Department Cell and Developmental Biology
Dr. Luca Gentile
E-Mail: luca.gentile (at) mpi-muenster.mpg.de
Tel: 0251 83 46870
Fax: 0251 83 46900
Profile
Planarians are free-living members of the phylum Platyhelminthes and among the simplest bilaterian animals. Yet they possesses derivatives of all the three germ layers organized in complex organ systems and unreached regeneration ability.
Such extraordinary body plasticity relies on a large population of planarian adult stem cells (pASCs) – historically referred to as neoblasts – distributed throughout the planarian body. In wounded or amputated animals, these cells give rise to a structure called blastema, where stem cell progeny differentiates and missing tissues regenerate.
It is comprehensible why planarians have recently become a new powerful model organism for stem cell research. The possibility of studying stem cell biology in vivo and in an adult organism, without the need to use developmental stages or in vitro systems, has re-kindled interest in these fascinating organisms.
Our principal goal is to understand the cellular and molecular bases of planarian stem cell pluripotency. In doing so, we focus on one of the most peculiar characteristic of these stem cells, a ribonucleoprotein particle called chromatoid body, and the way it is linked to pluripotency. We also invest resources on developing important molecular tools that are currently missing; above the others, the production of antibodies against pASCs surface markers and a reliable protocol for the production of transgenic animals.
The development of ambitious projects, like the establishment of planarian stem cell culture conditions or the production of transgenic animals, needs a considerable effort. For this reason, one of the first priorities was to promote and support the creation of a European network of planarian laboratories (EuroPlanNet), with the intent of pursuing large projects with a shared contribution. EuroPlanNet, founded in early 2010, nowadays accounts for 13 members – all involved in leading planarian research – and can already account its first success, the organization of the 1st International Meeting on Planarian Biology, which was held in Münster on May 25-28, 2010.
Planarian stem cell surface-specific antibody
Few antibodies are currently available for planarian, which severely limits any application involving protein localization and protein expression quantification. The isolation of planarian stem cells currently relies on the morphological features of pASCs – namely size and DNA content – which does not allow isolating those stem cells in the G1 phase of their cell cycle. Moreover, planarian stem cells are thought to be a homogeneous population of pluripotent cells, but a definitive prove of this is still missing. The availability of antibodies that specifically cross-react with stem cell membrane antigens not only allows drawing a clear boundary between stem and differentiated cells, but might also unravel a scenario in which different subpopulations of stem cells (early committed or uni- or multi-potent progenitors that are still able to cycle) express different sets of markers. Based on a new protocol for FAC-sorting developed in house, we used the purified microsomal fraction of the sorted stem cells to immunize mice for the production of stem cell-specific monoclonal antibodies directed against the membrane proteins. As a result, thousands of hybridoma clones are currently under investigation.
Chromatoid body and stemness
In collaboration with Emili Saló’s lab in Barcelona, we produced by RNA interference (RNAi) against Smed-SmB, a stem cell-free animal in which differentiated cells are virtually unaltered, in contrast to irradiated worms. Smed-SmB, a member of the LSm protein superfamily, is expressed in stem cells and neurons and localizes to the nucleus and the chromatoid body (CB). Smed-SmB(RNAi) animals fail to regenerate after amputation and die within two weeks. Our studies showed that this severe phenotype is due to the disorganization of the CB, which leads to the inability to process the planarian cyclinB transcripts and results in the proliferative failure of the stem cells. Indeed, the majority of stem cell-specific genes are involved in the RNA metabolism and some, like Smed-SmB, Spol-tudor and DjCBC1 localize to the CB. Thus the CB seems to play an important role for maintaining the identity of the planarian stem cells and we are currently investigating on the other components of this particle and their link to stemness.
Blastema whole transcriptome
One of the dogmas of planarian biology is that blastema is devoid of proliferating stem cells, which otherwise accumulate in the adjacent region called post-blastema. Therefore, the blastema is the perfect playground to study the dynamics of the molecular mechanisms triggering planarian stem cells differentiation. Combining laser-based microdissection with whole-transcriptome deep sequencing, we are generating the molecular signature of the blastemata at different time points after amputation. In this way we will be able to dissect the transcriptional networks that generate the differentiated cell types that regenerate the missing tissues and organs. Conserved pathways and novel candidates will then be validated by RNAi and the function of newly found transcription factors will be investigated in mammalian models. Eventually, also the targets of the transcription factorswill be studied at the global scale, combining chromatin immuno-precipitation (ChIP) with targeted deep sequencing (ChIP-seq).
Selected publications
Gentile L*, Cebrià F*, Bartscherer K (2011). The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration. Disease Models & Mechanisms 4: ePub.
Wu G, Gentile L, Fuchikami T, Sutter J, Psathaki K, Esteves TC, Araúzo-Bravo MJ, Ortmeier C, Verberk G, Abe K, Schöler HR (2010). Initiation of trophectoderm lineage specification in mouse embryos is independent of Cdx2. Development 137:4159-69.
Sebastiano V*, Dalvai M*, Gentile L*, Schubart K, Sutter J, Wu G, Tapia N, Esch D, Ju JY, Hübner K, Arauzo Bravo M, Schöler HR, Cavaleri F, Matthias P (2010). Oct1 regulates trophoblast development during early mouse embryogenesis. Development 137:3551-3560.
Fernandéz-Taboada E, Moritz S, Zeuschner D, Stehling M, Schöler HR, Saló E, Gentile L (2010). Smed-SmB, a member of the LSm protein superfamily, is essential for chromatoid body organization and planarian stem cell proliferation. Development 137:1055-1065.
Kim JB, Sebastiano V, Wu G, Araúzo-Bravo MJ, Sasse P, Gentile L, Ko K, Ruau D, Ehrich M, van den Boom D, Meyer J, Hübner K, Bernemann C, Ortmeier C, Zenke M, Fleischmann BK, Zaehres H, Schöler HR (2009). Oct4-induced pluripotency in adult neural stem cells. Cell 136:411-9.
Kim JB, Zaehres H, Wu G, Gentile L, Ko K, Sebastiano V, Araúzo-Bravo M, Ruau D, Han DW, Zenke M, Schöler HR (2008). Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454:646-650.
Boiani M*, Gentile L*, Gambles VV, Cavaleri F, Redi CA, Schöler HR (2005). Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments. Stem Cells 23:1089-104.
* equal contributions
