Europeari Journal zyxwvutsrqp of Neuroscience, zyxwvutsrqpo Vol. 6, zyxwvutsrq pp. 1801-18l0, zyxwvut 1994 0 European Neuroscience Association zy Insulin and Insulin-like Growth Factor System Components Gene Expression in the Chicken Retina From Early Neurogenesis Until Late Development and Their Effect on Neuroepithelial Cells Enrique J. de la Rosa1r2, Carolyn A. Bondy’, Catalina Hernandez-Sanchez2, Xin Wul, Jian Zhou’, Ana Lopez-Carranza2, Louis M. Scavo3 and Flora de Pablo2 ’Developmental Endocrinology Branch, NICHD, NIH, Bethesda, MD 20892, USA ‘Department Biologia Celular y del Desarrollo, Centro des lnvestigaciones Biologicas, CSIC, E-28006 Madrid, Spain 3University of California San Francisco, San Francisco, CA 94143, USA Key words: IGF-I receptor, IGF binding protein, insulin receptor, PM1 neural precursors, retinal ganglion cell, retinal culture Abstract To better understand the role of insulin-related growth factors in neural development, we have characterized by in sifu hybridization in chicken embryonic retina the patterns of gene expression for insulin, insulin-like growth factor zyxwvutsrqpon I (IGF-I), their respective receptors and the IGF binding protein 5 (IGFBP5) from early stages (E6) until late stages (El8)-an analysis not performed yet in any species. In addition, we studied the effect of insulin and IGF-I on cultured neuroepithelial cells. Insulin receptor mRNA and IGF-I receptor mRNA were both present and showed a similar, widespread pattern throughout retina development. Insulin mRNA could be detected only by reverse transcription coupled to polymerase chain reaction. IGF-I mRNA was concentrated in the ciliary processes and extraocular muscles early in development (embryonic day 6; E6) and in maturing retinal ganglion cells subsequently (E9-15). IGFBP5 mRNA was preferentially localized in the more differentiated central retinal zone and was maximally concentrated in the inner nuclear and ganglion cell layers at E9. These findings suggest a near constitutive expression of insulin receptor and IGF-I receptor genes, while IGF-I and IGFBP5 showed a highly focal spatiotemporal regulation of gene expression. Insulin and IGF-I, already at lo4 M, increased the proportion of PM1 -positive neuroepithelial cells found in E5 retinal cultures without affecting significantly the total number of proliferating cells. Together, these data support the finding that, during early neurogenesis in chicken retina, insulin and IGF-I have a specific paracrine/autocrineaction. This action, as well as possible effects elicited subsequently, may be dictated by restricted local synthesis of the ligands and limited access to the factors contained in the vitreous humour. In the case of IGF’s role, local IGFBPs expression can contribute to the fine modulation. Introduction Multiple environmental signals, including growth factors, potentially influence the processes of neuroepithelial cell proliferation and differentiation during early neurogenesis. The precise localization zyxwvuts of the sites of gene expression of factors and their receptors in develop- ment, together with the determination of specific effects on proliferat- ing and differentiating cell populations, should help to understand better the regulation of early neurogenesis. The present study was undertaken as a part of a comprehensive approach to further clarify the role of insulin and insulin-like growth factor I (IGF-I) in early neural development, using the chicken retina as a model system. The family of insulin and IGFs includes in vertebrates three structurally and functionally related polypeptides: insulin, IGF-I and IGF-II. One or more of these polypeptides and their mRNAs are expressed during embryonic development of the nervous system in several species (Sara et al., 1986; Rotwein et al., 1988; Serrano et al., 1990; Bondy, 1991). They have been shown to have multiple effects on cultured primary neural cells and neural cell lines at the level of proliferation, differentiation, maturation and survival (Lenoir and Honegger, 1983; Kyriakis et al., 1987; Shemer et al., 1987; McMoms and Dubois-Dalcq, 1988; Recio-Pinto and Ishii, 1988; Saneto et al., 1988; DiCicco-Bloom and Black, 1989; Grant et al., 1990; Knusel et al., 1990; Torres-Alemin et al., 1990, 1992; Pahlman et al., 1991; Barres et al., 1992; Tesoriere et al., 1992; Meghani et al., 1993), suggesting a major involvement of these polypeptidic factors in neural development. The biological actions of insulin and IGF-I are mediated by their specific binding to membrane tyrosine kinase insulin receptors and IGF-I receptors (see De Pablo et al., 1990, and references therein), which are structurally and functionally related. Both types Correspondence lo: zyxwvutsrqpo E. J. de la Rosa, Centro de Investigaciones Biol6gicas. CSIC, Velbzquez 144, E-28006 Madrid, Spain Received 21 February 1994. revised 27 April 1994, accepred 13 June 1994