WOOD, AW; DUAN, C; Univ of Michigan, Ann Arbor: Targeted gene knockdown reveals an important role for insulin-like growth factor binding protein-2 in zebrafish embryonic development

The insulin-like growth factor (IGF) system plays a central role in mediating fetal and postnatal growth in vertebrates. The major players in this hormone system are the peptide growth factors IGF-I and IGF-II, their cell surface receptors, and a family of IGF binding proteins (IGFBP-1 to -6). These IGFBPs bind to IGFs with high affinity and can modulate their actions by regulating their availability to IGF receptors. The biological effects of IGFBPs have been studied almost exclusively in mammalian cell culture systems; their in vivo physiological functions, in particular their roles during fetal development, are poorly understood. We have employed the zebrafish as a model to investigate the role of selected IGFBPs during vertebrate embryogenesis. Our previous work has shown zebrafish IGFBP-2 to be an evolutionarily-conserved growth inhibitory protein; herein, we demonstrate that IGFBP-2 mRNA is present as a maternal and zygotic transcript in zebrafish embryos between 1 and 24 hours post-fertilization (hpf), but at levels below in situ hybridization detection limits. Between 25 and 32 hpf, IGFBP-2 mRNA expression is detectable in the developing lens in situ, but after 48 hpf, mRNA expression predominates in the cranial vasculature. By 72 hpf, the liver becomes the primary site of IGFBP-2 mRNA expression. Targeted knockdown of IGFBP-2 protein levels via microinjection of antisense morpholino oligonucleotides at the 2-4 cell stage resulted in marginally delayed development, severe hindbrain edema, and a persistent reduction in the number of circulating blood cells. The tissue-specific expression patterns, and the phenotypic consequences of targeted gene knockdown, suggest that IGFBP-2 is an important regulator of early embryonic development in a vertebrate model organism.