• nothobranchius
• diapause
• regeneration
• piRNAs
• macrostomum

The PIWI interacting RNAs (piRNAs) are small (28-33nt), single stranded RNA species associated with Piwi proteins. They have been extensively characterized as major players in the silencing of retro-transposable elements during gametogenesis, thus preserving the genome integrity of the gametes. The PIWI/piRNAs pathway has been often called “the genome immune system”. PiRNAs could, however, play crucial roles in regeneration and development as well, both processes being deeply affected by genome instability. Recently, Macrostomum lignano and Nothobranchius furzeri have been validated as novel and powerful animal models for the investigation and dissection of regeneration and development&aging, respectively. In this thesis we investigated the presence the PIWI/piRNA pathway in both these species, with a special focus on its eventual role in regeneration and diapause.
Macrostomum lignano is a hermaphrodite flatworm closely related to the more widely known planarians. Amongst the several shared features between these two models, we took advantage of the conserved presence of neoblasts, a cell population that accounts for the high regenerative potential of both animals. Neoblasts closely resemble germinal cells, since they both are mitotically active and express unique proteins of the Piwi clade. The Macrostomum lignano genome codes for four different PIWI proteins. Although specific antibodies have been purified in the course of this thesis for the C- and N-terminal domains of all four of them, we focused on the sole characterization of MacPiwi1 and MacPiwi2. Both these proteins are expressed in the gonads, where Piwi1 in particular strongly co-localizes with neoblasts (EdU+ cells). We were able to clone and sequence smallRNA species from whole worms; one of the population of smallRNAs indeed showed all the features usually associated with piRNAs, and they will be hereby defined as “putative piRNAs”. Putative piRNAs range 28-30nt in length, have a sequence bias toward a 5’Uridine and
co-immunoprecipitate with Piwi proteins. This led us to hypothesize a possible role of the piRNA pathway in regeneration.
In order to get a better insight of the neoblasts biology, we developed a protocol to purify neoblasts based on DNA content (2c) and/or progress in S-phase by FACS [Fluorescence Activated Cell Sorting], from cell suspensions of both fixed and live cells. We were able to clone and retrieve the putative piRNAs only from sorted neoblasts and not from post-mitotic cells, thus demonstrating that all the components of the Piwi/piRNA pathway are exclusively present in neoblasts. Moreover we irradiated the worms with lethal dosage of γ-rays. This treatment completely depleted the neoblasts population and steeply decreased the expression of Piwi mRNAs and proteins, consistently with the yet described tight relation between neoblasts and piRNA pathway; phenotypically, the irradiated worms showed impaired homeostasis and eventually all of them died.
Despite a draft of the genome is publically available, the genome of Macrostomum lignano is still not fully assembled nor is the transcriptome adequately assembled and annotated. We therefore invested plenty of resources for the preparation diverse libraries from gDNA and cDNA from whole worms or sorted neoblasts. These libraries have been sequenced on a High-seq Illumina or PacBio platform. These strategies allowed improving the genome assembly as well as to successfully assemble and annotate the transcriptome.
Since piRNAs are highly expressed in the gametes, it is likely for them to be maternally inherited and to have a role as fundamental regulators of the progression through early development, while the zygotic genome is not yet transcribed. Some animals are indeed able, as early zygotes, to enter a peculiar physiological condition, the diapause, which is characterized by an overall decrease in metabolic activity and an almost complete growth and developmental arrest.
Nothobranchius furzeri is a teleost fish that belongs to the group of killifishes. It populates ephemeral ponds in the savannah of South Mozambique and Zimbabwe. Because of the peculiarity of its habitat, N.furzeri has evolved a bi-phasic life-cycle that consists of a short free-living phase during the rainy season followed by three different diapause phases during the dry season, which allow the embryos to survive even in extreme environmental conditions. This species has been recently confirmed to be the vertebrate with the shortest lifespan and the fastest growth speed.
As parts of the same life cycle, aging and development are highly linked: the diapause, in this context, allows entangling the network of relations between the two. We cloned and sequenced small RNA libraries from embryos at the 1- or 2- cells stage and diapause from two strains of N.furzeri with a slightly different lifespans (pL and GRZ). We retrieved a population of putative small RNAs, showing all the piRNA-specific features as well as all the hallmarks of the “Ping-Pong” amplification loop, in all the libraries. Moreover, the longer living strain showed a significant enrichment in maternally deposited piRNAs as compared to the shorter living one. We then broadened the study to other killifish species, either with or without a diapause phase, looking for a correlation between the diapause and levels of piRNAs expression. Because of the poor quality of the sequencing data we were not able to draw any significant conclusion.
In conclusion of this thesis, under the light of the obtained results, we can state that a piRNA pathway is present and active not only during gametogenesis but also during regeneration and diapause. While a complete understanding of its role in both these phenomena still needs further in-depth analyses, the techniques and data generated in this thesis will hopefully be used as a starting point in the fulfillment of this goal.