Proliferative response of the stem cell system during regeneration of the rostrum in Macrostomum Lignano

Introduction

Figure 1.The anatomy of M. lignano

Figure 1.The anatomy of M. lignano

Macrostomum lignano ( Marine flatworm), belonging to the largest genus of the Macrostomorpha, is a transparent flatworm which is only 1.5 mm in size. This species has extraordinary ability to regenerate lost body parts after amputation, in which neoblasts (adult stem cells) plays an indispensable role. As long as the brain and pharynx are present, M. ligano has the ability to regenerate all lost body parts (Egger et al. 2006). In previous studies of neoblasts, triclads is the main model organism. However, M. lignano has become more popular because of its small size, which is easy to treat with color pigments and analysis. Also, the generation time of M. lignano is only 18 days. These attractive features make M. lignano becomes the new powerful model organism for neoblast studies.

In the past, there are a lot studies concerned about the regeneration of tail plate in M. lignano, but the reaction of neoblasts in regeneration of rostrum part (located in front of the eyes) as shown in Figure 1, in M. lignano is unknown. The eyes are used as marginal points of brain. Since there is no neoblasts in the region anterior to the eyes, the process of regeneration will be different with the previous study about the tail parts regeneration and may involve the migration of the adult stem cells.

In order to analyze the process, the anterior to the brain of M. lignano is amputated. In this study, live squeeze observation were used to describe the regenration process morphologically and determine its time course (Verdoodt et al., 2011). Furthermore, BrdU (thymidine analogue 5-bromo-2′-deoxyuridine) and anti-phos-H3 (anti-phospho-histone H3) mitosis marker were used as immunolabels to study the cellular dynamics of the neoblasts after amputation.

Results

FIgure 2. The growth curve of the rostrum during the regeneration process.

FIgure 2. The growth curve of the rostrum during the regeneration process.

After amputation of rostrum (parts anterior to the eyes), a ratio of the length of the the rostrum to that of the mouth opening is measured at different post-amputation days (p-a= 0, 2, 4, 6, 8, 10, 12). In control animals, the ratio is constant. After amputation, the ratio is at a low point at first and gradually increasing as new tissues formed as shown in Figure 2. By p-a day 6, the ratio is similar to the value of control animals.

The M. lignano are observed after amputation using in vivo squeeze preparations. Some visible morphological structures were recovered in eight days post-amputation (p-a) resembling control animals, such as rhammites, rhabdites and cilia of sensory receptors as shown in figure 3. However, the formation of an extension of unpigmented blastema cells at the level of the wound, which is formed during the regeneration of tail plate, was not able to be observed.

Figure 3. The comparison of the front margins of s-phase cells in control and experiment animals.

Figure 3. The comparison of the front margins of s-phase cells in control and experiment animals.

By using BrdU to label s-phase cells, the response of the adult stem cells (neoblasts) in both anterior and posterior regions is observed and measured after amputation of the rostrum as data shown in the Figure 3, 4 and 5. As shown in Figure 3, there is no s-phase cells in the anterior parts in control animals. However, after amputation of experiment animals, the s-phase cells are appeared near the wound, which means the s-phase cells are migrating to the anterior of the eyes. As seen in Figures 4&5, there is a fast response of S-phase cells in both anterior segment (near wound region) and posterior segment (far from wound region) at first; Later, s-phase cells activity is only restricted to the anterior segment (near wound region).

Figure 3. Graphs representing quantification data of S-phase cells during regeneration in the anterior and posterior segments.

Figure 4. The quantification data of S-phase cells during regeneration in the anterior segments.

Figure 5. The quantification data of S-phase cells during regeneration in the posterior segments.

Figure 6. The migration route of adult stem cells.

We may wondering how the adult stem cells appeared in the anterior part of M. lignano? Since there is no neoblasts at the anterior part before amputation, BrdU pulse-chase technique is used to study the migration of adult stem cells. In the Figure 6B, we can see that there is no s-phase cells in the anterior part of the eyes indicating by the white arrow as a mark of the marginal line. As shown in Figure 6C, 8 hours post-amputation, adult stem cells were migrating towards the wound region at the lateral sides of the rostrum. Twelve hours after amputation, the s-phase cells were reaching the margin of the wound as shown in Figure 6D, where they were never been to before. And after two days of amputation, the cells were accumulating in the margin region of the woud as shown in Figure 6E, which is the part anterior to the eyes. Based on the data, the minimum average migration speed was calculated and estimated as 9.7 μm/h, which is faster than cell migration speed during homeostasis.

Conclusion & Discussion

One of the interesting found in the experiment is that the activity of S-phase adult stem cells shows a biphasic pattern in response to amputation of the rostrum. In the first phase, the s-phase cells were increasing rapidly both in the region near the wound (anterior region) and region far from the wound (posterior region). Following the first phase, there is a second phase during which the s-phase cells were observed to increase only in the region near the wound (anterior region). The biphasic responses of s-phase cells were observed and found in the experiment; however, the underneath mechanisms of the two phses are unknown. Comparing the amputation of anterior and posterior parts in M. lignano, it is interesting that amputation of tail parts only cause a local proliferative response (Nimeth et al. 2007), which results a increasing in number of s-phase cells in the posterior part.

Another interesting found in this paper is the migration of adult stem cells. In order to regenerate rostrum, the adult stem cells need to migrate to the wound part following the routes at the lateral sides of the rostrum. Furthermore,  regeneration of anterior part can accelerate cell migration speed. This is interesting and different with previous ones because it shows a new pattern of how the adult stem cells working in the regeneration of body parts. Maybe in the future, there is more examples of migration of adult stem cells could be found and tested based on the results in this paper. This study also provided a foundational and essential understanding of the adult stem cells in the model organism M. lignano.

Reference

Egger B, Ladurner P, Nimeth K, Gschwentner R, Rieger R (2006) The regeneration capacity of the flatworm Macrostomum lignano – on repeated regeneration, rejuvenation, and the minimal size needed for regeneration. Dev Genes Evol 216:565–577.

Nimeth KT, Egger B, Rieger R, Salvenmoser W, Peter R, Gschwentner R (2007) Regeneration in Macrostomum lignano (Platyhel- minthes): cellular dynamics in the neoblast stem cell system. Cell Tissue Res 327:637–646.

Verdoodt, Freija, Wim Bert, Marjolein Couvreur, Katrien De Mulder, and Maxime Willems. “Proliferative Response of the Stem Cell System during Regeneration of the Rostrum in Macrostomum Lignano.” Cell Tissue Res 347 (2012): 397-406. Web.

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