INTRODUCTION During embryonic development of the vertebrate nervous system a variety of neurons, with characteristic morphol- ogy, axonal projections and connectivity, arise at pre- dictable times and locations. For many neuronal types a wealth of information is available on the processes of axonal pathfinding and synapse formation, but less is known about the regulatory processes and molecules that are involved in the generation of different neurons. To under- stand the molecular mechanisms participating in the gener- ation of specific neuronal cell types we need to identify reg- ulatory proteins that are expressed at the initial stages of differentiation in these various neurons. One such class of proteins are the homeodomain proteins and a number of homeobox genes are expressed during the formation of the vertebrate central nervous system (CNS; Kessel and Gruss, 1990). The Hox genes are expressed at high levels in the mouse spinal cord and hindbrain and the temporal and spatial patterns of expression of these genes are consistent with a role in the definition of spatial domains in the CNS (Kessel and Gruss, 1991; Hunt et al., 1991). In contrast to the broad expression patterns of the Hox genes, a few homeobox genes show a much more restricted pat- tern of expression in the adult brain including the mid- and forebrain. These include members of the Dlx (Price et al., 1991), Pax (Krauss et al., 1991), Nkx (Price et al., 1992), Otx (Simeone et al., 1992a), and Emx families of genes (Simeone et al., 1992b). Islet-1 (Isl-1) is one of the three original members of the LIM homeodomain protein family (Way and Chalfie, 1988; Freyd et al., 1990; Karlsson et al., 1990). This family of proteins has now expanded and includes an increasing number of new members from many different species (Bourgouin et al., 1992, Cohen et al., 1992, Taira et al., 1992, Xu et al., 1993). A variety of proteins containing one or two LIM motifs but which lack a homeodomain have also been described (Greenberg et al., 1990). Isl-1 was orig- inally isolated as a protein that could bind to the insulin gene enhancer. We have previously analyzed the pattern of expression of Isl-1 in the adult rat using anti-Isl-1 antiserum (Thor et al., 1991). Isl-1 was found to be expressed in a subset of endocrine cells and neurons. In the central ner- vous system (CNS), Isl-1 expression is restricted to the parts of the brain involved in autonomic and endocrine control. Isl-1 is not expressed in regions involved in processing of sensory information and neocortical areas. Thus, Isl-1 appears to be selectively expressed in the phylogenetically old parts of the brain. In the chicken spinal cord Isl-1 has been identified as the earliest known marker of developing motor neurons and the onset of Isl-1 expression in the motor neurons can be 417 Development 118, 417-425 (1993) Printed in Great Britain © The Company of Biologists Limited 1993 Isl-1 has previously been established as the earliest marker of developing chicken spinal motor neurons where it is regulated by inductive signals from the floor- plate and notochord. We now report that, in zebrafish, the expression of Isl-1 is initiated in Rohon-Beard cells, primary motor neurons, interneurons and cranial gan- glia, hours before the neural tube itself is formed. The expression is initiated simultaneously in the Rohon- Beard cells and the primary motor neurons, at the axial level of the presumptive first somite. The Isl-1-express- ing motor neurons appear on either side of the ventral midline whereas the interneurons and Rohon-Beard cells initiate expression while located at the edge of the germinal shield. Isl-1 expression is initiated in these cells before the formation of a differentiated notochord. Isl-1 is expressed in the various functional classes of primary neurons at 24 hours postfertilization. This selective expression of a homeodomain protein in the primary neurons implies that these neurons share a common program of early development and that they have evolved and been selected for as a coordinated system. One of the functions of the primary neurons is to send long axons which pioneer the major axon tracts in the zebrafish embryo. An evolutionary conserved functional role for Isl-1 in the expression of the pio- neering phenotype of the primary neurons is suggested. Key words: LIM homeodomain, Islet-1, zebrafish, pioneering neurons, primary neurons SUMMARY Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation V. Korzh, T. Edlund* and S. Thor Department of Microbiology, University of Umeå, S-90187 Umeå, Sweden *Author for correspondence