DING, J; DUAN, C: Looking at IGF binding protein-2 actions through the zebrafish eyes

Insulin-like growth factors (IGFs) are evolutionarily conserved peptides that are essential for normal animal growth. Recent studies utilizing cell cultures suggest that the bioactivity of IGFs could be modulated by specific, high-affinity IGF-binding proteins (IGFBPs). The in vivo roles of these IGFBP during embryonic stage, however, are poorly defined. To study the IGFBP functions in vivo using the free-living and transparent zebrafish embryos, we have cloned and characterized the full-length zebrafish IGFBP-2 cDNA. To determine whether the encoded protein is a functional IGFBP, the IGFBP-2 cDNA was inserted in the CMV2 expression vector and introduced into CHO cells. A novel 30 kDa protein was found in the conditioned medium of the transfected cells. Ligand blot and affinity cross-linking studies revealed that this protein binds to IGF-I and IGF-II with high affinity and specificity. To determine how IGFBPs regulate IGF activities in vivo, IGFBP-2 was ectopically expressed in zebrafish using an expression construct driven by the crystallin gene promoter. When introduced into fertilized eggs by microinjection, this construct directed targeted expression of the transgene in the lens of developing eyes. After confirming the transgene expression, the mitotic cells were visualized by immunostaining using a mitotic marker (phospho-histone) and the apoptotic retinal cells were identified by TUNEL assay. While ectopical expression of IGFBP-2 had no effect on cell proliferation, it significantly increased the number of apoptotic cells in the transgenic fish. Co-expression of IGF-I reduced the retinal cell apoptosis to the control levels. These results suggest that IGFBP-2 can influence retinal cell apoptosis in vivo and this action is ligand-dependent (Supported by NSF Grant IBN-9728911 and 0110864).