ONTOGENY OF CALMODULIN GENE EXPRESSION IN RAT BRAIN E. KORTVELY, 1 A. PALFI, 1 L. BAKOTA and K. GULYA  Department of Zoology and Cell Biology, University of Szeged, 2 Egyetem Street, P.O. Box 659, Szeged H-6722, Hungary AbstractöCalmodulin (CaM), a multifunctional intracellular calcium receptor, is a key element in signaling mechanisms. It is encoded in vertebrates by multiple apparently redundant genes (CaM I, II, III). To investigate whether di¡erential expression takes place in the developing rat brain, a quantitative in situ hybridization analysis was carried out involving 15brainareasatsixagesbetweenembryonicday19andpostnatalday20(PD20)withgene-speci¢c[ 35 S]cRNAprobes.A widespread,developmentalstage-speci¢canddi¡erentialexpressionofthethreeCaMgeneswasobserved.Thecharacter- istic changes in the CaM mRNA levels in the examined time frame allowed the brain regions to be classi¢ed into three categories. For the majority of the areas (e.g. the piriform cortex for CaM III), the signal intensities peaked at around PD10 and the expression pro¢le was symmetric (type 1). Other regions (e.g. the cerebral cortex, layer 1 for CaM II) displayed their highest signal intensities at the earliest age measured, followed by a gradual decrease (type 2). The signal intensities in the regions in the third group (e.g. the hypothalamus for CaM III) £uctuated from age to age (type 3). Marked CaM mRNA levels were measured for each transcript corresponding to the three CaM genes in the molecular layers of the cerebral and cerebellar cortici and hippocampus, suggesting their dendritic translocation. The highest signal intensity was measured for CaM II mRNA, followed by those for CaM III and CaM I mRNAs on PD1. However, the CaM II and CaM III mRNAs subsequently decreased steeply, while the CaM I mRNAs were readily detected even on PD20. Our results suggest that during development (1) the transcription of the CaM genes is under di¡erential, area- speci¢c control, and (2) a large population of CaM mRNAs is targeted to the dendritic compartment in a gene-speci¢c manner. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: development, quantitative in situ hybridization. Calmodulin(CaM)isamultifunctional,highlyconserved calcium ion-binding protein found in all eukaryotic cells (Cheung, 1980; Means et al., 1982; Chin and Means, 2000).Itregulatesseveralbasiccellularfunctions,includ- ing cell division, di¡erentiation, signal transduction, etc. (Meansetal.,1991;SweitzerandHanover,1996;Luket al., 1999), by interacting with a large and heterogenous array of target proteins (Kennedy, 1989). It is particu- larly abundant in the nervous tissue, where it is also involvedinthesynthesisandreleaseofneurotransmitters (DeLorenzo, 1980),inaxonaltransport(Kennedy, 1989), in long-term potentiation (Malenka et al., 1989) and in neuronal maturation (Polak et al., 1991). Although cer- tain glial cells contain considerable levels of CaM (Sola et al., 1997b, 1999), there is general agreement that within the CNS the vast majority of the protein is syn- thesized by neurons (Zhou et al., 1985), where it resides in several di¡erent intracellular pools (Caceres et al., 1983; Goldenring et al., 1984). In higher vertebrates, multiple CaM genes encode a single, highly conserved CaM protein. Three bona ¢de CaM genes (CaM I, CaM II and CaM III) expressed at high levels in the brain have been described in the rat (Nojima and Sokabe, 1986, 1987, 1989; Nojima et al., 1987; Sherbany et al., 1987; Nojima, 1989; Mori et al., 1994). The three CaM genes transcribe eight major mRNA species of di¡erent sizes by means of alternative polyadenylation, i.e. 4.0 kb, 1.7 kb, 1.65 kb, and 0.8 kb for CaM I, 1.4 kb for CaM II, and 2.3 kb, 1.9 kb and 0.9kbforCaMIII(Nojima,1989;Ikeshimaetal.,1993; Gannon and McEwen, 1994). Although the coding regions of the transcripts diverge only within the limits of degeneracy of the genetic code, their 5P- and 3P-untranslated regions are markedly di¡erent, suggest- ing a di¡erential regulation for the expression of the three CaM genes (Ni et al., 1992; Toutenhoofd and Strehler, 2000). Much e¡ort has gone into describing the exact pattern of the expression of the CaM genes. For example, their physiological (Sola et al., 1996; Pal¢ et al., 1999) and experimentally modi¢ed (Barron et al., 1995; Sola et al., 1997a; Pal¢ and Gulya, 1999; Vizi et al., 2000) expressions have been analyzed in adult (MacManus et al.,1989;GannonandMcEwen,1994;Pal¢etal.,1999) and embryonic tissues (MacManus et al., 1989; Cimino et al., 1990) and in di¡erent in vitro systems (Bai and Weiss, 1991; Bai et al., 1992; Zhang et al., 1993). Addi- tionally, stage-speci¢c changes in gene expression inten- sities have been detected during development and di¡erentiation (Slaughter and Means, 1989; Cimino et al., 1990; Weinman et al., 1991). For example, Cimino 301 1 These authors contributed equally to this work. *Corresponding author. Tel.: +36-62-544048; fax: +36-62-544049. E-mail address: gulyak@bio.u-szeged.hu (K. Gulya). Abbreviations: CaM, calmodulin; CaM-BP, calmodulin-binding protein;dlu,digitallightunit;E19,embryonicday19;PD,post- natal day; RT, room temperature; SSC, saline sodium citrate bu¡er. NSC 5750 15-8-02 www.neuroscience-ibro.com Neuroscience Vol. 114, No. 2, pp. 301^316, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain PII:S0306-4522(02)00317-2 0306-4522/02 $22.00+0.00