DEVELOPMENTAL NEUROSCIENCE NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 11 No 17 27 November 2000 3823 Cx36 is dynamically expressed during early development of mouse brain and nervous system Massimo Gulisano  , Rosalba Parenti  , Francesca Spinella and Federico Cicirata CA Dipartimento di Scienze Fisiologiche, Universita` di Catania, Viale A. Doria 6, 95125, Catania, Italy CA Corresponding Author.  M. G. and R. P. contributed equally to this work. Received 22 August 2000; accepted 15 September 2000 Connexins are structural proteins that are part of the gap junctional channels which couple cells in different tissues. Connexin36 (Cx36) is a new member of the connexin gene family, found to be expressed essentially if not exclusively in neuronal cells in adult CNS of mouse, rat and man. Here we have studied Cx36 expression during murine embryonic development. Cx36 shows a highly dynamic pattern of expres- sion. It is first (E9.5) evident in the forebrain and later its expression expand caudally in the midbrain. At E12.5 its expression correlates with major morphogenetic boundaries in the developing mouse brain, specifically with the dorsoventral telencephalic boundary and the Zona Limitans Intrathalamica. Starting at midgestation (E12.5), it is also expressed in both sympathetic and spinal ganglia, and in two longitudinal stripes along the spinal cord. NeuroReport 11:3823–3828 & 2000 Lippincott Williams & Wilkins. Key words: Boundary; Connexin; Forebrain; Gap junction; Intercellular communication; Mouse embryogenesis; Spinal ganglia; ZLI INTRODUCTION Multicellular organisms require the coordinated response of group of cells to environmental stimuli. Intercellular channels present in gap junctions (GJs) provide a simple method of synchronizing response through the direct exchange of ions, metabolites and other messenger mole- cules between adjacent cells. These signaling pathways permit coordinated activities in rapid physiologic pro- cesses, such as the transmission of signals at electrical synapses [1,2], and in slower processes, such as cell growth and development [3]. GJs have been assumed to play a key role in neuromeric subdivision in the vertebrate neural tube [4], specifically in intercellular communication within the neuroepithelium [5]. In insects, segmental boundaries form barriers that isolate adjoining embryonic compartments [6]. GJs partici- pate in the early patterning of the amphibian neural tube [7] and it has been proposed that they may be responsible for the intercellular diffusion of morphogenetic molecules to one compartment [4]. Another possibility is that GJs are modulated, either in number or state, at newly formed boundaries, thus redefining pre-existent morphogenetic fields, and perhaps contributing to position-dependent fate specification. Thus, the analysis of topographic distribution of the neuronal gap junction in the neuroepithelium bears on the related mechanism of clonal restriction. The findings waited from the analysis, when compared to the stages of developing brain, could show a relationship between them. Gap junction channels are composed by two hemichan- nels, named connexons, each formed by six structural units called connexins (Cx). Each connexin protein forms gap junction that shows different electrophysiological proper- ties, channel permeability and phosphorylation. Recently a new member of the connexin gene family has been cloned and named Cx36 [8–10]. This Cx has been localized mainly in neuronal cells, unlike the other Cxs that are mainly localised in glial cells. For example Cx43 was reported in astrocytes and some neurons [11–13], Cx32 in oligodendro- cytes and some neurons [14] and Cx45 in oligodendrocytes [15]. Analysis of Cx36 mRNA distribution in adult animals revealed its intense expression in different brain regions and in the retina [8,9] with a precise topographic pattern [16]. In the present study, time of appearance, time-related changes and topographic distribution of the Cx36 during a time period ranging from blastula (E3,5) to midgestation were studied by using both radioactive and digoxigenin in situ hybridization on mouse embryos as well as by RT-PCR and Northern blotting, on total RNA. MATERIALS AND METHODS Embryos: Outbred CD-1 mice (Charles River) were mated between 21.00 h and 10.00 h. Day 0.5 pc was assumed to begin at the middle of the day of vaginal plugging. To enable a comparison of data collected by in situ and the Northern blotting, for each time interval, embryos used in the two experiments were taken from a single pregnant mice. RT-PCR analysis: Total RNA from mouse brain of the developmental stages analyzed were extracted using TRI-