Dysregulation of FAS and FASL in Chordoma: Study of their Role in Zebrafish Notochord Development and Regression

Chordoma is a rare malignant bone tumor arising from notochord remnants, characterized by local invasiveness and variable tendency for recurrence. As the apoptotic pathway mediated by FAS-FASL was found to be involved in no-tochordal cells regression, we studied their expression in 34 chordomas and observed that most of them express FAS, but not FASL. To verify a possible implication of FAS/FASL pathway dysfunction during development leading to tumorigenesis, we started in vivo studies on zebrafish, firstly investiga-ting fas-fasl horthologue genes expression by RT-PCR in whole embryos and larvae. While fas was maternally and zigotycally expressed, fasl showed a stage-specific expression. Following microinjection of a GFP-construct acti-vated by the twhh promoter, we specifically sorted by FACS the GFP-positive notochordal cells at different developmental stages in which we studied fas-fasl expression by means of RT-PCR. We observed that fas was expressed at all the analyzed stages, while fasl is modulated during development. Immu-nohistochemical experiments showed notochord fasl expression at 48 hpf, 3 and 5 days post fertilization (dpf) and at 9 mm. fasl expression seems to decrease when notochordal cells disappear during chord regression, sug-gesting the activity of apoptotic mechanisms fas-fasl-mediated. Studies on fas and fasl silencing, by means of morpholino oligos injections, allowed us to observe notochord anomalies such as packed notochordal cells, curved body and bent tail. Further analysis will be performed to verify whether the reactivation of fas-fasl pathway might rescue the normal phenotype, allo-wing to unravel the role of this mechanism in chordoma development.