic epithelia of the rudiment of the 5-dpe animal with only one cell being labeled in the rudiment of the 2-dpe specimen. Bar= 50 μ m. (C) The percentage of BrdU-labeled cells or proliferation index was measured in the mesothelium (black) and connective tissue (gray) compartments of the regenerating intestinal rudiment. Each point represents the mean ± S.E. of at least three animals. p < 0.05, *p < 0.01.

References [+] :

Akimenko, Old questions, new tools, and some answers to the mystery of fin regeneration. 2003, Pubmed