Journal of Neurochemistry, 2001, 76, 806±814 Identi®cation of genes up-regulated by retinoic-acid-induced differentiation of the human neuronal precursor cell line NTERA-2 cl.D1 Frank Leypoldt,* Jan Lewerenz² and Axel Methner² *Zentrum fu Èr Molekulare Neurobiologie, Hamburg, Germany ²University Hospital Hamburg, Department of Neurology, Hamburg, Germany Abstract The human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up-regulated by this paradigm of neuronal differentiation. Micro®bril-associated glycoprotein 2 was found to be drastically up-regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identi®ed DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in speci®c defects in the development of the ¯y central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an ef®cient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identi®ed genes and expressed sequence tags. Keywords: dyrk4, gng3, magp2, neuronal differentiation, NTERA-2 cl.D1 (NT2) cells, suppression subtractive hybridization. J. Neurochem. (2001) 76, 806±814. Neurones and glia are derived from pluripotent precursor cells and differentiate in response to environmental and intrinsic factors. This process can be studied in vitro by retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells). NT2 cells remain as undifferentiated, but determined mitotic neuronal precursor cells, when maintained in medium supplemented only with fetal calf serum. Upon exposure to RA and the use of differential adhesion matrices and mitotic inhibitors, the cells develop the morphological and cytoskeletal characteristics of postmitotic CNS neurones (Pleasure and Lee 1993). A 4-week treatment with RA and successive replating in the presence of mitotic inhibitors for an additional 4 weeks resulted in the isolation of puri®ed neurones expressing neuronal markers like neuro®lament H, together with the loss of expression of neuroepithelial markers like nestin, present in the undifferentiated cells (Pleasure et al. 1992). This seems to model the in vivo situation, as high concentrations of RA have been detected in the embryonic CNS and the developing spinal cord (Rossant et al. 1991; Wagner et al. 1992; Horton and Maden 1995). RA binding proteins and its receptors are present in the developing nervous system, suggesting a role in neurogenesis. (Lyn and Giguere 1994; Maden et al. 1990; Ruberte et al. 1993; Zetterstrom et al. 1999). A drawback of the model is the time-consuming process and the tedious procedures of frequent dislodgement and replating, possibly leading to loss of neurone subtypes, such as those expressing tyrosine hydroxylase found in newly differentiated NT2 806 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 806±814 Received July 19, 2000; revised manuscript received September 13, 2000; accepted September 15, 2000. Abbreviations used: AP, alkaline phosphatase; CPH B, cyclophilin B; DMEM, Dulbecco's modi®ed Eagle's medium; EST, expression sequence tag; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GEF2, ganglioside expression factor 2; GNG3, guanine nucleotide binding protein g23 subunit; I, induced NT2 cells; MAGP2, micro®bril-associated glycoprotein 2; MMLV, moloney murine leukaemia virus; NT2, NTERA-2 cl.D1; RA, all trans retinoic acid; SDS, sodium dodecyl sulfate; SSH, suppression subtractive hybridization; U, uninduced NT2 cells.