• molecular biology
• planarian
• polyamines
• regeneration
• stem cells

Polyamines are small positively charged molecules essential for cell growth from bacteria to mammalian cells. An increasing body of evidence indicates that the regulation of cellular polyamines putrescine, spermidine and spermine is a central convergence point for the multiple signaling pathways driving various cellular functions. Among the roles that polyamines have in the support of cell growth, and in some cases survival, there is association with nucleic acids, maintenance of chromatin conformation, regulation of specific gene expression, cell motility, ion-channel regulation, provision of a precursor in the synthesis of eukaryotic translation initiation factor 5A (eIF5A) and free-radical scavenging.. Ornithine decarboxylase (ODC) is the rate limiting enzyme in polyamine biosynthesis and, together with the complex network of its negative regulators, provides the maintenance of polyamines proper concentration within the cell. Due to polyamines high pleiotropic activity, it is very challenging to understand their precise role in a specific cellular context. In this thesis I chose to study the polyamine pathway enzymes (in particular ODC) using planarians as model system. Planarians are free-living members of the order Tricladida known for their extraordinary regenerative abilities, food supply dependent scale of body size and great abundance of adult stem cells, which are called “neoblasts”. Thus, planarians are considered a very well-suited animal system to uncover unknown mechanisms of both regeneration and stem cell biology.
Through bioinformatic analysis we found six different isoforms homologous to human ornithine decarboxylase (called DjODC1-6), and several homologs of other genes taking part in polyamine pathway (antizyme, arginase, agmatinase and spermidine/spermine-N1-acetyltransferase) in the planarian species Dugesia japonica. The analysis of their expression pattern using in situ hybridization techniques revealed a complex scenario involving different cell types, with ODC being present both in differentiated tissues (gut and epidermis) and x-ray sensitive neoblast daughter cells, resembling the so called “late neoblast progeny”. To understand ODC function in tissue homeostasis and regeneration we silenced ODC genes one by one by RNA interference. Following RNAi treatment, only DjODC5 and DjODC6 showed significant and severe defects both in regenerating fragments (displaying delays in blastemal formation) and in intact organisms (showing different degrees of morphological changes and bringing animals to death). In particular, the longer the dsRNA was injected in animals, the more acute the phenotype was, resulting in an extremely severe phenotype represented by head absorption backwards into the trunk, which we called the blemmy phenotype.
In situ hybridization analysis of different cell subpopulation markers as those for neoblasts and neoblast progeny, together with mitotic index analysis in DjODC5 RNAi treated animals showed a global response to DjODC5 loss resulting in an increase in both mitosis and stem cell marker expression. On the other hand, DjODC5 (RNAi) animals showed a significant decrease in expression of the muscle cell marker DjMHC-B. Subepidermal muscle cells have been recently regarded as the cellular sources of positional control assuring a proper tissue homeostasis and regeneration in planarians. Given that DjODC5-expressing cells are subepidermal and undifferentiated, here we propose DjODC5 to mark specifically a subpopulation of muscle cell precursors, in which polyamine presence might play fundamental roles in achieving a proper differentiation into muscular cells. Further studies are necessary to confirm DjODC5 positive cells belonging to this lineage.