1) A proteomic and genomic study of the neoblasts, the totipotent planarian stem cells

The neoblasts are the only undifferentiated cell type and the only one with proliferative capacity present in the adult Platyhelminth. This cell behaviour is unique for the animal kingdom and is responsible for the high morphological plasticity in these organisms. The characterization of proteins and genes specific for the neoblasts will allow us to study molecules related to the maintenance of the undifferentiated stage, molecules related to the proliferation capacity and proteins responsible for the determination of the neoblasts into the different cell types present in the adult planarian. Earlier results on differential proteomics between organisms irradiated with X-rays and non-irradiated control organisms, together with other genomic results, have permitted us to detect the first differential gene products. We found for instance proteins that belong to different families, including heat shock proteins (Chaperones of 40, 60 and 70 Kd), small GTPases, small nuclear ribonucleoproteins, and other interesting families such as signalling pathway kinases and proteins implicated in cell cycle control. We propose then to continue the characterization of new proteins by proteomic strategies together with genomic strategies, verifying their function by RNA interference or gain-of-function phenotypes.

Figure 1. Protein analysis in bi-dimensional gel, SDS/PAGE.
The protein spots specific for the protein extraction
from non-irradiated planarians is marked in red.

Staff
Dr. Emili Saló


Postdocs
Dr. Josep Francesc Abril
Dr. Teresa Adell
Dr. Francesc Cebrià


Predocs
Gustavo Rodriguez-Esteban


Collaborators
Prof. Hans Schöler and Dr. Luca Gentile (Max Planck Institute of Munster)




2) Functional characterization of genes responsible for the reconstitution of the pattern during the planarian regeneration

The great morphologic plasticity of the flatworms is accompanied by an infinite capacity to reconstitute the pattern from any small fragment of the organism. Previous studies made in our laboratory in the last 30 years, together with recent studies have shown that the BMP and Wnt pathways are also responsible for the establishment and maintenance of the D-V and A-P axis. However, we still don't know some of the essential elements of these processes, as well as its regulation as a whole. In this objective, we propose the functional characterization of the Wnt canonical and non-canonical and BMP2-4 basic elements of that signaling pathways and also the characterization of new pathways, like the Hh, FGF or Insulin. To end with an integrated study of that pathways and neoblast markers.

Figure 2. Wild type Schmidthea mediterranea and a hypercephalized mutant induced by interfering with the function of Smed b-catenin-1 gene. Such mutant has lost the A-P axis and the tail and lateral regions became heads. The radial symmetry is due to the maintenance of the dorso-ventral axis, which is not affected.

The great morphological plasticity of the Platyhelmintes permits them to regenerate the eyes in the short period of 5 days. To the present time we have characterized the majority of genes of the eye network for the initial development of the eyes. We do not know still some of the elements, as well as the interactions among all these genes. Taking advantage of the accessibility of the complete genome of the species Schmidtea mediterranea, we designed an “Smed-microchip” that represents an almost complete transcriptome for this species and marks an important qualitative step forward in the analysis of global gene expression in planarians. The “Smed-microchips” were hybridized with cDNAs corresponding to head blastemas of normal regenerating organisms or organisms without eyes (Smed-six1 -) at different stages of regeneration. With that strategy, we propose to study the key genes of this process that have not yet been isolated. At the same time, we will try to define the type of interactions that exist among these genes by means of the use of transgenesis and RNAi.

Figure 3. Inhibition of eye regeneration by Gtsix-1 RNAi. Three weeks head regenerating planarians viewed dorsally. A) Bright field image shows the differentiated eyes in the regenerated head of a control organism. B) Bright field image shows the absence of eye differentiation in dsRNA Gtsix-1 injected at postblastema level after 3 weeks of regeneration. a, auricle; e, eye spot; pg, preiglobular unpigmented area. (Bars, 0,4 mm)