Invivo effects of insulin-like growth factor-I (IGF-I) on prenatal and early postnatal development of the central nervous system GregoryJ.Popken, 1 RebeccaD.Hodge, 2 PingYe, 1 Jihui Zhang, 1 WinnieNg, 2 JohnR.O'Kusky 2 and A.JosephD'Ercole 1 1 Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7039, USA 2 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada Keywords: embryology, gene expression, neurogenesis, postnatal development, stereology, transgenic mice Abstract The in vivo actions of insulin-like growth factor-I (IGF-I) on prenatal and early postnatal brain development were investigated in transgenic (Tg) mice that overexpress IGF-I prenatally under the control of regulatory sequences from the nestin gene. Tg mice demonstrated increases in brain weight of 6% by embryonic day (E) 18 and 27% by postnatal day (P) 12. In Tg embryos at E16, the volume of the cortical plate was signi®cantly increased by 52% and total cell number was increased by 54%. S-phase labeling with 5- bromo-2 0 -deoxyuridine revealed a 13±15% increase in the proportion of labeled neuroepithelial cells in Tg embryos at E14. In Tg mice at P12, signi®cant increases in regional tissue volumes were detected in the cerebral cortex (29%), subcortical white matter (52%), caudate-putamen (37%), hippocampus (49%), dentate gyrus (71%) and habenular complex (48%). Tg mice exhibited signi®cant increasesinthetotalnumberofneuronsinthecerebralcortex(27%),caudate-putamen(27%),dentategyrus(69%),medialhabenular nucleus(61%)andlateralhabenularnucleus(36%).InthecerebralcortexandsubcorticalwhitematterofTgmice,thetotalnumbersof glial cells were signi®cantly increased by 37% and 42%, respectively. The numerical density of apoptotic cells in the cerebral cortex, labeled by antibodies against active caspase-3, was reduced by 26% in Tg mice at P7. Our results demonstrate that IGF-I can both promote proliferation of neural cells in the embryonic central nervous system in vivo and inhibit their apoptosis during postnatal life. Introduction Insulin-like growth factor-I (IGF-I) has been shown to promote the growth and development of the central nervous system (CNS) (for reviews, see Feldman etal., 1997; D'Ercole & Roberts, 1999; Anderson et al., 2002). IGF-I can in¯uence the proliferation (Ye et al., 1995a, 1996), differentiation (Brooker et al., 2000; Schechter & Abboud, 2001) and survival (Li et al., 1994; Gutierrez-Ospina et al., 1996; Chrysis et al., 2001) of neural cells. The majority of in vivo studies, using transgenic (Tg) mice with genetic alterations in the expression of IGF-I, the type 1 IGF receptor (IGF1R) or IGF binding proteins, have focused on IGF-I actions in the brain during postnatal development (for review, see D'Ercole et al., 2002). Multiple lines of Tg mice with increased expression of IGF-I exhibit brain overgrowth characterized by increased neuron and synapse number, increased myelination and enlarged neuropil volume (D'Ercole et al., 2002). Mice with null mutations in the IGF-I gene, as well as those with ablated expression of the IGF1R, exhibit both somatic and brain growth retardation initiated during prenatal development (Baker et al., 1993; Liu et al., 1993). IGF-I expression in the rodent brain begins on or before embryonic day (E) 14 (Bach et al., 1991). IGF-I is expressed by neural stem cells, even in the adult brain (Brooker etal., 2000). In early postimplantation embryos, IGF-I mRNA is highly expressed in undifferentiated mesen- chymal tissues in the vicinity of sprouting nerves (Bondy et al., 1990). Several regions of the CNS in rodents exhibit peak IGF-I mRNA expression from E16 to E21 (Bondy, 1991). Each of these studies points to IGF-I actions during embryonic nervous system development. To investigate potential IGF-I actions in vivo during embryonic and early postnatal brain development, we have developed a Tg mouse model that utilizes a regulatory sequence from the nestin gene to drive IGF-I overexpression from early in CNS development. Nestin, a 220-kDa class VI intermediate ®lament protein, is expressed by neural progenitor cells in most regions of the proliferating CNS (Hock®eld & McKay, 1985), and is dramatically reduced once cells have committed to a neuronal or glial fate (Zimmerman et al., 1994; Dahlstrand et al., 1995; Matsuda et al., 1996). Use of the nestin regulatory sequence is effective in directing the expression of transgenes from early in brain development. Stereological analyses were performed to investigate prenatal and early postnatal changes in selected brain regions of Tg and control mice. We found that IGF-I overexpression stimulates growth of the brain and increases neuron number, beginning prenatally and persist- ing in postnatal animals. Embryonic IGF-I transgene expression in these mice was initiated during the proliferative phase of neurogenesis and before the onset of neuron apoptosis. By labeling proliferating neuroepithelial cells in the ventricular and subventricular zones of E14 embryos with 5-bromo-2 0 -deoxyuridine (BrdU), we found that IGF-I accelerated mitosis in Tg mice. Judged by cells labeled with antibody against active caspase-3, IGF-I signi®cantly reduced naturally occur- ring neural cell death in the cerebral cortex of postnatal mice. Our results therefore demonstrate that IGF-I promotes brain growth during embryogenesis and early postnatal development apparently by both stimulating neural cell proliferation and inhibiting apoptosis. European Journal of Neuroscience, Vol. 19, pp. 2056±2068, 2004 ß Federation of European Neuroscience Societies doi:10.1111/j.1460-9568.2004.03320.x Correspondence: Dr A. J. D'Ercole, as above. E-mail: ajd@med.unc.edu Received 15 October 2003, revised 2 February 2004, accepted 10 February 2004