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In vivo analysis of mammalian adult stem cells is problematic, due to their
paucity and difficult experimental accessibility. For this reason, the use of model organisms can be useful for the study of molecular mechanisms governing stem cell biology. Planarians 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 advantage, coupled with successful application of molecular, cellular and genomic approaches, as well as the possibility to perform loss of function studies by RNA interference (RNAi) technique, makes planarians a suitable model system for in vivo investigations on adult stem cell biology.
Neoblasts are small cells (5-10 μm), with a large nucleus and a narrow rim of poorly differentiated cytoplasm. The distribution of neoblasts has been defined by the use of molecular markers such as DjMCM2 and DjPCNA capable to detect proliferating cells, or through BrdU incorporation. These cells are scattered throughout the parenchyma with the exception of the anterior end of the cephalic region and the pharynx, and are preferentially accumulated in the dorsal region, along the anteroposterior body axis. Although all neoblasts share a similar morphology, heterogeneity in their population has been hypothesized. The recent characterization of two genes (DjPiwi, a PIWI-PAZ family member, and Djnos, a planarian homologue of
nanos gene) expressed in different neoblast subpopulations, confirms this hypothesis.
The first part of my thesis work was aimed at investigating neoblast sub-populations, by revealing possible differences in their radiotolerance level. Animals were treated with increasing doses of X-rays and the stem cell fate was monitored by analyzing the expression of different neoblast markers (DjMCM2, DjPiwi-1 and DjNos) and BrdU incorporation, and by measuring the mitotic index Such analysis revealed the existence of sub-groups of neoblasts with different sensitivity to X-rays. In particular, a group of cells resistant to the irradiation with 5 Gy is preferentially distributed in the vicinity of the nervous system, and is able to proliferate and reconstitute the whole neoblast population.
A peculiar advantage to study planarian neoblasts is that these cells can be selectively destroyed by high dose (30 Gy) of X-ray irradiation while differentiated cells are not affected, thus offering the possibility to directly compare worms lacking stem
6 cells with wild-type controls. Taking advantage of the possibility to produce stem cell-depleted organisms by X-ray treatment, a microarray chip was recently produced to compare the transcriptional profiles of planarians exposed to different X-ray doses, and with untreated worms. The comparison of 30 Gy- with 5 Gy-treated animals revealed 44 genes specifically expressed in stem cells. Among these genes several putative homologues of factors involved in chromatin modeling were identified, in particular
RbAp48, a component of several chromatin remodeling systems, and PHB2, a
transcriptional repressor acting via recruitment of histone deacetylase. Several observations indicate that epigenetic factors modulate the properties of stem cells.
In the second part of this work, the in vivo function of these two genes was investigated. Firstly, the full-length sequence of the coding sequence was obtained using the 3’-5’ RACE technique. The gene function was then analyzed in both intact and regenerating planarians, using RNAi.
The results show that in vivo silencing of DjRbAp48 induces lethality and inability to regenerate, even though neoblasts proliferate and accumulate after wounding. Despite a partial reduction of neoblast number, a significant number of these cells was always detectable in DjRbAp48 RNAi animals. Parallel to the decrease of neoblasts, a reduction in the number of differentiated cells and the presence of apoptotic-like neoblasts were 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. We discuss our data taking into account the possibility that DjRbAp48 may control the expression of genes necessary for cell differentiation by influencing the chromatin architecture.
In vivo silencing of DjPHB2 brings both intact and regenerating planarians to death.
The combined analysis of mitotic rate, of neoblast markers distribution by immunofluorescence and whole mount in situ hybridizatin, and electron microscopy allowed to demonstrate a significant loss of neoblasts in intact planarians. This loss can be detected 30 days after the beginning of the treatment with RNAi. In particular, dispersed neoblasts and dorso-lateral clusters are reduced, whereas clustered patches of cells of the dorsal midline appear unaffected.
DjPHB2 RNAi planarians produce a normal blastema when amputated (first
regeneration), although the neoblast number is reduced. The dynamic of this reduction is similar to that observed in intact animals: neoblasts are progressively lost, except for those accumulated along the dorsal midline.
7 Conversely, upon amputation of the first blastema (second regeneration), DjPHB2 RNAi planarians can no longer form a regenerative blastema, or they undergo a delayed and anomalous regeneration, although the presence of neoblasts can still be detected in the dorsal midline.
DjPHB2 is specifically expressed in neoblasts, and is present in all neoblast
subpopulations. However, RNAi results suggest that DjPHB2 is involved in the maintenance of dispersed neoblasts and neoblasts of the dorso-lateral clusters, whereas it seems dispensable for the maintenance of neoblasts of the dorsal midline.
DjPHB2 therefore seems to play different roles in the different neoblast
subpopulations, although it cannot be excluded the presence, in the dorsal midline, of a sub-group of cells expressing DjMCM2 and DjPiwi-1 but not DjPHB2.
In conclusion, although additional studies are necessary to understand at which level these factors might affect chromatin structure, our findings represent the first evidence of the role of these factors in stem cell biology in vivo. As both genes are conserved in evolution, it would be worthed extending the analysis of their function to mammalian stem cells.
