Browsing by Author "Portela, Ana"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- Fish as a model to assess the effects of Thalidomide on limb developmentPublication . Portela, Ana; Rosa, Joana; Simão, MárcioThalidomide was an anti-emetic non-toxic sedative prescribed for morning sickness during late 50s, until its teratogenicity was revealed with more than 10,000 children affected by thalidomide embryopathy. Thalidomide molecular mechanisms have remained elusive for many years until Cereblon was identified the main target of thalidomide, promoting the degradation of key transcription factors involved in embryogenesis. We aimed to use zebrafish as a biological model to understand molecular events associated with thalidomide embryopathies and identify how bone metabolism could be affected. To optimize the thalidomide exposure methodology a series of in vivo experiments were done aiming to improve zebrafish embryo dechorionation, effective thalidomide concentrations and time window of exposure. The impact of thalidomide was evaluated by analyzing pectoral fin deformities in zebrafish larvae at 4 days post-fertilization using alcian blue staining and gene expression analysis by qPCR. Furthermore, to understand thalidomide effects on bone metabolism, in vitro assays were performed in VSa13, Sparus aurata vertebra derived cell line of progenitor osteo-chondrocytes within a context of cell differentiation and mineralization. Thalidomide exposure from 2 hours post-fertilization after 5 minutes treatment with 0.05 mg/mL pronase, leads to the phenotype of interest and 600μM was the most effective concentration on pectoral fin length shortening. Gene expression confirmed thalidomide embryotoxicity through downregulation of sall4 and sox9a transcription factors. Additionally, a downregulation of col1a1 and upregulation of spp1 suggest an impact on bone metabolism. In vitro experiments corroborated that thalidomide inhibited mineralization, possibly by blocking terminal differentiation attending to the downregulation of sp7 transcription factor. The anti-angiogenic properties of thalidomide may also contribute to limb malformations, and the results revealed upregulation of vegfaa mRNAs and FLI1 protein. We conclude that thalidomide molecular action is complex, and limb malformations could result from a combination between angiogenesis deregulation and impairment of key regulators of chondrogenesis and bone metabolism.