5629 Regulation of ngn 1 in the forebrain Materials and methods

In vertebrates, neurogenesis is a complex process that controls the position, number, connectivity and neurophysiological properties of thousands of different neurones. It has become obvious in recent years that many features of the molecular mechanisms and principles controlling neurogenesis have been conserved throughout animal evolution. The neurogenic processes in the peripheral nervous system of the fruit fly Drosophila melanogaster served as important paradigms to unravel the regulatory principles of vertebrate neurogenesis (Bertrand et al., 2002; Chitnis, 1999; Hassan and Bellen, 2000). The Drosophilaperipheral nervous system is specified by a hierarchical series of cell-fate decisions, in which prepattern genes first define regions, the so-called proneural clusters, that express proneural basic helix-loop-helix (bHLH) proteins and have the potential to develop into neurones (Chitnis, 1999). In the subsequent step of lateral inhibition that involves the Delta/Notch signalling system, committed neural precursors are selected from these proneural regions (Simpson, 1997). The regulatory events in the neural plate of lower vertebrates bear a strong resemblance to these patterning mechanisms in the imaginal discs of Drosophila. As in Drosophila, bHLH transcription factors related to the proneural genes demarcate regions of neuronal precursors, from which, by a Delta/Notch-mediated process, committed neurones are selected (Blader et al., 1997; Chitnis, 1999; Ma et al., 1996). Central, but less well understood questions concern how the spatial aspects of neurogenesis are controlled in the vertebrate neural plate/tube and how these mechanisms have been modified during vertebrate evolution. Neurogenins (ngns), which belong to the bHLH family and which are most closely related to the DrosophilabHLH factors Biparous and Atonal, demarcate the regions of primary neurogenesis in the neural plate of zebrafish and Xenopusembryos (Blader et al., 1997; Ma et al., 1996). Accumulating evidence suggests that these ngn1-expressing regions in the neural plate of lower vertebrates are specified by pre-pattern genes that provide positional codes with their large and overlapping domains of expression (BallyCuif and Hammerschmidt, 2003). Genes of the iroquoisfamily, members of which are involved in positioning the expression f the proneural genes achaete and scute in the Drosophila imaginal disc, have been suggested to play similar roles in the vertebrate neural plate (Bellefroid et al., 1998; Cheng et al., The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. Here, we show that the upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. Our data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6expression was crucial in neocortex evolution.