ELSEVIER NeuroscienceLetters 200 (1995) 117-120 NEUROSCIEHC[ LETI[IS Expression of a MADS box gene, MEF2D, in neurons of the mouse central nervous system: implication of its binary function in myogenic and neurogenic cell lineages Hiroko Ikeshima a,*, Shin-ichiro Imai a, Kouji Shimoda c, Jun-ichi Hata b, Toshiya Takano a aDepartment of Microbiology, Keio UniversitySchool of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan bDepartment of Pathology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan CLaboratory of Animal Center, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Received 11 September 1995;revised version received6 October 1995;accepted9 October 1995 Abstract MEF2D, a member of myocyte-specific enhancer binding factor 2 (MEF2) gene family, was shown by Northern blot hybridization to be strongly expressed in the head portion of mouse embryos at later stages of ontogenesis, in the cerebellum and the cerebrum of adult mice, in cultured cell lines of neuronal origin, and in skeletal and cardiac muscles. During ontogenesis, MEF2D transcripts were detected by in situ hybridization in the olfactory bulb, entorhinal cortex, pyriform cortex, and hippocampus, in Purkinje and granule cells, and in large neurons in both the ventral and dorsal horns of the spinal cord. Adult mice continued to express MEF2D in these limited areas of the central nervous system. Thus, MEF2D seems to be involved in either the differentiation process or the function of these neurons. Keywords: In situ hybridization; MADS box; MEF2D; Neurogenesis; Northern blot hybridization; Central nervous system Members of the myocyte-specific enhancer binding factor 2 (MEF2) gene family have been identified in Dro- sophila, Xenopus, mouse, and human, and share signifi- cant homologies of amino acid sequences in MADS boxes, 80-amino acid domains conserved in MCM1, A gamous, Deficiens, and serum response factor, and in MEF2 domains [9,13-15,17,19]. Mhox, the mouse ho- mologue of the Phoxl homeodomain protein, and MEF2 seem to be involved in the regulation of the muscle cre- atine kinase gene [6,7]. The MADS box of MEF2A is necessary for binding to MyoD, a muscle-specific mem- ber of the helix-loop-helix protein family, and for deter- mining the myogenic lineage of cells [12]. Thus, MADS box proteins seem to regulate certain sets of cell type- specific genes by interacting with certain homeodomain proteins or other essential transcriptional factors, and the interaction of homeodomain and MADS box proteins seems to determine cellular identity during ontogenesis. The structure and function of the MEF2 gene family has * Corresponding author. Tel.: +81 3 33531211, extn. 2692; fax: +81 3 53601508. been extensively studied in cells of myogenic lineage [14, 17,19], but not in cells of neuronal lineage. To investigate MEF2D function during the development of neuronal tissue, we studied MEF2D expression in the central nerv- ous system (CNS) of embryonic, postnatal, and adult mice. The 1.0-kb MEF2D-specific region downstream of the MADS box and the MEF2 domain was amplified from the poly(A)÷ RNA of 8.5-embryonic day (E8.5) mouse embryos by polymerase chain reaction as described pre- viously [16] with the following primers: the 5' primer, 5'- AT AGA GAA qq'C CAT CTG TTC CGG CCC CCA ACT-3', and the 3' primer, 5'-TT TAA CTC GAG CAA AGT CCC CCC GTC CAT CAT-3'. The amplified frag- ment was used as the MEF2D-specific probe after label- ing with 32p as described previously [16]. Total RNA prepared from mouse embryos after shearing, from post- natal and adult mice after homogenizing, and from cul- tured cell lines by the guanidine-isothiocyanate method [5] was subjected to Northern blot analysis as described previously [16]. Although the amounts of 6.5-, 3.5- and 2.5-kb MEF2D RNA were less during early gestation 0304-3940/95/$09.50 © 1995Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95) 12092-F