Introduction Neurogenesis is considered to be the most complex event of organogenesis during embryonic development involving a precise signalling and cellular interaction cascade in order to generate a functional neural network. This comprises various subtypes of neurons, astroglia and oligodendroglia. Unlike the earlier belief that mammalian nervous system stem cells are confined to the embryo and that adult brain lacks the capacity to regenerate, recent studies demonstrate the presence of neural stem cells in both fetal and adult hypothalamus, olfactory bulb, subventricular zone and the dentate gyrus of hippocampus (Reynolds and Weiss, 1992; Gage, 2000; Bjornson et al., 1999; Doetsch et al., 1999; van der Kooy and Weiss, 2000). Since there is generally a recurrence of embryonic phenotypes in adults after injury (Laywell et al., 1992; Lendahl, 1997; Rossi et al., 1997), whether as a means of repair or merely a default state, it is mandatory to investigate the early embryonic events in order to understand the significance of this phenomenon. Hence, pluripotent embryonic stem (ES) cells recapitulating the in vivo events in a relatively precise manner may serve as an ideal model system for the investigation of early embryonic developmental processes (Okabe et al., 1996). In particular, tissue-specific promoter-mediated targeting is an efficient approach in the identification, isolation and functional characterization of lineage-specific populations in the heterogeneous cell mass of the ES-cell system (Kolossov et al., 1998). In the present study the ES cells have been targeted with neural-specific enhancer driving live reporter expression in order to understand early neurulation events. The intermediate filament protein, nestin, a well known marker for neural stem cells, is expressed in the majority of mitotically active CNS and PNS progenitors (Lendahl et al., 1990; Lendahl, 1997; Cattaneo and McKay, 1990; Mujtaba et al., 1998); it is downregulated upon differentiation (Zimmerman et al., 1994; Lothian and Lendahl, 1997) and reported to reappear upon injury (Lendahl, 1997; Krum and Rosenstein, 1999; Namiki and Tator, 1999; Pekny et al., 1999). Thus, the cells expressing nestin show all the characteristic features of stem cells such as multipotency, self-renewal and regeneration. Hence, nestin could serve as an efficient candidate marker gene in order to unravel early neurogenic proceedings from ES cells in vitro. Unlike Tα1 tubulin, the unipotent neuronal progenitor marker whose expression is confined only to the pre- and post-mitotic neurons (Wang et al., 1998; Roy et al., 2000a; Roy et al., 2000b), nestin represents a more broad spectral, multipotent neural lineage marker expressing not only in neurons but in glia as well (Hockfield and McKay, 1985; Messam et al., 2000). Thus, the 1471 To gain insight into early events of neurogenesis, transgenic embryonic stem (ES) cells were generated using the enhanced green fluorescence protein (EGFP) reporter gene under the regulatory control of the neural stem cell marker, nestin. The expression of EGFP in undifferentiated ES cells suggested that the onset of endogenous nestin occurred before neurulation. Upon differentiation of ES cells, the EGFP expression became confined to the neural lineage and asynchrony in ES-cell-derived neural differentiation was evident. The EGFP intensity was prominent in the proliferative progenitors and unipolar neurons, whereas downregulation occurred in differentiating bi- and multipolar neurons. This was corroborated quantitatively using flow cytometry where maximal generation of neural progenitors was observed 4-12 days post-plating. The proliferative potential of neural progenitors as well as glia, in contrast to post-mitotic neurons, was also evident by time-lapse microscopy. The functional characterization of progenitors revealed an absence of voltage-activated inward currents, whereas the Na + current (I Na ) was detected prior to Ca 2+ currents (I Ca ) in differentiating neurons. Additionally, inhibitory receptor-operated channels could be detected at these early stages of development in bi- and multipolar neurons suggesting that the pre-committed progenitors had retained their intrinsic ability to give rise to functional neurons. This study sheds new light on early events of neurogenesis defining the quantitative and qualitative aspects and demarcating the functional neural cell types from ES cells in vitro. Movies available on-line Key words: ES cells, Nestin, EGFP, Neural progenitor, Neurogenesis Summary Quantitation and functional characterization of neural cells derived from ES cells using nestin enhancer- mediated targeting in vitro Nibedita Lenka* ,‡ , Zhong J. Lu, Philipp Sasse, Jürgen Hescheler and Bernd K. Fleischmann Institute of Neurophysiology, University of Cologne, Cologne, Germany *Present address: National Centre For Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, Maharashtra, India ‡ Author for correspondence (e-mail: nibedital@yahoo.com) Accepted 13 January 2002 Journal of Cell Science 115, 1471-1485 (2002) © The Company of Biologists Ltd Research Article