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ABSTRACT
In vivo analysis of mammalian adult stem cells is problematic, due to their paucity and difficult experimental accessibility. For this reason, model organisms can be useful for the study of molecular mechanisms governing stem cell biology in vivo. One such model organism is represented by planarians: these animals possess experimentally accessible adult pluripotent stem cells, called neoblasts, that can give rise to all differentiated cell types of the planarian body and preside tissue homeostasis and regeneration. This feature, together with the possibility of applying molecular, cellular and genomic approaches, in particular to perform loss of function studies by RNA interference (RNAi), makes planarians a suitable model system for in vivo investigations on adult stem cell biology. The main feature of planarians is their ability to regenerate a complete organism after amputation. This extraordinary regeneration capability is due to the presence of neoblasts: this stem cell population is involved in both homeostasis and regeneration. During regeneration, neoblasts actively proliferate to create a blastema from which lost body parts are rebuilt.
Neoblasts are the only proliferating cells of asexual planarians; they are small cells (5-10µm diameter), with a large nucleus surrounded by a scanty rim of undifferentiated cytoplasm, in which there are typical structures called chromatoid bodies. These cells are spread throughout the parenchyma with the exception of the anterior end of the cephalic region and the pharynx, and are also accumulated in clusters along the midline and the dorso-lateral parenchyma. Although all neoblasts share a similar morphology, heterogeneity in their population has been hypothesized.
Neoblasts can be selectively destroyed by high dose (30 Gy) of X-ray irradiation, while differentiated cells are not affected, thus offering the
5 possibility to directly compare worms lacking stem cells with wild-type controls. Taking advantage of this opportunity, the transcriptional profiles of planarians deprived of stem cells was compared with that of wild-type animals.
This led to identify a number neoblast-specific genes, most of which, belong to the chromatin modeling and post-trascriptional regulation functional categories. One of these genes is RbAp48, a component of several chromatin remodeling complex. To date, no information is available about the role of this factor in stem cell biology in vivo.
The expression of DjRbAp48, the planarian homologue of mammalian RbAp48, is restricted to neoblasts.
The aim of this work was to investigate the function of DjRbAp48 in vivo.
Initially, the complete DjRbAp48 gene sequence was obtained using the 5’- and 3’- RACE technique, then DjRbAp48 function was analyzed in both intact and regenerating planarians using RNAi.
In vivo silencing of DjRbAp48 was lethal for both intact and regenerating planarians. Although a partial reduction of neoblast number was demonstrated by analysis of the mitotic index, immunohistochemistry, in situ hybridization experiments and electron microscopy, a significant number of these cells could always be detected in DjRbAp48 RNAi animals.
RNAi planarians at the first regeneration created a blastema from which lost body parts were rebuilt; however, in RNAi planarians at the second regeneration, neoblasts proliferated and accumulated after wounding, but they were unable to regenerate lost body parts. Moreover, apoptotic-like cells were detected below the epithelium after wounding. Parallel to the decrease of neoblasts, a reduction in the number of differentiated cells was detected in RNAi animals.
These findings suggest that DjRbAp48 is not involved in neoblast maintenance, but rather in the regulation of stem cell progeny differentiation.
Data are discussed, taking into account the possibility that DjRbAp48 may control the expression of genes necessary for cell differentiation by influencing chromatin architecture.
In conclusion, our findings represent the first evidence of a role of this factor in stem cell biology in vivo. As the sequence of RbAp48 is conserved in
6 evolution, it would be worthed extending the analysis of its function to mammalian stem cells.
