The development of the mammalian central nervous system (CNS) is a remarkable phenomenon. Since the CNS is composed of millions of distinct neural cells, its complex and accurate functions must depend on the highly organized architecture of the cells that assemble in precise circuits. These distinct classes of cells composing the functional networks, are positioned at specific coordinates, in a precise number, with a spatial and temporal hierarchy. Researchers working in developmental neuroscience are still seeking to answer certain questions involving the control of diversity, migration, and connection of neural cells . Following this direction, investigators believe that the way to understand these mechanisms requires the analysis of differential gene expression in different developmental stages of the brain.
Among the large number of differential genes identified as important factors for regulating brain development at certain stages, we have found that the gene for insulin-like growth factor binding protein-4 (IGFBP-4) is expressed more highly in mature neurons than in neural precursors, indicating that IGFBP-4 may potentially act as a proneuronal differentiation factor . It is already known that IGFBP-4 is one member of the IGFBP family, which is mainly composed of six highly homologous proteins that bind insulin-like growth factors (IGFs) with high affinity to regulate its activity .
IGFBP-4 is the smallest IGFBP and is unique in having two extra Cys residues in the variable L-domain encoded by exon 2 . IGFBP-4 also contains an N-linked glycosylation site and commonly exists in biological fluids as a doublet: a 24-kDa nonglycosylated form and a 28-kDa glycosylated form . Authentic rat IGFBP-4 was a mixture of about 20% glycosylated and 80% non-glycosylated forms, and the glycosylation of IGFBP-4 does not affect its binding to IGFs . The physiological significance of the glycosylation in IGFBP-4 is unknown.
IGFBP-4 is an important physiological regulator of IGF actions in bone cells and other cell types as well [7, 8]. IGFBP-4 usually inhibits IGFs effects, but IGFs can decreased IGFBP-4 levels by activating an IGFBP-4-specific protease-PAPP-A, so IGFBP-4 may act as a potent inhibitor of the anabolic effects of IGF-I or -II by regulating IGF bioavailability . Previous data showed that IGFBP-4 is a pro- or anti-apoptotic factor that binds to an unknown membrane receptor and negatively or positively regulates apoptosis-induced factors. IGFBP-4 also may act by modulating the expression and secretion of other IGFBPs, such as IGFBP-3 and IGFBP-6 . Although still preliminary, until now, IGFBP-4 is the only IGFBP that, when deleted, alters cell growth .
IGF-IGFBP system is already recognized as central to processes of cell growth, differentiation, and migration [9, 12]. The clear importance of IGFs in the CNS development underscores the need for examining the expression and action of molecules, which are capable of regulating and mediating IGF activities. IGFBP-2, -4, and -5 are the most predominant IGFBPs in the brain . In general, IGFBP-2 and -5 were detected in same cells, whereas IGFBP-4 and -5 were expressed mainly in different cells, which suggests IGFBP-4 may have a specific function .
In view of the predominantly local activity of various proteins, the distribution of expression of IGFBP-4 may have a particular significance.
In the rat embryos of embryonic day 14 (E14), IGFBP-4 transcripts are expressed widely in non-neural areas of the head . In E15 rat embryos, IGFBP-4 transcripts are undetectable in other regions of brain except in the choroid plexus primordium and the meninges; the appearance of IGFBP-4 in brain parenchyma is observed from E20, and its expression is more widespread with increasing age . IGFBP-4 was found in all gross anatomical divisions of rat brain from E15 until adulthood (at E15, E20, postnatal day 1 (P1), P5, Adult), and IGFBP-4 mRNA tends to be more abundant at the youngest ages .
In the mouse embryo, IGFBP-4 transcripts can be detected as early as E11 in different regions, including the telencephalon, mesencephalon, snout, tongue, and differentiating sclerotomes; its mRNA is undetected in the brain after E14, but clearly detectable in the lung, liver, kidneys, intestine, and vertebrae (from E11 to E18). At E18, IGFBP-4 transcripts cannot be detected in choroid plexus and meninges . IGFBP-4 mRNA and protein are detected in the telencephalon, mesencephalon of E13.5 mouse embryos, and the consistence of localization patterns between IGFBP mRNA and protein may suggest that the IGFBP-4 functions in an autocrine or paracrine manner .
Previous findings suggest that IGFBP-4 expression may be regulated during brain development, but unfortunately, the precise contributions of IGFBP-4 to brain development are still not clear, since previous studies only selected limited time-points of IGFBP-4 temporal expression, and/or did not quantify IGFBP-4 expression. The present study therefore, aimed to examine precisely the temporal expression of IGFBP-4 at different developmental time-points in the rat brain, by using immunohistochemistry, quantitative real-time PCR, and Western blot. We hope this observation could provide a foundation for understanding the role of IGFBP-4 in brain development.