The recently cloned human DRO1/CL2 gene is expressed in almost all adult tissues and is downregulated in several cancer cell lines and solid tumors. DRO1/CL2 is indeed considered a potential oncosoppressor gene. To understand the physiological role of DRO1/CL2, we decided to clone its zebrafish homolog and to analyse its function during embryonic development. The dro1/cl2 zebrafish gene shares high homology with the mammalian counterpart and the translated protein (867 aa) presents the same structure, which is characterized by the simultaneous presence of a signal peptide and multiple nuclear localization signals as well as by three highly similar domains of still unknown function. dro1/cl2 is maternally supplied. Temporal expression analysis performed by RT-PCR showed that it is downregulated during gastrulation. Its expression level starts increasing from the initial steps of segmentation up to the late somitogenesis, when it reaches the highest value. Whole mount in situ hybridization experiments show a diffuse dro1/cl2 expression during cleavage and epiboly. Interestingly, during somitogenesis, the dro1/ cl2 expression becomes mainly restricted to the notochord. Finally, in later developmental stages it becomes detectable in the muscles of both trunk and head, and in the heart. To investigate the functional role of the dro1/cl2 gene, we performed loss- and gain-of-function experiments by injecting a specific dro1/cl2 MO and the full length mRNA, respectively. The MO injected embryos presented with abnormally shaped somites in the early somitogenesis, while curved tails were evident at the end of this developmental phase. dro1/cl2 overexpression also impaired somitogenesis. To determine whether the observed defects are a result of a direct activity of dro1/cl2 on the different phases of somitogenesis rather than an indirect consequence of a disruption of nothocordal signals, we evaluated the expression of notochordal and somite markers in the injected embryos. We excluded a dro1/cl2 functional role in the developing notochord. Indeed, no tail and sonic hedgehog were unaffected. By converse, the block of the Hh signals indicated dro1/cl2 as a target of the Hh pathway. The expression pattern of the myogenic markers myod and myogenin resulted heavily disrupted in the lateral domain without affecting the adaxial one. The segmentation clock marker her1, and the segment polarization markers mesp-a and mesp-b showed no significant alteration. Interestingly, while no apparent effect was seen on the expression pattern of gadd45beta2 in the furrow, we observed the disappearance of its expression in the already formed somites. The demonstration of a specific link between dro1/cl2 and gadd45beta2 expression derived from rescue experiments, showing a significant phenotype recovery in dro1/cl2 morphants injected with gadd45beta2 mRNA. In our knowledge, this is the first demonstration of a notochordal signal controlling the lateral somitic maturation through gadd45beta2.
|Titolo:||Expression analysis and functional characterization of the dro1/cl2 gene during zebrafish somitogenesis|
|Data di pubblicazione:||2009|
|Settore Scientifico Disciplinare:||Settore BIO/06 - Anatomia Comparata e Citologia|
|Citazione:||Expression analysis and functional characterization of the dro1/cl2 gene during zebrafish somitogenesis / I. Della Noce, A. Pistocchi, E. Turola, C. Brusegan, S. Carra, R. Critelli, C. De Lorenzo, P. Sordino, E. Villa, F. Cotelli, F. Schepis. ((Intervento presentato al 6. convegno European Zebrafish Genetics and Development Meeting tenutosi a Roma nel 2009.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|