Insulin-like Growth Factor-binding Protein-3 Plays an Important Role in Regulating Pharyngeal Skeleton and Inner Ear Formation and Differentiation

Abstract

Insulin-like growth factor-binding protein (IGFBP)-3 is the major insulin-like growth factor (IGF) carrier protein in the bloodstream. IGFBP-3 prolongs the half-life of circulating IGFs and prevents their potential hypoglycemic effect. IGFBP-3 is also expressed in many peripheral tissues in fetal and adult stages. In vitro, IG-FBP-3 can inhibit or potentiate IGF actions and even possesses IGF-independent activities, suggesting that local IGFBP-3 may also have paracrine/autocrine function(s). The in vivo function of IGFBP-3, however, is unclear. In this study, we elucidate the developmental role of IGFBP-3 using the zebrafish model. IGFBP-3 mRNA expression is first detected in the migrating cranial neural crest cells and subsequently in pharyngeal arches in zebrafish embryos. IGFBP-3 mRNA is also persistently expressed in the developing inner ears. To determine the role of IGFBP-3 in these tissues, we ablated the IGFBP-3 gene product using morpholino-modified antisense oligonucleotides (MOs). The IGFBP-3 knocked down embryos had delayed pharyngeal skeleton morphogenesis and greatly reduced pharyngeal cartilage differentiation. Knockdown of IGFBP-3 also significantly decreased inner ear size and disrupted hair cell differentiation and semicircular canal formation. Furthermore, reintroduction of a MO-resistant form of IG-FBP-3 "rescued" the MO-induced defects. These findings suggest that IGFBP-3 plays an important role in regulating pharyngeal cartilage and inner ear development and growth in zebrafish.

Insulin-like growth factor signaling regulates zebrafish embryonic growth and development by promoting cell survival and cell cycle progression

“…37 The above constructs were linearized with NotI digests, and capped mRNAs encoding these proteins were synthesized using T7 polymerase, as previously reported. 38 Microinjection. MO, plasmid DNA or mRNA solutions were injected into 1-2 cell embryos as reported previously.…”

“…MO, plasmid DNA or mRNA solutions were injected into 1-2 cell embryos as reported previously. 38 To knockdown igf1r expression, we injected a mixture of all four MOs in the experiments described here. Each MO was injected at a final concentration of 1.25 ng/embryo, for a total of 5 ng MO/embryo.…”

“…The beads were pelleted again and the supernatant was subjected to Western blot analysis, which was performed as described previously. 38 The anti-phosphotyrosine, Tubulin and GFP antibodies were used at 1 : 1000 dilution, and the anti-Phospho-Akt, total Akt and IGF1R antibodies were used at 1 : 500 dilution.…”

Phylogenetic analysis of the insulin-like growth factor binding protein (IGFBP) and IGFBP-related protein gene families

“…Low stringency BLASTP analysis of the zebrafish EST database with the amino terminal domain of human IGFBP-3 (first 150 amino acids) retrieved all of the previously identified zebrafish IGFBPs (Duan et al, 1999;Li et al, 2005;Maures and Duan, 2002) and in addition, a tankyrase homolog (TANK1; GID, 68397005) that also contained a carboxy terminal IGFBP motif (Fig. 2, shaded residues 60-70).…”

An analysis of IGFBP evolution

“…Dr Cunming Duan has repeatedly demonstrated this phenomenon in zebrafish, where mutations in IGFBPs 1, 2 and 3 result in profoundly perturbed phenotypes in tissues that express a single IGFBP subfamily. [23][24][25] In contrast, mammals tend to express multiple IGFBPs in every tissue, and thus Dr John Pintar's six strains of IGFBP knock out mice demonstrate relatively normal phenotypes. [26][27][28] This might explain why six IGFBPs is the magic number.…”